Similarly, targeted depletion of PPAR�� from smooth muscle cells (22) or from endothelial this site cells (21) resulted in spontaneous pulmonary hypertension in mice. On the other hand, activation of PPAR�� with TZDs attenuated pulmonary hypertension or vascular remodeling caused by monocrotaline (35) or hypobaric hypoxia (8) in rats. TZDs also reduced pulmonary hypertension in ApoE-deficient mice fed high-fat diets (23) and attenuated hypoxia-induced pulmonary hypertension and Nox4 expression and activity in mice (39). Therefore, the current study was designed to more carefully examine the molecular mechanisms by which PPAR�� activation modulates Nox4 expression. Our results demonstrate that PPAR�� regulates hypoxia-induced Nox4 expression, cell proliferation, and ROS generation in human pulmonary artery smooth muscle cells (HPASMC) by suppressing binding of NF-��B to the Nox4 promoter.

MATERIALS AND METHODS Cell culture. HPASMC (Lonza, Basel, Switzerland) monolayers were grown and maintained at 37��C in a 5% CO2 atmosphere in SmGM-2 media (Lonza) containing 2% fetal calf serum, 10 ng/ml human epidermal growth factor, 1.0 mg/ml hydrocortisone, 12 mg/ml bovine brain extract, 50 mg/ml gentamicin, and 50 ng/ml amphotericin B. In selected studies, because of the ease with which they can be transfected, multipotent mesenchymal stem cells [C3H/10T1/2; American Type Culture Collection (ATCC), Manassas, VA] were maintained in basal modified Eagle’s medium with 10% fetal bovine serum. HPASMC or C3H/10T1/2 cells were placed into a hypoxia chamber (1% O2-5% CO2; BioSpherix, Lacona, NY) or into a cell culture incubator (21% O2-5% CO2) maintained at 37��C for 72 h.

During the final 24 h of this exposure, selected cells were treated with either vehicle (0.5% methyl cellulose) or rosiglitazone Anacetrapib (10 ��M). All manipulations of hypoxic cells were performed in a glove box (BioSpherix) that maintains oxygen (1%) and CO2 (5%) levels to avoid effects of hypoxia and reoxygenation. Measurements of H2O2 production and cell proliferation. HPASMC were propagated into 24-well plates (4 �� 104 cells/well). After control or hypoxic exposure for 72 h and treatment with or without rosiglitazone, samples of HPASMC media (50 ��l) were collected, and H2O2 concentrations were analyzed with the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes, Eugene, OR) following the manufacturer’s recommendations. H2O2 concentrations were determined by measuring the horseradish peroxidase-catalyzed, H2O2-dependent oxidation of the nonfluorescent Amplex Red reagent to fluorescent resorufin red.