Role of Endothelial and Macrophage ApoER2 in Atherosclerosis Modulation

描述（由申请人提供）：全基因组关联分析表明，LRP8基因的变体，包括R952Q LRP8变体，与人类过早动脉粥样硬化和急性心肌梗死有关，表明其编码的蛋白质apoE受体-2 （apoER2）对心血管健康和疾病有重大影响。apoER2如何影响心血管生物学是完全未知的。我们最近发现，在LDL受体缺失的小鼠中，apoER2缺失会导致动脉粥样硬化的明显加速，并促进病变坏死，而不影响血浆脂蛋白代谢。这个多pi项目的总体目标是确定内皮细胞和巨噬细胞中的apoER2如何促进心血管保护。这将由两个目前在脂蛋白、脂蛋白受体和血管生物学研究方面具有互补专业知识的合作实验室完成。我们最近的细胞培养工作表明，载脂蛋白E3 （apoE3）通过apoER2激活内皮NO合成酶（eNOS），从而促进内皮细胞迁移，并减弱内皮细胞-单核细胞粘附。相比之下，apoER2-R952Q对野生型受体激活eNOS具有显性负向作用。进一步发现Lrp8-/-小鼠的再内皮化受损。在Aim 1中，apoER2细胞质结构域突变体不能与接头蛋白相互作用，候选接头蛋白敲除将被用来确定apoER2在内皮中作用的生化基础。apoER2-R952Q功能障碍的基础也将被识别，表达apoER2-R952Q的内皮细胞对心脏保护行为的拯救将在NO供体中进行尝试。使用最近创建的floxed Lrp8小鼠，内皮细胞apoER2调节内皮细胞-白细胞粘附也将通过活体显微镜在体内进行研究。初步研究还表明，在巨噬细胞中，apoER2缺乏会导致氧化低密度脂蛋白诱导的过度中性脂质积累和细胞死亡/凋亡。巨噬细胞中缺乏apoER2也会导致PPAR¿和PPAR¿响应基因表达的增加。重点关注PPAR介导的过程，在Aim 2中，apoER2调节巨噬细胞细胞内信号，从而控制其功能和命运的机制将被识别。在Aim 3中，研究将使用Lrp8粘接小鼠或在LDL受体无背景下进行骨髓重构，以确定内皮细胞与巨噬细胞apoER2在防止动脉粥样硬化进展和斑块坏死方面的作用。计划中的研究将对apoER2及其主要配体apoE影响心血管健康的基础提供重要的新认识。获得的新信息有望改善以及个性化治疗，以对抗大量LRP8变异（~1%）和/或apoE多态性（~15%）个体的心血管疾病。一般人群也可能受益于利用这些过程来优化心血管保护的新疗法的发展。

DESCRIPTION (provided by applicant): Genome-wide association analyses have linked variants of the LRP8 gene, including the R952Q LRP8 variant, with premature atherosclerosis and acute myocardial infarction in humans, indicating that its encoded protein apoE receptor-2 (apoER2) has a major impact on cardiovascular health and disease. How apoER2 influences cardiovascular biology is entirely unknown. We recently discovered that apoER2 deletion in LDL receptor-null mice causes marked acceleration of atherogenesis and promotes lesion necrosis without influencing plasma lipoprotein metabolism. The overall goal of this multiple-PI project is to determine how apoER2 in endothelium and macrophages contributes to cardiovascular protection. This will be accomplished by two currently collaborating laboratories with complementary expertise in the study of lipoproteins, lipoprotein receptors and vascular biology. Our recent cell culture work has revealed that through apoER2, apolipoprotein E3 (apoE3) activates endothelial NO synthase (eNOS) and thereby promotes endothelial cell migration, and also blunts endothelial cell-monocyte adhesion. In contrast, apoER2-R952Q has dominant-negative effect on eNOS activation by wild-type receptor. It has further been found that Lrp8-/- mice have impaired reendothelialization. In Aim 1, an apoER2 cytoplasmic domain mutant incapable of adaptor protein interaction and candidate adaptor protein knockdown will be employed to determine the biochemical basis for apoER2 action in endothelium. The underpinnings of apoER2-R952Q dysfunction will also be discerned, and the rescue of cardioprotective behaviors by apoER2-R952Q- expressing endothelial cells will be attempted with NO donor. Using a recently-created floxed Lrp8 mouse, endothelial apoER2 regulation of endothelial cell-leukocyte adhesion will also be studied in vivo by intravital microscopy. Preliminary work has additionally shown that in macrophages apoER2 deficiency causes exaggerated oxLDL-induced neutral lipid accumulation and cell death/apoptosis. Lack of apoER2 in macrophages also results in increases in PPAR¿ and PPAR¿-responsive gene expression. With a focus on PPAR¿-mediated processes, in Aim 2 the mechanism(s) by which apoER2 modulates intracellular signaling in macrophages and thereby governs their function and fate will be discerned. In Aim 3, studies will be done using the Lrp8 floxed mouse or bone marrow reconstitution on LDL receptor-null background to determine the contributions of endothelial versus macrophage apoER2 to protection from atherosclerosis progression and plaque necrosis. The planned studies will provide critical new understanding of the basis by which apoER2 and its major ligand apoE influence cardiovascular health. The new information gained promises to improve as well as personalize therapies to combat cardiovascular disease in a large number of individuals with LRP8 variants (~1%) and/or apoE polymorphisms (~15%). The general population may also benefit by the development of novel therapies that harness these processes to optimize cardiovascular protection.