mTOR coordinates cell metabolism, growth and survival in pulmonary hypertension

描述（申请人提供）：肺动脉高压（PAH）是一种进行性致命性疾病，表现为肺动脉血管重构（PA）、右心室负荷升高、右心衰和死亡。肺动脉血管平滑肌细胞（PAVSMC）增殖增强和凋亡受损是PAH血管重构的关键病理生理成分，其分子机制尚不完全清楚。本研究主要关注哺乳动物雷帕霉素靶蛋白（mTOR）的两个功能不同的复合物mTORC1和mTORC2，它们作为PAH中PAVSMC代谢、生长和生存的新调节剂。通过使用特发性多环芳烃患者（人类多环芳烃PAVSMC）和未使用的供体肺的PAVSMC，我们发现培养的人类多环芳烃PAVSMC保留了体内多环芳烃肺中报道的分子和细胞异常，如增殖、存活升高，以及由于糖酵解代谢导致的细胞ATP水平改变，这为转化机制研究提供了独特的工具。我们发表的研究表明，慢性缺氧下PAVSMC增殖的增加需要mTORC1和mTORC2的激活。我们的初步数据显示，在体内和体外，特发性PAH受试者的小PA和慢性缺氧诱导的肺血管重构的大鼠的PAVSMC中，mTORC1和mTORC2信号通路上调。我们的数据还显示mTORC1和mTORC2都促进细胞增殖，但只有mTORC2调节细胞能量水平和细胞存活，这表明抑制mTORC1和mTORC2是抑制人PAH PAVSMC生长、增殖和促进细胞凋亡所必需的。基于已发表的研究和我们的数据，我们假设PAH中PAVSMC增殖和存活的增加需要激活mTORC1（促进蛋白质合成和细胞生长）和mTORC2（激活Akt，增加细胞能量水平，下调AMPK和增加细胞存活）。我们还提出mTORC1和mTORC2靶向mTOR对于抑制PAH中PAVSMC的生长、增殖、促进细胞凋亡以及防止或消除肺血管重构是必要的。为了验证这一假设，在Aim 1中，mTORC1和mTORC2信号通路的激活将使用来自PAH患者和健康供体的肺组织样本以及来自特发性PAH患者和未使用供体肺的培养PAVSMC进行严格测试；在Aim 2中，我们将确定mTORC1和mTORC2是否通过不同的分子机制来调节人类多环芳烃PAVSMC的生长、增殖和存活。mTORC1和mTORC2在调节细胞生长、细胞ATP水平和细胞凋亡中的具体作用将被研究；在Aim 3中，我们将评估mTORC1和mTORC2的抑制是否会在体外抑制人PAH PAVSMC的生长、细胞能量水平、增殖和诱导凋亡，并在大鼠慢性缺氧ph模型中阻止或消除体内肺血管重构。并将探索mTOR是否可以作为治疗人类多环芳烃的潜在分子靶点。

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH), a progressive fatal disease, manifests by vascular remodeling of pulmonary arteries (PA), elevated right ventricular afterload, right heart failure and death. Enhanced proliferation and impaired apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMC) are key pathophysiological components of vascular remodeling in PAH, the molecular mechanisms of which are not fully understood. This proposal focuses on two functionally distinct complexes of mammalian target of rapamycin (mTOR), mTORC1 and mTORC2, as novel regulators of PAVSMC metabolism, growth and survival in PAH. By using PAVSMC from subjects with idiopathic PAH (human PAH PAVSMC) and unused donor lungs, we show that cultured human PAH PAVSMC retain molecular and cellular abnormalities reported in PAH lungs in vivo, such as elevated proliferation, survival, and altered cellular ATP levels due to glycolytic metabolism that provides a unique tool for translational mechanistic studies. Our published study demonstrates that increased PAVSMC proliferation under chronic hypoxia requires activation of both mTORC1 and mTORC2. Our preliminary data show that mTORC1 and mTORC2 signaling pathways are up-regulated in vivo and in vitro in PAVSMC from small PA from subjects with idiopathic PAH and from rats with chronic hypoxia-induced pulmonary vascular remodeling. Our data also show that both mTORC1 and mTORC2 promote proliferation, but only mTORC2 modulates cellular energy levels and cell survival and suggest that inhibition of both mTORC1 and mTORC2 is required to suppress growth, proliferation and promote apoptosis in human PAH PAVSMC. Based on published studies and our data, we hypothesize that increased proliferation and survival of PAVSMC in PAH requires activation of both mTORC1 (promoting protein synthesis and cell growth) and mTORC2 (activating Akt, increasing cellular energy levels, down-regulating AMPK and increasing cell survival). We also propose that targeting mTOR in both mTORC1 and mTORC2 is necessary to inhibit PAVSMC growth, proliferation, promote apoptosis, and prevent or abrogate pulmonary vascular remodeling in PAH. To test this hypothesis, in Aim 1, activation of mTORC1 and mTORC2 signaling pathways will be critically tested using lung tissue samples from PAH patients and healthy donors and cultured PAVSMC from idiopathic PAH patients and unused donor lungs; in Aim 2, we will determine whether mTORC1 and mTORC2 employ differential molecular mechanisms to modulate growth, proliferation and survival of human PAH PAVSMC. Specific roles of mTORC1 and mTORC2 in regulating cell growth, cellular ATP levels and apoptosis will be examined; in Aim 3, we will evaluate whether suppression of both mTORC1 and mTORC2 inhibits growth, cellular energy levels, proliferation and induces apoptosis in vitro in human PAH PAVSMC and prevents or abrogates pulmonary vascular remodeling in vivo in a rat chronic hypoxia model of PH. Proposed studies will define the role of mTOR in regulating energy levels, growth, proliferation and survival of PAH PAVSMC and will explore whether mTOR could serve as a potential molecular target for treatment of human PAH.