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

Recently, we demonstrated that the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, either 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) or ciglitazone, increased endothelial nitric oxide (center dot NO) release without altering endothelial nitric oxide synthase ( eNOS) expression ( 4). However, the precise molecular mechanisms of PPAR-gamma-stimulated endothelial center dot NO release remain to be defined. Superoxide anion radical (O(2)(-)center dot) combines with center dot NO to decrease center dot NO bioavailability. NADPH oxidase, which produces O(2)(-)center dot, and Cu/Zn-superoxide dismutase ( Cu/Zn-SOD), which degrades O(2)(-)center dot, thereby contribute to regulation of endothelial cell center dot NO metabolism. Therefore, we examined the ability of PPAR-gamma ligands to modulate endothelial O(2)(-)center dot metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 mu M 15d-PGJ(2) or ciglitazone for 24 h decreased human umbilical vein endothelial cell ( HUVEC) membrane NADPH-dependent O(2)(-)center dot production detected with electron spin resonance spectroscopy. Treatment with 15d-PGJ(2) 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-PGJ(2) 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 center dot NO production, PPAR-gamma ligands enhance endothelial center dot NO bioavailability, in part by altering endothelial O(2)(-)center dot 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.