Carla S B Viegas1, Marta S Rafael1, José L Enriquez1, Alexandra Teixeira1, Rui Vitorino1, Inês M Luís1, Rúben M Costa1, Sofia Santos1, Sofia Cavaco1, José Neves1, Anjos L Macedo1, Brecht A G Willems1, Cees Vermeer1, Dina C Simes2. 1. 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.). 2. 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.
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. APPROACH AND 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.
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. APPROACH AND 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-MGPcalcification inhibitory system. GRP activity was found to be dependent on its γ-carboxylation status, with potential clinical relevance.
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