ABCG1 and endothelial function

描述（由申请人提供）：血浆高密度脂蛋白（HDL）在动脉粥样硬化中具有保护作用，但其潜在机制尚未完全了解。HDL的动脉粥样硬化保护作用的一部分可能与其逆转内皮功能障碍的能力有关，内皮功能障碍是早期动脉粥样硬化病变的特征，涉及eNOS衍生的NO的生物利用度降低以及细胞粘附分子和炎性趋化因子和细胞因子的表达增加。HDL的许多有益作用似乎与其通过ATP结合盒转运蛋白ABCA 1和ABCG 1促进胆固醇和毒性氧固醇从细胞流出的能力有关。ABCG 1在内皮细胞中高度表达，并在促进胆固醇和7-氧化固醇流出到HDL中具有重要作用。我们最近的研究表明，高胆固醇饮食喂养的Abcg 1-/-开发eNOS依赖性舒张动脉段的严重缺陷，与7-酮胆固醇的积累和破坏的活性，eNOS的二聚体形式。在胆固醇负载的人主动脉内皮细胞（HAECs）中，ABCG 1的敲低导致eNOS活性降低，这导致了ABCG 1在内皮中具有抗动脉粥样硬化作用的中心假设。在目的1中，我们建议调查负责在ABCG 1缺陷细胞中eNOS活性降低和细胞粘附分子表达增加的细胞机制。我们将测试的中心概念是是否存在抑制性eNOS/小窝蛋白复合物的形成增加、小窝定位增加和涉及NADPH氧化酶的信号传导复合物的激活。在目的2和3中，我们将使用最近开发的Abcglflox/flox小鼠来进行ABCG 1的内皮特异性敲除。将这些小鼠与Ldlr-/-小鼠杂交以确定是否存在eNOS活性降低、细胞粘附分子表达增加和动脉粥样硬化形成增加。我们还将确定这些小鼠是否对通过输注重组HDL或apoA-1转基因诱导的HDL水平升高的动脉粥样硬化保护作用具有抗性。这些研究将使用新的动物和细胞模型，以提供新的见解的动脉粥样硬化保护作用的HDL涉及动脉内皮细胞的机制。 公共卫生相关性：我们最近的研究表明，ABCG 1在促进胆固醇和7-氧固醇从内皮细胞流出到HDL中，从而保持eNOS活性和降低细胞粘附分子表达方面发挥重要作用。该提案将评估ABCG 1在内皮细胞中具有抗动脉粥样硬化作用的假设，使用内皮细胞特异性敲除ABCG 1的小鼠以及培养的内皮细胞。它还将使用这些小鼠来确定增加HDL水平对内皮功能和动脉粥样硬化形成的已知有益作用是否通过ABCG 1介导的固醇流出来介导。

DESCRIPTION (provided by applicant): Plasma high density lipoproteins (HDL) have a protective effect in atherosclerosis but the underlying mechanisms are incompletely understood. A part of the athero-protective effect of HDL may be related to its ability to reverse endothelial dysfunction, a characteristic feature of early atherosclerotic lesions involving decreased bioavailability of eNOS-derived NO and increased expression of cell adhesion molecules and inflammatory chemokines and cytokines. Many of the beneficial effects of HDL appear to be related to its ability to promote efflux of cholesterol and toxic oxysterols from cells via the ATP binding cassette transporters, ABCA1 and ABCG1. ABCG1 is highly expressed in endothelial cells and has an essential role in promoting efflux of cholesterol and 7- oxysterols to HDL. Our recent studies show that high cholesterol diet-fed Abcg1-/- develop a severe defect in eNOS-dependent relaxation of arterial segments, associated with accumulation of 7-ketocholesterol and disruption of the active, dimeric form of eNOS. In cholesterol-loaded human aortic endothelial cells (HAECs), knock-down of ABCG1 results in decreased eNOS activity and. This leads to the central hypothesis that ABCG1 has an anti-atherogenic role in endothelium. In Aim 1 we propose to investigate cellular mechanisms responsible for decreased eNOS activity and increased expression of cell adhesion molecules in ABCG1-deficient cells. The central concept we will test is whether there is increased formation of inhibitory eNOS/Caveolin complexes, increased caveolar localization and activation of signaling complexes involving NADPH oxidases. In Aims 2 and 3 we will use recently developed Abcg1flox/flox mice to carry out endothelial- specific knock-out of ABCG1. These mice will be crossed with Ldlr-/- mice to determine whether there is reduced eNOS activity, increased expression of cell adhesion molecules and increased atherogenesis. We will also determine if these mice are resistant to the athero-protective effects of increased HDL levels induced by infusions of reconstituted HDL or apoA-1 transgenesis. These studies will use novel animal and cellular models to provide new insights into the mechanisms of athero-protective effects of HDL involving arterial endothelium. PUBLIC HEALTH RELEVANCE: Our recent studies have shown a major role of ABCG1 in promoting efflux of cholesterol and 7-oxysterols from endothelial cells to HDL, and thereby preserving eNOS activity and decreasing expression of cell adhesion molecules. This proposal will evaluate the hypothesis that ABCG1 has anti-atherogenic effects in endothelial cells, using mice with endothelial cell-specific knock-out of ABCG1, as well as cultured endothelial cells. It will also use these mice to determine if known beneficial effects of increased HDL levels on endothelial functions and atherogenesis are mediated via ABCG1-mediated sterol efflux.