IMMUNOCHEMICAL STRUCTURE FUNCTION OF APOLIPOPROTEIN A-I

在动脉粥样硬化中，高密度脂蛋白（HDL）是治疗干预的关键目标。该提案将研究载脂蛋白（apo） AI如何促进多余胆固醇(C)从外周细胞有效转移到血浆HDL。通过小鼠动脉粥样硬化模型，我们将验证apoAI在介导巨噬细胞（Mphi） C外溢中的特异性是其与球形HDL分离能力的功能。解离产生稳定的低脂apoAI，可与细胞受体相互作用，介导能量依赖性C外排。第一个具体目标将检查C从胆固醇酯装载Mphi的能量依赖性运输的体外特异性。多种可交换的无脂和无脂载脂蛋白，包括apoAI， apoAII， apoAIV和apoE将进行比较。第二个目的是通过免疫化学和物理化学分析，确定存在于动脉粥样硬化病变内膜内的apoAI的形态。第三个目标将确定体内形成低脂apoAI的机制，并将研究清除率受体B1型、脂蛋白脂肪酶、肝脂肪酶、磷脂转移蛋白和apoAI的作用。第四个目标将验证一个假设，即通过在病变内提供低脂apoAI的替代来源，可以绕过apoAI的体内特异性要求。将产生表达mphi特异性apoAI的转基因小鼠，并将作为动脉粥样硬化易感小鼠骨髓重建研究的供体。这一假设驱动的建议将：1)提供对间质液脂蛋白组成的精确理解；2)确定apoAI从病变中清除C的独特能力的体内机制；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病程。

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.