Chronic hypoxia and pulmonary vascular smooth muscle

描述(由申请人提供)： 与许多肺部疾病一样，长时间暴露在氧分压降低的环境中，会导致肺动脉高压，显著恶化预后。这一过程的发病机制尚不清楚。慢性低氧(CH)引起的肺动脉平滑肌细胞(PASMC)收缩可能与细胞内钙离子浓度([Ca~(2+)]_i)升高有关。在PASMCs中，可观察到CH的去极化，这支持了电压门控钙通道激活或钠/钙交换增强导致[钙]i增加的猜测。内皮源性收缩因子-1(ET-1)可能参与了CHPH的发病机制，因为ET-1升高了[Ca~(2+)]_i。暴露于CH后，ET-1引起的[Ca~(2+)]_i升高减弱，但收缩仍维持，提示激活了非钙依赖性收缩通路。这可能是由于ET-1诱导酪氨酸激酶(TK)的激活所致。ET-1的低氧诱导是通过激活系统内皮细胞中的转录因子HIF-1而发生的，在部分缺乏HIF-1的小鼠中，CH诱导的肺动脉高压明显减轻。因此，我们假设HIF-1的诱导是肺动脉高压发展的起始步骤，导致ET-1水平升高。然后ET-1扩散到PASMC，激活三种收缩机制。首先，ET-1降低电压门控性钾通道的表达，导致去极化驱动的Na/Ca~(2+)交换激活和静息[Ca~(2+)]i升高。其次，ET-1通过L型钙通道引起TK介导的Ca~(2+)内流。这两种机制都增加了肌球蛋白轻链(MLCs)的磷酸化。最后，ET-1通过TK介导的肌动蛋白结合蛋白调节引起收缩机构对钙敏感性的改变。这最后一步允许肌动蛋白与步骤1和2中产生的磷酸化的MLCs相互作用，并导致收缩。为了验证这些假说，我们将在我们的缺氧性肺动脉高压模型中使用一系列技术，包括动脉节段的等长张力记录、Northern和Western印迹分析、全细胞膜片钳和显微荧光测量，以实现以下特定目的：1)确认缺氧诱导因子-1调节缺氧性肺血管系统内ET-1的诱导并鉴定所涉及的细胞类型(S)；2)确定CH诱导静息[Ca~(2+)]i升高的机制；以及3)确定ET-1导致CH期间收缩的机制。

DESCRIPTION (provided by applicant): Prolonged exposure to decreased oxygen tension, as occurs with many pulmonary diseases, results in pulmonary hypertension, significantly worsening prognosis. The mechanism underlying the pathogenesis of this process remains unknown. Pulmonary arterial smooth muscle cell (PASMC) contraction associated with chronic hypoxia (CH) may be caused by elevated intracellular Ca2+ concentration ([Ca2+]i). In PASMCs, depolarization is observed with CH, fueling speculation that [Ca2+]i is increased due to activation of voltage-gated Ca2+ channels or enhanced Na+/Ca2+ exchange. The endothelium-derived constricting factor, endothelin-1 (ET-1), may contribute to the pathogenesis of CHPH, since ET-1 increases [Ca2+]i. Following exposure to CH, the ET-1-induced rise in [Ca2+]i is reduced but contraction is maintained, suggesting activation of Ca2+-independent contractile pathways. This may be due to ET-1-induced activation of tyrosine kinases (TK). Hypoxic induction of ET-1 occurs via activation of the transcription factor, HIF-1, in systemic endothelium and in mice partially deficient for HIF-1, CH-induced pulmonary hypertension is markedly reduced. Therefore, we hypothesize induction of HIF-1 is an initiating step in the development of pulmonary hypertension, leading to elevated ET-1 levels. ET-1 then diffuses to PASMCs, activating three contractile mechanisms. First, ET-1 decreases voltage-gated K+ channel expression, leading to depolarization-driven activation of Na+/Ca2+ exchange and elevation of resting [Ca2+]i. Second, ET-1 causes TK-mediated Ca2+ influx through L-type Ca2+ channels. Both of these mechanisms increase phosphorylation of myosin light chains (MLCs). Finally, ET-1 causes changes in Ca2+-sensitivity of the contractile apparatus via TK-mediated regulation of actin binding proteins. This final step allows actin to interact with the phosphorylated MLCs generated in steps 1 and 2, and results in contraction. To test these hypotheses, we will use a combination of techniques in our model of hypoxic pulmonary hypertension, including isometric tension recording in arterial segments, Northern and Western blot analysis, whole-cell patch-clamp and microfluorescence measurements, to accomplish the following Specific Aims: 1) confirm that HIF-1 regulates hypoxic induction of ET-1 in the pulmonary vasculature and identify the cell type(s) involved; 2) determine the mechanisms responsible for the CH-induced increase in resting [Ca2+]i and 3) determine the mechanisms by which ET-1 causes contraction during CH.