Nitric oxide regulates vascular calcification by interfering with TGF-b signalling

Aims Vascular calcification often occurs with advancing age, atherosclerosis, and metabolic disorders such as diabetes mellitus and end-stage renal disease. Vascular calcification is associated with cardio-vascular events and increased mortality. Nitric oxide (NO) is crucial for maintaining vascular function, but little is known about how NO affects vascular calcification. The aim of this study was to examine the effect of NO on vascular calcification. Methods and results In this study, we examined the inhibitory effects of NO on calcification of murine vascular smooth muscle cells (VSMCs) in vitro. We measured calcium concentration, alizarin red staining, and alkaline phosphatase activity to examine the effect of NO on calcification of VSMCs and differentiation of VSMCs into osteoblastic cells. We also determined gene expression and levels of phosphorylation of Smad2/3 by RT–PCR and western blotting. NO inhibited calcification of VSMCs and differentiation of VSMCs into osteoblastic cells. An inhibitor of cyclic guanosine monophosphate (cGMP)-dependent protein kinase restored the inhibition by NO of osteoblastic differentiation and cal-cification of VSMCs. NO inhibited transforming growth factor-b (TGF-b)-induced phosphorylation of Smad2/3 and expression of TGF-b-induced genes such as plasminogen activator inhibitor-1. In addition, NO inhibited expression of the TGF-b receptor ALK5