title: Discoidin Domain Receptor-1 Regulates Calcific Extracellular Vesicle Release in Vascular Smooth Muscle Cell Fibrocalcific Response via Transforming Growth Factor-β Signaling.
abstract: OBJECTIVE
  Collagen accumulation and calcification are major determinants of atherosclerotic plaque stability. Extracellular vesicle (EV)-derived microcalcifications in the collagen-poor fibrous cap may promote plaque rupture. In this study, we hypothesize that the collagen receptor discoidin domain receptor-1 (DDR-1) regulates collagen deposition and release of calcifying EVs by vascular smooth muscle cells (SMCs) through the transforming growth factor-β (TGF-β) pathway.
  
  
  RESULTS
  SMCs from the carotid arteries of DDR-1(-/-) mice and wild-type littermates (n=5-10 per group) were cultured in normal or calcifying media. At days 14 and 21, SMCs were harvested and EVs isolated for analysis. Compared with wild-type, DDR-1(-/-) SMCs exhibited a 4-fold increase in EV release (P<0.001) with concomitantly elevated alkaline phosphatase activity (P<0.0001) as a hallmark of EV calcifying potential. The DDR-1(-/-) phenotype was characterized by increased mineralization (Alizarin Red S and Osteosense, P<0.001 and P=0.002, respectively) and amorphous collagen deposition (P<0.001). We further identified a novel link between DDR-1 and the TGF-β pathway previously implicated in both fibrotic and calcific responses. An increase in TGF-β1 release by DDR-1(-/-) SMCs in calcifying media (P<0.001) stimulated p38 phosphorylation (P=0.02) and suppressed activation of Smad3. Inhibition of either TGF-β receptor-I or phospho-p38 reversed the fibrocalcific DDR-1(-/-) phenotype, corroborating a causal relationship between DDR-1 and TGF-β in EV-mediated vascular calcification.
  
  
  CONCLUSIONS
  DDR-1 interacts with the TGF-β pathway to restrict calcifying EV-mediated mineralization and fibrosis by SMCs. We therefore establish a novel mechanism of cell-matrix homeostasis in atherosclerotic plaque formation.
authors.affiliation: From the Department of Medicine, Cardiovascular Division, Center for Excellence in Vascular Biology (J.B.K., E.M.-M., W.S.I., E.A.) and Center for Interdisciplinary Cardiovascular Sciences (J.D.H., E.A.), Harvard Medical School, Boston, MA; Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands (C.V.C.B.); Department of Medical Physics and Biomedical Engineering, University College London, London, United Kingdom (S.B.); and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada (M.P.B.).
authors.forename: Jona B
authors.initials: JB
authors.lastname: Krohn
authors.affiliation: From the Department of Medicine, Cardiovascular Division, Center for Excellence in Vascular Biology (J.B.K., E.M.-M., W.S.I., E.A.) and Center for Interdisciplinary Cardiovascular Sciences (J.D.H., E.A.), Harvard Medical School, Boston, MA; Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands (C.V.C.B.); Department of Medical Physics and Biomedical Engineering, University College London, London, United Kingdom (S.B.); and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada (M.P.B.).
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Hutcheson
authors.affiliation: From the Department of Medicine, Cardiovascular Division, Center for Excellence in Vascular Biology (J.B.K., E.M.-M., W.S.I., E.A.) and Center for Interdisciplinary Cardiovascular Sciences (J.D.H., E.A.), Harvard Medical School, Boston, MA; Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands (C.V.C.B.); Department of Medical Physics and Biomedical Engineering, University College London, London, United Kingdom (S.B.); and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada (M.P.B.).
authors.forename: Eduardo
authors.initials: E
authors.lastname: Martínez-Martínez
authors.affiliation: From the Department of Medicine, Cardiovascular Division, Center for Excellence in Vascular Biology (J.B.K., E.M.-M., W.S.I., E.A.) and Center for Interdisciplinary Cardiovascular Sciences (J.D.H., E.A.), Harvard Medical School, Boston, MA; Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands (C.V.C.B.); Department of Medical Physics and Biomedical Engineering, University College London, London, United Kingdom (S.B.); and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada (M.P.B.).
authors.forename: Whitney S
authors.initials: WS
authors.lastname: Irvin
authors.affiliation: From the Department of Medicine, Cardiovascular Division, Center for Excellence in Vascular Biology (J.B.K., E.M.-M., W.S.I., E.A.) and Center for Interdisciplinary Cardiovascular Sciences (J.D.H., E.A.), Harvard Medical School, Boston, MA; Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands (C.V.C.B.); Department of Medical Physics and Biomedical Engineering, University College London, London, United Kingdom (S.B.); and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada (M.P.B.).
authors.forename: Carlijn V C
authors.initials: CV
authors.lastname: Bouten
authors.affiliation: From the Department of Medicine, Cardiovascular Division, Center for Excellence in Vascular Biology (J.B.K., E.M.-M., W.S.I., E.A.) and Center for Interdisciplinary Cardiovascular Sciences (J.D.H., E.A.), Harvard Medical School, Boston, MA; Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands (C.V.C.B.); Department of Medical Physics and Biomedical Engineering, University College London, London, United Kingdom (S.B.); and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada (M.P.B.).
authors.forename: Sergio
authors.initials: S
authors.lastname: Bertazzo
authors.affiliation: From the Department of Medicine, Cardiovascular Division, Center for Excellence in Vascular Biology (J.B.K., E.M.-M., W.S.I., E.A.) and Center for Interdisciplinary Cardiovascular Sciences (J.D.H., E.A.), Harvard Medical School, Boston, MA; Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands (C.V.C.B.); Department of Medical Physics and Biomedical Engineering, University College London, London, United Kingdom (S.B.); and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada (M.P.B.).
authors.forename: Michelle P
authors.initials: MP
authors.lastname: Bendeck
authors.affiliation: From the Department of Medicine, Cardiovascular Division, Center for Excellence in Vascular Biology (J.B.K., E.M.-M., W.S.I., E.A.) and Center for Interdisciplinary Cardiovascular Sciences (J.D.H., E.A.), Harvard Medical School, Boston, MA; Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands (C.V.C.B.); Department of Medical Physics and Biomedical Engineering, University College London, London, United Kingdom (S.B.); and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada (M.P.B.). eaikawa@partners.org jdhutcheson@partners.org.
authors.forename: Elena
authors.initials: E
authors.lastname: Aikawa
mesh_terms: D000818:Animals; D001057:Apolipoproteins E / Q000172:deficiency / Q000235:genetics; D050197:Atherosclerosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D000070819:Discoidin Domain Receptor 1; D004195:Disease Models, Animal; D000067128:Extracellular Vesicles / Q000378:metabolism*; D005260:Female; D005355:Fibrosis; D020022:Genetic Predisposition to Disease; D008297:Male; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism* / Q000473:pathology; D010012:Osteogenesis; D010641:Phenotype; D010766:Phosphorylation; D058226:Plaque, Atherosclerotic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D061205:Vascular Calcification / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D001057:Apolipoproteins E; D018125:Receptors, Transforming Growth Factor beta; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C508961:Tgfb1 protein, mouse; D053773:Transforming Growth Factor beta1; D003094:Collagen; C083249:Ddr1 protein, mouse; D000070819:Discoidin Domain Receptor 1; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I
keywords: extracellular matrix; extracellular vesicles; fibrosis; smooth muscle; transforming growth factors