Oxidized Low Density Lipoprotein Displaces Endothelial Nitric-oxide Synthase (eNOS) from Plasmalemmal Caveolae and Impairs eNOS Activation

Abstract

Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide. Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveola protein, caveolin. In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveola cholesterol in endothelial cells. We found that treatment with 10 g/ml oxLDL for 60 min caused greater than 90% of eNOS and caveolin to leave caveolae. Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance. Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol. Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae. Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae. We conclude that oxLDL-induced depletion of caveola cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme. This process may be an important mechanism in the early pathogenesis of atherosclerosis.Studies in animal models and in humans have shown that hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by an early, selective impairment of endothelium-derived relaxation. This impairment is due to a decrease in bioavailable endothelium-derived nitric oxide (NO) 1 (1). In the initial phase of the disease process, there is impaired responsiveness to receptor-dependent stimuli, such as acetylcholine, whereas responsiveness to receptor-independent stimuli such as the calcium ionophore A23187 is not altered. As such, the early pathogenesis involves attenuated endothelial NO production in response to extracellular stimuli, but the capacity for maximal enzyme activation and the breakdown of NO are not affected. As the disease progresses, there is nonspecific inhibition of NO bioavailability. Although this later inhibition is most likely multifactorial in origin, it is at least partly due to enhanced inactivation of NO by superoxide anions (2-4). These processes result in increased neutrophil adherence to the endothelium, thereby promoting a key step in the pathogenesis of atherosclerosis (5). The chronic inhibition of NO synthesis in rabbit models of hypercholesterolemia accelerates the development of vascular dysfunction and intimal lesions, providing additional evidence that the impairment of NO synthesis promotes atherogenesis (6). In vitro investigations have further shown that oxidized LDL (oxLDL) inhibits NO-mediated responses. Antioxidants reduce both the formation of free radicals and the oxidative modification of LDL that lead to impaired NO-related responses (7). Thus, NO is critically involved ...

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

“…Similar results were reported in rabbit aorta (Darblade et al, 2001), whereas in guinea pig aorta, treatment with methyl-␤-cyclodextrin abolished the relaxation induced by ACh (Kaiser et al, 2002). It has been reported that caveolar cholesterol depletion is associated with a marked decline in ACh-induced eNOS activation (Blair et al, 1999). As far as protein composition, caveolae are characterized by the presence of a family of three proteins called caveolins.…”

“…Caveolae exist in most cell types and are particularly abundant in endothelial cells and smooth muscle cells (Galbiati et al, 1998;Voldstedlund et al, 2001). The absence of this organelle impairs NO and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone (Blair et al, 1999;Darblade et al, 2001;Je et al, 2004).…”

“…Cholesterol depletion leads to a marked decline in acetylcholine (ACh)-induced eNOS activation and smooth muscle relaxation (Blair et al, 1999;Darblade et al, 2001). AChinduced relaxation in rat aorta is mainly attributed to NO (Shimokawa et al, 1996).…”

The effect of cellular cholesterol on membrane-cytoskeleton adhesion

“…Conversely, cholesterol enrichment enhances endocytosis (Sharma et al, 2004). It is also important to note that although depleting cellular cholesterol with M␤CD is not physiological, it was shown that exposing endothelial cells to oxLDL results in cholesterol depletion from endothelial caveolae (Blair et al, 1999). Furthermore, the effects of M␤CD-induced cholesterol depletion on endothelial biomechanics are remarkably similar to those induced by oxLDL (Byfield et al, 2006).…”

Inability of HDL from type 2 diabetic patients to counteract the inhibitory effect of oxidised LDL on endothelium-dependent vasorelaxation

“…In contrast, no significant correlation was observed between the HDL fructosamine:total protein ratio or PON activity and Emax (ox-LDL+HDL) in type 2 diabetic patients or control subjects. The inhibitory effect of ox-LDL and some of their specific compounds such as derivatives of cholesterol oxidised in position 7 or lysophophatidylcholine on endothelium-dependent vasorelaxation is mainly related to a decreased bioavailability of NO [13][14][15][16][17]. HDL are likely to counteract the inhibitory effect of ox-LDL on endotheliumdependent vasorelaxation both by a direct and an indirect way.…”