Mitochondrial dynamics in human pulmonary hypertension: a new therapeutic target

描述(由申请人提供)：肺动脉高压(PAH)是一种致死性综合征，其特征是部分由于细胞过度增殖导致肺血管阻塞。我们最近发现，这种过度增殖的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，这种碎裂是由于线粒体融合和分裂的不平衡造成的，有利于分裂。在人和啮齿动物PAH PASMC中，介导融合的线粒体GTPase-2的表达减少，而介导分裂的线粒体GTPase的激活形式动力蛋白相关蛋白(DRP-1)的表达增加。其他融合/裂变介体的表达受到最小程度的干扰。通过增加丝裂原蛋白-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，可以产生一致的抗增殖结果。初步数据表明，虽然PAH中融合/分裂比率的降低有利于增殖，但强制融合抑制了细胞分裂所需的有丝分裂，并使细胞处于G2-M期。我们推测，有丝分裂是一个未被识别的有丝分裂检查点，如果被破坏(通过强制融合)，就会阻止或减缓有丝分裂。 假设：线粒体融合/分裂比率降低促进了人PAH血管细胞的增殖。 推论：融合线粒体网络可防止有丝分裂分裂，导致抗增殖的G2-M期停滞。由于人类细胞和组织的稀缺性，PAH血管细胞线粒体断裂的分子基础的定义和作为治疗靶点的作用的探索一直受到阻碍。这项为期两年的研究使用了来自15名WHO第1类PAH患者和15名正常受试者的组织/细胞/年，以确定人类PAH线粒体分裂和融合失衡的分子基础。我们还评估了针对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和内皮细胞的分裂和融合率通过双光子共聚焦显微镜进行量化，使用线粒体靶向、光激活的绿色荧光蛋白。用流式细胞仪定量检测增强丝裂原蛋白-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法测量细胞和肺中裂变和融合介体的完整轮廓，而通过激光捕获显微解剖来评估肺中线粒体异常的组织学区划。评估血液中丝裂原蛋白-2和DRP-1作为PAH的潜在生物标志物的价值。患者线粒体分裂/融合异常与他们的人口学和血流动力学相关，以评估他们的预后重要性。创新和影响：线粒体融合受损和分裂增强有助于患者的增殖素质 多环芳烃是一种新的环芳烃。在治疗方面，观察到丝裂原蛋白-2增强或DRP-1抑制导致G2-M期停滞，提示了一种新的抗增殖策略。从PHBI获得更多人类样本将催化我们设计新的线粒体靶向、抗增殖多环芳烃疗法的努力。

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.