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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01HL094622-07
Application #
8974429
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Harabin, Andrea L
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Belloli, Elizabeth A; Lama, Vibha N (2016) Spirometry States the Obvious: Recognizing Bronchiolitis Obliterans Syndrome Early after Hematopoietic Cell Transplantation. Ann Am Thorac Soc 13:1883-1884
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
Shah, R J; Diamond, J M; Cantu, E et al. (2015) Objective Estimates Improve Risk Stratification for Primary Graft Dysfunction after Lung Transplantation. Am J Transplant 15:2188-96
Dickson, Robert P; Erb-Downward, John R; Prescott, Hallie C et al. (2015) Intraalveolar Catecholamines and the Human Lung Microbiome. Am J Respir Crit Care Med 192:257-9
Mimura, Takeshi; Walker, Natalie; Aoki, Yoshiro et al. (2015) Local origin of mesenchymal cells in a murine orthotopic lung transplantation model of bronchiolitis obliterans. Am J Pathol 185:1564-74
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; Prescott, Hallie C et al. (2014) Analysis of culture-dependent versus culture-independent techniques for identification of bacteria in clinically obtained bronchoalveolar lavage fluid. J Clin Microbiol 52:3605-13
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
Walker, Natalie M; Badri, Linda N; Wadhwa, Anish et al. (2012) Prostaglandin E2 as an inhibitory modulator of fibrogenesis in human lung allografts. Am J Respir Crit Care Med 185:77-84
Badri, Linda; Lama, Vibha N (2012) Lysophosphatidic acid induces migration of human lung-resident mesenchymal stem cells through the β-catenin pathway. Stem Cells 30:2010-9

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