Exploiting novel valve interstitial cell lines to study calcific aortic valve disease

Calcific aortic valve disease (CAVD) involves progressive valve leaflet thickening and severe calcification, impairing leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to examine CAVD mechanisms. However, to date, no published studies have utilised cell lines to investigate this process. Our present study has therefore generated and evaluated the calcification potential of immortalized cell lines derived from sheep and rat VICs. We have produced immortalised sheep (SAVIC) and rat (RAVIC) cell lines by transduction with recombinant lentivirus encoding Simian virus (SV40) large and small T antigens (sheep), or large T antigen only (rat), which both express markers of VICs (vimentin and -SMA). Calcification was induced in the presence of calcium (Ca; 2.7 mM) in SAVICs (1.9 fold; p<0.001) and RAVICs (4.6 fold; p<0.01). Furthermore, a synergistic effect of calcium and phosphate was observed (2.7 mM Ca/2.0 mM Pi) on VIC calcification in both cell lines (p<0.001). Analysis of SAVICs revealed significant increases in the mRNA expression of two key genes associated with vascular calcification in cells cultured under calcifying conditions, RUNX2 (1.3 fold; p<0.05 in 4.5 mM Ca) and PiT1 (1.2 fold; p<0.05 in 5.4 mM Ca). A concomitant decrease in the expression of the calcification inhibitor MGP was noted at 3.6 mM Ca (1.3 fold; p<0.01). Assessment of RAVICs revealed changes in Runx2, Pit1 and Mgp mRNA expression (p<0.01). Furthermore, a significant reduction in calcification was observed in SAVICs following treatment with established calcification inhibitors, pyrophosphate (1.8 fold; p<0.01) and etidronate (3.2 fold; p<0.01). In conclusion, we have demonstrated that the use of immortalised sheep and rat VIC cell lines is a convenient and cost effective system to investigate CAVD in vitro, and will make a useful contribution to increasing our understanding of this pathophysiological process.