The Role of DRP-1 and Mitochondrial Fission in Pulmonary Arterial Hypertension

DRP-1 和线粒体裂变在肺动脉高压中的作用

基本信息

批准号：
8438976
负责人：
STEPHEN L ARCHER
金额：
$ 27万
依托单位：
QUEEN'S UNIVERSITY AT KINGSTON
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8438976
关键词：
Activity Cycles Angiography Blood Vessels CDC2 Protein Kinase Ca(2+)-Calmodulin Dependent Protein Kinase Calcineurin Calcium Catheters Cell Cycle Cell Cycle Progression Cell Proliferation Cells Confocal Microscopy Cyclin B Data Death Rate Dependence Disease susceptibility Dynamin Etiology Exercise Experimental Models Flow Cytometry Fluorescence Resonance Energy Transfer Green Fluorescent Proteins Growth Guanosine Triphosphate Phosphohydrolases Heart Heart failure Human Hypoxia Link Lung Lung diseases M Phase Arrest Measurement Measures Mediating Mediator of activation protein Metric Mitochondria Mitosis Mitotic Mitotic Checkpoint Modeling Modification Molecular Molecular Profiling Monocrotaline Nature Nifedipine Obstruction Pathologic Patients Performance Pharmaceutical Preparations Phenotype Phosphorylation Phosphotransferases Photons Physiological Post-Translational Protein Processing Process Protein Dephosphorylation Protein Kinase Proteins Reliance Right-On Rodent Rodent Model Role SU 5416 Serine Site Smooth Muscle Myocytes Structure Syndrome Testing Therapeutic Tissues Ultrasonography Vascular remodeling base cell growth cohort comparative efficacy cyclin B1 disability enzyme activity hemodynamics hypertension control improved in vivo inhibitor/antagonist innovation kinase inhibitor mortality novel novel strategies outcome forecast particle prevent public health relevance pulmonary arterial hypertension research study roscovitine small molecule voltage

项目摘要

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by obstruction of the pulmonary vasculature due (in part) to idiopathic hyper-proliferation of pulmonary arterial smooth muscle cells (PASMC). Preliminary data suggest that this proliferative diathesis relates to excessive mitochondrial fission caused by activation of dynamin related protein 1 (DRP-1). In this proposal we will define the molecular basis for dysregulation of fission in human PAH and explore the nature of the link between a mitochondrial fission-fusion cycle and the cell cycle. We also evaluate whether the requirement of these hyper-proliferative cells for high rates of mitochondrial fission presents an Achilles hee that can be therapeutically targeted. Experiments are performed in human PAH PASMC and lungs and in experimental models (induced by monocrotaline or SU5416 + hypoxia). Preliminary data indicate that human PAH PASMC have fragmented mitochondria, largely due to increased fission. We have developed metrics that use mitochondrial-targeted, photoactivated, green fluorescent protein (mito-paGFP) and 2-photon confocal microscopy to quantify mitochondrial fission. Increased fission in PAH appears to result from phosphorylation of DRP-1 at Serine 616 by the key mitotic regulatory kinase, Cyclin B1- CDK1, thus linking fission and mitosis. Elevated cytosolic calcium in PAH also promotes DRP-1-mediated fission by: a) activation of calmodulin kinase and b) calcineurin-mediated dephosphorylation of an inhibitory form of DRP- 1 (Phos-Ser637). Thus, increased activity of calcineurin, cyclin B1-CDK1 and DRP-1 promote fission and proliferation in human PAH PASMC; conversely, DRP-1 inhibition (via the small molecule inhibitor, mdivi-1, or siDRP-1) decreases PASMC proliferation and, in the case of mdivi-1, regresses PAH in vivo. Hypothesis: Pathologic DRP-1 activation increases fission and promotes excessive PASMC proliferation in PAH. Corollary: DRP-1 inhibition prevents cell cycle progression, causing G2-M phase arrest. Anti-fission therapies may constitute an antiproliferative therapy for PAH. Aim 1) Are DRP-1 activation and fission required for the hyperproliferation of PASMC in human PAH? Aim 2) Does post-translational DRP-1 modification regulate PASMC proliferation in PAH? Aim 3) Does inhibition of mitochondrial fission have therapeutic benefit in experimental PAH? Innovation and Impact: This proposal is innovative in recognizing that "mitochondrial checkpoints", related to DRP-1 activation and fission, regulate cell cycle progression. The concept that mitochondrial fission is tightly linked to cell cycle progression via shared regulatory kinases is also novel. The discovery that inhibiting DRP-1 (directly or by targeting its regulatory kinases) is anti-proliferative offers a new, "anti-fission profusion", therapeutic paradigm for PAH. The translational potential of this proposal is enhanced by careful correlation of results in rodent PAH models with findings in human PAH lungs/cells from a well-characterized cohort. This is the first study to exploit PAH's reliance on rapid fission to devise novel anti-fission, anti-proliferative therapies for PAH.

描述（由申请人提供）：肺动脉高压（PAH）是一种致命综合征，其特征是（部分）由于肺动脉平滑肌细胞（PASMC）的特发性过度增殖而导致肺脉管系统阻塞。初步数据表明，这种增殖素质与动力相关蛋白 1 (DRP-1) 激活引起的线粒体过度分裂有关。在本提案中，我们将定义人类 PAH 裂变失调的分子基础，并探讨线粒体裂变融合周期与细胞周期之间联系的本质。我们还评估了这些过度增殖细胞对高线粒体裂变率的需求是否存在可作为治疗目标的致命弱点。实验在人 PAH PASMC 和肺以及实验模型（由野百合碱或 SU5416 + 缺氧诱导）中进行。初步数据表明，人类 PAH PASMC 具有线粒体碎片，这主要是由于裂变增加所致。我们开发了使用线粒体靶向、光激活、绿色荧光蛋白 (mito-paGFP) 和 2 光子共聚焦显微镜来量化线粒体裂变的指标。 PAH 中裂变的增加似乎是由于关键有丝分裂调节激酶 Cyclin B1-CDK1 将 DRP-1 在丝氨酸 616 处磷酸化所致，从而将裂变和有丝分裂联系起来。 PAH 中胞质钙含量升高还通过以下方式促进 DRP-1 介导的裂变：a) 钙调蛋白激酶的激活和 b) 钙调磷酸酶介导的 DRP-1 抑制形式 (Phos-Ser637) 的去磷酸化。因此，钙调神经磷酸酶、细胞周期蛋白 B1-CDK1 和 DRP-1 活性的增加可促进人 PAH PASMC 的裂变和增殖；相反，DRP-1 抑制（通过小分子抑制剂 mdivi-1 或 siDRP-1）可减少 PASMC 增殖，并且在 mdivi-1 的情况下，可在体内消退 PAH。假设：PAH 中病理性 DRP-1 激活会增加裂变并促进 PASMC 过度增殖。推论：DRP-1 抑制可阻止细胞周期进展，导致 G2-M 期停滞。抗裂变疗法可能构成 PAH 的抗增殖疗法。目标 1) DRP-1 激活和裂变是否是人类 PAH 中 PASMC 过度增殖所必需的？目标 2) 翻译后 DRP-1 修饰是否调节 PAH 中的 PASMC 增殖？目标 3) 抑制线粒体裂变对实验性 PAH 是否有治疗作用？创新和影响：该提案的创新之处在于认识到与 DRP-1 激活和裂变相关的“线粒体检查点”调节细胞周期进程。线粒体裂变通过共享的调节激酶与细胞周期进程紧密相关的概念也是新颖的。抑制 DRP-1（直接或通过靶向其调节激酶）具有抗增殖作用的发现提供了一种新的“抗裂变” profusion”，PAH 的治疗范例。通过将啮齿动物 PAH 模型的结果与来自特征明确的队列的人类 PAH 肺/细胞的发现仔细关联，增强了该提案的转化潜力。这是第一项利用 PAH 依赖的研究快速裂变设计针对 PAH 的新型抗裂变、抗增殖疗法。