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ajrccm-conference.2017.195.1 MeetingAbstracts.A4663

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B97 FLIPPING THE SWITCH: DETERMINANTS OF FIBROSIS / Mini Symposium / Monday, May 22/2:15 PM-4:15 PM / Marquis Ballroom 6 (Level M2) Marriott Marquis Washington
Adam10-Mediated Ephrin-B2 Shedding Drives Myofibroblast Activation And Tissue Fibrosis
D. Lagares1, P. Ghassemi-Kakaroodi2, C. Tremblay2, D. Santos1, P. Grasberger1, N. Ahluwalia1, C. Probst1, S. Montesi1, B. Shea3,
K. E. Black1, R. Knipe1, A. Pardo4, M. Selman5, A. M. Tager6, M. Kapoor2
1Massachusetts General Hospital - Harvard Medical School, Boston, MA, 2University of Toronto, Toronto, ON, Canada, 3Brown University
School of Medicine, Providence, RI, 4Universidad Nacional Autónoma de México, Mexico City, Mexico, 5National Institute of Respiratory
Diseases, Mexico City, Mexico, 6Massachusetts General Hospital, Charlestown, MA
Corresponding author's email: [email protected]
Rationale: Excessive lung extracellular matrix deposition by activated myofibroblasts characterizes idiopathic pulmonary fibrosis (IPF), but
the molecular mediators directing myofibroblast activation in IPF remain to be fully elucidated. Analysis of publicly available microarray
data of lung fibroblasts isolated from IPF and control subjects indicated Efnb2 is upregulated in IPF lung fibroblasts. Efnb2 encodes a
transmembrane protein ephrin-B2 that controls cell motility and adhesion-induced signaling, which has not previously been implicated in
pulmonary fibrosis.
Methods: Ephrin-B2 globally-deficient mice die at mid-gestation. To evaluate the contribution of ephrin-B2 expressed by fibroblasts to
pulmonary fibrosis, we generated fibroblast-specific ephrin-B2 knockout (KO) mice and assessed them for fibrosis induced by intratracheal
injection of bleomycin (1.2 U/kg). Ephrin-B2 levels were assessed by western blot and ELISA in lung homogenates and bronchoalveolar
fluid (BALF). To identify the sheddase for a novel soluble form of ephrin-B2 that we identified, we performed a functional siRNA screen of
metalloproteinases, measuring the effects of individual protease knockdowns on human lung fibroblast ephrin-B2 shedding.
Results: Fibroblast-specific ephrin-B2 KO mice were significantly protected from bleomycin-induced pulmonary fibrosis. Following
pro-fibrotic injury, proteolytic cleavage of ephrin-B2 generated a novel soluble form of ephrin-B2’s ectodomain (sEphrin-B2) that was
present in greatly increased amounts in BALF from bleomycin-challenged mice. sEphrin-B2 mediated myofibroblast differentiation
through EphB3/EphB4 receptor signaling in vitro, and was sufficient to induce marked dermal fibrosis when injected subcutaneously into
naive mice. BALF sEphrin-B2 levels were reduced in bleomycin-challenged fibroblast specific-ephrin-B2 KO mice, indicating that
fibroblasts are major source of sEphrin-B2. We identified ADAM10 as the major protease responsible for fibroblast ephrin-B2 shedding.
ADAM10 and ephrin-B2 shedding were both induced by stimulating fibroblasts with transforming growth factor-beta (TGF-β1), whereas
ADAM10 pharmacological inhibition (with GX254023X) or genetic knockdown inhibited TGF-β-mediated myofibroblast differentiation,
indicating that ADAM10-mediated sEphrin-B2 generation is required for this process. ADAM10 expression and sEphrin-B2 generation were
significantly increased in human lung fibroblasts from IPF patients compared to controls. ADAM10 inhibition significantly inhibited the
increased sEphrin-B2 production, as well as the increased collagen type I and a-SMA expression levels, observed with IPF fibroblasts.
Conclusions: Proteolytic shedding of ephrin-B2 by ADAM10 represents a novel autocrine molecular mechanism that drives fibroblast
differentiation into myofibroblasts. The requirement of ADAM10-induced sEphrin-B2 shedding for TGF-β-induced myofibroblast
differentiation suggests that this process may be centrally involved in tissue fibrogenesis, and that sEphin-B2, its receptors EphB3/EphB4
and its sheddase ADAM10, may be able to serve as novel therapeutic targets for fibrotic diseases.
This abstract is funded by: The authors gratefully acknowledge funding support by CR-CHUM and The Arthritis Program (University Health
Network) grant (M.K) and by NIH-HL108975 and a grant from the Scleroderma Research Foundation (A.M.T).
Am J Respir Crit Care Med 2017;195:A4663
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ajrccm, a4663, 195, 2017, conference, meetingabstracts
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