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.

Public Health Relevance

Long term survival after lung transplantation is the worst among all solid organ transplants with less than 20% survival at 10 years. The major cause of these poor outcomes is fibrotic remodeling of the transplanted organ which results from unrelenting collagen deposition and fibro-proliferation. This study will investigate a pathway, amenable to therapeutic intervention, which we have found to be activated in mesenchymal cells derived directly from human fibrotic lung allografts. Studies of human cells will be combined with animal studies to further elucidate the signaling mechanisms upstream and downstream of mTOR in mesenchymal cells during graft fibrogenesis. More importantly we will investigate effectiveness of pharmacological inhibitors of this pathway in preventing development or progression of fibrosis in a mouse lung transplant model. This pre-clinical work will help guide future human studies investigating drug targeting this pathway in lung transplant recipients.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
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Craig, Matt
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University of Michigan Ann Arbor
Internal Medicine/Medicine
Schools of Medicine
Ann Arbor
United States
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Walker, Natalie M; Mazzoni, Serina M; Vittal, Ragini et al. (2018) c-Jun N-terminal kinase (JNK)-mediated induction of mSin1 expression and mTORC2 activation in mesenchymal cells during fibrosis. J Biol Chem 293:17229-17239
Cao, Pengxiu; Aoki, Yoshiro; Badri, Linda et al. (2017) Autocrine lysophosphatidic acid signaling activates ?-catenin and promotes lung allograft fibrosis. J Clin Invest 127:1517-1530
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Walker, Natalie M; Belloli, Elizabeth A; Stuckey, Linda et al. (2016) Mechanistic Target of Rapamycin Complex 1 (mTORC1) and mTORC2 as Key Signaling Intermediates in Mesenchymal Cell Activation. J Biol Chem 291:6262-71
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Belloli, Elizabeth A; Wang, Xin; Murray, Susan et al. (2015) Longitudinal Forced Vital Capacity Monitoring as a Prognostic Adjunct after Lung Transplantation. Am J Respir Crit Care Med 192:209-18
Dickson, Robert P; Erb-Downward, John R; Freeman, Christine M et al. (2014) Changes in the lung microbiome following lung transplantation include the emergence of two distinct Pseudomonas species with distinct clinical associations. PLoS One 9:e97214

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