Cholesterol Regulation of Endothelial K+ Channels

描述（申请人提供）：高胆固醇血症诱导的内皮功能障碍是心血管疾病发病的一个主要因素，但内皮损伤的机制尚不清楚。我们的一般假设是内皮细胞K+通道的抑制在胆固醇诱导的内皮功能障碍中起关键作用。我们的研究表明，膜胆固醇的增加强烈抑制内皮向内校正K+ (Kir)通道，这是主动脉内皮细胞和假定的流量传感器中主要的K+通道。此外，我们已经证明内皮Kir通道在体外被致动脉粥样硬化脂蛋白和体内血浆高胆固醇血症抑制，导致内皮膜去极化。基于这些发现，我们假设胆固醇诱导的内皮细胞Kir的抑制及其对血流敏感性的损害导致内皮功能障碍和血流诱导的内皮依赖性血管舒张功能的丧失。本研究的目的是研究胆固醇和低密度脂蛋白诱导的Kir抑制的分子机制，并确定其在血流诱导的血管舒张中的作用。为了解决这一假设，我们提出了三个具体目标：(1)通过鉴定特定的脂质-蛋白（PIP2，通道跨膜结构域的脂质-蛋白界面）和/或蛋白质-蛋白质（caveolin，支架蛋白）相互作用来阐明内皮Kir通道对胆固醇敏感性的分子基础。(2)探讨低密度脂蛋白诱导的Kir抑制对不同血管床内皮细胞的影响，并评估挽救当前的潜在策略。具体来说，我们将测试Kir活性是否可以通过HDL、胆固醇结构类似物和胆固醇不敏感的Kir突变体来挽救。(3)确定体内血浆高胆固醇血症对内皮细胞Kir对血流敏感性和血流诱导内皮膜超极化的影响，阐明不同胆固醇条件下Kir通道在血流诱导的导管和小冠状动脉血管舒张中的作用。这些研究将在饮食诱导的高胆固醇血症猪模型中进行。我们相信，实现这些目标将对理解胆固醇诱导的内皮功能障碍以及离子通道-胆固醇相互作用做出重大贡献。我们也相信这些研究可以为开发新的治疗策略提供基础。公共卫生相关性：心血管疾病（CVD）占所有死亡人数的40%，并导致男性和女性的严重发病率。众所周知，心血管疾病发展的关键早期步骤是血管内层单细胞薄层功能障碍，称为内皮。我们的目标是确定高胆固醇血症导致内皮功能受损的机制，高胆固醇血症是心血管疾病发展的主要危险因素。我们的研究集中在高胆固醇血症引起的内皮细胞对血流变化的反应能力丧失，这是内皮功能障碍的一个标志。我们打算研究血浆和细胞胆固醇增加抑制内皮离子通道的机制，这些通道参与血管张力的内皮依赖性控制和内皮细胞对血流的反应能力。我们相信，阐明这些机制将有助于了解与高胆固醇血症相关的内皮功能障碍，并为新的治疗方法提供基础。

DESCRIPTION (provided by applicant): Hypercholesterolemia-induced endothelial dysfunction is a major factor in the pathogenesis of cardiovascular disease but the mechanisms of endothelial injury are not well understood. Our general hypothesis is that suppression of endothelial K+ channels plays a key role in cholesterol-induced endothelial dysfunction. Our studies have shown that an increase in membrane cholesterol strongly suppresses endothelial inwardly-rectifying K+ (Kir) channels, the dominant K+ channels in aortic endothelial cells and putative flow sensors. Furthermore, we have shown that endothelial Kir channels are suppressed by atherogenic lipoproteins in vitro and by plasma hypercholesterolemia in vivo leading to endothelial membrane depolarization. Based on these findings, we hypothesize that cholesterol-induced suppression of endothelial Kir and impairment of its sensitivity to flow result in endothelial dysfunction and the loss of flow-induced endothelium-dependent vasodilatation. The goal of this study is to investigate molecular mechanisms responsible for cholesterol- and LDL-induced suppression of Kir and to determine its role in flow-induced vasodilatation. To address this hypothesis, we propose three specific aims: (1) To elucidate the molecular basis of the sensitivity of endothelial Kir channels to cholesterol by identifying specific lipid-protein (PIP2, lipid-protein interface of the transmembrane domains of the channels) and/or protein-protein (caveolin, scaffolding proteins) interactions responsible for the sensitivity of the channels to cholesterol. (2) To investigate the impact of LDL-induced Kir suppression in endothelial cells derived from different vascular beds and to evaluate potential strategies to rescue the current. Specifically, we will test whether Kir activity can be rescued by HDL, by cholesterol structural analogues and by cholesterol-insensitive Kir mutants. (3) To determine the impact of in vivo plasma hypercholesterolemia on the sensitivity of endothelial Kir to flow and on flow-induced endothelial membrane hyperpolarization and to elucidate the role of Kir channels in flow-induced vasodilatation of conduit and small coronary arteries under different cholesterol conditions. These studies will be performed in a porcine model of diet-induced hypercholesterolemia. We believe that achieving these goals will make a major contribution to the understanding of cholesterol-induced endothelial dysfunction, as well as of ion channel-cholesterol interactions in general. We also believe that these studies may provide the basis for developing new therapeutic strategies. PUBLIC HEALTH RELEVANCE: Cardiovascular disease (CVD) results in 40% of all deaths and results in serious morbidity in both men and women. It is well known that a key early step in the development of CVD is dysfunction of a thin single-cell layer of the inner lining of the blood vessels, called endothelium. Our goal is to determine the mechanisms responsible for the impairment of endothelial function by hypercholesterolemia, a major risk factor for the development of CVD. Our studies focus on hypercholesterolemia-induced loss of the ability of endothelial cells to respond to changes in blood flow, a hallmark of endothelial dysfunction. We intend to investigate the mechanisms by which an increase in plasma and cellular cholesterol suppress endothelial ion channels that are involved in endothelial-dependent control of vascular tone and the ability of endothelial cells to respond to blood flow. We believe that elucidating these mechanisms will contribute to the understanding of endothelial dysfunction that is associated with hypercholesterolemia and provide the basis for new therapeutic approaches.