A role for calcium in the release of matrix vesicles during aortic valve calcification

Calcific aortic valve disease (CAVD) is characterised by the progressive thickening of the valve leaflets. The 5-year survival rate of inoperable patients with aortic stenosis is lower than many metastatic cancers, and there is no medication therapy that can stop its progression1. Vascular calcification is particularly widespread amongst patients with chronic kidney disease (CKD), as elevated serum calcium (Ca) and phosphate (Pi) levels have reported to enhance vascular smooth muscle cell (VSMC) calcification via the release of matrix vesicles (MVs), which are nano-structures that initiate mineralisation during skeletogenesis2,3,4. In this study, we determined whether Ca and/or Pi induce valve interstitial cell (VIC)-derived MVs to mineralise. In vitro studies employing the SV40T rat VIC cell line revealed that elevated Ca induced calcium deposition at a minimum concentration of 2.7mM (4.5 fold; P<0.01), as determined by quantitative calcium assay. Moreover, 3.6mM Ca treatment significantly increased the mRNA expression of the osteogenic markers PiT-1 (2 fold, P<0.001), Runx2 (1.2 fold, P<0.05) and Msx2 (2 fold, P<0.001). While no effect of Pi treatment alone was observed, treatment of Ca (2.7mM) and Pi (2.5mM) synergistically induced calcium deposition (414 fold; P<0.001). In further studies, MVs were harvested using ultracentrifugation from primary rat VICs that were cultured with control medium or calcifying medium containing 2.7mM Ca and 2.5mM Pi for 16 hrs. Their composition was assessed using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Mass Spectrometry to identify potential mediators of mineralisation. The results revealed that VIC-MVs share similarities with both chondrocyte-derived and VSMC-derived MVs, including enrichment of the calcium-binding proteins: annexins (Anx) A2 (4.8 fold), A5 (4.6 fold) and A6 (4.3 fold). These findings were further validated by western blotting analysis. Additionally, immunohistochemical studies were performed on human valves showing different degrees of calcification. Increased expression of Anx A6 in the severely calcified valve sample was observed when compared to the uncalcified control. To determine whether MVs mediate calcification in vivo, transmission electron microscopy (TEM) was used to examine MV deposition in human valves. MV-like structures were observed in the extracellular matrix of heavily calcified valve tissue, suggesting MV plays a role in the pathogenesis of CAVD. Our data establish calcium as a novel trigger of VIC calcification. These studies are the first to report extracellular vesicles resembling MVs in calcified human aortic valve tissue, suggesting CAVD is a cell mediated process regulated by vesicle release.