ABCG1 and endothelial function

描述(申请人提供)：血浆高密度脂蛋白(高密度脂蛋白)在动脉粥样硬化中具有保护作用，但其潜在机制尚不完全清楚。高密度脂蛋白的部分动脉粥样硬化保护作用可能与其逆转内皮功能障碍有关，这是早期动脉粥样硬化病变的一个特征，涉及eNOS来源的NO生物可利用性降低，细胞黏附分子以及炎性趋化因子和细胞因子的表达增加。高密度脂蛋白的许多有益作用似乎与其通过三磷酸腺苷结合盒转运体ABCA1和Abcg1促进胆固醇和有毒氧固醇从细胞外流的能力有关。Abcg1在血管内皮细胞中高表达，在促进胆固醇和7-羟基固醇向高密度脂蛋白外流方面具有重要作用。我们最近的研究表明，高胆固醇饮食喂养的Abcg1-/-发展了eNOS依赖的动脉段松弛的严重缺陷，与7-酮胆固醇的积累和eNOS活跃的二聚体形式的破坏有关。在胆固醇负荷的人主动脉内皮细胞(HAECs)中，Abcg1基因敲除会导致eNOS活性降低。这导致了一个中心假设，即Abcg1在血管内皮细胞中具有抗动脉粥样硬化作用。在目标1中，我们建议研究在Abcg1缺陷细胞中eNOS活性降低和细胞黏附分子表达增加的细胞机制。我们将测试的中心概念是，是否有更多的抑制性eNOS/小窝复合体的形成，更多的空泡定位和涉及NADPH氧化酶的信号复合体的激活。在目标2和目标3中，我们将使用最近开发的Abcg1Flox/FLOX小鼠来进行内皮特异性Abcg1的敲除。这些小鼠将与Ldlr-/-小鼠杂交，以确定是否存在eNOS活性降低、细胞黏附分子表达增加和动脉粥样硬化增加。我们还将确定这些小鼠是否对输注重组高密度脂蛋白或apoA-1转基因引起的高密度脂蛋白水平升高的动脉粥样硬化保护作用具有抵抗力。这些研究将使用新的动物和细胞模型，为高密度脂蛋白涉及动脉内皮细胞的动脉粥样硬化保护作用的机制提供新的见解。 公共卫生相关性：我们最近的研究表明，Abcg1在促进胆固醇和7-羟基固醇从内皮细胞向高密度脂蛋白外流，从而保持eNOS活性和减少细胞黏附分子的表达方面发挥了重要作用。这项建议将评估Abcg1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Abcg1基因敲除的小鼠以及培养的内皮细胞。它还将利用这些小鼠来确定已知的高密度脂蛋白水平增加对内皮功能和动脉粥样硬化的有益影响是否通过Abcg1介导的类固醇外流介导。

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.