Gla-rich protein acts as a calcification inhibitor in the human cardiovascular system

Arterioscler Thromb Vasc Biol. 2015 Feb;35(2):399-408. doi: 10.1161/ATVBAHA.114.304823. Epub 2014 Dec 23.

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.

Keywords: aortic valve, calcification of; gene expression; multivesicular bodies; vascular calcification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Adult
  • Aged
  • Aged, 80 and over
  • Aorta / metabolism
  • Aorta / pathology
  • Aortic Valve / metabolism
  • Aortic Valve / pathology*
  • Aortic Valve Stenosis / genetics
  • Aortic Valve Stenosis / metabolism
  • Aortic Valve Stenosis / pathology
  • Aortic Valve Stenosis / prevention & control*
  • Calcinosis / genetics
  • Calcinosis / metabolism
  • Calcinosis / pathology
  • Calcinosis / prevention & control*
  • Calcium / metabolism*
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism
  • Case-Control Studies
  • Coronary Artery Disease / genetics
  • Coronary Artery Disease / metabolism
  • Coronary Artery Disease / pathology
  • Coronary Artery Disease / prevention & control*
  • Coronary Vessels / metabolism
  • Coronary Vessels / pathology
  • Extracellular Matrix Proteins / genetics
  • Extracellular Matrix Proteins / metabolism
  • Female
  • Gene Expression Regulation
  • Humans
  • Intercellular Signaling Peptides and Proteins
  • Intracellular Signaling Peptides and Proteins
  • Male
  • Middle Aged
  • Osteocalcin / genetics
  • Osteocalcin / metabolism
  • Proteins / genetics
  • Proteins / metabolism*
  • Tissue Culture Techniques
  • Vascular Calcification / genetics
  • Vascular Calcification / metabolism
  • Vascular Calcification / pathology
  • Vascular Calcification / prevention & control*
  • alpha-2-HS-Glycoprotein / metabolism

Substances

  • ACTA2 protein, human
  • AHSG protein, human
  • Actins
  • Calcium-Binding Proteins
  • Extracellular Matrix Proteins
  • Intercellular Signaling Peptides and Proteins
  • Intracellular Signaling Peptides and Proteins
  • Proteins
  • Ucma protein, human
  • alpha-2-HS-Glycoprotein
  • matrix Gla protein
  • Osteocalcin
  • Calcium

Supplementary concepts

  • Aortic Valve, Calcification of