Role of Resident Mesenchymal Stem Cells in Lung Allograft Rejection

描述(申请人提供)：肺移植后的长期存活受限于称为闭塞性细支气管炎综合征(BOS)的进行性阻塞性呼吸缺陷的发展。BOS是由小气道纤维性闭塞或闭塞性细支气管炎(BO)引起的。我们实验室的研究表明，移植物驻留的间充质细胞(MCs)在BO的纤维增殖中起着重要作用。令人振奋的新的初步数据表明，进化保守的蛋白激酶mTOR(雷帕霉素的机械靶点)在mTOR复合体1(MTORC1)和mTORC2中都参与了BOS中间充质细胞胶原合成功能的增强。从BOS患者来源的MCs被发现同时具有mTORC1和mTORC2的结构性激活。SiRNA介导的mTORC2结合伴侣Rictor的下调抑制了mTORC1底物的磷酸化，逆转了MCs的纤维化表型，提示mTORC2在活化的纤维化MCs中起着重要作用。针对mTORC1和mTORC2效应器的选择性ATP竞争性mTOR抑制剂被发现是BOS MCs胶原表达的有效抑制剂。这些数据使我们假设mTOR复合体1(MTORC1)和mTORC2通过介导移植物驻留的MC的翻译激活在BO的发病机制中发挥关键作用。在这一应用中，我们提出了在体内研究mTOR通路在BO发病机制中的作用的实验，目的是确定ATP竞争性mTOR抑制剂是否可以作为一种潜在的治疗选择。为了实现这些目标，我们建立了一种全肺小鼠原位肺移植模型，在该模型中，中度MHC不匹配会导致第28天前发生呼吸道纤维化。在目标1中，我们建议使用这个小鼠模型来研究体内常驻MC中的mTORC1/2信号。我们将利用BAC荧光标记Foxf1的小鼠，Foxf1是一种在肺间充质细胞中高表达的胚胎肺间充质相关转录因子，以特异性地鉴定和研究移植物驻留的MC。本课程将探讨BOS中单核细胞结构性mTOR1/2激活的上游信号机制(S)。我们将集中在G蛋白偶联受体，溶血磷脂酸(LPA)1，信号在提出的自分泌激活机制中的作用。目的2将剖析下游mTORC1效应器和异常翻译在维持BOS中单核细胞纤维化功能中的作用。此外，我们将利用最先进的核糖体图谱(Ribo-seq)来确定BOS和非BOS MC在翻译方面的基因组差异，并通过研究完全或部分抑制mTOR后的BOS细胞来描绘mTORC1翻译控制的基因表达网络。在目标3中，我们将测试ATP竞争性mTOR抑制剂是否可以在小鼠原位单肺移植模型中预防同种异体移植纤维化的发生或进展。这项拟议的工作将是首次研究mTORC1/2信号在移植肺纤维化中的作用，并将为未来肺移植中针对这一途径的临床试验提供重要的理论基础。

DESCRIPTION (provided by applicant): Long term survival post-lung transplantation is limited by development of progressive obstructive ventilatory defect termed bronchiolitis obliterans syndrome (BOS). BOS results from fibrotic obliteration of the small airways or bronchiolitis obliterans (BO). Studies from our laboratory have demonstrated that graft-resident mesenchymal cells (MCs) play an important role in fibroproliferation of BO. Exciting new preliminary data suggest that the evolutionary conserved protein kinase mTOR (mechanistic target of rapamycin) within both mTOR complex 1 (mTORC1) and mTORC2 contributes to the augmented collagen synthetic function of mesenchymal cell in BOS. MCs derived from patients with BOS were found to possess constitutive activation of both mTORC1 and mTORC2. siRNA-mediated knockdown of rictor, an mTORC2 binding partner, inhibited mTORC1 substrate phosphorylation and reversed the fibrotic phenotype of MCs, suggesting an important role for mTORC2 upstream of mTORC1 in activated fibrotic MCs. Selective ATP competitive mTOR inhibitors which target both mTORC1 and mTORC2 effectors were found to be potent inhibitors of collagen expression in BOS MCs. These data lead us to hypothesize that mTOR complex 1 (mTORC1) and mTORC2 play critical roles in BO pathogenesis by mediating translational activation of graft-resident MCs. In this application we propose experiments investigating in vivo role of mTOR pathway in pathogenesis of BO and aim to determine if ATP competitive mTOR inhibitors can be a potential therapeutic option in this disease. To achieve these aims we have established a whole lung mouse orthotopic lung transplant model where a moderate MHC mismatch leads to development of airway fibrosis by day 28. In Aim 1 we propose to investigate mTORC1/2 signaling in the resident MCs in vivo using this murine model. We will utilize mice with BAC florescent labeling of Foxf1, an embryonic lung mesenchyme-associated transcription factor which is highly expressed in lung mesenchymal cells, to specifically identify and study graft-resident MCs. Upstream signaling mechanism(s) that contribute to constitutive mTOR1/2 activation of MCs in BOS will be studied. We will focus on the role of G-protein coupled receptor, lysophosphatidic acid (LPA)1, signaling in a proposed autocrine mechanism of activation. Aim 2 will dissect the role of downstream mTORC1 effectors and dysregulated translation in sustaining fibrotic functions of MCs in BOS. Furthermore, we will utilize state-of-the-art ribosomal profiling (Ribo-seq) to identify genome wide differences in translation between BOS and non-BOS MCs and to delineate the mTORC1 translation controlled gene expression network by studying BOS cells upon complete or partial mTOR inhibition. In Aim 3 we will test if ATP competitive mTOR inhibitors can prevent onset or progression of allograft fibrosis in the murine orthotopic single lung transplant model. The proposed work will be the first investigation of role of mTORC1/2 signaling in lung allograft fibrogenesis and will provide important rationale for future clinical trials targeting this pathway in lung transplantation.