Mitochondrial dynamics in human pulmonary hypertension: a new therapeutic target

描述（由申请方提供）：肺动脉高压（PAH）是一种致死性综合征，其特征为部分由于细胞过度增殖导致的肺血管阻塞。我们最近发现，这种过度增殖表型与肺动脉平滑肌细胞（PASMC）线粒体网络的碎片化有关。初步数据表明，分裂的结果从线粒体融合和分裂的不平衡，有利于分裂。在人类和啮齿动物PAH PASMC中，线粒体融合蛋白-2（一种介导融合的线粒体GT3）的表达降低，而激活形式的动力蛋白相关蛋白（DRP-1）（一种介导分裂的线粒体GT3）的表达增加。其他融合/裂变介质的表达受到最小干扰。通过增加线粒体融合蛋白-2（使用腺病毒基因转移）或抑制DRP-1（使用小模块抑制剂mdivi-1或DRP-1 siRNA）来增加线粒体融合，产生一致的抗增殖结果。初步数据表明，PAH中融合/分裂比降低有利于增殖，而强制融合抑制细胞分裂所需的有丝分裂，并在G2-M期停止。我们推测，有丝分裂是一个未被识别的有丝分裂检查点，如果违反（通过强制融合），将阻止或减缓有丝分裂。 假设：线粒体融合/分裂比率降低促进人PAH血管细胞增殖。 推论：融合线粒体网络阻止有丝分裂，导致抗增殖G2-M期停滞。PAH血管细胞中线粒体片段化的分子基础的定义及其作为治疗靶点的作用的探索一直受到人类细胞和组织稀缺的阻碍。这项为期2年的研究每年使用15名WHO 1类PAH患者与15名正常受试者的组织/细胞，以确定人类PAH中线粒体分裂和融合失衡的分子基础。我们还评估了靶向线粒体分裂和融合的治疗是否可以纠正人类PAH的增殖素质。PASMC和内皮细胞中的分裂和融合率通过双光子共聚焦显微镜使用靶向细胞的光活化绿色荧光蛋白定量。通过流式细胞术定量增强丝裂融合蛋白-2或抑制DRP-1对细胞周期进程和增殖的影响。通过免疫印迹或免疫荧光在细胞和肺中测量裂变和融合介质的完整概况，而通过激光捕获显微切割评估肺中线粒体异常的组织学区室化。评估了血液中线粒体融合蛋白-2和DRP-1作为PAH潜在生物标志物的价值。患者线粒体分裂/融合异常与其人口统计学和血流动力学相关，以评估其预后重要性。 创新和影响：发现受损的线粒体融合和增强的裂变有助于增殖素质， PAH是新的。在治疗上，观察到mitofusin-2增强或DRP-1抑制诱导G2-M期阻滞提示了一种新的抗增殖策略。获得更多来自PHBI的人类样本将促进我们努力设计新的靶向组织的抗增殖PAH疗法。 公共卫生相关性：肺动脉高压（PAH）是一种破坏性的肺血管疾病，可导致残疾和过早死亡。我们已经确定了平滑肌细胞（PASMC）中线粒体的异常，导致这些细胞生长非常迅速，导致血管阻塞。正常情况下，PASMC中的线粒体形成网络;然而，在PAH中，线粒体融合蛋白-2减少，通常导致线粒体连接成链，动力蛋白相关蛋白-1增加，通常导致线粒体碎片化。我们将确定是否恢复mitofusin-2或抑制DRP-1提供了一种新的治疗人类PAH。这种RO 3为我们提供了罕见的人类PAH组织，这是我们治愈人类PAH目标的宝贵催化剂。

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is lethal syndrome characterized by obstruction of the pulmonary vasculature due, in part, to excessive cell proliferation. We recently discovered that this hyperproliferative phenotype is associated with fragmentation of the mitochondrial network in pulmonary arterial smooth muscle cells (PASMC). Preliminary data indicate that the fragmentation results from an imbalance in mitochondrial fusion and fission, favoring fission. In human and rodent PAH PASMC there is decreased expression of mitofusin-2, a mitochondrial GTPase that mediates fusion, and increased expression of the activated form of dynamin-related protein (DRP-1), a mitochondrial GTPase that mediates fission. Expression of other fusion/fission mediators is minimally perturbed. Increasing mitochondrial fusion, by augmenting mitofusin-2 (using adenoviral gene transfer), or inhibiting DRP-1 (using a small module inhibitor, mdivi-1, or DRP-1 siRNA), yields concordant antiproliferative results. Preliminary data suggest that whereas a decreased fusion/fission ratio in PAH favors proliferation, forced fusion inhibits the mitotic fission requird for cell division and arests cels in G2-M phase. We speculate that mitotic fission is an unrecognized mitotic checkpoint which if violated (by forced fusion) prevents or slows mitosis. Hypothesis: A decreased mitochondrial fusion/fission ratio promotes proliferation of human PAH vascular cells. Corollary: Fusing the mitochondrial network prevents mitotic fission, causing antiproliferative G2-M phase arrest. Definition of the molecular basis for mitochondrial fragmentation in PAH vascular cells and exploration of its role as a therapeutic target has been hampered by the scarcity of human cells and tissues. The 2-year study uses tissues/cells from 15 WHO category 1 PAH patients vs 15 normal subjects/year to determine the molecular basis for the imbalance of mitochondrial fission and fusion in human PAH. We also assess whether therapies targeting mitochondrial fission and fusion can correct the proliferation diathesis in human PAH. Fission and fusion rates in PASMC and endothelial cells are quantified by 2-photon confocal microscopy, using mitochondrial-targeted, photoactivated green fluorescent protein. The effects of enhancing mitofusin-2 or inhibiting DRP-1 on cell cycle progression and proliferation are quantified by flow cytometry. A complete profile of fission and fusion mediators is measured in cells and lungs by immunoblot or immunofluorescence, while histologic compartmentalization of mitochondrial abnormalities in the lung is assessed by laser capture microdissection. The value of mitofusin-2 and DRP-1 in the blood as potential biomarkers of PAH is assessed. Patient mitochondrial fission/fusion abnormalities are correlated with their demographics and hemodynamics to assess their prognostic importance. Innovation and Impact: The discovery that impaired mitochondrial fusion and enhanced fission contributes to a proliferative diathesis in PAH is novel. Therapeuticaly, the observation that mitofusin-2 augmentation or DRP-1 inhibition induces G2-M arrest suggests a new antiproliferative strategy. Access to more human samples from the PHBI will catalyze our efforts to devise new mitochondrial-targeted, antiproliferative PAH therapies. PUBLIC HEALTH RELEVANCE: Pulmonary arterial hypertension (PAH) is a devastating disease of the lung's blood vessels that causes disability and premature death. We have identified an abnormality of the mitochondria in smooth muscle cells (PASMC) that causes these cells to grow very rapidly, contributing to the obstruction of blood vessels. Normally mitochondria in PASMC form a network; however, in PAH there is decreased mitofusin-2, which normally causes mitochondria to join in chains, and increased dynamin-related protein-1, which normally causes mitochondrial fragmentation. We will determine whether restoring mitofusin-2 or inhibiting DRP-1 offers a new treatment for human PAH. This RO3 offers us rare access to human PAH tissues, an invaluable catalyst to our goal of curing human PAH.