title: Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.
abstract: BACKGROUND
  Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood.
  
  
  OBJECTIVE
  The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification.
  
  
  RESULTS
  Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions.
  
  
  CONCLUSIONS
  This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Kapustin
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Martijn L L
authors.initials: ML
authors.lastname: Chatrou
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Ignat
authors.initials: I
authors.lastname: Drozdov
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Ying
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Sean M
authors.initials: SM
authors.lastname: Davidson
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Daniel
authors.initials: D
authors.lastname: Soong
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Furmanik
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Pilar
authors.initials: P
authors.lastname: Sanchis
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Rafael Torres Martin
authors.initials: RT
authors.lastname: De Rosales
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Daniel
authors.initials: D
authors.lastname: Alvarez-Hernandez
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Rukshana
authors.initials: R
authors.lastname: Shroff
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Xiaoke
authors.initials: X
authors.lastname: Yin
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Karin
authors.initials: K
authors.lastname: Muller
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Jeremy N
authors.initials: JN
authors.lastname: Skepper
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Manuel
authors.initials: M
authors.lastname: Mayr
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Chris P
authors.initials: CP
authors.lastname: Reutelingsperger
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Adrian
authors.initials: A
authors.lastname: Chester
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Sergio
authors.initials: S
authors.lastname: Bertazzo
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.).
authors.forename: Leon J
authors.initials: LJ
authors.lastname: Schurgers
authors.affiliation: From the British Heart Foundation Centre of Excellence, Cardiovascular Division, King's College London, The James Black Centre, London, United Kingdom (A.N.K., I.D., D.S., M.F., P.S., D.A.-H., X.Y., M.M., C.M.S.); Department of Biochemistry-Vascular Aspects, Faculty of Medicine, Health and Life Science, Maastricht University, Maastricht, The Netherlands (M.L.L.C., C.P.R., L.J.S.); Hatter Cardiovascular Institute, University College London, London, United Kingdom (Y.Z., S.M.D.); Department of Imaging, King's College London, London, United Kingdom (R.T.M.D.R.); Great Ormond Street Hospital, London, United Kingdom (R.S.); Department of Anatomy, Multi-Imaging Centre, Cambridge, United Kingdom (K.M., J.N.S.); Heart Science Centre, Harefield, United Kingdom (A.C.); and Department of Materials, Imperial College London, London, United Kingdom (S.B.). cathy.shanahan@kcl.ac.uk alexander.kapustin@kcl.ac.uk.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Shanahan
mesh_terms: D000293:Adolescent; D000328:Adult; D002118:Calcium / Q000378:metabolism*; D016022:Case-Control Studies; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D005089:Exocytosis; D055354:Exosomes / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism* / Q000473:pathology; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D022142:Secretory Vesicles / Q000378:metabolism* / Q000473:pathology; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism; D059470:Tetraspanins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D061205:Vascular Calcification / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D055815:Young Adult; D060749:alpha-2-HS-Glycoprotein / Q000378:metabolism
chemical_list: C556354:AHSG protein, human; D016207:Cytokines; D036341:Intercellular Signaling Peptides and Proteins; D059470:Tetraspanins; D060749:alpha-2-HS-Glycoprotein; C491484:SMPD3 protein, human; D013108:Sphingomyelin Phosphodiesterase; D002118:Calcium
keywords: exosomes; extracellular matrix; vascular calcification