Platelet-associated NAD(P)H oxidase contributes to the thrombogenic phenotype induced by hypercholesterolemia

Abstract

Elevated cholesterol levels promote proinflammatory and prothrombogenic responses in venules and impaired endothelium-dependent arteriolar dilation. Although NAD(P)H oxidase-derived superoxide has been implicated in the altered vascular responses to hypercholesterolemia, it remains unclear whether this oxidative pathway mediates the associated arteriolar dysfunction and platelet adhesion in venules. Platelet and leukocyte adhesion in cremasteric postcapillary venules and arteriolar dilation responses to acetylcholine were monitored in wild-type (WT), Cu,Zn-superoxide dismutase transgenic (SOD-TgN), and NAD(P)H oxidase-knockout (gp91 phox−/−) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. HC elicited increased platelet and leukocyte adhesion in WT mice versus ND. Cytosolic subunits of NAD(P)H oxidase (p47phox and p67phox) were expressed in platelets. This was not altered by hypercholesterolemia; however, platelets and leukocytes from HC mice exhibited elevated generation of reactive oxygen species compared to ND mice. Hypercholesterolemia-induced leukocyte recruitment was attenuated in SOD-TgN–HC and gp91 phox−/−–HC mice. Recruitment of platelets derived from WT–HC mice in venules of SOD-TgN–HC or gp91 phox−/−–HC recipients was comparable to ND levels. Adhesion of SOD-TgN–HC platelets paralleled the leukocyte response and was attenuated in SOD-TgN–HC recipients, but not in WT–HC recipients. However, gp91 phox−/−–HC platelets exhibited low levels of adhesion comparable to those of WT–ND in both hypercholesterolemic gp91 phox−/− and WT recipients. Arteriolar dysfunction was evident in WT–HC mice, compared to WT–ND. Overexpression of SOD or, to a lesser extent, gp91 phox deficiency restored arteriolar vasorelaxation responses toward WT–ND levels. These findings reveal a novel role for platelet-associated NAD(P)H oxidase in producing the thrombogenic phenotype in hypercholesterolemia and demonstrate that NAD(P)H oxidase-derived superoxide mediates the HC-induced arteriolar dysfunction.