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.).
Arterioscler Thromb Vasc Biol. 2015 Feb;35(2):399-408. doi: 10.1161/ATVBAHA.114.304823. Epub 2014 Dec 23.
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.
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.
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.
血管和瓣膜钙化是受驻留细胞调节的病理过程，取决于钙化促进剂和抑制剂之间的复杂相互作用，类似于骨骼代谢。在这里，我们研究维生素 K 依赖性 Gla 丰富蛋白 (GRP) 在血管和瓣膜钙化过程中的作用。
免疫组织化学和定量聚合酶链反应显示，GRP 表达和积累与钙化同时上调，与骨钙素和基质 Gla 蛋白 (MGP) 同时上调。使用构象特异性抗体，发现γ-羧化 GRP 和未羧化 GRP 两种形式都在矿物质沉积部位积累，而未羧化 GRP 在钙化性主动脉瓣疾病瓣膜间质细胞中占优势。通过质谱鉴定了与矿物结合的 GRP、MGP 和胎球蛋白-A。在血管钙化的体外模型中，γ-羧化 GRP 而非未羧化 GRP 被证明通过上调α-平滑肌肌动蛋白和下调骨桥蛋白来抑制钙化和骨软骨分化。免疫沉淀实验表明，GRP 是瓣膜钙化部位 MGP-胎球蛋白-A 复合物的一部分。此外，正常血管平滑肌细胞释放的细胞外囊泡中含有 GRP、MGP 和胎球蛋白-A，而在钙化条件下，释放的细胞外囊泡显示出钙负荷增加和 GRP 和 MGP 耗竭。
GRP 是参与钙稳态的血管和瓣膜钙化的抑制剂。其功能可能与预防钙诱导的信号通路和直接结合矿物质抑制晶体形成/成熟有关。我们的数据表明，GRP 是细胞外囊泡矿化能力的新成员，可能与胎球蛋白-A-MGP 钙化抑制系统有关。GRP 活性依赖于其γ-羧化状态，具有潜在的临床相关性。
