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Epithelial Specific ROCK1 Deletion Protects from Bleomycin-Induced Pulmonary Fibrosis
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A4363 - Epithelial Specific ROCK1 Deletion Protects from Bleomycin-Induced Pulmonary Fibrosis
Author Block: R. S. Knipe1, J. J. Spinney1, A. Franklin1, C. K. Probst1, A. Pardo Saganta2, B. D. Medoff1, J. K. Liao3, A. M. Tager1, Dr. Tager passed away August 11, 2017; 1Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, MA, United States, 2Center for Applied Medical Research, Universidad de Navarra, Navarra, Spain, 3University of Chicago, Chicago, IL, United States.
Rationale:
Idiopathic pulmonary fibrosis is a progressive scarring disease of the lungs which leads to lung destruction and respiratory failure. Epithelial cell injury has long been considered an early event in the cycle of dysregulated wound repair leading to fibrosis. The actin cytoskeleton has been shown to play a role in epithelial cell responses to injury, including cell detachment (anoikis) and apoptosis. The Rho-associated coiled coil forming kinases ROCK1 and ROCK2 regulate the actin cytoskeleton, and we have previously shown that mice haploinsufficient for either of these enzymes are both protected from bleomycin-induced pulmonary fibrosis (RS Knipe et al, AJRCCM 2014;189:A1951). In this study, we determine the pro-fibrotic role of ROCK1 and ROCK2 in epithelial cells using a cell specific genetic deletion approach.
Methods:
Mice with the ROCK1 or ROCK2 gene floxed (ROCK1 f/f or ROCK2 f/f) were bred to mice which express an alveolar epithelial cell specific Cre recombinase (SPC-Cre). SPC-Cre/ROCK1 f/f, SPC-Cre/ROCK2 f/f and littermate control mice were challenged with intratracheal (IT) bleomycin (1.0 U/kg) or PBS, and the amount of pulmonary fibrosis was determined at day 14.
Results:
At day 14, there was significantly less fibrosis in the lungs of the mice with epithelial ROCK1 deletion but not epithelial ROCK2 deletion. Hydroxyproline for SPC-Cre/ROCK1 f/f mice at day 14 was 340.1 +/- 17.6 mcg/lungs as compared to Cre- mice, which was 413.4 +/- 24.4 mcg/lungs, p = 0.03. In contrast, hydroxyproline for SPC-Cre/ROCK2 f/f mice at day 14 was 394.4 +/- 24.7 mcg/lungs as compared to Cre- mice, which was 412.9 +/- 26.9 mcg/lungs, p = 0.62.
Conclusions:
Epithelial cell specific deletion of ROCK1 reduced the fibrotic response to IT bleomycin challenge. This suggests that ROCK1 but not ROCK2 is critical for pro-fibrotic epithelial cell functions. We are currently exploring the mechanisms for this protection, including epithelial cell detachment (anoikis) and apoptosis, activation of αvβ6 integrins, and activation of TGF-β.
Author Block: R. S. Knipe1, J. J. Spinney1, A. Franklin1, C. K. Probst1, A. Pardo Saganta2, B. D. Medoff1, J. K. Liao3, A. M. Tager1, Dr. Tager passed away August 11, 2017; 1Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, MA, United States, 2Center for Applied Medical Research, Universidad de Navarra, Navarra, Spain, 3University of Chicago, Chicago, IL, United States.
Rationale:
Idiopathic pulmonary fibrosis is a progressive scarring disease of the lungs which leads to lung destruction and respiratory failure. Epithelial cell injury has long been considered an early event in the cycle of dysregulated wound repair leading to fibrosis. The actin cytoskeleton has been shown to play a role in epithelial cell responses to injury, including cell detachment (anoikis) and apoptosis. The Rho-associated coiled coil forming kinases ROCK1 and ROCK2 regulate the actin cytoskeleton, and we have previously shown that mice haploinsufficient for either of these enzymes are both protected from bleomycin-induced pulmonary fibrosis (RS Knipe et al, AJRCCM 2014;189:A1951). In this study, we determine the pro-fibrotic role of ROCK1 and ROCK2 in epithelial cells using a cell specific genetic deletion approach.
Methods:
Mice with the ROCK1 or ROCK2 gene floxed (ROCK1 f/f or ROCK2 f/f) were bred to mice which express an alveolar epithelial cell specific Cre recombinase (SPC-Cre). SPC-Cre/ROCK1 f/f, SPC-Cre/ROCK2 f/f and littermate control mice were challenged with intratracheal (IT) bleomycin (1.0 U/kg) or PBS, and the amount of pulmonary fibrosis was determined at day 14.
Results:
At day 14, there was significantly less fibrosis in the lungs of the mice with epithelial ROCK1 deletion but not epithelial ROCK2 deletion. Hydroxyproline for SPC-Cre/ROCK1 f/f mice at day 14 was 340.1 +/- 17.6 mcg/lungs as compared to Cre- mice, which was 413.4 +/- 24.4 mcg/lungs, p = 0.03. In contrast, hydroxyproline for SPC-Cre/ROCK2 f/f mice at day 14 was 394.4 +/- 24.7 mcg/lungs as compared to Cre- mice, which was 412.9 +/- 26.9 mcg/lungs, p = 0.62.
Conclusions:
Epithelial cell specific deletion of ROCK1 reduced the fibrotic response to IT bleomycin challenge. This suggests that ROCK1 but not ROCK2 is critical for pro-fibrotic epithelial cell functions. We are currently exploring the mechanisms for this protection, including epithelial cell detachment (anoikis) and apoptosis, activation of αvβ6 integrins, and activation of TGF-β.
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