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

https://doi.org/10.1016/j.freeradbiomed.2007.02.027Get rights and content

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 (gp91phox−/−) 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 gp91phox−/−–HC mice. Recruitment of platelets derived from WT–HC mice in venules of SOD-TgN–HC or gp91phox−/−–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, gp91phox−/−–HC platelets exhibited low levels of adhesion comparable to those of WT–ND in both hypercholesterolemic gp91phox−/− and WT recipients. Arteriolar dysfunction was evident in WT–HC mice, compared to WT–ND. Overexpression of SOD or, to a lesser extent, gp91phox 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.

Section snippets

Animals

Male wild-type C57BL/6J (WT), B6.129S6-Cybbtm1Din/J (gp91phox−/−), and breeder stocks for C57BL/6-Tg(SOD1)3Cje/J (SOD-TgN) mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The SOD-TgN mice were identified by qualitative demonstration of Cu,Zn-SOD using nondenaturing gel electrophoresis followed by nitroblue tetrazolium staining. Nontransgenic littermates were used as controls (SOD-nonTgN). At 6–8 weeks of age the mice were placed on either a normal

Blood cholesterol concentration and wall shear rate

Placement of WT and mutant mice on a cholesterol-enriched diet for 2 weeks led to a two- to threefold increase in blood cholesterol concentration, with no statistical difference between the HC groups (Table 1). Wall shear rate was comparable between all groups examined, suggesting this was not a factor in producing the differences in blood cell recruitment. Furthermore, no differences in circulating leukocyte or platelet counts were noted between any of the experimental groups.

Leukocyte recruitment in postcapillary venules

Discussion

Hypercholesterolemia elicits an inflammatory response in the microvasculature of many organs, including skeletal muscle, intestine, brain, and mesentery [3], [4], [11], [12], [14]. This inflammatory response is characterized by endothelial dysfunction/activation and manifests as impaired endothelium-dependent vasodilation in arterioles [3], [4] and leukocyte and platelet recruitment in postcapillary venules [4], [14], [22]. In hypercholesterolemic humans and animals, oxidative stress has been

Acknowledgments

This work was supported by a grant from the National Heart, Lung, and Blood Institute (HL26441). The authors thank Dr. Kevin Pruitt and Ms. Sherry Jackson for their technical assistance.

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