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Osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK) axis, and TNF-related apoptosis-inducing ligand (TRAIL) participate in vascular calcification process including atherosclerosis, but their contributions under high glucose (HG) and phosphate (HP) condition for a long-term period (more than 2 weeks) have not been fully determined. In this study, we evaluated the effects of HG and HP levels over 2 or 4 weeks on the progression of vascular calcification in rat vascular smooth muscle cells (VSMCs). Calcium deposition in VSMCs was increased in medium containing HG (30 mmol/L D-glucose) with β -glycerophosphate (β-GP, 12 mmol/L) after 2 weeks and increased further after 4 weeks. OPG mRNA and protein expressions were unchanged in HG group with or without β -GP after 2 weeks. However, after 4 weeks, OPG mRNA and protein expressions were significantly lower in HG group with β -GP. No significant expression changes were observed in RANKL, RANK, or TRAIL during the experiment. After 4 weeks of treatment in HG group containing β -GP and rhBMP-7, an inhibitor of vascular calcification, OPG expressions were maintained. Furthermore, mRNA expression of alkaline phosphatase (ALP), a marker of vascular mineralization, was lower in the presence of rhBMP-7. These results suggest that low OPG levels after long term HG and phosphate stimulation might reduce the binding of OPG to RANKL and TRAIL, and these changes could increase osteo-inductive VSMC differentiation, especially vascular mineralization reflected by increased ALP activity during vascular calcification. [ABSTRACT FROM AUTHOR]

Vascular calcification refers to the deposition of calcium phosphate in cardiovascular tissues, including arteries and myocardium. Vascular calcification is frequently associated with cardiovascular disease. Recently, bone morphgenetic protein-7 (BMP-7) has been proposed to play an inhibitory role in vascular calcification, but its inhibitory effect has not been fully elucidated. We therefore tested the hypothesis that BMP-7 inhibits vascular calcification by using two conditions, high levels of vitamin D and phosphate, each of which could enhance vascular calcification. C57BL/6 mice were treated for 3 days with high vitamin D (500,000 IU/kg/day) in the presence or absence of recombinant human BMP-7 (rhBMP-7). Expression levels of osteopontin and osteocalcin, markers of the osteoblastic phenotype, were assessed by immunohistochemical staining or Western blotting analysis. Vitamin D increased calcium staining in thoracic aortas and hearts and the expression levels of osteopontin and osteocalcin in mice. Importantly, pretreatment for 7 days and subsequent treatment for 3 days with rhBMP-7 (10 μg/kg/day) abolished the vitamin D-mediated increases in the above parameters. In addition, human aortic smooth muscle cells (HASMCs) were cultured with high β-glycerophosphate, a phosphate donor, for 2 weeks in the presence or absence of rhBMP-7. High β-glycerophosphate increased expression levels of osteopontin and osteocalcin as well as calcium staining in HASMCs, but these changes were attenuated by treatment with BMP-7. Thus, BMP-7 inhibits vascular calcification associated with high levels of vitamin D or phosphate. We propose that BMP-7 treatment may be helpful in reducing the risks of cardiovascular disease related to vascular calcification. [ABSTRACT FROM AUTHOR]