PPARγ Ligands Attenuate Hypoxia-Induced Proliferation in Human Pulmonary Artery Smooth Muscle Cells through Modulation of MicroRNA-21

<div><p>Pulmonary hypertension (PH) is a progressive and often fatal disorder whose pathogenesis involves pulmonary artery smooth muscle cell (PASMC) proliferation. Although modern PH therapies have significantly improved survival, continued progress rests on the discovery of novel therapies and molecular targets. MicroRNA (miR)-21 has emerged as an important non-coding RNA that contributes to PH pathogenesis by enhancing vascular cell proliferation, however little is known about available therapies that modulate its expression. We previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) agonists attenuated hypoxia-induced HPASMC proliferation, vascular remodeling and PH through pleiotropic actions on multiple targets, including transforming growth factor (TGF)-β1 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN). PTEN is a validated target of miR-21. We therefore hypothesized that antiproliferative effects conferred by PPARγ activation are mediated through inhibition of hypoxia-induced miR-21 expression. Human PASMC monolayers were exposed to hypoxia then treated with the PPARγ agonist, rosiglitazone (RSG,10 μM), or in parallel, C57Bl/6J mice were exposed to hypoxia then treated with RSG. RSG attenuated hypoxic increases in miR-21 expression in vitro and in vivo and abrogated reductions in PTEN and PASMC proliferation. Antiproliferative effects of RSG were lost following siRNA-mediated PTEN depletion. Furthermore, miR-21 mimic decreased PTEN and stimulated PASMC proliferation, whereas miR-21 inhibition increased PTEN and attenuated hypoxia-induced HPASMC proliferation. Collectively, these results demonstrate that PPARγ ligands regulate proliferative responses to hypoxia by preventing hypoxic increases in miR-21 and reductions in PTEN. These findings further clarify molecular mechanisms that support targeting PPARγ to attenuate pathogenic derangements in PH.</p></div

Hypoxia-induced alterations in miR-21 and PTEN that mediate proliferative responses of HPASMC to hypoxia are modulated by RSG.

<p>The schema depicts the proposed signaling interactions involved in hypoxia-induced vascular SMC proliferation. Hypoxia exerts mitogenic effects on HPASMC through stimulation of miR-21 which and reciprocally reduces PTEN. TGF-β1 is a recognized mediator of HPASMC proliferation that may exert effects upstream of miR-21. Inhibitory actions of the PPARγ ligands are due to RSG effects on multiple mediators involved in proliferative responses to hypoxia.</p

Rosiglitazone attenuates hypoxia-induced TGF-β1 expression.

<p>(A) Mice were exposed to normoxia or hypoxia for 3 weeks and treated with vehicle or RSG during the last 10 days, and (A) TGF-β1 mRNA was quantified. (B) HPASMC pre-treated with TGF-β1 neutralizing antibodies (1 μg/ml) for four hours were placed into a hypoxia chamber for 48 hours and miR-21 expression was quantified. Each bar represents mean ± SEM (A) TGF-β1 mRNA or (B) miR-21 levels. *P< 0.05 vs Normoxia (-), ^#P< 0.01 vs Hypoxia (-) (n = 5–7)</p

Rosiglitazone does not attenuate HPASMC proliferation in the absence of PTEN.

<p>HPASMC were transfected with PTEN small interfering RNA constructs. (A) PTEN mRNA and (B) PTEN protein levels were examined after transfection with PTEN siRNA. A representative immunoblot can be found in panel B. (C) HPASMC proliferation was assessed in PTEN-depleted HPASMC. In selected groups, RSG (10 μM) was added during the last 24 hours of the 72-hour incubation period prior to conducting the MTT proliferation assay. Each bar represents mean ± SEM HPASMC (A) PTEN mRNA, (B) PTEN protein, or (C) proliferation. *P< 0.05 vs Scrambled, ***P< 0.0001 vs Scrambled, ⁺P< 0.01 vs Scrambled-Vehicle, ^#P< 0.05 vs Scrambled-RSG (n = 3–4).</p

Depletion of miR-21 attenuates hypoxia-induced HPASMC proliferation.

<p>HPASMC were transfected with locked nucleic acid (LNA)-scrambled sequences or LNA-antimiR-21 (100–1000 pM), and (A) miR-21 levels were assessed. (B) PTEN protein, and (C) proliferation (MTT) were measured following LNA-antimiR-21 (1 nM) transfection. A representative immunoblot is shown in panel B. Each bar represents mean ± SEM miR-21 or PTEN expression or proliferation. *P< 0.05 vs Scr, **P< 0.001 vs Scr, ***P< 0.0001 vs Scr, ⁺P< 0.05 vs Normoxia (-), ^#P< 0.05 vs Hypoxia (-). A,C. (n = 3–4), B. (n = 6).</p

Hypoxia increases miR-21 and reduces PTEN levels in HPASMC in vitro and in the mouse lung in vivo.

<p>HPASMC were exposed to normoxic or hypoxic conditions for 24–72 hours (A,C,D) and mice were exposed to normoxia (21% O₂) or hypoxia (10% O₂) for 3 weeks (B,E). Large and small mRNA species were isolated from HPASMC and mouse lung lysates, and miR-21 and PTEN levels were quantified. A representative immunoblot is shown in panel D. Each bar represents mean ± SEM miR-21 or PTEN levels. *P< 0.05 vs Normoxia-24 Hr, ^#P< 0.05 vs Normoxia-72 Hr, ⁺P< 0.05 vs Normoxia (Lung), ***P< 0.0001 vs Normoxia (HPASMC). A. (n = 6, 24 hr; n = 10, 72 hr); B. (n = 3); C,D. (n = 6); E. (n = 8).</p

Rosiglitazone attenuates hypoxia-induced increases in miR-21 levels.

<p>(A) Hypoxia-exposed HPASMC were treated with RSG (10 μM) during the last 24 hours of the 72 hour study period. (B) RSG (10 mg/kg/day) was administered to mice daily during the last 10 days of the 3 week hypoxia exposure period, and miR-21 expression was quantified. Bars represent mean ± SEM miR-21 levels. *P< 0.05 vs Normoxia (-), ⁺P< 0.01 vs Normoxia (-), ^#P< 0.01 vs Hypoxia (-) (n = 5–8).</p

Rosiglitazone attenuates hypoxia-induced reductions in HPASMC PTEN expression.

<p>HPASMC were exposed to hypoxic conditions for 72 hours, and RSG was administered to selected monolayers during the last 24 hours of exposure. PTEN mRNA and protein levels were determined in all groups. A representative immunoblot is shown in panel B. Each bar represents mean ± SEM HPASMC PTEN (A) mRNA or (B) protein levels. *P< 0.01 vs Normoxia (-), ^#P< 0.01 vs Hypoxia (-) (n = 3–4).</p

Hypoxia exposure stimulates endothelial ROS release.

<p>Human pulmonary artery endothelial cells were exposed to normoxic or hypoxic (1% O₂) conditions for 24-, 48- or 72-hours. Following exposure, HPAEC ROS release was assessed by DCF staining (A, n = 3) and Amplex Red assay (C, n = 4). Results demonstrate that prolonged hypoxia exposure significantly increases endothelial ROS production whereas administration with the antioxidants, PEG- catalase or superoxide dismutase reduces these effects (B). Amplex Red Assay indicates that chronic hypoxia exposure promotes H₂O₂ release (C). * p<0.0001 when compared to normoxic controls.</p

Chronic hypoxia exposure increases endothelial ALOX5 expression.

<p>Seventy two hours of hypoxia exposure significantly stimulates endothelial ALOX5 expression when compared to all other groups. HPAEC were exposed to normoxic or hypoxic conditions for 24-, 48-, or 72 hours. Following exposure, cells were collected, and total RNA and protein were isolated for expression analyses via quantitative real time PCR and Western blot respectively. Results indicate that ALOX5 mRNA levels are significantly increased following hypoxia exposure (A, n = 5). Chronic hypoxia exposure also causes a 3-fold elevation in ALOX5 protein expression levels (B, n = 4). Endothelial FLAP expression is also increased when compared to all other groups (C, n = 5–7). Values are expressed as percent of control. * p<0.001 when compared to all other groups.</p