In order to investigate the roles of Wnt signal pathway in transformation of cardiac valvular myofibroblasts to the osteoblast-like phenotype, the primary cultured porcine aortic valve myofibroblasts were incubated with oxidized low density lipoprotein(ox-LDL, 50 mg/L), and divided into four groups according to the ox-LDL treatment time: control group, ox-LDL 24-h group, ox-LDL 48-h group, and ox-LDL 72-h group. Wnt signal pathway blocker Dickkopf-1(DDK-1, 100 μg/L) was added in ox-LDL 72-h group. The expression of α-smooth muscle actin(α-SMA), bone morphogenetic protein 2(BMP2), alkaline phosphatase(ALP), and osteogenic transcription factor Cbfa-1 was detected by Western blotting, and that of β-catenin, a key mediator of Wnt signal pathway by immunocytochemical staining method. The Wnt/β-catenin was observed and the transformation of myofibroblasts to the osteoblast-like phenotype was examined. The expression of α-SMA, BMP2, ALP and Cbfa-1 proteins in the control group was weaker than in the ox-LDL-treated groups. In ox-LDL-treated groups, the protein expression of α-SMA, BMP2, ALP, and Cbfa-1 was significantly increased in a time-dependent manner as compared with the control group, and there was significant difference among the three ox-LDL-treated groups(P<0.05 for all); β-catenin protein was also up-regulated in the ox-LDL-treated groups in a time-dependent manner as compared with the control group(P<0.05), and its transfer from cytoplasm to nucleus and accumulation in the nucleus were increased in the same fashion(P<0.05). After addition of DKK-1, the expression of α-SMA, bone-related proteins and β-catenin protein was significantly reduced as compared with ox-LDL 72-h group(P<0.05). The Wnt/ β-catenin signaling pathway may play an important role in transformation of valvular myofibroblasts to the osteoblast-like phenotype.
Aortic valve calcification is a common disease in the elderly,but its cellular and molecular mechanisms are not clear.In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve,porcine aortic valve interstitial cells(VICs)were isolated,cultured and stimulated with oxidized low density lipoprotein(ox-LDL)for 48 h to induce the differentiation of VICs into osteoblast-like cells.The key proteins and genes of Wnt/β-catenin signaling pathway,such as glycogen synthase kinase 3β(GSK-3β)andβ-catenin,were detected by using Western blotting and real-time polymerase chain reaction(PCR).The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3βandβ-catenin were increased significantly in VICs after stimulation for 48 h(P<0.05).It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.
