Novel signaling in chronic hypoxic responses in pulmonary arteries

描述(申请人提供)：慢性低氧(CH)可见于许多心肺疾病和高原居住地。这种常见的临床应激导致肺动脉(PA)重塑、血管收缩和高血压。这些细胞反应主要是由于PA平滑肌细胞(PASMCs)细胞内钙离子浓度([Ca~(2+)]i，即Ca~(2+)信号)增加所致。然而，潜在的分子机制还不完全清楚，目前对肺动脉高压(PH)的治疗选择有限。基于我们的初步数据和以前的文献，我们提出了一个非常创新的中心假说，即CH导致Rieske Iron-Sulve Protein(RISP)介导的线粒体活性氧物种(ROS)的产生，扰乱FK506结合蛋白12.6(FKBP12.6)/ryanodine Receptor-2(RyR2)复合体，导致RyR2功能亢进，增加钙释放，激活钙调神经磷酸酶(Calcineurin)、活化T细胞胞浆核因子(NFATc)和核因子-kB(NFkB)，并增加PASMCs周期蛋白的表达，导致PA重塑、收缩和高血压。为了验证这一令人兴奋的假说，我们将使用互补的、最先进的激光扫描共聚焦显微镜、膜片钳记录、基因操作、双重免疫荧光染色、荧光共振能量转移和其他方法来解决以下基本问题(特定目的)：(1)FKBP12.6/RyR2复合体是否由于RISP介导的线粒体ROS产生而被破坏；(2)FKBP12.6/RyR2复合体的破坏是否介导了CH-诱导的PA重构、收缩和高血压；(3)FKBP12.6/RyR2复合体的破坏是否通过钙调神经磷酸酶依赖的、NF-kB/NFATc介导的细胞周期蛋白信号转导途径在CH诱导的PAS反应中起作用？我们完全相信，这些研究结果将极大地提高我们目前对PA重构、收缩和高血压的细胞分子机制的认识，并有助于全面了解钙离子下游信号轴是什么以及如何处于低氧状态。预计这一结果还可能有助于为PAH和其他相关肺血管疾病的治疗创造新的、特定的和更有效的治疗靶点，具有极大的变革临床实践的潜力。

DESCRIPTION (provided by applicant): Chronic hypoxia (CH) occurs in many cardiopulmonary diseases and high altitude residency. This common clinical stress causes the remodeling, vasoconstriction and hypertension in the pulmonary artery (PA). These cellular responses primarily result from an increase in intracellular Ca concentration ([Ca2+]i, i.e., Ca2+ signaling) in PA smooth muscle cells (PASMCs). However, the underlying molecular mechanisms are not fully understood, and current therapeutic options for pulmonary hypertension (PH) are limited. Based on our preliminary data and previous publications, we propose a very innovative central hypothesis that CH causes Rieske iron-sulfur protein (RISP)-mediated mitochondrial reactive oxygen species (ROS) production, disrupts FK506 binding protein 12.6 (FKBP12.6)/ryanodine receptor-2 (RyR2) complex, causes RyR2 hyperfunction, increases Ca2+ release, activates calcineurin, cytoplasmic nuclear factor of activated T- cells (NFATc) and nuclear factor-kB (NFkB), and increases cyclin expression in PASMCs, leading to PA remodeling, contraction and hypertension. To test this exciting hypothesis, we will employ complementary, state-of-the-art laser scanning confocal microscopy, patch clamp recording, gene-manipulation, double immunofluorescence staining, florescent resonance energy transfer, and other approaches to address the following fundamental questions (Specific Aims): (1) is the FKBP12.6/RyR2 complex disrupted as a result of RISP-mediated mitochondrial ROS production in PASMCs from mice following CH; (2) does the disruption of the FKBP12.6/RyR2 complex mediate CH-induced PA remodeling, contraction and hypertension; and (3) is the role of FKBP12.6/RyR2 complex disruption in CH-induced responses in PAs mediated by the calcineurin-dependent, NF-kB/NFATc-mediated cyclin signaling axis? We fully believe that the findings from the proposed studies will greatly improve our current knowledge of the cellular molecular mechanisms for PA remodeling, constriction and hypertension, and help to fully understand what and how Ca2+ downstream signaling axis is in hypoxic. It is expected that the results may also aid in the creation of novel, specific and more effective therapeutic targets for the treatment of PAH and other relevant pulmonary vascular diseases, with a great potential for revolutionizing clinical practices.