Nitric oxide regulates vascular calcification by interfering with TGF- signalling.
OBJECTIVE
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 cardiovascular 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.


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 calcification of VSMCs. NO inhibited transforming growth factor-beta (TGF-beta)-induced phosphorylation of Smad2/3 and expression of TGF-beta-induced genes such as plasminogen activator inhibitor-1. In addition, NO inhibited expression of the TGF-beta receptor ALK5.


CONCLUSIONS
Our data show that NO prevents differentiation of VSMCs into osteoblastic cells by inhibiting TGF-beta signalling through a cGMP-dependent pathway. Our findings suggest that NO may play a beneficial role in atherogenesis in part by limiting vascular calcification.
Kanno
Yosuke
Y
Department of Oral Disease Research, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka-cho, Obu, Aichi 474-8511, Japan.
Into
Takeshi
T
Lowenstein
Charles J
CJ
Matsushita
Kenji
K
D000818:Animals; D002114:Calcinosis / Q000517:prevention & control*; D002454:Cell Differentiation; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000473:pathology; D009569:Nitric Oxide / Q000502:physiology*; D010006:Osteoblasts / Q000166:cytology; D015398:Signal Transduction / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology*; D014652:Vascular Diseases / Q000517:prevention & control*
D016212:Transforming Growth Factor beta; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP