Hydrogen sulfide attenuates calcification of vascular smooth muscle cells via KEAP1/NRF2/NQO1 activation

- Sulfide attenuates CPP-induced VSMC calcification.
- NQO1 is a potential mediator of the calcification inhibitory properties of sulfide in VSMC.
- Sulfide prevents the mineralization process by an antioxidant-like effect via the KEAP1/NRF2/NQO1 signaling pathway.

Background and aimsVascular calcification is a common health problem related to oxidative stress, inflammation, and circulating calciprotein particles (CPP). Hydrogen sulfide is an endogenous signaling molecule with antioxidant properties and potential for drug development targeting redox signaling. Yet, its molecular mechanisms of action in vascular smooth muscle cell (VSMC) calcification have not been delineated. We therefore sought to identify key pathways involved in the calcification-inhibitory properties of sulfide employing our recently developed CPP-induced VSMC calcification model.MethodsUsing next-generation sequencing, we investigated the transcriptomic changes of sodium hydrosulfide-treated versus non-treated calcifying VSMCs. The potential role of candidate genes and/or regulatory pathways in prevention of calcification was investigated by small interfering RNA (siRNA).ResultsCPP led to a pronounced accumulation of cell-associated calcium, which was decreased by sulfide in a concentration-dependent manner. Both, CPP-induced hydrogen peroxide production and enhanced pro-inflammatory/oxidative stress-related gene expression signatures were attenuated by sulfide-treatment. Gene ontology enrichment and in silico pathway analysis of our transcriptome data suggested NAD(P)H dehydrogenase [quinone] 1 (NQO1) as potential mediator. Corroborating these findings, silencing of Kelch-like ECH-associated protein 1 (KEAP1), an inhibitor of nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear activity, enhanced NQO1 expression, whereas NRF2 silencing reduced the expression of NQO1 and abrogated the calcification-suppressing activity of sulfide. Moreover, immunofluorescence microscopy and Western blot analysis confirmed nuclear translocation of NRF2 by sulfide in VSMC.ConclusionsSulfide attenuates CPP-induced VSMC calcification in vitro via the KEAP1-NRF2 redox sensing/stress response system by enhancing NQO1 expression.