This is an interdisciplinary program aimed at elucidating the mechanisms regulating the transport of lipoprotein through the endothelium in relation to the accumulation of lipid-containing particles (liposomes) in the intima. In this renewal grant proposal we shall test a new hypothesis that the initiation and growth of subendothelial liposomes are dependent on the spatial and temporal characteristics of lipoprotein loading from localized endothelial leakage sites at the cellular level. Our proposed theoretical and experimental studies are designed with the following three specific aims: (1) To elucidate the factors that control the size of leakage spots in the intima, especially the roles of IEL fenestra and transendothelial water fluxes in modulating local convection, (2) to quantify the relative fluxes of LDL that enter the intima through transcytosis and leaky junction pathways, and (3) to demonstrate that there is a co-localization of cellular level leakage sites and liposome formation and to establish the relationship between these two processes. Experimental studies will be performed on New Zealand White rabbits and Sprague-Dawley rats following the intravenous injection of low density lipoproteins labeled with fluorescent markers and with 125I. Other tracers to be used are Evans Blue labeled albumin and horseradish peroxidase. The aortic tree will be systematically scanned to determine the size and frequency of LDL leakage spots as a function of time and location (including branch vs. nonbranch areas).
Under specific aim 1, we will perform parallel theoretical and experimental studies to analyze the effects of the structural features of the internal elastic lamina fenestrae and transmural pressure on the size and frequency of leakage spots.
Under specific aim 2, the relative contributions of the two pathways to LDL fluxes will be calculated from the relationship between number of leakage spots and the 125I counts of aortic specimens, and the experimental findings will be used as a guide in theoretical modeling and numerical computation.
Under specific aim 3, the frequency, size and duration of LDL leakage spots after cholesterol feeding will be determined, and these data will be used to derive the temporal and spatial distribution of LDL leakage waves and the probability of recurrence of leakage in a given area; the co-localization of leakage sites with liposomes will be tested directly by electron microscopic studies following LDL infusion. The findings in these theoretical and experimental studies will allow us to test our hypothesis on the initiation and growth of liposomes in relation to endothelial LDL leakage sites at the cellular level. These results will provide new insights into the mechanism of the initiation of atherogenesis involving intimal lipid accumulation.
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