Pulmonary Endothelial-Mesenchymal Crosstalk in Hyperactive mTOR Lung Diseases

项目摘要/摘要 肺淋巴管肌瘤病(LAM)是一种罕见的肺部疾病，由肺淋巴管肌瘤基因突变失活引起。 结节性硬化症复合体(TSC1/TSC2)基因，导致机械靶的结构性激活 雷帕霉素(MTOR)途径。LAM表现为弥漫性实质囊肿合并继发性 自发性气胸、乳糜性胸腔积液和肾血管平滑肌脂肪瘤。此外，LAM的一个子集 患者还会出现肺血管重塑和肺动脉高压。最近，mTOR已经 与其他增殖性肺部疾病有牵连，包括慢性阻塞性肺疾病(COPD)， 特发性肺纤维化(IPF)和肺动脉高压(PAH)。尽管越来越重要的是 在肺部疾病中mTOR的激活，mTOR的过度激活对肺细胞的影响仍然存在 不完全理解。 为了研究mTOR激活对肺血管重构的影响，我将利用一只新的小鼠 人LAM肺TSC2缺失模型及单细胞RNA测序。我发现了肺脏 TSC2基因缺失小鼠的血管重塑与人类LAM的组织病理学发现相一致。而且，我们的 实验室最近发现LAM细胞劫持肺转录组，导致LAM中WNT途径上调 阿龙。这项提案将研究mTOR过度激活的肺中WNT的激活如何促进肺 TSC2基因缺失(mTOR激活)转基因小鼠的血管重塑 用CTNNB1(β-catenin)WNT途径激活的小鼠株系。 这个项目将扩展我的培训，将蜂窝串扰和信令中的关键方法和概念包括在内。 我的培训将包括个性化指导、流式细胞仪课程作业/研讨会、 生物信息学和动物模型。我的训练将在维拉·克里姆斯卡娅博士的赞助下进行， 在LAM研究中处于领先地位。Krymskaya实验室是第一个建立人类LAM细胞培养以证明 雷帕霉素抑制mTORC1和抑制LAM细胞生长的作用。这项工作将在 宾夕法尼亚大学，一所拥有丰富知识环境的世界级研究机构， 合作研究人员和广泛的资源，以追求生物医学研究。团结在一起， 在此提出的研究和培训计划将有助于更好地了解肺 微环境和内皮-间充质的串扰，同时为我未来的职业生涯做好准备 肺部生物学领域的独立调查者。

Project Summary/Abstract Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by inactivating mutations in the tuberous sclerosis complex (TSC1/TSC2) gene which results in constitutive activation of the mechanistic target of rapamycin (mTOR) pathway. LAM manifests with diffuse parenchymal cysts complicated by secondary spontaneous pneumothorax, chylous pleural effusions and renal angiomyolipoma. In addition, a subset of LAM patients also develops pulmonary vascular remodeling and pulmonary hypertension. More recently, mTOR has been implicated in other proliferative pulmonary diseases including chronic obstructive lung disease (COPD), idiopathic pulmonary fibrosis (IPF) and pulmonary arterial hypertension (PAH). Despite the growing importance of mTOR activation in pulmonary diseases, the effects of mTOR hyperactivation on pulmonary cells remains incompletely understood. To study the effects of mTOR activation on pulmonary vascular remodeling, I will utilize a novel mouse model with tsc2 deletion as well as single cell RNA sequencing of human LAM lungs. I have found pulmonary vascular remodeling in the tsc2-null mice that mirrors histopathological findings in human LAM. Moreover, our lab recently found that LAM cells hijack the lung transcriptome leading to WNT pathway upregulation in the LAM lung. This proposal will examine how WNT activation in mTOR hyperactivated lungs contributes to pulmonary vascular remodeling with transgenic mice created by the cross of our novel tsc2-null (mTOR activated) mouse with Ctnnb1 (β-catenin) WNT pathway-activated mouse lines. This project will expand my training to include key methods and concepts in cellular crosstalk and signaling. My training will comprise of individualized mentorship, coursework/workshops in flow cytometry, training in bioinformatics and animal models. My training will take place under the sponsorship of Dr. Vera Krymskaya, a leader in LAM research. The Krymskaya lab was the first to establish human LAM cell cultures to demonstrate efficacy of rapamycin for inhibition of mTORC1 and abrogating LAM cell growth. This work will be conducted at the University of Pennsylvania, a world-class research institution with a rich intellectual environment, collaborative investigators and extensive resources for the pursuit of biomedical research. Together, the research and training plans proposed herein will facilitate a better understanding of the pulmonary microenvironment and endothelial-mesenchymal crosstalk while preparing me for my future career as an independent investigator in the field of pulmonary biology.