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

肺动脉高压中的线粒体自噬：PTEN 诱导的激酶 1 在肺动脉平滑肌细胞增殖和线粒体功能障碍病理学中的新作用

• 批准号: 10266041
• 负责人: C MICHAEL HART
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10266041
• 关键词: Affect; Apoptosis; Apoptotic; Autophagocytosis; Binding Sites; Biological; Biology; Blood flow; Cell Proliferation; Chronic; Chronic Obstructive Airway Disease; Chronic lung disease; Complication; Coupled; Cyclic AMP-Responsive DNA-Binding Protein; Data; Detection; Development; Disease; Exhibits; Exposure to; Failure; Gene Expression Regulation; Genetic; Genetic Transcription; Genus Hippocampus; Glycolysis; Goals; Hospital Costs; Hospitalization; Hypoxemia; Hypoxia; Imaging Techniques; Impairment; In Vitro; Lead; Length of Stay; Luciferases; Lung; Lung diseases; Mediating; Metabolic; Metabolism; MicroRNAs; Mitochondria; Molecular; Morbidity - disease rate; Mus; Oxygen Consumption; PTEN gene; PTEN-induced putative kinase; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Phenotype; Phosphotransferases; Post-Transcriptional Regulation; Predisposition; Prognosis; Proliferating; Protein phosphatase; Pulmonary Hypertension; Pulmonary artery structure; Quality Control; Rattus; Regulation; Reporter; Reporting; Research; Research Project Grants; Respiration; Risk; Rodent; Rodent Model; Role; Signal Transduction; Smooth Muscle Myocytes; Source; Structure of parenchyma of lung; Talents; Techniques; Testing; Therapeutic; Tissues; Transcriptional Regulation; Transfection; Transgenic Mice; Transgenic Organisms; Tumor Suppressor Proteins; Vascular Diseases; Vascular remodeling; Ventricular; Veterans; Work; base; bioinformatics tool; cancer cell; cellular imaging; comorbidity; differential expression; diminished oxidative phosphorylation; exercise capacity; exposed human population; human tissue; improved; improved functioning; in silico; in vivo; innovation; insight; loss of function; lung hypoxia; military veteran; mitochondrial dysfunction; mortality; neuroblastoma cell; new therapeutic target; novel; novel therapeutic intervention; promoter; pulmonary arterial hypertension; reduce symptoms; smoking-related lung disease; transcription factor; translational impact; vector

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.

肺高血压（pH）是一种与多种疾病相关的进行性疾病 受苦的老兵患者。新兴的证据表明pH和肺的过量 动脉平滑肌细胞（PASMC）是由线粒体功能的进化引起的 线粒体呼吸和改变线粒体代谢编程的方式以增强糖酵解的方式 代谢并减少氧化磷酸化。但是，线粒体质量控制异常 调节线粒体的机制尚未作为线粒体功能障碍的来源和 pH中的PASMC过度增殖，并将在拟议的研究中详细探讨。 拟议的研究将研究线粒体引发剂蛋白，PTEN诱导的推定的作用 pH和PASMC增殖的发病机理中的激酶-1（pink1）。我们的初步数据表明 在体外和肺部和肺动脉组织中降低了暴露于低氧PASMC的PINK1 在低氧条件下发展pH。在拟议的研究中，我们将研究丧失的假设 PINK1导致线粒体和线粒体代谢编程的损害，导致 pH中的PASMC增殖过多。其他研究将定义调节pink1的机制 低氧条件下的损失。我们将通过执行以下特定目的来检验我们的假设： AIM 1将研究通过调节Pink1的转录和转录后机制 microRNA（mir）。在计算机分析中确定的几种miR中，miR-27a和miR-516a暴露了 低氧啮齿动物肺和低氧PASMC的最大差异表达变化。因此，初始 研究将确定pink1是否通过miR-27a-或miR-516a介导的转录后发生了 抑制Pink1。为了确定调节PINK1转录的因素，我们采用了生物信息学工具 定义PINK1启动子中的预测转录因子（TF）结合位点。在确定的几个TF中， 营地反应元件结合蛋白（CREB）和PINK1之间的调节相互作用尚未 先前已验证。因此，我们将使用PINK1启动子荧光素酶报告媒介进行研究 确定推定的CREB结合位点功能是否功能。为了增强翻译影响，我们将 确定使用特发性肺动脉调节PINK1表达和活性的机制 高血压（IPAH）PASMC和肺组织。 AIM 2将表征PINK1改变线粒体代谢的功能后果 pH中的重编程，线粒体和PASMC增殖。在分离的大鼠PASMC中，缺氧或 在线粒体氧消耗和糖酵解方面的PINK1增益和功能丧失将使用 Seahorse XF96 Bioanalyzer™。其他研究将确定缺氧或PINK1改变的影响 使用MT-Keima转基因小鼠和从大鼠和IPAH分离的PASMC中的MT-Keima转基因小鼠在体内的线粒体上 在体外使用Mkeima-Red®线粒体检测载体的供体。 该提案的基本目标是为线粒体的各个方面提供新颖的见解 pH功能障碍通过PINK1和 线粒体。这项工作的成功执行将扩大我们对线粒体在 pH并奠定基础，以研究针对PINK1的新型疗法。