Salusin-β Promotes Vascular Calcification via Nicotinamide Adenine Dinucleotide Phosphate/Reactive Oxygen Species-Mediated Klotho Downregulation

Aims: Vascular calcification (VC) is a hallmark feature of cardiovascular disease and a significant risk factor for morbidity and mortality. Salusin-beta exerts cardiovascular regulating effects in hypertension, atherosclerosis, and diabetes. The present study was designed to examine the roles of salusin-beta in the progression of VC and its downstream signaling mechanisms. Results: Salusin-beta expression in both the aortas of VC rats induced by vitamin D3 and nicotine and vascular smooth muscle cells (VSMCs) incubated with calcifying media was increased. Salusin-beta knockdown remarkably reduced VC, whereas overexpression of salusin-beta exacerbated VC both in vitro and in vivo. Overexpression of salusin-beta promoted the VSMC osteochondrogenic transition, decreased Klotho protein levels, enhanced Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the translocation of p47phox to the membrane, increased the expression of nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase subunits and the production of reactive oxygen species (ROS) with or without calcifying media; however, salusin-beta deficiency played the opposite roles. The calcification and downregulated Klotho protein levels induced by salusin-beta were restored by ROS scavenger N-acetyl-l-cysteine, diphenyleneiodonium chloride [an inhibitor of flavin-containing enzyme, including NAD(P)H oxidase], or gene knockdown of NAD(P)H oxidase (NOX)-2, p22phox, or p47phox but were not affected by NOX-1 and NOX-4 knockdown. Klotho knockdown attenuated the protective effect of salusin-beta deficiency on VSMC calcification. By contrast, exogenous Klotho ameliorated the development of VC and ROS generation induced by salusin-beta overexpression. Innovation: Salusin-beta is a critical modulator in VC. Conclusion: Salusin-beta regulates VC through activation of NAD(P)H/ROS-mediated Klotho downregulation, suggesting that salusin-beta may be a novel target for treatment of VC.