Protective effect of niclosamide on calcification of aortic valve interstitial cells and its underlying molecular mechanism

Background and Aims: The most common heart valvular disorder is calcific aortic valve stenosis (CAVS), which is characterized by a narrowing of the aortic valve. CAVS is known to impose a huge social and economic burden on patients. However, no pharmacotherapy for this condition has yet been established. Aortic valve replacement is the only treatment option, although its lifelong efficacy is not guaranteed and involves inevitable complications. Therefore, the purpose of this study is to see if niclosamide can reduce calcification in aortic valve interstitial cells (VICs). Methods: Porcine hearts were obtained immediately after sacrifice and VICs are isolated using collagenase. To induce calcification, cells were treated with a pro-calcifying medium (PCM), different concentrations of niclosamide were added to the PCM-treated cells, and the level of calcification, mRNA, and protein expression of calcification markers was measured. The level of intracellular reactive oxygen species (ROS) was measured using a redox-sensitive cell-permeable dye, 2',7'- dichlorodihydrofluorescein diacetate (DCF-DA). Results: Niclosamide reduced the level of calcium deposition in calcified VICs, along with reductions in the gene and protein expression of the calcification markers Runx2 and osteopontin. Niclosamide also decreased the formation of reactive oxygen species and the expression of Nox2 and p22phox. Furthermore, in calcified VICs, niclosamide inhibited the expression of β-catenin and phosphorylated glycogen synthase kinase (GSK-3β), as well as the phosphorylation of AKT and ERK. Conclusions: Our findings suggest that niclosamide may alleviate PCM-induced calcification, at least in part, by inhibiting oxidative stress mediated activation of the GSK-3β/β-catenin/AKT/ERK signaling pathway, and may be a potential treatment for CAVS.