title: End stage renal disease-induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived-matrix vesicles.
abstract: Patients with end-stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4-fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8-fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP-1, and LAMP-2 and a concomitant up-regulation of the Annexin family of calcium-binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC-derived MVs (51.9-fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up-regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC-derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co-localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC-derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD.
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Edinburgh, United Kingdom.
authors.forename: Lin
authors.identifier: http://orcid.org/0000-0001-9319-0824
authors.initials: L
authors.lastname: Cui
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Edinburgh, United Kingdom.
authors.forename: Nabil A
authors.initials: NA
authors.lastname: Rashdan
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Edinburgh, United Kingdom.
authors.forename: Dongxing
authors.initials: D
authors.lastname: Zhu
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Edinburgh, United Kingdom.
authors.forename: Elspeth M
authors.initials: EM
authors.lastname: Milne
authors.affiliation: Gemini Biosciences Ltd, Liverpool Science Park, Liverpool, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Ajuh
authors.affiliation: Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.
authors.forename: Gillian
authors.initials: G
authors.lastname: Milne
authors.affiliation: Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.
authors.forename: Miep H
authors.initials: MH
authors.lastname: Helfrich
authors.affiliation: Department of Cardiothoracic Surgery, Royal Infirmary Hospital of Edinburgh (NHS Lothian), The University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Kelvin
authors.initials: K
authors.lastname: Lim
authors.affiliation: Department of Cardiothoracic Surgery, Royal Infirmary Hospital of Edinburgh (NHS Lothian), The University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Sai
authors.initials: S
authors.lastname: Prasad
authors.affiliation: Department of Cardiothoracic Surgery, Royal Infirmary Hospital of Edinburgh (NHS Lothian), The University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Lerman
authors.affiliation: University/BHF Center for Cardiovascular Sciences, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, United Kingdom.
authors.forename: Alex T
authors.initials: AT
authors.lastname: Vesey
authors.affiliation: University/BHF Center for Cardiovascular Sciences, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, United Kingdom.
authors.forename: Marc R
authors.initials: MR
authors.lastname: Dweck
authors.affiliation: University/BHF Center for Cardiovascular Sciences, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, United Kingdom.
authors.forename: William S
authors.initials: WS
authors.lastname: Jenkins
authors.affiliation: University/BHF Center for Cardiovascular Sciences, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, United Kingdom.
authors.forename: David E
authors.initials: DE
authors.lastname: Newby
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Edinburgh, United Kingdom.
authors.forename: Colin
authors.initials: C
authors.lastname: Farquharson
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Edinburgh, United Kingdom.
authors.forename: Vicky E
authors.initials: VE
authors.lastname: Macrae
mesh_terms: D000368:Aged; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017317:Annexin A6 / Q000378:metabolism*; D001021:Aortic Valve / Q000378:metabolism* / Q000473:pathology* / Q000648:ultrastructure; D001024:Aortic Valve Stenosis / Q000209:etiology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002114:Calcinosis / Q000209:etiology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism* / Q000648:ultrastructure; D000067128:Extracellular Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006934:Hypercalcemia / Q000175:diagnosis / Q000209:etiology*; D007676:Kidney Failure, Chronic / Q000150:complications* / Q000175:diagnosis; D008297:Male; D046529:Microscopy, Electron, Transmission; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation
chemical_list: D017317:Annexin A6; D050659:Core Binding Factor Alpha 1 Subunit; D018398:Homeodomain Proteins; C084119:MSX2 protein; D012333:RNA, Messenger; C493717:Runx2 protein, rat; D000469:Alkaline Phosphatase; D002118:Calcium
keywords: Annexin VI; calcific aortic valve disease; calcification; matrix vesicles