IMMUNOCHEMICAL STRUCTURE FUNCTION OF APOLIPOPROTEIN A-I

在动脉粥样硬化中，高密度脂蛋白(HDL)是治疗干预的关键靶点。这项建议将研究载脂蛋白(Apo)AI如何促进过量胆固醇(C)从外周细胞有效转移到血浆高密度脂蛋白。利用动脉粥样硬化的小鼠模型，我们将检验这一假设，即ApoAI在介导巨噬细胞(Mphi)C外流中的特异性是其从球形高密度脂蛋白解离能力的函数。解离可以产生稳定的、缺乏脂质的载脂蛋白AI，它可以与细胞受体相互作用，介导能量依赖的C外流。第一个特定的目标将检查从胆固醇酯负载的Mphi中能量依赖的C转运的体外特异性。将比较多种可交换的无脂和贫脂载脂蛋白，包括apoAI、apoAII、apoAIV和apoE。第二个目标是通过免疫化学和物理化学分析来鉴定动脉粥样硬化病变内膜中存在的载脂蛋白AI的形式(S)。第三个目标将确定体内形成低脂载脂蛋白AI的机制(S)，并将研究清道夫受体B1、脂蛋白脂肪酶、肝脂酶、磷脂转移蛋白和载脂蛋白II的作用。第四个目标将检验这样的假设，即通过在病变内提供低脂载脂蛋白AI的替代来源，可以绕过载脂蛋白AI的体内特异性要求。将产生表达Mphi特异性载脂蛋白AI的转基因小鼠，并将作为供体用于动脉粥样硬化倾向小鼠的骨髓重建研究。这一假说驱动的建议将：1)提供对间质液脂蛋白组成的准确了解；2)确定体内机制来解释ApoAI从病变中去除C的独特能力；以及3)使用永久性基因转移来改变动脉粥样硬化性血管疾病的进程。

In atherosclerosis high density lipoproteins (HDL) are a key target for therapeutic intervention. This proposal will examine how apolipoprotein (apo) AI promotes efficient transfer of excess cholesterol (C) from peripheral cells to plasma HDL. Using mouse models of atherosclerosis, we will test the hypothesis that specificity for apoAI in mediating macrophage (Mphi) C efflux is a function of its ability to dissociate from spherical HDL. Dissociation gives rise to a stable, lipid-poor apoAI that can interact with cellular receptors to mediate energy-dependent C efflux. The first specific aim will examine the in vitro specificity of energy-dependent transport of C from cholesteryl ester loaded Mphi. Multiple exchangeable lipid-free and lipid-poor apoproteins including apoAI, apoAII, apoAIV, and apoE will be compared. The second aim will identify the form(s) of apoAI present in vivo in the intima of an atherosclerotic lesion using immunochemical and physicochemical analyses. The third aim will identify the in vivo mechanism(s) for formation of lipid-poor apoAI and will examine the roles of scavenger receptor-type B1, lipoprotein lipase, hepatic lipase, phospholipid transfer protein and apoAII. The fourth aim will test the hypothesis that the in vivo specificity requirement for apoAI can be bypassed by providing an alternate source of lipid-poor apoAI within lesions. Transgenic mice expressing Mphi-specific apoAI will be produced and will serve as donors in bone marrow reconstitution studies of atherosclerosis-prone mice. This hypothesis-driven proposal will: 1) provide a precise understanding of the lipoprotein composition of interstitial fluids; 2) identify in vivo mechanisms to explain the unique capacity of apoAI to remove C from lesions; and 3) use permanent gene transfer to alter the course of atherosclerotic vascular disease.