Bio

Clinical Focus


  • Intensive Care, Pediatric
  • Pediatric Critical Care Medicine

Academic Appointments


Honors & Awards


  • Outstanding Junior Investigator Award, American Journal of Physiology-Lung Molecular and Cellular Physiology (2013)
  • Young Investigator Coaching Program, Society for Pediatric Research (2012)
  • Fellow to Faculty Transition Grant, American Heart Association (2008-2013)
  • Pediatric Clerkship Honor Roll for Teaching, Lucile Packard Children's Hospital at Stanford (2007)
  • Travel Award, Society for Pediatric Research (2004)
  • Travel Award, Western Society for Clinical Investigation (2004)
  • Excellence in Clinical Medicine, Louis Weinstein Prize (1999)
  • Member, Alpha Omega Alpha National Honor Society (1998)

Professional Education


  • Fellowship:Stanford University Medical Center (2005) CA
  • Internship:Stanford University Medical Center (2000) CA
  • Residency:Stanford University Medical Center (2002) CA
  • Medical Education:Tufts University (1999) MA
  • Board Certification: Pediatric Critical Care Medicine, American Board of Pediatrics (2006)
  • BS, Tufts University, Biology (1995)
  • MD, Tufts University, Medicine (1999)

Research & Scholarship

Current Research and Scholarly Interests


Secondary septation, the process that marks the alveolar phase of lung development,involves the coordinated activities of multiple different cell types within the lung. Secretion of extracellular matrix components, proliferation and migration of myofibroblasts and epithelial cells, and pulmonary capillary angiogenesis, have been identified as key players in this process. However, in contrast to the identification of multiple transcription factors controlling branching morphogenesis during the early stages of lung development, the regulators that control and coordinate the individual components of alveolarization remain unknown. The nuclear factor kappa-B (NFkB) family of transcription factors plays a key role in regulating cell survival, differentiation, and inflammation, however, a role in lung development has not been previously identified. A main focus of our work is to define a novel function for NFkB in regulating postnatal lung development using mouse models and primary cell lines.

Postnatal pulmonary angiogenesis is essential for alveolarization. We have recently demonstrated a high degree of constitutive NFkB signaling in primary pulmonary endothelial cells (PEC) isolated from neonatal mice as compared to those isolated from adult mice. Furthermore, inhibiting constitutive NFkB activity in the neonatal PEC with either pharmacologic inhibitors or RNA interference, blocked PEC survival, decreased proliferation, and impaired in vitro angiogenesis. In this project we are utilizing RNAi to block the individual components of the NFkB pathway, gene expression analysis, and endothelial specific conditional knock-out mice in order to identify novel NFkB mediated targets that are essential for postnatal pulmonary angiogenesis.

In a separate but related project, we are exploring pathways which help to preserve normal lung development in the setting of lung injury. Both local and systemic infections can injure the lung. Clinical and experimental evidence suggests that unique pathways may exist that serve to protect the immature lung from severe inflammation, and potentially allow for a greater regeneration after injury. Using a murine model of acute respiratory distress syndrome induced by the administration of systemic lipopolysaccharide, we are exploring the molecular mechanisms that serve to protect the lung against injury, and identify how these mechanisms are distinct in immature and mature animals. We believe that the information learned from these studies will be clinically relevant to a broad number of pulmonary diseases including bronchopulmonary dysplasia, asthma, ARDS, and emphysema.

Teaching

2013-14 Courses


Publications

Journal Articles


  • Voltage-Dependent Anion Channel-2 Interaction with Nitric Oxide Synthase Enhances Pulmonary Artery Endothelial Cell Nitric Oxide Production AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Alvira, C. M., Umesh, A., Husted, C., Ying, L., Hou, Y., Lyu, S., Nowak, J., Cornfield, D. N. 2012; 47 (5): 669-678

    Abstract

    Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.

    View details for DOI 10.1165/rcmb.2011-0436OC

    View details for Web of Science ID 000314406400014

    View details for PubMedID 22842492

  • Inhibiting NF-kappa B in the developing lung disrupts angiogenesis and alveolarization AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Iosef, C., Alastalo, T., Hou, Y., Chen, C., Adams, E. S., Lyu, S., Cornfield, D. N., Alvira, C. M. 2012; 302 (10): L1023-L1036

    Abstract

    Bronchopulmonary dysplasia (BPD), a chronic lung disease of infancy, is characterized by arrested alveolar development. Pulmonary angiogenesis, mediated by the vascular endothelial growth factor (VEGF) pathway, is essential for alveolarization. However, the transcriptional regulators mediating pulmonary angiogenesis remain unknown. We previously demonstrated that NF-?B, a transcription factor traditionally associated with inflammation, plays a unique protective role in the neonatal lung. Therefore, we hypothesized that constitutive NF-?B activity is essential for postnatal lung development. Blocking NF-?B activity in 6-day-old neonatal mice induced the alveolar simplification similar to that observed in BPD and significantly reduced pulmonary capillary density. Studies to determine the mechanism responsible for this effect identified greater constitutive NF-?B in neonatal lung and in primary pulmonary endothelial cells (PEC) compared with adult. Moreover, inhibiting constitutive NF-?B activity in the neonatal PEC with either pharmacological inhibitors or RNA interference blocked PEC survival, decreased proliferation, and impaired in vitro angiogenesis. Finally, by chromatin immunoprecipitation, NF-?B was found to be a direct regulator of the angiogenic mediator, VEGF-receptor-2, in the neonatal pulmonary vasculature. Taken together, our data identify an entirely novel role for NF-?B in promoting physiological angiogenesis and alveolarization in the developing lung. Our data suggest that disruption of NF-?B signaling may contribute to the pathogenesis of BPD and that enhancement of NF-?B may represent a viable therapeutic strategy to promote lung growth and regeneration in pulmonary diseases marked by impaired angiogenesis.

    View details for DOI 10.1152/ajplung.00230.2011

    View details for Web of Science ID 000304357600005

    View details for PubMedID 22367785

  • Inhibition of Transforming Growth Factor beta Worsens Elastin Degradation in a Murine Model of Kawasaki Disease AMERICAN JOURNAL OF PATHOLOGY Alvira, C. M., Guignabert, C., Kim, Y., Chen, C., Wang, L., Duong, T. T., Yeung, R. S., Li, D. Y., Rabinovitch, M. 2011; 178 (3): 1210-1220

    Abstract

    Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) ? increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF-? would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF-? signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF-? by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF-? suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-?, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental.

    View details for DOI 10.1016/j.ajpath.2010.11.054

    View details for Web of Science ID 000288185600028

    View details for PubMedID 21356372

  • Rho kinase modulates postnatal adaptation of the pulmonary circulation through separate effects on pulmonary artery endothelial and smooth muscle cells AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Alvira, C. M., Sukovich, D. J., Lyu, S., Cornfield, D. N. 2010; 299 (6): L872-L878

    Abstract

    At birth, pulmonary vasodilation occurs concomitant with the onset of air-breathing life. Whether and how Rho kinase (ROCK) modulates the perinatal pulmonary vascular tone remains incompletely understood. To more fully characterize the separate and interactive effects of ROCK signaling, we hypothesized that ROCK has discrete effects on both pulmonary artery (PA): 1) endothelial cell (PAEC) nitric oxide (NO) production and contractile state; and 2) smooth muscle cell tone independent of endothelial NO synthase (eNOS) activity. To test these hypotheses, NO production and endothelial barrier function were determined in fetal PAEC under baseline hypoxia and following exposure to normoxia with and without treatment with Y-27632, a specific pharmacological inhibitor of ROCK. In acutely instrumented, late-gestation ovine fetuses, eNOS was inhibited by nitro-l-arginine infusion into the left PA (LPA). Subsequently, fetal lambs were mechanically ventilated (MV) with 100% oxygen in the absence (control period) and presence of Y-27632. In PAEC, treatment with Y-27632 had no effect on cytosolic calcium but did increase normoxia-induced NO production. Moreover, acute normoxia increased PAEC barrier function, an effect that was potentiated by Y-27632. In fetal lambs, MV during the control period had no effect on LPA flow. In contrast, MV after Y-27632 increased LPA flow and fetal arterial P(O)? (Pa(O?)) and decreased PA pressure. In conclusion, ROCK activity modulates vascular tone in the perinatal pulmonary circulation via combined effects on PAEC NO production, barrier function, and smooth muscle tone. ROCK inhibition may represent a novel treatment strategy for neonatal pulmonary vascular disease.

    View details for DOI 10.1152/ajplung.00199.2010

    View details for Web of Science ID 000284941600016

    View details for PubMedID 20709731

  • Nuclear factor-kappa B activation in neonatal mouse lung protects against lipopolysaccharide-induced inflammation AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Alvira, C. M., Abate, A., Yang, G., Dennery, P. a., Rabinovitch, M. 2007; 175 (8): 805-815

    Abstract

    Injurious agents often cause less severe injury in neonates as compared with adults.We hypothesized that maturational differences in lung inflammation induced by lipopolysaccharide (LPS) may be related to the nature of the nuclear factor (NF)-kappaB complex activated, and the profile of target genes expressed.Neonatal and adult mice were injected with intraperitoneal LPS. Lung inflammation was assessed by histology, and apoptosis was determined by TUNEL (terminal deoxynucleotidyl transferase UTP nick-end labeling). The expression of candidate inflammatory and apoptotic mediators was evaluated by quantitative real-time polymerase chain reaction and Western immunoblot.Neonates demonstrated reduced inflammation and apoptosis, 24 hours after LPS exposure, as compared with adults. This difference was associated with persistent activation of NF-kappaB p65p50 heterodimers in the neonates in contrast to early, transient activation of p65p50 followed by sustained activation of p50p50 in the adults. Adults had increased expression of a panel of inflammatory and proapoptotic genes, and repression of antiapoptotic targets, whereas no significant changes in these mediators were observed in the neonates. Inhibition of NF-kappaB activity in the neonates decreased apoptosis, but heightened inflammation, with increased expression of the same inflammatory genes elevated in the adults. In contrast, inhibition of NF-kappaB in the adults resulted in partial suppression of the inflammatory response.NF-kappaB activation in the neonatal lung is antiinflammatory, protecting against LPS-mediated lung inflammation by repressing similar inflammatory genes induced in the adult.

    View details for DOI 10.1164/rccm.200608-11620C

    View details for Web of Science ID 000245724700012

    View details for PubMedID 17255561

  • Disrupted lung development and bronchopulmonary dysplasia: opportunities for lung repair and regeneration. Current opinion in pediatrics Baker, C. D., Alvira, C. M. 2014; 26 (3): 306-314

    Abstract

    Advances in medical therapy have increased survival of extremely premature infants and changed the pathology of bronchopulmonary dysplasia (BPD) from one of acute lung injury to a disease of disrupted lung development. With this evolution, new questions emerge regarding the molecular mechanisms that control postnatal lung development, the effect of early disruptions of postnatal lung development on long-term lung function, and the existence of endogenous mechanisms that permit lung regeneration after injury.Recent data demonstrate that a significant component of alveolarization, the final stage of lung development, occurs postnatally. Further, clinical and experimental studies demonstrate that premature birth disrupts alveolarization, decreasing the gas exchange surface area of the lung and causing BPD. BPD is associated with significant short-term morbidity, and new longitudinal, clinical data demonstrate that survivors of BPD have long-standing deficits in lung function and may be at risk for the development of additional lung disease as adults. Unfortunately, current care is mainly supportive with few effective therapies that prevent or treat established BPD. These studies underscore the need to further elucidate the mechanisms that direct postnatal lung growth and develop innovative strategies to stimulate lung regeneration.Despite significant improvements in the care and survival of extremely premature infants, BPD remains a major clinical problem. Although efforts should remain focused on the prevention of preterm labor and BPD, novel research aimed at promoting postnatal alveolarization offers a unique opportunity to develop effective strategies to treat established BPD.

    View details for DOI 10.1097/MOP.0000000000000095

    View details for PubMedID 24739494

  • Nuclear factor-kappa-B signaling in lung development and disease: One pathway, numerous functions. Birth defects research. Part A, Clinical and molecular teratology Alvira, C. M. 2014; 100 (3): 202-216

    Abstract

    In contrast to other organs, the lung completes a significant portion of its development after term birth. During this stage of alveolarization, division of the alveolar ducts into alveolar sacs by secondary septation, and expansion of the pulmonary vasculature by means of angiogenesis markedly increase the gas exchange surface area of the lung. However, postnatal completion of growth renders the lung highly susceptible to environmental insults such as inflammation that disrupt this developmental program. This is particularly evident in the setting of preterm birth, where impairment of alveolarization causes bronchopulmonary dysplasia, a chronic lung disease associated with significant morbidity. The nuclear factor ?-B (NF?B) family of transcription factors are ubiquitously expressed, and function to regulate diverse cellular processes including proliferation, survival, and immunity. Extensive evidence suggests that activation of NF?B is important in the regulation of inflammation and in the control of angiogenesis. Therefore, NF?B-mediated downstream effects likely influence the lung response to injury and may also mediate normal alveolar development. This review summarizes the main biologic functions of NF?B, and highlights the regulatory mechanisms that allow for diversity and specificity in downstream gene activation. This is followed by a description of the pro and anti-inflammatory functions of NF?B in the lung, and of NF?B-mediated angiogenic effects. Finally, this review summarizes the clinical and experimental data that support a role for NF?B in mediating postnatal angiogenesis and alveolarization, and discusses the challenges that remain in developing therapies that can selectively block the detrimental functions of NF?B yet preserve the beneficial effects. Birth Defects Research (Part A) 100:202-216, 2014. 2014 Wiley Periodicals, Inc.

    View details for DOI 10.1002/bdra.23233

    View details for PubMedID 24639404

  • Chronic Lung Disease in the Preterm Infant Lessons Learned from Animal Models AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Hilgendorff, A., Reiss, I., Ehrhardt, H., Eickelberg, O., Alvira, C. M. 2014; 50 (2): 233-245

    Abstract

    Neonatal chronic lung disease, also known as bronchopulmonary dysplasia (BPD), is the most common complication of premature birth, affecting up to 30% of very low birth weight infants. Improved medical care has allowed for the survival of the most premature infants and has significantly changed the pathology of BPD from a disease marked by severe lung injury to the "new" form characterized by alveolar hypoplasia and impaired vascular development. However, increased patient survival has led to a paucity of pathologic specimens available from infants with BPD. This, combined with the lack of a system to model alveolarization in vitro, has resulted in a great need for animal models that mimic key features of the disease. To this end, a number of animal models have been created by exposing the immature lung to injuries induced by hyperoxia, mechanical stretch, and inflammation and most recently by the genetic modification of mice. These animal studies have 1) allowed insight into the mechanisms that determine alveolar growth, 2) delineated factors central to the pathogenesis of neonatal chronic lung disease, and 3) informed the development of new therapies. In this review, we summarize the key findings and limitations of the most common animal models of BPD and discuss how knowledge obtained from these studies has informed clinical care. Future studies should aim to provide a more complete understanding of the pathways that preserve and repair alveolar growth during injury, which might be translated into novel strategies to treat lung diseases in infants and adults.

    View details for DOI 10.1165/rcmb.2013-0014TR

    View details for Web of Science ID 000331795600001

    View details for PubMedID 24024524

  • Hypoxia-inducible factor-1a in pulmonary artery smooth muscle cells lowers vascular tone by decreasing Myosin light chain phosphorylation. Circulation research Kim, Y., Barnes, E. A., Alvira, C. M., Ying, L., Reddy, S., Cornfield, D. N. 2013; 112 (9): 1230-1233

    Abstract

    Hypoxia-inducible factor-1? (HIF-1?), an oxygen (O2)-sensitive transcription factor, mediates transcriptional responses to low-O2 tension states. Although acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia can cause vascular remodeling and pulmonary hypertension, conflicting data exist on the role of HIF-1? in modulating pulmonary vascular tone.To investigate the role of smooth muscle cell (SMC)-specific HIF-1? in regulating pulmonary vascular tone. Methods andMice with an SMC-specific deletion of HIF-1? (SM22?-HIF-1?(-/-)) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1? modulates pulmonary vascular tone and the response to hypoxia. SM22?-HIF-1?(-/-) mice exhibited significantly higher right ventricular systolic pressure compared with wild-type littermates under normoxia and with exposure to either acute or chronic hypoxia in the absence of histological evidence of accentuated vascular remodeling. Moreover, myosin light chain phosphorylation, a determinant of SMC tone, was higher in PASMCs isolated from SM22?-HIF-1?(-/-) mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1? decreased myosin light chain phosphorylation in HIF-1?-null SMCs.In both normoxia and hypoxia, PASMC HIF-1? maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1? expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.

    View details for DOI 10.1161/CIRCRESAHA.112.300646

    View details for PubMedID 23513056

  • Hypoxia-inducible factor-1 alpha regulates KCNMB1 expression in human pulmonary artery smooth muscle cells AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Ahn, Y., Kim, Y., Adams, E., Lyu, S., Alvira, C. M., Cornfield, D. N. 2012; 302 (3): L352-L359

    Abstract

    Previously, we observed that hypoxia increases the expression of the ?1-subunit (KCNMB1) of the calcium-sensitive potassium channel (BK(Ca)). Herein, we elucidate the mechanism whereby hypoxia increases KCNMB1 expression in human pulmonary artery smooth muscle cells (hPASMC). In response to hypoxia, the expression of both the transcription factor hypoxia-inducible factor 1-? (HIF-1?) and KCNMB1 are increased. Knockdown of HIF-1? using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1? binding to three discrete regions of the human KCNMB1 promoter known to contain hypoxia response elements (HREs). A KCNMB1 promoter reporter assay combined with site-directed mutagenesis identified two adjacent HREs located between -3,540 bp and -3,311 bp that are essential for the hypoxic induction of KCNMB1 promoter activity. Furthermore, additional ChIP assays demonstrated recruitment of the HIF-1? transcriptional coactivator, p300, to this same promoter region. Treatment of hPASMC with the histone deacetylase inhibitor, trichostatin, prolonged the increase in KCNMB1 observed with hypoxia, suggesting that alterations in chromatin remodeling function to limit the hypoxic induction of KCNMB1. Finally, KCNMB1 knockdown potentiated the hypoxia-induced increase in cytosolic calcium in hPASMC, highlighting the contribution of the ?1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1? increases KCNMB1 expression in response to hypoxia in hPASMC by binding to two HREs located at -3,540 to -3,311 of the KCNMB1 promoter. We speculate that selective modulation of KCNMB1 expression may serve as a novel therapeutic approach to address diseases characterized by an increase in vascular tone.

    View details for DOI 10.1152/ajplung.00302.2011

    View details for Web of Science ID 000300245600009

    View details for PubMedID 22114151

  • 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-?

    Abstract

    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-? (TGF-?) signaling. Although TGF-? blockade prevents aneurysms in MFS mouse models, the mechanisms through which excessive TGF-? 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 ?B, a repressor of miR-29b, and a factor suppressed by TGF-?, was also observed in Fbn1(C1039G/+) aorta. Furthermore, administration of a nuclear factor ?B inhibitor increased miR-29b levels, whereas TGF-? 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

  • Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions AMERICAN JOURNAL OF PATHOLOGY Kim, Y., Haghighat, L., Spiekerkoetter, E., Sawada, H., Alvira, C. M., Wang, L., Acharya, S., Rodriguez-Colon, G., Orton, A., Zhao, M., Rabinovitch, M. 2011; 179 (3): 1560-1572

    Abstract

    Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease.

    View details for DOI 10.1016/j.ajpath.2011.05.051

    View details for Web of Science ID 000298307300049

    View details for PubMedID 21763677

  • Prolonged mechanical ventilation with air induces apoptosis and causes failure of alveolar septation and angiogenesis in lungs of newborn mice AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Mokres, L. M., Parai, K., Hilgendorff, A., Ertsey, R., Alvira, C. M., Rabinovitch, M., Bland, R. D. 2010; 298 (1): L23-L35

    Abstract

    Defective lung septation and angiogenesis, quintessential features of neonatal chronic lung disease (CLD), typically result from lengthy exposure of developing lungs to mechanical ventilation (MV) and hyperoxia. Previous studies showed fewer alveoli and microvessels, with reduced VEGF and increased transforming growth factor-beta (TGFbeta) signaling, and excess, scattered elastin in lungs of premature infants and lambs with CLD vs. normal controls. MV of newborn mice with 40% O(2) for 24 h yielded similar lung structural abnormalities linked to impaired VEGF signaling, dysregulated elastin production, and increased apoptosis. These studies could not determine the relative importance of cyclic stretch vs. hyperoxia in causing these lung growth abnormalities. We therefore studied the impact of MV for 24 h with air on alveolar septation (quantitative lung histology), angiogenesis [CD31 quantitative-immunohistochemistry (IHC), immunoblots], apoptosis [TdT-mediated dUTP nick end labeling (TUNEL), active caspase-3 assays], VEGF signaling [VEGF-A, VEGF receptor 1 (VEGF-R1), VEGF-R2 immunoblots], TGFbeta activation [phosphorylated Smad2 (pSmad2) quantitative-IHC], and elastin production (tropoelastin immunoblots, quantitative image analysis of Hart's stained sections) in lungs of 6-day-old mice. Compared with unventilated controls, MV caused a 3-fold increase in alveolar area, approximately 50% reduction in alveolar number and endothelial surface area, >5-fold increase in apoptosis, >50% decrease in lung VEGF-R2 protein, 4-fold increase of pSmad2 protein, and >50% increase in lung elastin, which was distributed throughout alveolar walls rather than at septal tips. This study is the first to show that prolonged MV of developing lungs, without associated hyperoxia, can inhibit alveolar septation and angiogenesis and increase apoptosis and lung elastin, findings that could reflect stretch-induced changes in VEGF and TGFbeta signaling, as reported in CLD.

    View details for DOI 10.1152/ajplung.00251.2009

    View details for Web of Science ID 000272827900005

    View details for PubMedID 19854954

  • Tie2-mediated loss of peroxisome proliferator-activated receptor-gamma in mice causes PDGF receptor-beta-dependent pulmonary arterial muscularization AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Guignabert, C., Alvira, C. M., Alastalo, T., Sawada, H., Hansmann, G., Zhao, M., Wang, L., El-Bizri, N., Rabinovitch, M. 2009; 297 (6): L1082-L1090

    Abstract

    Peroxisome proliferator-activated receptor (PPAR)-gamma is reduced in pulmonary arteries (PAs) of patients with PA hypertension (PAH), and we reported that deletion of PPARgamma in smooth muscle cells (SMCs) of transgenic mice results in PAH. However, the sequelae of loss of PPARgamma in PA endothelial cells (ECs) are unknown. Therefore, we bred Tie2-Cre mice with PPARgamma(flox/flox) mice to induce EC loss of PPARgamma (Tie2 PPARgamma(-/-)), and we assessed PAH by right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and muscularized distal PAs in room air (RA), after chronic hypoxia (CH), and after 4 wk of recovery in RA (Rec-RA). The Tie2 PPARgamma(-/-) mice developed spontaneous PAH in RA with increased RVSP, RVH, and muscularized PAs vs. wild type (WT); both genotypes exhibited a similar degree of PAH following chronic hypoxia, but Tie2 PPARgamma(-/-) mice had more residual PAH compared with WT mice after Rec-RA. The Tie2 PPARgamma(-/-) vs. WT mice in RA had increased platelet-derived growth factor receptor-beta (PDGF-Rbeta) expression and signaling, despite an elevation in the PPARgamma target apolipoprotein E, an inhibitor of PDGF signaling. Inhibition of PDGF-Rbeta signaling with imatinib, however, was sufficient to reverse the PAH observed in the Tie2 PPARgamma(-/-) mice. Thus the disruption of PPARgamma signaling in EC is sufficient to cause mild PAH and to impair recovery from CH-induced PAH. Inhibition of heightened PDGF-Rbeta signaling is sufficient to reverse PAH in this genetic model.

    View details for DOI 10.1152/ajplung.00199.2009

    View details for Web of Science ID 000272017900009

    View details for PubMedID 19801450

  • LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor JOURNAL OF CELL SCIENCE Ying, L., Lau, A., Alvira, C. M., West, R., Cann, G. M., Zhou, B., Kinnear, C., Jan, E., Sarnow, P., van de Rijn, M., Rabinovitch, M. 2009; 122 (9): 1441-1451

    Abstract

    Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3' UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types.

    View details for DOI 10.1242/jcs.025957

    View details for Web of Science ID 000265443300020

    View details for PubMedID 19366727

  • An antiproliferative BMP-2/PPAR gamma/apoE axis in human and murine SMCs and its role in pulmonary hypertension JOURNAL OF CLINICAL INVESTIGATION Hansmann, G., de Jesus Perez, V. A., Alastalo, T., Alvira, C. M., Guignabert, C., Bekker, J. M., Schellong, S., Urashima, T., Wang, L., Morrell, N. W., Rabinovitch, M. 2008; 118 (5): 1846-1857

    Abstract

    Loss-of-function mutations in bone morphogenetic protein receptor II (BMP-RII) are linked to pulmonary arterial hypertension (PAH); the ligand for BMP-RII, BMP-2, is a negative regulator of SMC growth. Here, we report an interplay between PPARgamma and its transcriptional target apoE downstream of BMP-2 signaling. BMP-2/BMP-RII signaling prevented PDGF-BB-induced proliferation of human and murine pulmonary artery SMCs (PASMCs) by decreasing nuclear phospho-ERK and inducing DNA binding of PPARgamma that is independent of Smad1/5/8 phosphorylation. Both BMP-2 and a PPARgamma agonist stimulated production and secretion of apoE by SMCs. Using a variety of methods, including short hairpin RNAi in human PASMCs, PAH patient-derived BMP-RII mutant PASMCs, a PPARgamma antagonist, and PASMCs isolated from PPARgamma- and apoE-deficient mice, we demonstrated that the antiproliferative effect of BMP-2 was BMP-RII, PPARgamma, and apoE dependent. Furthermore, we created mice with targeted deletion of PPARgamma in SMCs and showed that they spontaneously developed PAH, as indicated by elevated RV systolic pressure, RV hypertrophy, and increased muscularization of the distal pulmonary arteries. Thus, PPARgamma-mediated events could protect against PAH, and PPARgamma agonists may reverse PAH in patients with or without BMP-RII dysfunction.

    View details for DOI 10.1172/JCI32503

    View details for Web of Science ID 000255490100028

    View details for PubMedID 18382765

  • Reactivation of gamma HV68 induces neointimal lesions in pulmonary arteries of S100A4/Mts1-overexpressing mice in association with degradation of elastin AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Spiekerkoetter, E., Alvira, C. M., Kim, Y., Bruneau, A., Pricola, K. L., Wang, L., Ambartsumian, N., Rabinovitch, M. 2008; 294 (2): L276-L289

    Abstract

    S100A4/Mts-overexpressing mice have thick elastic laminae and mild pulmonary arterial hypertension (PAH), and the occasional older mouse develops occlusive neointimal lesions and perivascular inflammation. We hypothesized that a vasculotropic virus could induce neointimal lesions in the S100A4/Mts1 mouse by facilitating breakdown of elastin and migration and proliferation of smooth muscle cells. To test this hypothesis, we infected S100A4/Mts1 mice with gammaherpesvirus 68 (gammaHV68). We observed, 6 mo after gammaHV68 [4 x 10(3) plaque-forming units (PFU)], perivascular inflammation in 10/15 S100A4/Mts1 mice and occlusive neointimal formation in 3/10 mice, accompanied by striking degradation of elastin. We then compared the early response after high-dose gammaHV68 (4 x 10(6) PFU) in C57Bl/6 and S100A4/Mts1 mice. In S100A4/Mts1 mice only, significant PAH, muscularization of distal vessels, and elastase activity were observed 6 wk after gammaHV68. These features resolved by 3 mo without neointimal formation. We therefore infected mice with the M1-gammaHV68 strain that reactivates from latency with higher efficiency and observed neointimal lesions at 3 mo in 2/5 C57Bl/6 (5-9% of vessels) and in 5/5 S100A4/Mts1 mice (13-40% of vessels) accompanied by mild PAH, heightened lung elastase activity, and intravascular viral expression. This suggested that enhanced generation of elastin peptides in S100A4/Mts1 mice may promote increased viral entry in the vessel wall. Using S100A4/Mts1 PA organ culture, we showed, in response to elastase activity, heightened production of elastin peptides associated with invasion of inflammatory cells and intravascular viral antigen. We therefore propose that early viral access to the vessel wall may be a critical determinant of the extent of vascular pathology following reactivation.

    View details for DOI 10.1152/ajplung.00414.2007

    View details for Web of Science ID 000253067400017

    View details for PubMedID 18083765

  • Mechanical ventilation uncouples synthesis and assembly of elastin and increases apoptosis in lungs of newborn mice. Prelude to defective alveolar septation during lung development? AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Bland, R. D., Ertsey, R., Mokres, L. M., Xu, L., Jacobson, B. E., Jiang, S., Alvira, C. M., Rabinovitch, M., Shinwell, E. S., Dixit, A. 2008; 294 (1): L3-L14

    Abstract

    Prolonged mechanical ventilation (MV) with O2-rich gas inhibits lung growth and causes excess, disordered accumulation of lung elastin in preterm infants, often resulting in chronic lung disease (CLD). Using newborn mice, in which alveolarization occurs postnatally, we designed studies to determine how MV with either 40% O2 or air might lead to dysregulated elastin production and impaired lung septation. MV of newborn mice for 8 h with either 40% O2 or air increased lung mRNA for tropoelastin and lysyl oxidase, relative to unventilated controls, without increasing lung expression of genes that regulate elastic fiber assembly (lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, emilin-1). Serine elastase activity in lung increased fourfold after MV with 40% O2, but not with air. We then extended MV with 40% O2 to 24 h and found that lung content of tropoelastin protein doubled, whereas lung content of elastin assembly proteins did not change (lysyl oxidases, fibrillins) or decreased (fibulin-5, emilin-1). Quantitative image analysis of lung sections showed that elastic fiber density increased by 50% after MV for 24 h, with elastin distributed throughout the walls of air spaces, rather than at septal tips, as in control lungs. Dysregulation of elastin was associated with a threefold increase in lung cell apoptosis (TUNEL and caspase-3 assays), which might account for the increased air space size previously reported in this model. Our findings of increased elastin synthesis, coupled with increased elastase activity and reduced lung abundance of proteins that regulate elastic fiber assembly, could explain altered lung elastin deposition, increased apoptosis, and defective septation, as observed in CLD.

    View details for DOI 10.1152/ajplung.00362.2007

    View details for Web of Science ID 000252398600002

    View details for PubMedID 17934062

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