Tumor necrosis factor alpha promotes an osteoblast-like phenotype in human aortic valve myofibroblasts: A potential regulatory mechanism of valvular calcification

Valvular calcification during calcific aortic stenosis is associated with morphological features of bone formation and expression of various bone-associated proteins, which are both associated with marked leukocyte infiltration of the calcified valve areas. The pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) is abundantly present in areas of leukocyte infiltration in stenotic aortic valves. We therefore hypothesized that valvular calcification might be actively regulated by an inflammatory process involving TNF-α. Upon stimulation with TNF-α, human aortic valve myofibroblasts cultured under mineralizing conditions showed an increased formation of calcified, alkaline phosphatase (ALP)-enriched cell nodules, ALP activity, concentration of the bone-type ALP isoenzyme, and concentration of osteocalcin, all of which are markers of an osteoblast-like cellular phenotype. By electrophoretic mobility shift assay, DNA binding of the essential osteoblastic transcription factor Cbfa-1 was increased compared to untreated controls. These results support the concept that aortic valve calcification is associated with an osteoblast-like phenotype of local myofibroblasts. In addition, the data demonstrate direct mechanistic evidence that aortic valve calcification may be actively regulated by an inflammatory process involving TNF-α.