Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs

Abstract

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding αv integrin subunit because various αv-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride–induced hepatic fibrosis, whereas global loss of β3, β5 or β6 integrins or conditional loss of β8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the αv integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of αv-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

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Figure 1: Pdgfrb-Cre effectively targets recombination in quiescent and activated HSCs.
Figure 2: Depletion of the αv integrin on HSCs protects mice from CCl4-induced hepatic fibrosis.
Figure 3: αv integrin depletion on HSCs inhibits profibrotic gene expression through a reduction in TGF-β activation.
Figure 4: Global loss of β3, β5 or β6 integrins or conditional loss of β8 integrins on HSCs does not protect mice from CCl4-induced hepatic fibrosis.
Figure 5: Pdgfrb-Cre–mediated depletion of the αv integrin is protective in multiple models of solid organ fibrogenesis.
Figure 6: Blockade of αv integrins by a small molecule (CWHM 12) attenuates liver and lung fibrosis.

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Acknowledgements

This work was supported by a Wellcome Trust Intermediate Clinical Fellowship (ref. 085187) to N.C.H., National Institutes of Health grants HL102292, HL53949 and AI077439 (to D.S.), a University of California, San Francisco (UCSF) Liver Center Tool and Technology grant (to N.C.H) and P30 DK026743 (UCSF Liver Center). We thank K. Thorn at the UCSF Nikon Imaging Center for assistance with image analysis. We also thank C. Her, N. Wu, S. Huling, D. Rodrigues and R. Aucott for expert technical assistance. We also acknowledge the contribution of M. Singh (chemical synthesis of compounds CWHM 12 and CWHM 96), D. Tajfirouz, S. Freeman and M. Yates at Saint Louis University for technical assistance in conducting integrin functional assays to characterize compound activities. L. Reichardt (UCSF) provided Itgb8flox/flox mice and R. Hynes (Massachusetts Institute of Technology) provided Itgb3−/− mice on 129/svJae background. S. Violette (Biogen Idec) provided antibody to αvβ6 (human/mouse chimeric 2A1), W. Stallcup (Sanford-Burnham Medical Research Institute) provided antibody to PDGFR-β and H. Yagita (Juntendo University) provided antibody to αv integrin (clone RMV-7).

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N.C.H. and D.S. conceived and designed the project. N.C.H. performed the experiments with assistance from T.D.A., Y.K., M.M.G., J.D.R. and A.P.; J.H.M. contributed reagents; P.G.R., D.W.G. and M.J.P. designed and synthesized the small molecule αv integrin inhibitor (CWHM 12) and performed the ligand-binding studies to characterize the in vitro potency of CWHM 12; J.J.M. and J.P.I. contributed reagents and provided substantial intellectual contribution; E.R. and C.B. contributed Pdgfrb-BAC-eGFP knock-in reporter mice; A.L.-H. contributed Itgavflox/flox mice; R.H.A. contributed Pdgfrb-Cre mice; N.C.H., T.D.A., Y.K., M.M.G. and D.S. analyzed data and N.C.H., J.P.I. and D.S. wrote the manuscript.

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Correspondence to Neil C Henderson or Dean Sheppard.

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Competing interests

P.G.R. and D.W.G. hold equity in Antegrin Therapeutics, LLC.

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Henderson, N., Arnold, T., Katamura, Y. et al. Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nat Med 19, 1617–1624 (2013). https://doi.org/10.1038/nm.3282

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