Microvascular endothelial Kir channels in flow-induced dilation and hypertension

摘要： 血流诱导的血管扩张(FIV)是血管内皮细胞对流和血管内皮细胞反应的标志 控制血液流向微循环的基本机制。它由来已久。 FIV的一个关键机制是一氧化氮(NO)的产生。我们最近的研究 发现FIV和血流诱导的小鼠阻力动脉中NO的产生和 人类严重依赖内皮细胞内向整流钾通道(Kir2.1)。我们也 确定Kir2.1通过丝氨酸/苏氨酸激酶调节内皮型一氧化氮合酶(ENOS) AKT1。这一点特别有趣和重要，因为KIR频道长期以来 已知对剪应力敏感，但它们在血管内皮细胞对血流反应中的作用仍然存在 未知。这项提案的目标是确定Kir2.1通道的机制 血流动力学切应力对内皮型一氧化氮合酶(ENOS)激活的耦合作用 一氧化氮的产生及内皮KIR通道在人血管反应性中的作用 高血压中的血管。我们的第一个目标是阐明导致 Kir2.1通道对剪应力的敏感性，目前完全未知。我们的 初步数据表明，Kir2.1的流动敏感性被酶降解所消除 从Sydecan1-/-小鼠分离的内皮细胞中被还原的硫酸乙酰肝素(HS)-Glyocalyx。我们 因此，我们认为血流诱导的KIR通道激活是由内皮细胞介导的。 尤其是Syndecan-1，可能还有HS-Glyocalyx的其他成分。我们也 提出Kir2.1与Syndecan-1直接相互作用，并阐明其作用机制 互动。我们的第二个目标是研究Kir2.1与下游连接的机制 AKT1信号通路。众所周知，AKT1的流动诱导激活需要其 通过与磷脂PIP3结合而移位和募集到膜上。我们 建议KIR加强Akt1与PIP3的联系，从而促进其招募 与膜结合，导致Akt1磷酸化增加。我们还探索了各种可能性 流动诱导的Kir2.1激活可能调节上游事件，如激活PI3K 及其与VEGFR2机械传感复合体的募集或抑制磷酸酶PTEN 将PIP3转换为PIP2。这种信号机制在原代内皮细胞中和在 新鲜取自小鼠的完整阻力动脉。一种新的内皮细胞特异性诱导物 为了实现这些目标，我们实验室已经建立了Kir2.1缺乏症小鼠模型。在AIM 3、我们建议检验微血管内皮细胞KIR功能受抑的假设 在人类高血压期间。这一目标是基于我们的初步数据显示减少 高血压患者先导队列中Kir2.1对FIV的贡献。在这项研究中，我们将招募 3组受试者，包括高血压前期或1期高血压患者和 健康对照组。我们还将确定是否应该损失基尔2.1对FIV的贡献 可归因于Kir2.1通道功能表达的丧失或其偶联功能受损 到下行信令。最后，我们还将确定受损的FIV在 通过恢复Kir2.1的活动，高血压患者可能会获救。

Abstract: Flow-induced vasodilation (FIV) is a hallmark of the endothelial response to flow and an essential mechanism for the control of blood flow to the microcirculation. It is well established that a key mechanism responsible for FIV is generation of nitric oxide (NO). Our recent study discovered that FIV and flow-induced generation of NO in resistance arteries of mice and humans critically depend on endothelial inwardly-rectifying K+ channels (Kir2.1). We also established that Kir2.1 regulate endothelial NO synthase (eNOS) via a serine/threonine kinase Akt1. This was particularly interesting and important because Kir channels have long been known to be sensitive to shear stress but their role in endothelial responses to flow remained unknown. The goals of this proposal are to determine the mechanisms by which Kir2.1 channels couple hemodynamic shear stress forces to activation of endothelial NO synthase (eNOS) and NO production and to evaluate the role of endothelial Kir channels in vasoreactivity of human vessels in hypertension. Our first aim is to elucidate the mechanism responsible for the sensitivity of Kir2.1 channels to shear stress, which is currently completely unknown. Our preliminary data show that flow-sensitivity of Kir2.1 is abrogated by enzymatic degradation of Heparan Sulphate (HS)-Glycocalyx and reduced in ECs isolated from Sydecan1-/- mice. We propose, therefore, that flow-induced activation of Kir channels is mediated by the endothelial Glycocalyx, specifically Syndecan-1, and possibly other elements of HS-Glycocalyx. We also propose that Kir2.1 interacts directly with Syndecan-1, and elucidate the mechanism of this interaction. Our second aim focuses on the mechanism that couples Kir2.1 to the downstream Akt1 signaling pathway. It is well-known that flow-induced activation of AKT1 requires its translocation and recruitment to the membrane via association with a phospholipid PIP3. We propose that Kir enhances the association of Akt1 with PIP3 and thus facilitates its recruitment to the membrane, resulting in increased Akt1 phosphorylation. We also explore the possibilities that flow-induced activation of Kir2.1 may regulate the upstream events, such as activation PI3K and its recruitment to VEGFR2 mechanosensing complex or inhibit a phosphatase PTEN that converts PIP3 to PIP2. This signaling mechanism is explored in primary endothelial cells and in intact resistance arteries freshly-harvested from mice. A new endothelial-specific inducible mouse model of Kir2.1 deficiency has been generated in our lab to achieve these goals. In aim 3, we propose to test the hypothesis that microvascular endothelial Kir function is depressed during human hypertension. This aim is based on our preliminary data showing decreased contribution of Kir2.1 to FIV in a pilot cohort of hypertensive patients. In this study, we will recruit 3 groups of subjects that include patients with pre-hypertension or stage 1 hypertension and healthy controls. We will also determine whether the loss of Kir2.1 contribution to FIV should be attributed to the loss of the functional expression of Kir2.1 channels or to their impaired coupling to the downstream signaling. Finally, we will also determine whether impaired FIV in hypertensive patients may be rescued by restoring Kir2.1 activity.