Study Of Vascular Smooth Muscle Cell Calcification Induced By Hyperphosphate And Intervented By Phosphonoformic Acid

Chronic renal failure (CRF) is a common disease.There is an excess mortality in patients with end-stage renal disease(ESRD),despite measures to optimize dialysis treatment.Approximately 50% of all deaths are attributed to cardiovascular disease(CVD).Elevated serum phosphorus is a predictable consequence of ESRD and is present in most patients receiving dialysis. In resent studies, elevated serum P levels increase death risk in prevalent hemodialysis patients.Hyperphosphatemia and subsequently elevated calcium X phosphate product promote vascular calcification. Vascular calcification is positively correlatd with atherosclerotic plaque burden,increased risk of myocardial infarction,and increased ischemic episodes in peripheral vascular disease and is a strong independent marker of future cardiovascular events in diabetic patients.Recent studies indicated with and/or predictive of sudden cardiac death.lt is meaningful to explain the mechanism of vascular calcification induced by hyperphosphatemia and take active measures to treat vascular calcification,so we designed and carried out this study. Objective To evaluate the effects of different concentrations of phosphate on calcium deposition and osteocalcin level in bovine aortic smooth muscle cell cultures and investigate the mechanism of hyperphosphatemia to evoke calcification of vascular smooth muscle cell and observe the effects of phosphonoformic acid(PFA) in different concentrations on vascular calcification induced by elevated phosphate. Methods The culture of bovine aortic smooth muscle cells (BASMC) were used.Calcium deposition and the expression of osteocalcin indifferent concentrations of phosphate (1.5 mmol/L and 2.0 mmol/L) and PFA were determined by o-cresolphthalein complexone and radioimmunity methods respectively, Osteocalcin mRNA egression were determined by RT-PCR.Results 1 After six or nine days of BASMC culture ,the calcium deposition in Pi 2.0 mmol/L group was more than that in Pi 1.5 mmol/L group (77.187 + 11.692ug/mg protein vs 25.768 + 1.750ug/mg protein,p<0.01 and 125.399 + 16.677ug/mg protein vs 29.046 + 2.635 ug/mg protein,p<0.01 respectively). The calcium deposition was dependent on time and dosage of phosphate treatment. After 72 h culture the osteocalcin in Pi 2.0 mmol/L group was more than that in Pi 1.5 mmol/L group (in supernatant ,1.503x 102+2.601 X 10-3 ng/ug protein vs 2.981 X 10-3 + 8.382 X 10-4ng/ug protein,p<0.001),the same as osteocalcin mRNA expression (OC/GAPDH, 1.906 +0.132 vs 0.748 + 0.0366, p<0.001 ) .Results 2 Compared to Pi 1.5mmol/L group,bovine smooth muscle cells(BSMC) cultured in media containing Pi 2.0mmol/L phosphate levels increased calcium deposition (On day 6, 77.187+11.692ug/mg protein vs 25.768 +1.750ug/mg protein,p<0.001). Elevated phosphate treatment of BSMCs also enhanced the expression of the osteoblastic differentiation marker osteocalcin(On day 3, Pi 2.0mmol/L group vs Pi 1.5mmol/L group,1.503 x 10-2 +2.601 x 10-3ng/ug protein vs 2.981 x 10-3 + 8.382 x 10-4ng/ug protein,p<0.001).PFA decreased ciacium deposition and osteocalcin expression statistically [Pi 2.0mmol/L+PFA1.0 mmol/L group vs Pi 2.0mmol/L group ,ciacium deposition, 37.729+5.899ug/mg protein vs 77.187 + 11.692ug/mg protein, p<0.001; osteocalcin in supernatant, 4.529 x 10-3 + 1.250 x 10-3ng/ug protein vs 1.503 X 10-2+ 2.601 X 10-3 ng/ug protein, p<0.001;osteocalcin mRNA expression , OC/GAPDH,0.642 + 0.092 vs 1.89 + 0.165, p<0.01].Conclusion Hyperphosphate may directly promote calcium deposition and the osteocalcin expression of BASMCs,it may be a new explanation of the phenomenon on vascular calcification under hyperphosphatemic conditions. Hyperphosphatemia is an independent factor to stimulate vascular calcification.PFA can inhibit calcium deposition and osteocalcin expression induced by elevated phosphate.PFA may be a new medicine to treat vascular calcification induced by elevated phosphate.