Upregulation of steroidogenic acute regulatory protein by hypoxia stimulates aldosterone synthesis in pulmonary artery endothelial cells to promote pulmonary vascular fibrosis

Abstract

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.

Keywords: aldosterone; cell hypoxia; fibrosis; hypertension, pulmonary.

Figure 1. Hypoxia increases aldosterone levels in human pulmonary artery endothelial cells

(A) Human pulmonary artery endothelial cells (HPAECs) were exposed to normoxia (21% FiO₂) or hypoxia (2.0% FiO₂) for 24 h (n=5), and enzyme immunoassay was performed to quantify aldosterone (ALDO) levels in the culture medium. (B) Liquid chromatography-mass spectrometry (LC-MS) was performed as a second methodology to confirm our findings that hypoxia increased ALDO levels in HPAECs. The total ion chromatogram with peak intensities normalized to protein content demonstrated a peak at a retention time of 3.68 min that corresponds to (C) the correct MS-MS spectrum identifying the aldosterone parent ion at 359 m/z [M-H⁻¹], as well as the three daughter ions at m/z 189, 297, and 331. This peak was not observed in unconditioned medium (i.e., culture medium not used to treat HPAECs). a.u., arbitrary units. Data are presented as mean ± S.E..

Figure 2. Hypoxia increases StAR protein expression in pulmonary artery endothelial cells

(A) Human pulmonary artery endothelial cells were exposed to normoxia (21% FiO₂) or hypoxia (2.0% FiO₂) for 24 h and protein levels of StAR were assessed by immunoblotting (n=3) and (B) anti-StAR immunohistochemistry (n=3). Data are presented as mean ± S.E. a.u., arbitrary units. Representative blots and photomicrographs (at 400× magnification) are shown.

Figure 3. Hypoxia increases StAR activity to promote aldosterone synthesis in pulmonary artery endothelial cells (HAPECs)

(A) HPAECs were exposed to normoxia (21% FiO₂) or hypoxia (2.0% FiO₂) for 15, 30, or 60 min and protein levels of c-Fos (n=3) and c-Jun (n=3), which are known to influence StAR activity in adrenal cells, were assessed by immunoblotting. (B) The effect of hypoxia on c-Fos/c-Jun association with the StAR promoter was assessed by chromatin immunoprecipitation (n=3). Compared to normoxia, association with StAR was increased by hypoxia for 1 hr for c-Jun and c-Fos. IgG served as negative control. (C) The functional effect of increased c-Fos/c-Jun association with the StAR promoter to changes in aldosterone (ALDO) levels mediated by hypoxia was assessed next. To accomplish this, StAR expression was inhibited using StAR siRNA (40 nM)(Si-StAR). Cells transfected with si-StAR and stimulated with hypoxia demonstrated decreased StAR expression compared to hypoxia-stimulated cells treated with vehicle control (n=3), which was associated with (D) a significant decrease in ALDO levels in normoxia-treated cells and in cells stimulated with hypoxia (n=6). a.u., arbitrary units. NS, not statistically significant; V, vehicle control, which was OptiMEM I media; SSc, negative (scrambled) control siRNA; Lipo, Lipofectamine™ 2000; ANOVA, analysis of variance. Data are presented as mean ± S.E.; Representative blots are shown.

Figure 4. Hypoxia increases StAR activity to increase progesterone and aldosterone levels in pulmonary artery endothelial cells

Human pulmonary artery endothelial cells were treated with vehicle (V) control or 20-α-hydroxycholesterol (20-α-OH-C) (5 μg/ml) at the time of exposure to normoxia (21% FiO₂) or hypoxia (2.0% FiO₂) for 24 h. As a soluble cholesterol derivative, 20-α-OH-C is a bioavailable substrate for steroidogenic acute regulatory protein (StAR) activation; thus, supplementing cells with 20-α-OH-C affords assessment of maximal StAR activity. The effect of 20-α-OH-C and hypoxia on levels of (A) progesterone (n=4) and (B) aldosterone (ALDO)(n=3) in the cell culture medium was assessed by enzyme immunoassay. ANOVA, analysis of variance. Data are presented as mean ± S.E.

Figure 5. Aldosterone promotes hypoxia-induced pulmonary vascular remodeling and fibrosis

Human pulmonary artery endothelial cells were treated with vehicle (V) control or the mineralocorticoid receptor antagonist spironolactone (SP)(10μM) at the time of exposure to normoxia (21% FiO₂) or hypoxia (2.0% FiO₂) for 24 h, and expression levels of collagen III and connective tissue growth factor (CTGF), which are associated with hypoxia-mediated vascular fibrosis, and matrix-metalloproteinase (MMP)-2 and MMP-9, which are associated with hypoxia-mediated vascular remodeling, were assessed by Western immunoblot. n=3 for each immunoblot. Data are presented as mean ± S.E. Representative blots are shown.

Figure 6. Increased aldosterone levels stimulated by hypoxia in HPAECs are sufficient to promote CTGF upregulation in pulmonary artery smooth muscle cells (PASMCs) in vitro

(A) Cultured human PASMCs were exposed for 24 hr to normoxia (21% FiO₂) or hypoxia (2.0% FiO₂) in the presence or absence of standard culture medium or conditioned medium (CM) from HPAECs treated with normoxia or hypoxia for 24 hr, and (B) connective tissue growth factor (CTGF) expression levels were assessed by Western immunoblot (IB) (n=3). Data are presented as mean ± S.E. Representative blots are shown.

Figure 7. StAR is increased in experimental pulmonary arterial hypertension (PAH) and in PAH patients in vitro

(A) Paraffin-embedded lung sections were harvested from PAH patients (n=4) or age-matched controls (n=4) and anti-StAR Vector red immunohistochemical staining was performed on pulmonary arterioles. (B) In a prevention study, Sugen-5416/hypoxia3-treated rats were randomized to receive standard chow or the mineralocorticoid receptor antagonist eplerenone (EPL)(0.6 mg/1g chow) immediately following exposure to hypoxia until completion of the study 21 days later. Severe pulmonary hypertension and hyperaldosteronism in lung homogenates was confirmed (ref. 7), and the effect of EPL on vascular fibrosis in paraffin-embedded pulmonary arterioles specimens (20-50 μm in diameter) was assessed using anti-connective tissue growth factor (CTGF) and anti-collagen III immunohistochemistry (n=4-6 rats/condition). a.u., arbitrary units. Data are presented as mean ± S.E. Representative photomicrographs (400× magnification) are shown.