HEPATIC METABOLISM OF DIET DERIVED LIPOPROTIENS

总的目的是了解乳糜微粒残余物如何促进动脉粥样硬化。目前的建议的目标是阐明这些颗粒的肝脏去除的机制。残余物在餐后期间积聚在血液中，这意味着有时它们的去除变得有限。待检验的假设是乳糜微粒残留物去除的初始速率由LDL受体的数量和肝脏在内化颗粒之前隔离颗粒的能力（隔离空间）决定。隔离空间的大小由硫酸乙酰肝素蛋白聚糖定义，并且通过肝脂肪酶的存在来修改空间对残余物的亲和力。通过从螯合转移到LDL受体和LRP来确定稳态去除速率。后者部分由辅因子，特别是肝apoE决定。提出了三个具体目标。首先，完成利用离体灌流小鼠肝脏来表征残余物去除过程的研究。小鼠离体肝脏灌注作为一种研究残留物清除的技术得到了验证。数据表明，LDL受体和LRP都是该过程的饱和组分，并且可能存在额外的清除机制; apoE是快速清除残留物所绝对需要的，但apoE的肝脏分泌仅是该过程的一个组分所需要的。LDL受体和LRP以及apoE缺陷的小鼠将用于完成这些研究。第二，将制定各种方法，以确定封存空间的大小和从这一空间中撤离的速度。使用荧光标记的残余物和荧光标记的内皮细胞和肝细胞的共聚焦显微镜将允许确定残余物在哪里积累，积累的残余物中的哪一部分在Disse空间中，以及它们离开这个空间的速率。目前正在开发一个用于分析同位素去除数据的多隔室模型，以确定去除过程各组成部分的亲和力和能力。使用第三个特定目标的动物，这将确定哪些分子有助于这些成分。作为备用策略，细胞分离技术可用于实现前两种方法的目标。第三个具体目标将审查进程决定因素的性质。表达不同固定水平LDL受体的小鼠将用于研究这种分子在初始去除以及从隔离中排出中的作用。类似的策略将通过产生截短但功能性的LRP样分子而用于LRP。将研究具有不同apoE同种型的小鼠，以评估这如何影响其作为辅因子和作为捕获机制的一部分的功能。这组研究还将评估LPL在肝脏残留清除中的作用。在最后一组中，肝脂肪酶在肝脏中的量、位置和酶活性将变化，以了解它是否作为LRP的辅因子，或者它是否作为隔离空间的决定因素，或者两者兼而有之。此外，将尝试改变肝脏的多配体蛋白聚糖含量，以测试该HSPG作为隔离空间的主要决定因素的作用。总而言之，预计这些研究将提供残留物去除的具体模型，并确定该机制中的关键要素，并可能针对这些要素进行治疗干预。

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.