Chronic hypoxia and pulmonary vascular smooth muscle

描述（由申请人提供）：暴露于慢性缺氧（CH）会导致许多肺部疾病，并导致肺动脉高压的发展。本实验室和其他实验室的研究表明，转录因子低氧诱导因子-1（HIF-1）在低氧性肺动脉高压的发生中起关键作用。众所周知，HIF-1 α（HIF-1的氧敏感亚基）的表达与编码参与肺动脉高压发展的因子（包括内皮素-1（ET-1），一种有效的血管收缩和促有丝分裂剂）的基因的缺氧诱导相关。在上一个资助期间，我们确定了CH和ET-1改变肺血管张力的几种机制。最近，我们的研究揭示了ET-1调节HIF-1表达的新模式。我们的初步数据显示，ET-1增加了肺动脉平滑肌细胞（PASMC）中HIF-1的氧敏感1亚基HIF-1a的表达，即使在常氧条件下，也减少了脯氨酰羟化酶的表达，脯氨酰羟化酶是负责靶向HIF-1a快速降解的关键酶。这些数据表明，虽然在内皮细胞中通过缺氧激活HIF-1可能导致ET-1产生升高，但PASMC中随后的ET-1信号传导有助于维持HIF-1的上调，从而产生正反馈或前馈过程。相反，ET-1水平的升高，如在许多疾病状态中发生的那样，可能导致在不存在相关缺氧的情况下HIF-1表达增加。基于这些新的发现，我们假设在中度缺氧期间，肺ET-1产生的增加和PASMCs上ET-1受体的激活导致HIF-11蛋白积累和增强的HIF-1依赖性基因转录的正反馈或前馈机制。这导致PASMC功能的改变，这有助于肺动脉高压的发展。为了验证这一假设，我们将使用包括转基因动物、显微荧光测量、全细胞膜片钳和分子生物学在内的技术的组合来实现以下具体目的：1）确定特异性来源于内皮细胞的ET-1是否是CH在肺循环中的HIF依赖性病理生理作用所必需的和/或加速CH的HIF依赖性病理生理作用; 2）阐明ET-1调节HIF-1表达的机制和3）确定HIF-1是否是介导低氧诱导的PASMC稳态改变的下游效应分子。 公共卫生相关性：该提案中的实验将探索参与肺动脉高压发展的细胞机制，肺动脉高压是一种治疗选择有限的毁灭性疾病。了解随着肺动脉高压的发展在肺血管中发生的细胞变化是推进治疗和治疗选择的关键。

DESCRIPTION (provided by applicant): Exposure to chronic hypoxia (CH) occurs with many pulmonary diseases and results in the development of pulmonary hypertension. Studies from our lab and others demonstrated a key role for the transcription factor, hypoxia-inducible factor-1 (HIF-1) in the development of hypoxic pulmonary hypertension. It is well recognized that expression of HIF-1a, the oxygen-sensitive subunit of HIF-1, correlates with hypoxic induction of genes encoding factors implicated in development of pulmonary hypertension, including endothelin-1 (ET-1), a potent vasoconstrictive and mitogenic agent. During the previous funding period, we defined several mechanisms by which CH and ET-1 alter pulmonary vasomotor tone. Recently, our studies revealed a new paradigm where ET-1 regulates HIF-1 expression. Our preliminary data show that ET-1 increased expression of the oxygen-sensitive 1 subunit of HIF-1, HIF-1a, in pulmonary arterial smooth muscle cells (PASMCs), even under normoxic conditions, and reduced expression of prolyl hydroxylases, key enzymes that are responsible for targeting HIF-1a for rapid degradation. These data suggest that while activation of HIF-1 by hypoxia in endothelial cells might cause elevated ET-1 production, subsequent ET-1 signaling in PASMCs contributes to maintained upregulation of HIF-1, creating a positive feedback, or feed-forward, process. Conversely, an elevation in ET-1 levels, as occurs in numerous disease states, may result in increased HIF-1 expression in the absence of associated hypoxia. Based on these new findings, we hypothesize that during moderate hypoxia, increased pulmonary ET-1 production and activation of ET-1 receptors on PASMCs leads to a positive feedback, or feed- forward, mechanism of HIF-11 protein accumulation and enhanced HIF-1-dependent gene transcription. This results in alterations in PASMC function which contribute to the development of pulmonary hypertension. To test this hypothesis, we will use a combination of techniques including transgenic animals, microfluorescence measurements, whole-cell patch-clamp, and molecular biology, to accomplish the following Specific Aims: 1) determine whether ET-1 derived specifically from endothelial cells is required for and/or accelerates HIF-dependent pathophysiological effects of CH in the pulmonary circulation; 2) elucidate the mechanism(s) by which ET-1 modulates HIF-1 expression and 3) determine whether HIF-1 is the downstream effector molecule mediating hypoxia- induced alterations in PASMC homeostasis. PUBLIC HEALTH RELEVANCE: The experiments in this proposal will explore cellular mechanisms involved in the development of pulmonary hypertension, a devastating disease with limited treatment options. Understanding the cellular changes that occur in the pulmonary vasculature with development of pulmonary hypertension is key to advancing treatment and therapeutic options.