Long Term Effect of High Glucose and Phosphate Levels on the OPG/RANK/RANKL/TRAIL System in the Progression of Vascular Calcification in rat Aortic Smooth Muscle Cells

Abstract

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.

Keywords: High glucose; Osteoprotegerin; Phosphate; TNF-related apoptosis-inducing ligand; Vascular calcification.

Fig. 1. Calcium stains and total calcium deposition in rat aortic smooth muscle cells. RASMCs were treated with normal glucose (5.5 mmol/L D-glucose) or high glucose (30 mmol/L D-glucose) with or without β-glycerophosphate (12 mmol/L) for 2 weeks (A, C) and 4 weeks (B, D). In some experiments, cells were treated with 25 mmol/L D- mannitol as osmolality control. (A, B) Upper panel: von Kossa stain, lower panel: alizarin red stain. Magnification, ×100 in all panels. (C, D) Data are shown as mean±SD from three experiments. NG, normal glucose; OC, osmolality control; HG, high glucose; NG+Pi, normal glucose with β-glycerophosphate; OC+Pi, osmolality control with β-glycerophosphate; HG+Pi, high glucose with β-glycerophosphate. ^*p<0.05 vs NG, OC and HG.

Fig. 2. mRNA (A) and protein (B) expressions of OPG, RANK, RANKL, and TRAIL in rat aortic smooth muscle cells after 2 and 4 weeks. NG, normal glucose; NG+Pi, normal glucose with β-glycerophosphate; HG, high glucose; HG+Pi, high glucose with β-glycerophosphate. ^*p<0.05 vs HG.

Fig. 3. Calcium stains (A: upper; von Kossa stain, lower; alizarin red stain. Magnification, ×100) and total calcium deposition (B) in after 4 weeks stimulation with high glucose with β-glycerophosphate (12 mmol/L) in the absence or presence of rhBMP-7 (200 ng/mL) in rat aortic smooth muscle cells. (B) Data are shown as mean±SD from three experiments. HG+Pi, high glucose with β-glycerophosphate; HG+Pi+BMP-7, high glucose with β-glycerophosphate in the presence of BMP-7. ^*p<0.05 vs HG+Pi.

Fig. 4. mRNA (A) and protein (B) expressions of OPG, RANK, RANKL, and TRAIL after 4 weeks stimulation with high glucose with β-glycerophosphate (12 mmol/L) in the absence or presence of rhBMP-7 (200 ng/mL) in RASMCs. Data are shown as mean±SD from three experiments. HG, high glucose; HG+Pi, high glucose with β-glycerophosphate; HG+Pi+BMP-7, high glucose with β-glycerophosphate in the presence of BMP-7. ^*p<0.05 vs HG and HG+Pi.

Fig. 5. mRNA expression of alkaline phosphatase (ALP) after 4 weeks stimulation with high glucose with β-glycerophosphate (12 mmol/L) in the absence or presence of rhBMP-7 (200 ng/mL) in rat aortic smooth muscle cells. Data are shown as mean±SD from three experiments. HG+Pi, high glucose with β-glycerophosphate; HG+Pi+BMP-7, high glucose with β-glycerophosphate in the presence of BMP-7. ^*p<0.05 vs HG+Pi.