Tumor Necrosis Factor-α Accelerates the Calcification of Human Aortic Valve Interstitial Cells Obtained from Patients with Calcific Aortic Valve Stenosis via the BMP2-Dlx5 Pathway

Calcific aortic valve stenosis (CAS) is the most frequent heart valve disease in the elderly, accompanied by valve calcification. Tumor necrosis factor-alpha (TNF-alpha), a pleiotropic cytokine secreted mainly from macrophages, has been detected in human calcified valves. However, the role of TNF-alpha in valve calcification remains unclear. To clarify whether TNF-alpha accelerates the calcification of aortic valves, we investigated the effect of TNF-alpha on human aortic valve interstitial cells (HAVICs) obtained from patients with CAS (CAS group) and with aortic regurgitation or aortic dissection having a noncalcified aortic valve (control group). HAVICs (2 x 10(4)) were cultured in a 12-well dish in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The medium containing TNF-alpha (30 ng/ml) was replenished every 3 days after the cells reached confluence. TNF-alpha significantly accelerated the calcification and alkaline phosphatase (ALP) activity of HAVICs from CAS but not the control group after 12 days of culture. Furthermore, gene expression of calcigenic markers, ALP, bone morphogenetic protein 2 (BMP2), and distal-less homeobox 5 (Dlx5) were significantly increased after 6 days of TNF-alpha treatment in the CAS group but not the control group. Dorsomorphin, an inhibitor of mothers against decapentaplegic homologs (Smads) 1/5/8 phosphorylation, significantly inhibited the enhancement of TNF-alpha -induced calcification, ALP activity, Smad phosphorylation, and Dlx5 gene expression of HAVICs from the CAS group. These results suggest that HAVICs from the CAS group have greater sensitivity to TNF-alpha, which accelerates the calcification of aortic valves via the BMP2-Dlx5 pathway.