Mitochondrial dynamics in human pulmonary hypertension: a new therapeutic target

描述（由申请人提供）：肺动脉高压（PAH）是一种致命综合征，其特征是肺血管阻塞，部分原因是细胞过度增殖。我们最近发现，这种高增殖表型与肺动脉平滑肌细胞（PASMC）线粒体网络的断裂有关。初步数据表明，分裂是由于线粒体融合和裂变的不平衡，有利于裂变。在人类和啮齿动物PAH PASMC mitofusin-2的表达减少,线粒体GTPase介导融合,并增加表达的活化形式dynamin-related蛋白质(DRP-1),线粒体GTPase产生裂变。其他聚变/裂变介质的表达受到的干扰最小。通过增加mitofusin-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，虽然PAH中融合/裂变比的降低有利于增殖，但强制融合抑制细胞分裂所需的有丝分裂，并使细胞在G2-M期停滞。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）阻止或减缓有丝分裂。

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.