Hypercholesterolemia, 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. Recent evidence in the literature and from our laboratory indicates that hypercholesterolemia profoundly exaggerates the microvascular dysfunction, inflammatory cell infiltration, and cellular necrosis associated with reperfusion of ischemic tissues. The work proposed in this application will extend our effort to define the mechanisms that underlie these exaggerated responses to ischemia/reperfusion (I/R) in hypercholesterolemic animals. We propose to assess the contributions of lymphocytes, platelets, and enhanced oxidant production to the leukocyte-endothelial cell adhesion, capillary malperfusion, tissue hypoxia, cytokine production, and cellular necrosis observed in an established model of I/R induced liver injury. Wild-type and LDL-receptor knockout mice with normal and elevated serum cholesterol levels will be studied. Intravital fluorescence microscopy will be used to monitor the number of perfused sinusoids and the accumulation of total leukocytes, lymphocytes, and platelets in liver sinusoids and hepatic venules after I/R. NADH autofluorescence will be monitored as an index of oxidative stress (tissue hypoxia) while the oxidation of dihyrorhodamine 123 will be used to monitor oxidant production. Plasma levels of liver enzymes will be used to assess hepatocellular injury, while a radiolabelled monoclonal antibody technique will be used to quantify the expression of P-selectin in the liver vasculature.
One specific aim will focus on the influence of varying durations of ischemia, followed by reperfusion on the microvascular accumulation of total leukocytes, lymphocytes (T- and B-cells), and platelets in the postischemic liver of normal and hypercholesterolemic mice. A second specific aim will focus on the contributions of lymphocytes and platelets to the exaggerated responses of the liver microvasculature to I/R. SCID mice, reconstituted with specific lymphocyte populations from wild-type and mutant mice, will be used to address the role of lymphocytes in I/R-induced liver injury. Platelet-directed interventions (including neutralizing antibodies and mutant mice) will be used to assess the contribution of platelet accumulation. A third specific aim will determine whether the accumulation of these inflammatory cells and subsequent tissue injury is linked to an enhanced production of oxidants in hypercholesterolemic mice. Different mutant mice and oxidant-directed reagents will be used to assess the contribution and source of oxidants in normal and hypercholesterolemic mice. The results obtained from the proposed studies should lead to an improved understanding of the mechanisms by which hypercholesterolemia exacerbates ischemic tissue injury.
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