Peroxisome proliferator-activated receptor-γ ligands regulate endothelial membrane superoxide production

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Abstract
Recently, we demonstrated that the peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands, either 15-deoxy-Δ12,14-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-γ-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-γ ligands to modulate endothelial O2−· metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 μM 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, gp91 phox (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-γ 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-γ 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-γ ligands directly alter vascular endothelial function.