Elena Arons

Impaired Vasoconstriction and Nitric Oxide-Mediated Relaxation in Pulmonary Arteries of Hypoxia- and Monocrotaline-Induced Pulmonary Hypertensive Rats

Pulmonary hypertension (PH) is a life-threatening disease with unclear vascular mechanisms. We tested whether PH involves abnormal pulmonary vasoconstriction and impaired vasodilation. Male Sprague-Dawley rats were exposed to hypoxia (9% O2) for 2 weeks or injected with single dose of monocrotaline (MCT, 60 mg/kg s.c.). Control rats were normoxic or injected with saline. After the hemodynamic measurements were performed, pulmonary and mesenteric arteries were isolated for measurement of vascular function. Hematocrit was elevated in hypoxic rats. Right ventricular systolic pressure and Fultons Index [right/(left + septum) ventricular weight] were greater in hypoxic and MCT-treated rats than in normoxic rats. Pulmonary artery contraction by phenylephrine and 96 mM KCl was less in hypoxic and MCT-treated rats than in normoxic rats. Acetylcholine-induced relaxation was less in the pulmonary arteries of hypoxic and MCT-treated rats than of normoxic rats, suggesting reduced effects of endothelium-derived vasodilators. The nitric oxide synthase inhibitor, Nω-nitro-l-arginine methyl ester, and the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, inhibited acetylcholine relaxation, suggesting that it was mediated by nitric oxide (NO)-cGMP. The NO donor sodium nitroprusside caused less relaxation in the pulmonary arteries of hypoxic and MCT-treated than of normoxic rats, suggesting decreased responsiveness of vascular smooth muscle cells (VSMCs) to vasodilators. Phenylephrine and KCl contraction and acetylcholine and sodium nitroprusside relaxation were not different in the mesenteric arteries from all groups. In lung tissue sections, the wall thickness of pulmonary arterioles was greater in hypoxic and MCT-treated rats than in normoxic rats. The specific reductions in pulmonary, but not systemic, arterial vasoconstriction and vasodilation in hypoxia- and MCT-induced PH are consistent with the possibility of de-differentiation of pulmonary VSMCs to a more proliferative/synthetic and less contractile phenotype in PH.

Upregulation of Steroidogenic Acute Regulatory Protein by Hypoxia Stimulates Aldosterone Synthesis in Pulmonary Artery Endothelial Cells to Promote Pulmonary Vascular Fibrosis

Background— The molecular mechanism(s) regulating hypoxia-induced vascular fibrosis are unresolved. Hyperaldosteronism correlates positively with vascular remodeling in pulmonary arterial hypertension, suggesting that aldosterone may contribute to the pulmonary vasculopathy of hypoxia. The hypoxia-sensitive transcription factors c-Fos/c-Jun regulate steroidogenic acute regulatory protein (StAR), which facilitates the rate-limiting step of aldosterone steroidogenesis. We hypothesized that c-Fos/c-Jun upregulation by hypoxia activates StAR-dependent aldosterone synthesis in human pulmonary artery endothelial cells (HPAECs) to promote vascular fibrosis in pulmonary arterial hypertension. Methods and Results— Patients with pulmonary arterial hypertension, rats with Sugen/hypoxia–pulmonary arterial hypertension, and mice exposed to chronic hypoxia expressed increased StAR in remodeled pulmonary arterioles, providing a basis for investigating hypoxia–StAR signaling in HPAECs. Hypoxia (2.0% FiO2) increased aldosterone levels selectively in HPAECs, which was confirmed by liquid chromatography–mass spectrometry. Increased aldosterone by hypoxia resulted from enhanced c-Fos/c-Jun binding to the proximal activator protein-1 site of the StAR promoter in HPAECs, which increased StAR expression and activity. In HPAECs transfected with StAR–small interfering RNA or treated with the activator protein-1 inhibitor SR-11302 [3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid], hypoxia failed to increase aldosterone, confirming that aldosterone biosynthesis required StAR activation by c-Fos/c-Jun. The functional consequences of aldosterone were confirmed by pharmacological inhibition of the mineralocorticoid receptor with spironolactone or eplerenone, which attenuated hypoxia-induced upregulation of the fibrogenic protein connective tissue growth factor and collagen III in vitro and decreased pulmonary vascular fibrosis to improve pulmonary hypertension in vivo. Conclusion— Our findings identify autonomous aldosterone synthesis in HPAECs attributable to hypoxia-mediated upregulation of StAR as a novel molecular mechanism that promotes pulmonary vascular remodeling and fibrosis.

The p38 Mitogen-Activated Protein kinase pathway is involved in the regulation of Heme Oxygenase-1 by acidic extracellular pH in aortic smooth muscle cells

Extracellular acidosis (EA) regulates Heme Oxygenase‐1 (HO‐1) expression in vascular smooth muscle cells via transcriptional and posttranscriptional mechanisms but the signaling pathways involved are not known. We examined the role of Mitogen‐Activated Protein Kinase (MAPK) pathways in HO‐1 regulation by EA. Primary rat aortic smooth muscle cells were exposed to EA or physiologic pH. Levels of the total and phosphorylated forms of p38, extracellular signal‐regulated protein kinases1/2 (ERK1/2), c‐Jun N‐terminal kinases/stress‐activated protein kinases (JNK1/2), and HO‐1 protein were assessed by Western analysis and HO‐1 mRNA levels were assessed by quantitative PCR. Inhibition of p38 MAPK was achieved with the chemical inhibitor SB203580, or adenoviral infection of a dominant‐negative form of p38α. Phospho p38 MAPK activity was evaluated with an in vitro kinase activity assay. Binding of Activator Protein‐1 (AP‐1), a known target of MAPK pathways, was assessed by Electromobility shift assay (EMSA). EA induced phosphorylation of p38 MAPK in a biphasic manner while total p38 was unchanged. EA did not alter levels of phospho ERK 1/2 and phospho JNK 1/2. There was increased phospho p38 MAPK activity in the setting of EA which preceded the induction of HO‐1. Inhibition of phospho p38 activity with either SB20358 or a dominant negative p38α oligonucleotide abrogated the induction of HO‐1 by EA. Increased specific binding of AP‐1 in the setting of EA was shown by EMSA. Increased phospho p38 activity precedes and likely mediates HO‐1 induction by EA. Increased AP‐1 binding may underlie the transcriptional regulation of HO‐1 by EA. J. Cell. Biochem. 105: 1298–1306, 2008.

Heme Oxygenase-1 Does Not Mediate the Effects of Extracellular Acidosis on Vascular Smooth Muscle Cell Proliferation, Migration, and Susceptibility to Apoptosis

Background: Unbalanced vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis contribute to vascular disorders such as atherosclerosis, restenosis, and pulmonary hypertension. The effect of extracellular acidosis (EA) on VSMC homeostasis is incompletely understood but we previously reported that EA increases heme oxygenase-1 (HO-1) expression in VSMCs. Since HO-1 regulates VSMC proliferation and apoptosis we sought to define the role of HO-1 in VSMC responses to EA. Methods: Mouse aortic smooth muscle cells (MASMCs) were isolated from wild-type and HO-1-null mice. Cell proliferation and migration assays were done in a physiologic pH (7.4) or EA (pH 6.8). VSMC apoptosis in response to hydrogen peroxide was assessed by JC-1 staining, caspase-3 cleavage, annexin V, and Hoechst staining. Results: Wild-type MASMCs showed decreased proliferation and migration at pH 6.8 compared to pH 7.4. This observation was also true in HO-1-null MASMCs. Although wild-type and HO-1-null cells showed differences in the mode and kinetics of cell death, both genotypes exhibited increased susceptibility to hydrogen peroxide-induced apoptosis at pH 6.8 compared to 7.4. Conclusions: EA inhibits VSMC proliferation and migration and increases susceptibility to oxidant-induced apoptosis. These effects of acidosis on VSMC homeostasis are independent of HO-1.

Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension

Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone‐Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU‐5416/hypoxia‐PAH rats and monocrotaline‐PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone‐Raptor signaling in human PASMCs. Aldosterone (10‐9 to 10‐7 M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor‐small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro. Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine‐monomethoxy polyethylene glycol that was formulated with Raptor‐small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo. Up‐regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.—Aghamohammadzadeh, R., Zhang, Y.‐Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.‐M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up‐regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension. FASEB J. 30, 2511‐2527 (2016). www.fasebj.org

Improved pulmonary vascular reactivity and decreased hypertrophic remodeling during nonhypercapnic acidosis in experimental pulmonary hypertension

Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in decreased responsiveness of pulmonary circulation to vasodilator therapies. We have shown that extracellular acidosis inhibits VSMC proliferation and migration in vitro. Here we tested whether induction of nonhypercapnic acidosis in vivo ameliorates PH and the underlying pulmonary vascular remodeling and dysfunction. Adult male Sprague-Dawley rats were exposed to hypoxia (8.5% O(2)) for 2 wk, or injected subcutaneously with monocrotaline (MCT, 60 mg/kg) to develop PH. Acidosis was induced with NH(4)Cl (1.5%) in the drinking water 5 days prior to and during the 2 wk of hypoxic exposure (prevention protocol), or after MCT injection from day 21 to 28 (reversal protocol). Right ventricular systolic pressure (RVSP) and Fultons index were measured, and pulmonary arteriolar remodeling was analyzed. Pulmonary and mesenteric artery contraction to phenylephrine (Phe) and high KCl, and relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) were examined ex vivo. Hypoxic and MCT-treated rats demonstrated increased RVSP, Fultons index, and pulmonary arteriolar thickening. In pulmonary arteries of hypoxic and MCT rats there was reduced contraction to Phe and KCl and reduced vasodilation to ACh and SNP. Acidosis prevented hypoxia-induced PH, reversed MCT-induced PH, and resulted in reduction in all indexes of PH including RVSP, Fultons index, and pulmonary arteriolar remodeling. Pulmonary artery contraction to Phe and KCl was preserved or improved, and relaxation to ACh and SNP was enhanced in NH(4)Cl-treated PH animals. Acidosis alone did not affect the hemodynamics or pulmonary vascular function. Phe and KCl contraction and ACh and SNP relaxation were not different in mesenteric arteries of all groups. Thus nonhypercapnic acidosis ameliorates experimental PH, attenuates pulmonary arteriolar thickening, and enhances pulmonary vascular responsiveness to vasoconstrictor and vasodilator stimuli. Together with our finding that acidosis decreases VSMC proliferation, the results are consistent with the possibility that nonhypercapnic acidosis promotes differentiation of pulmonary VSMCs to a more contractile phenotype, which may enhance the effectiveness of vasodilator therapies in PH.

NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension

TGF-β–independent oxidative modification of NEDD9 at Cys18 promotes vascular fibrosis and the pathobiology of pulmonary arterial hypertension. In silico sleuthing for pulmonary hypertension Cell proliferation and fibrosis (the accumulation of excess connective tissue) cause arterioles to thicken during pulmonary arterial hypertension (PAH). Aldosterone, a hormone involved in reactive oxygen species generation, wound healing, and fibrosis, is also increased in PAH. Samokhin et al. used in silico analysis to identify NEDD9 as a critical protein involved in vascular remodeling, fibrosis, and pulmonary hypertension. Oxidative modification of NEDD9 impaired its degradation and promoted collagen production. Mice lacking NEDD9 were protected from pulmonary hypertension, and collagen deposition, vascular remodeling, and cardiopulmonary metrics were normalized in a rat model of PAH treated with siRNA targeting NEDD9. This work suggests that therapeutic targeting of NEDD9 may be effective for combating vascular fibrosis in PAH. Germline mutations involving small mothers against decapentaplegic–transforming growth factor–β (SMAD–TGF-β) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). We developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys18 in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to COL3A1. Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-β ligand trap and, thus, was not contingent on TGF-β signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-β–independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH.