End stage renal disease-induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived-matrix vesicles

Lin Cui, Nabil A Rashdan, Dongxing Zhu, Elspeth M Milne, Paul Ajuh, Gillian Milne, Miep H Helfrich, Kelvin Lim, Sai Prasad, Daniel A Lerman, Alex T Vesey, Marc R Dweck, William S Jenkins, David E Newby, Colin Farquharson, Vicky E Macrae

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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.

Original languageEnglish
Pages (from-to)2985-2995
Number of pages11
JournalJournal of Cellular Physiology
Issue number11
Early online date24 May 2017
Publication statusPublished - 1 Nov 2017

Bibliographical note

We would like to thank Emma McDermott and Stephen Mitchell for their assistance with the electron microscopy sample preparation, and for the use of the (JEOL JEM-1400 Plus) transmission electron microscope enabled by Wellcome Trust Multi User Equipment Grant (WT104915MA).
Funding information
Wellcome Trust, Grant number: WT103782AIA; British Heart Foundation,
Grant numbers: CH/09/002, FS/12/84; Biotechnology and Biological Sciences Research Council, Grant numbers: BB/F023928/1, BB/ J004316/1, BB/K011618/1


  • Annexin VI
  • calcific aortic valve disease
  • calcification
  • matrix vesicles


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