Abstract
BackgroundThe aging gene p66Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66Shc 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 p66Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66Shc activation.Methods and ResultsIncubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66Shc at Ser-36, resulting in an enhanced production of superoxide anion (O2 -). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66Shc phosphorylation, suggesting that basal NO production inhibits O2 - production. oxLDL-induced, p66Shc-mediated O2- 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 p66Shc activation and reduced O2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66Shc activation, and reduced O2- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species.ConclusionsThe present study reports that in primary cultured HAEC treated with oxLDL, p66Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66Shc 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.
Highlights
The present study reports that in primary cultured human aortic endothelial cells (HAEC) treated with oxidized low density lipoprotein (oxLDL), p66Shc-mediated oxidative stress is derived from endothelial nitric oxide synthase (eNOS) uncoupling
Endothelial nitric oxide synthase produces nitric oxide (NO), a key factor involved in maintaining endothelial homeostasis [1]
In primary HAECs stimulated by oxLDL, eNOS uncoupled and acted as the primary source of p66Shcmediated ROS
Summary
Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), a key factor involved in maintaining endothelial homeostasis [1]. The p66Shc adaptor protein is an important mediator of oxidative stress-induced vascular dysfunction [10], acting as a redox enzyme implicated in mitochondrial ROS generation and the translation of oxidative signals into apoptosis [11,12,13,14,15]. The aging gene p66Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. The putative role of the NO pathway on p66Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66Shc activation
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