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: 10881631
• 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 2024-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10881631
• 关键词: Affect; Apoptosis; Apoptotic; Autophagocytosis; Binding Sites; Biological; Biology; Blood flow; Cell Proliferation; Cell Separation; Chronic; Chronic Obstructive Pulmonary Disease; Chronic lung disease; Complication; Coupled; Cyclic AMP-Responsive DNA-Binding Protein; Data; Detection; Development; Disease; Exhibits; Exposure to; Genetic; Genetic Transcription; Genus Hippocampus; Glycolysis; Goals; Hospital Costs; Hospitalization; Human; Hypoxemia; Hypoxia; Imaging Techniques; Impairment; In Vitro; Length of Stay; Luciferases; Lung; Lung diseases; Mediating; Metabolic; Metabolism; MicroRNAs; Mitochondria; Molecular; Morbidity - disease rate; Mus; Oxygen Consumption; PINK1 gene; PTEN gene; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Phenotype; Phosphotransferases; Post-Transcriptional Regulation; Predisposition; Proliferating; Proteins; 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; Transfection; Transgenic Mice; Transgenic Organisms; Tumor Suppressor Proteins; Vascular Diseases; Vascular remodeling; Veterans; Work; bioinformatics tool; cancer cell; cellular imaging; comorbidity; differential expression; diminished oxidative phosphorylation; disease prognosis; exercise capacity; exposed human population; functional improvement; gain of function; human tissue; hypoxia-induced pulmonary hypertension; improved; 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; posttranscriptional; promoter; pulmonary arterial hypertension; reduce symptoms; right ventricular failure; 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.

肺动脉高压 (PH) 是一种与多种常见疾病相关的进行性疾病 新出现的证据表明，PH 与肺部过度增殖有关。 动脉平滑肌细胞 (PASMC) 是由线粒体功能紊乱引起的，线粒体功能受损 线粒体呼吸并以增强糖酵解的方式改变线粒体代谢程序 代谢并减少氧化磷酸化然而，线粒体质量控制异常。 调节线粒体自噬的机制尚未被研究为线粒体功能障碍的根源 PASMC 在 PH 中过度增殖，并将在拟议的研究中详细探讨。 拟议的研究将调查线粒体自噬起始蛋白 PTEN 诱导的假定作用 激酶 1 (PINK1) 在 PH 和 PASMC 增殖的发病机制中的作用 我们的初步数据表明。 PINK1 在体外缺氧暴露的 PASMC 以及啮齿动物的肺和肺动脉组织中减少 在缺氧条件下发展 PH 在拟议的研究中，我们将调查以下假设： PINK1 会导致线粒体自噬和线粒体代谢编程受损，从而导致 其他研究将确定 PINK1 的调节机制。 我们将通过执行以下具体目标来检验我们的假设： 目标 1 将研究通过以下方式调节 PINK1 的转录和转录后机制： 在通过计算机分析鉴定的几种 miR 中，miR-27a 和 miR-516a 显示了 缺氧啮齿动物肺和缺氧 PASMC 中的差异表达变化最大，因此，最初。 研究将确定 PINK1 丢失是否通过 miR-27a 或 miR-516a 介导的转录后发生 为了识别调节 PINK1 转录的因子，我们使用了生物信息学工具。 定义 PINK1 启动子中预测的转录因子 (TF) 结合位点 在已识别的几个 TF 中， cAMP 反应元件结合蛋白 (CREB) 和 PINK1 之间的调节相互作用尚未被证实 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性 为了增强翻译影响，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影响 PH 中的重编程、线粒体自噬和 PASMC 增殖 在离体大鼠 PASMC 中，缺氧或缺氧的影响。 将使用以下方法分析 PINK1 对线粒体耗氧量和糖酵解功能的获得和丧失 Seahorse XF96 Bioanalyzer™ 的其他研究将确定缺氧或 PINK1 改变的影响。 使用 Mt-Keima 转基因小鼠体内以及从大鼠和 IPAH 分离的 PASMC 中对肺体内线粒体自噬的影响 捐赠者在体外使用 mKeima-Red® 线粒体自噬检测载体。 该提案的基本目标是为线粒体的各个方面提供新的见解 PH 功能障碍，通过 PINK1 和 PINK1 的改变导致 PASMC 过度增殖和 PH 这项工作的成功执行将拓宽我们对线粒体自噬作用的理解。 PH 并为研究针对 PINK1 的新疗法奠定了基础。