Microvascular endothelial Kir channels in flow-induced dilation and hypertension

摘要： 血流诱导的血管舒张（FIV）是内皮细胞对血流反应的标志， 控制血液流向微循环的基本机制。公认的情况是 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和流动诱导小鼠阻力动脉中NO的产生， 人类严重依赖于内皮内向整流K+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NO合酶（eNOS Akt1。这是特别有趣和重要的，因为基尔渠道一直是 已知其对剪切应力敏感，但其在内皮对流动的反应中的作用仍然存在 未知该提案的目标是确定Kir2.1通道 将血流动力学剪切应力力与内皮NO合酶（eNOS）的激活相结合， 内皮细胞Kir通道在血管反应性中的作用 高血压的血管我们的第一个目标是阐明负责的机制， Kir2.1通道对剪切应力的敏感性，这是目前完全未知的。我们 初步数据表明，Kir2.1的流动敏感性被酶促降解所消除， 硫酸乙酰肝素（HS）-糖萼和从Sydecan 1-/-小鼠分离的EC中还原。我们 因此，我们提出，流动诱导的Kir通道激活是由内皮细胞介导的。 糖萼，特别是Syndecan-1，以及可能的HS-糖萼的其他元件。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集中在将Kir2.1与下游分子偶联的机制上。 Akt 1信号通路。众所周知，流动诱导的AKT 1激活需要其自身的免疫反应。 通过与磷脂PIP 3缔合，转运和募集到膜。我们 我认为Kir增强了Akt 1与PIP 3的结合，从而促进了其募集 导致Akt 1磷酸化增加。我们还探索了 流动诱导的Kir2.1激活可能调节上游事件，如激活PI 3 K 并将其募集到VEGFR 2机械感应复合物或抑制磷酸酶PTEN， 将PIP 3转换为PIP 2。这种信号传导机制在原代内皮细胞和 从小鼠新鲜收获的完整阻力动脉。一种新的内皮特异性诱导因子 为了实现这些目标，我们在实验室中建立了Kir2.1缺陷的小鼠模型。在aim中 3、我们提出了微血管内皮细胞Kir功能被抑制的假设， 在人类高血压期间。这一目标是基于我们的初步数据显示， Kir2.1对高血压患者先导队列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， 健康对照我们还将确定是否应该失去Kir2.1对FIV的贡献 这归因于Kir2.1通道功能表达的丧失或其偶联受损 到下游信令。最后，我们还将确定是否受损的FIV， 高血压患者可以通过恢复Kir2.1活性来挽救。

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.