Apolipoprotein F enhances HDL function

项目摘要 HDL对心血管疾病风险的有益作用的一个主要机制是它能够 通过胆固醇逆向转运（RCT）促进胆固醇排泄。HDL升高 胆固醇水平通常与降低疾病风险相关。然而，最近的高密度脂蛋白升高试验 药物未能观察到临床益处。这导致了一个修正的HDL假设，其中功能性 HDL促进外周胆固醇流出的能力和RCT，而不是HDL浓度，被认为是最重要的。 重要的是它的保护作用。胆固醇酯转运蛋白（CETP）促进细胞的净运动， 胆固醇酯（CE）。在人类中，随着组织来源的 胆固醇转化为HDL上的CE，70%的HDL CE在被去除之前被CETP转化为VLDL和LDL 由肝。因此，CETP是HDL介导的胆固醇排泄途径中的中心参与者。我们有 在体外显示载脂蛋白F（ApoF）通过抑制涉及LDL的脂质转移来调节CETP活性 但刺激HDL和VLDL之间的脂质转移。我们假设ApoF是作为一种代谢调节因子， 通过将HDL衍生CE重定向为VLDL而不是LDL，从而降低血浆LDL胆固醇 水平和刺激RCT。在这里，我们测试这一假设，并调查机制控制载脂蛋白F 活动目的1 -确定ApoF在定义HDL衍生CE结局中的作用-使用已建立的 在仓鼠中siRNA介导的ApoF敲低模型中，我们将量化这种关键蛋白的丢失 改变HDL CE向LDL和VLDL的流出，测量VLDL和LDL CE的肝脏清除率，和 确定ApoF对饲料喂养和脂肪喂养动物中HDL功能和RCT的影响。目标2 -定义 将ApoF转化为其活性形式并量化高脂血症对ApoF影响的机制 血浆ApoF以活性和非活性形式存在;活性ApoF是 与LDL结合。高胆固醇血症LDL富含ApoF。我们假设， LDL表面脂质控制ApoF结合。我们将确定LDL的分子特性， 增强ApoF结合，并严格测试它们在此过程中的作用。总ApoF和活性ApoF浓度 将在高脂血症人类受试者中定量，以确定它们对异常代谢的可能贡献。 脂蛋白水平目的3 -确定控制血浆ApoF水平的因素-在脂肪喂养的动物中，血浆 ApoF增加，但肝APOF mRNA水平降低。为了理解这些不和谐的观察- 因此，将确定导致APOF mRNA减少的分子机制， 将比较血脂正常和高胆固醇血症仓鼠中血浆ApoF的浓度。我们预计会找到- ApoF与LDL的更大结合延长了其血浆停留时间。总的来说，这些研究将 为ApoF如何调节CETP活性和增强HDL的功能特性提供了新的见解。

PROJECT SUMMARY A major mechanism proposed for HDL's beneficial effect on cardiovascular disease risk is its capacity to promote cholesterol excretion through a process called reverse cholesterol transport (RCT). Elevated HDL cholesterol levels are generally correlated with reduced disease risk. However, recent trials of HDL-raising drugs failed to observe a clinical benefit. This has led to a revised HDL hypothesis where the functional capacity of HDL to promote peripheral cholesterol efflux and RCT, not HDL concentration, is deemed most important for its protective role. Cholesteryl ester transfer protein (CETP) facilitates the net movement of cholesteryl ester (CE) between plasma lipoproteins. In humans, following the conversion of tissue-derived cholesterol to CE on HDL, 70% of HDL CE is transferred by CETP to VLDL and LDL prior to being removal by the liver. Thus, CETP is a central player in the HDL-mediated cholesterol excretion pathway. We have shown in vitro that apolipoprotein F (ApoF) modulates CETP activity by inhibiting lipid transfers involving LDL but stimulating lipid transfer between HDL and VLDL. We hypothesize that ApoF operates as a metabolic switch by redirecting HDL-derived CE to VLDL instead of LDL, and that this reduces plasma LDL cholesterol levels and stimulates RCT. Here, we test this hypothesis and investigate mechanisms controlling ApoF activity. Aim 1 – Determine the role of ApoF in defining the fate of HDL-derived CE – With an established model of siRNA-mediated ApoF knockdown in hamsters, we will quantify how the loss of this key protein modifies the efflux of HDL CE to LDL and VLDL, measure the hepatic clearance of VLDL and LDL CE, and determine the impact of ApoF on HDL function and RCT in chow-fed and fat-fed animals. Aim 2 – Define the mechanism that converts ApoF to its active form and quantify the effect of hyperlipidemia on ApoF concentration and its activation status – Plasma ApoF exists in both active and inactive forms; active ApoF is bound to LDL. Hypercholesterolemic LDL is enriched in ApoF. We hypothesize that the molecular packing of LDL surface lipids controls ApoF binding. We will identify the molecular properties of LDL that correlate with enhanced ApoF binding and rigorously test their role in this process. Total and active ApoF concentrations will be quantified in hyperlipidemic human subjects to determine their possible contribution to aberrant lipoprotein levels. Aim 3 – Determine the factors that control plasma ApoF levels – In fat-fed animals, plasma ApoF is increased but hepatic APOF mRNA levels are decreased. To understand these discordant observa- tions, the molecular mechanisms causing this decrease in APOF mRNA will be determined, and the turnover of plasma ApoF in normolipidemic and hypercholesterolemic hamsters will be compared. We anticipate find- ing that greater association of ApoF with LDL lengthens its plasma residence time. Overall, these studies will provide novel insight into how ApoF regulates CETP activity and enhances the functional properties of HDL.