16 Investigating calcific aortic valve disease using novel immortalised sheep and rat valve interstitial cell lines

Calcific aortic valve disease (CAVD) involves progressive valve leaflet thickening and severe calcification, resulting in impaired leaflet motion. Although much research has advanced our knowledge of this disease, the mechanisms underlying the initiation and progression of CAVD are still unclear, necessitating further studies to elucidate the underpinning processes in the early stages of this disorder. Our present study aimed to (i) generate and (ii) evaluate the calcification potential of immortalised cell lines derived from sheep and rat aortic valve interstitial cells (VICs). We show that both sheep (SAVIC) and rat (RAVIC) cell lines expressed markers of VICs (vimentin and α-SMA). We also established that sheep VIC (SAVIC) calcification can be induced in the presence of increased calcium only (2.7 mM; 1.9 fold; p<0.001), with a synergistic effect of calcium and phosphate on VIC calcification noted at 2.7 mM and 2.0 mM, respectively (22.2 fold; p<0.001). Significant increases in mRNA expression of key genes associated with valve calcification were observed (RUNX2 and PiT1) when SAVICs were cultured under increasing calcium conditions. Contrastingly, the mRNA level of a potential calcification inhibitor (MGP) decreased under these conditions. Interestingly, we found very little alkaline phosphatase (ALPL) expression in these cells. Comparable data were observed in the RAVIC studies. Furthermore, SAVIC calcification levels were significantly reduced in the presence of known inhibitors of calcification, pyrophosphate (PPi; 1.7 fold; p<0.01) and etidronate (3.2 fold; p<0.01). In conclusion, the use of immortalised sheep and rat VICs can provide a reliable model system to investigate aortic valve calcification in vitro, which will assist in our understanding of this pathophysiological process