Pulmonary Vasoreactivity Following Chronic Hypoxia

描述（由申请人提供）：慢性缺氧（CH）与阻塞性肺疾病和睡眠呼吸暂停相关，可导致肺动脉高压。基底血管平滑肌（VSM）张力升高和血管收缩剂反应性增强在ch诱导的肺动脉高压中起重要作用。然而，CH引起这些血管收缩反应的机制尚不清楚。初步研究揭示了CH在肺VSM中通过RhoA/Rho激酶依赖性Ca2+敏化途径促进肌原性张力和增强血管收缩剂反应性的新作用。这些研究进一步表明，ch诱导的VSM膜去极化刺激RhoA/Rho激酶，而不依赖于VSM游离Ca2+水平的变化，并为NADPH氧化酶（NOX）衍生的活性氧（ROS）对这一反应的主要贡献提供了有趣的证据。因此，拟议的研究将探讨CH-诱导的VSM膜去极化介导RhoA/Rho激酶途径的nox依赖性刺激，导致肌原性和激动剂诱导的肌丝Ca2+敏感性增加的中心假设。这一假设将通过以下具体目标来解决：具体目标#1：确定细胞内信号机制，导致CH后肌原性和激动剂依赖性收缩升高。我们假设CH通过RhoA/ rok诱导的VSM Ca2+致敏，增强了肺动脉肌原性张力和激动剂介导的收缩。具体目标#2：确定膜去极化对CH后基础和激动剂诱导的肺动脉收缩增加的贡献。基于下面概述的试验数据，该目标的工作假设是CH诱导的VSM膜去极化刺激RhoA/Rho激酶途径，导致基础和激动剂诱导的肌丝Ca2+敏感性增加。具体目标3：建立NOX衍生的ROS在CH后介导增强的肌原性和激动剂依赖的肺血管收缩剂反应性中的作用。我们假设由NOX产生的去极化诱导的ROS上调CH后肺VSM中基础和激动剂诱导的RhoA/Rho激酶信号。拟议的研究将采用全动物、离体肺、离体血管和分子方法来建立VSM膜去极化、一氧化氮衍生的ROS生成，以及高血压肺循环中RhoA/ROK信号传导。总的来说，这些结果有望从根本上推进我们对ROK调节血管张力的信号机制以及导致肺动脉高压发展的血管收缩机制的理解。

DESCRIPTION (provided by applicant): Chronic hypoxia (CH) associated with obstructive lung diseases and sleep apnea leads to pulmonary hypertension. Elevated basal vascular smooth muscle (VSM) tone and enhanced vasoconstrictor reactivity play a prominent role in mediating CH-induced pulmonary hypertension. However, the mechanisms by which CH elicits these vasoconstrictor responses are not understood. Pilot studies have revealed a novel effect of CH to promote myogenic tone and enhance vasoconstrictor reactivity through a RhoA/Rho kinase-dependent Ca2+-sensitization pathway in pulmonary VSM. These studies further suggest that CH-induced VSM membrane depolarization stimulates RhoA/Rho kinase independent of changes in VSM free Ca2+ levels, and provide intriguing evidence for a major contribution of NADPH oxidase (NOX)-derived reactive oxygen species (ROS) to this response. Therefore, the proposed studies will investigate the central hypothesis that CH- induced VSM membrane depolarization mediates NOX-dependent stimulation the RhoA/Rho kinase pathway leading to increased myogenic and agonist-induced myofilament Ca2+ sensitivity. This hypothesis will be addressed by the following specific aims: Specific Aim #1: Identify the intracellular signaling mechanism responsible for elevated myogenic and agonist-dependent constriction following CH. We hypothesize that CH augments pulmonary arterial myogenic tone and agonist-mediated constriction via RhoA/ROK-induced VSM Ca2+ sensitization. Specific Aim #2: Determine the contribution of membrane depolarization to increased basal and agonist-induced pulmonary arterial constriction following CH. Based on pilot data outlined below, the working hypothesis for this aim is that CH-induced VSM membrane depolarization stimulates the RhoA/Rho kinase pathway leading to increased basal and agonist-induced myofilament Ca2+ sensitivity. Specific Aim #3: Establish the role of NOX-derived ROS in mediating enhanced myogenic and agonist-dependent pulmonary vasoconstrictor reactivity following CH. We hypothesize that depolarization-induced ROS-generation by NOX upregulates basal and agonist-induced RhoA/Rho kinase signaling in pulmonary VSM following CH. The proposed research will employ whole animal, isolated lung, isolated vessel and molecular approaches to establish a novel mechanistic link between VSM membrane depolarization, NOX-derived ROS generation, and RhoA/ROK signaling in the hypertensive pulmonary circulation. Collectively, these outcomes are expected to fundamentally advance our understanding of signaling mechanisms by which ROK regulates vascular tone, as well as vasoconstrictor mechanisms that contribute to the development of pulmonary hypertension. PUBLIC HEALTH RELEVANCE: Chronic obstructive pulmonary diseases (COPD) such as emphysema and chronic bronchitis are established major causes of mortality and morbidity in the U.S. COPD is currently the fourth leading cause of death in the U.S. and is predicted to be the third leading cause of death by 2020 (Sin, D.D. and Man, S.F. Proc Am Thorac Soc 2:8-11, 2005). The aim of this study is to determine the factors responsible for the development of pulmonary hypertension in a rat model of COPD. Such disease states cause poor oxygenation of the blood (hypoxia), and the resultant pulmonary hypertension leads to right heart failure and mortality in humans.