The overall objective is to understand how chylomicron remnants contribute to atherosclerosis. The goal of the current proposal is to elucidate the mechanisms of the hepatic removal of these particles. Remnants accumulate in the blood during the postprandial period implying that there are times when their removal becomes limiting. The hypothesis to be tested is that the initial rate of chylomicron remnant removal is determined by the number of LDL receptors, and the capacity of the liver to sequester the particles before internalizing them (sequestration space). The size of the sequestration space is defined by heparan-sulfate-proteoglycans, and the affinity of the space for remnants is modified by the presence of hepatic lipase. The steady state rate of removal is determined by transfer from sequestration to LDL receptors and the LRP. The latter, is determined in part, by co-factors, particularly hepatic apoE.
Three specific aims are proposed. First, to complete studies utilizing the isolated perfused mouse liver to characterize the remnant removal process. Isolated mouse liver perfusion was validated as a technique for studying remnant removal. Data demonstrates that both the LDL receptor and the LRP are saturable components of this process, and that there may be an additional mechanism for removal; apoE is absolutely required for rapid remnant removal, but the hepatic secretion of apoE is required for only one component of the process. Mice deficient in LDL receptors and the LRP, as well as apoE, will be utilized to complete these studies. Second, methodologies will be developed to determine the size of the sequestration space and the rate of egress from this space. The use of confocal microscopy with fluorescent labeled remnants and fluorescent markers for both endothelial cells and hepatocytes will allow determination of where remnants are accumulating, what fraction of the accumulated remnants are in the space of Disse, and the rate at which they leave this space. A multi-compartment model for analysis of the isotope removal data is being developed to determine the affinities and capacities of the components of the removal process. Using the animals of the third specific aim, this will identify which molecules contribute to these components. As a back up strategy, cell separation techniques can be used to accomplish the goals of the first two methods. The third specific aim will examine the nature of the determinants of the process. Mice that express varying fixed levels of LDL receptors will be used to study the role of this molecule in initial removal, as well as egress from sequestration. A similar strategy will be utilized for the LRP by creating truncated but functional, LRP-like molecule. Mice with different isoforms of apoE will be studied to evaluate how this affects its function as a co- factor, and as a portion of the capture mechanism. This group of studies will also evaluate the role of LPL in remnant removal in the liver. In the last group, the amount, location and enzymatic activity of hepatic lipase in the liver will be varied, to learn whether it serves as a co-factor for the LRP, or whether it serves as a determinant for the sequestration space, or both. In addition, attempts will be made to vary the syndecan content of the liver to test the role of this HSPG as a major determinant of the sequestration space. Together, it is anticipated that these studies will provide a concrete model for remnant removal, as well as identify the key elements in this mechanism, at which therapeutic intervention might be directed.
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