It is becoming increasingly clear that elevated blood cholesterol levels result in alterations in arterial wall function, quite apart from its role in the etiology of atherosclerosis. This is indicated by arterial hypersensitivity and reduced organ blood flows reported during dietary hypercholesterolemia. Excess cholesterol has been shown to alter membrane mediated functions in several cell types and in this way may contribute to altered cell function. Based on data from this and other laboratories, we have developed the hypothesis that elevated cholesterol levels result in the incorporation of excess cholesterol into membranes of the arterial wall cells, thereby altering membrane fluidity and transmembrane ion movements. We believe that these changes alter excitation-contraction coupling and contribute to the arterial hypersensitivity complicating hypercholesterolemia. The broad aim of this proposal is to determine the mechanism by which cholesterol alters cell membranes and contributes to abnormal cell function in arterial smooth muscle cells (SMC) and endothelial cells (EC). This proposal reflects the long-term goal of this laboratory which is in study the effects of altering membrane lipid composition on cell function in arterial SMC's and EC's.
The specific aims of this study are to determine: 1, the effects of excess cholesterol on the physical state (fluidity) of the plasmalemma membrane of SMC and EC, and 2, the effects of these membrane alteration on transmembrane calcium, potassium and sodium movements and function in smooth muscle cells, and 3, the effects of these membrane alterations on transmembrane calcium, potassium and sodium movements and function in endothelial cells. Two general and complimentary methodological approaches to this problem will be used. First, rabbit arteries will be perfused in vitro, and cholesterol enrichment of the cellular plasmalemma membranes of the vessel will be accomplished with cholesterol rich liposomes. Ring segments of these vessels will be used to study smooth muscle contraction in parallel with ion flux studies. Secondly, we will examine the effects of cholesterol enrichment on endothelial cell function (EDRF release) and transmembrane ion movement using primary cell cultures of rabbit endothelium. In addition, ion flux studies will also be performed in cultured rabbit SMC to define the details of transmembrane ion movements not otherwise discernable in intact vessel segments. The effects of cholesterol enrichment on the physical state (fluidity) of the membranes measured by fluorescence anisotropy will help define the molecular mechanism for cholesterol's effect. Integrating the results of these methodological approaches will permit a thorough and broad analysis of the molecular mechanisms by which cholesterol alters arterial reactivity. This study will provide new insights into the interplay between altered membrane cholesterol composition and cell function and will contribute to our understanding of the cellular mechanisms underlying inappropriate vasomotion associated with hypercholesterolemia.
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