Cristina M. Alvira

An antiproliferative BMP-2/PPARγ/apoE axis in human and murine SMCs and its role in pulmonary hypertension

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.

miR-29b Participates in Early Aneurysm Development in Marfan Syndrome

Rationale: 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-&bgr; (TGF-&bgr;) signaling. Although TGF-&bgr; blockade prevents aneurysms in MFS mouse models, the mechanisms through which excessive TGF-&bgr; causes aneurysms remain ill-defined. Objective: We investigated the role of microRNA-29b (miR-29b) in aneurysm formation in MFS. Methods and Results: 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 (Fbn1C1039G/+) 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 Fbn1C1039G/+ aorta. Histological evidence of decreased and fragmented elastin was observed exclusively in the Fbn1C1039G/+ 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 &kgr;B, a repressor of miR-29b, and a factor suppressed by TGF-&bgr;, was also observed in Fbn1C1039G/+ aorta. Furthermore, administration of a nuclear factor &kgr;B inhibitor increased miR-29b levels, whereas TGF-&bgr; blockade or losartan effectively decreased miR-29b levels in Fbn1C1039G/+ mice. Finally, miR-29b blockade by locked nucleic acid antisense oligonucleotides prevented early aneurysm development, aortic wall apoptosis, and extracellular matrix deficiencies. Conclusions: 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.

Tie2-mediated loss of peroxisome proliferator-activated receptor-γ in mice causes PDGF receptor-β-dependent pulmonary arterial muscularization

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.

Prolonged mechanical ventilation with air induces apoptosis and causes failure of alveolar septation and angiogenesis in lungs of newborn mice

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 Harts 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.

Inhibiting NF-κB in the developing lung disrupts angiogenesis and alveolarization.

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-κB, a transcription factor traditionally associated with inflammation, plays a unique protective role in the neonatal lung. Therefore, we hypothesized that constitutive NF-κB activity is essential for postnatal lung development. Blocking NF-κB 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-κB in neonatal lung and in primary pulmonary endothelial cells (PEC) compared with adult. Moreover, inhibiting constitutive NF-κB 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-κB 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-κB in promoting physiological angiogenesis and alveolarization in the developing lung. Our data suggest that disruption of NF-κB signaling may contribute to the pathogenesis of BPD and that enhancement of NF-κB may represent a viable therapeutic strategy to promote lung growth and regeneration in pulmonary diseases marked by impaired angiogenesis.

Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions

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.

Hypoxia Inducible Factor-1α in Pulmonary Artery Smooth Muscle Cells Lowers Vascular Tone by Decreasing Myosin Light Chain Phosphorylation

Rationale: Hypoxia-inducible factor-1&agr; (HIF-1&agr;), 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&agr; in modulating pulmonary vascular tone. Objective: To investigate the role of smooth muscle cell (SMC)–specific HIF-1&agr; in regulating pulmonary vascular tone. Methods and Results: Mice with an SMC-specific deletion of HIF-1&agr; (SM22&agr;-HIF-1&agr;−/−) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1&agr; modulates pulmonary vascular tone and the response to hypoxia. SM22&agr;-HIF-1&agr;−/− 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&agr;-HIF-1&agr;−/− mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1&agr; decreased myosin light chain phosphorylation in HIF-1&agr;–null SMCs. Conclusions: In both normoxia and hypoxia, PASMC HIF-1&agr; maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1&agr; expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.

Nuclear Factor-Kappa-B Signaling in Lung Development and Disease: One Pathway, Numerous Functions

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 κ-B (NFκB) 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 NFκB is important in the regulation of inflammation and in the control of angiogenesis. Therefore, NFκB-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 NFκB, 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 NFκB in the lung, and of NFκB-mediated angiogenic effects. Finally, this review summarizes the clinical and experimental data that support a role for NFκB in mediating postnatal angiogenesis and alveolarization, and discusses the challenges that remain in developing therapies that can selectively block the detrimental functions of NFκB yet preserve the beneficial effects. Birth Defects Research (Part A) 100:202–216, 2014.

Disrupted lung development and bronchopulmonary dysplasia: opportunities for lung repair and regeneration

Purpose of review 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 findings 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. Summary 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.

Hypoxia-inducible factor-1α regulates KCNMB1 expression in human pulmonary artery smooth muscle cells

Previously, we observed that hypoxia increases the expression of the β1-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-α (HIF-1α) and KCNMB1 are increased. Knockdown of HIF-1α using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1α 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-1α 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 β1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1α 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.