FUNCTIONAL HIERARCHY OF REMNANT LIPOPROTEIN RECEPTORS

描述(改编自申请人的摘要)： 这项拨款的重点是阐明 肝脏清除残存脂蛋白颗粒的机制 感受器。首席调查员和他的同事M·林顿博士 率先使用骨髓移植来研究载脂蛋白E的传递 通过移植的巨噬细胞进入基因改变的小鼠。关键人物 初步数据中的观察，作为赠款的基础 建议，骨髓移植进入载脂蛋白E缺陷小鼠能够完全 恢复正常的血脂水平，但同样的移植不能 使载脂蛋白E和低密度脂蛋白均缺乏的动物的血脂正常化 受体。低密度脂蛋白受体纯合子的载脂蛋白E缺乏的动物有 对骨髓移植的反应良好，但血脂值高于接受两次骨髓移植的动物 编码低密度脂蛋白-R的完整基因重要的是，体内的载脂蛋白E水平 双Ko动物甚至比正常小鼠的Ko还要高，而且数据显示 表明这个载脂蛋白E存在于 肝脏病变，其中脂蛋白被肝脂蛋白捕获 受体应该会出现。这些发现表明，载脂蛋白E由 巨噬细胞不能完全替代内源性肝载脂蛋白E 产生一条可以代谢残留颗粒的途径。 PI列出了三个主要的具体目标，每个目标分为三个 子目标。第一个目标是评估低密度脂蛋白受体的作用。 LRP受体在用骨清除残留脂蛋白中的作用 部分表达载脂蛋白E巨噬细胞的骨髓移植 重组载脂蛋白E基因缺陷小鼠的载脂蛋白E表达 低密度脂蛋白-受体。载脂蛋白B-48和载脂蛋白B-100残留物的周转研究将 移植前后及脂蛋白去向的分析 被LRP或LDL-R内化后的相关载脂蛋白E将是 学习。在具体目标2中，研究人员将检验他们的假设 Disse间隙中脂蛋白的分泌-捕获对于 残留物摄取，这一过程需要当地的载脂蛋白E生产 肝细胞。这将通过使用腺病毒来表达E 在E缺乏的动物中，在骨髓移植前后的局部。载脂蛋白E的作用 LRP活性的表达将通过放射性标记抗体进行评估 针对体内LRP，辅之以载脂蛋白E的体外研究 浓缩的B-极低密度脂蛋白与低密度脂蛋白受体或低密度脂蛋白缺乏的成纤维细胞结合 LRP。第三个具体目标将集中在载脂蛋白E和LRP在 巨噬细胞泡沫细胞形成与动脉粥样硬化BMT入低密度脂蛋白受体/载脂蛋白E 双重Ko将允许评估巨噬细胞特异性的E表达 血脂持续升高动物动脉粥样硬化发展的研究。 胎肝细胞移植是一种产生LRP的方法 以确定野生型巨噬细胞的活性 可归因于LRP。最后，将进行体外研究，以 评估LRP在结合和降解B-极低密度脂蛋白中的作用。

DESCRIPTION (Adapted from applicant's abstract): 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 is 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.