title: Gla-rich protein acts as a calcification inhibitor in the human cardiovascular system.
abstract: OBJECTIVE
  Vascular and valvular calcifications are pathological processes regulated by resident cells, and depending on a complex interplay between calcification promoters and inhibitors, resembling skeletal metabolism. Here, we study the role of the vitamin K-dependent Gla-rich protein (GRP) in vascular and valvular calcification processes.
  
  
  RESULTS
  Immunohistochemistry and quantitative polymerase chain reaction showed that GRP expression and accumulation are upregulated with calcification simultaneously with osteocalcin and matrix Gla protein (MGP). Using conformation-specific antibodies, both γ-carboxylated GRP and undercarboxylated GRP species were found accumulated at the sites of mineral deposits, whereas undercarboxylated GRP was predominant in calcified aortic valve disease valvular interstitial cells. Mineral-bound GRP, MGP, and fetuin-A were identified by mass spectrometry. Using an ex vivo model of vascular calcification, γ-carboxylated GRP but not undercarboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation through α-smooth muscle actin upregulation and osteopontin downregulation. Immunoprecipitation assays showed that GRP is part of an MGP-fetuin-A complex at the sites of valvular calcification. Moreover, extracellular vesicles released from normal vascular smooth muscle cells are loaded with GRP, MGP, and fetuin-A, whereas under calcifying conditions, released extracellular vesicles show increased calcium loading and GRP and MGP depletion.
  
  
  CONCLUSIONS
  GRP is an inhibitor of vascular and valvular calcification involved in calcium homeostasis. Its function might be associated with prevention of calcium-induced signaling pathways and direct mineral binding to inhibit crystal formation/maturation. Our data show that GRP is a new player in mineralization competence of extracellular vesicles possibly associated with the fetuin-A-MGP calcification inhibitory system. GRP activity was found to be dependent on its γ-carboxylation status, with potential clinical relevance.
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Carla S B
authors.initials: CS
authors.lastname: Viegas
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Marta S
authors.initials: MS
authors.lastname: Rafael
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: José L
authors.initials: JL
authors.lastname: Enriquez
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Alexandra
authors.initials: A
authors.lastname: Teixeira
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Rui
authors.initials: R
authors.lastname: Vitorino
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Inês M
authors.initials: IM
authors.lastname: Luís
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Rúben M
authors.initials: RM
authors.lastname: Costa
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Sofia
authors.initials: S
authors.lastname: Santos
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Sofia
authors.initials: S
authors.lastname: Cavaco
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: José
authors.initials: J
authors.lastname: Neves
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Anjos L
authors.initials: AL
authors.lastname: Macedo
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Brecht A G
authors.initials: BA
authors.lastname: Willems
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.).
authors.forename: Cees
authors.initials: C
authors.lastname: Vermeer
authors.affiliation: From the Centre of Marine Sciences (CCMAR) (C.S.B.V., M.S.R., I.M.L., R.M.C., S.S., S.C., D.C.S.), GenoGla Diagnostics (C.S.B.V., D.C.S.), University of Algarve, Faro, Portugal; Department of Histopathology, Algarve Medical Centre, Faro, Portugal (J.L.E., A.T.); Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal (R.V.); Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal (J.N.); UCIBIO@REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (A.L.M.); VitaK, Maastricht University, Maastricht, The Netherlands (B.A.G.W., C.V.); and Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, The Netherlands (B.A.G.W.). dsimes@ualg.pt.
authors.forename: Dina C
authors.initials: DC
authors.lastname: Simes
mesh_terms: D000199:Actins / Q000378:metabolism; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001011:Aorta / Q000378:metabolism / Q000473:pathology; D001021:Aortic Valve / Q000378:metabolism / Q000473:pathology*; D001024:Aortic Valve Stenosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D002114:Calcinosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016022:Case-Control Studies; D003324:Coronary Artery Disease / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D003331:Coronary Vessels / Q000378:metabolism / Q000473:pathology; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008875:Middle Aged; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D046509:Tissue Culture Techniques; D061205:Vascular Calcification / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D060749:alpha-2-HS-Glycoprotein / Q000378:metabolism; D000097568:Matrix Gla Protein
chemical_list: D000199:Actins; D002118:Calcium; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D015675:Osteocalcin; D011506:Proteins; D060749:alpha-2-HS-Glycoprotein; D000097568:Matrix Gla Protein; C541116:ACTA2 protein, human; C556354:AHSG protein, human; C528959:Ucma protein, human
keywords: aortic valve, calcification of; gene expression; multivesicular bodies; vascular calcification