Treatment with Vitamin K₂ Combined with Bisphosphonates Synergistically Inhibits Calcification in Cultured Smooth Muscle Cells

Abstract

Aim: Vascular calcification is a common feature in patients with advanced atherosclerosis, postmenopausal women and patients with renal failure, which results in reduced elasticity of arteries. Pamidronate, a bisphosphonate, is used as a therapeutic agent for anti-osteoporosity, although there are adverse side effects, such as renal damage and aortic inflamed plaque rupture. In the present study, we demonstrated the effects of vitamin K₂ alone or in combination with pamidronate in an arterial calcification model induced using inorganic phosphate in cultured bovine aortic smooth muscle cells (BASMCs).
Methods: Calcification was induced by the addition of Pi (3 mM) in BASMCs. Calcium deposition was determined by Calcium C-test Wako and von Kossa staining. mRNA expression was assessed by semi-quantitative reverse transcription-polymerase chain reaction.
Results: Calcium deposition assay and von Kossa staining showed that calcification could be inhibited in a dose-dependent manner by treatment with vitamin K₂ alone, and that its inhibitory effect was enhanced when combined with pamidronate. It was found that the expression of tropoelastin mRNA was synergistically enhanced by combined treatment with vitamin K₂ and pamidronate, and the expression matrix Gla protein mRNA and osteopontin mRNA expression were also enhanced and suppressed, respectively, by treatment with vitamin K₂ or pamidronate. Moreover, our data showed that the suppression of TE expression by siRNA significantly increased Pi-induced vascular calcification.
Conclusion: Taken together, our study suggests that vitamin K₂ in combination with pamidronate synergistically inhibits arterial calcification via the increased expression of tropoelastin, which would be a useful marker for developing effective therapeutic or prophylactic agents for arterial calcification.