HEPATIC METABOLISM OF DIET DERIVED LIPOPROTIENS

总的目标是了解乳糜微粒残留物是如何导致动脉粥样硬化的。本研究的目的是阐明肝清除这些颗粒的机制。剩余物在餐后期间积聚在血液中，这意味着有时它们的清除变得有限。待验证的假设是，乳糜微粒残余清除的初始速率取决于LDL受体的数量，以及肝脏在将其内化（隔离空间）之前将其隔离的能力。隔离空间的大小由肝素-硫酸酯-蛋白聚糖决定，而残体空间的亲和力则由肝脂肪酶的存在而改变。稳态脱除速率由从固存到LDL受体和LRP的转移决定。后者部分由辅助因素决定，尤其是肝载脂蛋白e。提出了三个具体目标。首先，完成利用离体灌注小鼠肝脏表征残体去除过程的研究。小鼠离体肝脏灌注是研究残体清除的有效方法。数据表明LDL受体和LRP都是该过程的可饱和组分，并且可能存在另一种去除机制；快速清除残体绝对需要apoE，但肝脏分泌apoE只需要这个过程的一个组成部分。LDL受体和LRP以及apoE缺乏的小鼠将被用来完成这些研究。第二，将制定方法来确定封存空间的大小和从该空间流出的速度。对内皮细胞和肝细胞使用荧光标记残留物和荧光标记的共聚焦显微镜，可以确定残留物积聚的位置，积聚残留物在Disse空间中的比例，以及它们离开该空间的速率。目前正在开发一种多室模型，用于分析同位素去除数据，以确定去除过程各组成部分的亲和力和能力。使用第三种特定目标的动物，这将确定哪些分子有助于这些成分。作为一种后备策略，细胞分离技术可以用来实现前两种方法的目标。第三个具体目标将审查这一进程的决定因素的性质。表达不同固定水平LDL受体的小鼠将被用于研究该分子在初始去除以及从隔离中退出的作用。类似的策略将被用于LRP，通过创建截断但功能性的LRP样分子。具有不同apoE亚型的小鼠将被研究，以评估这如何影响其作为辅助因子的功能，以及作为捕获机制的一部分。这组研究还将评估LPL在肝脏残体清除中的作用。在最后一组中，将改变肝脏中脂肪酶的数量、位置和酶活性，以了解它是否作为LRP的辅助因素，还是作为封存空间的决定因素，或者两者兼而有之。此外，将尝试改变肝脏中syndecan的含量，以测试HSPG作为隔离空间的主要决定因素的作用。总之，预计这些研究将为残体清除提供一个具体的模型，并确定该机制中的关键因素，从而指导治疗干预。

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.