Chronic hypoxia contributes to pulmonary hypertension through complex mechanisms that include enhanced NADPH oxidase expression and reactive oxygen species (ROS) generation in the lung. Stimulation of peroxisome proliferator-activated receptor g (PPARg) reduces the expression and activity of NADPH oxidase. Therefore, we hypothesized that activating PPARg with rosiglitazone would attenuate chronic hypoxia-induced pulmonary hypertension, in part, through suppressing NADPH oxidase-derived ROS that stimulate proliferative signaling pathways. Male C57Bl/6 mice were exposed to chronic hypoxia (CH, FI O 2 10%) or room air for 3 or 5 weeks. During the last 10 days of exposure, each animal was treated daily by gavage with either the PPARg ligand, rosiglitazone (10 mg/kg/d) or with an equal volume of vehicle. CH increased: (1) right ventricular systolic pressure (RVSP), (2) right ventricle weight, (3) thickness of the walls of small pulmonary vessels, (4) superoxide production and Nox4 expression in the lung, and (5) platelet-derived growth factor receptor b (PDGFRb) expression and activity and reduced phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression. Treatment with rosiglitazone prevented the development of pulmonary hypertension at 3 weeks; reversed established pulmonary hypertension at 5 weeks; and attenuated CH-stimulated Nox4 expression and superoxide production, PDGFRb activation, and reductions in PTEN expression. Rosiglitazone also attenuated hypoxia-induced increases in Nox4 expression in pulmonary endothelial cells in vitro despite hypoxia-induced reductions in PPARg expression. Collectively, these findings indicate that PPARg ligands attenuated hypoxia-induced pulmonary vascular remodeling and hypertension by suppressing oxidative and proliferative signals providing novel insights for mechanisms underlying therapeutic effects of PPARg activation in pulmonary hypertension.

Recently, we demonstrated that the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, either 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, increased endothelial nitric oxide (.NO) release without altering endothelial nitric oxide synthase (eNOS) expression (4). However, the precise molecular mechanisms of PPAR-gamma-stimulated endothelial.NO release remain to be defined. Superoxide anion radical (O2-.) combines with .NO to decrease.NO bioavailability. NADPH oxidase, which produces O2-., and Cu/Zn-superoxide dismutase (Cu/Zn-SOD), which degrades O2-., thereby contribute to regulation of endothelial cell.NO metabolism. Therefore, we examined the ability of PPAR-gamma ligands to modulate endothelial O2-. metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 microM 15d-PGJ2 or ciglitazone for 24 h decreased human umbilical vein endothelial cell (HUVEC) membrane NADPH-dependent O2-. production detected with electron spin resonance spectroscopy. Treatment with 15d-PGJ2 or ciglitazone also reduced relative mRNA levels of the NADPH oxidase subunits, nox-1, gp91phox (nox-2), and nox-4, as measured using real-time PCR analysis. Concordantly, Western blot analysis demonstrated that 15d-PGJ2 or ciglitazone decreased nox-2 and nox-4 protein expression. PPAR-gamma ligands also stimulated both activity and expression of Cu/Zn-SOD in HUVEC. These data suggest that in addition to any direct effects on endothelial.NO production, PPAR-gamma ligands enhance endothelial.NO bioavailability, in part by altering endothelial O2-. metabolism through suppression of NADPH oxidase and induction of Cu/Zn-SOD. These findings further elucidate the molecular mechanisms by which PPAR-gamma ligands directly alter vascular endothelial function.

Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2(-).) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidase-derived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor beta and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidase-derived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH.

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