Vascular Smooth Muscle Function in Pulmonary Hypertension

描述(申请人提供)：慢性低氧(CH)与阻塞性肺疾病和睡眠呼吸暂停相关，通常会导致全身性肺动脉收缩和血管重构，随后的肺动脉高压和右心衰竭。肺动脉平滑肌细胞(PASMC)细胞内钙离子([Ca~(2+)]i)在建立肺血管阻力中起着重要作用，而且在CH诱导的肺动脉高压中，Ca~(2+)内流的增加在血管收缩和血管重塑反应中的作用日益明显。我们的实验室发现了酸敏感离子通道1(ASIC1)在介导动脉粥样硬化后PASMC内钙离子内流(SOCE)中的新作用。然而，对控制PASMC中ASIC1的运输、稳定和激活的机制(S)知之甚少。此外，CH如何改变这些机制(S)以增加质膜上功能的ASIC1还不清楚。这项应用的总体目标是确定ASIC1在CH诱导的肺动脉高压的发展过程中的重要作用以及参与这一反应的潜在机制。我们将验证这一中心假设，即ASIC1通过PASMC中SOCE的一种新机制，有助于CH诱导的血管反应性和肺动脉高压的增加，具体目的如下：1)确定ASIC1在CH诱导的肺动脉高压中的作用。我们将通过评估活体测量的肺动脉压、动脉重塑和血管反应性来验证ASIC1参与CH诱导的肺动脉高压的活性血管收缩成分的假设。2)确定ASIC1膜转运的机制(S)以及CH如何改变这一机制。我们将使用细胞表面生物素化分析、[Ca~(2+)]i成像和荧光标记的ASIC1蛋白的活细胞共聚焦成像来验证这一假设，即CH通过增加RhoA介导的肌动蛋白聚合来促进依赖于PICK1(与C-激酶1相互作用的蛋白质)的ASIC1转运到膜上。3)检测细胞氧化还原电位对ASIC1激活的影响。我们假设，CH后过氧化氢(H_2O_2)的减少增加了ASIC1的表面表达和通道活性。我们将评估血管反应性，并进行[Ca~(2+)]i成像和电生理学研究，以检测还原/氧化剂和H_2O_2对ASIC1通道活性和PASMC转运的作用。这项研究的创新之处在于，它聚焦于ASIC1膜转运、通道调节和正常肺循环中钙离子内流的未知机制。此外，我们的工作处于确定低氧如何影响高血压循环中ASIC1功能的前沿。这项拟议研究的成功完成将提供对ASIC1如何在CH诱导的肺动脉高压中起作用的机制的了解，并将极大地促进我们对PASMC钙稳态改变和高血压肺循环中血管收缩的细胞机制的了解。最终，这些知识有可能为肺动脉高压的治疗提供新的方向。

DESCRIPTION (provided by applicant): Chronic hypoxia (CH) associated with obstructive pulmonary disease and sleep apnea often results in generalized pulmonary arterial constriction and vascular remodeling, subsequent pulmonary hypertension and right heart failure. Pulmonary arterial smooth muscle cell (PASMC) intracellular calcium ([Ca2+]i) plays a vital role in establishing pulmonary vascular resistance and it has become increasingly evident that increased Ca2+ influx contributes to both the vasoconstrictor and vascular remodeling responses in CH-induced pulmonary hypertension. Our laboratory has found a novel role for acid sensing ion channel 1 (ASIC1) in mediating store-operated Ca2+ entry (SOCE) in PASMC following CH. However, little is known about the mechanism(s) that govern ASIC1 trafficking, stability, and activation in PASMC. In addition, it is unclear how CH alters these mechanism(s) to increase functional ASIC1 at the plasma membrane. The overall objective of this application is to establish an important role for ASIC1 in the development of CH-induced pulmonary hypertension and potential mechanisms involved in this response. We will test the central hypothesis that ASIC1, through a novel mechanism of SOCE in PASMC, contributes to CH-induced increases in vascular reactivity and pulmonary hypertension with the following specific aims: 1) Determine the contribution of ASIC1 to CH-induced pulmonary hypertension. We will test the hypothesis that ASIC1 contributes to the active vasoconstrictor component of CH-induced pulmonary hypertension by assessing in vivo measurements of pulmonary arterial pressure, arterial remodeling, and vasoreactivity. 2) Identify the mechanism(s) responsible for ASIC1 membrane trafficking and how this is altered by CH. We will test the hypothesis that CH promotes PICK1 (protein interacting with C- kinase 1)-dependent ASIC1 trafficking to the membrane through increased RhoA-mediated actin polymerization by use of cell surface biotinylation assays, [Ca2+]i imaging, and live-cell confocal imaging of a fluorescently- labeled ASIC1 protein. 3) Examine the effect of cellular redox potential on ASIC1 activation. We hypothesize that decreased hydrogen peroxide (H2O2) following CH increases ASIC1 surface expression and channel activity. We will assess vasoreactivity and conduct [Ca2+]i imaging and electrophysiology studies to examine the role of reducing/oxidizing agents and H2O2 on ASIC1 channel activity and trafficking in PASMC. The proposed research is innovative through its focus on the previously undefined mechanisms of ASIC1 membrane trafficking, channel regulation and Ca2+ influx in the normal pulmonary circulation. In addition, our work is at the forefront of determining how hypoxia affects ASIC1 function in the hypertensive circulation. Successful completion of the proposed research will provide a mechanistic-based understanding of how ASIC1 contributes to CH- induced pulmonary hypertension and will significantly advance our knowledge of the cellular mechanisms responsible for altered PASMC Ca2+ homeostasis and vasoconstriction in the hypertensive pulmonary circulation. Ultimately, such knowledge has the potential to provide new directions in pulmonary hypertension therapy.