Hypoxic Pulmonary Hypertension:Contractile Mechanisms

描述（由申请人提供）： 长时间暴露于氧分压降低的环境中， 疾病，导致肺动脉高压（CHPH）。 新出现的证据 表明持续的肺动脉平滑肌细胞（PASMC）收缩 与慢性缺氧（CH）相关的可能与K通道的变化有关， 膜电位和细胞内Ca2+浓度（[Ca2 +] i）;然而， 这一过程的确切机制和介导因素仍然存在， 未知 缺氧诱导因子1（HIF-1）是一种转录因子， 对缺氧的适应性反应 HIF-1控制缺氧诱导的 许多基因可能在CHPH的发展中起重要作用，包括内皮素- I（ET-12）. ET-1受体拮抗剂预防CHPH，ET-1抑制CH。 此外，在慢性缺氧动物的PASMC中，ET-1信号 转导途径被改变，使得ET-1诱导的收缩被抑制。 增强，似乎主要是通过激活Ca2+非依赖性 收缩途径，可能是由于蛋白激酶C（PKC）的激活， 丝裂原活化蛋白激酶（MAPK）。 收缩增强， ET-1水平升高可能导致正反馈机制， 维持持续的血管收缩。 我们建立了CHPH的小鼠模型 与部分缺陷的转基因小鼠一起， 针对HIF-1亚基的变化，创建了一个独特的动物模型系统， 使我们能够特别针对HIF-1在这种疾病过程中的作用。 我们将使用该模型来检验假设，即在CH期间， HIF-1是CHPH发展的关键起始步骤， 低氧诱导HIF-1导致ET-1水平升高， PASMQ中收缩机制的组合，包括：a）去极化 由于K+通道的减少; B）去极化驱动的 静息[Ca2 +] i和c）收缩细胞Ca2+敏感性的变化 由于PKC和MAPK依赖性途径的激活， 测试 这个假设，我们将使用技术的组合，包括等距 动脉段的张力记录和蛋白质印迹分析，以及 PASMCs的全细胞膜片钳和微荧光测量， 实现以下具体目的：1）确定HIF-1是否调节 在PASM中观察到ET-1水平和介导CH诱导的改变; 2）确定 ET-1是否介导CH诱导的PASM改变; 3）确定 CH是否降低K+通道蛋白表达，以及这是否是 4）确定CH诱导的升高是否受HIF-1和ET-1的调节; 静息[Ca2 +] i是由于去极化驱动的Ca2+内流增强 通过Na +/Ca~（2+）交换和5）确定PKC和MAPK在ET-1 在暴露于CH后，-诱导的PASM中Ca2+敏感性增加。 摘要）

DESCRIPTION (provided by applicant): Prolonged exposure to decreased oxygen tension, as occurs in many pulmonary diseases, results in pulmonary hypertension (CHPH). Emerging evidence indicates sustained pulmonary arterial smooth muscle cell (PASMC) contraction associated with chronic hypoxia (CH) may be related to changes in K channels, membrane potential and intracellular Ca2+ concentration ([Ca2+]i); however, the exact mechanisms underlying, and factors mediating, this process remain unknown. Hypoxia-inducible factor 1 (HIF-1), a transcription factor, mediates numerous adaptive responses to hypoxia. HIF-1 controls hypoxic induction of many genes that may be important in development of CHPH, including endothelin- I (ET-12). ET-1 receptor antagonists prevent CHPH and ET-1 inhibits CH. Furthermore, in PASMC from chronically hypoxic animals, ET-1 signal transduction pathways are altered such that ET-1 -induced contraction is enhanced and appears to occur primarily via activation of Ca2+ -independent contractile pathways, possibly due to activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Enhanced contraction coupled with elevated ET-1 levels could result in a positive feedback mechanism by which sustained vasoconstriction is maintained. We developed a murine model of CHPH that, together with the generation of transgenic mice with partial deficiency for the varies as subunit of HIF-1, creates a unique animal model system that allows us to specifically target the role of HIF-1 in this disease process. We will use this model to test the hypothesis that during CH, induction of HIF-1 is a critical initiating step in the development of CHPH, and that hypoxic induction of HIF-1 results in elevated ET-1 levels which activate a combination of contractile mechanisms in PASMQs, including: a) depolarization due to reduction of K+ channels; b) depolarization-driven elevation of resting [Ca2+]i and c) changes in Ca2+ -sensitivity of the contractile apparatus due to activation of PKC- and MAPK- dependent pathways. To test this hypothesis, we will use a combination of techniques, including isometric tension recording and Western blot analysis in arterial segments, and whole-cell patch-clamp and microfluorescence measurement in PASMCs, to accomplish the following Specific Aims: 1) determine whether HIF-1 regulates ET-1 levels and mediates CH-induced alterations observed in PASM; 2) determine whether ET-1 mediates CH-induced alterations observed in PASM; 3) determine whether CH decreases K+ channel protein expression and whether this is regulated by HIF-1 and ET-1; 4) determine whether the CH-induced elevation in resting [Ca 2+]i is due to depolarization-driven enhancement of Ca2+ influx through Na+/Ca2+ exchange and 5) determine the role of PKC and MAPK in ET-1 -induced increase in Ca2+ sensitivity in PASM following exposure to CH. (End of Abstract)