Abstract 1667: Angiotensin II and Free Cholesterol Synergistically Impaired eNOS Bioavailability via Lipid Raft
Both hypercholesterolemia and angiotensin II (Ang II) are potent factors inducing endothelial dysfunction that leads to atherosclerosis. It has been reported that coexistence of hypercho-lesterolemia and Ang II accelerates atherogenesis markedly. Several studies showed that elevated cellular cholesterol affected cellular function by modifying lipid raft. We investigated the contribution of lipid raft to bridging hypercholesterolemia and Ang II in endothelial cells. Free cholesterol (Chol) loading induced 2–3 fold increase in cellular cholesterol content. Co-treatment of Ang II and Chol markedly impaired eNOS phosphorylation and dimerization than Chol or Ang II loading alone. This inactivation of eNOS corresponded to eNOS translocation to Chol-rich fractions in density centrifugation. In this fraction, eNOS phosphorylation and dimerization were markedly suppressed. Olmesartan or ROS inhibitor suppressed this eNOS trafficking and reversed the impairment of eNOS bioavailability. In immunohistochemistry, the target of this eNOS trafficking was identified as Chol-rich vesicle structure at the perinuclear area and those were fused with lysosomal membrane associated protein-1 (LAMP-1), a lysosome marker, labeled endosome. These results suggested that in cholesterol enriched condition, AngII induced eNOS trafficking to Chol-rich vesicles that were fused with lysosome at the perinuclear area and keeps eNOS inactive. We next investigated the association between these Chol-rich vesicles and lipid raft. GM1, phospho-caveilin1 and Rac, which are all lipid raft actors, also shifted to Chol-rich fractions in density centrifugation and assembled at LAMP-1 labeled endosome at the perinuclear area after Ang II and Chol loading. Therefore, LAMP-1 labeled endosome was considered to be the intracellular target of lipid raft in that condition. In electron microscopy, Chol-rich vesicles corresponded to multivesicular bodies and these converged to lysosome with Ang II addition, which were consistent with immunohistochemical findings. These studies show the important crosstalk between AngII and Chol signaling leading to endothelial dysfunction and provide a novel insight into the mechanisms of endothelial dysfunction by hypercholesterolemia.