The methylation of Notch1 promoter mediates the osteogenesis differentiation in human aortic valve interstitial cells through Wnt/β‐catenin signaling

: Aortic valve interstitial cells (AVICs) have the potential to undergo calcification, which has been regarded as a critical issue during the pathology of calcific aortic valve disease (CAVD). In the past decade, epigenetics, in particular, DNA methylation dysregulation, has been reported to play a vital role in the occurrence and development of CAVD. In the present study, the expression of Notch1, which can inhibit the osteogenesis differentiation of valve interstitial cells, was downregulated whereas the expression of methyltransferases was upregulated in CAVD tissues, suggesting the potential role of DNA methylation in Notch1 expression and CAVD progression. As revealed by DNA extraction and bisulfite sequencing polymerase chain reaction (PCR), the methylation level in Notch1 promoter was much higher in CAVD tissues and human AVICs on Day 14 of osteogenesis differentiation induction. The silence of Notch1 intercellular domain (NICD) promoted while the treatment of demethylation agent, 5-Aza-dC, inhibited the osteogenesis differentiation. Moreover, NICD overexpression significantly suppressed the transcriptional activity of β-catenin on TCF4, and the expression of osteogenesis differentiation factors, indicating the involvement of Wnt/β-catenin signaling in Notch1 modulating the osteogenesis differentiation in human AVICs (hAVICs). Taken together, Notch1 promoter methylation leads to a decreased Notch1 expression and subsequent decreased release of NICD in the nucleus of hAVICs, therefore promoting the activation of Wnt/β-catenin signaling and the expression of osteogenesis differentiation factors, finally promoting the osteogenesis differentiation in hAVICs. DNA methylation might act as an important bridge to link epigenetic variation and CAVD progression.