Mitophagy in pulmonary hypertension: Novel roles of PTEN-Induced Kinase-1 in the pathobiology of pulmonary artery smooth muscle cell proliferation and mitochondrial dysfunction

肺动脉高压是一种进行性疾病，通常与多种疾病相关。 折磨着老病人。新的证据表明，PH与肺过度增殖有关 动脉平滑肌细胞(PASMC)是由线粒体功能紊乱引起的 线粒体呼吸和改变线粒体代谢程序以增强糖酵解的方式 新陈代谢，减少氧化磷酸化。然而，线粒体质量控制的异常 调控有丝分裂的机制尚未被作为线粒体功能障碍的来源进行调查 PASMC在PH中过度增殖，并将在拟议的研究中详细探讨。 拟议中的研究将调查吞噬有丝分裂的启动蛋白PTEN诱导的假定的作用 PINK1在PH和PASMC增殖中的作用我们的初步数据表明 PINK1在体外低氧暴露的PASMC以及啮齿类动物的肺和肺动脉组织中减少 在低氧条件下发生肺高压。在拟议的研究中，我们将调查以下假设： PINK1导致有丝分裂和线粒体代谢编程受损，导致 PH组PASMC过度增殖。其他研究将确定调节PINK1的机制 在低氧条件下的损失。我们将通过执行以下具体目标来验证我们的假设： 目标1将研究转录和转录后调节PINK1的机制 MicroRNA(MiR)。在In Silico分析鉴定的几种MIR中，MIR-27a和MIR-516a显示 低氧鼠肺和低氧PASMC的差异表达变化最大。因此，首字母 研究将确定PINK1丢失是通过miR-27a还是miR-516a介导的转录后转录发生的 抑制PINK1。为了确定调控PINK1转录的因素，我们使用了生物信息学工具 确定PINK1启动子中预测的转录因子(Tf)结合位点。在确定的几个TF中， CAMP反应元件结合蛋白(CREB)与PINK1之间的调控相互作用尚未见报道 之前已经过验证。因此，我们将使用PINK1启动子荧光素酶报告载体进行研究 确定假定的CREB结合位点是否具有功能。为了增强翻译效果，我们将 利用特发性肺动脉确定调节PINK1表达和活性的机制 高血压(IPAH)、PASMC和肺组织。 目标2将描述PINK1改变对线粒体代谢的功能后果 PH时重编程、有丝分裂和PASMC增殖。在分离的大鼠PASMC中，缺氧或缺氧的影响 PINK1功能的获得和丧失对线粒体耗氧量和糖酵解的影响将使用 海马XF96生物分析仪™。其他研究将确定缺氧或PINK1改变的影响 Mt-kema转基因小鼠体内肺内有丝分裂吞噬作用及大鼠和IPAH分离的PASMC的研究 在体外使用mKeima-Red®有丝分裂检测载体的供者。 这项提案的基本目标是为线粒体的各个方面提供新的见解 通过PINK1和PINK1的改变促进PASMC过度增殖和PH的PH功能障碍 有丝分裂。这项工作的成功实施将扩大我们对吞噬丝裂原在 为研究针对PINK1的新疗法奠定了基础。

Pulmonary hypertension (PH) is a progressive disorder associated with a variety of diseases commonly afflicting veteran patients. Emerging evidence demonstrates that PH and excessive proliferation of pulmonary artery smooth muscle cells (PASMC) are caused by derangements in mitochondrial function that impair mitochondrial respiration and alter mitochondrial metabolic programming in a manner that enhances glycolytic metabolism and diminishes oxidative phosphorylation. However, abnormalities in mitochondrial quality control mechanisms that regulate mitophagy have not been investigated as a source of mitochondrial dysfunction and PASMC hyperproliferation in PH, and will be explored in detail in the proposed studies. The proposed studies will investigate the role of the mitophagy initiator protein, PTEN-induced putative kinase-1 (PINK1) in the pathogenesis of PH and PASMC proliferation. Our preliminary data demonstrate that PINK1 is reduced in hypoxia-exposed PASMC in vitro and in the lungs and pulmonary artery tissues of rodents that develop PH in hypoxic conditions. In the proposed studies, we will investigate the hypothesis that loss of PINK1 causes impairments in mitophagy and mitochondrial metabolic programming that lead to excessive PASMC proliferation in PH. Additional studies will define the mechanisms that regulate PINK1 loss in hypoxic conditions. We will test our hypothesis through the execution of the following Specific Aims: Aim 1 will investigate transcriptional and posttranscriptional mechanisms that regulate PINK1 through microRNA (miR). Among several miRs identified by in silico analysis, miR-27a and miR-516a exhibited the greatest differential expression changes in the hypoxic rodent lung and in hypoxic PASMC. Therefore, initial studies will determine if PINK1 loss occurs through miR-27a- or miR-516a-mediated posttranscriptional suppression of PINK1. To identify factors that regulate PINK1 transcription, we employed bioinformatics tools to define predicted transcription factor (TF) binding sites in the PINK1 promoter. Among several TFs identified, regulatory interactions between cAMP response element-binding protein (CREB) and PINK1 had not been previously validated. Therefore, we will conduct studies using a PINK1 promoter luciferase reporter vector to determine whether putative CREB binding sites are functional. To enhance the translational impact, we will determine mechanisms that regulate PINK1 expression and activity using idiopathic pulmonary artery hypertension (IPAH) PASMC and lung tissue. Aim 2 will characterize the functional consequences of PINK1 alterations on mitochondrial metabolic reprogramming, mitophagy, and PASMC proliferation in PH. In isolated rat PASMC, the impact of hypoxia or PINK1 gain and loss of function on mitochondrial oxygen consumption and glycolysis will be analyzed using the Seahorse XF96 Bioanalyzer™. Additional studies will determine the effects of hypoxia or PINK1 alterations on mitophagy in the lung in vivo using Mt-Keima transgenic mice and in PASMC isolated from rats and IPAH donors using the mKeima-Red® mitophagy detection vector in vitro. The fundamental goals of this proposal are to provide novel insights into aspects of mitochondrial dysfunction in PH that contribute to PASMC hyperproliferation and PH through alterations in PINK1 and mitophagy. The successful execution of this work will broaden our understanding of the role of mitophagy in PH and lay the groundwork to investigate novel therapies that target PINK1.