Comparison of Non-Coding RNAs in Exosomes and Functional Efficacy of Human Embryonic Stem Cell- Versus Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
Stem cells (Dayton, Ohio)
DNA damage-associated biomarkers in studying individual sensitivity to low-dose radiation from cardiovascular imaging.
European heart journal
2016; 37 (40): 3075-3080
Assessment of the Radiation Effects of Cardiac CT Angiography Using Protein and Genetic Biomarkers
2015; 8 (8): 873-884
Variable activation of the DNA damage response pathways in patients undergoing single-photon emission computed tomography myocardial perfusion imaging.
Circulation. Cardiovascular imaging
2015; 8 (2)
Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can serve as unlimited cell sources for cardiac regenerative therapy. However, the functional equivalency between human ESC-CMs and iPSC-CMs for cardiac regenerative therapy has not been demonstrated. Here we performed a head-to-head comparison of ESC-CMs and iPSC-CMs in their ability to restore cardiac function in a rat myocardial infarction (MI) model as well as their exosomal secretome.Human ESCs and iPSCs were differentiated into cardiomyocytes using small molecule inhibitors. Fluorescence-activated cell sorting (FACS) analysis confirmed ?85% and ?83% of CMs differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T. At a single-cell level, both cell types displayed similar calcium handling and electrophysiological properties, with gene expression comparable to the human fetal heart marked by striated sarcomeres. Sub-acute transplantation of ESC-CMs and iPSC-CMs into nude rats post-MI improved cardiac function, which was associated with increased expression of angiogenic genes in vitro following hypoxia. Profiling of exosomal microRNAs (miRs) and long non-coding RNAs (lncRNAs) revealed that both groups contain an identical repertoire of miRs and lncRNAs, including some that are known to be cardioprotective.We demonstrate for the first time that both ESC-CMs and iPSC-CMs can facilitate comparable cardiac repair. This is advantageous because unlike allogeneic ESC-CMs used in therapy, autologous iPSC-CMs could potentially avoid immune rejection when used for cardiac cell transplantation in the future. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/stem.2669
View details for PubMedID 28710827
Mining Exosomal MicroRNAs from Human-Induced Pluripotent Stem Cells-Derived Cardiomyocytes for Cardiac Regeneration.
Methods in molecular biology (Clifton, N.J.)
2018; 1733: 127?36
Although single-photon emission computed tomography myocardial perfusion imaging (SPECT MPI) has improved the diagnosis and risk stratification of patients with suspected coronary artery disease, it remains a primary source of low-dose radiation exposure for cardiac patients. To determine the biological effects of low-dose radiation from SPECT MPI, we measured the activation of the DNA damage response pathways using quantitative flow cytometry and single-cell gene expression profiling.Blood samples were collected from patients before and after SPECT MPI (n=63). Overall, analysis of all recruited patients showed no marked differences in the phosphorylation of proteins (H2AX, protein 53, and ataxia telangiectasia mutated) after SPECT. The majority of patients also had either downregulated or unchanged expression in DNA damage response genes at both 24 and 48 hours post-SPECT. Interestingly, a small subset of patients with increased phosphorylation had significant upregulation of genes associated with DNA damage, whereas those with no changes in phosphorylation had significant downregulation or no difference, suggesting that some patients may potentially be more sensitive to low-dose radiation exposure.Our findings showed that SPECT MPI resulted in a variable activation of the DNA damage response pathways. Although only a small subset of patients had increased protein phosphorylation and elevated gene expression postimaging, continued care should be taken to reduce radiation exposure to both the patients and operators.
View details for DOI 10.1161/CIRCIMAGING.114.002851
View details for PubMedID 25609688
View details for PubMedCentralID PMC4354894
Role of NADPH Oxidase in Radiation-induced Pro-oxidative and Pro-inflammatory Pathways in Mouse Brain.
International journal of radiation biology
Myocardial infarction is the leading cause of morbidity and mortality worldwide. Recent advances in cardiac regenerative therapy have allowed for novel modalities in replenishing the damaged myocardium. However, poor long-term engraftment and survival of transplanted cells have largely precluded effective cell replacement. As an alternative to direct cell replacement, the release of paracrine protective factors may be a more plausible effector for cardioprotection which may partially be mediated through secretion of microvesicles, or exosomes, that contribute to cell-cell communication. In this chapter, we describe the isolation of exosomes from induced pluripotent stem cells-derived cardiomyocytes for subsequent microRNA profiling for a better understanding of the biological cargo contained within exosomes.
View details for DOI 10.1007/978-1-4939-7601-0_10
View details for PubMedID 29435928
Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant Human Hypoxia-Inducible Factor 1-a (HIF1-a) in an Ovine Model of Acute Myocardial Infarction.
Journal of the American Heart Association
2016; 5 (7)
The present study was designed to investigate our hypothesis that NADPH oxidase plays a role in radiation-induced pro-oxidative and pro-inflammatory environments in brain.C57BL/6 mice received either fractionated whole brain irradiation or sham-irradiation. The mRNA expression levels of pro-inflammatory mediators, such as TNF-? and MCP-1, were determined by quantitative real-time RT-PCR. The protein expression levels of TNF-?, MCP-1, NOX-2 and Iba1 were detected by immunofluorescence staining. The levels of ROS were visualized by in situ DHE fluorescence staining.A significant up-regulation of mRNA and protein expression levels of TNF-? and MCP-1 was observed in irradiated mouse brains. Additionally, immunofluorescence staining of Iba1 showed a marked increase of microglial activation in mouse brain after irradiation. Moreover, in situ DHE fluorescence staining revealed that fractionated whole brain irradiation significantly increased production of ROS. Furthermore, a significant increase in immunoreactivity of NOX-2 was detected in mouse brain after irradiation. On the other hand, an enhanced ROS generation in mouse brain after irradiation was markedly attenuated in the presence of NOX inhibitors or NOX-2 neutralizing antibody.These results suggest that NOX-2 may play a role in fractionated whole brain irradiation-induced pro-oxidative and pro-inflammatory pathways in mouse brain.
View details for DOI 10.1080/09553002.2017.1377360
View details for PubMedID 28880721
Effects of cellular origin on differentiation of human induced pluripotent stem cell-derived endothelial cells.
2016; 1 (8)
Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia-inducible factor 1-? (HIF1-?), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen-resistant HIF1-? would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI.Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1-? (BMMSC-HIF) were injected in the peri-infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P<0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2-fold (P<0.001) in the presence of markedly decreased end-systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo-treated animals (n=6), neither parameters changed over time. HIF1-?-transfected BMMSCs (BMMSC-HIF) induced angio-/arteriogenesis and decreased apoptosis by HIF1-mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin-1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long-term retention of BMMSC-HIF.Intramyocardial delivery of BMMSC-HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1-mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.
View details for DOI 10.1161/JAHA.116.003714
View details for PubMedID 27385426
Comparison of Magnetic Resonance Imaging and Serum Biomarkers for Detection of Human Pluripotent Stem Cell-Derived Teratomas
STEM CELL REPORTS
2016; 6 (2): 176-187
Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC-derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31(+) population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC-ECs maintained a higher CD31(+) population than FB-iPSC-ECs and CPC-iPSC-ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation.
View details for PubMedID 27398408
Microfluidic Single-Cell Analysis of Transplanted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes After Acute Myocardial Infarction.
2015; 132 (8): 762-771
The use of cells derived from pluripotent stem cells (PSCs) for regenerative therapies confers a considerable risk for neoplastic growth and teratoma formation. Preclinical and clinical assessment of such therapies will require suitable monitoring strategies to understand and mitigate these risks. Here we generated human-induced pluripotent stem cells (iPSCs), selected clones that continued to express reprogramming factors after differentiation into cardiomyocytes, and transplanted these cardiomyocytes into immunocompromised rat hearts post-myocardial infarction. We compared magnetic resonance imaging (MRI), cardiac ultrasound, and serum biomarkers for their ability to delineate teratoma formation and growth. MRI enabled the detection of teratomas with a volume >8 mm(3). A combination of three plasma biomarkers (CEA, AFP, and HCG) was able to detect teratomas with a volume >17 mm(3) and with a sensitivity of more than 87%. Based on our findings, a combination of serum biomarkers with MRI screening may offer the highest sensitivity for teratoma detection and tracking.
View details for DOI 10.1016/j.stemcr.2015.12.008
View details for Web of Science ID 000369726500003
View details for PubMedID 26777057
MicroRNA-mediated regulation of differentiation and trans-differentiation in stem cells
ADVANCED DRUG DELIVERY REVIEWS
2015; 88: 3-15
Human induced pluripotent stem cells (iPSCs) are attractive candidates for therapeutic use, with the potential to replace deficient cells and to improve functional recovery in injury or disease settings. Here, we test the hypothesis that human iPSC-derived cardiomyocytes (iPSC-CMs) can secrete cytokines as a molecular basis to attenuate adverse cardiac remodeling after myocardial infarction.Human iPSCs were generated from skin fibroblasts and differentiated in vitro with a small molecule-based protocol. Troponin(+) iPSC-CMs were confirmed by immunohistochemistry, quantitative polymerase chain reaction, fluorescence-activated cell sorting, and electrophysiological measurements. Afterward, 2×10(6) iPSC-CMs derived from a cell line transduced with a vector expressing firefly luciferase and green fluorescent protein were transplanted into adult NOD/SCID mice with acute left anterior descending artery ligation. Control animals received PBS injection. Bioluminescence imaging showed limited engraftment on transplantation into ischemic myocardium. However, magnetic resonance imaging of animals transplanted with iPSC-CMs showed significant functional improvement and attenuated cardiac remodeling compared with PBS-treated control animals. To understand the underlying molecular mechanism, microfluidic single-cell profiling of harvested iPSC-CMs, laser capture microdissection of host myocardium, and in vitro ischemia stimulation were used to demonstrate that the iPSC-CMs could release significant levels of proangiogenic and antiapoptotic factors in the ischemic microenvironment.Transplantation of human iPSC-CMs into an acute mouse myocardial infarction model can improve left ventricular function and attenuate cardiac remodeling. Because of limited engraftment, most of the effects are possibly explained by paracrine activity of these cells.
View details for DOI 10.1161/CIRCULATIONAHA.114.015231
View details for PubMedID 26304668
Response to letter regarding article, "Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer".
2015; 131 (12)
HIF-1 reduces ischaemia-reperfusion injury in the heart by targeting the mitochondrial permeability transition pore.
2014; 104 (1): 24-36
MicroRNAs (miRNAs) are key components of a broadly conserved post-transcriptional mechanism that controls gene expression by targeting mRNAs. miRNAs regulate diverse biological processes, including the growth and differentiation of stem cells as well as the regulation of both endogenous tissue repair that has critical implications in the development of regenerative medicine approaches. In this review, we first describe key features of miRNA biogenesis and their role in regulating self-renewal, and then discuss the involvement of miRNAs in the determination of cell fate decisions. We highlight the role of miRNAs in the emergent field of reprogramming and trans-differentiation of somatic cells that could further our understanding of miRNA biology and regenerative medicine applications. Finally, we describe potential techniques for proper delivery of miRNAs in target cells.
View details for DOI 10.1016/j.addr.2015.04.004
View details for Web of Science ID 000358628200002
Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer.
2014; 130 (11): S60-9
Hypoxia inducible factor-1 (HIF-1) has been reported to promote tolerance against acute myocardial ischemia-reperfusion injury (IRI). However, the mechanism through which HIF-1 stabilization actually confers this cardioprotection is not clear. We investigated whether HIF-1¦Á stabilization protects the heart against acute IRI by preventing the opening of the mitochondrial permeability transition pore (MPTP) and the potential mechanisms involved.Stabilization of myocardial HIF-1 was achieved by pharmacological inhibition of prolyl hydroxylase domain-containing enzyme (PHD) using GSK360A or using cardiac-specific ablation of von-Hippel Lindau protein (VHL(fl/fl)) in mice. Treatment of HL-1 cardiac cells with GSK360A stabilized HIF-1, increased the expression of HIF-1 target genes pyruvate dehydrogenase kinase-1 and hexokinase II (HKII) and reprogrammed cell metabolism to aerobic glycolysis, thereby resulting in the production of less mitochondrial oxidative stress during IRI, and less MPTP opening, effects which were shown to be dependent on HKII. These findings were further confirmed when HIF-1 stabilization in the rat and murine heart resulted in smaller myocardial infarct sizes (both in and ex vivo), decreased mitochondrial oxidative stress, and inhibited MPTP opening following IRI, effects which were also found to be dependent on HKII.We have demonstrated that acute HIF-1¦Á stabilization using either a pharmacological or genetic approach protected the heart against acute IRI by promoting aerobic glycolysis, decreasing mitochondrial oxidative stress, activating HKII, and inhibiting MPTP opening.
View details for DOI 10.1093/cvr/cvu172
View details for PubMedID 25063991
Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation
2012; 111 (7): 882-893
Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs.After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes.In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.
View details for DOI 10.1161/CIRCULATIONAHA.113.007917
View details for PubMedID 25200057
Whole Brain Radiation-Induced Cognitive Impairment: Pathophysiological Mechanisms and Therapeutic Targets
BIOMOLECULES & THERAPEUTICS
2012; 20 (4): 357-370
Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models.To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia.Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery.In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.
View details for DOI 10.1161/CIRCRESAHA.112.269001
View details for Web of Science ID 000308868800015
View details for PubMedID 22821929
View details for PubMedCentralID PMC3473073
Irradiation Alters MMP-2/TIMP-2 System and Collagen Type IV Degradation in Brain
INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS
2012; 82 (5): 1559-1566
Radiation therapy, the most commonly used for the treatment of brain tumors, has been shown to be of major significance in tu-mor control and survival rate of brain tumor patients. About 200,000 patients with brain tumor are treated with either partial large field or whole brain radiation every year in the United States. The use of radiation therapy for treatment of brain tumors, however, may lead to devastating functional deficits in brain several months to years after treatment. In particular, whole brain radiation therapy results in a significant reduction in learning and memory in brain tumor patients as long-term consequences of treatment. Although a number of in vitro and in vivo studies have demonstrated the pathogenesis of radiation-mediated brain injury, the cel-lular and molecular mechanisms by which radiation induces damage to normal tissue in brain remain largely unknown. Therefore, this review focuses on the pathophysiological mechanisms of whole brain radiation-induced cognitive impairment and the iden-tification of novel therapeutic targets. Specifically, we review the current knowledge about the effects of whole brain radiation on pro-oxidative and pro-inflammatory pathways, matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) system and extracellular matrix (ECM), and physiological angiogenesis in brain. These studies may provide a foundation for defin-ing a new cellular and molecular basis related to the etiology of cognitive impairment that occurs among patients in response to whole brain radiation therapy. It may also lead to new opportunities for therapeutic interventions for brain tumor patients who are undergoing whole brain radiation therapy.
View details for DOI 10.4062/biomolther.2012.20.4.357
View details for Web of Science ID 000307680000001
View details for PubMedCentralID PMC3762274
Radiation Attenuates Physiological Angiogenesis by Differential Expression of VEGF, Ang-1, Tie-2 and Ang-2 in Rat Brain
2011; 176 (6): 753-760
Blood-brain barrier (BBB) disruption is one of the major consequences of radiation-induced normal tissue injury in the central nervous system. We examined the effects of whole-brain irradiation on matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in the brain.Animals received either whole-brain irradiation (a single dose of 10 Gy ?-rays or a fractionated dose of 40 Gy ?-rays, total) or sham-irradiation and were maintained for 4, 8, and 24 h following irradiation. mRNA expression levels of MMPs and TIMPs in the brain were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR). The functional activity of MMPs was measured by in situ zymography, and degradation of ECM was visualized by collagen type IV immunofluorescent staining.A significant increase in mRNA expression levels of MMP-2, MMP-9, and TIMP-1 was observed in irradiated brains compared to that in sham-irradiated controls. In situ zymography revealed a strong gelatinolytic activity in the brain 24 h postirradiation, and the enhanced gelatinolytic activity mediated by irradiation was significantly attenuated in the presence of anti-MMP-2 antibody. A significant reduction in collagen type IV immunoreactivity was also detected in the brain at 24 h after irradiation. In contrast, the levels of collagen type IV were not significantly changed at 4 and 8 h after irradiation compared with the sham-irradiated controls.The present study demonstrates for the first time that radiation induces an imbalance between MMP-2 and TIMP-2 levels and suggests that degradation of collagen type IV, a major ECM component of BBB basement membrane, may have a role in the pathogenesis of brain injury.
View details for DOI 10.1016/j.ijrobp.2010.12.032
View details for Web of Science ID 000301891300019
View details for PubMedID 22429332
AT1 Receptor Antagonism Does Not Influence Early Radiation-Induced Changes in Microglial Activation or Neurogenesis in the Normal Rat Brain
2011; 176 (1): 71-83
The etiology of radiation-induced cerebrovascular rarefaction remains unknown. In the present study, we examined the effect of whole-brain irradiation on endothelial cell (EC) proliferation/apoptosis and expression of various angiogenic factors in rat brain. F344 × BN rats received either whole-brain irradiation (a single dose of 10 Gy ? rays) or sham irradiation and were maintained for 4, 8 and 24 h after irradiation. Double immunofluorescence staining was employed to visualize EC proliferation/apoptosis in brain. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), endothelial-specific receptor tyrosine kinase (Tie-2), and Ang-2 in brain were determined by real-time RT-PCR and immunofluorescence staining. A significant reduction in CD31-immunoreactive cells was detected in irradiated rat brains compared with sham-irradiated controls. Whole-brain irradiation significantly suppressed EC proliferation and increased EC apoptosis. In addition, a significant decrease in mRNA and protein expression of VEGF, Ang-1 and Tie-2 was observed in irradiated rat brains. In contrast, whole-brain irradiation significantly upregulated Ang-2 expression in rat brains. The present study provides novel evidence that whole-brain irradiation differentially affects mRNA and protein expression of VEGF, Ang-1, Tie-2 and Ang-2. These changes are closely associated with decreased EC proliferation and increased EC apoptosis in brain.
View details for DOI 10.1667/RR2647.1
View details for Web of Science ID 000297904000007
View details for PubMedID 21962003
Role of NADPH oxidase in interleukin-4-induced monocyte chemoattractant protein-1 expression in vascular endothelium
2010; 59 (9): 755-765
Blockers of the renin-angiotensin-aldosterone system (RAAS) ameliorate cognitive deficits and some aspects of brain injury after whole-brain irradiation. We investigated whether treatment with the angiotensin II type 1 receptor antagonist L-158,809 at a dose that protects cognitive function after fractionated whole-brain irradiation reduced radiation-induced neuroinflammation and changes in hippocampal neurogenesis, well-characterized effects that are associated with radiation-induced brain injury. Male F344 rats received L-158,809 before, during and after a single 10-Gy dose of radiation. Expression of cytokines, angiotensin II receptors and angiotensin-converting enzyme 2 was evaluated by real-time PCR 24 h, 1 week and 12 weeks after irradiation. At the latter times, microglial density and proliferating and activated microglia were analyzed in the dentate gyrus of the hippocampus. Cell proliferation and neurogenesis were also quantified in the dentate subgranular zone. L-158,809 treatment modestly increased mRNA expression for Ang II receptors and TNF-? but had no effect on radiation-induced effects on hippocampal microglia or neurogenesis. Thus, although L-158,809 ameliorates cognitive deficits after whole-brain irradiation, the drug did not mitigate the neuroinflammatory microglial response or rescue neurogenesis. Additional studies are required to elucidate other mechanisms of normal tissue injury that may be modulated by RAAS blockers.
View details for DOI 10.1667/RR2560.1
View details for Web of Science ID 000292441900008
View details for PubMedID 21545290
Aging attenuates radiation-induced expression of pro-inflammatory mediators in rat brain
2010; 476 (2): 89-93
The pro-oxidative and pro-inflammatory pathways in vascular endothelium have been implicated in the development of atherosclerosis. In the present study, we investigated effect of interleukin-4 (IL-4) on monocyte chemoattractant protein-1 (MCP-1) expression in vascular endothelium and examined the role of distinct sources of reactive oxygen species (ROS) in this process.Real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay showed that IL-4 significantly up-regulated mRNA and protein expression of MCP-1 in human aortic endothelial cells (HAEC) and C57BL/6 mice. A significant and dose-dependent inhibition of IL-4-induced MCP-1 expression was observed in HAEC pre-treated with antioxidants, such as pyrrolidine dithiocarbamate and epigallocatechin gallate, indicating that IL-4-induced MCP-1 expression is mediated via a ROS-dependent mechanism. Additionally, pharmacological inhibitors of NADPH oxidase (NOX) significantly attenuated IL-4-induced MCP-1 expression in HAEC. Furthermore, the disruption of the NOX gene dramatically reduced IL-4-induced MCP-1 expression in NOX knockout mice (B6.129S6-Cybb(tm1Din)/J). In contrast, overexpression of MCP-1 in IL-4-stimulated HAEC was not affected by inhibiting other ROS generating pathways, such as xanthine oxidase and the mitochondrial electron transport chain.These results demonstrate that IL-4 up-regulates MCP-1 expression in vascular endothelium through NOX-mediated ROS generation.
View details for DOI 10.1007/s00011-010-0187-3
View details for Web of Science ID 000280728100008
View details for PubMedID 20349326
Interleukin-4, Oxidative Stress, Vascular Inflammation and Atherosclerosis
BIOMOLECULES & THERAPEUTICS
2010; 18 (2): 135-144
The present study was designed to examine the effect of aging on radiation-induced expression of pro-inflammatory mediators in rat brain. Male F344xBN rats (4, 16, and 24 months of age) received either whole brain irradiation with a single dose of 10Gy gamma-rays or sham-irradiation, and were maintained for 4, 8, and 24h post-irradiation. The mRNA expression levels of various pro-inflammatory mediators such as cytokines, adhesion molecules, chemokine, and matrix metalloproteinase were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The acute inflammatory responses to irradiation, including overexpression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, monocyte chemoattractant protein-1 (MCP-1), and matrix metalloproteinase-9 (MMP-9) were markedly attenuated in the hippocampus of middle-aged and old rats compared with young groups. Specifically, a significant age-dependent decrease in TNF-alpha expression was detected 8 and 24h after irradiation and a similar age-related attenuation was observed in IL-1beta, ICAM-1, and VCAM-1 expression 4 and 8h post-irradiation. MCP-1 expression was reduced 4h post-irradiation and MMP-9 expression at 8h post-irradiation. These results provide evidence for the first time that radiation-induced pro-inflammatory responses in the brain are suppressed in aged animals.
View details for DOI 10.1016/j.neulet.2010.04.009
View details for Web of Science ID 000278605100009
View details for PubMedID 20385203
Irradiation induces regionally specific alterations in pro-inflammatory environments in rat brain
INTERNATIONAL JOURNAL OF RADIATION BIOLOGY
2010; 86 (2): 132-144
The pro-oxidative and pro-inflammatory pathways in vascular endothelium have been implicated in the initiation and progression of atherosclerosis. In fact, inflammatory responses in vascular endothelium are primarily regulated through oxidative stress-mediated signaling pathways leading to overexpression of pro-inflammatory mediators. Enhanced expression of cytokines, chemokines and adhesion molecules in endothelial cells and their close interactions facilitate recruiting and adhering blood leukocytes to vessel wall, and subsequently stimulate transendothelial migration, which are thought to be critical early pathologic events in atherogenesis. Although interleukin-4 (IL-4) was traditionally considered as an anti-inflammatory cytokine, recent in vitro and in vivo studies have provided robust evidence that IL-4 exerts pro-inflammatory effects on vascular endothelium and may play a critical role in the development of atherosclerosis. The cellular and molecular mechanisms responsible for IL-4-induced atherosclerosis, however, remain largely unknown. The present review focuses on the distinct sources of IL-4-mediated reactive oxygen species (ROS) generation as well as the pivotal role of ROS in IL-4-induced vascular inflammation. These studies will provide novel insights into a clear delineation of the oxidative mechanisms of IL-4-mediated stimulation of vascular inflammation and subsequent development of atherosclerosis. It will also contribute to novel therapeutic approaches for atherosclerosis specifically targeted against pro-oxidative and pro-inflammatory pathways in vascular endothelium.
View details for DOI 10.4062/biomolther.2010.18.2.135
View details for Web of Science ID 000277582000002
Oxidative mechanisms of IL-4-induced IL-6 expression in vascular endothelium
2010; 49 (1): 73-79
Pro-inflammatory environments in the brain have been implicated in the onset and progression of neurological disorders. In the present study, we investigate the hypothesis that brain irradiation induces regionally specific alterations in cytokine gene and protein expression.Four month old F344 x BN rats received either whole brain irradiation with a single dose of 10 Gy gamma-rays or sham-irradiation, and were maintained for 4, 8, and 24 h following irradiation. The mRNA and protein expression levels of pro-inflammatory mediators were analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. To elucidate the molecular mechanisms of irradiation-induced brain inflammation, effects of irradiation on the DNA-binding activity of pro-inflammatory transcription factors were also examined.A significant and marked up-regulation of mRNA and protein expression of pro-inflammatory mediators, including tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and monocyte chemoattractant protein-1 (MCP-1), was observed in hippocampal and cortical regions isolated from irradiated brain. Cytokine expression was regionally specific since TNF-alpha levels were significantly elevated in cortex compared to hippocampus (57% greater) and IL-1beta levels were elevated in hippocampus compared to cortical samples (126% greater). Increases in cytokine levels also were observed after irradiation of mouse BV-2 microglial cells. A series of electrophoretic mobility shift assays (EMSA) demonstrated that irradiation significantly increased activation of activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), and cAMP response element-binding protein (CREB).The present study demonstrated that whole brain irradiation induces regionally specific pro-inflammatory environments through activation of AP-1, NF-kappaB, and CREB and overexpression of TNF-alpha, IL-1beta, and MCP-1 in rat brain and may contribute to unique pathways for the radiation-induced impairments in tissue function.
View details for DOI 10.3109/09553000903419346
View details for Web of Science ID 000274629400006
View details for PubMedID 20148699
A novel in vitro ischemia/reperfusion injury model
ARCHIVES OF PHARMACAL RESEARCH
2009; 32 (3): 421-429
The present study is designed to investigate the effects of interleukin-4 (IL-4) on expression of interleukin-6 (IL-6), as well as to examine the role of distinct sources of reactive oxygen species (ROS) in this process. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that IL-4 significantly up-regulated the mRNA and protein expression of IL-6 in human aortic endothelial cells (HAEC) and C57BL/6 mice. Dihydroethidium (DHE) and dichlorofluorescein (DCF) fluorescence staining demonstrated that IL-4 significantly increased ROS generation in HAEC. A significant and dose-dependent inhibition of IL-4-induced IL-6 expression was observed in HAEC pre-treated with antioxidants, such as pyrrolidine dithiocarbamate (PDTC) and epigallocatechin gallate (EGCG), indicating that IL-4-induced IL-6 expression is mediated via an ROS-dependent mechanism. Additionally, pharmacological inhibitor of NADPH oxidase (NOX) significantly attenuated IL-4-induced ROS generation and IL-6 expression in HAEC. Furthermore, the disruption of NOX gene dramatically and significantly reduced IL-4-induced IL-6 expression in NOX knockout mice (B6.129S6-Cybb(tm1Din)/J). In contrast, overexpression of IL-6 in IL-4-activated HAEC was not affected by inhibiting other ROS generating pathways, such as xanthine oxidase, arachidonic acid metabolism, and the mitochondrial electron transport chain. These results demonstrate that IL-4 up-regulates IL-6 expression in vascular endothelium through NOX-mediated ROS generation.
View details for DOI 10.1016/j.cyto.2009.08.009
View details for Web of Science ID 000273864000011
View details for PubMedID 19822443
Protective effects of genistein on proinflammatory pathways in human brain microvascular endothelial cells
JOURNAL OF NUTRITIONAL BIOCHEMISTRY
2008; 19 (12): 819-825
The reperfusion of blood flow occurred in a number of conditions such as stroke and organ transplantation immensely augments tissue injury and causes more severe damage than prolonged ischemia. In the present study, we designed a novel double-layer parallel-plate flow chamber (PPFC) to develop an in vitro ischemia/reperfusion (I/R) injury model and examined the effects of I/R on inflammatory responses in human microvascular endothelial cells (HMEC-1). The expression of pro-inflammatory mediators, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1) in HMEC-1 was measured by quantitative real-time RT-PCR. The cells were also pre-treated with antioxidant pyrrolidine dithiocarbamate (PDTC) to verify involvement of an oxidative mechanism in I/R injury in vitro. The morphological changes and attenuated expression of pro-inflammatory mediators were observed in HMCE-1 exposed to the physiological flow. In contrast, I/R markedly and significantly up-regulated expression of pro-inflammatory mediators in HMEC-1. Additionally, pretreatment with PDTC significantly reduced I/R-mediated overexpression of pro-inflammatory mediators. The data from the present study provide evidence demonstrating that our newly designed PPFC can be utilized as an effective in vitro cell culture model system to develop new drugs specifically targeting against ischemia/reperfusion (I/R) injury.
View details for DOI 10.1007/s12272-009-1316-9
View details for Web of Science ID 000265440100018
View details for PubMedID 19387587
A novel double-layer parallel-plate flow chamber
2007 IEEE 33RD ANNUAL NORTHEAST BIOENGINEERING CONFERENCE
Proinflammatory cerebromicrovascular environment has been implicated in the critical early pathologic events in a variety of neurodegenerative diseases. Recent studies also have demonstrated the potential beneficial effects of soy isoflavones. However, cellular and molecular mechanisms underlying these processes are not fully understood. The present study was designed to examine the hypothesis that soy isoflavone genistein may attenuate cytokine-induced proinflammatory pathways in human brain microvascular endothelial cells. The quantitative real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay showed that pretreatment of HBMEC with increasing concentrations of genistein significantly and dose-dependently inhibited cytokine-induced up-regulation of mRNA and protein expression of proinflammatory mediators such as tumor necrosis factor-alpha, interleukin-1beta, monocyte chemoattractant protein-1, interleukin-8, and intercellular adhesion molecule-1. In addition, genistein pretreatment significantly reduced cytokine-mediated up-regulation of transmigration of blood leukocytes in a dose-dependent manner. Our results suggest that genistein may attenuate proinflammatory pathways through inhibition of cytokine-induced overexpression of proinflammatory mediators and inflammatory reactions in human brain microvascular endothelial cells.
View details for DOI 10.1016/j.jnutbio.2007.10.006
View details for Web of Science ID 000261253700004
View details for PubMedID 18479900