TY - JOUR
T1 - Gla-Rich Protein Acts as a Calcification Inhibitor in the Human Cardiovascular System
AU - Viegas, Carla S. B.
AU - Rafael, Marta S.
AU - Enriquez, Jose L.
AU - Teixeira, Alexandra
AU - Vitorino, Rui
AU - Luis, Ines M.
AU - Costa, Ruben M.
AU - Santos, Sofia
AU - Cavaco, Sofia
AU - Neves, José
AU - Macedo, Maria dos Anjos López de
AU - Willems, Brecht A. G.
AU - Vermeer, Cees
AU - Simes, Dina C.
N1 - Sem PDF
PY - 2015/2
Y1 - 2015/2
N2 - 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 gamma-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, gamma-carboxylated GRP but not undercarboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation through alpha-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 gamma-carboxylation status, with potential clinical relevance.
AB - 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 gamma-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, gamma-carboxylated GRP but not undercarboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation through alpha-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 gamma-carboxylation status, with potential clinical relevance.
KW - aortic valve, calcification of
KW - gene expression
KW - multivesicular bodies
KW - vascular calcification
KW - MUSCLE-CELL CALCIFICATION
KW - VASCULAR CALCIFICATION
KW - AORTIC-STENOSIS
KW - MATRIX VESICLES
KW - VITAMIN-K
KW - ATHEROSCLEROTIC PLAQUES
KW - GAMMA-CARBOXYLATION
KW - FETUIN-A
KW - DISEASE
KW - CARTILAGE
U2 - 10.1161/ATVBAHA.114.304823
DO - 10.1161/ATVBAHA.114.304823
M3 - Article
C2 - 25538207
SN - 1079-5642
VL - 35
SP - 399
EP - 408
JO - Arteriosclerosis, thrombosis, and vascular biology
JF - Arteriosclerosis, thrombosis, and vascular biology
IS - 2
ER -