Chronic hypoxia and pulmonary vascular smooth muscle

描述（由申请人提供）：暴露于慢性缺氧（CH）发生在许多肺部疾病中，并导致肺动脉高压的发展。我们和其他实验室的研究表明，转录因子缺氧诱导因子-1 （HIF-1）在低氧性肺动脉高压的发展中起着关键作用。众所周知，HIF-1的氧敏感亚基HIF-1a的表达与肺动脉高压相关的基因编码因子的缺氧诱导有关，包括内皮素-1 (ET-1)，一种有效的血管收缩和有丝分裂剂。在之前的资助期内，我们确定了CH和ET-1改变肺血管舒缩张力的几种机制。最近，我们的研究揭示了ET-1调节HIF-1表达的新范式。我们的初步数据显示，即使在常氧条件下，ET-1也增加了HIF-1的氧敏感1亚基HIF-1a在肺动脉平滑肌细胞（PASMCs）中的表达，并降低了脯氨酸羟化酶的表达，脯氨酸羟化酶是负责靶向HIF-1a快速降解的关键酶。这些数据表明，内皮细胞缺氧激活HIF-1可能导致ET-1产生升高，PASMCs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，在许多疾病状态下，ET-1水平升高可能导致HIF-1在缺乏相关缺氧的情况下表达增加。基于这些新发现，我们假设在中度缺氧时，肺部ET-1生成的增加和PASMCs上ET-1受体的激活导致HIF-11蛋白积累和hif -1依赖性基因转录增强的正反馈或前反馈机制。这导致PASMC功能的改变，从而导致肺动脉高压的发生。为了验证这一假设，我们将使用包括转基因动物、微荧光测量、全细胞膜片钳和分子生物学在内的技术组合来实现以下具体目标：1)确定是否需要来自内皮细胞的ET-1来促进和/或加速肺循环中hif依赖性CH的病理生理作用；2)阐明ET-1调节HIF-1表达的机制；3)确定HIF-1是否是介导缺氧诱导的PASMC稳态改变的下游效应分子。

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.