Academic Appointments

Administrative Appointments

  • Executive Committee, Stanford Cardiovascular Institute (2010 - Present)
  • Co-Director, Cardiovascular Pulmonary Sciences Application (2005 - Present)

Honors & Awards

  • Department of Medicine Teaching Award, Stanford (2003)
  • Fellow, Arteriosclerosis, Thrombosis, and Vascular Biology Council of the American Heart Association (2003)
  • Established Investigator Award, American Heart Association (2008)

Professional Education

  • PhD, Thomas Jefferson University, Cardiovascular Physiology (1991)

Research & Scholarship

Current Research and Scholarly Interests

Our primary interests are in understanding the molecular underpinnings of vascular disease as well as assessing disease risk. We use a wide range of biochemical, molecular and physiological techniques to make primary observations in cell systems as well as preclinical models. Furthermore, we continue to extend our findings to human subjects in order to confirm their clinical applicability. Current research projects include:

Mechanisms regulating atherosclerosis and abdominal aortic aneurysm disease: While single genes can have dramatic effects in cellular biology, it is becoming increasingly clear that vascular disease (and health) is regulated by the coordinated expression of gene cassettes or pathways. By monitoring expression patterns of the entire genome simultaneously, we can begin to identify networks of genes that work in concert to affect disease progression. Moreover, this approach can often implicate specific nexus genes that are at the center of larger networks and/or participate in multiple pathways. Additionally, we are investigating the role microRNAs, a newly discovered class of small RNA molecues, in orchestrating the activity of multiple genes during the course of disease.

Role of insulin resistance: Reduced activity of the endogenous hormone, insulin, is now recognized as a cardinal feature of type 2 diabetes and an independent risk factor for cardiovascular disease. We have investigated the effects of insulin resistance in several tissues and have recently focused our attention on adipose tissue biology and how it relates to CVD. Long known as a storage vehicle for excess calories, the fat cell is now recognized to be a factory of different products that can not only affect local activity, but can circulate in the blood as hormones and regulate many biological processes. For example, we have recently reported that the novel hormone, apelin, is produced by fat tissue and has important effects upon insulin resistance, obesity and diabetes, all of which have significant implications for cardiovascular disease.

Biomarkers for risk assessment: In addition to target identification, we are applying transcriptional profiling and pathway analysis for another important aspect of cardiovascular disease management--biomarker discovery. As the name connotes, a biomarker should be a good indication of the disease state and thereby allow for early detection as well as monitoring disease progression and, hopefully, efficacy of an applied therapy. Biomarkers can encompass a wide range of molecules including DNA variants, RNA, proteins, as well as lipids. They can even encompass modalities such as molecular imaging. We are engaged in not only identifying novel biomarkers for cardiovascular disease, but also in producing algorithms that combine multiple biomarkers to optimally assess risk.

Clinical Trials

  • Exercise Therapy to Treat Adults With Abdominal Aortic Aneurysms Not Recruiting

    An abdominal aortic aneurysm (AAA) is a weakened and enlarged area in the abdominal aorta, which is a large blood vessel in the abdomen. If an AAA ruptures, it can be life-threatening. Research has shown that sedentary individuals are at increased risk of developing AAAs. This study will evaluate the effectiveness of an exercise program at limiting the growth of small AAAs in older individuals.

    Stanford is currently not accepting patients for this trial. For more information, please contact Ronald Dalman, (650) 723 - 2169.

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  • Effects of Glutathione (an Antioxidant) and N-Acetylcysteine on Inflammation Not Recruiting

    The rationale for the potential role of antioxidants in the prevention of cardiovascular diseases (CVD) remains strong despite the disappointing results of recent trials with a few select antioxidant vitamins. Glutathione (GSH) is one of the body's most powerful antioxidant agents but there is a surprising paucity of data on its use as an interventional therapy. Glutathione, when taken orally, is immediately broken down into its constituent amino acids, of which cysteine is the only one to be essential. Available cysteine is the critical determinant of intracellular GSH concentrations. N-acetyl cysteine (NAC) is an antioxidant supplement that has been used to provide a source of cysteine to replete GSH levels. By replenishing endogenous glutathione, it is possible that NAC would exert the same effect(s) as exogenous GSH. However, there is a new delivery system, liposomal GSH, which keeps glutathione intact. In this study, the investigators propose to match the cysteine content of NAC and GSH and compare the effects of these two supplements, at two different doses, on markers of inflammation and oxidative stress.

    Stanford is currently not accepting patients for this trial. For more information, please contact Antonella Dewell, (650) 736 - 8577.

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  • Effects of Omega-3 Fatty Acids on Markers of Inflammation Not Recruiting

    The major purpose of this study is to examine the effect of two sources of dietary omega-3 fatty acids, each given at two doses, on potential health benefits related to cardiovascular disease prevention. The two sources of dietary omega-3 fatty acids will be fish oil, and flaxseed oil.

    Stanford is currently not accepting patients for this trial. For more information, please contact Antonella Dewell, (650) 736 - 8577.

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  • Permission to Collect Blood Over Time for Research Not Recruiting

    To determine whether biomarkers assessed in blood samples can be used to detect individuals at risk for developing blood clots or worsening of their underlying disease. The ultimate goal of the study is to identify key biomarkers derived from blood that are most characteristic and informative of individuals who will go on to develop a clotting complication.

    Stanford is currently not accepting patients for this trial. For more information, please contact Fizaa Ahmed, 650-725-6409.

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  • Effects of Dietary Antioxidants on Cardiovascular Risk Factors Not Recruiting

    The aim of the Antioxidant Study was to compare the efficacy of foods naturally rich in antioxidants with that of antioxidants in a pill form on markers of inflammation and plasma cholesterol in healthy adults at risk of cardiovascular disease.

    Stanford is currently not accepting patients for this trial. For more information, please contact Antonella Dewell, (650) 736 - 8577.

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2017-18 Courses

Stanford Advisees

Graduate and Fellowship Programs


All Publications

  • Segmental Aortic Stiffening Contributes to Experimental Abdominal Aortic Aneurysm Development CIRCULATION Raaz, U., Zoellner, A. M., Schellinger, I. N., Toh, R., Nakagami, F., Brandt, M., Emrich, F. C., Kayama, Y., Eken, S., Adam, M., Maegdefessel, L., Hertel, T., Deng, A., Jagger, A., Buerke, M., Dalman, R. L., Spin, J. M., Kuhl, E., Tsao, P. S. 2015; 131 (20): 1783-1795


    Stiffening of the aortic wall is a phenomenon consistently observed in age and in abdominal aortic aneurysm (AAA). However, its role in AAA pathophysiology is largely undefined.Using an established murine elastase-induced AAA model, we demonstrate that segmental aortic stiffening precedes aneurysm growth. Finite-element analysis reveals that early stiffening of the aneurysm-prone aortic segment leads to axial (longitudinal) wall stress generated by cyclic (systolic) tethering of adjacent, more compliant wall segments. Interventional stiffening of AAA-adjacent aortic segments (via external application of surgical adhesive) significantly reduces aneurysm growth. These changes correlate with the reduced segmental stiffness of the AAA-prone aorta (attributable to equalized stiffness in adjacent segments), reduced axial wall stress, decreased production of reactive oxygen species, attenuated elastin breakdown, and decreased expression of inflammatory cytokines and macrophage infiltration, and attenuated apoptosis within the aortic wall, as well. Cyclic pressurization of segmentally stiffened aortic segments ex vivo increases the expression of genes related to inflammation and extracellular matrix remodeling. Finally, human ultrasound studies reveal that aging, a significant AAA risk factor, is accompanied by segmental infrarenal aortic stiffening.The present study introduces the novel concept of segmental aortic stiffening as an early pathomechanism generating aortic wall stress and triggering aneurysmal growth, thereby delineating potential underlying molecular mechanisms and therapeutic targets. In addition, monitoring segmental aortic stiffening may aid the identification of patients at risk for AAA.

    View details for DOI 10.1161/CIRCULATIONAHA.114.012377

    View details for Web of Science ID 000354610300015

    View details for PubMedID 25904646

    View details for PubMedCentralID PMC4439288

  • miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development NATURE COMMUNICATIONS Maegdefessel, L., Spin, J. M., Raaz, U., Eken, S. M., Toh, R., Azuma, J., Adam, M., Nagakami, F., Heymann, H. M., Chernugobova, E., Jin, H., Roy, J., Hultgren, R., Caidahl, K., Schrepfer, S., Hamsten, A., Eriksson, P., McConnell, M. V., Dalman, R. L., Tsao, P. S. 2014; 5


    Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.

    View details for DOI 10.1038/ncomms6214

    View details for Web of Science ID 000343982800003

    View details for PubMedCentralID PMC4217126

  • Pathogenesis of Abdominal Aortic Aneurysms: MicroRNAs, Proteases, Genetic Associations. Annual review of medicine Maegdefessel, L., Dalman, R. L., Tsao, P. S. 2014; 65: 49-62


    Abdominal aortic aneurysm (AAA) disease is a common, morbid, and highly lethal pathology. Extraordinary efforts have been launched to determine the molecular and pathophysiological characteristics of AAAs. Although surgery is highly effective in preventing death by rupture for larger AAAs, no guidance or preventive therapy is currently available for the >90% of patients whose aneurysms are below the surgical threshold. Predictive animal models of AAA as well as human pathological samples have revealed a complex circuit of AAA formation and progression. The proteolytic destruction of matrix components of the aorta by different proteases has been extensively studied over many years. Recently, a novel class of small noncoding RNAs, called microRNAs, was identified as "fine-tuners" of the translational output of target genes; they act by promoting mRNA degradation. Their therapeutic potential in limiting AAA development appears very intriguing. Further, current studies assessing genetic and heritable associations for AAA disease have provided great insight into its pathogenesis, potentially enabling us to better clinically manage affected patients.

    View details for DOI 10.1146/annurev-med-101712-174206

    View details for PubMedID 24274177

  • MicroRNA-21 Blocks Abdominal Aortic Aneurysm Development and Nicotine-Augmented Expansion SCIENCE TRANSLATIONAL MEDICINE Maegdefessel, L., Azuma, J., Toh, R., Deng, A., Merk, D. R., Raiesdana, A., Leeper, N. J., Raaz, U., Schoelmerich, A. M., McConnell, M. V., Dalman, R. L., Spin, J. M., Tsao, P. S. 2012; 4 (122)


    Identification and treatment of abdominal aortic aneurysm (AAA) remains among the most prominent challenges in vascular medicine. MicroRNAs are crucial regulators of cardiovascular pathology and represent possible targets for the inhibition of AAA expansion. We identified microRNA-21 (miR-21) as a key modulator of proliferation and apoptosis of vascular wall smooth muscle cells during development of AAA in two established murine models. In both models (AAA induced by porcine pancreatic elastase or infusion of angiotensin II), miR-21 expression increased as AAA developed. Lentiviral overexpression of miR-21 induced cell proliferation and decreased apoptosis in the aortic wall, with protective effects on aneurysm expansion. miR-21 overexpression substantially decreased expression of the phosphatase and tensin homolog (PTEN) protein, leading to increased phosphorylation and activation of AKT, a component of a pro-proliferative and antiapoptotic pathway. Systemic injection of a locked nucleic acid-modified antagomir targeting miR-21 diminished the pro-proliferative impact of down-regulated PTEN, leading to a marked increase in the size of AAA. Similar results were seen in mice with AAA augmented by nicotine and in human aortic tissue samples from patients undergoing surgical repair of AAA (with more pronounced effects observed in smokers). Modulation of miR-21 expression shows potential as a new therapeutic option to limit AAA expansion and vascular disease progression.

    View details for DOI 10.1126/scitranslmed.3003441

    View details for Web of Science ID 000300952100004

    View details for PubMedID 22357537

  • Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development JOURNAL OF CLINICAL INVESTIGATION Maegdefessel, L., Azuma, J., Toh, R., Merk, D. R., Deng, A., Chin, J. T., Raaz, U., Schoelmerich, A. M., Raiesdana, A., Leeper, N. J., McConnell, M. V., Dalman, R. L., Spin, J. M., Tsao, P. S. 2012; 122 (2): 497-506


    MicroRNAs (miRs) regulate gene expression at the posttranscriptional level and play crucial roles in vascular integrity. As such, they may have a role in modifying abdominal aortic aneurysm (AAA) expansion, the pathophysiological mechanisms of which remain incompletely explored. Here, we investigate the role of miRs in 2 murine models of experimental AAA: the porcine pancreatic elastase (PPE) infusion model in C57BL/6 mice and the AngII infusion model in Apoe-/- mice. AAA development was accompanied by decreased aortic expression of miR-29b, along with increased expression of known miR-29b targets, Col1a1, Col3a1, Col5a1, and Eln, in both models. In vivo administration of locked nucleic acid anti-miR-29b greatly increased collagen expression, leading to an early fibrotic response in the abdominal aortic wall and resulting in a significant reduction in AAA progression over time in both models. In contrast, overexpression of miR-29b using a lentiviral vector led to augmented AAA expansion and significant increase of aortic rupture rate. Cell culture studies identified aortic fibroblasts as the likely vascular cell type mediating the profibrotic effects of miR-29b modulation. A similar pattern of reduced miR-29b expression and increased target gene expression was observed in human AAA tissue samples compared with that in organ donor controls. These data suggest that therapeutic manipulation of miR-29b and its target genes holds promise for limiting AAA disease progression and protecting from rupture.

    View details for DOI 10.1172/JCI61598

    View details for Web of Science ID 000299765800016

    View details for PubMedID 22269326

  • Non-coding RNAs in aneurysmal aortopathy. Vascular pharmacology Spin, J. M., Li, D. Y., Maegdefessel, L., Tsao, P. S. 2018


    Aortic aneurysms represent a major public health burden, and currently have no medical treatment options. The pathophysiology behind these aneurysms is complex and variable, depending on location and underlying cause, and generally involves progressive dysfunction of all elements of the aortic wall. Changes in smooth muscle behavior, endothelial signaling, extracellular matrix remodeling, and to a variable extent inflammatory signaling and cells, all contribute to the dilation of the aorta, ultimately resulting in high mortality and morbidity events including dissection and rupture. A large number of researchers have identified non-coding RNAs as crucial regulators of aortic aneurysm development, both in humans and in animal models. While most work to-date has focused on microRNAs, intriguing information has also begun to emerge regarding the role of long-non-coding RNAs. This review summarizes the currently available data regarding the involvement of non-coding RNAs in aneurysmal aortopathies. Going forward, these represent key potential therapeutic targets that might be leveraged in the future to slow or prevent aortic aneurysm formation, progression and rupture.

    View details for DOI 10.1016/j.vph.2018.06.008

    View details for PubMedID 29909014

  • Apelin and APJ orchestrate complex tissue-specific control of cardiomyocyte hypertrophy and contractility in the hypertrophy-heart failure transition. American journal of physiology. Heart and circulatory physiology Parikh, V. N., Liu, J., Shang, C., Woods, C., Chang, A. C., Zhao, M., Charo, D. N., Grunwald, Z., Huang, Y., Seo, K., Tsao, P. S., Bernstein, D., Ruiz-Lozano, P., Quertermous, T., Ashley, E. A. 2018


    The G protein coupled receptor APJ is a promising therapeutic target for heart failure. Constitutive deletion of APJ in the mouse is protective against the hypertrophy-heart failure transition via elimination of ligand-independent, beta-arrestin dependent stretch transduction. However, the cellular origin of this stretch transduction and the details of its interaction with apelin signaling remain unknown. We generated mice with conditional elimination of APJ in the endothelium (APJendo-/-) and myocardium (APJmyo-/-). No baseline difference was observed in LV function in APJendo-/-, APJmyo-/- or controls (APJendo+/+, APJmyo+/+). After exposure to transaortic constriction (TAC), APJendo-/- animals developed left ventricular failure while APJmyo-/- were protected. At the cellular level, carbon fiber stretch of freshly isolated single cardiomyocytes demonstrated decreased contractile response to stretch in APJ-/- cardiomyocytes compared to APJ+/+ cardiomyocytes. Calcium transient did not change with stretch in either APJ-/- or APJ+/+ cardiomyocytes. Application of apelin to APJ+/+ cardiomyocytes resulted in decreased calcium transient. Further, hearts of mice treated with apelin exhibited decreased phosphorylation at Troponin I (cTnI) N-terminal residues (Ser 22,23), consistent with increased calcium sensitivity. These data establish that APJ stretch transduction is mediated specifically by myocardial APJ, that APJ is necessary for stretch-induced increases in contractility, and that apelin opposes APJ's stretch-mediated hypertrophy signaling by lowering calcium transient while maintaining contractility through myofilament calcium sensitization. These findings underscore apelin's unique potential as a therapeutic agent that can simultaneously support cardiac function and protect against the hypertrophy-heart failure transition.

    View details for DOI 10.1152/ajpheart.00693.2017

    View details for PubMedID 29775410

  • MicroRNA-Mediated Therapy Modulating Blood-Brain Barrier Disruption Improves Vascular Cognitive Impairment. Arteriosclerosis, thrombosis, and vascular biology Toyama, K., Spin, J. M., Deng, A. C., Huang, T., Wei, K., Wagenhauser, M. U., Yoshino, T., Nguyen, H., Mulorz, J., Kundu, S., Raaz, U., Adam, M., Schellinger, I. N., Jagger, A., Tsao, P. S. 2018


    OBJECTIVE: There are currently no effective treatments for the prevention of dementia associated with vascular cognitive impairment. MicroRNAs regulate gene expression at the post-transcriptional level and play key roles in vascular disorders. TNFalpha (tumor necrosis factor-alpha) regulates blood-brain barrier breakdown through modification of cerebral tight junctions. Here, we sought key TNFalpha-responsive microRNAs that might influence blood-brain barrier breakdown via cerebral tight junction disruption in vascular cognitive impairment.APPROACH AND RESULTS: Using a mouse model of vascular cognitive impairment, chronic cerebral hypoperfusion within the white matter was induced with bilateral common carotid artery stenosis (BCAS) surgery. TNFalpha gene expression was increased in white matter post-BCAS surgery, and TNFalpha stimulation decreased claudin-5, ZO-1 (tight-junction protein 1), and occludin gene expression in murine brain endothelial cells. In silico analysis predicted 8 candidate microRNAs as regulators of claudin-5, ZO-1, and occludin gene expression. Of these, only miR-501-3p was upregulated by TNFalpha in vitro and was upregulated in the white matter after BCAS surgery. Further, miR-501-3p directly bound to the 3'-untranslated region of human ZO-1 and downregulated transendothelial electric resistance. In vivo administration of a locked nucleic acid -modified antisense oligonucleotide versus miR-501-3p suppressed BCAS-induced reduction of ZO-1 gene expression and blood-brain barrier disruption within the white matter and significantly ameliorated working memory deficits after BCAS surgery.CONCLUSIONS: We here provide the first evidence that the TNFalpha-miR-501-3p-ZO-1 axis plays an important role in the pathogenesis of cerebral hypoperfusion-induced working memory deficits and white matter lesions, as a result of blood-brain barrier breakdown via tight junction disruption. Therapeutic manipulation of miR-501-3p holds promise for limiting vascular cognitive impairment progression.

    View details for DOI 10.1161/ATVBAHA.118.310822

    View details for PubMedID 29650692

  • Antioxidants from diet or supplements do not alter inflammatory markers in adults with cardiovascular disease risk. A pilot randomized controlled trial NUTRITION RESEARCH Dewell, A., Tsao, P., Rigdon, J., Gardner, C. D. 2018; 50: 63–72


    Antioxidants have been reported to have anti-inflammatory effects, but there is a lack of research comparing food to supplement antioxidant sources. The aim of this study was to determine if increases in intake of foods naturally rich in antioxidants would lower blood levels of inflammatory markers more than consuming antioxidant supplements among adults with cardiovascular disease risk factors. Eighty-eight generally healthy adults with ≥1 elevated risk factor for cardiovascular disease were randomized in a single-blind (diets)/double-blind (supplements), parallel-group study for 8 weeks. Participants consumed (1) usual diet and placebo pills (n = 29), (2) usual diet and antioxidant supplements (n = 29), or (3) antioxidant-rich foods closely matched to antioxidant content of supplements and placebo (n = 30). Usual diet combined with antioxidant supplements or increased antioxidant-rich food intake was designed to approximately double daily habitual antioxidant intake. Antioxidant pills included carotenoids, mixed tocopherols, vitamin C, and selenium. Fasting blood samples were analyzed for inflammatory marker concentrations of interleukin-6, monocyte chemotactic protein-1, and soluble intercellular adhesion molecule-1. Participants in the intervention groups successfully doubled most antioxidants as verified by diet records and elevated blood concentrations in treatment groups. Baseline levels of inflammatory markers for the entire study group were 110 ± 65 pg/mL for monocyte chemotactic protein-1, 0.9 ± 0.7 pg/mL for interleukin-6, and 217 ± 56 ng/mL for soluble intercellular adhesion molecule-1 (means ± standard deviation) and did not differ by treatment arm. After 8 weeks, there were no significant within-group changes or between-group 8-week change differences in inflammatory marker concentrations. In conclusion, no beneficial effects were detected on the inflammatory markers investigated in response to antioxidants from foods or supplements.

    View details for DOI 10.1016/j.nutres.2017.10.017

    View details for Web of Science ID 000428942800007

    View details for PubMedID 29540273

    View details for PubMedCentralID PMC5858717

  • GWAS of epigenetic aging rates in blood reveals a critical role for TERT NATURE COMMUNICATIONS Lu, A. T., Xue, L., Salfati, E. L., Chen, B. H., Ferrucci, L., Levy, D., Joehanes, R., Murabito, J. M., Kiel, D. P., Tsai, P., Yet, I., Bell, J. T., Mangino, M., Tanaka, T., McRae, A. F., Marioni, R. E., Visscher, P. M., Wray, N. R., Deary, I. J., Levine, M. E., Quach, A., Assimes, T., Tsao, P. S., Absher, D., Stewart, J. D., Li, Y., Reiner, A. P., Hou, L., Baccarelli, A. A., Whitsel, E. A., Aviv, A., Cardona, A., Day, F. R., Wareham, N. J., Perry, J. B., Ong, K. K., Raj, K., Lunetta, K. L., Horvath, S. 2018; 9: 387


    DNA methylation age is an accurate biomarker of chronological age and predicts lifespan, but its underlying molecular mechanisms are unknown. In this genome-wide association study of 9907 individuals, we find gene variants mapping to five loci associated with intrinsic epigenetic age acceleration (IEAA) and gene variants in three loci associated with extrinsic epigenetic age acceleration (EEAA). Mendelian randomization analysis suggests causal influences of menarche and menopause on IEAA and lipoproteins on IEAA and EEAA. Variants associated with longer leukocyte telomere length (LTL) in the telomerase reverse transcriptase gene (TERT) paradoxically confer higher IEAA (P < 2.7 × 10-11). Causal modeling indicates TERT-specific and independent effects on LTL and IEAA. Experimental hTERT-expression in primary human fibroblasts engenders a linear increase in DNA methylation age with cell population doubling number. Together, these findings indicate a critical role for hTERT in regulating the epigenetic clock, in addition to its established role of compensating for cell replication-dependent telomere shortening.

    View details for DOI 10.1038/s41467-017-02697-5

    View details for Web of Science ID 000423430900007

    View details for PubMedID 29374233

    View details for PubMedCentralID PMC5786029

  • Systemic Upregulation of IL-10 (Interleukin-10) Using a Nonimmunogenic Vector Reduces Growth and Rate of Dissecting Abdominal Aortic Aneurysm. Arteriosclerosis, thrombosis, and vascular biology Adam, M., Kooreman, N., Jagger, A., Wagenhaeuser, M. U., Mehrkens, D., Wang, Y., Kayama, Y., Toyama, K., Raaz, U., Schellinger, I. N., Maegdefessel, L., Spin, J. M., Hamming, J. F., Quax, P. H., Baldus, S., Wu, J. C., Tsao, P. S. 2018


    Recruitment of immunologic competent cells to the vessel wall is a crucial step in formation of abdominal aortic aneurysms (AAA). Innate immunity effectors (eg, macrophages), as well as mediators of adaptive immunity (eg, T cells), orchestrate a local vascular inflammatory response. IL-10 (interleukin-10) is an immune-regulatory cytokine with a crucial role in suppression of inflammatory processes. We hypothesized that an increase in systemic IL-10-levels would mitigate AAA progression.Using a single intravenous injection protocol, we transfected an IL-10 transcribing nonimmunogenic minicircle vector into the Ang II (angiotensin II)-ApoE-/- infusion mouse model of AAA. IL-10 minicircle transfection significantly reduced average aortic diameter measured via ultrasound at day 28 from 166.1±10.8% (control) to 131.0±5.8% (IL-10 transfected). Rates of dissecting AAA were reduced by IL-10 treatment, with an increase in freedom from dissecting AAA from 21.5% to 62.3%. Using flow cytometry of aortic tissue from minicircle IL-10-treated animals, we found a significantly higher percentage of CD4+/CD25+/Foxp3 (forkhead box P3)+ regulatory T cells, with fewer CD8+/Granzyme B+ cytotoxic T cells. Furthermore, isolated aortic macrophages produced less TNF-α (tumor necrosis factor-α), more IL-10, and were more likely to be MRC1 (mannose receptor, C type 1)-positive alternatively activated macrophages. These results concurred with gene expression analysis of LPS-stimulated and Ang II-primed human peripheral blood mononuclear cells.Taken together, we provide an effective gene therapy approach to AAA in mice by enhancing antiinflammatory and dampening proinflammatory pathways through minicircle-induced augmentation of systemic IL-10 expression.

    View details for DOI 10.1161/ATVBAHA.117.310672

    View details for PubMedID 29880489

  • Exome-wide association study of plasma lipids in > 300,000 individuals NATURE GENETICS Liu, D. J., Peloso, G. M., Yu, H., Butterworth, A. S., Wang, X., Mahajan, A., Saleheen, D., Emdin, C., Alam, D., Alves, A., Amouyel, P., Di Angelantonio, E., Arveiler, D., Assimes, T. L., Auer, P. L., Baber, U., Ballantyne, C. M., Bang, L. E., Benn, M., Bis, J. C., Boehnke, M., Boerwinkle, E., Bork-Jensen, J., Bottinger, E. P., Brandslund, I., Brown, M., Busonero, F., Caulfield, M. J., Chambers, J. C., Chasman, D. I., Chen, Y., Chen, Y., Chowdhury, R., Christensen, C., Chu, A. Y., Connell, J. M., Cucca, F., Cupples, L., Damrauer, S. M., Davies, G., Deary, I. J., Dedoussis, G., Denny, J. C., Dominiczak, A., Dube, M., Ebeling, T., Eiriksdottir, G., Esko, T., Farmaki, A., Feitosa, M. F., Ferrario, M., Ferrieres, J., Ford, I., Fornage, M., Franks, P. W., Frayling, T. M., Frikke-Schmidt, R., Fritsche, L. G., Frossard, P., Fuster, V., Ganesh, S. K., Gao, W., Garcia, M. E., Gieger, C., Giulianini, F., Goodarzi, M. O., Grallert, H., Grarup, N., Groop, L., Grove, M. L., Gudnason, V., Hansen, T., Harris, T. B., Hayward, C., Hirschhorn, J. N., Holmen, O. L., Huffman, J., Huo, Y., Hveem, K., Jabeen, S., Jackson, A. U., Jakobsdottir, J., Jarvelin, M., Jensen, G. B., Jorgensen, M. E., Jukema, J., Justesen, J. M., Kamstrup, P. R., Kanoni, S., Karpe, F., Kee, F., Khera, A. V., Klarin, D., Koistinen, H. A., Kooner, J. S., Kooperberg, C., Kuulasmaa, K., Kuusisto, J., Laakso, M., Lakka, T., Langenberg, C., Langsted, A., Launer, L. J., Lauritzen, T., Liewald, D. M., Lin, L., Linneberg, A., Loos, R. F., Lu, Y., Lu, X., Magi, R., Malarstig, A., Manichaikul, A., Manning, A. K., Mantyselka, P., Marouli, E., Masca, N. D., Maschio, A., Meigs, J. B., Melander, O., Metspalu, A., Morris, A. P., Morrison, A. C., Mulas, A., Mueller-Nurasyid, M., Munroe, P. B., Neville, M. J., Nielsen, J. B., Nielsen, S. F., Nordestgaard, B. G., Ordovas, J. M., Mehran, R., O'Donnell, C. J., Orho-Melander, M., Molony, C. M., Muntendam, P., Padmanabhan, S., Palmer, C. A., Pasko, D., Patel, A. P., Pedersen, O., Perola, M., Peters, A., Pisinger, C., Pistis, G., Polasek, O., Poulter, N., Psaty, B. M., Rader, D. J., Rasheed, A., Rauramaa, R., Reilly, D. F., Reiner, A. P., Renstrom, F., Rich, S. S., Ridker, P. M., Rioux, J. D., Robertson, N. R., Roden, D. M., Rotter, J. I., Rudan, I., Salomaa, V., Samani, N. J., Sanna, S., Sattar, N., Schmidt, E. M., Scott, R. A., Sever, P., Sevilla, R. S., Shaffer, C. M., Sim, X., Sivapalaratnam, S., Small, K. S., Smith, A. V., Smith, B. H., Somayajula, S., Southam, L., Spector, T. D., Speliotes, E. K., Starr, J. M., Stirrups, K. E., Stitziel, N., Strauch, K., Stringham, H. M., Surendran, P., Tada, H., Tall, A. R., Tang, H., Tardif, J., Taylor, K. D., Trompet, S., Tsao, P. S., Tuomilehto, J., Tybjaerg-Hansen, A., van Zuydam, N. R., Varbo, A., Varga, T. V., Virtamo, J., Waldenberger, M., Wang, N., Wareham, N. J., Warren, H. R., Weeke, P. E., Weinstock, J., Wessel, J., Wilson, J. G., Wilson, P. F., Xu, M., Yaghootkar, H., Young, R., Zeggini, E., Zhang, H., Zheng, N. S., Zhang, W., Zhang, Y., Zhou, W., Zhou, Y., Zoledziewska, M., Howson, J. M., Danesh, J., McCarthy, M. I., Cowan, C. A., Abecasis, G., Deloukas, P., Musunuru, K., Willer, C. J., Kathiresan, S., Charge Diabet Working Grp, EPIC-InterAct Consortium, EPIC-CVD Consortium, GOLD Consortium, VA Million Veteran Program 2017; 49 (12): 1758-+


    We screened variants on an exome-focused genotyping array in >300,000 participants (replication in >280,000 participants) and identified 444 independent variants in 250 loci significantly associated with total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C), low-density-lipoprotein cholesterol (LDL-C), and/or triglycerides (TG). At two loci (JAK2 and A1CF), experimental analysis in mice showed lipid changes consistent with the human data. We also found that: (i) beta-thalassemia trait carriers displayed lower TC and were protected from coronary artery disease (CAD); (ii) excluding the CETP locus, there was not a predictable relationship between plasma HDL-C and risk for age-related macular degeneration; (iii) only some mechanisms of lowering LDL-C appeared to increase risk for type 2 diabetes (T2D); and (iv) TG-lowering alleles involved in hepatic production of TG-rich lipoproteins (TM6SF2 and PNPLA3) tracked with higher liver fat, higher risk for T2D, and lower risk for CAD, whereas TG-lowering alleles involved in peripheral lipolysis (LPL and ANGPTL4) had no effect on liver fat but decreased risks for both T2D and CAD.

    View details for DOI 10.1038/ng.3977

    View details for Web of Science ID 000416480600016

    View details for PubMedID 29083408

    View details for PubMedCentralID PMC5709146

  • Phenotype and genotype analysis of metabolic liver disease in half million US Veterans enrolled in the Million Veteran Program (MVP) megabiobank Serper, M., Kaplan, D. E., Lynch, J., Lee, J., Damrauer, S., Miller, D. R., Shao, Q., Lee, K. M., Reaven, P., Huang, J., Wilson, P. W., O'Donnell, C. J., Rader, D. J., Tsao, P., Saleheen, D., Chang, K., VA Million Vet Program WILEY. 2017: 90A–91A
  • Hypoxia inducible factor stabilization improves defective ischemia-induced angiogenesis in a rodent model of chronic kidney disease. Kidney international Schellinger, I. N., Cordasic, N., Panesar, J., Buchholz, B., Jacobi, J., Hartner, A., Klanke, B., Jakubiczka-Smorag, J., Burzlaff, N., Heinze, E., Warnecke, C., Raaz, U., Willam, C., Tsao, P. S., Eckardt, K., Amann, K., Hilgers, K. F. 2017; 91 (3): 616-627


    Chronic kidney disease (CKD) is associated with increased risk and worse prognosis of cardiovascular disease, including peripheral artery disease. An impaired angiogenic response to ischemia may contribute to poor outcomes of peripheral artery disease in patients with CKD. Hypoxia inducible factors (HIF) are master regulators of angiogenesis and therefore represent a promising target for therapeutic intervention. To test this we induced hind-limb ischemia in rats with CKD caused by 5/6 nephrectomy and administered two different treatments known to stabilize HIF protein in vivo: carbon monoxide and a pharmacological inhibitor of prolyl hydroxylation 2-(1-chloro-4- hydroxyisoquinoline-3-carboxamido) acetate (ICA). Expression levels of pro-angiogenic HIF target genes (Vegf, Vegf-r1, Vegf-r2, Ho-1) were measured by qRT-PCR. Capillary density was measured by CD31 immunofluorescence staining and HIF expression was evaluated by immunohistochemistry. Capillary density in ischemic skeletal muscle was significantly lower in CKD animals compared to sham controls. Rats with CKD showed significantly lower expression of HIF and all measured pro-angiogenic HIF target genes, including VEGF. Both HIF stabilizing treatments rescued HIF target gene expression in animals with CKD and led to significantly higher ischemia-induced capillary sprouting compared to untreated controls. ICA was effective regardless of whether it was administered before or after induction of ischemia and led to a HIF expression in skeletal muscle. Thus, impaired ischemia-induced angiogenesis in rats with CKD can be improved by HIF stabilization, even if started after onset of ischemia.

    View details for DOI 10.1016/j.kint.2016.09.028

    View details for PubMedID 27927598

  • Epigenetic clock analysis of diet, exercise, education, and lifestyle factors. Aging Quach, A., Levine, M. E., Tanaka, T., Lu, A. T., Chen, B. H., Ferrucci, L., Ritz, B., Bandinelli, S., Neuhouser, M. L., Beasley, J. M., Snetselaar, L., Wallace, R. B., Tsao, P. S., Absher, D., Assimes, T. L., Stewart, J. D., Li, Y., Hou, L., Baccarelli, A. A., Whitsel, E. A., Horvath, S. 2017; 9 (2): 419-446


    Behavioral and lifestyle factors have been shown to relate to a number of health-related outcomes, yet there is a need for studies that examine their relationship to molecular aging rates. Toward this end, we use recent epigenetic biomarkers of age that have previously been shown to predict all-cause mortality, chronic conditions, and age-related functional decline. We analyze cross-sectional data from 4,173 postmenopausal female participants from the Women's Health Initiative, as well as 402 male and female participants from the Italian cohort study, Invecchiare nel Chianti.Extrinsic epigenetic age acceleration (EEAA) exhibits significant associations with fish intake (p=0.02), moderate alcohol consumption (p=0.01), education (p=3x10(-5)), BMI (p=0.01), and blood carotenoid levels (p=1x10(-5))-an indicator of fruit and vegetable consumption, whereas intrinsic epigenetic age acceleration (IEAA) is associated with poultry intake (p=0.03) and BMI (p=0.05). Both EEAA and IEAA were also found to relate to indicators of metabolic syndrome, which appear to mediate their associations with BMI. Metformin-the first-line medication for the treatment of type 2 diabetes-does not delay epigenetic aging in this observational study. Finally, longitudinal data suggests that an increase in BMI is associated with increase in both EEAA and IEAA.Overall, the epigenetic age analysis of blood confirms the conventional wisdom regarding the benefits of eating a high plant diet with lean meats, moderate alcohol consumption, physical activity, and education, as well as the health risks of obesity and metabolic syndrome.

    View details for DOI 10.18632/aging.101168

    View details for PubMedID 28198702

    View details for PubMedCentralID PMC5361673

  • Effect of Pioglitazone on Cardiometabolic Risk in Patients With Obstructive Sleep Apnea. American journal of cardiology Liu, A., Abbasi, F., Kim, S. H., Ariel, D., Lamendola, C., Cardell, J., Xu, S., Patel, S., Tomasso, V., Mojaddidi, H., Grove, K., Tsao, P. S., Kushida, C. A., Reaven, G. M. 2017


    Prevalence of insulin resistance is increased in patients with obstructive sleep apnea (OSA). Because insulin resistance is an independent predictor of cardiovascular disease (CVD), this study was initiated to see if pioglitazone administration would improve insulin sensitivity and thereby decrease risk of CVD in overweight/obese, nondiabetic, insulin-resistant patients with untreated OSA. Patients (n = 30) were administered pioglitazone (45 mg/day) for 8 weeks, and measurements were made before and after intervention of insulin action (insulin-mediated glucose uptake by the insulin suppression test), C-reactive protein, lipid/lipoprotein profile, and gene expression profile of periumbilical subcutaneous fat tissue. Insulin sensitivity increased 31% (p <0.001) among pioglitazone-treated subjects, associated with a decrease in C-reactive protein concentration (p ≤0.001), a decrease in plasma triglyceride, and increase in high-density lipoprotein cholesterol concentrations (p ≤0.001), accompanied by significant changes in apolipoprotein A1 and B concentrations and lipoprotein subclasses known to decrease CVD risk. In addition, subcutaneous adipose tissue gene expression profile showed a 1.6-fold (p <0.01) increase in GLUT4 expression and decreased expression in 5 of 9 inflammatory genes (p <0.05). In conclusion, enhanced insulin sensitivity can significantly decrease multiple cardiometabolic risk factors in patients with untreated OSA, consistent with the view that coexisting insulin resistance plays an important role in the association between OSA and increased risk of CVD.

    View details for DOI 10.1016/j.amjcard.2016.12.034

    View details for PubMedID 28219664

    View details for PubMedCentralID PMC5386603

  • A Pilot Study: The Beneficial Effects of Combined Statinexercise Therapy on Cognitive Function in Patients with Coronary Artery Disease and Mild Cognitive Decline INTERNAL MEDICINE Toyama, K., Sugiyama, S., Oka, H., Hamada, M., Iwasaki, Y., Horio, E., Rokutanda, T., Nakamura, S., Spin, J. M., Tsao, P. S., Ogawa, H. 2017; 56 (6): 641-649


    Objective Hypercholesterolemia, a risk factor in cognitive impairment, can be treated with statins. However, cognitive decline associated with "statins" (HMG-CoA reductase inhibitors) is a clinical concern. This pilot study investigated the effects of combining statins and regular exercise on cognitive function in coronary artery disease (CAD) patients with prior mild cognitive decline. Methods We recruited 43 consecutive CAD patients with mild cognitive decline. These patients were treated with a statin and weekly in-hospital aerobic exercise for 5 months. We measured serum lipids, exercise capacity, and cognitive function using the mini mental state examination (MMSE). Results Low-density lipoprotein cholesterol levels were significantly decreased, and maximum exercise capacity (workload) was significantly increased in patients with CAD and mild cognitive decline after treatment compared with before. Combined statin-exercise therapy significantly increased the median (range) MMSE score from 24 (22-25) to 25 (23-27) across the cohort (p<0.01). Changes in body mass index (BMI) were significantly and negatively correlated with changes in the MMSE. After treatment, MMSE scores in the subgroup of patients that showed a decrease in BMI were significantly improved, but not in the BMI-increased subgroup. Furthermore, the patients already on a statin at the beginning of the trial displayed a more significant improvement in MMSE score than statin-naïve patients, implying that exercise might be the beneficial aspect of this intervention as regards cognition. In a multivariate logistic regression analysis adjusted for age >65 years, sex, and presence of diabetes mellitus, a decrease in BMI during statin-exercise therapy was significantly correlated with an increase in the MMSE score (odds ratio: 4.57, 95% confidence interval: 1.05-20.0; p<0.05). Conclusion Statin-exercise therapy may help improve cognitive dysfunction in patients with CAD and pre-existing mild cognitive decline.

    View details for DOI 10.2169/internalmedicine.56.7703

    View details for Web of Science ID 000398893200010

    View details for PubMedID 28321063

  • Cloud-based Interactive Analytics for Terabytes of Genomic Variants Data Bioinformatics Pan, C., McInnes, G., Deflaux, N., Snyder, M. P., Bingham, J., Datta, S., Tsao, P. S. 2017: 3709–15


    Large scale genomic sequencing is now widely used to decipher questions in diverse realms such as biological function, human diseases, evolution, ecosystems, and agriculture. With the quantity and diversity these data harbor, a robust and scalable data handling and analysis solution is desired.We present interactive analytics using a cloud-based columnar database built on Dremel to perform information compression, comprehensive quality controls, and biological information retrieval in large volumes of genomic data. We demonstrate such Big Data computing paradigms can provide orders of magnitude faster turnaround for common genomic analyses, transforming long-running batch jobs submitted via a Linux shell into questions that can be asked from a web browser in seconds. Using this method, we assessed a study population of 475 deeply sequenced human genomes for genomic call rate, genotype and allele frequency distribution, variant density across the genome, and pharmacogenomic information.Our analysis framework is implemented in Google Cloud Platform and BigQuery. Codes are available at or data are available at Bioinformatics online.

    View details for DOI 10.1093/bioinformatics/btx468

    View details for PubMedCentralID PMC5860318

  • DNA methylation signatures of chronic low-grade inflammation are associated with complex diseases GENOME BIOLOGY Ligthart, S., Marzi, C., Aslibekyan, S., Mendelson, M. M., Conneely, K. N., Tanaka, T., Colicino, E., Waite, L. L., Joehanes, R., Guan, W., Brody, J. A., Elks, C., Marioni, R., Jhun, M. A., Agha, G., Bressler, J., Ward-Caviness, C. K., Chen, B. H., Huan, T., Bakulski, K., Salfati, E. L., Wahl, S., Schramm, K., Sha, J., Hernandez, D. G., Just, A. C., Smith, J. A., Sotoodehnia, N., Pilling, L. C., Pankow, J. S., Tsao, P. S., Liu, C., Zhao, W., Guarrera, S., Michopoulos, V. J., Smith, A. K., Peters, M. J., Melzer, D., Vokonas, P., Fornage, M., Prokisch, H., Bis, J. C., Chu, A. Y., Herder, C., Grallert, H., Yao, C., Shah, S., McRae, A. F., Lin, H., Horvath, S., Fallin, D., Hofman, A., Wareham, N. J., Wiggins, K. L., Feinberg, A. P., Starr, J. M., Visscher, P. M., Murabito, J. M., Kardia, S. L., Absher, D. M., Binder, E. B., Singleton, A. B., Bandinelli, S., Peters, A., Waldenberger, M., Matullo, G., Schwartz, J. D., Demerath, E. W., Uitterlinden, A. G., van Meurs, J. B., Franco, O. H., Chen, Y. I., Levy, D., Turner, S. T., Deary, I. J., Ressler, K. J., Dupuis, J., Ferrucci, L., Ong, K. K., Assimes, T. L., Boerwinkle, E., Koenig, W., Arnett, D. K., Baccarelli, A. A., Benjamin, E. J., Dehghan, A. 2016; 17


    Chronic low-grade inflammation reflects a subclinical immune response implicated in the pathogenesis of complex diseases. Identifying genetic loci where DNA methylation is associated with chronic low-grade inflammation may reveal novel pathways or therapeutic targets for inflammation.We performed a meta-analysis of epigenome-wide association studies (EWAS) of serum C-reactive protein (CRP), which is a sensitive marker of low-grade inflammation, in a large European population (n = 8863) and trans-ethnic replication in African Americans (n = 4111). We found differential methylation at 218 CpG sites to be associated with CRP (P < 1.15 × 10(-7)) in the discovery panel of European ancestry and replicated (P < 2.29 × 10(-4)) 58 CpG sites (45 unique loci) among African Americans. To further characterize the molecular and clinical relevance of the findings, we examined the association with gene expression, genetic sequence variants, and clinical outcomes. DNA methylation at nine (16%) CpG sites was associated with whole blood gene expression in cis (P < 8.47 × 10(-5)), ten (17%) CpG sites were associated with a nearby genetic variant (P < 2.50 × 10(-3)), and 51 (88%) were also associated with at least one related cardiometabolic entity (P < 9.58 × 10(-5)). An additive weighted score of replicated CpG sites accounted for up to 6% inter-individual variation (R2) of age-adjusted and sex-adjusted CRP, independent of known CRP-related genetic variants.We have completed an EWAS of chronic low-grade inflammation and identified many novel genetic loci underlying inflammation that may serve as targets for the development of novel therapeutic interventions for inflammation.

    View details for DOI 10.1186/s13059-016-1119-5

    View details for Web of Science ID 000390199000001

    View details for PubMedID 27955697

    View details for PubMedCentralID PMC5151130

  • Transcriptomic Profiling Maps Anatomically Patterned Subpopulations among Single Embryonic Cardiac Cells DEVELOPMENTAL CELL Li, G., Xu, A., Sim, S., Priest, J. R., Tian, X., Khan, T., Quertermous, T., Zhou, B., Tsao, P. S., Quake, S. R., Wu, S. M. 2016; 39 (4): 491-507


    Embryonic gene expression intricately reflects anatomical context, developmental stage, and cell type. To address whether the precise spatial origins of cardiac cells can be deduced solely from their transcriptional profiles, we established a genome-wide expression database from 118, 949, and 1,166 single murine heart cells at embryonic day 8.5 (e8.5), e9.5, and e10.5, respectively. We segregated these cells by type using unsupervised bioinformatics analysis and identified chamber-specific genes. Using a random forest algorithm, we reconstructed the spatial origin of single e9.5 and e10.5 cardiomyocytes with 92.0% ± 3.2% and 91.2% ± 2.8% accuracy, respectively (99.4% ± 1.0% and 99.1% ± 1.1% if a ±1 zone margin is permitted) and predicted the second heart field distribution of Isl-1-lineage descendants. When applied to Nkx2-5(-/-) cardiomyocytes from murine e9.5 hearts, we showed their transcriptional alteration and lack of ventricular phenotype. Our database and zone classification algorithm will enable the discovery of novel mechanisms in early cardiac development and disease.

    View details for DOI 10.1016/j.devcel.2016.10.014

    View details for Web of Science ID 000389162800013

    View details for PubMedID 27840109

  • DNA methylation-based measures of biological age: meta-analysis predicting time to death. Aging Chen, B. H., Marioni, R. E., Colicino, E., Peters, M. J., Ward-Caviness, C. K., Tsai, P., Roetker, N. S., Just, A. C., Demerath, E. W., Guan, W., Bressler, J., Fornage, M., Studenski, S., Vandiver, A. R., Moore, A. Z., Tanaka, T., Kiel, D. P., Liang, L., Vokonas, P., Schwartz, J., Lunetta, K. L., Murabito, J. M., Bandinelli, S., Hernandez, D. G., Melzer, D., Nalls, M., Pilling, L. C., Price, T. R., Singleton, A. B., Gieger, C., Holle, R., Kretschmer, A., Kronenberg, F., Kunze, S., Linseisen, J., Meisinger, C., Rathmann, W., Waldenberger, M., Visscher, P. M., Shah, S., Wray, N. R., McRae, A. F., Franco, O. H., Hofman, A., Uitterlinden, A. G., Absher, D., Assimes, T., Levine, M. E., Lu, A. T., Tsao, P. S., Hou, L., Manson, J. E., Carty, C. L., LaCroix, A. Z., Reiner, A. P., Spector, T. D., Feinberg, A. P., Levy, D., Baccarelli, A., van Meurs, J., Bell, J. T., Peters, A., Deary, I. J., Pankow, J. S., Ferrucci, L., Horvath, S. 2016; 8 (9): 1844-1865


    Estimates of biological age based on DNA methylation patterns, often referred to as "epigenetic age", "DNAm age", have been shown to be robust biomarkers of age in humans. We previously demonstrated that independent of chronological age, epigenetic age assessed in blood predicted all-cause mortality in four human cohorts. Here, we expanded our original observation to 13 different cohorts for a total sample size of 13,089 individuals, including three racial/ethnic groups. In addition, we examined whether incorporating information on blood cell composition into the epigenetic age metrics improves their predictive power for mortality. All considered measures of epigenetic age acceleration were predictive of mortality (p≤8.2x10(-9)), independent of chronological age, even after adjusting for additional risk factors (p<5.4x10(-4)), and within the racial/ethnic groups that we examined (non-Hispanic whites, Hispanics, African Americans). Epigenetic age estimates that incorporated information on blood cell composition led to the smallest p-values for time to death (p=7.5x10(-43)). Overall, this study a) strengthens the evidence that epigenetic age predicts all-cause mortality above and beyond chronological age and traditional risk factors, and b) demonstrates that epigenetic age estimates that incorporate information on blood cell counts lead to highly significant associations with all-cause mortality.

    View details for DOI 10.18632/aging.101020

    View details for PubMedID 27690265

    View details for PubMedCentralID PMC5076441

  • Role of microRNAs on Blood Brain Barrier Dysfunction in Vascular Cognitive Impairment. Current drug delivery Toyama, K., Spin, J. M., Tsao, P. S. 2016: -?


    Dementia cases are increasing as the population ages, leading to increased financial costs. Several neuronal diseases including ischemic and hemorrhagic stroke involve cerebrovascular injury or pathophysiology. Cerebrovascular injury is closely tied to blood brain barrier (BBB) disruption. Many studies have shown a significant association between BBB dysfunction and neurological diseases. Therefore, an understanding of the molecular mechanisms which regulate BBB permeability and disruption is essential for establishing future therapeutic strategies to alter dementia disease progression related to cerebrovascular injury,so-called vascular cognitive impairment (VCI). microRNAs (miRs) are small noncodingRNAs that regulate gene expression through targeting of mRNA transcripts.miRs have been implicated in the development and progression of various illnesses, including vascular disease. However, the role of miRs in BBB breakdown or permeability and VCI development has not yet been well clarified.Research content related to the origins of VCI and the role of the BBB in pathologic development and therapeutic targeting are reviewed, including current relevant animal models. We draw from the published literature regarding microRNA candidates that are associated with modulation of BBB structure and function.In this review, we summarize the current knowledge about VCI, explore the potential role of miRs in BBB breakdown and VCI progression, and identify potential candidate miRs for development of new treatment strategies.miRs constitute a promising novel avenue as future therapeutic options for alteration of both BBB permeability and development of VCI.

    View details for PubMedID 27572324

  • Does enhanced insulin sensitivity improve sleep measures in patients with obstructive sleep apnea: a randomized, placebo-controlled pilot study. Sleep medicine Liu, A., Kim, S. H., Ariel, D., Abbasi, F., Lamendola, C., Cardell, J., Xu, S., Patel, S., Tomasso, V., Mojaddidi, H., Grove, K., Tsao, P. S., Kushida, C. A., Reaven, G. M. 2016; 22: 57-60


    High fasting insulin levels have been reported to predict development of observed apneas, suggesting that insulin resistance may contribute to the pathogenesis of obstructive sleep apnea (OSA). The aim of this study was to determine whether enhancing insulin sensitivity in individuals with OSA would improve sleep measures.Insulin-resistant, nondiabetic individuals with untreated OSA were randomized (2:1) to pioglitazone (45 mg/day) or placebo for eight weeks in this single-blind study. All individuals had repeat measurements pertaining to sleep (overnight polysomnography and functional outcomes of sleep questionnaire) and insulin action (insulin suppression test).A total of 45 overweight/obese men and women with moderate/severe OSA were randomized to pioglitazone (n = 30) or placebo (n = 15). Although insulin sensitivity increased 31% among pioglitazone-treated compared with no change among individuals receiving placebo (p <0.001 for between-group difference), no improvement in quantitative or qualitative sleep measurements was observed.Pioglitazone administration increased insulin sensitivity in otherwise untreated individuals with OSA, without any change in polysomnographic sleep measures over an eight-week period. These findings do not support a causal role for insulin resistance in the pathogenesis of OSA.

    View details for DOI 10.1016/j.sleep.2016.06.005

    View details for PubMedID 27544837

  • Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene. journal of clinical investigation Knowles, J. W., Xie, W., Zhang, Z., Chennamsetty, I., Assimes, T. L., Paananen, J., Hansson, O., Pankow, J., Goodarzi, M. O., Carcamo-Orive, I., Morris, A. P., Chen, Y. I., Mäkinen, V., Ganna, A., Mahajan, A., Guo, X., Abbasi, F., Greenawalt, D. M., Lum, P., Molony, C., Lind, L., Lindgren, C., Raffel, L. J., Tsao, P. S., Schadt, E. E., Rotter, J. I., Sinaiko, A., Reaven, G., Yang, X., Hsiung, C. A., Groop, L., Cordell, H. J., Laakso, M., Hao, K., Ingelsson, E., Frayling, T. M., Weedon, M. N., Walker, M., Quertermous, T. 2016; 126 (1): 403-?

    View details for DOI 10.1172/JCI85921

    View details for PubMedID 26727231

  • Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression. PloS one Azuma, J., Wong, R. J., Morisawa, T., Hsu, M., Maegdefessel, L., Zhao, H., Kalish, F., Kayama, Y., Wallenstein, M. B., Deng, A. C., Spin, J. M., Stevenson, D. K., Dalman, R. L., Tsao, P. S. 2016; 11 (2)


    Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.

    View details for DOI 10.1371/journal.pone.0149288

    View details for PubMedID 26894432

    View details for PubMedCentralID PMC4760983

  • An epigenetic clock analysis of race/ethnicity, sex, and coronary heart disease. Genome biology Horvath, S., Gurven, M., Levine, M. E., Trumble, B. C., Kaplan, H., Allayee, H., Ritz, B. R., Chen, B., Lu, A. T., Rickabaugh, T. M., Jamieson, B. D., Sun, D., Li, S., Chen, W., Quintana-Murci, L., Fagny, M., Kobor, M. S., Tsao, P. S., Reiner, A. P., Edlefsen, K. L., Absher, D., Assimes, T. L. 2016; 17 (1): 171-?


    Epigenetic biomarkers of aging (the "epigenetic clock") have the potential to address puzzling findings surrounding mortality rates and incidence of cardio-metabolic disease such as: (1) women consistently exhibiting lower mortality than men despite having higher levels of morbidity; (2) racial/ethnic groups having different mortality rates even after adjusting for socioeconomic differences; (3) the black/white mortality cross-over effect in late adulthood; and (4) Hispanics in the United States having a longer life expectancy than Caucasians despite having a higher burden of traditional cardio-metabolic risk factors.We analyzed blood, saliva, and brain samples from seven different racial/ethnic groups. We assessed the intrinsic epigenetic age acceleration of blood (independent of blood cell counts) and the extrinsic epigenetic aging rates of blood (dependent on blood cell counts and tracks the age of the immune system). In blood, Hispanics and Tsimane Amerindians have lower intrinsic but higher extrinsic epigenetic aging rates than Caucasians. African-Americans have lower extrinsic epigenetic aging rates than Caucasians and Hispanics but no differences were found for the intrinsic measure. Men have higher epigenetic aging rates than women in blood, saliva, and brain tissue.Epigenetic aging rates are significantly associated with sex, race/ethnicity, and to a lesser extent with CHD risk factors, but not with incident CHD outcomes. These results may help elucidate lower than expected mortality rates observed in Hispanics, older African-Americans, and women.

    View details for DOI 10.1186/s13059-016-1030-0

    View details for PubMedID 27511193

  • Dietary fructose in pregnancy induces hyperglycemia, hypertension, and pathologic kidney and liver changes in a rodent model PREGNANCY HYPERTENSION-AN INTERNATIONAL JOURNAL OF WOMENS CARDIOVASCULAR HEALTH Shortliffe, L. M., Hammam, O., Han, X., Kouba, E., Tsao, P. S., Wang, B. 2015; 5 (4): 308-314

    View details for DOI 10.1016/j.preghy.2015.08.002

    View details for Web of Science ID 000366078600010

    View details for PubMedID 26597746

  • Diabetic Cardiovascular Disease Induced by Oxidative Stress INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Kayama, Y., Raaz, U., Jagger, A., Adam, M., Schellinger, I. N., Sakamoto, M., Suzuki, H., Toyama, K., Spin, J. M., Tsao, P. S. 2015; 16 (10): 25234-25263

    View details for DOI 10.3390/ijms161025234

    View details for Web of Science ID 000364232100112

    View details for PubMedID 26512646

  • Human Engineered Heart Muscles Engraft and Survive Long Term in a Rodent Myocardial Infarction Model. Circulation research Riegler, J., Tiburcy, M., Ebert, A., Tzatzalos, E., Raaz, U., Abilez, O. J., Shen, Q., Kooreman, N. G., Neofytou, E., Chen, V. C., Wang, M., Meyer, T., Tsao, P. S., Connolly, A. J., Couture, L. A., Gold, J. D., Zimmermann, W. H., Wu, J. C. 2015; 117 (8): 720-730


    Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure.Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model.We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring.EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

    View details for DOI 10.1161/CIRCRESAHA.115.306985

    View details for PubMedID 26291556

  • Local MicroRNA Modulation Using a Novel Anti-miR21-Eluting Stent Effectively Prevents Experimental In-Stent Restenosis ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Wang, D., Deuse, T., Stubbendorff, M., Chernogubova, E., Erben, R. G., Eken, S. M., Jin, H., Li, Y., Busch, A., Heeger, C., Behnisch, B., Reichenspurner, H., Robbins, R. C., Spin, J. M., Tsao, P. S., Schrepfer, S., Maegdefessel, L. 2015; 35 (9): 1945-1953
  • Local MicroRNA Modulation Using a Novel Anti-miR-21-Eluting Stent Effectively Prevents Experimental In-Stent Restenosis. Arteriosclerosis, thrombosis, and vascular biology Wang, D., Deuse, T., Stubbendorff, M., Chernogubova, E., Erben, R. G., Eken, S. M., Jin, H., Li, Y., Busch, A., Heeger, C., Behnisch, B., Reichenspurner, H., Robbins, R. C., Spin, J. M., Tsao, P. S., Schrepfer, S., Maegdefessel, L. 2015; 35 (9): 1945-1953


    Despite advances in stent technology for vascular interventions, in-stent restenosis (ISR) because of myointimal hyperplasia remains a major complication.We investigated the regulatory role of microRNAs in myointimal hyperplasia/ISR, using a humanized animal model in which balloon-injured human internal mammary arteries with or without stenting were transplanted into Rowett nude rats, followed by microRNA profiling. miR-21 was the only significantly upregulated candidate. In addition, miR-21 expression was increased in human tissue samples from patients with ISR compared with coronary artery disease specimen. We systemically repressed miR-21 via intravenous fluorescein-tagged-locked nucleic acid-anti-miR-21 (anti-21) in our humanized myointimal hyperplasia model. As expected, suppression of vascular miR-21 correlated dose dependently with reduced luminal obliteration. Furthermore, anti-21 did not impede reendothelialization. However, systemic anti-miR-21 had substantial off-target effects, lowering miR-21 expression in liver, heart, lung, and kidney with concomitant increase in serum creatinine levels. We therefore assessed the feasibility of local miR-21 suppression using anti-21-coated stents. Compared with bare-metal stents, anti-21-coated stents effectively reduced ISR, whereas no significant off-target effects could be observed.This study demonstrates the efficacy of an anti-miR-coated stent for the reduction of ISR.

    View details for DOI 10.1161/ATVBAHA.115.305597

    View details for PubMedID 26183619

    View details for PubMedCentralID PMC4552606

  • Transcription Factor Runx2 Promotes Aortic Fibrosis and Stiffness in Type 2 Diabetes Mellitus CIRCULATION RESEARCH Raaz, U., Schellinger, I. N., Chernogubova, E., Warnecke, C., Kayama, Y., Penov, K., Hennigs, J. K., Salomons, F., Eken, S., Emrich, F. C., Zheng, W. H., Adam, M., Jagger, A., Nakagami, F., Toh, R., Toyama, K., Deng, A., Buerke, M., Maegdefessel, L., Hasenfuss, G., Spin, J. M., Tsao, P. S. 2015; 117 (6): 513-524
  • Transcription Factor Runx2 Promotes Aortic Fibrosis and Stiffness in Type 2 Diabetes Mellitus. Circulation research Raaz, U., Schellinger, I. N., Chernogubova, E., Warnecke, C., Kayama, Y., Penov, K., Hennigs, J. K., Salomons, F., Eken, S., Emrich, F. C., Zheng, W. H., Adam, M., Jagger, A., Nakagami, F., Toh, R., Toyama, K., Deng, A., Buerke, M., Maegdefessel, L., Hasenfuß, G., Spin, J. M., Tsao, P. S. 2015; 117 (6): 513-524


    Accelerated arterial stiffening is a major complication of diabetes mellitus with no specific therapy available to date.The present study investigates the role of the osteogenic transcription factor runt-related transcription factor 2 (Runx2) as a potential mediator and therapeutic target of aortic fibrosis and aortic stiffening in diabetes mellitus.Using a murine model of type 2 diabetes mellitus (db/db mice), we identify progressive structural aortic stiffening that precedes the onset of arterial hypertension. At the same time, Runx2 is aberrantly upregulated in the medial layer of db/db aortae, as well as in thoracic aortic samples from patients with type 2 diabetes mellitus. Vascular smooth muscle cell-specific overexpression of Runx2 in transgenic mice increases expression of its target genes, Col1a1 and Col1a2, leading to medial fibrosis and aortic stiffening. Interestingly, increased Runx2 expression per se is not sufficient to induce aortic calcification. Using in vivo and in vitro approaches, we further demonstrate that expression of Runx2 in diabetes mellitus is regulated via a redox-sensitive pathway that involves a direct interaction of NF-κB with the Runx2 promoter.In conclusion, this study highlights Runx2 as a previously unrecognized inducer of vascular fibrosis in the setting of diabetes mellitus, promoting arterial stiffness irrespective of calcification.

    View details for DOI 10.1161/CIRCRESAHA.115.306341

    View details for PubMedID 26208651

    View details for PubMedCentralID PMC4553105

  • Levosimendan displays anti-inflammatory effects and decreases MPO bioavailability in patients with severe heart failure SCIENTIFIC REPORTS Adam, M., Meyer, S., Knors, H., Klinke, A., Radunski, U. K., Rudolph, T. K., Rudolph, V., Spin, J. M., Tsao, P. S., Costard-Jaeckle, A., Baldus, S. 2015; 5

    View details for DOI 10.1038/srep09704

    View details for Web of Science ID 000352724400001

    View details for PubMedID 25867530

  • Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene JOURNAL OF CLINICAL INVESTIGATION Knowles, J. W., Xie, W., Zhang, Z., Chennemsetty, I., Assimes, T. L., Paananen, J., Hansson, O., Pankow, J., Goodarzi, M. O., Carcamo-Orive, I., Morris, A. P., Chen, Y. I., Maekinen, V., Ganna, A., Mahajan, A., Guo, X., Abbasi, F., Greenawalt, D. M., Lum, P., Molony, C., Lind, L., Lindgren, C., Raffel, L. J., Tsao, P. S., Schadt, E. E., Rotter, J. I., Sinaiko, A., Reaven, G., Yang, X., Hsiung, C. A., Groop, L., Cordell, H. J., Laakso, M., Hao, K., Ingelsson, E., Frayling, T. M., Weedon, M. N., Walker, M., Quertermous, T. 2015; 125 (4): 1739-1751


    Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.

    View details for DOI 10.1172/JCI74592

    View details for Web of Science ID 000352248600037

    View details for PubMedID 25798622

  • MicroRNAs in Abdominal Aortic Aneurysm. Current vascular pharmacology Adam, M., Raaz, U., Spin, J. M., Tsao, P. S. 2015; 13 (3): 280-290


    Abdominal aortic aneurysms (AAA) are an important source of morbidity and mortality in the U.S. and worldwide. Treatment options are limited, with open surgery or endovascular repair remaining the only curative treatments. Classical cardiovascular medications have generally failed to prevent or significantly alter AAA formation or progression. Therefore, there is a tremendous need for better therapeutic approaches. With increasing knowledge of microRNA (miR) regulation in the context of cardiovascular disease, and with improving technical options permitting alteration of miR-expression levels in vitro and in vivo, we are offered a glimpse into the diagnostic and therapeutic possibilities of using miRs to treat vascular pathobiology. This review focuses on the role of miRs in aneurysmal disease of the abdominal aorta, summarizing recent publications regarding this topic, and outlining known effects of relevant miRs in AAA formation, including miR-21 and miR-29b. Despite there being only limited studies available, several other miRs also display clear potential for alteration of the disease process including miR-26a, the miR-17-92-cluster, miRs-221/222, miR-133 and miR-146a. While studies have shown that miRs can regulate the activity and interplay of vascular inflammatory cells, endothelial cells, smooth muscle cells and fibroblasts, all key elements leading to AAA formation, much work remains to be done.

    View details for PubMedID 23713862

  • Levosimendan displays anti-inflammatory effects and decreases MPO bioavailability in patients with severe heart failure. Scientific reports Adam, M., Meyer, S., Knors, H., Klinke, A., Radunski, U. K., Rudolph, T. K., Rudolph, V., Spin, J. M., Tsao, P. S., Costard-Jäckle, A., Baldus, S. 2015; 5: 9704-?


    Treatment of decompensated heart failure often includes administration of levosimendan. Myeloperoxidase (MPO) is released during polymorphonuclear neutrophil (PMN) degranulation, and mediates dysregulation of vascular tone in heart failure. We evaluated the effects of levosimendan-treatment on MPO in patients with acute decompensation of chronic heart failure over a one week course. Plasma MPO levels were significantly decreased after levosimendan treatment (from 252.1 ± 31.1 pmol/l at baseline to 215.02 ± 27.96 pmol/l at 6 h, p < 0.05). Ex vivo incubation of whole blood with levosimendan decreased MPO release after PMN-stimulation (8.2 ± 1.4-fold increase at baseline vs. 6.0 ± 1.1-fold increase with levosimendan). MPO levels also significantly correlated with diastolic blood pressure over the time course. In a multivariate linear model, the main contributor to systolic, diastolic and mean blood pressure was level of PMN elastase. MPO contributed only in heparin-treated patients, suggesting a more significant role for endothelial-bound MPO than for circulating MPO or elastase with respect to blood pressure regulation. We here provide the first evidence that levosimendan treatment inhibits MPO release by PMNs in decompensated heart failure patients. This mechanism may regulate endothelial function and vascular tone in heart failure patients.

    View details for DOI 10.1038/srep09704

    View details for PubMedID 25867530

    View details for PubMedCentralID PMC4394753

  • Erratum: miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development. Nature communications Maegdefessel, L., Spin, J. M., Raaz, U., Eken, S. M., Toh, R., Azuma, J., Adam, M., Nakagami, F., Heymann, H. M., Chernogubova, E., Jin, H., Roy, J., Hultgren, R., Caidahl, K., Schrepfer, S., Hamsten, A., Eriksson, P., McConnell, M. V., Dalman, R. L., Tsao, P. S. 2015; 6: 6506-?

    View details for DOI 10.1038/ncomms7506

    View details for PubMedID 25716653

  • Red blood cells serve as intravascular carriers of myeloperoxidase. Journal of molecular and cellular cardiology Adam, M., Gajdova, S., Kolarova, H., Kubala, L., Lau, D., Geisler, A., Ravekes, T., Rudolph, V., Tsao, P. S., Blankenberg, S., Baldus, S., Klinke, A. 2014; 74: 353-363


    Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localized MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme.

    View details for DOI 10.1016/j.yjmcc.2014.06.009

    View details for PubMedID 24976018

  • New ways to dismantle a ticking time bomb: microRNA 712/205 and abdominal aortic aneurysm development. Arteriosclerosis, thrombosis, and vascular biology Maegdefessel, L., Spin, J. M., Tsao, P. S. 2014; 34 (7): 1339-1340

    View details for DOI 10.1161/ATVBAHA.114.303952

    View details for PubMedID 24951652

  • Dichloroacetate prevents restenosis in preclinical animal models of vessel injury. Nature Deuse, T., Hua, X., Wang, D., Maegdefessel, L., Heeren, J., Scheja, L., Bolaños, J. P., Rakovic, A., Spin, J. M., Stubbendorff, M., Ikeno, F., Länger, F., Zeller, T., Schulte-Uentrop, L., Stoehr, A., Itagaki, R., Haddad, F., Eschenhagen, T., Blankenberg, S., Kiefmann, R., Reichenspurner, H., Velden, J., Klein, C., Yeung, A., Robbins, R. C., Tsao, P. S., Schrepfer, S. 2014; 509 (7502): 641-644


    Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (ΔΨm) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ΔΨm hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ΔΨm and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.

    View details for DOI 10.1038/nature13232

    View details for PubMedID 24747400

  • Dichloroacetate prevents restenosis in preclinical animal models of vessel injury. Nature Deuse, T., Hua, X., Wang, D., Maegdefessel, L., Heeren, J., Scheja, L., Bolaños, J. P., Rakovic, A., Spin, J. M., Stubbendorff, M., Ikeno, F., Länger, F., Zeller, T., Schulte-Uentrop, L., Stoehr, A., Itagaki, R., Haddad, F., Eschenhagen, T., Blankenberg, S., Kiefmann, R., Reichenspurner, H., Velden, J., Klein, C., Yeung, A., Robbins, R. C., Tsao, P. S., Schrepfer, S. 2014; 509 (7502): 641-644

    View details for DOI 10.1038/nature13232

    View details for PubMedID 24747400

  • Hemodynamic regulation of reactive oxygen species: implications for vascular diseases. Antioxidants & redox signaling Raaz, U., Toh, R., Maegdefessel, L., Adam, M., Nakagami, F., Emrich, F. C., Spin, J. M., Tsao, P. S. 2014; 20 (6): 914-928


    Significance: Arterial blood vessels functionally and structurally adapt to altering hemodynamic forces in order to accommodate changing needs and to provide stress homeostasis. This ability is achieved at the cellular level by converting mechanical stimulation into biochemical signals (i.e., mechanotransduction). Whereas physiological mechanical stress helps to maintain vascular structure and function, pathologic or aberrant stress may impair cellular mechano-signaling, and initiate or augment cellular processes which drive disease. Recent advances: Reactive oxygen species (ROS) may represent an intriguing class of mechanically- regulated second messengers. Chronically enhanced ROS-generation may be induced by adverse mechanical stresses, and is associated with a multitude of vascular diseases. Although a causal relationship has clearly been demonstrated in large numbers of animal studies, an effective ROS-modulating therapy still remains to be established by clinical studies. Critical issues and Future directions: This review article focuses on the role of various mechanical forces (in the form of laminar shear stress, oscillatory shear stress or cyclic stretch) as modulators of ROS- driven signaling, and their subsequent effects on vascular biology and homeostasis, as well as on specific diseases such as arteriosclerosis, hypertension and abdominal aortic aneurysms. Specifically, it highlights the significance of the various NADPH oxidase (NOX) isoforms as critical ROS generators in the vasculature. Directed targeting of defined components in the complex network of ROS (mechano)signaling may represent a key for successful translation of experimental findings into clinical practice.

    View details for DOI 10.1089/ars.2013.5507

    View details for PubMedID 23879326

  • MicroRNA-29b regulation of abdominal aortic aneurysm development TRENDS IN CARDIOVASCULAR MEDICINE Maegdefessel, L., Azuma, J., Tsao, P. S. 2014; 24 (1): 1-6


    Tremendous efforts have been initiated to elucidate the molecular and pathophysiological characteristics of abdominal aortic aneurysm (AAA) disease, which is a significant contributor to morbidity and mortality in the Western world. Recently, a novel class of small noncoding RNAs, called microRNAs, was identified as important transcriptional and posttranscriptional inhibitors of gene expression thought to simultaneously "fine tune" the translational output of multiple target messenger RNAs (mRNAs) by promoting mRNA degradation or inhibiting translation. Several research groups were able to identify the miR-29 family, and miR-29b in particular, as crucial regulators of-not only vascular fibrosis-but also cardiac-, kidney-, liver-, and skin-fibrosis. The current review briefly points out data indicating a causal role for miR-29 in various diseases, while focusing on its potential benefit during AAA initiation and propagation.

    View details for DOI 10.1016/j.tcm.2013.05.002

    View details for Web of Science ID 000329384000001

    View details for PubMedID 23871588

  • miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development. Nature communications Maegdefessel, L., Spin, J. M., Raaz, U., Eken, S. M., Toh, R., Azuma, J., Adam, M., Nakagami, F., Heymann, H. M., Chernogubova, E., Jin, H., Roy, J., Hultgren, R., Caidahl, K., Schrepfer, S., Hamsten, A., Eriksson, P., McConnell, M. V., Dalman, R. L., Tsao, P. S. 2014; 5: 5214-?


    Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.

    View details for DOI 10.1038/ncomms6214

    View details for PubMedID 25358394

    View details for PubMedCentralID PMC4217126

  • Micromanaging Abdominal Aortic Aneurysms INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Maegdefessel, L., Spin, J. M., Adam, M., Raaz, U., Toh, R., Nakagami, F., Tsao, P. S. 2013; 14 (7): 14374-14394


    The contribution of abdominal aortic aneurysm (AAA) disease to human morbidity and mortality has increased in the aging, industrialized world. In response, extraordinary efforts have been launched to determine the molecular and pathophysiological characteristics of the diseased aorta. This work aims to develop novel diagnostic and therapeutic strategies to limit AAA expansion and, ultimately, rupture. Contributions from multiple research groups have uncovered a complex transcriptional and post-transcriptional regulatory milieu, which is believed to be essential for maintaining aortic vascular homeostasis. Recently, novel small noncoding RNAs, called microRNAs, have been identified as important transcriptional and post-transcriptional inhibitors of gene expression. MicroRNAs are thought to "fine tune" the translational output of their target messenger RNAs (mRNAs) by promoting mRNA degradation or inhibiting translation. With the discovery that microRNAs act as powerful regulators in the context of a wide variety of diseases, it is only logical that microRNAs be thoroughly explored as potential therapeutic entities. This current review summarizes interesting findings regarding the intriguing roles and benefits of microRNA expression modulation during AAA initiation and propagation. These studies utilize disease-relevant murine models, as well as human tissue from patients undergoing surgical aortic aneurysm repair. Furthermore, we critically examine future therapeutic strategies with regard to their clinical and translational feasibility.

    View details for DOI 10.3390/ijms140714374

    View details for Web of Science ID 000322171700085

    View details for PubMedID 23852016

  • Measurement of insulin-mediated glucose uptake: direct comparison of the modified insulin suppression test and the euglycemic, hyperinsulinemic clamp. Metabolism Knowles, J. W., Assimes, T. L., Tsao, P. S., Natali, A., Mari, A., Quertermous, T., Reaven, G. M., Abbasi, F. 2013; 62 (4): 548-553


    Two direct measurements of peripheral insulin sensitivity are the M value derived from the euglycemic, hyperinsulinemic clamp (EC) and the steady-state plasma glucose (SSPG) concentration derived from the insulin suppression test (IST). Prior work suggests that these measures are highly correlated, but the agreement between them is unknown. To determine the agreement between SSPG and M and to develop transformation equations to convert SSPG to M and vice versa, we directly compared these two measurements in the same individuals.A total of 15 nondiabetic subjects (9 women and 6 men) underwent both an EC and a modified version of the IST within a median interval of 5days. We performed standard correlation metrics of the two measures and developed transformation regression equations for the two measures.The mean±SD age of the subjects was 57±7years and body mass index, 27.7±3.9kg/m(2). The median (interquartile range) SSPG concentration was 6.7 (5.1, 9.8) mmol/L and M value, 49.6 (28.9, 64.2) μmol/min/kg-LBM. There was a highly significant correlation between SSPG and M (r=-0.87, P <0.001). The relationship was best fit by regression models with exponential/logarithmic functions (R(2)=0.85). Bland-Altman plots demonstrated an excellent agreement between these measures of insulin action.The SSPG and M are highly related measures of insulin sensitivity and the results provide the means to directly compare the two measurements.

    View details for DOI 10.1016/j.metabol.2012.10.002

    View details for PubMedID 23151437

  • MicroRNA-24 controls abdominal aortic aneurysm development through regulation of YKL-40 Maegdefessel, L., Raaz, U., Adam, M., Spin, J., Eriksson, P., Hamsten, A., Tsao, P. SPRINGER. 2013: 10–10
  • Loss of CDKN2B promotes p53-dependent smooth muscle cell apoptosis and aneurysm formation. Arteriosclerosis, thrombosis, and vascular biology Leeper, N. J., Raiesdana, A., Kojima, Y., Kundu, R. K., Cheng, H., Maegdefessel, L., Toh, R., Ahn, G., Ali, Z. A., Anderson, D. R., Miller, C. L., Roberts, S. C., Spin, J. M., de Almeida, P. E., Wu, J. C., Xu, B., Cheng, K., Quertermous, M., Kundu, S., Kortekaas, K. E., Berzin, E., Downing, K. P., Dalman, R. L., Tsao, P. S., Schadt, E. E., Owens, G. K., Quertermous, T. 2013; 33 (1): e1-e10


    Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear.Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow-derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model.These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

    View details for DOI 10.1161/ATVBAHA.112.300399

    View details for PubMedID 23162013

    View details for PubMedCentralID PMC3569043

  • Loss of CDKN2B Promotes p53-Dependent Smooth Muscle Cell Apoptosis and Aneurysm Formation ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Leeper, N. J., Raiesdana, A., Kojima, Y., Kundu, R. K., Cheng, H., Maegdefessel, L., Toh, R., Ahn, G., Ali, Z. A., Anderson, D. R., Miller, C. L., Roberts, S. C., Spin, J. M., de Almeida, P. E., Wu, J. C., Xu, B., Cheng, K., Quertermous, M., Kundu, S., Kortekaas, K. E., Berzin, E., Downing, K. P., Dalman, R. L., Tsao, P. S., Schadt, E. E., Owens, G. K., Quertermous, T. 2013; 33 (1): E1-?


    Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear.Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow-derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model.These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

    View details for DOI 10.1161/ATVBAHA.112.300399

    View details for Web of Science ID 000312392500001

    View details for PubMedID 23162013

    View details for PubMedCentralID PMC3569043

  • Prospective Transcriptomic Pathway Analysis of Human Lymphatic Vascular Insufficiency: Identification and Validation of a Circulating Biomarker Panel PLOS ONE Lin, S., Kim, J., Lee, M., Roche, L., Yang, N. L., Tsao, P. S., Rockson, S. G. 2012; 7 (12)


    In our previous transcriptional profiling of a murine model, we have identified a remarkably small number of specific pathways with altered expression in lymphedema. In this investigation, we utilized microarray-based transcriptomics of human skin for an unbiased a priori prospective candidate identification, with subsequent validation of these candidates through direct serum assay. The resulting multi-analyte biomarker panel sensitively should sensitively discriminate human lymphedema subjects from normal individuals.We enrolled 63 lymphedema subjects and 27 normals in our attempt to discover protein analytes that can distinguish diseased individuals from controls. To minimize technical and biologically irrelevant variation, we first identified potential candidates by performing transcriptional microarray analysis on paired diseased and normal skin specimens sampled from the same individuals. We focused our attention on genes with corresponding protein products that are secreted and took these candidates forward to a protein multiplex assay applied to diseased and normal subjects. We developed a logistic regression-based model on an eventual group of six proteins and validated our system on a separate cohort of study subjects. The area under the receiver operating characteristic curve was calculated to be 0.87 (95% CI : 0.75 to 0.97).We have developed an accurate bioassay utilizing proteins representing four central pathogenetic modalities of the disease: lymphangiogenesis, inflammation, fibrosis, and lipid metabolism, suggesting that these proteins are directly related to the pathogenesis of the tissue pathology in lymphatic vascular insufficiency. Further studies are warranted to determine whether this newly-identified biomarker panel will possess utility as an instrument for in vitro diagnosis of early and latent disease; the ultimate applicability to risk stratification, quantitation of disease burden, and response to therapy can easily be envisioned.

    View details for DOI 10.1371/journal.pone.0052021

    View details for Web of Science ID 000312483300046

    View details for PubMedID 23272198

    View details for PubMedCentralID PMC3525657

  • Coronary risk assessment among intermediate risk patients using a clinical and biomarker based algorithm developed and validated in two population cohorts CURRENT MEDICAL RESEARCH AND OPINION Cross, D. S., McCarty, C. A., Hytopoulos, E., Beggs, M., Nolan, N., Harrington, D. S., Hastie, T., Tibshirani, R., Tracy, R. P., Psaty, B. M., McClelland, R., Tsao, P. S., Quertermous, T. 2012; 28 (11): 1819-1830


    Many coronary heart disease (CHD) events occur in individuals classified as intermediate risk by commonly used assessment tools. Over half the individuals presenting with a severe cardiac event, such as myocardial infarction (MI), have at most one risk factor as included in the widely used Framingham risk assessment. Individuals classified as intermediate risk, who are actually at high risk, may not receive guideline recommended treatments. A clinically useful method for accurately predicting 5-year CHD risk among intermediate risk patients remains an unmet medical need.This study sought to develop a CHD Risk Assessment (CHDRA) model that improves 5-year risk stratification among intermediate risk individuals.Assay panels for biomarkers associated with atherosclerosis biology (inflammation, angiogenesis, apoptosis, chemotaxis, etc.) were optimized for measuring baseline serum samples from 1084 initially CHD-free Marshfield Clinic Personalized Medicine Research Project (PMRP) individuals. A multivariable Cox regression model was fit using the most powerful risk predictors within the clinical and protein variables identified by repeated cross-validation. The resulting CHDRA algorithm was validated in a Multiple-Ethnic Study of Atherosclerosis (MESA) case-cohort sample.A CHDRA algorithm of age, sex, diabetes, and family history of MI, combined with serum levels of seven biomarkers (CTACK, Eotaxin, Fas Ligand, HGF, IL-16, MCP-3, and sFas) yielded a clinical net reclassification index of 42.7% (p < 0.001) for MESA patients with a recalibrated Framingham 5-year intermediate risk level. Across all patients, the model predicted acute coronary events (hazard ratio = 2.17, p < 0.001), and remained an independent predictor after Framingham risk factor adjustments.These include the slightly different event definition with the MESA samples and inability to include PMRP fatal CHD events.A novel risk score of serum protein levels plus clinical risk factors, developed and validated in independent cohorts, demonstrated clinical utility for assessing the true risk of CHD events in intermediate risk patients. Improved accuracy in cardiovascular risk classification could lead to improved preventive care and fewer deaths.

    View details for DOI 10.1185/03007995.2012.742878

    View details for Web of Science ID 000310985600009

    View details for PubMedID 23092312

  • Wall shear stress is decreased in the pulmonary arteries of patients with pulmonary arterial hypertension: An image-based, computational fluid dynamics study. Pulmonary circulation Tang, B. T., Pickard, S. S., Chan, F. P., Tsao, P. S., Taylor, C. A., Feinstein, J. A. 2012; 2 (4): 470-476


    Previous clinical studies in pulmonary arterial hypertension (PAH) have concentrated predominantly on distal pulmonary vascular resistance, its contribution to the disease process, and response to therapy. However, it is well known that biomechanical factors such as shear stress have an impact on endothelial health and dysfunction in other parts of the vasculature. This study tested the hypothesis that wall shear stress is reduced in the proximal pulmonary arteries of PAH patients with the belief that reduced shear stress may contribute to pulmonary endothelial cell dysfunction and as a result, PAH progression. A combined MRI and computational fluid dynamics (CFD) approach was used to construct subject-specific pulmonary artery models and quantify flow features and wall shear stress (WSS) in five PAH patients with moderate-to-severe disease and five age- and sex-matched controls. Three-dimensional model reconstruction showed PAH patients have significantly larger main, right, and left pulmonary artery diameters (3.5 ± 0.4 vs. 2.7 ± 0.1 cm, P = 0.01; 2.5 ± 0.4 vs. 1.9 ± 0.2 cm, P = 0.04; and 2.6 ± 0.4 vs. 2.0 ± 0.2 cm, P = 0.01, respectively), and lower cardiac output (3.7 ± 1.2 vs. 5.8 ± 0.6 L/min, P = 0.02.). CFD showed significantly lower time-averaged central pulmonary artery WSS in PAH patients compared to controls (4.3 ± 2.8 vs. 20.5 ± 4.0 dynes/cm(2), P = 0.0004). Distal WSS was not significantly different. A novel method of measuring WSS was utilized to demonstrate for the first time that WSS is altered in some patients with PAH. Using computational modeling in patient-specific models, WSS was found to be significantly lower in the proximal pulmonary arteries of PAH patients compared to controls. Reduced WSS in proximal pulmonary arteries may play a role in the pathogenesis and progression of PAH. This data may serve as a basis for future in vitro studies of, for example, effects of WSS on gene expression.

    View details for DOI 10.4103/2045-8932.105035

    View details for PubMedID 23372931

    View details for PubMedCentralID PMC3555417

  • In vivo, ex vivo, and in vitro studies on apelin's effect on myocardial glucose uptake PEPTIDES Xu, S., Han, P., Huang, M., Wu, J. C., Chang, C., Tsao, P. S., Yue, P. 2012; 37 (2): 320-326


    Apelin is an endogenous peptide hormone recently implicated in glucose homeostasis. However, whether apelin affects glucose uptake in myocardial tissue remains undetermined. In this study, we utilized in vivo, ex vivo and in vitro methods to study apelin's effect on myocardial glucose uptake. Pyroglutamated apelin-13 (2mg/kg/day) was administered to C57BL6/J mice for 7 days. In vivo myocardial glucose uptake was measured by FDG-PET scanning, and GLUT4 translocation was assessed by immunofluorescence imaging. For in vitro studies, differentiated H9C2 cardiomyoblasts were exposed to pyroglutamated apelin-13 (100 nM) for 2h. To test their involvement in apelin-stimulated myocardial glucose uptake, the energy sensing protein kinase AMPK were inhibited by pharmacologic inhibition (compound C) and RNA interference. IRS-1 phosphorylation was assessed by western blotting using an antibody directed against IRS-1 Ser-789-phosphorylated form. We found that apelin increased myocardial glucose uptake and GLUT4 membrane translocation in C57BL6/J mice. Apelin was also sufficient to increase glucose uptake in H9C2 cells. Apelin-mediated glucose uptake was significantly decreased by AMPK inhibition. Finally, apelin increased IRS-1 Ser-789 phosphorylation in an AMPK-dependent manner. The results of our study demonstrated that apelin increases myocardial glucose uptake through a pathway involving AMPK. Apelin also facilitates IRS-1 Ser-789 phosphorylation, suggesting a novel mechanism for its effects on glucose uptake.

    View details for DOI 10.1016/j.peptides.2012.08.004

    View details for Web of Science ID 000310047700020

    View details for PubMedID 22906703

  • Apolipoprotein(a) Genetic Sequence Variants Associated With Systemic Atherosclerosis and Coronary Atherosclerotic Burden But Not With Venous Thromboembolism JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Helgadottir, A., Gretarsdottir, S., Thorleifsson, G., Holm, H., Patel, R. S., Gudnason, T., Jones, G. T., van Rij, A. M., Eapen, D. J., Baas, A. F., Tregouet, D., Morange, P., Emmerich, J., Lindblad, B., Gottsater, A., Kiemeny, L. A., Lindholt, J. S., Sakalihasan, N., Ferrell, R. E., Carey, D. J., Elmore, J. R., Tsao, P. S., Grarup, N., Jorgensen, T., Witte, D. R., Hansen, T., Pedersen, O., Pola, R., Gaetani, E., Magnadottir, H. B., Wijmenga, C., Tromp, G., Ronkainen, A., Ruigrok, Y. M., Blankensteijn, J. D., Mueller, T., Wells, P. S., Corral, J., Manuel Soria, J., Carlos Souto, J., Peden, J. F., Jalilzadeh, S., Mayosi, B. M., Keavney, B., Strawbridge, R. J., Sabater-Lleal, M., Gertow, K., Baldassarre, D., Nyyssonen, K., Rauramaa, R., Smit, A. J., Mannarino, E., Giral, P., Tremoli, E., de Faire, U., Humphries, S. E., Hamsten, A., Haraldsdottir, V., Olafsson, I., Magnusson, M. K., Samani, N. J., Levey, A. I., Markus, H. S., Kostulas, K., Dichgans, M., Berger, K., Kuhlenbaeumer, G., Ringelstein, E. B., Stoll, M., Seedorf, U., Rothwell, P. M., Powell, J. T., Kuivaniemi, H., Onundarson, P. T., Valdimarsson, E., Matthiasson, S. E., Gudbjartsson, D. F., Thorgeirsson, G., Quyyumi, A. A., Watkins, H., Farrall, M., Thorsteinsdottir, U., Stefansson, K. 2012; 60 (8): 722-729
  • Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism. Journal of the American College of Cardiology Helgadottir, A., Gretarsdottir, S., Thorleifsson, G., Holm, H., Patel, R. S., Gudnason, T., Jones, G. T., van Rij, A. M., Eapen, D. J., Baas, A. F., Tregouet, D., Morange, P., Emmerich, J., Lindblad, B., Gottsäter, A., Kiemeny, L. A., Lindholt, J. S., Sakalihasan, N., Ferrell, R. E., Carey, D. J., Elmore, J. R., Tsao, P. S., Grarup, N., Jørgensen, T., Witte, D. R., Hansen, T., Pedersen, O., Pola, R., Gaetani, E., Magnadottir, H. B., Wijmenga, C., Tromp, G., Ronkainen, A., Ruigrok, Y. M., Blankensteijn, J. D., Mueller, T., Wells, P. S., Corral, J., Soria, J. M., Souto, J. C., Peden, J. F., Jalilzadeh, S., Mayosi, B. M., Keavney, B., Strawbridge, R. J., Sabater-Lleal, M., Gertow, K., Baldassarre, D., Nyyssönen, K., Rauramaa, R., Smit, A. J., Mannarino, E., Giral, P., Tremoli, E., de Faire, U., Humphries, S. E., Hamsten, A., Haraldsdottir, V., Olafsson, I., Magnusson, M. K., Samani, N. J., Levey, A. I., Markus, H. S., Kostulas, K., Dichgans, M., Berger, K., Kuhlenbäumer, G., Ringelstein, E. B., Stoll, M., Seedorf, U., Rothwell, P. M., Powell, J. T., Kuivaniemi, H., Onundarson, P. T., Valdimarsson, E., Matthiasson, S. E., Gudbjartsson, D. F., Thorgeirsson, G., Quyyumi, A. A., Watkins, H., Farrall, M., Thorsteinsdottir, U., Stefansson, K. 2012; 60 (8): 722-729


    The purpose of this study is investigate the effects of variants in the apolipoprotein(a) gene (LPA) on vascular diseases with different atherosclerotic and thrombotic components.It is unclear whether the LPA variants rs10455872 and rs3798220, which correlate with lipoprotein(a) levels and coronary artery disease (CAD), confer susceptibility predominantly via atherosclerosis or thrombosis.The 2 LPA variants were combined and examined as LPA scores for the association with ischemic stroke (and TOAST [Trial of Org 10172 in Acute Stroke Treatment] subtypes) (effective sample size [n(e)] = 9,396); peripheral arterial disease (n(e) = 5,215); abdominal aortic aneurysm (n(e) = 4,572); venous thromboembolism (n(e) = 4,607); intracranial aneurysm (n(e) = 1,328); CAD (n(e) = 12,716), carotid intima-media thickness (n = 3,714), and angiographic CAD severity (n = 5,588).LPA score was associated with ischemic stroke subtype large artery atherosclerosis (odds ratio [OR]: 1.27; p = 6.7 × 10(-4)), peripheral artery disease (OR: 1.47; p = 2.9 × 10(-14)), and abdominal aortic aneurysm (OR: 1.23; p = 6.0 × 10(-5)), but not with the ischemic stroke subtypes cardioembolism (OR: 1.03; p = 0.69) or small vessel disease (OR: 1.06; p = 0.52). Although the LPA variants were not associated with carotid intima-media thickness, they were associated with the number of obstructed coronary vessels (p = 4.8 × 10(-12)). Furthermore, CAD cases carrying LPA risk variants had increased susceptibility to atherosclerotic manifestations outside of the coronary tree (OR: 1.26; p = 0.0010) and had earlier onset of CAD (-1.58 years/allele; p = 8.2 × 10(-8)) than CAD cases not carrying the risk variants. There was no association of LPA score with venous thromboembolism (OR: 0.97; p = 0.63) or intracranial aneurysm (OR: 0.85; p = 0.15).LPA sequence variants were associated with atherosclerotic burden, but not with primarily thrombotic phenotypes.

    View details for DOI 10.1016/j.jacc.2012.01.078

    View details for PubMedID 22898070

  • Vascular smooth muscle cell phenotypic plasticity: focus on chromatin remodelling CARDIOVASCULAR RESEARCH Spin, J. M., Maegdefessel, L., Tsao, P. S. 2012; 95 (2): 147-155


    Differentiated vascular smooth muscle cells (SMCs) retain the capacity to modify their phenotype in response to inflammation or injury. This phenotypic switching is a crucial component of vascular disease, and is partly dependent on epigenetic regulation. An appreciation has been building in the literature for the essential role chromatin remodelling plays both in SMC lineage determination and in influencing changes in SMC behaviour and state. This process includes numerous chromatin regulatory elements and pathways such as histone acetyltransferases, deacetylases, and methyltransferases and other factors that act at SMC-specific marker sites to silence or permit access to the cellular transcriptional machinery and on other key regulatory elements such as myocardin and Kruppel-like factor 4 (KLF4). Various stimuli known to alter the SMC phenotype, such as transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), oxidized phospholipids, and retinoic acid, appear to act in part through effects upon SMC chromatin structure. In recent years, specific covalent histone modifications that appear to establish SMC determinacy have been identified, while others alter the differentiation state. In this article, we review the mechanisms of chromatin remodelling as it applies to the SMC phenotype.

    View details for DOI 10.1093/cvr/cvs098

    View details for Web of Science ID 000306141100004

    View details for PubMedID 22362814

  • Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Hua, X., Deuse, T., Michelakis, E. D., Haromy, A., Tsao, P. S., Maegdefessel, L., Erben, R. G., Bergow, C., Behnisch, B. B., Reichenspurner, H., Robbins, R. C., Schrepfer, S. 2012

    View details for DOI 10.3791/3663

    View details for Web of Science ID 000209223000016

  • Three-Dimensional Microstructural Changes in Murine Abdominal Aortic Aneurysms Quantified Using Immunofluorescent Array Tomography JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY Saatchi, S., Azuma, J., Wanchoo, N., Smith, S. J., Yock, P. G., Taylor, C. A., Tsao, P. S. 2012; 60 (2): 97-109


    This study investigated the spatial and temporal remodeling of blood vessel wall microarchitecture and cellular morphology during abdominal aortic aneurysm (AAA) development using immunofluorescent array tomography (IAT), a high-resolution three-dimensional (3D) microscopy technology, in the murine model. Infrarenal aortas of C57BL6 mice (N=20) were evaluated at 0, 7, and 28 days after elastase or heat-inactivated elastase perfusion. Custom algorithms quantified volume fractions (VF) of elastin, smooth muscle cell (SMC) actin, and adventitial collagen type I, as well as elastin thickness, elastin fragmentation, non-adventitial wall thickness, and nuclei amount. The 3D renderings depicted elastin and collagen type I degradation and SMC morphological changes. Elastin VF decreased 37.5% (p<0.01), thickness decreased 48.9%, and fragmentation increased 449.7% (p<0.001) over 28 days. SMC actin VF decreased 78.3% (p<0.001) from days 0 to 7 and increased 139.7% (p<0.05) from days 7 to 28. Non-adventitial wall thickness increased 61.1%, medial nuclei amount increased 159.1% (p<0.01), and adventitial collagen type I VF decreased 64.1% (p<0.001) over 28 days. IAT and custom image analysis algorithms have enabled robust quantification of vessel wall content, microstructure, and organization to help elucidate the dynamics of vascular remodeling during AAA development.

    View details for DOI 10.1369/0022155411433066

    View details for Web of Science ID 000299481300001

    View details for PubMedID 22140132

    View details for PubMedCentralID PMC3351119

  • miR-29b Participates in Early Aneurysm Development in Marfan Syndrome CIRCULATION RESEARCH Merk, D. R., Chin, J. T., Dake, B. A., Maegdefessel, L., Miller, M. O., Kimura, N., Tsao, P. S., Iosef, C., Berry, G. J., Mohr, F. W., Spin, J. M., Alvira, C. M., Robbins, R. C., Fischbein, M. P. 2012; 110 (2): 312-?


    Marfan syndrome (MFS) is a systemic connective tissue disorder notable for the development of aortic root aneurysms and the subsequent life-threatening complications of aortic dissection and rupture. Underlying fibrillin-1 gene mutations cause increased transforming growth factor-β (TGF-β) signaling. Although TGF-β blockade prevents aneurysms in MFS mouse models, the mechanisms through which excessive TGF-β causes aneurysms remain ill-defined.We investigated the role of microRNA-29b (miR-29b) in aneurysm formation in MFS.Using quantitative polymerase chain reaction, we discovered that miR-29b, a microRNA regulating apoptosis and extracellular matrix synthesis/deposition genes, is increased in the ascending aorta of Marfan (Fbn1(C1039G/+)) mice. Increased apoptosis, assessed by increased cleaved caspase-3 and caspase-9, enhanced caspase-3 activity, and decreased levels of the antiapoptotic proteins, Mcl-1 and Bcl-2, were found in the Fbn1(C1039G/+) aorta. Histological evidence of decreased and fragmented elastin was observed exclusively in the Fbn1(C1039G/+) ascending aorta in association with repressed elastin mRNA and increased matrix metalloproteinase-2 expression and activity, both targets of miR-29b. Evidence of decreased activation of nuclear factor κB, a repressor of miR-29b, and a factor suppressed by TGF-β, was also observed in Fbn1(C1039G/+) aorta. Furthermore, administration of a nuclear factor κB inhibitor increased miR-29b levels, whereas TGF-β blockade or losartan effectively decreased miR-29b levels in Fbn1(C1039G/+) mice. Finally, miR-29b blockade by locked nucleic acid antisense oligonucleotides prevented early aneurysm development, aortic wall apoptosis, and extracellular matrix deficiencies.We identify increased miR-29b expression as key to the pathogenesis of early aneurysm development in MFS by regulating aortic wall apoptosis and extracellular matrix abnormalities.

    View details for DOI 10.1161/CIRCRESAHA.111.253740

    View details for Web of Science ID 000299432600015

    View details for PubMedID 22116819

  • Human internal mammary artery (IMA) transplantation and stenting: a human model to study the development of in-stent restenosis. Journal of visualized experiments : JoVE Hua, X., Deuse, T., Michelakis, E. D., Haromy, A., Tsao, P. S., Maegdefessel, L., Erben, R. G., Bergow, C., Behnisch, B. B., Reichenspurner, H., Robbins, R. C., Schrepfer, S. 2012: e3663-?


    Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches (1,2). The commonly used animal models include mice, rats, rabbits, and pigs (3-5). However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models (6,7). In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents. This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

    View details for DOI 10.3791/3663

    View details for PubMedID 22617624

  • Low- and High-Dose Plant and Marine (n-3) Fatty Acids Do Not Affect Plasma Inflammatory Markers in Adults with Metabolic Syndrome JOURNAL OF NUTRITION Dewell, A., Marvasti, F. F., Harris, W. S., Tsao, P., Gardner, C. D. 2011; 141 (12): 2166-2171


    Chronic inflammation is considered to play a role in the development of cardiovascular disease. Various (n-3) fatty acids (FA) have been reported to have antiinflammatory effects, but there is a lack of consensus in this area, particularly in regard to optimal source(s) and dose(s). This study aimed to determine the effects of high and low doses of (n-3) FA from plant and marine sources on plasma inflammatory marker concentrations. One-hundred adults with metabolic syndrome were randomly assigned to a low or high dose of plant- (2.2 or 6.6 g/d α-linolenic acid) or marine- (1.2 or 3.6 g/d EPA and DHA) derived (n-3) FA or placebo for 8 wk, using a parallel arm design (n = 20/arm). Fasting blood samples collected at 0, 4, and 8 wk were analyzed for concentrations of monocyte chemotactic protein-1 (MCP-1), IL-6, and soluble intercellular adhesion molecule-1 (sICAM-1) and for cardiovascular risk factors. Baseline concentrations across all 5 groups combined were (mean ± SD) 103 ± 32 ng/L for MCP-1, 1.06 ± 0.56 ng/L for IL-6, and 0.197 ± 0.041 ng/L for sICAM-1. There were no significant differences in 8-wk changes in plasma inflammatory marker concentrations among the 5 groups. Plasma TG and blood pressure decreased significantly more and the LDL cholesterol concentration increased more in the high-dose fish oil group compared to the 8-wk changes in some of the other 4 groups (P ≤ 0.04). In conclusion, no beneficial effects were detected for any of the 3 inflammatory markers investigated in response to (n-3) FA in adults with metabolic syndrome regardless of dose or source.

    View details for DOI 10.3945/jn.111.142240

    View details for Web of Science ID 000297387200011

    View details for PubMedID 22031659

    View details for PubMedCentralID PMC3223874

  • In Vivo Bioluminescence Imaging of Inducible Nitric Oxide Synthase Gene Expression in Vascular Inflammation MOLECULAR IMAGING AND BIOLOGY Terashima, M., Ehara, S., Yang, E., Kosuge, H., Tsao, P. S., Quertermous, T., Contag, C. H., McConnell, M. V. 2011; 13 (6): 1061-1066


    Inflammation plays a critical role in atherosclerosis and is associated with upregulation of inducible nitric oxide synthase (iNOS). We studied bioluminescence imaging (BLI) to track iNOS gene expression in a murine model of vascular inflammation.Macrophage-rich vascular lesions were induced by carotid ligation plus high-fat diet and streptozotocin-induced diabetes in 18 iNOS-luc reporter mice. In vivo iNOS expression was imaged serially by BLI over 14 days, followed by in situ BLI and histology.BLI signal from ligated carotids increased over 14 days (9.7 ± 4.4 × 10(3 ) vs. 4.4 ± 1.7 × 10(3) photons/s/cm(2)/sr at baseline, p < 0.001 vs. baseline, p < 0.05 vs. sham controls). Histology confirmed substantial macrophage infiltration, with iNOS and luciferase expression, only in ligated left carotid arteries and not controls.BLI allows in vivo detection of iNOS expression in murine carotid lesions and may provide a valuable approach for monitoring vascular gene expression and inflammation in small animal models.

    View details for DOI 10.1007/s11307-010-0451-5

    View details for Web of Science ID 000296794400002

    View details for PubMedID 21057879

  • Microrna-21 Regulates Expansion of Abdominal Aortic Aneurysms Through the PTEN/PI3K/AKT Pathway Scientific Sessions of the American-Heart-Association/Resuscitation Science Symposium Maegdefessel, L., Azuma, J., Deng, A., Toh, R. M., Merk, D. R., Raiesdana, A., Leeper, N. J., Spin, J. M., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2011
  • Nicotine-Augmented Abdominal Aortic Aneurysms are Regulated by MicroRNA-29b Scientific Sessions of the American-Heart-Association/Resuscitation Science Symposium Maegdefessel, L., Azuma, J., Merk, D. R., Toh, R. M., Deng, A., Chin, J. P., Spin, J. M., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2011
  • Human Leukocyte Antigen I Knockdown Human Embryonic Stem Cells Induce Host Ignorance and Achieve Prolonged Xenogeneic Survival Annual Meeting of the American-Heart-Association Deuse, T., Seifert, M., Phillips, N., Fire, A., Tyan, D., Kay, M., Tsao, P. S., Hua, X., Velden, J., Eiermann, T., Volk, H., Reichenspurner, H., Robbins, R. C., Schrepfer, S. LIPPINCOTT WILLIAMS & WILKINS. 2011: S3–S9


    Although human embryonic stem cells (hESC) have enormous potential for cell replacement therapy of heart failure, immune rejection of hESC derivatives inevitably would occur after transplantation. We therefore aimed to generate a hypoantigeneic hESC line with improved survival characteristics.Using various in vivo, nonischemic, hindlimb xenotransplant models (immunocompetent and defined immunodefective mouse strains) as well as human in vitro T-cell and natural killer (NK)-cell assays, we revealed a central role for T cells in mediating hESC rejection. The NK-cell susceptibility of hESC in vivo was found to be low, and the NK response to hESC challenge in vitro was negligible. To reduce the antigenicity of hESC, we successfully generated human leukocyte antigen (HLA) I knockdown cells (hESC(siRNA+IB)) using both HLA I RNA interference (siRNA) and intrabody (IB) technology. HLA I expression was ≈99% reduced after 7 days and remained low for weeks. Cellular immune recognition of these hESC(siRNA+IB) was strongly reduced in both xenogeneic and allogeneic settings. Immune rejection was profoundly mitigated after hESC(siRNA+IB) transplantation into immunocompetent mice, and even long-term graft survival was achieved in one third of the animals without any immunosuppression. The survival benefit of hESC(siRNA+IB) was further confirmed under ischemic conditions in a left anterior descending coronary artery ligation model.HLA I knockdown hESC(siRNA+IB) provoke T-cell ignorance and experience largely mitigated xenogeneic rejection. By generating hypoantigeneic hESC lines, the generation of acceptable hESC derivatives may become a practical concept and push cell replacement strategies forward.

    View details for DOI 10.1161/CIRCULATIONAHA.111.020727

    View details for Web of Science ID 000294782800001

    View details for PubMedID 21911816

  • Transcriptional profiling and network analysis of the murine angiotensin II-induced abdominal aortic aneurysm PHYSIOLOGICAL GENOMICS Spin, J. M., Hsu, M., Azuma, J., Tedesco, M. M., Deng, A., Dyer, J. S., Maegdefessel, L., Dalman, R. L., Tsao, P. S. 2011; 43 (17): 993-1003


    We sought to characterize temporal gene expression changes in the murine angiotensin II (ANG II)-ApoE-/- model of abdominal aortic aneurysm (AAA). Aortic ultrasound measurements were obtained over the 28-day time-course. Harvested suprarenal aortic segments were evaluated with whole genome expression profiling at 7, 14, and 28 days using the Agilent Whole Mouse Genome microarray platform and Statistical Analysis of Microarrays at a false discovery rate of <1%. A group of angiotensin-treated mice experienced contained rupture (CR) within 7 days and were analyzed separately. Progressive aortic dilatation occurred throughout the treatment period. However, the numerous early expression differences between ANG II-treated and control were not sustained over time. Ontologic analysis revealed widespread upregulation of inflammatory, immune, and matrix remodeling genes with ANG II treatment, among other pathways such as apoptosis, cell cycling, angiogenesis, and p53 signaling. CR aneurysms displayed significant decreases in TGF-β/BMP-pathway signaling, MAPK signaling, and ErbB signaling genes vs. non-CR/ANG II-treated samples. We also performed literature-based network analysis, extracting numerous highly interconnected genes associated with aneurysm development such as Spp1, Myd88, Adam17 and Lox. 1) ANG II treatment induces extensive early differential expression changes involving abundant signaling pathways in the suprarenal abdominal aorta, particularly wide-ranging increases in inflammatory genes with aneurysm development. 2) These gene expression changes appear to dissipate with time despite continued growth, suggesting that early changes in gene expression influence disease progression in this AAA model, and that the aortic tissue adapts to prolonged ANG II infusion. 3) Network analysis identified nexus genes that may constitute aneurysm biomarkers or therapeutic targets.

    View details for DOI 10.1152/physiolgenomics.00044.2011

    View details for Web of Science ID 000294730000002

    View details for PubMedID 21712436

  • Apelin and insulin resistance: Another arrow for the quiver? JOURNAL OF DIABETES Xu, S., Tsao, P. S., Yue, P. 2011; 3 (3): 225-231


    Apelin is a newly discovered peptide hormone that has recently been linked to insulin resistance and obesity. Data collected from both the clinical and basic research settings show that apelin: (i) is correlated with the states of insulin resistance and obesity; (ii) stimulates glucose utilization; (iii) decreases insulin secretion; and (iv) negatively regulates catecholamine-mediated lipolysis. These and other lines of evidence demonstrate that apelin may be a potentially viable candidate in the search for treatments for Type 2 diabetes and the insulin resistance (metabolic syndrome). The present review summarizes the literature on the regulation by apelin of glucose and lipid metabolism and the signaling pathways involved.

    View details for DOI 10.1111/j.1753-0407.2011.00132.x

    View details for Web of Science ID 000307064700009

    View details for PubMedID 21631898

  • Immunobiology of naive and genetically modified HLA-class-I-knockdown human embryonic stem cells JOURNAL OF CELL SCIENCE Deuse, T., Seifert, M., Phillips, N., Fire, A., Tyan, D., Kay, M., Tsao, P. S., Hua, X., Velden, J., Eiermann, T., Volk, H., Reichenspurner, H., Robbins, R. C., Schrepfer, S. 2011; 124 (17): 3029-3037


    Human embryonic stem cells (hESCs) can serve as a universal cell source for emerging cell or tissue replacement strategies, but immune rejection of hESC derivatives remains an unsolved problem. Here, we sought to describe the mechanisms of rejection for naïve hESCs and upon HLA class I (HLA I) knockdown (hESC(KD)). hESCs were HLA I-positive but negative for HLA II and co-stimulatory molecules. Transplantation of naïve hESC into immunocompetent Balb/c mice induced substantial T helper cell 1 and 2 (Th1 and Th2) responses with rapid cell death, but hESCs survived in immunodeficient SCID-beige recipients. Histology revealed mainly macrophages and T cells, but only scattered natural killer (NK) cells. A surge of hESC-specific antibodies against hESC class I, but not class II antigens, was observed. Using HLA I RNA interference and intrabody technology, HLA I surface expression of hESC(KD) was 88%-99% reduced. T cell activation after hESC(KD) transplantation into Balb/c was significantly diminished, antibody production was substantially alleviated, the levels of graft-infiltrating immune cells were reduced and the survival of hESC(KD) was prolonged. Because of their very low expression of stimulatory NK ligands, NK-susceptibility of naïve hESCs and hESC(KD) was negligible. Thus, HLA I recognition by T cells seems to be the primary mechanism of hESC recognition, and T cells, macrophages and hESC-specific antibodies participate in hESC killing.

    View details for DOI 10.1242/jcs.087718

    View details for Web of Science ID 000294419200015

    View details for PubMedID 21878509

  • Detecting Drug Interactions From Adverse-Event Reports: Interaction Between Paroxetine and Pravastatin Increases Blood Glucose Levels CLINICAL PHARMACOLOGY & THERAPEUTICS Tatonetti, N. P., Denny, J. C., Murphy, S. N., Fernald, G. H., Krishnan, G., Castro, V., Yue, P., Tsau, P. S., Kohane, I., Roden, D. M., Altman, R. B. 2011; 90 (1): 133-142


    The lipid-lowering agent pravastatin and the antidepressant paroxetine are among the most widely prescribed drugs in the world. Unexpected interactions between them could have important public health implications. We mined the US Food and Drug Administration's (FDA's) Adverse Event Reporting System (AERS) for side-effect profiles involving glucose homeostasis and found a surprisingly strong signal for comedication with pravastatin and paroxetine. We retrospectively evaluated changes in blood glucose in 104 patients with diabetes and 135 without diabetes who had received comedication with these two drugs, using data in electronic medical record (EMR) systems of three geographically distinct sites. We assessed the mean random blood glucose levels before and after treatment with the drugs. We found that pravastatin and paroxetine, when administered together, had a synergistic effect on blood glucose. The average increase was 19 mg/dl (1.0 mmol/l) overall, and in those with diabetes it was 48 mg/dl (2.7 mmol/l). In contrast, neither drug administered singly was associated with such changes in glucose levels. An increase in glucose levels is not a general effect of combined therapy with selective serotonin reuptake inhibitors (SSRIs) and statins.

    View details for DOI 10.1038/clpt.2011.83

    View details for Web of Science ID 000291853800023

    View details for PubMedID 21613990

    View details for PubMedCentralID PMC3216673

  • Asymmetric dimethylarginine impairs fibrinolytic activity in human umbilical vein endothelial cells via p38 MAPK and NF-kappa B pathways THROMBOSIS RESEARCH Zhang, Q., Chen, N., Qiu, W., Xu, X., Wang, D., Tsao, P. S., Jin, H. 2011; 128 (1): 42-46


    Asymmetric dimethylarginine (ADMA) is a potent endogenous inhibitor of nitric oxide (NO) synthase. An increased synthesis and/or a reduced catabolism of ADMA might contribute to the onset and progression of thrombosis. The present study was designed to evaluate the effect of ADMA on fibrinolytic factors in endothelial cells, and to investigate the cellular mechanisms.Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of ADMA for various periods; Then HUVECs were preincubated with NO precursor (L-arginine), MAPK inhibitors, or NF-κB inhibitor (PDTC) before ADMA treatment to repeat the experiment. Protein levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and NF-κB activity were measured by ELISA; mRNA levels of tPA and PAI-1 were assayed by qRT-PCR; The activation of MAPK was characterized by western blot analysis.(1) ADMA decreased tPA antigen levels in time- and concentration-dependent manners, with the maximum effect of 30 μmol/L ADMA for 48h (control 109.01 ± 4.15 ng/ml vs ADMA 86.76 ± 5.95 ng/ml, p<0.01); (2) 30 μmol/L ADMA elevated antigen levels of PAI-1 in a time-dependent manner, with the maximum effect of 30 μmol/L ADMA for 48 h (control 2721.12 ± 278.02 ng/ml vs. ADMA 3435.78 ± 22.33ng/ml, p<0.05); (3) ADMA reduced tPA mRNA levels and increased PAI-1 mRNA levels; (4) L-arginine, SB203580 (p38 MAPK inhibitor) and PDTC attenuated the effects of ADMA on tPA and PAI-1 significantly. (5) ADMA induced a rapid phosphorylation of p38 MAPK, and stimulated NF-κB activity greatly.ADMA may accelerate thrombosis development by impairing fibrinolytic activity in vascular via inhibiting nitric oxide production and then activating its downstream p38 MAPK and NF-κB pathways.

    View details for DOI 10.1016/j.thromres.2011.02.013

    View details for Web of Science ID 000291383500008

    View details for PubMedID 21429569

  • The Use of Immunofluorescent Array Tomography to Study the Three-Dimensional Microstructure of Murine Blood Vessels CELLULAR AND MOLECULAR BIOENGINEERING Saatchi, S., Wanchoo, N., Azuma, J., Smith, S. J., Tsao, P. S., Yock, P. G., Taylor, C. A. 2011; 4 (2): 311-323
  • MicroRNA-26a Is a Novel Regulator of Vascular Smooth Muscle Cell Function JOURNAL OF CELLULAR PHYSIOLOGY Leeper, N. J., Raiesdana, A., Kojima, Y., Chun, H. J., Azuma, J., Maegdefessel, L., Kundu, R. K., Quertermous, T., Tsao, P. S., Spin, J. M. 2011; 226 (4): 1035-1043


    Aberrant smooth muscle cell (SMC) plasticity has been implicated in a variety of vascular disorders including atherosclerosis, restenosis, and abdominal aortic aneurysm (AAA) formation. While the pathways governing this process remain unclear, epigenetic regulation by specific microRNAs (miRNAs) has been demonstrated in SMCs. We hypothesized that additional miRNAs might play an important role in determining vascular SMC phenotype. Microarray analysis of miRNAs was performed on human aortic SMCs undergoing phenotypic switching in response to serum withdrawal, and identified 31 significantly regulated entities. We chose the highly conserved candidate miRNA-26a for additional studies. Inhibition of miRNA-26a accelerated SMC differentiation, and also promoted apoptosis, while inhibiting proliferation and migration. Overexpression of miRNA-26a blunted differentiation. As a potential mechanism, we investigated whether miRNA-26a influences TGF-β-pathway signaling. Dual-luciferase reporter assays demonstrated enhanced SMAD signaling with miRNA-26a inhibition, and the opposite effect with miRNA-26a overexpression in transfected human cells. Furthermore, inhibition of miRNA-26a increased gene expression of SMAD-1 and SMAD-4, while overexpression inhibited SMAD-1. MicroRNA-26a was also found to be downregulated in two mouse models of AAA formation (2.5- to 3.8-fold decrease, P < 0.02) in which enhanced switching from contractile to synthetic phenotype occurs. In summary, miRNA-26a promotes vascular SMC proliferation while inhibiting cellular differentiation and apoptosis, and alters TGF-β pathway signaling. MicroRNA-26a represents an important new regulator of SMC biology and a potential therapeutic target in AAA disease.

    View details for DOI 10.1002/jcp.22422

    View details for Web of Science ID 000287258800019

    View details for PubMedID 20857419

  • Influences of Aortic Motion and Curvature on Vessel Expansion in Murine Experimental Aneurysms ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Goergen, C. J., Azuma, J., Barr, K. N., Magdefessel, L., Kallop, D. Y., Gogineni, A., Grewall, A., Weimer, R. M., Connolly, A. J., Dalman, R. L., Taylor, C. A., Tsao, P. S., Greve, J. M. 2011; 31 (2): 270-U102


    To quantitatively compare aortic curvature and motion with resulting aneurysm location, direction of expansion, and pathophysiological features in experimental abdominal aortic aneurysms (AAAs).MRI was performed at 4.7 T with the following parameters: (1) 3D acquisition for vessel geometry and (2) 2D cardiac-gated acquisition to quantify luminal motion. Male 24-week-old mice were imaged before and after AAA formation induced by angiotensin II (AngII)-filled osmotic pump implantation or infusion of elastase. AngII-induced AAAs formed near the location of maximum abdominal aortic curvature, and the leftward direction of expansion was correlated with the direction of suprarenal aortic motion. Elastase-induced AAAs formed in a region of low vessel curvature and had no repeatable direction of expansion. AngII significantly increased mean blood pressure (22.7 mm Hg, P<0.05), whereas both models showed a significant 2-fold decrease in aortic cyclic strain (P<0.05). Differences in patterns of elastin degradation and localization of fluorescent signal from protease-activated probes were also observed.The direction of AngII aneurysm expansion correlated with the direction of motion, medial elastin dissection, and adventitial remodeling. Anterior infrarenal aortic motion correlated with medial elastin degradation in elastase-induced aneurysms. Results from both models suggest a relationship between aneurysm pathological features and aortic geometry and motion.

    View details for DOI 10.1161/ATVBAHA.110.216481

    View details for Web of Science ID 000286376800010

    View details for PubMedID 21071686

    View details for PubMedCentralID PMC3024449

  • Human ferritin cages for imaging vascular macrophages BIOMATERIALS Terashima, M., Uchida, M., Kosuge, H., Tsao, P. S., Young, M. J., Conolly, S. M., Douglas, T., McConnell, M. V. 2011; 32 (5): 1430-1437


    Atherosclerosis is a leading cause of death worldwide. Macrophages are key components of vascular inflammation, which contributes to the development and complications of atherosclerosis. Ferritin, an iron storage and transport protein, has been found to accumulate in macrophages in human atherosclerotic plaques. We hypothesized that ferritin could serve as an intrinsic nano-platform to target delivery of imaging agents to vascular macrophages to detect high-risk atherosclerotic plaques. Here we show that engineered human ferritin protein cages, either conjugated to the fluorescent Cy5.5 molecule or encapsulating a magnetite nanoparticle, are taken up in vivo by macrophages in murine atherosclerotic carotid arteries and can be imaged by fluorescence and magnetic resonance imaging. These results indicate that human ferritin can serve as a nanoparticle platform to image vascular inflammation in vivo.

    View details for DOI 10.1016/j.biomaterials.2010.09.029

    View details for Web of Science ID 000287073000020

    View details for PubMedID 21074263

  • Selective Glucocorticoid Receptor (GR-II) Antagonist Reduces Body Weight Gain in Mice. Journal of nutrition and metabolism Asagami, T., Belanoff, J. K., Azuma, J., Blasey, C. M., Clark, R. D., Tsao, P. S. 2011; 2011: 235389-?


    Previous research has shown that mifepristone can prevent and reverse weight gain in animals and human subjects taking antipsychotic medications. This proof-of-concept study tested whether a more potent and selective glucocorticoid receptor antagonist could block dietary-induced weight gain and increase insulin sensitivity in mice. Ten-week-old, male, C57BL/6J mice were fed a diet containing 60% fat calories and water supplemented with 11% sucrose for 4 weeks. Groups (n = 8) received one of the following: CORT 108297 (80 mg/kg QD), CORT 108297 (40 mg/kg BID), mifepristone (30 mg/kg BID), rosiglitazone (10 mg/kg QD), or vehicle. Compared to mice receiving a high-fat, high-sugar diet plus vehicle, mice receiving a high-fat, high-sugar diet plus either mifepristone or CORT 108297 gained significantly less weight. At the end of the four week treatment period, mice receiving CORT 108297 40 mg/kg BID or CORT 108297 80 mg/kg QD also had significantly lower steady plasma glucose than mice receiving vehicle. However, steady state plasma glucose after treatment was not highly correlated with reduced weight gain, suggesting that the effect of the glucocorticoid receptor antagonist on insulin sensitivity may be independent of its mitigating effect on weight gain.

    View details for DOI 10.1155/2011/235389

    View details for PubMedID 21811679

  • Apelin Decreases Lipolysis via G(q), G(i), and AMPK-Dependent Mechanisms ENDOCRINOLOGY Yue, P., Jin, H., Xu, S., Aillaud, M., Deng, A. C., Azuma, J., Kundu, R. K., Reaven, G. M., Quertermous, T., Tsao, P. S. 2011; 152 (1): 59-68


    The release of free fatty acids (FFAs) from adipocytes (i.e. lipolysis) is increased in obesity and is a contributory factor to the development of insulin resistance. A recently identified adipokine, apelin, is up-regulated in states of obesity. Although apelin is secreted by adipocytes, its functions in them remain largely unknown. To determine whether apelin affects lipolysis, FFA, glycerol, and leptin levels, as well as abdominal adiposity, were measured at baseline and after reintroduction of exogenous apelin in apelin-null mice. To examine apelin's effects in vitro, isoproterenol-induced FFA/glycerol release, and hormone-sensitive lipase (HSL) and acetyl CoA carboxylase phosphorylation were investigated in 3T3-L1 cells and isolated wild-type adipocytes. Serum FFA, glycerol, and leptin concentrations, as well as abdominal adiposity, were significantly increased in apelin-null vs. wild-type mice; these changes were ameliorated in response to exogenous apelin. Apelin also reduced isoproterenol-induced FFA release in adipocytes isolated from wild-type but not APJ-null mice. In 3T3-L1 cells and isolated adipocytes, apelin attenuated isoproterenol-induced FFA/glycerol release. Apelin's inhibition was reversed by pertussis toxin, the G(q) inhibitor glycoprotein antagonist 2A, and the AMP-activated protein kinase inhibitors compound C and dorsomorphin. Apelin increased HSL phosphorylation at Ser-565 and also abrogated isoproterenol-induced HSL phosphorylation at Ser-563. Notably, apelin increased acetyl CoA carboxylase phosphorylation, suggesting AMPK activation. In conclusion, apelin negatively regulates lipolysis. Its actions may be mediated by pathways involving G(q), G(i), and AMP-activated protein kinase.

    View details for DOI 10.1210/en.2010-0576

    View details for Web of Science ID 000285573000008

    View details for PubMedID 21047945

  • Three-Dimensional Hemodynamics in the Human Pulmonary Arteries Under Resting and Exercise Conditions ANNALS OF BIOMEDICAL ENGINEERING Tang, B. T., Fonte, T. A., Chan, F. P., Tsao, P. S., Feinstein, J. A., Taylor, C. A. 2011; 39 (1): 347-358


    The biomechanical forces associated with blood flow have been shown to play a role in pulmonary vascular cell health and disease. Therefore, the quantification of human pulmonary artery hemodynamic conditions under resting and exercise states can be useful in investigating the physiology of disease development and treatment outcomes. In this study, a combined magnetic resonance imaging and computational fluid dynamics approach was used to quantify pulsatile flow fields, wall shear stress (WSS), oscillations in WSS (OSI), and energy efficiency in six subject-specific models of the human pulmonary vasculature with high spatial and temporal resolution. Averaging over all subjects, WSS was found to increase from 19.8±4.0 to 51.8±6.7 dynes/cm2, and OSI was found to decrease from 0.094±0.016 to 0.081±0.015 in the proximal pulmonary arteries between rest and exercise conditions (p<0.05). These findings demonstrate the localized, biomechanical effects of exercise. Furthermore, an average decrease of 10% in energy efficiency was noted between rest and exercise. These data indicate the amount of energy dissipation that typically occurs with exercise and may be useful in future surgical planning applications.

    View details for DOI 10.1007/s10439-010-0124-1

    View details for Web of Science ID 000287213100030

    View details for PubMedID 20640512

  • Assessment of Elastase-Induced Murine Abdominal Aortic Aneurysms: Comparison of Ultrasound Imaging with In Situ Video Microscopy JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY Azuma, J., Maegdefessel, L., Kitagawa, T., Dalman, R. L., McConnell, M. V., Tsao, P. S. 2011


    The aim of this study was to definitively assess the validity of noninvasive high-frequency ultrasound (US) measurements of aortic luminal diameter (ALD) in a murine model of elastase-induced abdominal aortic aneurysm in comparison with in situ video microscopy (VM).C57BL/6 mice underwent transient perfusion of the aorta with either elastase (n = 20: Elastase group) or saline (n = 10: Sham). Unoperated mice (n = 10) were also studied.ALD measurements by US had excellent linear correlation and absolute agreement with that by VM in both Control (unoperated or sham-operated mice) and elastase groups (r = 0.96, intraclass correlation coefficient (ICC) = 0.88 and r = 0.93, ICC = 0.92, resp.). Bland-Altman analysis of US compared with VM measurements in both groups indicated good agreement, however US measurements were slightly but significantly higher than VM measurements in the control group (mean bias 0.039 mm, P < .05). Linear regression analysis revealed excellent correlation between US and VM measurements in both groups. (R² = 0.91 in Control group, R² = 0.85 in elastase group.) The reliability of US measurements was also confirmed by ex vivo histological measurements.High-frequency US provides reliable ALD measurements in developing murine abdominal aortic aneurysms.

    View details for DOI 10.1155/2011/252141

    View details for Web of Science ID 000289091200001

    View details for PubMedID 21331328

  • Chromatin Remodeling Pathways in Smooth Muscle Cell Differentiation, and Evidence for an Integral Role for p300 PLOS ONE Spin, J. M., Quertermous, T., Tsao, P. S. 2010; 5 (12)


    Phenotypic alteration of vascular smooth muscle cells (SMC) in response to injury or inflammation is an essential component of vascular disease. Evidence suggests that this process is dependent on epigenetic regulatory processes. P300, a histone acetyltransferase (HAT), activates crucial muscle-specific promoters in terminal (non-SMC) myocyte differentiation, and may be essential to SMC modulation as well.We performed a subanalysis examining transcriptional time-course microarray data obtained using the A404 model of SMC differentiation. Numerous chromatin remodeling genes (up to 62% of such genes on our array platform) showed significant regulation during differentiation. Members of several chromatin-remodeling families demonstrated involvement, including factors instrumental in histone modification, chromatin assembly-disassembly and DNA silencing, suggesting complex, multi-level systemic epigenetic regulation. Further, trichostatin A, a histone deacetylase inhibitor, accelerated expression of SMC differentiation markers in this model. Ontology analysis indicated a high degree of p300 involvement in SMC differentiation, with 60.7% of the known p300 interactome showing significant expression changes. Knockdown of p300 expression accelerated SMC differentiation in A404 cells and human SMCs, while inhibition of p300 HAT activity blunted SMC differentiation. The results suggest a central but complex role for p300 in SMC phenotypic modulation.Our results support the hypothesis that chromatin remodeling is important for SMC phenotypic switching, and detail wide-ranging involvement of several epigenetic modification families. Additionally, the transcriptional coactivator p300 may be partially degraded during SMC differentiation, leaving an activated subpopulation with increased HAT activity and SMC differentiation-gene specificity.

    View details for DOI 10.1371/journal.pone.0014301

    View details for Web of Science ID 000285246900013

    View details for PubMedID 21179216

  • Nicotine Accelerates the Expansion of Abdominal Aortic Aneurysms in Mice; A Potential Role for miR-21 and miR-26a Maegdefessel, L., Azuma, J., Spin, J. M., Deng, A., McConnell, M. V., Dalman, R. L., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2010
  • Apelin Inhibits Lipolysis in a Gq-, Gi-, and AMPK-Dependent Manner Yue, P., Jin, H., Aillaud, M., Deng, A., Azuma, J., Kundu, R., Reaven, G., Quertermous, T., Tsao, P. LIPPINCOTT WILLIAMS & WILKINS. 2010
  • Novel 3-Dimensional Microscopy Methodology Development to Study Regional Microstructural Changes over the Time Course of Abdominal Aortic Aneurysm Development Scientific Sessions on Arteriosclerosis, Thrombosis and Vascular Biology Saatchi, S., Azuma, J., Wanchoo, N., Tsao, P. S., Smith, S. J., Yock, P. G., Taylor, C. A. LIPPINCOTT WILLIAMS & WILKINS. 2010: E277–E277
  • Microarray Analysis Identifies miRNA-26a as a Regulator of Vascular Smooth Muscle Cell Phenotypic Modulation Scientific Sessions on Arteriosclerosis, Thrombosis and Vascular Biology Leeper, N. J., Raiesdana, A., Kojima, Y., Chun, H. J., Azuma, J., Kundu, R. K., Quertermous, T., Tsao, P. S., Spin, J. M. LIPPINCOTT WILLIAMS & WILKINS. 2010: E244–E244
  • In Vivo Quantification of Murine Aortic Cyclic Strain, Motion, and Curvature: Implications for Abdominal Aortic Aneurysm Growth JOURNAL OF MAGNETIC RESONANCE IMAGING Goergen, C. J., Barr, K. N., Huynh, D. T., Eastham-Anderson, J. R., Choi, G., Hedehus, M., Dalman, R. L., Connolly, A. J., Taylor, C. A., Tsao, P. S., Greve, J. M. 2010; 32 (4): 847-858


    To develop methods to quantify cyclic strain, motion, and curvature of the murine abdominal aorta in vivo.C57BL/6J and apoE(-/-) mice underwent three-dimensional (3D) time-of-flight MR angiography to position cardiac-gated 2D slices at four locations along the abdominal aorta where circumferential cyclic strain and lumen centroid motion were calculated. From the 3D data, a centerline through the aorta was created to quantify geometric curvature at 0.1-mm intervals. Medial elastin content was quantified with histology postmortem. The location and shape of abdominal aortic aneurysms (AAAs), created from angiotensin II infusion, were evaluated qualitatively.Strain waveforms were similar at all locations and between groups. Centroid motion was significantly larger and more leftward above the renal vessels than below (P < 0.05). Maximum geometric curvature occurred slightly proximal to the right renal artery. Elastin content was similar around the circumference of the vessel. AAAs developed in the same location as the maximum curvature and grew in the same direction as vessel curvature and motion.The methods presented provide temporally and spatially resolved data quantifying murine aortic motion and curvature in vivo. This noninvasive methodology will allow serial quantification of how these parameters influence the location and direction of AAA growth.

    View details for DOI 10.1002/jmri.22331

    View details for Web of Science ID 000282764800010

    View details for PubMedID 20882615

    View details for PubMedCentralID PMC2975391

  • Differential adipogenic and inflammatory properties of small adipocytes in Zucker Obese and Lean rats DIABETES & VASCULAR DISEASE RESEARCH Liu, A., Sonmez, A., Yee, G., Bazuine, M., Arroyo, M., Sherman, A., McLaughlin, T., Reaven, G., Cushman, S., Tsao, P. 2010; 7 (4): 311-318


    We recently reported that a preponderance of small adipose cells, decreased expression of cell differentiation markers, and enhanced inflammatory activity in human subcutaneous whole adipose tissue were associated with insulin resistance. To test the hypothesis that small adipocytes exhibited these differential properties, we characterised small adipocytes from epididymal adipose tissue of Zucker Obese (ZO) and Lean (ZL) rats. Rat epididymal fat pads were removed and adipocytes isolated by collagenase digestion. Small adipocytes were separated by sequential filtration through nylon meshes. Adipocytes were fixed in osmium tetroxide for cell size distribution analysis via Beckman Coulter Multisizer. Quantitative real-time PCR for cell differentiation and inflammatory genes was performed. Small adipocytes represented a markedly greater percentage of the total adipocyte population in ZO than ZL rats (58±4% vs. 12±3%, p<0.001). In ZO rats, small as compared with total adipocytes had 4-fold decreased adiponectin, and 4-fold increased visfatin and IL-6 levels. Comparison of small adipocytes in ZO versus ZL rats revealed 3-fold decreased adiponectin and PPARγ levels, and 2.5-fold increased IL-6. In conclusion, ZO rat adipose tissue harbours a large proportion of small adipocytes that manifest impaired cell differentiation and pro-inflammatory activity, two mechanisms by which small adipocytes may contribute to insulin resistance.

    View details for DOI 10.1177/1479164110386126

    View details for Web of Science ID 000285080700008

    View details for PubMedID 20961992

  • Pioglitazone Increases the Proportion of Small Cells in Human Abdominal Subcutaneous Adipose Tissue OBESITY McLaughlin, T. M., Liu, T., Yee, G., Abbasi, F., Lamendola, C., Reaven, G. M., Tsao, P., Cushman, S. W., Sherman, A. 2010; 18 (5): 926-931


    Rodent and in vitro studies suggest that thiazolidinediones promote adipogenesis but there are few studies in humans to corroborate these findings. The purpose of this study was to determine whether pioglitazone stimulates adipogenesis in vivo and whether this process relates to improved insulin sensitivity. To test this hypothesis, 12 overweight/obese nondiabetic, insulin-resistant individuals underwent biopsy of abdominal subcutaneous adipose tissue at baseline and after 12 weeks of pioglitazone treatment. Cell size distribution was determined via the Multisizer technique. Insulin sensitivity was quantified at baseline and postpioglitazone by the modified insulin suppression test. Regional fat depots were quantified by computed tomography (CT). Insulin resistance (steady-state plasma insulin and glucose (SSPG)) decreased following pioglitazone (P < 0.001). There was an increase in the ratio of small-to-large cells (1.16 +/- 0.44 vs. 1.52 +/- 0.66, P = 0.03), as well as a 25% increase in the absolute number of small cells (P = 0.03). The distribution of large cell diameters widened (P = 0.009), but diameter did not increase in the case of small cells. The increase in proportion of small cells was associated with the degree to which insulin resistance improved (r = -0.72, P = 0.012). Visceral abdominal fat decreased (P = 0.04), and subcutaneous abdominal (P = 0.03) and femoral fat (P = 0.004) increased significantly. Changes in fat volume were not associated with SSPG change. These findings demonstrate a clear effect of pioglitazone on human subcutaneous adipose cells, suggestive of adipogenesis in abdominal subcutaneous adipose tissue, as well as redistribution of fat from visceral to subcutaneous depots, highlighting a potential mechanism of action for thiazolidinediones. These findings support the hypothesis that defects in subcutaneous fat storage may underlie obesity-associated insulin resistance.

    View details for DOI 10.1038/oby.2009.380

    View details for Web of Science ID 000277234800013

    View details for PubMedID 19910937

  • Using Pre-existing Microarray Datasets to Increase Experimental Power: Application to Insulin Resistance PLOS COMPUTATIONAL BIOLOGY Daigle, B. J., Deng, A., McLaughlin, T., Cushman, S. W., Cam, M. C., Reaven, G., Tsao, P. S., Altman, R. B. 2010; 6 (3)


    Although they have become a widely used experimental technique for identifying differentially expressed (DE) genes, DNA microarrays are notorious for generating noisy data. A common strategy for mitigating the effects of noise is to perform many experimental replicates. This approach is often costly and sometimes impossible given limited resources; thus, analytical methods are needed which increase accuracy at no additional cost. One inexpensive source of microarray replicates comes from prior work: to date, data from hundreds of thousands of microarray experiments are in the public domain. Although these data assay a wide range of conditions, they cannot be used directly to inform any particular experiment and are thus ignored by most DE gene methods. We present the SVD Augmented Gene expression Analysis Tool (SAGAT), a mathematically principled, data-driven approach for identifying DE genes. SAGAT increases the power of a microarray experiment by using observed coexpression relationships from publicly available microarray datasets to reduce uncertainty in individual genes' expression measurements. We tested the method on three well-replicated human microarray datasets and demonstrate that use of SAGAT increased effective sample sizes by as many as 2.72 arrays. We applied SAGAT to unpublished data from a microarray study investigating transcriptional responses to insulin resistance, resulting in a 50% increase in the number of significant genes detected. We evaluated 11 (58%) of these genes experimentally using qPCR, confirming the directions of expression change for all 11 and statistical significance for three. Use of SAGAT revealed coherent biological changes in three pathways: inflammation, differentiation, and fatty acid synthesis, furthering our molecular understanding of a type 2 diabetes risk factor. We envision SAGAT as a means to maximize the potential for biological discovery from subtle transcriptional responses, and we provide it as a freely available software package that is immediately applicable to any human microarray study.

    View details for DOI 10.1371/journal.pcbi.1000718

    View details for Web of Science ID 000278125200026

    View details for PubMedID 20361040

    View details for PubMedCentralID PMC2845644

  • Inflammation in subcutaneous adipose tissue: relationship to adipose cell size DIABETOLOGIA McLaughlin, T., Deng, A., Yee, G., Lamendola, C., Reaven, G., Tsao, P. S., Cushman, S. W., Sherman, A. 2010; 53 (2): 369-377


    Inflammation is associated with increased body mass and purportedly with increased size of adipose cells. We sought to determine whether increased size of adipose cells is associated with localised inflammation in weight-stable, moderately obese humans.We recruited 49 healthy, moderately obese individuals for quantification of insulin resistance (modified insulin suppression test) and subcutaneous abdominal adipose tissue biopsy. Cell size distribution was analysed with a multisizer device and inflammatory gene expression with real-time PCR. Correlations between inflammatory gene expression and cell size variables, with adjustment for sex and insulin resistance, were calculated.Adipose cells were bimodally distributed, with 47% in a 'large' cell population and the remainder in a 'small' cell population. The median diameter of the large adipose cells was not associated with expression of inflammatory genes. Rather, the fraction of small adipose cells was consistently associated with inflammatory gene expression, independently of sex, insulin resistance and BMI. This association was more pronounced in insulin-resistant than insulin-sensitive individuals. Insulin resistance also independently predicted expression of inflammatory genes.This study demonstrates that among moderately obese, weight-stable individuals an increased proportion of small adipose cells is associated with inflammation in subcutaneous adipose tissue, whereas size of mature adipose cells is not. The observed association between small adipose cells and inflammation may reflect impaired adipogenesis and/or terminal differentiation. However, it is unclear whether this is a cause or consequence of inflammation. This question and whether small vs large adipose cells contribute differently to inflammation in adipose tissue are topics for future research. Trial registration: NCT00285844.

    View details for DOI 10.1007/s00125-009-1496-3

    View details for Web of Science ID 000273084400019

    View details for PubMedID 19816674

  • New options with dabigatran etexilate in anticoagulant therapy. Vascular health and risk management Maegdefessel, L., Spin, J. M., Azuma, J., Tsao, P. S. 2010; 6: 339-349


    Thrombosis, the localized clotting of blood, occurs in both the arterial and venous circulation, and has a major impact on health outcomes. The primary etiology of myocardial infarctions, and approximately 80% of strokes, is acute arterial thrombosis. In combination this represents the most common cause of death in the Western world, while the third leading cause of cardiovascular-associated death is venous thromboembolism. An understanding of the pathogenic changes in the vessel wall and the blood that result in thrombosis is crucial for developing safer and more effective antithrombotic drugs. Dabigatran etexilate belongs to a new class of direct thrombin inhibitors. Following oral administration, dabigatran reaches peak plasma concentrations within 2 hours, shows linear pharmacokinetics, and a limited (but important) amount of direct drug interactions. Given once daily at 150 mg or 220 mg, it has proven to be competitive with enoxaparin in the prevention of venous thromboembolism after major orthopedic surgery, with a comparable safety profile. For stroke prevention in patients suffering from atrial fibrillation, dabigatran administered at a dose of 110 mg twice daily was associated with rates of stroke and systemic embolism that were similar to those associated with warfarin, as well as lower rates of hemorrhage. Dabigatran given at a dose of 150 mg twice daily, as compared with warfarin, was associated with lower rates of stroke and systemic embolism but similar rates of major hemorrhage. Oral bioavailability of dabigatran, together with a rapid onset and offset of action and predictable anticoagulation response, makes this newly available antithrombotic drug an attractive alternative to traditional anticoagulant therapies for numerous thrombosis-related indications.

    View details for PubMedID 20531953

  • Apelin is necessary for the maintenance of insulin sensitivity AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Yue, P., Jin, H., Aillaud, M., Deng, A. C., Azuma, J., Asagami, T., Kundu, R. K., Reaven, G. M., Quertermous, T., Tsao, P. S. 2010; 298 (1): E59-E67


    The recently discovered peptide apelin is known to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role in the chronic setting. Fasting glucose, insulin, and adiponectin levels were determined on mice with generalized deficiency of apelin (APKO). Additionally, insulin (ITT) and glucose tolerance tests (GTT) were performed. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 wk. Following the infusion, ITT/GTTs were repeated and the animals euthanized. Soleus muscles were harvested and homogenized in lysis buffer, and insulin-induced Akt phosphorylation was determined by Western blotting. Apelin-13 infusion and ITTs/GTTs were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelin's effects, apelin-13 was also delivered to cultured C2C12 myotubes. 2-[3H]deoxyglucose uptake and Akt phosphorylation were assessed in the presence of various inhibitors. APKO mice had diminished insulin sensitivity, were hyperinsulinemic, and had decreased adiponectin levels. Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to APKO and db/db mice resulted in improved insulin sensitivity. In C2C12 myotubes, apelin increased glucose uptake and Akt phosphorylation. These events were fully abrogated by pertussis toxin, compound C, and siRNA knockdown of AMPKalpha1 but only partially diminished by LY-294002 and not at all by L-NAME. We conclude that apelin is necessary for the maintenance of insulin sensitivity in vivo. Apelin's effects on glucose uptake and Akt phosphorylation are in part mediated by a G(i) and AMPK-dependent pathway.

    View details for DOI 10.1152/ajpendo.00385.2009

    View details for Web of Science ID 000272793700007

    View details for PubMedID 19861585

  • Modern role for clopidogrel in management of atrial fibrillation and stroke reduction. Vascular health and risk management Maegdefessel, L., Azuma, J., Tsao, P. S. 2010; 6: 95-103


    Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. The prevalence of AF increases sharply in old age (prevalence approximately 10% among persons 80 years of age and older). The expected risk for ischemic stroke is increased five-fold by the presence of AF, primarily as a result of cardiogenic embolism. Multiple large-scale, randomized trials have been completed or are still underway to find optimal, efficacious, and relatively safe ways to reduce the risk of ischemic stroke and other systemic thromboembolic events related to AF. Antithrombotic strategies are accompanied by serious bleeding complications that threaten patients in need of medical stroke prevention. Treatment regimens for preventing thromboembolism in AF patients range from vitamin K antagonists such as warfarin or coumadins, antiplatelet drugs like aspirin or clopidogrel, to newly developed orally available antithrombotics like the direct thrombin inhibitor dabigatran, or the Factor Xa-inhibitor rivaroxaban. The available anticoagulant and antiplatelet drugs have different advantages and disadvantages. This review attempts to delineate the specific role of clopidogrel in patients with AF and at risk of stroke, taking into consideration new and ongoing trials in this important field of medical practice.

    View details for PubMedID 20234784

  • Distribution of Asymmetric Dimethylarginine among 980 Healthy, Older Adults of Different Ethnicities CLINICAL CHEMISTRY Sydow, K., Fortmann, S. P., Fair, J. M., Varady, A., Hlatky, M. A., Go, A. S., Iribarren, C., Tsao, P. S. 2010; 56 (1): 111-120


    Endothelium-derived nitric oxide plays a crucial role in the regulation of vascular tone and the development of cardiovascular disease. The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) has emerged as a novel cardiovascular risk factor. ADMA appears to be an independent predictor for cardiovascular and overall mortality. However, the majority of studies investigating the clinical role of ADMA were performed in European study populations with few individuals of other ethnicities.We performed a cross-sectional study of 980 healthy, older (age 60-72 years) individuals of different ethnicities living in the San Francisco Bay area and analyzed ADMA plasma concentrations and their relationship to other cardiovascular risk factors. Plasma ADMA concentrations were measured using a recently developed, highly sensitive ELISA.In our entire sample, we were able to define a reference interval for ADMA plasma concentrations of 0.47 (90% CI 0.46-0.48) mumol/L to 0.85 (0.84-0.89) mumol/L. The mean ADMA concentration was 0.63 (SD 0.11) mumol/L (median 0.61 mumol/L). Mean ADMA concentrations were significantly lower in African Americans (0.60 mumol/L; P < 0.01) and mixed non-Hispanics (0.60 mumol/L; P < 0.05) compared with whites (0.63 mumol/L). ADMA was positively correlated with cystatin-C in both men (rho = 0.29) and women (rho = 0.37), and median plasma ADMA concentrations increased across cystatin-C quintiles.ADMA varies nearly 2-fold across a healthy sample of older men and women, correlates with age, body mass index, and renal function, and is different across ethnic groups. Additional studies in a wider age range and including larger ethnic subgroups would be useful.

    View details for DOI 10.1373/clinchem.2009.136200

    View details for Web of Science ID 000273466300017

    View details for PubMedID 19892843

  • Apelin is Necessary for the Maintenance of Insulin Sensitivity 82nd National Conference and Exhibitions and Scientific Sessions of the American-Heart-Association Yue, P., Jin, H., Aillaud-Manzanera, M., Deng, A. C., Azuma, J., Kundu, R. K., Reaven, G. M., Quertermous, T., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2009: S1114–S1115
  • Differential Intra-abdominal Adipose Tissue Profiling in Obese, Insulin-resistant Women OBESITY SURGERY Liu, A., McLaughlin, T., Liu, T., Sherman, A., Yee, G., Abbasi, F., Lamendola, C., Morton, J., Cushman, S. W., Reaven, G. M., Tsao, P. S. 2009; 19 (11): 1564-1573


    We recently identified differences in abdominal subcutaneous adipose tissue (SAT) from insulin-resistant (IR) as compared to obesity-matched insulin sensitive individuals, including accumulation of small adipose cells, decreased expression of cell differentiation markers, and increased inflammatory activity. This study was initiated to see if these changes in SAT of IR individuals were present in omental visceral adipose tissue (VAT); in this instance, individuals were chosen to be IR but varied in degree of adiposity. We compared cell size distribution and genetic markers in SAT and VAT of IR individuals undergoing bariatric surgery.Eleven obese/morbidly obese women were IR by the insulin suppression test. Adipose tissue surgical samples were fixed in osmium tetroxide for cell size analysis via Beckman Coulter Multisizer. Quantitative real-time polymerase chain reaction for genes related to adipocyte differentiation and inflammation was performed.While proportion of small cells and expression of adipocyte differentiation genes did not differ between depots, inflammatory genes were upregulated in VAT. Diameter of SAT large cells correlated highly with increasing proportion of small cells in both SAT and VAT (r = 0.85, p = 0.001; r = 0.72, p = 0.01, respectively). No associations were observed between VAT large cells and cell size variables in either depot. The effect of body mass index (BMI) on any variables in both depots was negligible.The major differential property of VAT of IR women is increased inflammatory activity, independent of BMI. The association of SAT adipocyte hypertrophy with hyperplasia in both depots suggests a primary role SAT may have in regulating regional fat storage.

    View details for DOI 10.1007/s11695-009-9949-9

    View details for Web of Science ID 000271282900016

    View details for PubMedID 19711137

    View details for PubMedCentralID PMC3181138

  • Hypercholesterolemia impairs exercise capacity in mice VASCULAR MEDICINE Maxwell, A. J., Niebauer, J., Lin, P. S., Tsao, P. S., Bernstein, D., Cooke, J. P. 2009; 14 (3): 249-257


    We previously reported an attenuation of both exercise hyperemia and measures of aerobic capacity in hypercholesterolemic mice. In this study, we expanded upon the previous findings by examining the temporal and quantitative relationship of hypercholesterolemia to aerobic and anaerobic capacity and by exploring several potential mechanisms of dysfunction. Eight-week-old wild type (n = 123) and apoE knockout (n = 79) C57BL/6J mice were divided into groups with distinct cholesterol levels by feeding with regular or high-fat diets. At various ages, the mice underwent treadmill ergospirometry. To explore mechanisms, aortic ring vasodilator function and nitrate (NO(x)) activity, urinary excretion of NO(x), running muscle microvascular density and citrate synthase activity, as well as myocardial mass and histologic evidence of ischemia were measured. At 8 weeks of age, all mice had similar measures of exercise capacity. All indices of aerobic exercise capacity progressively declined at 12 and 20 weeks of age in the hypercholesterolemic mice as cholesterol levels increased while indices of anaerobic capacity remained unaffected. Across the four cholesterol groups, the degree of aerobic dysfunction was related to serum cholesterol levels; a relationship that was maintained after correcting for confounding factors. Associated with the deterioration in exercise capacity was a decline in measures of nitric oxide-mediated vascular function while there was no evidence of aberrations in functional or oxidative capacities or in other components of transport capacity. In conclusion, aerobic exercise dysfunction is observed in murine models of genetic and diet-induced hypercholesterolemia and is associated with a reduction in vascular nitric oxide production.

    View details for DOI 10.1177/1358863X08100040

    View details for Web of Science ID 000268568300008

    View details for PubMedID 19651675

    View details for PubMedCentralID PMC3140166

  • Apelin prevents aortic aneurysm formation by inhibiting macrophage inflammation AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Leeper, N. J., Tedesco, M. M., Kojima, Y., Schultz, G. M., Kundu, R. K., Ashley, E. A., Tsao, P. S., Dalman, R. L., Quertermous, T. 2009; 296 (5): H1329-H1335


    Apelin is a potent inodilator with recently described antiatherogenic properties. We hypothesized that apelin might also attenuate abdominal aortic aneurysm (AAA) formation by limiting disease-related vascular wall inflammation. C57BL/6 mice implanted with osmotic pumps filled with apelin or saline were treated with pancreatic elastase to create infrarenal AAAs. Mice were euthanized for aortic PCR analysis or followed ultrasonographically and then euthanized for histological analysis. The cellular expression of inflammatory cytokines and chemokines in response to apelin was also assessed in cultured macrophages, smooth muscle cells, and fibroblasts. Apelin treatment resulted in diminished AAA formation, with a 47% reduction in maximal cross-sectional area (0.74 vs. 1.39 mm(2), P < 0.03) and a 57% reduction in macrophage infiltrate (113 vs. 261.3 cells/high-power field, P < 0.0001) relative to the saline-treated group. Apelin infusion was also associated with significantly reduced aortic macrophage colony-stimulating factor expression and decreased monocyte chemattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, interleukin (IL)-6, and tumor necrosis factor (TNF)-alpha mean mRNA levels. Apelin stimulation of cultured macrophages significantly reduced MCP-1 and TNF-alpha mRNA levels relative to baseline (2.03- and 1.89-fold reduction, P < 0.03, respectively) but did not affect intimal adhesion molecule expression or medial or adventitial cell cytokine production. Apelin significantly reduces aneurysm formation in the elastase model of human AAA disease. The mechanism appears to be decreased macrophage burden, perhaps related to an apelin-mediated decrease in proinflammatory cytokine and chemokine activation.

    View details for DOI 10.1152/ajpheart.01341.2008

    View details for Web of Science ID 000265659100020

    View details for PubMedID 19304942

  • Creation of murine experimental abdominal aortic aneurysms with elastase. Journal of visualized experiments : JoVE Azuma, J., Asagami, T., Dalman, R., Tsao, P. S. 2009


    Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.(1) The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.(2) The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

    View details for DOI 10.3791/1280

    View details for PubMedID 19629030

  • Robust Automatic Feature Extraction for Protein Microarrays 26th IEEE International Instrumentation and Measurement Technology Conference Ahmed, M. O., Dyer, J. S., Hytopoulos, E., Itakura, H., Tsao, P. S. IEEE. 2009: 1721–1726
  • Insulin resistance is associated with a modest increase in inflammation in subcutaneous adipose tissue of moderately obese women DIABETOLOGIA McLaughlin, T., Deng, A., Gonzales, O., AILLAUD, M., Yee, G., Lamendola, C., Abbasi, F., Connolly, A. J., Sherman, A., Cushman, S. W., Reaven, G., Tsao, P. S. 2008; 51 (12): 2303-2308


    We have previously described differences in adipose cell size distribution and expression of genes related to adipocyte differentiation in subcutaneous abdominal fat obtained from insulin-sensitive (IS) and -resistant (IR) persons, matched for degree of moderate obesity. To determine whether other biological properties also differ between IR and IS obese individuals, we quantified markers of inflammatory activity in adipose tissue from overweight IR and IS individuals.Subcutaneous abdominal tissue was obtained from moderately obese women, divided into IR (n = 14) and IS (n = 19) subgroups by determining their steady-state plasma glucose (SSPG) concentrations during the insulin suppression test. Inflammatory activity was assessed by comparing expression of nine relevant genes and by immunohistochemical quantification of CD45- and CD68-containing cells.SSPG concentrations were approximately threefold higher in IR than in IS individuals. Expression levels of CD68, EMR1, IL8, IL6 and MCP/CCL2 mRNAs were modestly but significantly increased (p < 0.05) in IR compared with IS participants. Results of immunohistochemical staining were consistent with gene expression data, demonstrating modest differences between IR and IS individuals. Crown-like structures, in which macrophages surround single adipocytes, were rarely seen in tissue from either subgroup.A modest increase in inflammatory activity was seen in subcutaneous adipose tissue from IR compared with equally obese IS individuals. Together with previous evidence of impaired adipose cell differentiation in IR vs equally obese individuals, it appears that at least two biological processes in subcutaneous adipose tissue characterize the insulin-resistant state independent of obesity per se.

    View details for DOI 10.1007/s00125-008-1148-z

    View details for Web of Science ID 000260686000019

    View details for PubMedID 18825363

  • A Human Ferritin Iron Oxide Nano-composite Magnetic Resonance Contrast Agent MAGNETIC RESONANCE IN MEDICINE Uchida, M., Terashima, M., Cunningham, C. H., Suzuki, Y., Willits, D. A., Willis, A. F., Yang, P. C., Tsao, P. S., McConnell, M. V., Young, M. J., Douglas, T. 2008; 60 (5): 1073-1081


    Macrophages play important roles in the immunological defense system, but at the same time they are involved in inflammatory diseases such as atherosclerosis. Therefore, imaging macrophages is critical to assessing the status of these diseases. Toward this goal, a recombinant human H chain ferritin (rHFn)-iron oxide nano composite has been investigated as an MRI contrast agent for labeling macrophages. Iron oxide nanoparticles in the form of magnetite (or maghemite) with narrow size distribution were synthesized in the interior cavity of rHFn. The composite material exhibited the R(2) relaxivity comparable to known iron oxide MRI contrast agents. Furthermore, the mineralized protein cages are readily taken up by macrophages in vitro and provide significant T2* signal loss of the labeled cells. These results encourage further investigation into the development of the rHFn-iron oxide contrast agent to assess inflammatory disease status such as macrophage-rich atherosclerotic plaques in vivo.

    View details for DOI 10.1002/mrm.21761

    View details for Web of Science ID 000260341700008

    View details for PubMedID 18956458

  • Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis JOURNAL OF CLINICAL INVESTIGATION Chun, H. J., Ali, Z. A., Kojima, Y., Kundu, R. K., Sheikh, A. Y., Agrawal, R., Zheng, L., Leeper, N. J., Pearl, N. E., Patterson, A. J., Anderson, J. P., Tsao, P. S., Lenardo, M. J., Ashley, E. A., Quertermous, T. 2008; 118 (10): 3343-3354


    Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function and a vasodepressor function in the systemic circulation. The apelin-APJ pathway appears to have opposing physiological roles to the renin-angiotensin system. Here we investigated whether the apelin-APJ pathway can directly antagonize vascular disease-related Ang II actions. In ApoE-KO mice, exogenous Ang II induced atherosclerosis and abdominal aortic aneurysm formation; we found that coinfusion of apelin abrogated these effects. Similarly, apelin treatment rescued Ang II-mediated increases in neointimal formation and vascular remodeling in a vein graft model. NO has previously been implicated in the vasodepressor function of apelin; we found that apelin treatment increased NO bioavailability in ApoE-KO mice. Furthermore, infusion of an NO synthase inhibitor blocked the apelin-mediated decrease in atherosclerosis and aneurysm formation. In rat primary aortic smooth muscle cells, apelin inhibited Ang II-mediated transcriptional regulation of multiple targets as measured by reporter assays. In addition, we demonstrated by coimmunoprecipitation and fluorescence resonance energy transfer analysis that the Ang II and apelin receptors interacted physically. Taken together, these findings indicate that apelin signaling can block Ang II actions in vascular disease by increasing NO production and inhibiting Ang II cellular signaling.

    View details for DOI 10.1172/JCI34871

    View details for Web of Science ID 000259828600016

    View details for PubMedID 18769630

  • Transcriptome Alteration in the Diabetic Heart by Rosiglitazone: Implications for Cardiovascular Mortality PLOS ONE Wilson, K. D., Li, Z., Wagner, R., Yue, P., Tsao, P., Nestorova, G., Huang, M., Hirschberg, D. L., Yock, P. G., Quertermous, T., Wu, J. C. 2008; 3 (7)


    Recently, the type 2 diabetes medication, rosiglitazone, has come under scrutiny for possibly increasing the risk of cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiac transcriptional profiling and imaging studies of a murine model of type 2 diabetes, the C57BL/KLS-lepr(db)/lepr(db) (db/db) mouse.We compared cardiac gene expression profiles from three groups: untreated db/db mice, db/db mice after rosiglitazone treatment, and non-diabetic db/+ mice. Prior to sacrifice, we also performed cardiac magnetic resonance (CMR) and echocardiography. As expected, overall the db/db gene expression signature was markedly different from control, but to our surprise was not significantly reversed with rosiglitazone. In particular, we have uncovered a number of rosiglitazone modulated genes and pathways that may play a role in the pathophysiology of the increase in cardiac mortality as seen in several recent meta-analyses. Specifically, the cumulative upregulation of (1) a matrix metalloproteinase gene that has previously been implicated in plaque rupture, (2) potassium channel genes involved in membrane potential maintenance and action potential generation, and (3) sphingolipid and ceramide metabolism-related genes, together give cause for concern over rosiglitazone's safety. Lastly, in vivo imaging studies revealed minimal differences between rosiglitazone-treated and untreated db/db mouse hearts, indicating that rosiglitazone's effects on gene expression in the heart do not immediately turn into detectable gross functional changes.This study maps the genomic expression patterns in the hearts of the db/db murine model of diabetes and illustrates the impact of rosiglitazone on these patterns. The db/db gene expression signature was markedly different from control, and was not reversed with rosiglitazone. A smaller number of unique and interesting changes in gene expression were noted with rosiglitazone treatment. Further study of these genes and molecular pathways will provide important insights into the cardiac decompensation associated with both diabetes and rosiglitazone treatment.

    View details for DOI 10.1371/journal.pone.0002609

    View details for Web of Science ID 000264065800015

    View details for PubMedID 18648539

    View details for PubMedCentralID PMC2481284

  • Asymmetrical dimethylarginine in renal disease: Limits of variation or variation limits? AMERICAN JOURNAL OF NEPHROLOGY Jacobi, J., Tsao, P. S. 2008; 28 (2): 224-237


    Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is increasingly recognized as a putative biomarker in cardiovascular and renal disease. Elevated plasma levels of ADMA are the consequence of increased synthesis, reduced renal clearance or reduced enzymatic degradation. Based upon the metabolic fate the highest plasma concentrations of ADMA have been reported in patients with renal failure in whom this molecule accumulates. However, the range of published ADMA levels in patients with chronic renal failure as well as in patients with end-stage renal failure undergoing maintenance hemodialysis, peritoneal dialysis or kidney transplant recipients is widely scattered and overlaps with the levels reported in healthy individuals. This wide distribution can in part be explained by different bioanalytical techniques and the lack of standardization of such assays. This review summarizes available literature on ADMA in patients with kidney disease and stresses the urgent need for a consensus regarding reference values for different analytical methods in order to appreciate the prognostic significance of elevated ADMA levels. At present, one cannot advocate this molecule for risk assessment or individual patient prognosis in the clinical work-up of patients with renal impairment.

    View details for DOI 10.1159/000110092

    View details for Web of Science ID 000251436000007

    View details for PubMedID 17960061

  • In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Sheikh, A. Y., Chun, H. J., Glassford, A. J., Kundu, R. K., Kutschka, I., Ardigo, D., Hendry, S. L., Wagner, R. A., Chen, M. M., Ali, Z. A., Yue, P., Huynh, D. T., Connolly, A. J., Pelletier, M. P., Tsao, P. S., Robbins, R. C., Quertermous, T. 2008; 294 (1): H88-H98


    Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse. Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.

    View details for DOI 10.1152/ajpheart.00935.2007

    View details for Web of Science ID 000252261200013

    View details for PubMedID 17906101

    View details for PubMedCentralID PMC2570026

  • HIF-1 regulates hypoxia- and insulin-induced expression of apelin in adipocytes AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Glassford, A. J., Yue, P., Sheikh, A. Y., Chun, H. J., Zarafshar, S., Chan, D. A., Reaven, G. M., Quertermous, T., Tsao, P. S. 2007; 293 (6): E1590-E1596


    Apelin, a novel peptide with significant cardioactive properties, is upregulated by insulin in adipocytes. However, the mechanism by which insulin promotes apelin production is unknown. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor involved in the angiogenic and metabolic responses to tissue hypoxia, has been shown to be activated by insulin in various settings. We therefore hypothesized that HIF-1 regulates insulin-mediated apelin expression in adipocytes. 3T3-L1 cells were differentiated into adipocytes in culture. For experiments, serum-starved 3T3-L1 cells were exposed to insulin and/or a 1% O(2) environment. Apelin expression was assessed using quantitative real-time PCR and ELISA. To directly assess the role of HIF-1 in apelin production, we differentiated mouse embryonic fibroblasts (MEFs) containing a targeted deletion of the HIF-1alpha gene into adipocytes and measured their response to insulin and hypoxia. Apelin expression in mature 3T3-L1 adipocytes was increased significantly by insulin and was attenuated by pharmacological inhibition of insulin signaling. Exposure of cells to either hypoxia or the chemical HIF activators cobalt chloride (CoCl(2)) and dimethyloxaloylglycine (DMOG) resulted in significant upregulation of apelin, consistent with a role for HIF in apelin induction. Moreover, hypoxia-, CoCl(2)-, DMOG-, and insulin-induced apelin expression were all attenuated in differentiated HIF-1alpha-deficient MEFs. In summary, in cultured 3T3-L1 adipocytes and differentiated MEFs, HIF-1 appears to be involved in hypoxia- and insulin-induced apelin expression.

    View details for DOI 10.1152/ajpendo.00490.2007

    View details for Web of Science ID 000251510200014

    View details for PubMedID 17878221

  • Circulating chemokines accurately identify individuals with clinically significant atherosclerotic heart disease PHYSIOLOGICAL GENOMICS Ardigo, D., Assimes, T. L., Fortmann, S. P., Go, A. S., Hlatky, M., Hytopoulos, E., Iribarren, C., Tsao, P. S., Tabibiazar, R., Quertermous, T. 2007; 31 (3): 402-409


    Serum inflammatory markers correlate with outcome and response to therapy in subjects with cardiovascular disease. However, current individual markers lack specificity for the diagnosis of coronary artery disease (CAD). We hypothesize that a multimarker proteomic approach measuring serum levels of vascular derived inflammatory biomarkers could reveal a "signature of disease" that can serve as a highly accurate method to assess for the presence of coronary atherosclerosis. We simultaneously measured serum levels of seven chemokines [CXCL10 (IP-10), CCL11 (eotaxin), CCL3 (MIP1 alpha), CCL2 (MCP1), CCL8 (MCP2), CCL7 (MCP3), and CCL13 (MCP4)] in 48 subjects with clinically significant CAD ("cases") and 44 controls from the ADVANCE Study. We applied three classification algorithms to identify the combination of variables that would best predict case-control status and assessed the diagnostic performance of these models with receiver operating characteristic (ROC) curves. The serum levels of six chemokines were significantly higher in cases compared with controls (P < 0.05). All three classification algorithms entered three chemokines in their final model, and only logistic regression selected clinical variables. Logistic regression produced the highest ROC of the three algorithms (AUC = 0.95; SE = 0.03), which was markedly better than the AUC for the logistic regression model of traditional risk factors of CAD without (AUC = 0.67; SE = 0.06) or with CRP (AUC = 0.68; SE = 0.06). A combination of serum levels of multiple chemokines identifies subjects with clinically significant atherosclerotic heart disease with a very high degree of accuracy. These results need to be replicated in larger cross-sectional studies and their prognostic value explored.

    View details for DOI 10.1152/physiolgenomics.00104.2007

    View details for Web of Science ID 000251780600005

    View details for PubMedID 17698927

  • The cardioactive peptide apelin increases adipocyte glucose uptake and is necessary for the maintenance of systemic insulin sensitivity 80th Annual Scientific Session of the American-Heart-Association (AHA) Yue, P., Asagami, T., Kundu, R. K., Yee, Y., Glassford, A. J., Azuma, J., Quertermous, T., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2007: 247–47
  • Small adipocytes: implications for inflammation and insulin resistance Sonmez, A., Sung, K., Yee, G., Deng, A., Mullen, S., McLaughlin, T., Cushman, S., Reaven, G., Tsao, P. OXFORD UNIV PRESS. 2007: 619–619
  • Enhanced proportion of small adipose cells in insulin-resistant vs insulin-sensitive obese individuals implicates impaired adipogenesis DIABETOLOGIA McLaugblin, T., Sherman, A., Tsao, P., Gonzalez, O., Yee, G., Lamendola, C., Reaven, G. M., Cusbman, S. W. 2007; 50 (8): 1707-1715


    The biological mechanism by which obesity predisposes to insulin resistance is unclear. One hypothesis is that larger adipose cells disturb metabolism via increased lipolysis. While studies have demonstrated that cell size increases in proportion to BMI, it has not been clearly shown that adipose cell size, independent of BMI, is associated with insulin resistance. The aim of this study was to test this widely held assumption by comparing adipose cell size distribution in 28 equally obese, otherwise healthy individuals who represented extreme ends of the spectrum of insulin sensitivity, as defined by the modified insulin suppression test.Subcutaneous periumbilical adipose tissue biopsy samples were fixed in osmium tetroxide and passed through the Beckman Coulter Multisizer to obtain cell size distributions. Insulin sensitivity was quantified by the modified insulin suppression test. Quantitative real-time PCR for adipose cell differentiation genes was performed for 11 subjects.All individuals exhibited a bimodal cell size distribution. Contrary to expectations, the mean diameter of the larger cells was not significantly different between the insulin-sensitive and insulin-resistant individuals. Moreover, insulin resistance was associated with a higher ratio of small to large cells (1.66 +/- 1.03 vs 0.94 +/- 0.50, p = 0.01). Similar cell size distributions were observed for isolated adipose cells. The real-time PCR results showed two- to threefold lower expression of genes encoding markers of adipose cell differentiation (peroxisome proliferator-activated receptor gamma1 [PPARgamma1], PPARgamma2, GLUT4, adiponectin, sterol receptor element binding protein 1c) in insulin-resistant compared with insulin-sensitive individuals.These results suggest that after controlling for obesity, insulin resistance is associated with an expanded population of small adipose cells and decreased expression of differentiation markers, suggesting that impairment in adipose cell differentiation may contribute to obesity-associated insulin resistance.

    View details for DOI 10.1007/s00125-007-0708-y

    View details for Web of Science ID 000248225100017

    View details for PubMedID 17549449

  • Asymmetric Dimethyl L-Arginine (ADMA) is a critical regulator of myocardial reperfusion injury CARDIOVASCULAR RESEARCH Stuehlinger, M. C., Conci, E., Haubner, B. J., Stocker, E., Schwaighofer, J., Cooke, J. P., Tsao, P. S., Pachinger, O., Metzler, B. 2007; 75 (2): 417-425


    Endothelial dysfunction by the loss of nitric oxide (NO) is a critical event during reperfusion of ischemic myocardium. Reduced NO availability signals important pathophysiological changes leading to myocardial reperfusion injury. We have recently shown that NO biosynthesis can be disturbed by the endogenous NO synthase (NOS) inhibitor ADMA and that these changes are mediated by an impairment of its metabolism by dimethylarginine dimethylaminohydrolase (DDAH). We therefore analyzed the role of ADMA and its metabolism in the setting of myocardial ischemia and reperfusion.C57-bl6 mice underwent myocardial ischemia for exactly 30 min followed by 2, 4, 8, 12, 24, and 72 h of reperfusion achieved by occlusion and re-opening of the left coronary artery. The reperfused left ventricle was subsequently homogenized for measurements of determinants of the NO synthase pathway. Furthermore, the effects and its mechanisms of ADMA on reperfusion injury were analyzed in a genetic mouse model.A significant accumulation of ADMA was found in myocardial tissue when mice were subjected to 30 min of ischemia followed by reperfusion in our in vivo model. The maximum increase of tissue ADMA at 4 h of reperfusion coincided with reductions of NO tissue concentrations and DDAH activity; protein expression of NOS isoforms, however, was not changed. Furthermore, DDAH overexpression in a genetic mouse model as well as treatment with oral L-arginine markedly reduced reperfusion injury by 40-50% at 4 h of reperfusion. The effects of ADMA on reperfusion injury were shown to be mediated by reduced eNOS activity and phosphorylation, expression of adhesion molecules, and leukocyte activity.Accumulation of tissue ADMA by impairment of DDAH was found to be a significant determinant of reperfusion injury. Our results indicate that ADMA could be a potential new target for the treatment of myocardial ischemia/reperfusion injury.

    View details for DOI 10.1016/j.cardiores.2007.04.030

    View details for Web of Science ID 000248296300022

    View details for PubMedID 17559823

  • Magnetic resonance imaging of progressive cardiomyopathic changes in the db/db mouse AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Yue, P., Arai, T., Terashima, M., Sheikh, A. Y., Cao, F., Charo, D., Hoyt, G., Robbins, R. C., Ashley, E. A., Wu, J., Yang, P. C., Tsao, P. S. 2007; 292 (5): H2106-H2118


    The db/db mouse is a well-established model of diabetes. Previous reports have documented contractile dysfunction (i.e., cardiomyopathy) in these animals, although the extant literature provides limited insights into cardiac structure and function as they change over time. To better elucidate the natural history of cardiomyopathy in db/db mice, we performed cardiac magnetic resonance (CMR) scans on these animals. CMR imaging was conducted with a 4.7-T magnet on female db/db mice and control db/+ littermates at 5, 9, 13, 17, and 22 wk of age. Gated gradient echo sequences were used to obtain cineographic short-axis slices from apex to base. From these images left ventricular (LV) mass (LVM), wall thickness, end-diastolic volume (LVEDV), and ejection fraction (LVEF) were determined. Additionally, cardiac [(18)F]fluorodeoxyglucose ([(18)F]FDG) PET scanning, pressure-volume loops, and real-time quantitative PCR on db/db myocardium were performed. Relative to control, db/db mice developed significant increases in LVM and wall thickness as early as 9 wk of age. LVEDV diverged slightly later, at 13 wk. Interestingly, compared with the baseline level, LVEF in the db/db group did not decrease significantly until 22 wk. Additionally, [(18)F]FDG metabolic imaging showed a 40% decrease in glucose uptake in db/db mice. Furthermore, contractile dysfunction was observed in 15-wk db/db mice undergoing pressure-volume loops. Finally, real-time quantitative PCR revealed an age-dependent recapitulation of the fetal gene program, consistent with a myopathic process. In summary, as assessed by CMR, db/db mice develop characteristic structural and functional changes consistent with cardiomyopathy.

    View details for DOI 10.1152/ajpheart.00856.2006

    View details for Web of Science ID 000247777200012

    View details for PubMedID 17122193

  • Adiponectin and insulin resistance in young and healthy smokers ENDOCRINE JOURNAL Sonmez, A., Dogru, T., Yilmaz, M. I., Tasci, I., Ocal, R., Ozgurtas, T., Kilic, S., Erbil, K., Erikci, S., Tsao, P. 2006; 53 (6): 729-734


    Smoking is closely associated with insulin resistance, though the mechanism is not clear. Adiponectin, a novel anti-atherosclerotic and anti-inflammatory adipose tissue product, which is closely associated with insulin resistance, was reported to be low in smokers with cofactors for atherosclerosis. However, the effects of smoking on circulating adiponectin levels in otherwise healthy people are unknown. In this study, a case control design was implemented to search for the effect of smoking on plasma adiponectin and insulin sensitivities in healthy people. Sixty-four healthy male smokers, with no family history of hypertension and diabetes mellitus were compared with appropriate 36 age and body mass index matched controls. Both the patients and controls were the soldiers of a troop with regular daily physical activity. Plasma adiponectin, high sensitive C-reactive protein (hsCRP), insulin and lipid levels, and insulin sensitivity as assessed by homeostasis model assessment index (HOMA) of the smokers were measured and compared with those of the controls. The plasma adiponectin, hsCRP, insulin levels and HOMA indexes of the two groups were similar. These parameters were not affected by the amount of cigarettes per day. HDL-cholesterol levels were lower (p = 0.01) and systolic blood pressures were higher (p = 0.02) in the smokers. These results indicate that smoking may not affect plasma adiponectin and insulin levels in young and healthy men with high exercise capacity.

    View details for Web of Science ID 000244725500003

    View details for PubMedID 16960399

  • Shear stress quantification and regulation of endothelial cell dysfunction in models of pulmonary arterial hypertension 79th Annual Scientific Session of the American-Heart-Association Tang, B. T., Fonte, T. A., Feinstein, J. A., Taylor, C. A., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2006: 81–81
  • Iron oxide cellular MRI of plaque macrophages has limited in vivo uptake in deep foam cells despite active uptake in vitro 79th Annual Scientific Session of the American-Heart-Association Terashima, M., Ikeda, K., Tsao, P. S., McConnell, M. V. LIPPINCOTT WILLIAMS & WILKINS. 2006: 40–40
  • Abdominal aortic hemodynamics in young healthy adults at rest and during lower limb exercise: quantification using image-based computer modeling AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Tang, B. T., Cheng, C. P., Draney, M. T., Wilson, N. M., Tsao, P. S., Herfkens, R. J., Taylor, C. A. 2006; 291 (2): H668-H676


    Localization of atherosclerotic lesions in the abdominal aorta has been previously correlated to areas of adverse hemodynamic conditions, such as flow recirculation, low mean wall shear stress, and high temporal oscillations in shear. Along with its many systemic benefits, exercise is also proposed to have local benefits in the vasculature via the alteration of these regional flow patterns. In this work, subject-specific models of the human abdominal aorta were constructed from magnetic resonance angiograms of five young, healthy subjects, and computer simulations were performed under resting and exercise (50% increase in resting heart rate) pulsatile flow conditions. Velocity fields and spatial variations in mean wall shear stress (WSS) and oscillatory shear index (OSI) are presented. When averaged over all subjects, WSS increased from 4.8 +/- 0.6 to 31.6 +/- 5.7 dyn/cm2 and OSI decreased from 0.22 +/- 0.03 to 0.03 +/- 0.02 in the infrarenal aorta between rest and exercise. WSS significantly increased, whereas OSI decreased between rest and exercise at the supraceliac, infrarenal, and suprabifurcation levels, and significant differences in WSS were found between anterior and posterior sections. These results support the hypothesis that exercise provides localized benefits to the cardiovascular system through acute mechanical stimuli that trigger longer-term biological processes leading to protection against the development or progression of atherosclerosis.

    View details for DOI 10.1152/ajpheart.01301.2005

    View details for Web of Science ID 000239020300021

    View details for PubMedID 16603687

  • Molecular signatures determining coronary artery and saphenous vein smooth muscle cell phenotypes - Distinct responses to stimuli ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Deng, D. X., Spin, J. M., Tsalenko, A., Vailaya, A., Ben-Dor, A., Yakhini, Z., Tsao, P., Bruhn, L., Quertermous, T. 2006; 26 (5): 1058-1065


    Phenotypic differences between vascular smooth muscle cell (VSMC) subtypes lead to diverse pathological processes including atherosclerosis, postangioplasty restenosis and vein graft disease. To better understand the molecular mechanisms underlying functional differences among distinct SMC subtypes, we compared gene expression profiles and functional responses to oxidized low-density lipoprotein (OxLDL) and platelet-derived growth factor (PDGF) between cultured SMCs from human coronary artery (CASM) and saphenous vein (SVSM).OxLDL and PDGF elicited markedly different functional responses and expression profiles between the 2 SMC subtypes. In CASM, OxLDL inhibited cell proliferation and migration and modified gene expression of chemokines (CXCL10, CXCL11 and CXCL12), proinflammatory cytokines (IL-1, IL-6, and IL-18), insulin-like growth factor binding proteins (IGFBPs), and both endothelial and smooth muscle marker genes. In SVSM, OxLDL promoted proliferation partially via IGF1 signaling, activated NF-kappaB and phosphatidylinositol signaling pathways, and upregulated prostaglandin (PG) receptors and synthases. In untreated cells, alpha-chemokines, proinflammatory cytokines, and genes associated with apoptosis, inflammation, and lipid biosynthesis were higher in CASM, whereas some beta-chemokines, metalloproteinase inhibitors, and IGFBPs were higher in SVSM. Interestingly, the basal expression levels of these genes seemed closely related to their responses to OxLDL and PDGF. In summary, our results suggest dramatic differences in gene expression patterns and functional responses to OxLDL and PDGF between venous and arterial SMCs, with venous SMCs having stronger proliferative/migratory responses to stimuli but also higher expression of atheroprotective genes at baseline.These results reveal molecular signatures that define the distinct phenotypes characteristics of coronary artery and saphenous vein SMC subtypes.

    View details for DOI 10.1161/01.ATV.0000208185.16371.97

    View details for Web of Science ID 000236942400017

    View details for PubMedID 16456091

  • Plasma asymmetric dimethylarginine concentrations are elevated in obese insulin-resistant women and fall with weight loss JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM McLaughlin, T., Stuhlinger, M., Lamendola, C., Abbasi, F., Bialek, J., Reaven, G. M., Tsao, P. S. 2006; 91 (5): 1896-1900


    Plasma asymmetric dimethylarginine (ADMA) concentrations are higher in apparently healthy, insulin-resistant (IR) individuals and decrease in response to thiazolidenedione treatment.The objective of the study was to determine whether ADMA concentrations would also fall when insulin sensitivity is enhanced with weight loss in obese individuals. DESIGN/SETTING/PATIENTS/INTERVENTION: Twenty obese women classified as IR or insulin sensitive (IS) on the basis of their steady-state plasma glucose (SSPG) concentration during the insulin suppression test underwent 12 wk of dietary weight loss.Plasma glucose, insulin, and ADMA were measured at baseline and after weight loss; change in insulin resistance was quantified by repeating the SSPG after the dietary intervention.Although weight loss was similar in the two groups, significant improvements in SSPG, glucose, and insulin concentrations were confined to the IR group. Baseline plasma ADMA concentrations (mean +/- sd) were higher in IR subjects (1.69 +/- 0.44 vs. 1.18 +/- 0.45 micromol/liter, P = 0.02) and decreased to 1.20 +/- 0.22 micromol/liter (P < 0.001) with weight loss. In contrast, ADMA levels did not change with a similar extent of weight loss in the IS group.Plasma ADMA levels are higher in obese, IR women than in equally obese, IS women and decrease in response to weight loss when associated with enhancement of insulin sensitivity.

    View details for DOI 10.1210/jc.2005-1441

    View details for Web of Science ID 000237330000043

    View details for PubMedID 16507636

  • Proteomic profiles of serum inflammatory markers accurately predict atherosclerosis in mice PHYSIOLOGICAL GENOMICS Tabibiazar, R., Wagner, R. A., Deng, A., Tsao, P. S., Quertermous, T. 2006; 25 (2): 194-202


    At a population level, inflammatory markers have been shown to predict outcome and response to therapy in patients with atherosclerotic cardiovascular disease. However, current markers are not sufficiently sensitive or specific to provide clinical utility for managing individual patients. We hypothesize that measurement of multiple circulating disease-related inflammatory factors will be more informative, allowing the early identification of vascular wall disease activity. We have investigated whether protein microarray-based abundance measurements of circulating proteins can predict the severity of atherosclerotic disease. Using a longitudinal experimental design with apolipoprotein E-deficient mice and control C57Bl/6J and C3H/HeJ wild-type mice, we measured the time-related serum protein expression of 30 inflammatory markers using a protein microarray. We were able to identify a subset of proteins that classify and predict the severity of atherosclerotic disease with a high level of accuracy. The time-specific vascular expression of these markers was verified by showing that their gene expression in the mouse aorta correlated closely to the temporal pattern of serum protein levels. In conclusion, these data suggest that quantification of multiple disease-related inflammatory proteins can provide a more sensitive and specific methodology for assessing atherosclerotic disease activity in humans, and identify candidate biomarkers for such studies.

    View details for DOI 10.1152/physiolgenomics.00240.2005

    View details for Web of Science ID 000236791300002

    View details for PubMedID 16418319

  • THR0921, a novel peroxisome proliferator-activated receptor gamma agonist, reduces the severity of collagen-induced arthritis ARTHRITIS RESEARCH & THERAPY Tomita, T., Kakiuchi, Y., Tsao, P. S. 2006; 8 (1)


    THR0921 is a novel peroxisome proliferator-activated receptor gamma (PPARgamma) agonist with potent anti-diabetic properties. Because of the proposed role of PPARgamma in inflammation, we investigated the potential of orally active THR0921 to inhibit the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in DBA/1J mice by the injection of bovine type II collagen in complete Freund's adjuvant on days 0 and 21. Mice were treated with THR0921 (50 mg/kg/day) starting on the day of the booster injection and throughout the remaining study period. Both clinical disease activity scores as well as histological scores of joint destruction were significantly reduced in mice treated with THR0921 compared to untreated mice. Proliferation of isolated spleen cells, as well as circulating levels of IgG antibody to type II collagen, was decreased by THR0921. Moreover, spleen cell production of IFN-gamma, tumor necrosis factor (TNF)-alpha and IL-1beta in response to exposure to lipopolysaccharide or type II collagen was reduced by in vivo treatment with THR0921. Steady state mRNA levels of TNF-alpha, IL-1beta, monocyte chemotactic protein-1 and receptor activator of nuclear factor kappaB ligand (RANKL) in isolated joints were all decreased in mice treated with THR0921. Finally, THR0921 inhibited osteoclast differentiation of bone marrow-derived cells stimulated with macrophage colony-stimulating factor and RANKL. In conclusion, THR0921 attenuates collagen-induced arthritis in part by reducing the immune response. As such, PPARgamma may be an important therapeutic target for rheumatoid arthritis.

    View details for DOI 10.1186/ar1856

    View details for Web of Science ID 000235803900016

    View details for PubMedID 16356194

  • Prolonged cold ischemia in rat cardiac allografts promotes ischemia-reperfusion injury and the development of graft coronary artery disease in a linear fashion JOURNAL OF HEART AND LUNG TRANSPLANTATION Tanaka, M., Mokhtari, G. K., Terry, R. D., Gunawan, F., Balsam, L. B., Hoyt, G., Lee, K. H., Tsao, P. S., Robbins, R. C. 2005; 24 (11): 1906-1914


    Prolonged cold ischemia is thought to exacerbate ischemia-reperfusion injury and graft coronary artery disease (GCAD). We investigated the effect of varying lengths of cold ischemia on inflammation and apoptosis during ischemia-reperfusion injury and correlated this with the degree of GCAD in rat cardiac allografts.PVG rat (RT1c) hearts subjected to 30, 60, 90, 120, or 150 minutes of cold ischemia were heterotopically transplanted into ACI rats (RT1a). Grafts were procured after 4 hours of reperfusion and analyzed for superoxide generation, myeloperoxidase activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and monocyte chemoattractant protein-1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2) production, cardiomyocyte apoptosis, and caspase-2, -3, -8, -9 activities. Additional transplanted animals received cyclosporine A (7.5 mg/kg/day) for 10 days as chronic rejection models. Indices of GCAD were determined at 90 days.A direct linear correlation was found between cold ischemic time, ischemia-reperfusion injury, and GCAD. Superoxide generation, myeloperoxidase activity, TNF-alpha, IL-1beta, MCP-1/CCL2 production, cardiomyocyte apoptosis, and caspase-2, -3, -8, and -9 activities increased with ischemic time, peaking at 120 minutes and plateauing at 150 minutes. GCAD, assessed by the percentage of luminal narrowing, the intima/media ratio, and the percentage of diseased vessels, worsened with increased ischemic time, peaking at 120 minutes and plateauing at 150 minutes. All tested variables in both the acute and chronic phases were significantly increased with 120-minute ischemia compared with 30-minute ischemia.These data indicate that the degree of cardiomyocyte apoptosis and inflammatory response in cardiac allografts during ischemia-reperfusion injury depends on the duration of cold ischemia. More important, that prolonged cold ischemia correlates with increased GCAD.

    View details for DOI 10.1016/j.healun.2004.06.007

    View details for Web of Science ID 000233412300031

    View details for PubMedID 16297799

  • Pathway analysis of coronary atherosclerosis PHYSIOLOGICAL GENOMICS King, J. Y., Ferrara, R., Tabibiazar, R., Spin, J. M., Chen, M. M., Kuchinsky, A., Vailaya, A., Kincaid, R., Tsalenko, A., Deng, D. X., Connolly, A., Zhang, P., Yang, E., Watt, C., Yakhini, Z., Ben-Dor, A., Adler, A., Bruhn, L., Tsao, P., Quertermous, T., Ashley, E. A. 2005; 23 (1): 103-118


    Large-scale gene expression studies provide significant insight into genes differentially regulated in disease processes such as cancer. However, these investigations offer limited understanding of multisystem, multicellular diseases such as atherosclerosis. A systems biology approach that accounts for gene interactions, incorporates nontranscriptionally regulated genes, and integrates prior knowledge offers many advantages. We performed a comprehensive gene level assessment of coronary atherosclerosis using 51 coronary artery segments isolated from the explanted hearts of 22 cardiac transplant patients. After histological grading of vascular segments according to American Heart Association guidelines, isolated RNA was hybridized onto a customized 22-K oligonucleotide microarray, and significance analysis of microarrays and gene ontology analyses were performed to identify significant gene expression profiles. Our studies revealed that loss of differentiated smooth muscle cell gene expression is the primary expression signature of disease progression in atherosclerosis. Furthermore, we provide insight into the severe form of coronary artery disease associated with diabetes, reporting an overabundance of immune and inflammatory signals in diabetics. We present a novel approach to pathway development based on connectivity, determined by language parsing of the published literature, and ranking, determined by the significance of differentially regulated genes in the network. In doing this, we identify highly connected "nexus" genes that are attractive candidates for therapeutic targeting and followup studies. Our use of pathway techniques to study atherosclerosis as an integrated network of gene interactions expands on traditional microarray analysis methods and emphasizes the significant advantages of a systems-based approach to analyzing complex disease.

    View details for DOI 10.1152/physiolgenomics.00101.2005

    View details for Web of Science ID 000232065200012

    View details for PubMedID 15942018

  • Dimethylarginine dimethylaminohydrolase overexpression suppresses graft coronary artery disease CIRCULATION Tanaka, M., Sydow, K., Gunawan, F., Jacobi, J., Tsao, P. S., Robbins, R. C., Cooke, J. P. 2005; 112 (11): 1549-1556


    Graft coronary artery disease (GCAD) is the leading cause of death after the first year of heart transplantation. The reduced bioavailability of endothelium-derived nitric oxide (NO) may play a role in endothelial vasodilator dysfunction and the structural changes that are characteristic of GCAD. A potential contributor to endothelial pathobiology is asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor. We hypothesized that lowering ADMA concentrations by dimethylarginine dimethylaminohydrolase (DDAH) overexpression in the recipient might suppress GCAD and long-term immune responses in murine cardiac allografts.In one series, donor hearts of C-H-2(bm12)KhEg (H-2(bm12)) wild-type (WT) mice were heterotopically transplanted into C57BL/6 (H-2b) transgenic mice overexpressing human DDAH-I or WT littermates and procured after 4 hours of reperfusion (WT and DDAH-I recipients, n=6 each). In a second series, donor hearts were transplanted into DDAH-I-transgenic or WT mice and procured 30 days after transplantation (n=7 each). In DDAH-I recipients, plasma ADMA concentrations were lower, in association with reduced myocardial generation of superoxide anion (WT versus DDAH-I, 465.7+/-79.8 versus 173.4+/-32.3 micromol.L(-1).mg(-1).h(-1); P=0.02), inflammatory cytokines, adhesion molecules, and chemokines. GCAD was markedly reduced in cardiac allografts of DDAH-I-transgenic recipients as assessed by luminal narrowing (WT versus DDAH, 79+/-2% versus 33+/-7%; P<0.01), intima-media ratio (WT versus DDAH, 1.1+/-0.1 versus 0.5+/-0.1; P<0.01), and the percentage of diseased vessels (WT versus DDAH, 100+/-0% versus 62+/-10%; P<0.01).Overexpression of DDAH-I attenuated oxidative stress, inflammatory cytokines, and GCAD in murine cardiac allografts. The effect of DDAH overexpression may be mediated by its reduction of plasma and tissue ADMA concentrations.

    View details for DOI 10.1161/CIRCULATIONAHA.105.537670

    View details for Web of Science ID 000231821200007

    View details for PubMedID 16144995

  • Transforming growth factor-beta receptors localize to caveolae and regulate endothelial nitric oxide synthase in normal human endothelial cells BIOCHEMICAL JOURNAL Schwartz, E. A., Reaven, E., Topper, J. N., Tsao, P. S. 2005; 390: 199-206


    Caveolae (sphingolipid- and cholesterol-rich, 100 nm flask-shaped invaginations of the cell membrane) serve as a nexus of cell signalling. In the present study caveolin-rich lipid raft domains were extracted from HUVEC (human umbilical-vein endothelial cells) using both density gradient and immunoprecipitation techniques, and demonstrated localization of the TGF-beta (transforming growth factor-beta) receptors TbetaRI and TbetaRII to the Cav-1 (caveolin-1)-enriched raft fractions of these normal, human endothelial cells. Immunoprecipitation demonstrated an association between TbetaRI and TbetaRII, as well as an association of the TbetaRs receptors with Cav-1 and eNOS (endothelial nitric oxide synthase), suggesting a mutual co-localization to caveolae; after treatment of HUVEC with 5 ng/ml TGF-beta1 for 15 min, however, co-precipitation of eNOS with TbetaRI, TbetaRII and Cav-1 was diminished. The loss of immunoprecipitable eNOS from Cav-1-enriched fractions was accompanied by a decrease both in phosphorylation of eNOS and in enzymatic activity (conversion of arginine into citrulline). No change in the localization of eNOS to morphologically distinct caveolae could be detected by electron microscopy after treatment of HUVEC with TGF-beta1 for 20 min. The results of these investigations provide evidence that TbetaRI interacts with eNOS in the caveolae of normal, human endothelial cells and has a regulatory function on basal eNOS enzymatic activity.

    View details for DOI 10.1042/BJ20041182

    View details for Web of Science ID 000231492800021

    View details for PubMedID 15819614

  • Increased aortic stiffness in the insulin-resistant Zucker fa/fa rat AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Sista, A. K., O'Connell, M. K., Hinohara, T., Oommen, S. S., Fenster, B. E., Glassford, A. J., Schwartz, E. A., Taylor, C. A., Reaven, G. M., Tsao, P. S. 2005; 289 (2): H845-H851


    Accumulating clinical evidence indicates increased aortic stiffness, an independent risk factor for cardiovascular and all-cause mortality, in type 2 diabetic and glucose-intolerant individuals. The present study sought to determine whether increased mechanical stiffness, an altered extracellular matrix, and a profibrotic gene expression profile could be observed in the aorta of the insulin-resistant Zucker fa/fa rat. Mechanical testing of Zucker fa/fa aortas showed increased vascular stiffness in longitudinal and circumferential directions compared with Zucker lean controls. Unequal elevations in developed strain favoring the longitudinal direction resulted in a loss of anisotropy. Real-time quantitative PCR and immunohistochemistry revealed increased expression of fibronectin and collagen IV alpha 3 in the Zucker fa/fa aorta. In addition, expression of transforming growth factor-beta and several Smad proteins was increased in vessels from insulin-resistant animals. In rat vascular smooth muscle cells, 12-18 h of exposure to insulin (100 nmol/l) enhanced transforming growth factor-beta1 mRNA expression, implicating a role for hyperinsulinemia in vascular stiffness. Thus there is mechanical, structural, and molecular evidence of arteriosclerosis in the Zucker fa/fa rat at the glucose-intolerant, hyperinsulinemic stage.

    View details for DOI 10.1152/ajpheart.00134.2005

    View details for Web of Science ID 000230458800045

    View details for PubMedID 15833807

  • Signature patterns of gene expression in mouse atherosclerosis and their correlation to human coronary disease PHYSIOLOGICAL GENOMICS Tabibiazar, R., Wagner, R. A., Ashley, E. A., King, J. Y., Ferrara, R., Spin, J. M., Sanan, D. A., Narasimhan, B., Tibshirani, R., Tsao, P. S., Efron, B., Quertermous, T. 2005; 22 (2): 213-226


    The propensity for developing atherosclerosis is dependent on underlying genetic risk and varies as a function of age and exposure to environmental risk factors. Employing three mouse models with different disease susceptibility, two diets, and a longitudinal experimental design, it was possible to manipulate each of these factors to focus analysis on genes most likely to have a specific disease-related function. To identify differences in longitudinal gene expression patterns of atherosclerosis, we have developed and employed a statistical algorithm that relies on generalized regression and permutation analysis. Comprehensive annotation of the array with ontology and pathway terms has allowed rigorous identification of molecular and biological processes that underlie disease pathophysiology. The repertoire of atherosclerosis-related immunomodulatory genes has been extended, and additional fundamental pathways have been identified. This highly disease-specific group of mouse genes was combined with an extensive human coronary artery data set to identify a shared group of genes differentially regulated among atherosclerotic tissues from different species and different vascular beds. A small core subset of these differentially regulated genes was sufficient to accurately classify various stages of the disease in mouse. The same gene subset was also found to accurately classify human coronary lesion severity. In addition, this classifier gene set was able to distinguish with high accuracy atherectomy specimens from native coronary artery disease vs. those collected from in-stent restenosis lesions, thus identifying molecular differences between these two processes. These studies significantly focus efforts aimed at identifying central gene regulatory pathways that mediate atherosclerotic disease, and the identification of classification gene sets offers unique insights into potential diagnostic and therapeutic strategies in atherosclerotic disease.

    View details for DOI 10.1152/physiolgenomics.00001.2005

    View details for Web of Science ID 000230987900011

    View details for PubMedID 15870398

  • Rosuvastatin attenuates monocyte-endothelial cell interactions and vascular free radical production in hypercholesterolemic mice JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Li, W., Asagami, T., Matsushita, H., Lee, K. H., Tsao, P. S. 2005; 313 (2): 557-562


    One of the earliest observable events in atherogenesis is enhanced monocyte adhesion to the endothelium. In addition to reducing circulating levels of cholesterol, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) are thought to have direct salutary effects upon vascular cells. We hypothesized that the new statin, rosuvastatin, would have anti-inflammatory effects on the vessel wall. Eight-week-old apolipoprotein E-deficient mice were fed a normal chow diet for a period of 12 weeks. During this time mice were administered vehicle or rosuvastatin at a dose of 0, 1, 5, or 20 mg/kg by subcutaneous injection at the same time daily for a period of 2 or 6 weeks prior to sacrifice. At the end of the study, rosuvastatin-treated animals displayed lower plasma total cholesterol levels, whereas showing little change in high-density lipoprotein cholesterol or triglycerides. Using a functional binding assay, we also demonstrated that endothelial adhesiveness for monocytes was significantly attenuated after 2 weeks of treatment with rosuvastatin. Quantitative real-time polymerase chain reaction determined that rosuvastatin reduced the expression of vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and metalloproteinase-9 in the vessel wall. In addition, rosuvastatin inhibited vascular expression of p22(phox) and superoxide production, as well as diminishing plasma 8-isoprostanes concentrations. Thus, treatment with rosuvastatin has acute anti-inflammatory actions that likely participate in its beneficial actions during atherogenesis.

    View details for DOI 10.1124/jpet.104.080002

    View details for Web of Science ID 000228357900009

    View details for PubMedID 15665143

  • Inhibition of heart transplant injury and graft coronary artery disease after prolonged organ ischemia by selective protein kinase C regulators JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Tanaka, M., Gunawan, F., Terry, R. D., Inagaki, K., Caffarelli, A. D., Hoyt, G., Tsao, P. S., Mochly-Rosen, D., Robbins, R. C. 2005; 129 (5): 1160-1167


    Transplanted hearts subjected to prolonged ischemia develop ischemia-reperfusion injury and graft coronary artery disease. To determine the effect of delta-protein kinase C and -protein kinase C on ischemia-reperfusion injury and the resulting graft coronary artery disease induced by prolonged ischemia, we used a delta-protein kinase C-selective inhibitor peptide and an -protein kinase C-selective activator peptide after 30 or 120 minutes of ischemia.Hearts of piebald viral glaxo (PVG) rats were heterotopically transplanted into allogeneic August Copenhagen Irish (ACI) rats. After cardioplegic arrest of the donor heart, -protein kinase C activator was injected antegrade into the coronary arteries. Hearts were procured and bathed in -protein kinase C activator, and before reperfusion, delta-protein kinase C inhibitor was injected into the recipient inferior vena cava. Controls were treated with saline. To analyze ischemia-reperfusion injury, grafts were procured at 4 hours after transplantation and analyzed for superoxide generation; myeloperoxidase activity; tumor necrosis factor alpha, interleukin 1beta, and monocyte/macrophage chemoattractant protein 1 production; and cardiomyocyte apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase 2, 3, 8, and 9 activity. To analyze graft coronary artery disease, another set of animals underwent equal ischemic times and treatment strategies and then after 90 days were analyzed for graft coronary artery disease indexes.All measures of ischemia-reperfusion injury and graft coronary artery disease after 120 minutes of ischemia in the saline-treated group were significantly increased relative to those observed after 30 minutes of ischemia. It is important to note that all ischemia-reperfusion injury parameters and graft coronary artery disease indexes decreased significantly in the protein kinase C regulator-treated group in comparison to saline-treated controls; additionally, these values were equivalent to those in saline-treated controls with 30 minutes of ischemia.Combined treatment with -protein kinase C activator and delta-protein kinase C inhibitor reduces ischemia-reperfusion injury and decreases the resulting graft coronary artery disease induced by prolonged ischemia.

    View details for DOI 10.1016/j.jtcvs.2004.09.015

    View details for Web of Science ID 000228947200029

    View details for PubMedID 15867794

  • Mouse strain-specific differences in vascular wall gene expression and their relationship to vascular disease ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Tabibiazar, R., Wagner, R. A., Spin, J. M., Ashley, E. A., Narasimhan, B., Rubin, E. M., Efron, B., Tsao, P. S., Tibshirani, R., Quertermous, T. 2005; 25 (2): 302-308


    Different strains of inbred mice exhibit different susceptibility to the development of atherosclerosis. The C3H/HeJ and C57Bl/6 mice have been used in several studies aimed at understanding the genetic basis of atherosclerosis. Under controlled environmental conditions, variations in susceptibility to atherosclerosis reflect differences in genetic makeup, and these differences must be reflected in gene expression patterns that are temporally related to the development of disease. In this study, we sought to identify the genetic pathways that are differentially activated in the aortas of these mice.We performed genome-wide transcriptional profiling of aortas from C3H/HeJ and C57Bl/6 mice. Differences in gene expression were identified at baseline as well as during normal aging and longitudinal exposure to high-fat diet. The significance of these genes to the development of atherosclerosis was evaluated by observing their temporal pattern of expression in the well-studied apolipoprotein E model of atherosclerosis.Gene expression differences between the 2 strains suggest that aortas of C57Bl/6 mice have a higher genetic propensity to develop inflammation in response to appropriate atherogenic stimuli. This study expands the repertoire of factors in known disease-related signaling pathways and identifies novel candidate genes for future study. To gain insights into the molecular pathways that are differentially activated in strains of mice with varied susceptibility to atherosclerosis, we performed comprehensive transcriptional profiling of their vascular wall. Genes identified through these studies expand the repertoire of factors in disease-related signaling pathways and identify novel candidate genes in atherosclerosis.

    View details for DOI 10.1161/011.ATV.0000151372.86863.a5

    View details for Web of Science ID 000226594000009

    View details for PubMedID 15550693

  • Effect of rosiglitazone treatment on circulating vascular and inflammatory markers in insulin-resistant subjects. Diabetes & vascular disease research Chu, J. W., Abbasi, F., Lamendola, C., McLaughlin, T., Reaven, G. M., Tsao, P. S. 2005; 2 (1): 37-41


    Thiazolidinedione (TZD) compounds enhance insulin sensitivity and attenuate inflammation. The effect of the TZD compound, rosiglitazone (RSG) on both actions was evaluated in two groups of insulin-resistant subjects with minimal elevations of fasting plasma glucose (PG) concentration: group A (n=15, PG < 7.0 mmol/L) and group B (n=14, PG 7.0-8.3 mmol/L). Insulin action, quantified by the insulin suppression test, improved after three months of treatment in both groups, and concentrations of C-reactive protein, plasminogen activator inhibitor-1 and Eselectin all fell. Significant decreases in L-selectin and P-selectin were confined to group B, and concentrations of interleukin-6, intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 did not fall in either group. Significant relationships were not discerned between enhanced insulin sensitivity and related variables and decreases in inflammatory/vascular markers, suggesting that RSG-induced changes in the latter variables in insulin-resistant individuals might be at least partly independent of the effects of the drug on insulin action.

    View details for PubMedID 16305071

  • Signature pattern of circulating chemokines can improve the identification of coronary artery disease 41st Annual Meeting of the European-Association-for-the-Study-of-Diabetes Ardigo, D., Tabibiazar, R., Olshen, R., Tsao, P. S., Quetermous, T. SPRINGER. 2005: A406–A407
  • Cardiomyocyte-specific Bcl-2 overexpression attenuates ischemia-reperfusion injury, immune response during acute rejection, and graft coronary artery disease BLOOD Tanaka, M., Nakae, S., Terry, P. D., Mokhtari, G. K., Gunawan, F., Balsam, L. B., Kaneda, H., Kofidis, T., Tsao, P. S., Robbins, R. C. 2004; 104 (12): 3789-3796


    After cardiac transplantation, graft damage occurs secondary to ischemia-reperfusion injury and acute rejection. This damage ultimately leads to the development of graft coronary artery disease (GCAD), which limits long-term graft survival. Apoptosis is directly involved in graft injury, contributing to the development of GCAD. To assess the role of the antiapoptotic factor Bcl-2 in the process of GCAD, we transplanted hearts from FVB transgenic mice overexpressing human Bcl-2 under the control of alpha-myosin heavy chain promoter into allogenic C57BL/6 mice. Bcl-2 overexpression led to reduced cytochrome c-mediated caspase-9-dependent cardiomyocyte apoptosis and local inflammation (neutrophil infiltration and proinflammatory cytokine production) in cardiac allografts during ischemia-reperfusion injury and also led to reduced immune responses (inflammatory cell infiltration, production of T(H)1 cytokines and chemokines, and expression of adhesion molecules) during acute and chronic rejection without affecting host CD4(+) and CD8(+) cell responses in the spleen. Thus, local Bcl-2 expression directly contributes to the modulation of local immune responses in allograft rejection, resulting in attenuated GCAD. In conclusion, our findings suggest that the modulation of Bcl-2 expression by pharmacologic up-regulation or gene transfer may be of clinical benefit in the short- and long-term function of cardiac allografts.

    View details for Web of Science ID 000225351600061

    View details for PubMedID 15280201

  • Low flow promotes instent intimal hyperplasia comparison with lumen loss in balloon-injured and uninjured vessels and the effects of the antioxidant pyrrolidine dithiocarbamate ATHEROSCLEROSIS Hanratty, C. G., Murrell, M., Khachigian, L. M., Tsao, P. S., Ward, M. R. 2004; 177 (2): 269-274


    Low flow (LF) promotes late lumen loss after angioplasty by exacerbating inward remodelling through redox-sensitive mechanisms. Stents eliminate inward remodelling and the effect of LF on in-stent restenosis is uncertain. We performed over-sized (1.3-1.5:1) stenting (S) and balloon injury (in the same vessel, B) to the carotid arteries of cholesterol-fed rabbits and compared 28-day late lumen loss with that in an uninjured segment in the same vessel (U). Vessels (n = 5 animals per group) were subjected to high (H), normal (N) and low (L) flow in animals fed either vehicle (V) or the antioxidant pyrrolidine dithiocarbamate, PDTC (P). LF significantly increased in-stent neointima formation relative to normal and high flow (SLV 0.72 +/- 0.07 mm(2) versus SNV 0.43 +/- 0.08 mm(2) versus SHV 0.28 +/- 0.04 mm(2), P < 0.05). However, LF resulted in greater lumen loss in segments from the same vessel subject to balloon injury (lumen SLV 5.18 +/- 0.40 mm(2) and SNV 5.32 +/- 0.40 mm(2) versus BLV 1.28 +/- 0.33 mm(2) and BNV 2.19 +/- 0.28 mm(2)), by greater enhancement of inward remodelling. In addition, inward remodelling and lumen loss due to LF were greater in balloon-injured segments than in adjacent uninjured segments where shear homeostatic remodelling occurs (lumen BLV 1.28 +/- 0.33 mm(2) versus ULV 1.52 +/- 0.22 mm(2)). Lastly, while PDTC effectively reduced intima formation and inward remodelling due to LF in balloon-injured vessels there was no effect on flow-dependent neointima formation in stented vessels. We conclude that LF accentuates in-stent neointima formation, but that flow-dependent lumen loss after stenting is less than that after balloon injury. When LF is present lumen loss can be minimised by antioxidants or stenting.

    View details for DOI 10.1016/j.atherosclerosis.2004.07.016

    View details for Web of Science ID 000225985600006

    View details for PubMedID 15530899

  • Transcriptional profiling of in vitro smooth muscle cell differentiation identifies specific patterns of gene and pathway activation PHYSIOLOGICAL GENOMICS Spin, J. M., Nallamshetty, S., Tabibiazar, R., Ashley, E. A., King, J. Y., Chen, M., Tsao, P. S., Quertermous, T. 2004; 19 (3): 292-302


    Mesodermal and epidermal precursor cells undergo phenotypic changes during differentiation to the smooth muscle cell (SMC) lineage that are relevant to pathophysiological processes in the adult. Molecular mechanisms that underlie lineage determination and terminal differentiation of this cell type have received much attention, but the genetic program that regulates these processes has not been fully defined. Study of SMC differentiation has been facilitated by development of the P19-derived A404 embryonal cell line, which differentiates toward this lineage in the presence of retinoic acid and allows selection for cells adopting a SMC fate through a differentiation-specific drug marker. We sought to define global alterations in gene expression by studying A404 cells during SMC differentiation with oligonucleotide microarray transcriptional profiling. Using an in situ 60-mer array platform with more than 20,000 mouse genes derived from the National Institute on Aging clone set, we identified 2,739 genes that were significantly upregulated after differentiation was completed (false-detection ratio <1). These genes encode numerous markers known to characterize differentiated SMC, as well as many unknown factors. We further characterized the sequential patterns of gene expression during the differentiation time course, particularly for known transcription factor families, providing new insights into the regulation of the differentiation process. Changes in genes associated with specific biological ontology-based pathways were evaluated, and temporal trends were identified for functional pathways. In addition to confirming the utility of the A404 model, our data provide a large-scale perspective of gene regulation during SMC differentiation.

    View details for DOI 10.1152/physiolgenomics.00148.2004

    View details for Web of Science ID 000225840800007

    View details for PubMedID 15340120

  • Overexpression of human copper/zinc superoxide dismutase (SOD1) suppresses ischemia-reperfusion injury and subsequent development of graft coronary artery disease in murine cardiac grafts CIRCULATION Tanaka, M., Mokhtari, G. K., Terry, R. D., Balsam, L. B., Lee, K. H., Kofidis, T., Tsao, P. S., Robbins, R. C. 2004; 110 (11): II200-II206


    Ischemia-reperfusion injury is an important risk factor for graft coronary artery disease (GCAD). We hypothesized that overexpression of SOD1 in donor hearts would suppress ischemia-reperfusion injury and thereby reduce GCAD.In one series, donor hearts of C57BL/6 (H-2b) transgenic mice overexpressing human SOD1 or C57BL/6 wild-type mice were heterotopically transplanted into C57BL/6 recipients and procured after 4 hours of reperfusion (n=6 each). Superoxide, TNF-alpha, and MCP-1/CCL2 production were significantly reduced in the SOD1 transgenic donor heart recipients, and graft injury determined by serum CPK-MB levels was significantly decreased. Cardiomyocyte apoptosis and caspase-3 and caspase-9 activities were significantly decreased in these recipients; caspase-8 activity was unchanged. Fas ligand but not Fas expression was also reduced. In a second series, transgenic and wild-type hearts were transplanted into C-H-2bm12KhEg (H-2bm12) recipients, and then procured on day 56 (n=7 each). Cardiac graft beating was significantly better in the SOD1 transgenic donor heart recipients on days 28, 42, and 56 (but not day 14). Significant reduction in luminal narrowing, the intima/media ratio, and the percentage of diseased vessels was seen in the SOD1 transgenic donor heart recipients, and MCP-1/CCL2, ICAM-1, and VCAM-1 production were significantly reduced.Overexpression of SOD1 attenuates both apoptosis and the inflammatory response during ischemia-reperfusion injury and therefore mitigates against the subsequent development of GCAD.

    View details for DOI 10.1161/01.CIR.0000138390.81640.54

    View details for Web of Science ID 000224023600035

    View details for PubMedID 15364863

  • Long-term effects of polymer-based, slow-release, sirolimus-eluting stents in a porcine coronary model CARDIOVASCULAR RESEARCH Carter, A. J., Aggarwal, M., Kopia, G. A., Tio, F., Tsao, P. S., Kolata, R., Yeung, A. C., Llanos, G., Dooley, L., Falotico, R. 2004; 63 (4): 617-624


    Stent-based delivery of sirolimus (SRL) has shown reduction in neointimal hyperplasia and restenosis. The purpose of this study was to evaluate the chronic vascular response and the expression of cell cycle regulators after SRL-eluting stent implantation in a porcine coronary model.Forty-nine pigs underwent placement of 109 oversized stents (control, n=54, SRL (140 microg/cm(2)), n=55) in the coronary arteries with histologic analysis and Western blot (PCNA, p27(kip1), CD45, MCP-1, IL-2, IL-6, TNF-beta) at 3, 30, 90 or 180 days.At 3 days, the mean thrombus area was similar for control (0.38+/-0.19 mm(2)) and SRL (0.29+/-0.09 mm(2)) stents. After 30 days, the mean neointimal area was significantly less for the SRL (1.40+/-0.35 mm(2)) versus the control stents (2.94+/-1.28 mm(2), p<0.001). At 90 and 180 days, the mean neointimal area was similar for the SRL (3.03+/-0.92 and 3.34+/-0.99 mm(2)) as compared with control stents (3.45+/-1.09 and 3.65+/-1.23 mm(2)). Western blot analysis demonstrated an increased expression of p27(kip1) in the vessel wall at 90 days for the SRL versus control stents (p=0.05) but with increased levels of PCNA in the SRL as compared with control stents (p=0.003).SRL-eluting stents favorably modulate neointimal formation for 30 days in the porcine coronary model. Long-term inhibition of neointimal hyperplasia is not sustained presumably due to delayed cellular proliferation despite increased levels of the cyclin-dependent kinase p27(kip1) in the vessel wall.

    View details for DOI 10.1016/j.cardiores.2004.04.029

    View details for Web of Science ID 000223591400008

    View details for PubMedID 15306217

  • The effect of VEGF-C-induced lymphangiogenesis on gene expression profiles in experimental lymphedema 5th Annual Conference on Arteriosclerosis, Thrombosis, and Vascular Biology Kiazand, A., Tsao, P., An, A. C., Han, J., Swanson, J., Berkowski, A., Karkkainen, M., Alitalo, K., Rockson, S. G. LIPPINCOTT WILLIAMS & WILKINS. 2004: E62–E62
  • The effect of VEGF-C-Induced Lymphangiogenesis on Expression profiles for Lymphangiogenesis-related genes in experimental lymphedema Experimental Biology 2004 Annual Meeting Kiazand, A., Berkowski, J. A., An, A. C., Swanson, J., Han, J., Tsao, P., Alitalo, K., Karkkainen, M., Rockson, S. G. FEDERATION AMER SOC EXP BIOL. 2004: A635–A635
  • Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis - Genetic and physiological evidence CIRCULATION Dayoub, H., Achan, V., Adimoolam, S., Jacobi, J., Stuehlinger, M. C., Wang, B. Y., Tsao, P. S., Kimoto, M., Vallance, P., Patterson, A. J., Cooke, J. P. 2003; 108 (24): 3042-3047


    NO is a major regulator of cardiovascular physiology that reduces vascular and cardiac contractility. Accumulating evidence indicates that endogenous inhibitors may regulate NOS. The NOS inhibitors asymmetric dimethylarginine (ADMA) and N-monomethylarginine are metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). This study was designed to determine if increased expression of DDAH could reduce tissue and plasma levels of the NOS inhibitors and thereby increase NO synthesis.We used gene transfer and transgenic approaches to overexpress human DDAH I in vitro and in vivo. The overexpression of DDAH in cultured endothelial cells in vitro induced a 2-fold increase in NOS activity and NO production. In the hDDAH-1 transgenic mice, we observed approximately 2-fold increases in tissue NOS activity and urinary nitrogen oxides, associated with a 2-fold reduction in plasma ADMA. The systolic blood pressure of transgenic mice was 13 mm Hg lower than that of wild-type controls (P<0.05). The systemic vascular resistance and cardiac contractility were decreased in response to the increase in NO production.DDAH I overexpression increases NOS activity in vitro and in vivo. The hDDAH-1 transgenic animal exhibits a reduced systolic blood pressure, systemic vascular resistance, and cardiac stroke volume. This study provides compelling evidence that the elaboration and metabolism of endogenous ADMA plays an important role in regulation of NOS activity.

    View details for DOI 10.1161/01.CIR.0000101924.04515.2E

    View details for Web of Science ID 000187286300018

    View details for PubMedID 14638548

  • Novel role for the potent endogenous inotrope apelin in human cardiac dysfunction CIRCULATION Chen, M. M., Ashley, E. A., Deng, D. X., Tsalenko, A., Deng, A., Tabibiazar, R., Ben-Dor, A., Fenster, B., Yang, E., King, J. Y., Fowler, M., Robbins, R., Johnson, F. L., Bruhn, L., McDonagh, T., Dargie, H., Yakhini, Z., Tsao, P. S., Quertermous, T. 2003; 108 (12): 1432-1439


    Apelin is among the most potent stimulators of cardiac contractility known. However, no physiological or pathological role for apelin-angiotensin receptor-like 1 (APJ) signaling has ever been described.We performed transcriptional profiling using a spotted cDNA microarray with 12 814 unique clones on paired samples of left ventricle obtained before and after placement of a left ventricular assist device in 11 patients. The significance analysis of microarrays and a novel rank consistency score designed to exploit the paired structure of the data confirmed that natriuretic peptides were among the most significantly downregulated genes after offloading. The most significantly upregulated gene was the G-protein-coupled receptor APJ, the specific receptor for apelin. We demonstrate here using immunoassay and immunohistochemical techniques that apelin is localized primarily in the endothelium of the coronary arteries and is found at a higher concentration in cardiac tissue after mechanical offloading. These findings imply an important paracrine signaling pathway in the heart. We additionally extend the clinical significance of this work by reporting for the first time circulating human apelin levels and demonstrating increases in the plasma level of apelin in patients with left ventricular dysfunction.The apelin-APJ signaling pathway emerges as an important novel mediator of cardiovascular control.

    View details for DOI 10.1161/01.CIR.0000091235.94914.75

    View details for Web of Science ID 000185467400005

    View details for PubMedID 12963638

  • Endothelial dysfunction: Clinical strategies for treating oxidant stress AMERICAN HEART JOURNAL Fenster, B. E., Tsao, P. S., Rockson, S. G. 2003; 146 (2): 218-226


    A growing body of evidence has demonstrated that oxidants play a critical role in the pathogenesis of endothelial dysfunction. Pathologic processes fundamental to development and progression of endothelial dysfunction such as the oxidation of LDL, the loss of bioavailable nitric oxide, and the vascular inflammatory response are all modulated by oxidant stress. Therapeutic strategies to reverse endothelial dysfunction have begun to focus on agents with the ability to ameliorate oxidant stress.Preclinical and clinical studies evaluating the actions of antioxidants as well as traditional cardiovascular therapies in ameliorating oxidative stress and endothelial dysfunction were reviewed through the use of a MEDLINE search of English language articles published between the years of 1992 and 2002.Antioxidants appear to be an attractive candidate therapy, yet despite compelling preclinical evidence supporting their benefits, efforts to validate the use of vitamins C and E in a clinical setting have been conflicting. In contrast, conventional cardiovascular therapies such as ACE inhibitors, statins, insulin-sensitizing agents, and estrogens have been shown to alleviate endothelial dysfunction, often independent of their effects on systemic disease processes.These agents restore endothelial function through their salutary effects on pathologic vascular oxidative processes.

    View details for DOI 10.1016/S0002-8703(03)00087-X

    View details for Web of Science ID 000184708400011

    View details for PubMedID 12891188

  • NOS inhibition accelerates atherogenesis: reversal by exercise AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Niebauer, J., Maxwell, A. J., Lin, P. S., Wang, D., Tsao, P. S., Cooke, J. P. 2003; 285 (2): H535-H540


    In this study, we assessed the effects of chronic exercise training (12 wk) on atherosclerotic lesion formation in hypercholesterolemic apolipoprotein E-deficient mice (n = 31). At the age of 9 wk, mice were assigned to the following groups: sedentary (Sed; n = 9); exercise (Ex; n = 12); sedentary and oral NG-nitro-L-arginine (L-NNA, Sed-NA; n = 4), or exercise and oral L-NNA (Ex-NA; n = 6). Chronic exercise training was performed on a treadmill for 12 wk (6 times/wk and twice for 1 h/day) at a final speed of 22 m/min, and an 8 degrees grade. L-NNA was discontinued 5 days before final treadmill testing. The farthest distance run to exhaustion was observed in Ex-NA mice (Sed: 306 +/- 32 m; Ex: 640 +/- 87; Sed-NA: 451 +/- 109 m; Ex-NA: 820 +/- 49 m; all P < 0.05). Lesion formation was assessed in the proximal ascending aorta by dissection microscopy after oil red O staining. The aortas of Sed-NA mice manifested a threefold increase in lesion formation compared with the other groups. This L-NNA-induced lesion formation was reduced by chronic exercise training (Sed, 786 +/- 144; Ex, 780 +/- 206; Sed-NA, 2,147 +/- 522; Ex-NA, 851 +/- 253; Sed-NA vs. all other groups: P < 0.001). In conclusion, treatment with oral L-NNA (an nitric oxide synthase antagonist) leads to accelerated atherogenesis in genetically determined hypercholesterolemic mice. This adverse effect can be overcome by chronic exercise training.

    View details for DOI 10.1152/ajpheart.00360.2001

    View details for Web of Science ID 000184153100011

    View details for PubMedID 12598230

  • Flow-responsive remodeling after angioplasty is enhanced by high cholesterol diet. Prevention with pyrrolidine dithiocarbamate ATHEROSCLEROSIS Ward, M. R., Tsao, P. S., Herity, N. A., Cooke, J. P., Yeung, A. C. 2003; 168 (2): 333-341


    We examined the effects of high cholesterol diet and pyrrolidine dithiocarbamate (PDTC) on flow-dependent remodeling after angioplasty. After right common carotid balloon-injury, the right external carotid (low flow) or left common carotid artery were ligated (high flow) in rabbits fed normal diet, 1% cholesterol diet without or with the antioxidant PDTC for 7 days pre- and 7-28 days post-injury. Angiographic lumen diameter was significantly greater at 28 days in high flow than low flow normal diet animals, attributable on perfusion-fixed vessel morphometry to altered remodeling (area within the external elastic lamina: high flow 1.85+/-0.24 vs. low flow 1.31+/-0.04 mm(2), P<0.05) rather than differences in neointima formation or vessel tone. In animals on 1% cholesterol diet high flow remodeling was significantly enhanced (area within the external elastic lamina 3.13+/-0.17 mm(2), P<0.05 vs. high flow normal diet) but low flow inward remodeling was similar (area within the external elastic lamina 1.29+/-0.07 mm(2)). Mean Doppler flow velocities (initial/post-ligation/28 day follow-up, cm/s) had almost normalized in normal diet animals (high flow 30/49/35, low flow 32/9/26) but showed overcompensation in 1% cholesterol diet animals (high flow 32/49/22, low flow 30/11/25). PDTC therapy markedly attenuated remodeling (area within the external elastic lamina: high flow 2.20+/-0.18, and low flow 2.00+/-0.11 both P<0.05 vs. 1% cholesterol diet alone) and flow velocities only partially normalized (high flow 26/42/34, low flow 27/7/16). We conclude that hypercholesterolemia enhances and PDTC attenuates flow-dependent remodeling after angioplasty.

    View details for DOI 10.1016/S0021-9150(03)00103-5

    View details for Web of Science ID 000183784900016

    View details for PubMedID 12801617

  • Identification of endothelial cell genes by combined database mining and microarray analysis PHYSIOLOGICAL GENOMICS Ho, M., Yang, E., Matcuk, G., Deng, D., Sampas, N., Tsalenko, A., Tabibiazar, R., Zhang, Y., Chen, M., Talbi, S., Ho, Y. D., Wang, J., Tsao, P. S., Ben-Dor, A., Yakhini, Z., Bruhn, L., Quertermous, T. 2003; 13 (3): 249-262


    Vascular endothelial cells maintain the interface between the systemic circulation and soft tissues and mediate critical processes such as inflammation in a vascular bed-selective fashion. To expand our understanding of the genetic pathways that underlie these specific functions, we have focused on the identification of novel genes that are differentially expressed in all endothelial cells, as well as restricted groups of this cell type. Virtual subtraction was conducted employing gene expression data deposited in public databases and 384 genes identified. These genes were spotted on custom microarrays, along with 288 genes identified through subtraction cloning from TGF-beta-stimulated endothelial cells. Arrays were evaluated with RNA samples representing endothelial cells cultured from four vascular sources and five non-endothelial cell types. These studies identified 64 pan-endothelial markers that were differentially expressed with at least a threefold difference (range 3- to 55-fold). In addition, differences in gene expression profiles among endothelial cells from different vascular beds were identified. Validation of these findings was performed by RNA blot expression studies, and a number of the novel genes were shown to be expressed under angiogenic conditions in the developing mouse embryo. The combined tools of database mining and transcriptional profiling thus provide expanded knowledge of endothelial cell gene expression and endothelial cell biology.

    View details for DOI 10.1152/physiolgenomics.00186.2002

    View details for Web of Science ID 000182853000008

    View details for PubMedID 12644598

  • Rosuvastatin attenuates the production of vascular reactive oxygen species in ApoE(-/-) mice 4th Annual Conference on Arteriosclerosis Thrombosis and Vascular Biology Asagami, T., Li, W., Lee, K. H., McTaggart, F., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2003: A59–A60
  • The effect of oxidant stress, injury and flow on c-jun and egr-1 expression 4th Annual Conference on Arteriosclerosis Thrombosis and Vascular Biology Murrell, M. J., Khachigian, L. M., Tsao, P., Ward, M. R. LIPPINCOTT WILLIAMS & WILKINS. 2003: A20–A20
  • Lymphangiogenesis in lymphatic insufficiency: Lymphatic endothelial and inflammatory RNA expression patterns 4th Annual Conference on Arteriosclerosis Thrombosis and Vascular Biology Shin, W. S., Rockson, N. B., Sanchez, D. R., Midde, R., Alitalo, K., Karkkainen, M., Tsao, P. S., Rockson, S. G. LIPPINCOTT WILLIAMS & WILKINS. 2003: A52–A52
  • Stent-based immunosuppressive therapies for the prevention of restenosis. Cardiovascular radiation medicine Aggarwal, M., Tsao, P. S., Yeung, A., Carter, A. J. 2003; 4 (2): 98-107

    View details for PubMedID 14581091

  • Insulin resistance and compensatory hyperinsulinemia - The key player between cigarette smoking and cardiovascular disease? JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Reaven, G., Tsao, P. S. 2003; 41 (6): 1044-1047


    Hyperinsulinemia, dyslipidemia, and endothelial dysfunction are characteristic findings in insulin-resistant individuals, and all of these abnormalities have been identified as increasing cardiovascular disease (CVD) risk. Smokers tend to be relatively insulin resistant, hyperinsulinemic, and dyslipidemic, with evidence of endothelial dysfunction, as compared with nonsmokers, and recent epidemiologic data have suggested that CVD in smokers is primarily seen in those individuals who also have the characteristic findings of insulin resistance. Based on these observations, it is argued that insulin resistance and its consequences represent a major mechanistic link between cigarette smoking and CVD. It is also postulated that the enhanced CVD risk in smokers, resulting from hyperinsulinemia, abnormalities of lipoprotein metabolism, and endothelial dysfunction, will primarily be present in those smokers who are insulin resistant. As a corollary, it is suggested that CVD risk in individuals who cannot, or will not, stop smoking can be reduced by therapeutic efforts aimed at attenuating the adverse effects of insulin resistance and its consequences.

    View details for DOI 10.1016/S0735-1097(02)02982-0

    View details for Web of Science ID 000181552800024

    View details for PubMedID 12651055

  • Reduced myocardial brain natriuretic peptide expression and collagen deposition following ventricular assist device support for heart failure 52nd Annual Scientific Session of the American-College-of-Cardiology Fenster, B. E., Fowler, M. B., Yee, Y. G., Connolly, A., Tsao, P. S. ELSEVIER SCIENCE INC. 2003: 165A–165A
  • Short polymers of arginine rapidly translocate into vascular cells - Effects on nitric oxide synthesis CIRCULATION JOURNAL Uemura, S., Rothbard, J. B., Matsushita, H., Tsao, P. S., Fathman, C. G., Cooke, J. P. 2002; 66 (12): 1155-1160


    The present study was designed to determine the efficiency of translocation of short polymers of arginine into vascular smooth muscle cells (VSMC) and to determine their effect on nitric oxide (NO) synthesis. Immunostaining revealed that heptamers of L-arginine (R7) rapidly translocated into the VSMC. This rapid transport was not observed with shorter polymers of L-arginine (R5) nor heptamers of lysine (K7). Translocation of R7 was not inhibited by the addition of free L-arginine into the media. When cells were transiently pretreated with R7 or a nonamer of arginine (R9), NO(2) production from cytokine stimulated VSMC was significantly increased, whereas incubation with R5 and K7 had no effect. Short polymers of arginine not only have a unique ability of rapid VSMC translocation but once internalized enhance NO production. Heptamers (or larger polypeptides) of arginine may be useful in therapy to enhance NO production in the vascular system.

    View details for Web of Science ID 000179496000015

    View details for PubMedID 12499624

  • Flow loading induces macrophage antioxidative gene expression in experimental aneurysms ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Nakahashi, T. K., Hoshina, K., Tsao, P. S., Sho, E., Sho, M., Karwowski, J. K., Yeh, C., Yang, R. B., Topper, J. N., Dalman, R. L. 2002; 22 (12): 2017-2022


    Reactive oxygen species may act as proinflammatory mediators in abdominal aortic aneurysm (AAA) disease. Flow loading increases antioxidative enzyme expression and limits reactive oxygen species production in vascular smooth muscle cells in vitro, limits experimental AAA enlargement in rodent models, and is indirectly associated with reduced clinical AAA risk. We attempted to determine the mechanism or mechanisms by which flow loading limits AAA enlargement.Rodent AAAs were flow loaded via femoral arteriovenous fistula creation. Aortic wall shear stress and relative wall strain were significantly higher in flow-loaded rodents. Flow loading reduced AAA diameter by 26% despite evidence of flow-mediated aortic enlargement proximal to the aneurysmal segment. Messenger RNA from AAA tissue was harvested for cDNA labeling and hybridization to a 384-clone DNA microarray. Twenty-nine genes were differentially expressed (relative intensity/relative intensity of control ratio >1.5 and <0.67) in flow-loaded compared with normal flow AAA tissue, including heme oxygenase 1 (HO-1). Increased HO-1 expression was confirmed via reverse transcriptase-polymerase chain reaction. Immunohistochemistry localized HO-1 expression to infiltrative macrophages. alpha-Tocopherol was found to be as effective as flow loading in limiting AAA enlargement. Flow loading and alpha-tocopherol therapy reduced AAA reactive oxygen species production.Flow loading may attenuate AAA enlargement via wall shear or strain-related reductions in oxidative stress.

    View details for DOI 10.1161/01.ATV.0000042082.38014.EA

    View details for Web of Science ID 000180046000014

    View details for PubMedID 12482828

  • Impaired nitric oxide synthase pathway in diabetes mellitus - Role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase CIRCULATION LIN, K. Y., Ito, A., Asagami, T., Tsao, P. S., Adimoolam, S., Kimoto, M., Tsuji, H., Reaven, G. M., Cooke, J. P. 2002; 106 (8): 987-992


    An endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), is elevated in patients with type 2 diabetes mellitus (DM). This study explored the mechanisms by which ADMA becomes elevated in DM.Male Sprague-Dawley rats were fed normal chow or high-fat diet (n=5 in each) with moderate streptozotocin injection to induce type 2 DM. Plasma ADMA was elevated in diabetic rats (1.33+/-0.31 versus 0.48+/-0.08 micromol/L; P<0.05). The activity, but not the expression, of dimethylarginine dimethylaminohydrolase (DDAH) was reduced in diabetic rats and negatively correlated with their plasma ADMA levels (P<0.05). DDAH activity was significantly reduced in vascular smooth muscle cells and human endothelial cells (HMEC-1) exposed to high glucose (25.5 mmol/L). The impairment of DDAH activity in vascular cells was associated with an accumulation of ADMA and a reduction in generation of cGMP. In human endothelial cells, coincubation with the antioxidant polyethylene glycol-conjugated superoxide dismutase (22 U/mL) reversed the effects of the high-glucose condition on DDAH activity, ADMA accumulation, and cGMP synthesis.A glucose-induced impairment of DDAH causes ADMA accumulation and may contribute to endothelial vasodilator dysfunction in DM.

    View details for DOI 10.1161/01.CIR.0000027109.14149.67

    View details for Web of Science ID 000177634600019

    View details for PubMedID 12186805

  • Impaired NOS pathway in diabetes mellitus: role of ADMA and DDAH 24th Annual Scientific Meeting of the European-Society-of-Cardiology (ESC) Lin, K., Tsao, P., Cooke, J. OXFORD UNIV PRESS. 2002: 493–493
  • Metformin treatment lowers asymmetric dimethylarginine concentrations in patients with type 2 diabetes METABOLISM-CLINICAL AND EXPERIMENTAL Asagami, T., Abbasi, F., Stuelinger, M., Lamendola, C., McLaughlin, T., Cooke, J. P., Reaven, G. M., Tsao, P. S. 2002; 51 (7): 843-846


    This study was initiated to see if plasma asymmetric dimethylarginine (ADMA) concentrations decreased in hyperglycemic patients with type 2 diabetes following metformin treatment, either as monotherapy or following its addition to sulfonylurea-treated patients. Fasting plasma glucose, dimethylarginine, and L-arginine concentrations were measured before and 3 months after the administration of a maximally effective dose of metformin to 31 patients with type 2 diabetes in poor glycemic control (fasting plasma concentrations > 9.7 mmol/L), while being treated with either diet (n = 16) or a maximal amount of a sulfonylurea compound (n = 15). Fasting plasma glucose concentration (mean +/- SEM) decreased to a similar degree (P <.01) in patients treated with either metformin alone (12.4 +/- 0.5 to 9.5 +/- 0.5 mmol/L) or when it was added to a sulfonylurea compound (14.1 +/- 0.5 to 10.6 +/- 0.9 mmol/L). The improvement in glycemic control was associated with similar decreases (P <.01) in ADMA concentrations in metformin (1.65 +/- 0.21 to 1.18 +/- 0.13 micromol/L) and sulfonylurea + metformin-treated patients (1.75 +/- 0.13 to 1.19 +/- 0.08 micromol/L). Plasma L-arginine concentrations were similar in the 2 groups at baseline and did not change in response to metformin. Thus, metformin treatment was associated with a favorable increase in the plasma L-arginine/ADMA ratio. These results provide the first evidence that plasma ADMA concentrations decrease in association with improved glycemic control in patients with type 2 diabetes and demonstrate that the magnitude of the change in metformin-treated patients was similar, irrespective of whether it was used as monotherapy or in combination with sulfonylurea treatment.

    View details for DOI 10.1053/meta.2002.33349

    View details for Web of Science ID 000176578200007

    View details for PubMedID 12077728

  • Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Stuhlinger, M. C., Abbasi, F., Chu, J. W., Lamendola, C., McLaughlin, T. L., Cooke, J. P., Reaven, G. M., Tsao, P. S. 2002; 287 (11): 1420-1426


    Increased levels of asymmetric dimethylarginine (ADMA) are associated with endothelial dysfunction and increased risk of cardiovascular disease. Several cardiovascular risk factors are associated with reduced sensitivity to insulin, but elevated ADMA concentrations have not been fully linked to the metabolic syndrome.To evaluate the relationship between insulin sensitivity and plasma ADMA concentrations, and to determine whether a pharmacological treatment that increases insulin sensitivity would also modulate ADMA concentrations.Cross-sectional study, containing a nonrandomized controlled trial component, of 64 healthy volunteers without diabetes (42 women, 22 men; 48 with normal blood pressure and 16 with hypertension), which was conducted at a university medical center between October 2000 and July 2001.Rosiglitazone (4 mg/d for 4 weeks and then 4 mg twice daily for 8 weeks), an insulin-sensitizing agent, was given to 7 insulin-resistant subjects with hypertension. These subjects were studied before and after 12-week treatment.Insulin sensitivity measured by the insulin suppression test, and fasting plasma levels of low-density lipoprotein cholesterol, triglycerides, high-density lipoprotein cholesterol, glucose, insulin, and ADMA concentrations.Plasma ADMA concentrations were positively correlated with impairment of insulin-mediated glucose disposal in nondiabetic, normotensive subjects (r = 0.73; P<.001). Consistent with the metabolic syndrome, ADMA levels were also positively correlated with fasting triglyceride levels (r = 0.52; P<.001) but not with low-density lipoprotein cholesterol levels (r = 0.19; P =.20). Plasma ADMA concentrations increased in insulin-resistant subjects independent of hypertension. Pharmacological treatment improved insulin sensitivity and reduced mean (SD) plasma ADMA concentrations from 1.50 (0.30) to 1.05 (0.33) micromol/L (P =.001).A significant relationship exists between insulin resistance and plasma concentrations of ADMA. Pharmacological intervention with rosiglitazone enhanced insulin sensitivity and reduced ADMA levels. Increases in plasma ADMA concentrations may contribute to the endothelial dysfunction observed in insulin-resistant patients.

    View details for Web of Science ID 000174465000024

    View details for PubMedID 11903029

  • Homocysteine impairs the nitric oxide synthase pathway - Role of asymmetric dimethylarginine CIRCULATION Stuhlinger, M. C., Tsao, P. S., Her, J. H., Kimoto, M., Balint, R. F., Cooke, J. P. 2001; 104 (21): 2569-2575


    Hyperhomocysteinemia is a putative risk factor for cardiovascular disease, which also impairs endothelium-dependent vasodilatation. A number of other risk factors for cardiovascular disease may exert their adverse vascular effects in part by elevating plasma levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase. Accordingly, we determined if homocysteine could increase ADMA levels.When endothelial or nonvascular cells were exposed to DL-homocysteine or to its precursor L-methionine, ADMA concentration in the cell culture medium increased in a dose- and time-dependent fashion. This effect was associated with the reduced activity of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA. Furthermore, homocysteine-induced accumulation of ADMA was associated with reduced nitric oxide synthesis by endothelial cells and segments of pig aorta. The antioxidant pyrrollidine dithiocarbamate preserved DDAH activity and reduced ADMA accumulation. Moreover, homocysteine dose-dependently reduced the activity of recombinant human DDAH in a cell free system, an effect that was due to a direct interaction between homocysteine and DDAH.Homocysteine post-translationally inhibits DDAH enzyme activity, causing ADMA to accumulate and inhibit nitric oxide synthesis. This may explain the known effect of homocysteine to impair endothelium-mediated nitric oxide-dependent vasodilatation.

    View details for Web of Science ID 000172307200013

    View details for PubMedID 11714652

  • Plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus AMERICAN JOURNAL OF CARDIOLOGY Abbasi, F., Asagmi, T., Cooke, J. P., Lamendola, C., McLaughlin, T., Reaven, G. M., Stuehlinger, M., Tsao, P. S. 2001; 88 (10): 1201-?

    View details for Web of Science ID 000172412300025

    View details for PubMedID 11703973

  • eNOS activity is reduced in senescent human endothelial cells - Preservation by hTERT immortalization CIRCULATION RESEARCH Matsushita, H., Chang, E., Glassford, A. J., Cooke, J. P., Chiu, C. P., Tsao, P. S. 2001; 89 (9): 793-798


    Advanced age is associated with endothelial dysfunction and increased risk for atherosclerosis. However, the mechanisms for these observed effects are not clear. To clarify the association between aging and loss of endothelial function, young human aortic endothelial cells (HAECs), senescent HAECs transfected with control vector, and immortalized HAECs containing human telomerase reverse transcriptase (hTERT) were compared for expression of endothelial nitric oxide synthase (eNOS) and production of NO. To investigate a specific function modulated by endothelial NO, adhesion of monocytes under basal conditions as well as after exposure to TNF-alpha was assessed. A decrease in eNOS mRNA, protein, and activity was observed in endothelial cells at senescence as compared with young HAEC; this effect was blunted in hTERT cells. In all cells, shear stress induced a greater increase in the expression of eNOS protein with the final result being higher levels in hTERT compared with senescent cells. Basal monocyte binding was significantly elevated on aged endothelial cells compared with parental and hTERT cells. Exposure of TNF-alpha resulted in a 2-fold increase in monocyte adhesion in senescent cells, whereas this effect was reduced in cells transfected with hTERT. Prior exposure to fluid flow significantly reduced subsequent monocyte adhesion in all groups. These studies demonstrate that replicative aging results in decreased endothelial expression of eNOS accompanied by enhanced monocyte binding. Stable expression of hTERT results in endothelial cells with a younger phenotype with greater amount of eNOS and NO activity. Thus, telomerase transfection may have important functional consequences on endothelial cells.

    View details for Web of Science ID 000171974800010

    View details for PubMedID 11679409

  • The novel HMG-CoA reducase inhibitor, Rosuvastatin, attenuates monocyte adhesion in a murine model of hypercholesterolemia Li, W., Asagami, T., McTaggart, F., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2001: 212–12
  • Mechanical stretch modulates TGF-beta/Smad signaling in human vascular cells via down regulation of Smad6 Hayashi, S. I., Matsushita, H., Glassford, A., Li, W., Topper, J. N., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2001: 273–73
  • A novel functional interaction of TGF-beta receptors with eNOS in caveolae Schwartz, E. A., Topper, J. N., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2001: 106–
  • Anti-angiogenic effects of thalidomide characterized by transciptional profiling of endothelial cell-specific gene expression Yang, E., Matcuk, G., Zhang, Y., Talbi, S., Chen, M., Ho, M., Liao, C., Tsao, P. S., Quertermous, T., Deng, D., Sampas, N., Ach, R., Love, W. LIPPINCOTT WILLIAMS & WILKINS. 2001: 123–23
  • Stent-based delivery of sirolimus reduces neointimal formation in a porcine coronary model CIRCULATION Suzuki, T., Kopia, G., Hayashi, S., Bailey, L. R., Llanos, G., Wilensky, R., Klugherz, B. D., Papandreou, G., Narayan, P., Leon, M. B., Yeung, A. C., Tio, F., Tsao, P. S., Falotico, R., Carter, A. J. 2001; 104 (10): 1188-1193


    The purpose of this study was to determine the efficacy of stent-based delivery of sirolimus (SRL) alone or in combination with dexamethasone (DEX) to reduce in-stent neointimal hyperplasia. SRL is a potent immunosuppressive agent that inhibits SMC proliferation by blocking cell cycle progression.Stents were coated with a nonerodable polymer containing 185 microgram SRL, 350 microgram DEX, or 185 microgram SRL and 350 microgram DEX. Polymer biocompatibility studies in the porcine and canine models showed acceptable tissue response at 60 days. Forty-seven stents (metal, n=13; SRL, n=13; DEX, n=13; SRL and DEX, n=8) were implanted in the coronary arteries of 16 pigs. The tissue level of SRL was 97+/-13 ng/artery, with a stent content of 71+/-10 microgram at 3 days. At 7 days, proliferating cell nuclear antigen and retinoblastoma protein expression were reduced 60% and 50%, respectively, by the SRL stents. After 28 days, the mean neointimal area was 2.47+/-1.04 mm(2) for the SRL alone and 2.42+/-1.04 mm(2) for the combination of SRL and DEX compared with the metal (5.06+/-1.88 mm(2), P<0.0001) or DEX-coated stents (4.31+/-3.21 mm(2), P<0.001), resulting in a 50% reduction of percent in-stent stenosis.Stent-based delivery of SRL via a nonerodable polymer matrix is feasible and effectively reduces in-stent neointimal hyperplasia by inhibiting cellular proliferation.

    View details for Web of Science ID 000170922100022

    View details for PubMedID 11535578

  • Cholesterol-induced upregulation of angiotensin II and its effects on monocyte-endothelial interaction and superoxide production VASCULAR MEDICINE Niebauer, J., Tsao, P. S., Lin, P. S., Pratt, R. E., Cooke, J. P. 2001; 6 (3): 133-138


    Atherogenesis involves an early endothelial dysfunction hallmarked by elevated free radical production and increased adhesiveness for monocytes. It was hypothesized that activation of the tissue renin angiotensin system may contribute to the endothelial alteration. To test this hypothesis, thoracic aortae were isolated from normocholesterolemic (NC; n = 6) and hypercholesterolemic (HC; n = 6; diet: 0.5% cholesterol; 6 weeks) New Zealand white rabbits, and incubated for 2 h with the angiotensin II (Ang II) receptor antagonist Sar-1,Ile-8-Ang II, the antioxidant pyrolidine dithiocarbamate (PDTC) and the protein kinase C (PKC) antagonist staurosporin. Superoxide production from aortic segments was measured by lucigenin-enhanced chemiluminescence. In comparison to the normocholesterolemic state, hypercholesterolemia led to a significant increase in superoxide production (221 +/- 44%, p < 0.02); this was reduced by ex vivo treatment of the vessel segment with Ang II-antagonist (to 130 +/- 29%; p < 0.04 vs HC), or PKC-antagonist (to 86 +/- 26%; p < 0.001 vs HC), or PDTC (to 103 +/- 27%; p < 0.02 vs HC). Monocyte-endothelial interaction was assessed by functional binding assay. When compared to normocholesterolemic rabbits, hypercholesterolemia led to a twofold increase in monocyte binding (74 +/- 13 vs 37 +/- 4 monocytoid cells per high power field (m/hpf); p < 0.03). The Ang II-antagonist and the PKC-antagonist led to a normalization of monocyte-endothelial binding (Ang II-antagonist: 37 +/- 9 m/hpf; PKC-antagonist: 41 +/- 17 m/hpf; p < 0.05). In conclusion, these results indicate that hypercholesterolemia activates the tissue renin angiotensin system, which results in an increased endothelial production of superoxide and monocyte adhesiveness. Ang II-antagonist inhibits free radical production and monocyte adhesion through a mechanism which may include PKC.

    View details for Web of Science ID 000172694700002

    View details for PubMedID 11789966

  • Effects of stenting on adjacent vascular distensibility and neointima formation: role of nitric oxide VASCULAR MEDICINE Schwarzacher, S. P., Tsao, P. S., Ward, M., Hayase, M., Niebauer, J., Cooke, J. P., Yeung, A. C. 2001; 6 (3): 139-144


    Intravascular stents increase long-term patency but their effects on the vascular mechanics of adjacent segments have not been studied. In this study, stents were deployed in the rabbit abdominal aorta after 1 week of normal diet, 1% cholesterol diet or 1% cholesterol diet with L-nitro arginine (L-NA 60 mg/l water). Intravascular ultrasound showed a small distal decrease in vessel distensibility (area/pressure * 100) before stenting. Distensibility was almost abolished by stenting (0.12 +/- 0.01, p < 0.001), but was increased proximal to the stent and decreased distal to the stent both acutely (proximal: 1.18 +/- 0.10 vs distal: 0.65 +/- 0.06, p < 0.001), and at 4 weeks (proximal: 1.05 +/- 0.08 vs distal: 0.37 +/- 0.07, p < 0.001). Nitric oxide (NO) activity was enhanced proximal to and within the stent, and remained constant distal to the stent, (versus control, proximal: 57 +/- 23%, stent: 136 +/- 35%, distal: 2 +/- 12%, p < 0.01). The I/M ratio was significantly higher proximal to and within the stent than in the distal segment (proximal: 0.40 +/- 0.10, stent: 0.37 +/- 0.12, distal: 0.12 +/- 0.11, p < 0.01). NO blockade with L-NA prevented hyperdistensibility proximally, and significantly increased the I/M ratio within the stent and distally (stent: 0.81 +/- 0.19, distal: 0.30 +/- 0.10, p < 0.05) but not proximally (0.38 +/- 0.09). In conclusion, aortic stenting increases proximal vascular distensibility and intimal lesion formation. Nitric oxide blockade augments intimal growth within but not proximal to the stent.

    View details for Web of Science ID 000172694700003

    View details for PubMedID 11789967

  • Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis NATURE MEDICINE Heeschen, C., Jang, J. J., Weis, M., Pathak, A., Kaji, S., Hu, R. S., Tsao, P. S., Johnson, F. L., Cooke, J. P. 2001; 7 (7): 833-839


    We provide anatomic and functional evidence that nicotine induces angiogenesis. We also show that nicotine accelerates the growth of tumor and atheroma in association with increased neovascularization. Nicotine increased endothelial-cell growth and tube formation in vitro, and accelerated fibrovascular growth in vivo. In a mouse model of hind-limb ischemia, nicotine increased capillary and collateral growth, and enhanced tissue perfusion. In mouse models of lung cancer and atherosclerosis, we found that nicotine enhanced lesion growth in association with an increase in lesion vascularity. These effects of nicotine were mediated through nicotinic acetylcholine receptors at nicotine concentrations that are pathophysiologically relevant. The endothelial production of nitric oxide, prostacyclin and vascular endothelial growth factor might have a role in these effects.

    View details for Web of Science ID 000169808600036

    View details for PubMedID 11433349

  • Diabetes mellitus enhances vascular matrix metalloproteinase activity - Role of oxidative stress CIRCULATION RESEARCH Uemura, S., Matsushita, H., Li, W., Glassford, A. J., Asagami, T., Lee, K. H., Harrison, D. G., Tsao, P. S. 2001; 88 (12): 1291-1298


    Diabetes mellitus (DM) is a primary risk factor for cardiovascular disease. Although recent studies have demonstrated an important role for extracellular matrix metalloproteinases (MMPs) in atherosclerosis, little is known about the effects of hyperglycemia on MMP regulation in vascular cells. Gelatin zymography and Western blot analysis revealed that the activity and expression of 92-kDa (MMP-9) gelatinase, but not of 72 kDa (MMP-2) gelatinase, were significantly increased in vascular tissue and plasma of two distinct rodent models of DM. Bovine aortic endothelial cells (BAECs) grown in culture did not express MMP-9 constitutively; however, chronic (2-week) incubation with high glucose medium induced MMP-9 promoter activity, mRNA and protein expression, and gelatinase activity in BAECs. On the other hand, high glucose culture did not change MMP-9 activity from vascular smooth muscle cells or macrophages. Electron paramagnetic resonance studies indicate that BAECs chronically grown in high glucose conditions produce 70% more ROS than do control cells. Enhanced MMP-9 activity was significantly reduced by treatment with the antioxidants polyethylene glycol-superoxide dismutase and N-acetyl-L-cysteine but not by inhibitors of protein kinase C. In conclusion, vascular MMP-9 activity is increased in DM, in part because of enhanced elaboration from vascular endothelial cells, and oxidative stress plays an important role. This novel mechanism of redox-sensitive MMP-9 expression by hyperglycemia may provide a rationale for antioxidant therapy to modulate diabetic vascular complications.

    View details for Web of Science ID 000169541600013

    View details for PubMedID 11420306

  • Alpha-tocopherol limits experimental aortic aneurysm enlargement YEH, C. C., Nakahashi, T., Hoshina, K., Xu, C. P., Tsao, P., Karwowski, J. K., Dalman, R. L. LIPPINCOTT WILLIAMS & WILKINS. 2001: 638–38
  • Low blood flow after angioplasty augments mechanisms of restenosis - Inward vessel remodeling, cell migration, and activity of genes regulating migration ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Ward, M. R., Tsao, P. S., Agrotis, A., Dilley, R. J., Jennings, G. L., Bobik, A. 2001; 21 (2): 208-213


    -The predominant cause of restenosis after angioplasty is now thought to be inward remodeling, but the mechanisms responsible are unknown. Remodeling in normal vessels is regulated by the endothelium in response to altered shear stress. Although the endothelium is often damaged by angioplasty, restenosis rates after angioplasty have been correlated with impaired coronary flow. Thus, we examined how increases or decreases in blood flow through balloon catheter-injured rat carotid arteries affect vessel morphometry (4, 10, and 28 days), cell migration (4 days), and levels of promigratory mRNAs (2 and 10 days). After 28 days, the luminal area in vessels with low blood flow was significantly less than in those with normal and high blood flow (0.17+/-0.01 [low] versus 0.24+/-0.06 [normal] versus 0.30+/-0.02 [high] mm(2), P:<0.01), predominantly because of accentuated inward remodeling (or reduced area within the external elastic lamina; 0.42+/-0.02 [low] versus 0.54+/-0.07 [normal] versus 0.53+/-0.04 [high] mm(2), P:<0.05). Low flow also enhanced smooth muscle cell migration 4 days after injury by 90% above normal and high flows (P:<0.01). Two days after injury, low flow significantly increased levels of mRNAs encoding promigratory peptides (integrin alpha(v)ss(3), transforming growth factor-ss(1), CD44v6, MDC9, urokinase plasminogen activator receptor, and ss-inducible gene h3); these changes persisted 10 days after injury and were localized to the neointima. Low blood flow may promote restenosis after angioplasty because of its adverse effect on vessel remodeling, and it is associated with the augmented expression of multiple genes central to cell migration and restenosis.

    View details for Web of Science ID 000167563200007

    View details for PubMedID 11156854

  • Cyclic strain induces reactive oxygen species production via an endothelial NAD(P)H oxidase JOURNAL OF CELLULAR BIOCHEMISTRY Matsushita, H., Lee, K. H., Tsao, P. S. 2001: 99-106


    Vascular endothelial cells are constantly subjected to pressure-induced cyclic strain. Reactive oxygen species (ROS) have been implicated in atherosclerosis and vascular remodeling. Recent evidence indicates that a vascular NAD(P)H oxidase may be an important source of ROS in both physiologic and pathophysiologic situations. The aim of this study was to investigate cyclic strain-induced NAD(P)H oxidase activity in endothelial cells. ROS production was examined by electron paramagnetic resonance and lucigenin chemiluminescence. Cyclic strain-induced NAD(P)H oxidase activity was quantified by activity assay while the expression of p22phox was monitored by Northern blotting. Endothelial cells produce basal amounts of ROS that were enhanced by cyclic strain. Moreover subsequent stimulation with TNF-alpha resulted in significantly greater ROS production in cells previously exposed to cyclic strain as compared to static conditions. Cyclic strain resulted in a significant increase in message for the p22phox subunit as well as activity of the NAD(P)H oxidase. The induced oxidative stress was accompanied by increased mobilization of the transcription factor NFkappaB, an effect that was blocked by a pharmacological inhibitor of NAD(P)H. These results demonstrate a pivotal role for NAD(P)H oxidase in cyclic strain-induced endothelial ROS production and may provide insight into the modulation of vascular disease by biomechanical forces. J. Cell. Biochem. Suppl. 36: 99-106, 2001.

    View details for Web of Science ID 000168543400011

    View details for PubMedID 11455575

  • Mechanotransduction of endothelial oxidative stress induced by cyclic strain ENDOTHELIUM-JOURNAL OF ENDOTHELIAL CELL RESEARCH Wang, D. S., Proffit, D., Tsao, P. S. 2001; 8 (4): 283-291


    Atherosclerotic lesions display a nonuniform distribution throughout the vascular tree. Mechanical forces produced by local alterations in blood flow may play an important role in the localization of atherosclerosis. One such force, cyclic strain, has been hypothesized to promote atherogenesis by inducing oxidative stress in endothelial cells, resulting in enhanced endothelial adhesiveness for monocytes. To investigate the signal transduction systems involved, human aortic endothelial cells were plated on flexible silicone strips that were either non-coated or adsorbed with poly-L-lysine, vitronectin, fibronectin, or collagen I. Cells were then subjected to uniform sinusoidal stretch (10%) for 6 h. Endothelial superoxide anion production was increased in cells exposed to cyclic strain compared to static conditions. Furthermore, endothelial oxidative response to stretch was matrix protein-dependent, whereas cells grown on fibronectin and collagen I produced significantly more superoxide. The oxidative response to cyclic strain was reduced by coincubation with RGD peptides, blocking antibodies to alpha2- and beta-integrins antibodies, as well as inhibitors of protein kinase C. To investigate the effect of oxidative stress on gene transcription, endothelial cells grown on collagen I were transfected with an NFkappaB-sensitive luciferase construct. Cells that underwent cyclic strain displayed a tenfold induction of NFkappaB activation compared to static controls. Strain-induced luciferase activity was blunted by coincubation with RGD peptides or calphostin C. Thus, exposure of endothelial cells to cyclic strain led to integrin activation of a PKC-sensitive pathway that results in increased superoxide anion production and mobilization of NFkappaB.

    View details for Web of Science ID 000173063400007

    View details for PubMedID 11824481

  • An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Boger, R. H., Bode-Boger, S. M., Tsao, P. S., Lin, P. S., Chan, J. R., Cooke, J. P. 2000; 36 (7): 2287-2295


    We sought to determine whether asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) elaboration in cultured human endothelial cells and whether this is associated with the activation of oxidant-sensitive signaling mediating endothelial adhesiveness for monocytes.Endothelial NO elaboration is impaired in hypercholesterolemia and atherosclerosis, which may be due to elevated concentrations of ADMA, an endogenous inhibitor of NO synthase.Human umbilical vein endothelial cells (ECV 304) and human monocytoid cells (THP-1) were studied in a functional binding assay. Nitric oxide and superoxide anion (O2-) were measured by chemiluminescence; ADMA by high pressure liquid chromatography; monocyte chemotactic protein-1 (MCP-1) by ELISA and NF-KB by electromobility gel shift assay.Incubation of endothelial cells with ADMA (0.1 microM to 100 microM) inhibited NO formation, which was reversed by coincubation with L-arginine (1 mM). The biologically inactive stereoisomer symmetric dimethylarginine did not inhibit NO release. Asymmetric dimethylarginine (10 microM) or native low-density lipoprotein cholesterol (100 mg/dL) increased endothelial O2- to the same degree. Asymmetric dimethylarginine also stimulated MCP-1 formation by endothelial cells. This effect was paralleled by activation of the redox-sensitive transcription factor NF-KB. Preincubation of endothelial cells with ADMA increased the adhesiveness of endothelial cells for THP-1 cells in a concentration-dependent manner. Asymmetric dimethylarginine-induced monocyte binding was diminished by L-arginine or by a neutralizing anti-MCP-1 antibody.We concluded that the endogenous NO synthase inhibitor ADMA is synthesized in human endothelial cells. Asymmetric dimethylarginine increases endothelial oxidative stress and potentiates monocyte binding. Asymmetric dimethylarginine may be an endogenous proatherogenic molecule.

    View details for Web of Science ID 000165600400041

    View details for PubMedID 11127475

  • Hyperglycemia induces matrix metalloproteinase activity in vascular cells: Role of oxidative stress Uemura, S., Matsushita, H. S., Asagami, T. K., Li, W., Lee, K. H., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2000: 174–74
  • Metformin attenuates plasma asymmetric dimethylarginine and monocyte adhesion in type 2 diabetes Asagami, T., Stuehlinger, M. C., Li, W., Abbasi, F. A., Tsao, P. S., Cooke, J. P., Reaven, G. M. LIPPINCOTT WILLIAMS & WILKINS. 2000: 232–32
  • Regulated expression of endothelial cell-derived lipase BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hirata, K., Ishida, T., Matsushita, H., Tsao, P. S., Quertermous, T. 2000; 272 (1): 90-93


    A lipoprotein lipase-like gene was recently cloned from endothelial cells. In vitro functional experiments have suggested that this endothelial-derived lipase (EDL) has phospholipase activity, and preliminary in vivo studies have suggested a role in the regulation of high-density lipoprotein metabolism. To investigate local control of lipase activity and lipid metabolism in the blood vessel wall, we have examined the regulation of EDL expression in cultured human umbilical vein and coronary artery endothelial cells. EDL mRNA levels were upregulated in both cell types by inflammatory cytokines implicated in vascular disease etiology, including TNF-alpha and IL-1beta. In addition, both fluid shear stress and cyclic stretch were found to increase the EDL mRNA levels in these cultured cells. This highly regulated expression of EDL in vascular endothelial cells suggests that this recently identified lipase is intricately involved in modulating vessel wall lipid metabolism and may play a role in vascular diseases such as atherosclerosis.

    View details for Web of Science ID 000087378900015

    View details for PubMedID 10872808

  • Asymmetric dimethylarginine increases mononuclear cell adhesiveness in hypercholesterolemic humans ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Chan, J. R., Boger, R. H., Bode-Boger, S. M., Tangphao, O., Tsao, P. S., Blaschke, T. F., Cooke, J. P. 2000; 20 (4): 1040-1046


    Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is elevated in hypercholesterolemia. This study was designed to determine the role of ADMA in the increased mononuclear cell adhesiveness observed in human hypercholesterolemia. In patient studies, plasma ADMA levels were determined by high-performance liquid chromatography. Functional mononuclear leukocyte adhesion assays were performed in parallel, and flow cytometry was used to characterize bound monocytes and T lymphocytes. Hypercholesterolemic patients were then placed on an oral L-arginine regimen of 14 or 21 g/d and studied over 12 weeks. In cell culture studies, bovine aortic endothelial cells were incubated with varied concentrations of ADMA. Monocytoid cells were cocultured with these bovine aortic endothelial cells, and their adhesiveness was assessed by use of a binding assay. Flow cytometry was used to quantify adhesion molecule expression. Plasma ADMA levels and adhesiveness of mononuclear cells (specifically, monocytes and T lymphocytes) were elevated in hypercholesterolemic patients. Adhesiveness was inversely correlated with the plasma L-arginine/ADMA ratio. Oral administration of L-arginine normalized plasma L-arginine/ADMA ratios and attenuated monocyte and T-lymphocyte adhesiveness. ADMA had no direct effect on the adhesiveness of mononuclear cells. However, monocytes became hyperadhesive when cocultured with ADMA-exposed endothelial cells. In human hypercholesterolemia, the plasma L-arginine/ADMA ratio is inversely correlated with mononuclear cell adhesiveness. Restoration of the L-arginine/ADMA ratio to control levels normalizes mononuclear cell adhesiveness. Our studies suggest that the elaboration of endothelium-derived nitric oxide affects the behavior of circulating T lymphocytes and monocytes.

    View details for Web of Science ID 000086468300020

    View details for PubMedID 10764670

  • Nicotine is an agent of angiogenesis: Role of nitric oxide and prostacyclin Heeschen, C., Ho, H. K., Jang, J., Kaji, S., Yang, P., Hu, B. S., Tsao, P., Cooke, J. P. ELSEVIER SCIENCE INC. 2000: 545A–546A
  • Flow-responsive remodeling after angioplasty is dependent on oxidant stress Ward, M. R., Tsao, P. S., Herity, N. A., Cooke, J. P., Yeung, A. C. LIPPINCOTT WILLIAMS & WILKINS. 1999: 699–99
  • Gene transfer of nitric oxide synthase - Effects on endothelial biology JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Niebauer, J., Dulak, J., Chan, J. R., Tsao, P. S., Cooke, J. P. 1999; 34 (4): 1201-1207


    The purpose of the study was to investigate the role of nitric oxide (NO) in monocyte-endothelial interaction by augmenting NO release via transfection of human endothelial cells (ECs) with EC NO synthase (eNOS) DNA.Enhancement of NO synthesis by L-arginine or shear stress reduces endothelial adhesiveness for monocytes and inhibits atherogenesis. To elucidate further the underlying mechanism, we augmented NO synthase expression by transfection of human EC.Liposome-mediated transfection of EC was performed with a plasmid construct containing the gene encoding eNOS. Expression of eNOS was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Endothelial cells were exposed to human monocytoid cells, and adherent cells were quantitated using a computer-assisted program. Nitric oxide was measured by chemiluminescence.The NO levels were not different in EC that were either not transfected, transfected with beta-gal or liposomes only. The nitric oxide synthase (NOS) transfection increased NO release by +60% (n = 6), which increased further when EC were stimulated by shear stress (24 h) by +137% (n = 5) as compared with untransfected, unstimulated EC (both p < 0.05). The RT-PCR revealed diminished monocyte chemotactic protein-1 (MCP-1) expression in eNOS transfected EC. There was an inverse relation between NO levels and monocyte binding (r = -0.5669, p < 0.002). Stimulation of EC with tumor necrosis factor-alpha (TNF-alpha; 250 U/ml) led to a decrease in NO synthesis, and an increase in monocyte binding. Cells transfected with NOS were resistant to both effects of TNF-alpha.Endothelial cells transfected with eNOS synthesize an increased amount of NO; this is associated with diminished MCP-1 expression and monocyte-endothelial binding. The reduction in monocyte-endothelial binding persists even after cytokine stimulation.

    View details for Web of Science ID 000082936900039

    View details for PubMedID 10520813

  • Enhanced monocyte adherence to thoracic aortae from rats with two forms of experimental hypertension AMERICAN JOURNAL OF HYPERTENSION Asagami, T., Reaven, G. M., Tsao, P. S. 1999; 12 (9): 890-893


    To extend our previous observation that thoracic aortae from rats with spontaneous hypertension (SHR) bind monocytoid cells with enhanced avidity, we isolated thoracic aortae from two different forms of rodent hypertension: Dahl salt-sensitive (Dahl-S) rats fed a high salt diet and Sprague-Dawley (S-D) rats fed a fructose-enriched diet. Blood pressure was determined 14 days after feeding normal chow or chow containing 8% NaCl to Dahl-S and Dahl salt-resistant (Dahl-R) rats, and either chow or a fructose-enriched diet to S-D and Fischer 344 (F-344) rats. Blood pressure was similar in Dahl-S and Dahl-R rats on the chow diet, but higher in Dahl-S rats in response to the 8% NaCl diet (188 +/- 7 v 137 +/- 3 mm Hg, P < .001). Blood pressure also increased when S-D rats consumed fructose as compared with chow (149 +/- 4 v 128 +/-2, P < .05), whereas blood pressure did not change with diet in F-344. Thoracic aortae were removed from rats in each experimental group, and their ability to bind murine monocytoid cells quantified. Measurements of monocyte binding were performed on one experimental and one control rat simultaneously, and results presented as the ratio of cells bound by thoracic aortae from the experimental compared with the control rat. With this approach, the ratio of monocyte binding (8% NaCl/chow) was increased in Dahl-S versus Dahl-R rats (1.7 +/- 0.1 v 1.3 +/- 0.1, P < .05), as well as in S-D as compared with F-344 rats (1.7 +/- 0.2 v 1.1 +/-0.1, P < .05). These results provide evidence that hypertension in Dahl-S and fructose-fed S-D rats was associated with changes in the endothelium that favor atherogenesis.

    View details for Web of Science ID 000082738300006

    View details for PubMedID 10509546

  • Mononuclear cell adherence to cultured endothelium is enhanced by hypertension and insulin resistance in healthy nondiabetic volunteers CIRCULATION Chen, N. G., Abbasi, F., Lamendola, C., McLaughlin, T., Cooke, J. P., Tsao, P. S., Reaven, G. M. 1999; 100 (9): 940-943


    This study was initiated to compare the adherence to cultured endothelial cells of mononuclear cells isolated from normotensive and hypertensive individuals.Mononuclear cell binding to endothelium was greater in patients with hypertension (32+/-1 versus 25+/-2; P<0.001) than in normal volunteers. There was a significant relationship (r=0.42, P<0. 01) between mononuclear cell binding and mean arterial pressure, independent of differences in age, sex, and body mass index. A significant relationship also existed between insulin resistance (estimated by the steady-state plasma glucose concentration during the insulin suppression test) and mononuclear cell binding in both the normotensive (r=0.86, P<0.001) and hypertensive (r=0.74, P<0. 001) groups. Furthermore, multiple regression analysis demonstrated an independent relationship (P<0.001) between mononuclear cell binding and both steady-state plasma glucose and hypertensive status.These results indicate that both hypertension and insulin resistance lead to changes in mononuclear cells that increase their adherence to cultured endothelial cells.

    View details for Web of Science ID 000082353000009

    View details for PubMedID 10468524

  • Novel mechanism for endothelial dysfunction - Dysregulation of dimethylarginine dimethylaminohydrolase CIRCULATION Ito, A., Tsao, P. S., Adimoolam, S., Kimoto, M., Ogawa, T., Cooke, J. P. 1999; 99 (24): 3092-3095


    Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). Plasma levels of ADMA are elevated in individuals with hypercholesterolemia or atherosclerosis. We postulated that reduced degradation of ADMA may play a role in the accumulation of ADMA in these individuals. Accordingly, we studied the effects of oxidized LDL (oxLDL) or tumor necrosis factor-alpha (TNF-alpha) on the accumulation of ADMA by transformed human umbilical vein endothelial cells (ECV304) and on the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which degrades ADMA.ECV304 were incubated with or without native LDL (100 micrograms/mL), oxLDL (100 micrograms/mL), or TNF-alpha (250 U/mL) for 48 hours. The concentration of ADMA in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. We assayed DDAH activity by determining L-citrulline formation from ADMA. The addition of oxLDL or TNF-alpha to ECV304 significantly increased the level of ADMA in the conditioned medium. The effect of oxLDL or TNF-alpha was not due to a change in DDAH expression but rather to the reduction of DDAH activity. To determine whether dysregulation of DDAH also occurred in vivo, New Zealand White rabbits were fed normal chow or a high-cholesterol diet. Hypercholesterolemia significantly reduced aortic, renal, and hepatic DDAH activity.These results suggest that the endothelial vasodilator dysfunction observed in hypercholesterolemia may be due to reduced degradation of ADMA, the endogenous inhibitor of NOS.

    View details for Web of Science ID 000080943900003

    View details for PubMedID 10377069

  • Impaired aerobic capacity in hypercholesterolemic mice: partial reversal by exercise training AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Niebauer, J., Maxwell, A. J., Lin, P. S., Tsao, P. S., Kosek, J., Bernstein, D., Cooke, J. P. 1999; 276 (4): H1346-H1354


    The present study assessed whether impaired aerobic capacity previously observed in hypercholesterolemic mice is reversible by exercise training. Seventy-two 8-wk-old female C57BL/6J wild-type (+, n = 42) and apolipoprotein E-deficient (-, n = 30) mice were assigned to the following eight interventions: normal chow, sedentary (E+, n = 17; E-, n = 8) or exercised (E+ex, n = 13; E-ex, n = 7) and high-fat chow, sedentary (E+chol, n = 6; E-chol, n = 8) or exercised (E+chol-ex, n = 6; E-chol-ex, n = 7). Mice were trained on a treadmill 2 x 1 h/day, 6 days/wk, for 4 wk. Cholesterol levels correlated inversely with maximum oxygen uptake (r = -0.35; P < 0. 02), which was blunted in all hypercholesterolemic sedentary groups (all P < 0.05). Maximum oxygen uptake improved in all training groups but failed to match E+ex (all P < 0.05). Vascular reactivity and nitric oxide (NO) synthesis correlated with anaerobic threshold (r = 0.36; P < 0.025) and maximal distance run (r = 0.59; P < 0.007). We conclude that genetically induced hypercholesterolemia impairs aerobic capacity. This adverse impact of hypercholesterolemia on aerobic capacity may be related to its impairment of vascular NO synthesis and/or vascular smooth muscle sensitivity to nitrovasodilators. Aerobic capacity is improved to the same degree by exercise training in normal and genetically hypercholesterolemic mice, although there remains a persistent difference between these groups after training.

    View details for Web of Science ID 000079554200030

    View details for PubMedID 10199861

  • Regression of atherosclerosis - Role of nitric oxide and apoptosis CIRCULATION Wang, B. Y., Ho, H. K., Lin, P. S., Schwarzacher, S. P., Pollman, M. J., Gibbons, G. H., Tsao, P. S., Cooke, J. P. 1999; 99 (9): 1236-1241


    We have recently found that administration of L-arginine to hypercholesterolemic rabbits induces regression of preexisting lesions. Others have previously shown that activation of the L-arginine/nitric oxide (NO) synthase pathway can induce apoptosis of vascular cells in vitro. Accordingly, the current study was designed to determine if dietary supplementation of L-arginine induces apoptosis of intimal lesions and if this effect is mediated through the NO synthase pathway.Male New Zealand White rabbits were fed a 0.5% cholesterol diet for 10 weeks and subsequently placed on 2.5% L-arginine HCl in the drinking water, and the cholesterol diet was continued for 2 weeks, at which time the aortas were harvested for histological studies. L-Arginine treatment increased the number of apoptotic cells (largely macrophages) in the intimal lesions by 3-fold (11.9+/-3.9 vs 3.9+/-1. 4 apoptotic cells/mm2, P<0.01). In subsequent studies, aortas were harvested for ex vivo studies. Aortic segments were incubated in cell culture medium for 4 to 24 hours with modulators of the NO synthase pathway. The tissues were then collected for histological studies and the conditioned medium collected for measurement of nitrogen oxides by chemiluminescence. Addition of sodium nitroprusside (10(-5) mol/L) to the medium caused a time-dependent increase in apoptosis of vascular cells (largely macrophages) in the intimal lesion. L-Arginine (10(-3) mol/L) had an identical effect on apoptosis, which was associated with an increase in nitrogen oxides released into the medium. These effects were not mimicked by D-arginine, and they were antagonized by the NO synthase inhibitor L-nitro-arginine (10(-4) mol/L). The effect of L-arginine was not influenced by an antagonist of cGMP-dependent protein kinase, nor was the effect mimicked by the agonist of protein kinase G or 8-BR cGMP.These results indicate that supplemental L-arginine induces apoptosis of macrophages in intimal lesions by its metabolism to NO, which acts through a cGMP-independent pathway. These studies are consistent with our previous observation that supplementation of dietary arginine induces regression of atheroma in this animal model. These studies provide a rationale for further investigation of the therapeutic potential of manipulating the NO synthase pathway in atherosclerosis.

    View details for Web of Science ID 000078978500016

    View details for PubMedID 10069793

  • Asymmetric dimethylarginine (ADMA): A novel risk factor for endothelial dysfunction - Its role in hypercholesterolemia CIRCULATION Boger, R. H., Bode-Boger, S. M., Szuba, A., Tsao, P. S., Chan, J. R., Tangphao, O., Blaschke, T. F., Cooke, J. P. 1998; 98 (18): 1842-1847


    Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase. Because endothelial NO elaboration is impaired in hypercholesterolemia, we investigated whether plasma concentrations of ADMA are elevated in young, clinically asymptomatic hypercholesterolemic adults. We further studied whether such elevation of ADMA levels was correlated with impaired endothelium-dependent, NO-mediated vasodilation and urinary nitrate excretion. In a randomized, double-blind, placebo-controlled study, we investigated whether these changes could be reversed with exogenous L-arginine.We measured plasma levels of L-arginine, ADMA, and symmetrical dimethylarginine (SDMA) by high-performance liquid chromatography in 49 hypercholesterolemic (HC) and 31 normocholesterolemic (NC) humans. In 8 HC subjects, endothelium-dependent forearm vasodilation was assessed before and after an intravenous infusion of L-arginine or placebo and compared with 8 NC control subjects. ADMA levels were significantly elevated by >100% (2.17+/-0.15 versus 1.03+/-0.09 micromol/L; P<0.05) in HC subjects compared with NC adults. L-Arginine levels were similar, resulting in a significantly decreased L-arginine/ADMA ratio in HC subjects (27.7+/-2.4 versus 55. 7+/-5.4; P<0.05). In 8 HC subjects, intravenous infusion of L-arginine significantly increased the L-arginine/ADMA ratio and normalized endothelium-dependent vasodilation and urinary nitrate excretion. ADMA levels were inversely correlated with endothelium-mediated vasodilation (R=0.762, P<0.01) and urinary nitrate excretion rates (R=0.534, P<0.01).We find that ADMA is elevated in young HC individuals. Elevation of ADMA is associated with impaired endothelium-dependent vasodilation and reduced urinary nitrate excretion. This abnormality is reversed by administration of L-arginine. ADMA may be a novel risk factor for endothelial dysfunction in humans.

    View details for Web of Science ID 000076718900005

    View details for PubMedID 9799202

  • Adherence of mononuclear cells to endothelium is increased in patients with hypertension Chen, N. G., Abbasi, F. A., CHEN, Y. D., Cooke, J. P., Tsao, P. S., Reaven, G. M. LIPPINCOTT WILLIAMS & WILKINS. 1998: 376–76
  • Modulation of the nitric oxide synthase pathway in atherosclerosis EXPERIMENTAL PHYSIOLOGY Maxwell, A. J., Tsao, P. S., Cooke, J. P. 1998; 83 (5): 573-584

    View details for Web of Science ID 000076498500001

    View details for PubMedID 9793778

  • Interaction of diabetes and hypertension on determinants of endothelial adhesiveness ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Tsao, P. S., Niebauer, J., Buitrago, R., Lin, P. S., Wang, B. Y., Cooke, J. P., CHEN, Y. D., Reaven, G. M. 1998; 18 (6): 947-953


    Epidemiological studies have established that diabetes mellitus and hypertension are independent risk factors for atherosclerosis. One of the earliest abnormalities seen in atherogenesis is enhanced monocyte adherence to the endothelium. The mechanisms by which diabetes mellitus or hypertension enhances monocyte-endothelial cell interactions are incompletely characterized. It is not known whether there are additive interactions between these risk factors on endothelial adhesiveness for monocytes. Male Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were fed a normal or fructose-enriched diet. In some cases, animals were injected with streptozotocin (35 mg/kg body weight) to induce diabetes. After 2 weeks, plasma was drawn for biochemical measurements, and thoracic aortas were harvested, opened longitudinally, and exposed to fluorescently labeled mouse monocytoid cells (WEHI 78/24, 2 x 10(6)/mL) for 30 minutes on a rocking platform. Adherent cells were counted by epifluorescence microscopy. WEHI 78/24 binding to aortic segments from SHR animals was elevated compared with segments from WKYs. Fructose feeding alone had no effect on endothelial adhesiveness. When WKYs were made hyperglycemic by STZ injection, monocyte binding was 160% of the control value. Elevated monocyte binding was also observed in aortas derived from SHR animals injected with STZ, indicating an additive effect of hypertension and hyperglycemia. To determine whether alterations in oxidative state played a role in the endothelial adhesiveness, aortic segments were exposed to lucigenin (250 micromol/L) for measurement of superoxide anion. Aortic segments from SHR elaborated 120% more superoxide anion than did controls. Elevated free-radical production was also observed in aortas from diabetic WKYs. Furthermore, thoracic aortas derived from diabetic SHR animals elaborated more superoxide anion than did any of the other groups (374%, P<0.05). Immunohistochemical staining for monocyte chemotactic protein-1 demonstrated increased expression in aortas isolated from diabetic WKY and SHR compared with control vessels. These studies demonstrate that both diabetes and hypertension lead to increased monocyte adherence to the endothelium. This abnormality is associated with increased vascular superoxide production and monocyte chemotactic protein-1 expression. Furthermore, there appears to be an additive interaction between hyperglycemia and hypertension in their effects on endothelial adhesiveness and its determinants.

    View details for Web of Science ID 000074175500014

    View details for PubMedID 9633936

  • Effect of cyclosporine on chronic graft vascular disease in a rat cardiac isograft model International Congress on Immunosuppression Teranishi, K., Poston, R. S., Tsao, P. S., Asagami, T., Cooke, J. P., Reitz, B. A., Robbins, R. C. ELSEVIER SCIENCE INC. 1998: 1012–13

    View details for Web of Science ID 000074150800034

    View details for PubMedID 9636409

  • Protein kinase C-epsilon mediates glucose-induced superoxide production and MCP-1 expression in endothelial cells Tsao, P. S., Heidary, S., Wang, A., Chan, J. R., Reaven, G. M., Cooke, J. P. FEDERATION AMER SOC EXP BIOL. 1998: A88–A88
  • Endothelial alterations in hypercholesterolemia: More than simply vasodilator dysfunction 1st Meeting of the International Scientific Faculty on Endothelial Function Tsao, P. S., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 1998: S48–S53


    Occlusive vascular disease begins with an alteration of the endothelium, which is characterized by a decrease in nitric oxide (NO) activity. Endogenous NO inhibits many key processes in atherogenesis, including monocyte adherence, platelet activation, and smooth muscle proliferation. The mechanism by which NO activity is reduced in hypercholesterolemia and in other metabolic disorders associated with atherogenesis appears to be multifactorial. It includes increased production of oxygen-derived free radicals, alterations in NO synthase, and the accumulation of endogenous inhibitors (ADMA) of NO synthase. Plasma concentrations of ADMA are elevated in hypercholesterolemic humans. Elevated ADMA concentrations are associated with impaired endothelium-dependent, NO-mediated vasodilatation and reduced urinary nitrate exertion. These effects of ADMA are counteracted by administration of the NO precursor L-arginine. It is likely that basic insights regarding the mechanisms of endothelial dysfunction will lead to new therapeutic strategies for atherosclerosis.

    View details for Web of Science ID 000077892100009

    View details for PubMedID 9883748

  • Adhesiveness of mononuclear cells in hypercholesterolemic humans is normalized by dietary L-arginine ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Theilmeier, G., Chan, J. R., Zalpour, C., Anderson, B., Wang, B. Y., Wolf, A., Tsao, P. S., Cooke, J. P. 1997; 17 (12): 3557-3564


    Hypercholesterolemia reduces vascular nitric oxide (NO) activity. This dysfunction may promote endothelial monocyte interaction, as NO is a potent inhibitor of cell adhesion. We have previously shown that in hypercholesterolemic (HC) rabbits, chronic oral supplementation of L-arginine (Arg) restores NO activity and inhibits monocyte-endothelial cell interaction, in association with a reduction in atherogenesis. We hypothesized that enhancement of endothelial NO activity in HC humans would reduce monocyte adhesiveness. We used a functional binding assay to assess the adhesiveness of human mononuclear cells (MNCs) ex vivo to determine the effects of hypercholesterolemia and L-arginine administration. MNCs from HC subjects adhered in greater numbers (50% more cells per high-power field; P < .0001) than cells derived from normocholesterolemic (NC) subjects. To determine whether enhancement of endogenous NO activity could inhibit mononuclear cell adhesiveness, in a double-blinded placebo-controlled study, oral arginine HCl (8.4 g/d) was administered to HC subjects. Over a course of 2 weeks, this treatment abolished the increased adhesiveness of HC MNCs (160 +/- 11% versus 104 +/- 5%; before and after 2 weeks of Arg treatment; results expressed as a percentage of the binding values obtained using cells derived from paired NC individuals). By contrast, MNC adhesion remained significantly elevated in placebo-treated HC subjects. To examine whether endothelium-derived NO could act as a paracrine modulator of monocyte behavior, monocytes were exposed to NO donors or cocultered in the presence of endothelial cells exposed to antagonists of NO synthase in the presence or absence of L-arginine. NO donors inhibited monocyte adhesiveness. Furthermore, the adhesiveness of monocytes cocultured with endothelial cells was increased by antagonists of NO synthase; this effect was reversed by L-arginine. This study shows that the adhesiveness of human MNCs is increased by hypercholesterolemia. The increase in adhesiveness was reversed in vivo by administration of the NO precursor L-arginine. NO donors or endothelium-derived NO inhibits the adhesiveness of monocytes in vitro, supporting the hypothesis that the effects of L-arginine are mediated by NO.

    View details for Web of Science ID 000072212600025

    View details for PubMedID 9437205

  • Anti-CD43 inhibits monocyte-endothelial adhesion in inflammation and atherogenesis BLOOD McEvoy, L. M., JUTILA, M. A., Tsao, P. S., Cooke, J. P., BUTCHER, E. C. 1997; 90 (9): 3587-3594


    Recruitment of blood monocytes into tissues is a central event in the inflammatory response and in atherogenesis. The mechanisms leading to monocyte adhesion and migration through endothelium are not completely defined. We recently reported that MAb L11, against the leukocyte sialomucin CD43, blocks T-lymphocyte binding to lymph node and Peyer's patch high endothelial venules (HEV) and inhibits T-cell extravasation from the blood into organized secondary lymphoid tissues. We have now assessed the ability of L11 to inhibit monocyte-endothelial (EC) interactions and trafficking. L11 blocks binding of WEHI78/24 cells, a murine monocytoid cell line, to inflamed lymph node HEV and inhibits recruitment of monocytes and neutrophils to thioglycollate-inflamed peritoneum. Because monocyte adhesion to the endothelium and diapedesis in lesion-prone regions of the vasculature is among the earliest events in atherogenesis, leading to formation of lipid-laden foam cells, the ability of L11 to block monocyte recognition of aortic endothelial cells was assessed in a novel ex vivo assay of monocyte binding to intact rabbit aortic endothelium. Cholesterol feeding of rabbits induces enhanced aortic adhesiveness for monocytes and WEHI78/24 monocytoid cells, and this adhesion is inhibited by L11. The inhibitory effect of L11 is additive with that of a cocktail of anti-L-selectin and anti-alpha4 and beta2 integrin monoclonal antibodies. Thus, CD43 represents a novel target for manipulation of monocyte recruitment in inflammation and atherogenesis.

    View details for Web of Science ID A1997YD10800034

    View details for PubMedID 9345042

  • Adherence of mononuclear cells to endothelium in vitro is increased in patients with NIDDM DIABETES CARE Carantoni, M., Abbasi, F., Chu, L., CHEN, Y. D., Reaven, G. M., Tsao, P. S., Varasteh, B., Cooke, J. P. 1997; 20 (9): 1462-1465


    To compare the binding to cultured endothelial cells of mononuclear cells isolated from healthy volunteers and patients with NIDDM.Mononuclear cells were isolated from healthy volunteers (n = 11) and patients with NIDDM (n = 14) and incubated with ECV 304 cells, a human umbilical endothelial cell-derived transformed cell line. Following a period of incubation, the adherence of mononuclear cells to endothelial cells was determined.Adherence of mononuclear cells from patients with NIDDM was significantly greater (P < 0.05) than that of cells isolated from the healthy volunteers, and this difference persisted when adjusted for age, sex, and degree of obesity. Mononuclear cell binding to ECV 304 cells correlated significantly with fasting plasma glucose (r = 0.52, P < 0.01), insulin (r = 0.51, P < 0.01), triglyceride (r = 0.54, P < 0.01), and VLDL (r = 0.54, P < 0.01) and HDL cholesterol (r = -0.45, P < 0.05) levels, but not with either total or LDL cholesterol levels or blood pressure.Since the adherence of mononuclear cells to the endothelium represents the earliest step in atherogenesis, the observation that mononuclear cells from patients with NIDDM bind more avidly to cultured endothelial cells may help explain why accelerated atherosclerosis occurs in patients with NIDDM. The metabolic abnormality, or abnormalities, present in patients with NIDDM that is responsible for the enhanced adhesiveness of mononuclear cells requires further examination.

    View details for Web of Science ID A1997XT09100023

    View details for PubMedID 9283798

  • Nitric oxide regulates monocyte chemotactic protein-1 CIRCULATION Tsao, P. S., Wang, B. Y., Buitrago, R., Shyy, J. Y., Cooke, J. P. 1997; 96 (3): 934-940


    Monocyte chemotactic protein-1 (MCP-1) is a 76-amino-acid chemokine thought to be the major chemotactic factor for monocytes. We and others have demonstrated that NO inhibits monocyte-endothelial cell interactions and atherogenesis. We hypothesize that the antiatherogenic effect of NO may be due in part to its inhibition of MCP-1 expression.Smooth muscle cells (SMCs) were isolated from normal rabbit aortas by the explant method. Cells were then exposed to LPS (10 microg/mL), native LDL, or oxidized LDL (30 microg/mL) for 6 hours. The expression of MCP-1 in SMCs and chemotactic activity in the conditioned medium were induced by lipopolysaccharide (LPS) or by oxidized LDL but not native LDL. The induction of MCP-1 by cytokines or oxidized lipoproteins was associated with an increased generation of superoxide anion by the SMCs and increased activity of the transcriptional protein nuclear factor-kappaB (NFkappaB). The induced expression of MCP-1 and activation of NFkappaB were reduced by previous exposure of the SMCs to the NO donor DETA-NONOate (100 micromol/L) (P<.05). To determine whether NO exerted its effect at a transcriptional level, SMCs and COS cells were transfected with a 400-bp fragment of the MCP-1 promoter. Promoter activity was enhanced by oxidized LDL, and LPS was inhibited by DETA-NO. Nuclear run-on assays confirmed that the effect of NO occurred at a transcriptional level. To investigate the role of endogenous NO in the regulation of MCP-1 in vivo, New Zealand White rabbits were fed normal chow, normal chow plus nitro-L-arginine (LNA), high-cholesterol diet (Chol), or high-cholesterol diet supplemented with L-arginine (Arg). After 2 weeks, thoracic aortas were harvested and total RNA was isolated. Northern analysis using full-length MCP-1 cDNA demonstrated increased expression in Chol and LNA aortas; this expression was decreased in aortas from Arg animals.These studies indicate that the antiatherogenic effect of NO may be mediated in part by its inhibition of MCP-1 expression.

    View details for Web of Science ID A1997XP13000035

    View details for PubMedID 9264504

  • Novel vascular molecule involved in monocyte adhesion to aortic endothelium in models of atherogenesis JOURNAL OF EXPERIMENTAL MEDICINE McEvoy, L. M., Sun, H. L., Tsao, P. S., Cooke, J. P., Berliner, J. A., BUTCHER, E. C. 1997; 185 (12): 2069-2077


    Adhesion of monocytes to the endothelium in lesion-prone areas is one of the earliest events in fatty streak formation leading to atherogenesis. The molecular basis of increased monocyte adhesion is not fully characterized. We have identified a novel vascular monocyte adhesion-associated protein, VMAP-1, that plays a role in adhesion of monocytes to activated endothelium. Originally selected for its ability to block binding of a mouse monocyte-like cell line (WEHI78/24) to cytokine- or LPS-stimulated cultured mouse endothelial cells in vitro, antiVMAP-1 mAb LM151 cross-reacts with rabbit endothelium and blocks binding of human monocytes to cultured rabbit aortic endothelial cells stimulated with minimally modified low density lipoprotein, thought to be a physiologically relevant atherogenic stimulus. Most importantly, LM151 prevents adhesion of normal monocytes and monocytoid cells to intact aortic endothelium from cholesterol-fed rabbits in an ex vivo assay. VMAP-1 is a 50-kD protein. Immunohistology of vessels reveals focal constitutive expression in aorta and other large vessels. VMAP-1 is thus a novel vascular adhesion-associated protein that appears to play a critical role in monocyte adhesion to aortic endothelial cells in atherogenesis in vivo.

    View details for Web of Science ID A1997XF79100005

    View details for PubMedID 9182678

    View details for PubMedCentralID PMC2196357

  • Cell cycle inhibition preserves endothelial function in genetically engineered rabbit vein grafts JOURNAL OF CLINICAL INVESTIGATION Mann, M. J., Gibbons, G. H., Tsao, P. S., VONDERLEYEN, H. E., Cooke, J. P., Buitrago, R., Kernoff, R., Dzau, V. J. 1997; 99 (6): 1295-1301


    We have recently shown that ex vivo gene therapy of rabbit autologous vein grafts with antisense oligodeoxynucleotides (AS ODN) blocking cell cycle regulatory gene expression inhibits not only neointimal hyperplasia, but also diet-induced, accelerated graft atherosclerosis. We observed that these grafts remained free of macrophage invasion and foam cell deposition. Since endothelial dysfunction plays an important role in vascular disease, the current study examined the effect of this genetic engineering strategy on graft endothelial function and its potential relationship to the engineered vessels' resistance to atherosclerosis. Rabbit vein grafts transfected with AS ODN against proliferating cell nuclear antigen (PCNA) and cell division cycle 2 (cdc2) kinase elaborated significantly more nitric oxide and exhibited greater vasorelaxation to both calcium ionophore and acetylcholine than did untreated or control ODN-treated grafts. This preservation of endothelial function was associated with a reduction in superoxide radical generation, vascular cell adhesion molecule-1 (VCAM-1) expression, and monocyte binding activity in grafts in both normal and hypercholesterolemic rabbits. Our data demonstrate that AS ODN arrest of vascular cell cycle progression results in the preservation of normal endothelial phenotype and function, thereby influencing the biology of the vessel wall towards a reduction of its susceptibility to occlusive disease.

    View details for Web of Science ID A1997WQ61300023

    View details for PubMedID 9077539

    View details for PubMedCentralID PMC507945

  • Dietary L-arginine supplementation normalizes platelet aggregation in hypercholesterolemic humans JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Wolf, A., Zalpour, C., Theilmeier, G., Wang, B. Y., Ma, A., Anderson, B., Tsao, P. S., Cooke, J. P. 1997; 29 (3): 479-485


    The present study was designed to test the hypothesis that long-term dietary supplementation with the nitric oxide precursor L-arginine would enhance vascular or platelet-derived nitric oxide activity, or both, and thereby inhibit platelet reactivity in hypercholesterolemic humans.We have shown that reduced vascular activity of nitric oxide in hypercholesterolemic rabbits can be restored by L-arginine supplementation. The improvement in nitric oxide activity is associated with an inhibition of platelet aggregation ex vivo. This effect is most likely due to increased elaboration of endothelium- or platelet-derived nitric oxide, or both, because the inhibition of platelet reactivity was associated with elevation of intraplatelet cyclic guanosine monophosphate and was reversed by the nitric oxide synthase antagonist N-methyl-arginine.In a double-blinded, randomized, placebo-controlled trial, hypercholesterolemic patients were assigned to L-arginine hydrochloride, 8.4 g/day orally, or placebo for 2 weeks. Platelet-rich plasma was obtained for aggregometry induced by collagen (1 to 10 micrograms/ml) at four points: baseline, after 2 weeks of treatment, after a 2-week washout and after a long-term washout of 16 weeks on average. Aggregation was quantified by light transmittance and expressed as a percent transmittance observed with platelet-poor plasma.Compared with normocholesterolemic control subjects, platelets from hypercholesterolemic subjects stimulated with 5 micrograms/ml of collagen showed increased aggregability (68.6% in hypercholesterolemic patients vs. 54.5% in normocholesterolemic control subjects, p < or = 0.02). After 2 weeks of treatment with L-arginine (but not placebo), platelet reactivity was modestly reduced; this effect persisted for 2 weeks after discontinuation of arginine (52.6% in arginine-treated patients vs. 65.1% in normocholesterolemic control subjects, p = 0.07). After 18 weeks (i.e., 16 weeks after discontinuing arginine treatment), the platelets of hypercholesterolemic patients once again became hyperaggregable, and the extent of platelet aggregation was significantly increased compared with the 4-week point (73.6% after vs. 52.6% during arginine treatment, p < 0.01). No significant change in platelet reactivity was seen in placebo-treated hypercholesterolemic patients throughout the study. L-Arginine treatment was well tolerated without side effects.This double-blinded, placebo-controlled study demonstrates that dietary supplementation with L-arginine can modestly attenuate the increased platelet reactivity seen in hypercholesterolemic patients. The data are consistent with our previous studies in hypercholesterolemic animals, demonstrating that L-arginine restores endogenous nitric oxide activity and inhibits platelet aggregation. Enhancement of endogenous nitric oxide activity is a potential novel therapeutic strategy worthy of further study.

    View details for Web of Science ID A1997WL49000002

    View details for PubMedID 9060881

  • The role of endothelium-derived nitric oxide in atherosclerosis European Congress of Angiology, 11th Meeting of the European Chapter (EUROCHAP 97) Cooke, J. P., Tsao, P. S. ELSEVIER SCIENCE BV. 1997: 3–14
  • Arginine restores nitric oxide activity and inhibits monocyte accumulation after vascular injury in hypercholesterolemic rabbits JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Wang, B. Y., Candipan, R. C., Arjomandi, M., HSIUN, P. T., Tsao, P. S., Cooke, J. P. 1996; 28 (6): 1573-1579


    This study sought to determine whether the alterations in vascular function and structure after balloon injury in hypercholesterolemic rabbits could be inhibited by dietary arginine.Administration of arginine (the nitric oxide [NO] precursor) restores vascular NO activity in hypercholesterolemic animals. We and other investigators have shown that enhancement of vascular NO activity can inhibit myointimal hyperplasia after vascular injury in normocholesterolemic animals.Twenty-eight New Zealand White rabbits received either normal rabbit chow, 0.5% cholesterol diet or 0.5% cholesterol diet plus L-arginine hydrochloride (2.25% wt/vol) in the drinking water. After 6 weeks of dietary intervention, the left iliac artery of each animal was subjected to a balloon injury. Four weeks later, the iliac arteries were harvested for vascular reactivity studies and immunohistochemical analysis.Vascular injury induced intimal thickening that was largely composed of vascular smooth muscle cells and extracellular matrix. In the setting of hypercholesterolemia, vascular injury induced an exuberant myointimal lesion that was augmented by the accumulation of lipid-laden macrophages. Dietary arginine reduced intimal thickening in the injured vessels of hypercholes-terolemic animals and substantially inhibited the accumulation of macrophages in the lesion (from 28% to 5% of the lesion area, p < 0.001).We report that lesions induced by vascular injury in hypercholesterolemic animals are markedly reduced by oral administration of arginine. Moreover, we find that the nature of the lesion is altered, with a striking reduction in the percentage of macrophages comprising the lesion.

    View details for Web of Science ID A1996VT31000020

    View details for PubMedID 8917274

  • Intravascular stenting changes vascular distensibility and upregulates nitric oxide activity Schwarzacher, S. P., Niebauer, J., Hayase, M., Uren, N. G., Tsao, P., Yock, P. G., Yeung, A. C. LIPPINCOTT WILLIAMS & WILKINS. 1996: 1526–26
  • Nitric oxide induces regression: Role of apoptosis Wang, B. Y., Lin, P. S., Tsao, P. S., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 1996: 900–900
  • Fluid flow inhibits endothelial adhesiveness - Nitric oxide and transcriptional regulation of VCAM-1 CIRCULATION Tsao, P. S., Buitrago, R., Chan, J. R., Cooke, J. P. 1996; 94 (7): 1682-1689


    In the arterial tree, regions exposed to reduced shear stress (low and/or disturbed flow) are predisposed to atherogenesis. Fluid flow is a potent stimulus for the release of endothelium-derived nitric oxide (NO). Because NO inhibits monocyte-endothelial cell interaction, we speculated that the effects of flow in inhibiting atherogenesis might be mediated in part by NO.Confluent monolayers of human aortic endothelial cells were exposed to static or fluid flow conditions for 4 hours. The medium was replaced, and cells were then incubated with native LDL (50 micrograms/mL), oxidized LDL (30 micrograms/mL), or lipopolysaccharide (LPS) (10 ng/mL)+tumor necrosis factor-alpha (TNF-alpha) (10 U/mL) for an additional 4 hours. Functional binding assays using THP-1 monocytes were then performed. Superoxide production by human aortic endothelial cells was monitored by lucigenin chemiluminescence, and expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 were quantified by flow cytometry. Whereas native LDL had little effect, incubation with either oxidized LDL or LPS/TNF-alpha significantly increased superoxide production, nuclear factor-kappa B activity, VCAM-1 expression, and endothelial adhesiveness for monocytes. Previous exposure to fluid flow inhibited these sequelae of exposure to cytokines or oxidized lipoprotein. The effect of fluid flow appears to be due in part to shear-induced release of NO, because coincubation with nitro-L-arginine completely abolished these effects of flow. Furthermore, the NO donor PAPA-NONO-ate and 8-Br-cGMP (but not 8-Br-cAMP) mimicked the effects of flow.Previous exposure to fluid flow decreased cytokine- or lipoprotein-stimulated endothelial cell superoxide production, VCAM-1 expression, and monocyte binding; the effects of flow appear to be due to NO. Flow-mediated NO-dependent regulation of oxidant-responsive transcription may influence the site of a lesion.

    View details for Web of Science ID A1996VJ97900030

    View details for PubMedID 8840861

  • Hypertension-enhanced monocyte adhesion in experimental atherosclerosis JOURNAL OF VASCULAR SURGERY Tropea, B. I., Huie, P., Cooke, J. P., Tsao, P. S., Sibley, R. K., Zarins, C. K. 1996; 23 (4): 596-605


    Hypertension is a known clinical risk factor for atherosclerosis. In experimental atherosclerosis, monocyte adhesion to the endothelial surface is enhanced and is considered to be an important early stage in plaque formation. We tested the hypothesis that hypertension enhances monocyte adhesion in experimental atherosclerosis.Twenty-two New Zealand White rabbits were fed an atherogenic diet for 3 weeks to induce plaque formation. Aortic coarctation was created in eight rabbits by wrapping a Dacron band around the midportion of the descending thoracic aorta (stenosis group), whereas six rabbits underwent banding without aortic constriction (no stenosis group). Eight rabbits served as nonoperated controls. Monocyte binding to the aortic endothelial surface was counted with epifluorescent microscopy on standard aortic segments proximal and distal to the band. Immunohistochemistry was performed for the following antibodies: VCAM-1, RAM11, CD11b, and factor VIII.Mean blood pressure was 89 +/- 3 mm Hg in the aorta proximal to the stenosis, compared with 64 +/- 4 mm Hg in the no stenosis group and 74 +/- 3 mm Hg in the control group (p < 0.01). The mean aortic blood pressure gradient across the stenosis was 16 +/- 2 mm Hg in the stenosis group, whereas the aortic blood pressure gradient was 0.2 +/- 0.6 mm Hg in the no stenosis group and -0.3 +/- 0.4 mm Hg in the control group (p < 0.001). Monocyte adhesion to the aortic endothelial surface proximal to the stenosis was increased twofold compared with adhesion to the aorta distal to the stenosis and compared with the proximal aorta in the control group (p < 0.02). The proximal-to-distal aortic ratio of monocyte binding was enhanced in the stenosis group (2.2) compared with the no stenosis (0.76) and control (0.83) groups (p < 0.01). The intima area of the aorta proximal to the stenosis was significantly increased compared with the proximal aortas in the no stenosis and control groups (p < 0.01). RAM11, CD11b, and endothelial VCAM-1 expression were enhanced in the hypertensive region proximal to the stenosis.In the hypertensive region in the aorta proximal to the stenosis, monocyte adhesion and endothelial VCAM-1 expression were increased, with intimal thickening and accumulation of macrophages. These findings suggest that hypertension may promote atherosclerotic plaque formation by enhancing monocyte adhesion.

    View details for Web of Science ID A1996UJ12600010

    View details for PubMedID 8627894

  • Expression of inducible nitric oxide synthase fin human heart failure CIRCULATION Haywood, G. A., Tsao, P. S., VONDERLEYEN, H. E., Mann, M. J., Kelling, P. J., Trindade, P. T., Lewis, N. P., Byrne, C. D., Rickenbacher, P. R., Bishopric, N. H., Cooke, J. P., McKenna, W. J., Fowler, M. B. 1996; 93 (6): 1087-1094


    There is increasing evidence that alterations in nitric oxide synthesis are of pathophysiological importance in heart failure. A number of studies have shown altered nitric oxide production by the endothelial constitutive isoform of nitric oxide synthase (NOS), but there is very little information on the role of the inducible isoform.We analyzed inducible NOS (iNOS) expression in ventricular myocardium taken from 11 control subjects (who had died suddenly from noncardiac causes), from 10 donor hearts before implantation, and from 51 patients with heart failure (24 with dilated cardiomyopathy [DCM], 17 with ischemic heart disease [IHD], and 10 with valvular heart disease [VHD]). Reverse transcription-polymerase chain reaction was used to confirm the presence of intact mRNA and to detect expression of iNOS and atrial natriuretic peptide (ANP). ANP was used as a molecular phenotypic marker of ventricular failure. iNOS was expressed in 36 of 51 biopsies (71%) from patients with heart failure and in none of the control patients (P<.0001). iNOS expression could also be detected in 50% of the donor hearts. All samples that expressed iNOS also expressed ANP. iNOS gene expression occurred in 67% of patients with DCM, 59% of patients with IHD, and 100% of patients with VHD. To determine whether iNOS protein was expressed in failing ventricles, immunohistochemistry was performed on three donor hearts and nine failing hearts with iNOS mRNA expression. Staining for iNOS was almost undetectable in the donor myocardium and in control sections, but all failing hearts showed diffuse cytoplasmic staining in cardiac myocytes. Expression of iNOS could be observed in all four chambers. Western blot analysis with the same primary antibody showed a specific positive band for iNOS protein in the heart failure specimens; minimal iNOS protein expression was seen in donor heart samples.iNOS expression occurs in failing human cardiac myocytes and may be involved in the pathophysiology of DCM, IHD, and VHD.

    View details for Web of Science ID A1996UA05300006

    View details for PubMedID 8653828

  • Induction of nitric oxide synthase in the human cardiac allograft is associated with contractile dysfunction of the left ventricle CIRCULATION Lewis, N. P., Tsao, P. S., Rickenbacher, P. R., Xue, C., Johns, R. A., Haywood, G. A., VONDERLEYEN, H., Trindade, P. T., Cooke, J. P., Hunt, S. A., Billingham, M. E., Valantine, H. A., Fowler, M. B. 1996; 93 (4): 720-729


    The mechanisms underlying cardiac contractile dysfunction after transplantation remain poorly defined. Previous work has revealed that inducible nitric oxide synthase (iNOS) is expressed in the rat heterotopic cardiac allograft during rejection; resultant overproduction of nitric oxide (NO) might cause cardiac contractile dysfunction via the negative inotropic and cytotoxic actions of NO. In this investigation, we tested the hypothesis that induction of iNOS may occur and be associated with cardiac allograft contractile dysfunction in humans.We prospectively studied 16 patients in the first year after cardiac transplantation at the time of serial surveillance endomyocardial biopsy. Clinical data, the results of biopsy histology, and echocardiographic and Doppler evaluation of left ventricular systolic and diastolic function were recorded. Total RNA was extracted from biopsy specimens, and mRNA for beta-actin, detected by reverse transcription-polymerase chain reaction (RT-PCR) using human specific primers, was used as a constitutive gene control; iNOS mRNA was similarly detected by RT-PCR using human specific primers. iNOS protein was detected in biopsy frozen sections by immunofluorescence. Myocardial cGMP was measured by radioimmunoassay, and serum nitrogen oxide levels (NOx = NO2 + NO3) were measured by chemiluminescence. iNOS mRNA was detected in allograft myocardium at some point in each patient and in 59 of 123 biopsies (48%) overall. In individual patients, iNOS mRNA expression was episodic and time dependent; the frequency of expression was highest during the first 180 days after transplant (P = .0006). iNOS protein associated with iNOS mRNA was detected by immunofluorescence in cardiac myocytes. iNOS mRNA expression was not related to the ISHLT histological grade of rejection or to serum levels of NOx but was associated with increased levels of myocardial cGMP (P = .01) and with both systolic (P = .024) and diastolic (P = .006) left ventricular contractile dysfunction measured by echocardiography and Doppler.These data support a relation between iNOS mRNA expression and contractile dysfunction in the human cardiac allograft.

    View details for Web of Science ID A1996TV05300015

    View details for PubMedID 8641001

  • Regression or progression - Dependency on vascular nitric oxide ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Candipan, R. C., Wang, B. Y., Buitrago, R., Tsao, P. S., Cooke, J. P. 1996; 16 (1): 44-50


    We have shown that chronic administration of the nitric oxide (NO) precursor L-arginine inhibits atherogenesis in the hypercholesterolemic rabbit. However, the effect of supplemental arginine on preexisting lesions is not known and was the focus of the present study. New Zealand White rabbits received normal chow or 0.5% cholesterol chow for 10 weeks. Subsequently, L-arginine (2.25% in drinking water; ARG group) or vehicle (CHOL group) was administered for an additional 13 weeks, while the high-cholesterol diet was continued. Thoracic aortae were harvested at weeks 10, 14, 18, or 23. Rings of aorta were used to assess NO-dependent vasodilation to acetylcholine. Maximal relaxation to acetylcholine in the CHOL rabbits became progressively attenuated from 53.4% (at week 10) to 17.4% (by week 23). Planimetry of the luminal surface of the aortae from CHOL animals revealed a progressive increase in lesion surface area from 30.3% (at week 10) to 56.5% (by week 23). By contrast, animals in the ARG groups manifested improved endothelium-dependent relaxation associated with a reduction of lesion surface area at 14 and 18 weeks. The arginine-induced improvement in endothelium-dependent relaxation was associated with an increased generation of vascular NO and a reduced generation of vascular superoxide anion. By 23 weeks, 3 of 7 ARG animals had persistent improvement in NO-dependent vasodilation and exhibited a further reduction of lesion surface area tc 5.4%. We conclude that hypercholesterolemia induces a progressive loss of NO-dependent vasodilation associated with progressive intimal lesion formation. Administration of L-arginine to animals with preexisting intimal lesions augments vascular NO elaboration, reduces superoxide anion generation, and is associated with a reduction in lesion surface area. This is the first demonstration that restoration of NO activity can induce regression of preexisting intimal lesions and provides evidence that L-arginine therapy may be of potential clinical benefit.

    View details for Web of Science ID A1996TP12400006

    View details for PubMedID 8548425

  • Felodipine inhibits intimal lesion formation in the hypercholesterolemic rabbit: differential effects on endothelial and monocyte determinants of atherogenesis. Vascular medicine Wang, B. Y., Niebauer, J., Singer, A. H., Tsao, P. S., Cooke, J. P. 1996; 1 (3): 173-179


    The purpose of this study was to determine if the calcium entry antagonist felodipine inhibited intimal lesion formation in hypercholesterolemic rabbits, and to determine if this was due to an effect upon monocyte and/or endothelial determinants of this interaction. Twenty-three male New Zealand White rabbits received the following treatment regimen for 10 weeks: normal chow (NP, n = 3); normal chow with felodipine infusion (NF, n = 6); 0.5% cholesterol chow (CP, n = 7); or 0.5% cholesterol chow and felodipine infusion (CF, n = 7). After 10 weeks blood was collected for biochemical measurements and mononuclear cell binding assays, and thoracic aortae were harvested for vascular reactivity studies and histomorphometry. In the animals receiving normal chow, felodipine did not significantly affect blood pressure, plasma cholesterol levels, binding studies, vascular reactivity, or structure; therefore these animals were analyzed as one group (N). Plasma cholesterol levels were significantly elevated in groups receiving the 0.5% cholesterol diet (N, 29 +/- 3 mg/dl; CP, 1221 +/- 73 mg/dl; CF, 979 +/- 108 mg/dl). High-density lipoprotein cholesterol was not different between the groups (25 +/- 4 vs 23 +/- 4 vs 27 +/- 4 mg/dl; N vs CF vs CP respectively; p = NS). Cholesterol feeding markedly augmented the adhesiveness of mononuclear cells, as demonstrated by a 250% increase in cell binding. Felodipine did not alter the adhesiveness of mononuclear cells in hypercholesterolemic animals. Cholesterol feeding significantly impaired endothelium-dependent relaxations. Endothelium-dependent relaxations were restored by felodipine treatment as reflected by the maximal responses to acetylcholine (40 +/- 7% vs 58 +/- 4% vs 67 +/- 5%; CP vs CF vs N respectively). The improvement in endothelium-dependent relaxation in the felodipine-treated animals was associated with a 2.2-fold reduction in lesion surface area of the thoracic aorta (8.2 +/- 6.3% vs 18.2 +/- 9.5%; CF vs CP; p < 0.01). Moreover, the intima/media ratio reflecting lesion thickness was substantially reduced by felodipine treatment (0.05 +/- 0.02 vs 0.20 +/- 0.07; CF vs CP; p = 0.006). Ex vivo studies revealed that felodipine inhibited the adhesiveness of vascular endothelium, but not mononuclear cells, derived from hypercholesterolemic animals. Low-dose felodipine appears to inhibit monocyte-endothelial interaction, as indicated by a reduction in the formation of lesions in hypercholesterolemic animals. This effect is not due to an alteration in adhesiveness of mononuclear cells. The salutary effect of felodipine is associated with an increase in vascular nitric oxide activity which may reduce endothelial adhesiveness.

    View details for PubMedID 9546935



    Shear stress increases the release of nitric oxide (NO) by endothelial cells (ECs). We and others have provided evidence that endothelium-derived NO inhibits monocyte adhesion to the vessel wall. We therefore hypothesized that previous exposure to shear stress would inhibit endothelial adhesiveness for monocytes by virtue of its effect to increase NO release.Confluent monolayers of bovine aortic endothelial cells, human aortic endothelial cells, or human venous endothelial cells were exposed to laminar fluid flow. Culture media were collected for measurement of NO (by chemiluminescence) and the prostacyclin metabolite 6-keto-prostaglandin F1 alpha. NOx and 6-keto-prostaglandin F1 alpha accumulated in the conditioned medium during laminar fluid flow from 30 minutes to 24 hours in a time-dependent fashion. In another set of studies, ECs previously exposed to flow or to static conditions were washed with Hanks' buffer and exposed to THP-1 cells for 30 minutes. Adherent cells were counted by microscopy. Previous exposure to flow reduced endothelial adhesiveness for monocytes by 50% (P < .05). The effect of flow on endothelial adhesiveness occurred within 30 minutes. This effect was abrogated by nitro-L-arginine (an antagonist of NO synthesis), as well as by tetraethylammonium ion (an antagonist of the flow-activated potassium channel); the effects of these inhibitors were reversed by the NO donor SPM-5185. Although the cyclo-oxygenase inhibitor indomethacin totally inhibited the flow-induced production of prostacyclin by ECs, it minimally affected adherence of THP-1 cells. The early effect of flow on endothelial adhesiveness was not mediated by alterations in the expression of the endothelial adhesion molecules VCAM-1 or ICAM-1 as assessed by fluorescent activated cell sorting.Shear stress alters endothelial adhesiveness for monocytes; at early time points, this effect is largely due to flow-stimulated release of NO and, to a lesser extent, prostacyclin. This effect of flow occurs within 30 minutes and is probably due to alterations in the signal transduction or activation state (rather than the expression) of endothelial adhesion molecules.

    View details for Web of Science ID A1995TJ65500026

    View details for PubMedID 8521574



    Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.

    View details for Web of Science ID A1995QX87600085

    View details for PubMedID 7753833



    We investigated the effect of dietary supplementation of L-arginine (L-Arg), the precursor of endothelial nitric oxide (NO), on endothelium-dependent and endothelium-independent vascular responses, as well as vascular structure, in the abdominal aorta of hypercholesterolemic rabbits. Rabbits were fed (a) normal rabbit chow, (b) 1% cholesterol diet, or (c) 1% cholesterol diet supplemented with 2.25% L-Arg HCl in drinking water. After 10 weeks, the abdominal aorta was harvested for study of vascular reactivity and histomorphometry. L-Arg did not affect serum cholesterol levels. Histomorphometric analysis demonstrated an eightfold reduction in intimal thickening in the abdominal aorta of the arginine-supplemented hypercholesterolemic rabbits. By contrast, the effects on vascular reactivity were subtle. Contraction to norepinephrine (NE) was not altered by hypercholesterolemia or L-Arg. Contraction to acetylcholine (ACh) was increased in hypercholesterolemic animals; this was normalized by dietary arginine supplementation. Relaxation to nitroglycerin (NTG) was not altered by hypercholesterolemia but was attenuated in the arginine-supplemented rabbits. Endothelium-dependent relaxation to ACh was impaired in both hypercholesterolemic groups. Dietary L-Arg has a dramatic antiatherogenic effect in hypercholesterolemic rabbits. This effect is associated with rather slight changes in vascular reactivity that are suggestive of a slight increase in NO elaboration by the endothelium. The discordance between the effects of dietary arginine on vascular structure and reactivity suggests that the antiatherogenic effects of the NO precursor may not be mediated entirely by its effect on the endothelium.

    View details for Web of Science ID A1995QV17000005

    View details for PubMedID 7630149



    Platelets are capable of producing nitric oxide (NO) through the L-arginine-NO synthase pathway. Acute exposure to supraphysiological concentrations of L-arginine in vitro increases the production of NO by platelets and is associated with an increase in platelet cyclic GMP (cGMP) levels and a reduction in platelet aggregation. The purpose of this study was to determine if chronic oral administration of L-arginine decreases platelet aggregation in hypercholesterolemic animals and to determine if this effect is mediated by the metabolism of L-arginine to NO. Male New Zealand White rabbits were fed normal chow (Con), a 1% cholesterol diet (Chol), or a 1% cholesterol diet supplemented with a sixfold enrichment of dietary L-arginine (Arg) or L-methionine (Met). After 10 weeks, cholesterol levels were equally increased in Chol and Arg animals, whereas plasma arginine levels were doubled in the Arg group. There was no difference in maximum aggregation initiated by ADP (100 mumol/L) between washed platelets from Con, Met, and Chol animals, but aggregation of platelets from Arg animals was significantly decreased (P < .05). In aggregating platelets from Arg animals, cGMP levels were significantly higher than the other groups (P < .05). When platelets were incubated ex vivo with the NO synthase inhibitor NG-monomethyl-L-arginine, the effects of dietary L-arginine were reversed. Chronic dietary supplementation of L-arginine decreases platelet aggregation in hypercholesterolemic rabbits. This effect appears to be due to the metabolism of L-arginine to NO.

    View details for Web of Science ID A1994PL39400002

    View details for PubMedID 7918301



    We have shown that chronic administration of the nitric oxide (NO) precursor L-arginine normalizes NO-dependent vasodilation and markedly inhibits atherogenesis in a hypercholesterolemic rabbit model. We hypothesized that this antiatherogenic effect is due to modulation of endothelial adhesiveness by endothelium-derived NO.New Zealand White rabbits were fed normal chow (Cont), a high-cholesterol diet (Chol), a high-cholesterol diet supplemented with L-arginine (Arg), or a normal diet supplemented with the NO synthase antagonist L-nitroarginine (L-NA) for 2 weeks. In additional studies, some animals receiving L-NA were also treated with hydralazine to normalize blood pressure. After 2 weeks, thoracic aortas were harvested, opened longitudinally, and placed in a culture dish with the endothelial surface exposed to medium containing WEHI 78/24 cells, a monocytoid cell line. After incubation with the monocytoid cells for 30 minutes on a rocking platform, the aortic segments were washed repeatedly to remove nonadherent cells and adherent cells counted by epifluorescent microscopy. Monocytoid cell binding to aortic endothelium was significantly increased in Chol (P < .001 versus Cont); binding was markedly reduced in arginine-fed hypercholesterolemic animals (P < .05, Arg versus Chol). Monocytoid cell binding to aortic endothelium was also significantly increased in L-NA (P < .05); hydralazine normalized blood pressure but did not reduce monocytoid cell binding. To confirm that alterations in NO activity modulate endothelial cell-monocyte interaction, the release of nitrogen oxides (NOx) by thoracic aortas was assessed by a chemiluminescent technique. The concentration of NOx in the conditioned medium from segments of Arg thoracic aortas was significantly greater than that from Cont aortas, whereas that from L-NA aortas was significantly less.Hypercholesterolemia enhances the adhesiveness of aortic endothelium for monocytes; this effect is attenuated by dietary L-arginine. Conversely, inhibition of NO synthesis enhances monocyte binding. The results suggest that endothelium-derived NO plays an important role in regulating the endothelial adhesiveness for monocytes. Alterations in NO activity may play a critical role in atherogenesis.

    View details for Web of Science ID A1994NL67500034

    View details for PubMedID 8181143



    This study was designed to test the hypothesis that long-term oral supplementation of dietary L-arginine (to provide a sustained elevation of nitric oxide activity) would inhibit atherogenesis in hypercholesterolemic rabbits, as assessed by histomorphometric measurements.Endothelium-derived nitric oxide inhibits a number of processes that are critical in atherogenesis. Hypercholesterolemia reduces endothelial nitric oxide activity, and we postulate that this may promote atherogenesis. This reduction in nitric oxide activity can be reversed acutely by intravenous infusion of L-arginine, the precursor of nitric oxide. We show that dietary supplementation of L-arginine abrogates the development of coronary atheroma in hypercholesterolemic rabbits.Male New Zealand White rabbits were fed normal rabbit chow, 1% cholesterol chow or 1% cholesterol chow with dietary arginine or methionine supplementation to increase their intake of these amino acids sixfold. After 1 or 10 weeks of dietary intervention, the left main and left anterior descending coronary arteries were harvested for histologic study. Plasma cholesterol measurements were elevated to the same degree in all groups of rabbits receiving the 1% cholesterol diet, whereas plasma arginine levels were doubled in the arginine-treated group. High density lipoprotein (HDL) cholesterol values were not affected by arginine treatment.In rabbits receiving the 1% cholesterol diet, with or without methionine supplementation, light and electron microscopy revealed a marked increase from 1 to 10 weeks in the intimal accumulation of macrophages, associated with an increase in the intimal area of the left main coronary artery. By contrast, in arginine-treated hypercholesterolemic rabbits, there was a near absence of adherent monocytes and tissue macrophages and no progression of intimal thickness from 1 to 10 weeks.Dietary supplements of L-arginine prevent intimal thickening in the coronary arteries of hypercholesterolemic rabbits. This antiatherogenic effect is not due to an alteration in plasma total cholesterol, HDL cholesterol or caloric or nitrogen balance. The data are consistent with the hypothesis that nitric oxide has antiatherogenic properties.

    View details for Web of Science ID A1994NR75200026

    View details for PubMedID 8294700

  • The role of endothelial dysfunction in restenosis. Revista portuguesa de cardiologia Cooke, J. P., Tsao, P. S. 1992; 11 (10): 889-892

    View details for PubMedID 1285965



    The purpose of this study was to determine if chronic administration of L-arginine, the precursor of endothelium-derived relaxing factor (EDRF), normalizes endothelium-dependent relaxation and decreases atherosclerosis in hypercholesterolemic animals. Male rabbits were fed (a) normal rabbit chow; (b) 1% cholesterol diet; or (c) 1% cholesterol diet supplemented by 2.25% L-arginine HCl in drinking water. Arginine supplementation doubled plasma arginine levels without affecting serum cholesterol values. After 10 wk, the thoracic aorta was harvested for studies of vascular reactivity and histomorphometry. Endothelium-dependent relaxations (to acetylcholine and calcium ionophore A23187) were significantly impaired in thoracic aortae from animals fed a 1% cholesterol diet. By contrast, vessels from hypercholesterolemic animals receiving L-arginine supplementation exhibited significantly improved endothelium-dependent relaxations. Responses to norepinephrine or nitroglycerin were not affected by either dietary intervention. Histomorphometric analysis revealed a reduction in lesion surface area and intimal thickness in thoracic aortae from arginine-supplemented animals compared to those from untreated hypercholesterolemic rabbits. This is the first study to demonstrate that supplementation of dietary L-arginine, the EDRF precursor, improves endothelium-dependent vasorelaxation. More importantly, we have shown that this improvement in EDRF activity is associated with a reduction in atherogenesis.

    View details for Web of Science ID A1992JN95900065

    View details for PubMedID 1522225