The focus of this grant is the elucidation of the mechanism by which remnant lipoprotein particles are cleared by hepatic receptors. The Principal Investigator and his colleague, Dr. M. Linton, have pioneered the use of bone marrow transplant to study apo E delivery into genetically altered mice via transplanted macrophages. The critical observation in the preliminary data, which serves as the basis for the grant proposal, is that BMT into apo E deficient mice is capable of fully restoring normal lipid levels, but the same transplantation does not normalize lipids in animals that are deficient in both apo E and the LDL receptor. Apo E deficient animals who are homozygous for the LDL-R have a good response to BMT, but lipid values are higher than in animals with two intact genes encoding the LDL-R. Importantly, the apo E levels in the double ko animals are even higher than those found in normal mice, and data presented in the grant indicates that this apo E is present in the space of Disse in the liver, where lipoprotein capture by hepatic lipoprotein receptors should occur. These findings indicate that the apo E provided by macrophages does not substitute fully for endogenous hepatic apo E in generating a pathway that can metabolize remnant particles. The PI lists three major specific aims, each of which is divided into three subaims.
The first aim i s designed to assess the roles of the LDL receptor and the LRP receptor in the clearance of remnant lipoproteins using bone marrow transplantation of apo E expressing macrophages to partially re-constitute apo E expression in mice genetically deficient in apo E and the LDL-R. Turnover studies of apo B-48 and apo B-100 remnants will be analyzed before and after transplantation and the fate of lipoprotein associated apo E after internalization by either the LRP or LDL-R will be studied.
In Specific Aim 2, the investigators will test their hypothesis that secretion-capture of lipoproteins in the space of Disse is critical to remnant uptake and that this process requires local apo E production by the hepatocyte. This will be accomplished using an adenovirus to express E locally in E deficient animals, before and after BMT. The effect of apo E expression on LRP activity will be assessed by radiolabeled antibodies directed against LRP in vivo, complemented by in vitro studies of apo E enriched B-VLDL binding to fibroblasts deficient in either the LDL-R or the LRP. The third specific aim will focus on the role of apo E and LRP in macrophage foam cell formation and atherosclerosis. BMT into LDL-R /apo E double ko's will permit the assessment of macrophage-specific E expression on atherosclerosis development in animals with persistently elevated lipids. Fetal hepatocyte transplantation is proposed as a method for generating LRP deficient macrophages to determine what activities of wild type macrophages are attributable to LRP. Finally, in vitro studies will be conducted to assess the role of LRP in the binding and degradation of B-VLDL.
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