Hypercholesterolemia (HCh), an established risk factor for ischemic diseases of the heart, brain, liver and other tissues, appears to alter the function of both circulating blood cells and microvascular endothelial cells. The microvascular dysfunction induced by HCh is associated with activation of endothelial cells and circulating blood cells (leukocytes and platelets), and is manifested as an oxidative stress in both arterioles and venules, an impairment of endothelium-dependent relaxation in arterioles, increased endothelial cell adhesion molecule expression, and the adhesion of leukocytes and platelets in postcapillary venules. The work outlined in this application will extend our effort to define the mechanisms that underlie the prooxidative, pro-inflammatory, and prothrombogenic phenotype that is assumed by the microvasculature in response to HCh. Preliminary evidence from our laboratory indicates that an immune cell-mediated, oxidant-dependent mechanism may account for the HCh-induced endothelial cell dysfunction that is exhibited in both arterioles and postcapillary venules. Intravital microscopy and the dual radiolabelled monoclonal antibody technique will be used to test the central hypothesis that hypercholesterolemia induces endothelium-dependent arteriolar and venular dysfunction via a mechanism that involves activation of circulating immune cells, the upregulation of angiotensin II, type la (ATla) receptors, activation of CD40/CD40L signaling, and the consequent induction of an oxidative stress in microvessels. Different components of this central hypothesis will be addressed by experiments outlined under 3 specific aims: 1) to define the contributions of leukocytes and immune cell-derived cytokines in producing the pro-inflammatory and prothrombogenic state in venules and defective endothelium dependent vasodilation in arterioles of HCh mice, 2) to determine whether CD40 - CD40 ligand signaling participates in the endothelium-dependent microvascular alterations associated with HCh, and 3) to assess the contribution of AT1a receptors towards the immune cell mediated, oxidant-dependent microvascular alterations that are induced by HCh. Different mutant mice and adoptive cell transfer strategies will be employed to assess the contribution of different immune cell-derived cytokines, AT1 receptors, and the CD40/CD40L dyad to the endothelium-dependent alterations in venular and arteriolar function during hypercholesterolemia. The proposed work should lead to an improved understanding of the microvascular alterations that precede the appearance of overt large artery pathology during hypercholesterolemia.
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