Dual Role of Endothelial Nitric Oxide Synthase in Oxidized LDL-Induced, p66Shc-Mediated Oxidative Stress in Cultured Human Endothelial Cells

Background: The aging gene p66(Shc), is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66(Shc) deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66(Shc) activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66(Shc) activation. Methods and Results: Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66(Shc) at Ser-36, resulting in an enhanced production of superoxide anion (O-2(-)). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66(Shc) phosphorylation, suggesting that basal NO production inhibits O-2(-) production. oxLDL-induced, p66(Shc)-mediated O-2(-) was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66(Shc) activation and reduced O-2(-) production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66(Shc) activation, and reduced O-2(-) generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species. Conclusions: The present study reports that in primary cultured HAEC treated with oxLDL, p66(Shc)-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66(Shc) activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease.