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.
Highlights
Calcific aortic valve disease (CAVD), and subsequent aortic valve stenosis is the most common heart valve disease in the Western world (Newby, Cowell, & Boon, 2006; Nkomo et al, 2006)
We identify Ca as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of end-stage renal disease (ESRD) patients to accelerated development of CAVD
Following recent reports establishing that Annexin VI is required for matrix vesicles (MVs) mediated Vascular smooth muscle cell (VSMC) calcification (Kapustin et al, 2011), we further investigated the role of this calcium-binding protein in valve interstitial cells (VICs) calcification
Summary
Calcific aortic valve disease (CAVD), and subsequent aortic valve stenosis is the most common heart valve disease in the Western world (Newby, Cowell, & Boon, 2006; Nkomo et al, 2006). CAVD is currently considered an actively regulated and progressive disease, characterized by a cascade of cellular changes that initially cause fibrotic thickening, followed by an extensive calcification of the aortic valve leaflets This in turn leads to significant aortic valve stenosis and eventual left ventricular outflow obstruction (Freeman & Otto, 2005), for which surgical replacement remains the only viable treatment option. Undertaken analysis of clinical CAVD tissues in conjunction with in vitro calcification studies in ratderived VICs to address the hypothesis that Ca and Pi induce aortic valve calcification through a MV-mediated mechanism These findings yield novel insights into the mechanisms of CAVD and highlight a critical role for the involvement of VIC-derived MVs. we identify Ca as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD
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