title: Calcium regulates key components of vascular smooth muscle cell-derived matrix vesicles to enhance mineralization.
abstract: BACKGROUND
  Matrix vesicles (MVs) are specialized structures that initiate mineral nucleation during physiological skeletogenesis. Similar vesicular structures are deposited at sites of pathological vascular calcification, and studies in vitro have shown that elevated levels of extracellular calcium (Ca) can induce mineralization of vascular smooth muscle cell (VSMC)-derived MVs.
  
  
  OBJECTIVE
  To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress.
  
  
  RESULTS
  Transmission electron microscopy showed that both nonmineralized and mineralized MVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites.
  
  
  CONCLUSIONS
  In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes.
authors.affiliation: British Heart Foundation Centre, Cardiovascular Division, Kings College London, London, United Kingdom.
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Kapustin
authors.forename: John D
authors.initials: JD
authors.lastname: Davies
authors.forename: Joanne L
authors.initials: JL
authors.lastname: Reynolds
authors.forename: Rosamund
authors.initials: R
authors.lastname: McNair
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Jones
authors.forename: Anissa
authors.initials: A
authors.lastname: Sidibe
authors.forename: Leon J
authors.initials: LJ
authors.lastname: Schurgers
authors.forename: Jeremy N
authors.initials: JN
authors.lastname: Skepper
authors.forename: Diane
authors.initials: D
authors.lastname: Proudfoot
authors.forename: Manuel
authors.initials: M
authors.lastname: Mayr
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Shanahan
mesh_terms: D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D017306:Annexin A2 / Q000502:physiology; D017317:Annexin A6 / Q000502:physiology; D001857:Bone Matrix / Q000502:physiology*; D002114:Calcinosis / Q000209:etiology*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000032:analysis; D002675:Child, Preschool; D019902:Chondrocytes / Q000166:cytology; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000032:analysis; D005260:Female; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010718:Phosphatidylserines / Q000502:physiology; D014652:Vascular Diseases / Q000209:etiology*; D000097568:Matrix Gla Protein
chemical_list: D000469:Alkaline Phosphatase; D017306:Annexin A2; D017317:Annexin A6; D002118:Calcium; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D020778:Matrix Metalloproteinase 2; D010718:Phosphatidylserines; D000097568:Matrix Gla Protein