Peroxisome proliferator-activated receptor gamma (PPARγ) regulates thrombospondin-1 and Nox4 expression in hypoxia-induced human pulmonary artery smooth muscle cell proliferation

Abstract

Transforming growth factor-β1 (TGF- β1) and thrombospondin-1 (TSP-1) are hypoxia-responsive mitogens that promote vascular smooth muscle cell (SMC) proliferation, a critical event in the pathogenesis of pulmonary hypertension (PH). We previously demonstrated that hypoxia-induced human pulmonary artery smooth muscle (HPASMC) cell proliferation and expression of the NADPH oxidase subunit, Nox4, were attenuated by the peroxisome proliferator-activated receptor γ (PPARγ) agonist, rosiglitazone. The current study examines the hypothesis that rosiglitazone regulates Nox4 expression and HPASMC proliferation by attenuating TSP-1 signaling. Selected HPASMC were exposed to normoxic or hypoxic (1% O(2)) environments or TSP-1 (0-1 μg/ ml) for 72 hours ± administration of rosiglitazone (10 μM). Cellular proliferation, Nox4, TSP-1, and TGF-β1 expression and reactive oxygen species generation were measured. Mice exposed to hypoxia (10% O(2)) for three weeks were treated with rosiglitazone (10 mg/kg/day) for the final 10 days, and lung TSP-1 expression was examined. Hypoxia increased TSP-1 and TGF-β1 expression and HPASMC proliferation, and neutralizing antibodies to TSP-1 or TGF-β1 attenuated proliferation. Rosiglitazone attenuated hypoxia-induced HPASMC proliferation and increases in mouse lung and HPASMC TSP-1 expression, but failed to reduce increases in TGF-β1 expression or Nox4 expression and activity caused by direct TSP-1 stimulation. Transfecting HPASMC with siRNA to Nox4 attenuated hypoxia- or TSP-1-stimulated HPASMC proliferation. These findings provide novel evidence that TSP-1-mediated Nox4 expression plays a critical role in hypoxia-induced HPASMC proliferation. PPARγ activation with exogenous ligands attenuates TSP-1 expression to reduce Nox4 expression. These results clarify mechanisms of hypoxia-induced SMC proliferation and suggest additional pathways by which PPARγ agonists may regulate critical steps in the pathobiology of PH.

Keywords: Nox4; PPARγ; hypoxia; rosiglitazone; thrombospondin-1.

Figure 1

Rosiglitazone or neutralizing antibodies to TSP-1 or TGF-β1 attenuated hypoxia-induced HPASMC proliferation. HPASMC exposed to normoxic (21% O₂) or hypoxic (1% O₂) conditions for 72 hours were treated with neutralizing antibodies to either TSP-1 (nTSP-1, 1 μg/ml) or TGF-β1 (nTGF-b1, 1 μg/ml) 3 hours prior to exposure, or rosiglitazone (Rosi, 10 μM) was administered during the last 24 hours of exposure. After treatment, an MTT assay was performed to measure cellular proliferation (n = 6). ^*P< 0.05 vs. Control-Normoxia, ⁺P< 0.05 vs. Control-Hypoxia, ^#P< 0.05 vs. Control-Normoxia.

Figure 2

Rosiglitazone treatment did not modulate hypoxia-induced increases in HPASMC TGF-β1 mRNA, protein or TGF-β1-induced activation of the canonical TGF-β1 signaling pathway. HPASMC were exposed to normoxic or hypoxic conditions for 72 hours ± rosiglitazone (Rosi, 10 μM). TGF-β1 expression was measured in cell lysates by (A) qRT-PCR (n = 12), and in the cell media by (B) ELISA (n = 3). (C) Smad 3 binding element luciferase reporter activity was measured in HPASMC treated with TGF-β1 (2 ng/ml) for 24 hours ± rosiglitazone (10 μM) (n = 3). ^*P< 0.05 vs. Control; ^**P< 0.01 vs. Control.

Figure 3

Rosiglitazone attenuated hypoxia-induced increases in TSP-1 protein expression. HPASMC were exposed to normoxic (control) or hypoxic conditions for 72 hours. During the final 24 hours of exposure, HPASMC were treated with rosiglitazone (Rosi 10 μM). HPASMC were collected, and proteins were isolated for Western blot analysis. Each bar represents the mean ± SEM TSP-1 band density normalized to CDK 4 and expressed relative to control samples (n=9). ^*P< 0.05 vs. Control. A representative TSP-1 immunoblot is presented below the bar graph.

Figure 4

TSP-1 caused dose and time-dependent increases in HPASMC Nox4 expression and ROS generation. Western blot to detect Nox4 protein was performed on protein isolated from HPASMC monolayers treated with (A) graded concentrations of TSP-1 for 72 hours (n = 3), or (B) 1 μg/ml of TSP-1 for 24-72 hours (n = 3). Nox4 immunoblots are presented below the bar graph. (C) HPASMC monolayers were treated with graded concentrations of TSP-1 for 72 hours and H₂O₂ production was measured by Amplex Red assay (n = 3). (A) ^*P< 0.01 vs. Control, (B) ^*P< 0.001 vs. Control, (C) ^*P< 0.01 vs. Control.

Figure 5

Nox4 siRNA decreased hypoxia or TSP-1-induced HPASMC proliferation. HPASMC were transfected with 35 nM siNox4 or a scrambled control (siCon). Twenty-four hours later, HPASMC were exposed to normoxic or hypoxic conditions for 72 hours ± TSP-1 (1 μg/ml). An MTT assay was then performed. Each bar represents mean ± SEM HPASMC proliferation expressed relative to control samples (n = 3). ^*P< 0.05 vs. Normoxia-siCon, +P< 0.05 vs. Hypoxia-siCon, #P< 0.05 vs. TSP-1.

Figure 6

TSP-1 induces Nox4 expression and rosiglitazone fails to attenuate this increase. HPASMC were treated with TSP-1 (1 μg/ml) for 72 hours and rosiglitazone (10 μM) was added to the cell culture media during the final 24 hours of treatment. Total RNA was collected and quantitative real-time PCR of Nox4 transcripts was performed. Each bar represents the mean ± SEM Nox4 transcripts normalized to 9S and expressed as fold change relative to control (n = 3-6). ^*P < 0.05 vs. Control.

Figure 7

Rosiglitazone attenuates chronic-hypoxia-induced TSP-1 protein expression. C57Bl/6 mice were exposed to normoxic (Control) or chronic hypoxic (CH) (10% O₂) conditions for three weeks. During the last 10 days of treatment, selected animals were gavaged with either rosiglitazone (Rosi, 10 mg/kg/day) or vehicle. Protein was prepared from whole lung homogenate for Western blot analysis of TSP-1 expression (n = 8). *P < 0.05 vs. Control. A representative TSP-1 immunoblot is presented below the bar graph.

Figure 8

Putative mechanisms by which PPARγ ligands attenuate hypoxia-induced HPASMC proliferation. The current schema depicts relationships that exist between TSP-1, Nox4, and ROS generation in hypoxia-induced HPASMC proliferation. Hypoxia enhances Nox4 expression and ROS generation in a TSP-1-mediated manner. Activation of PPARγ with rosiglitazone attenuates hypoxia-induced HPASMC proliferation through inhibitory effects on TSP-1 expression and signaling.