Niclosamide attenuates calcification in human heart valvular interstitial cells through inhibition of the AMPK/mTOR signaling pathway

Calcific aortic valve disease (CAVD) is a considerable health burden with a lack of effective therapeutic options. There is an urgent need to develop interventions that inhibit the osteogenic transformation of valvular interstitial cells (VICs) and delay the calcification process. Niclosamide, an FDA-approved anti-helminthic drug, has emerged as a promising candidate that demonstrates a negative regulatory effect on porcine VICs calcification. However, its molecular mechanism in human VICs (hVICs) remains to be investigated. In this study, high-resolution mass spectrometry-based proteomics and phosphoproteomics were employed, and 8373 proteins and 3697 phosphosites were identified in hVICs treated with a pro-calcifying medium and niclosamide. The quantitative proteomic and phosphoproteomic analysis resulted in the identification of calcification markers and osteogenesis-associated proteins. Bioinformatic analysis of the protein–protein interaction network and affected kinase prediction revealed that the AMPK/mTOR/p70S6K signaling cascade was altered upon calcific induction and niclosamide treatment. Further validation indicated that niclosamide inhibited the calcification of hVICs by targeting the mammalian target of the rapamycin (mTOR) signaling pathway. This study provides the first evidence that niclosamide could prevent osteoblastic differentiation in hVICs partially through the inhibition of the AMPK/mTOR/p70S6k signaling pathway, thereby mitigating hVICs calcification. These findings present a foundation for potential therapeutic strategies to impede the progression of CAVD and provide valuable insights into the pharmacological effects of niclosamide on human VICs.