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

Objective: To observe the effect on calcification and OPGmRNA expression in vascular smooth muscle cell induced by hyperphosphate; and investigate the effect and mechanism of Osteoprotegerin on vascular smooth muscle cell calcification induced by hyperphosphate.Methods: 1. Aortas were removed in a sterile manner from Sprague-Dawley rats that weighted 100 to 150 g. Aortic segments were used to primary culture rat vascular smooth muscle cells (RVSMCs). RVSMCs were placed in various culture media, including normal phosphate medium (Pi 1.4mmol/L), high phosphate medium (Pi 2.0mmol/L, Pi 2.6mmol/L), ZVAD.FMK medium(Pi2.6mmol/L+ZVAD.FMK0.1μmol/L,Pi2.6mmol/L+ ZVAD.FMK 1.0μmol/L,Pi2.6mmol/L+ ZVAD.FMK 2.0μmmol/L),osteoprotegerin medium (Pi2.6mmol/L + OPG1μg/ml,Pi2.6mmol/L + OPG10μg/ml,Pi2.6mmol/L + OPG100μg/ml). 2. We observed the cells with interted microscope, and the purity of culture was assessed by positive immunostaining forα-SMA. 3. We cultured RVSMCs in normal phosphate medium, high phosphate medium, and quantified calcium deposition on day 0, 3, 6, 9. Also, RVSMCs were cultured in osteoprotegerin medium for 6 days, and quantified calcium deposition. 4. Calcification was visualized by Von Kossa's method. 5. OPG mRNA levels in RVSMCs treated with normal and high phosphate medium was examined by RT-PCR analysis. 6. We cultured RVSMCs in normal phosphate medium, high phosphate medium (Pi 2.6mmol/L), Osteoprotegerin medium (Pi2.6mmol/L + OPG100ug/ml) and then quantified the apoptosis by flow cytometer on day 6.Result: 1. With invented microscope we found RVSMCs were spindle cells. RVSMCs immunostaining forα-SMA was positive. 2. Vascular calcification occurs frequently in individuals with hyperphosphatemia whose serum phosphate levels typically exceed 1.4mmol/L. In normal phosphate levels (Pi 1.4mmol/L), RVSMCs accumulated very little calcium mineral. In contrast, in the presence of high phosphate levels (Pi 2.0mmol/L, Pi 2.6mmol/L), calcium deposition dramatically increased in a concentration-and time-dependent manner. On day 6, we quantified calcification of RVSMCs which were culture in ZVAD.FMK medium. We found that ZVAD.FMK significantly inhibited Pi-induced apoptosis as well as calcification concentration-dependent manner. Furthermore, to investigate the effect of osteoprotegerin on Pi-induced calcification, RVSMCs were incubated with osteoprotegerin in the presence of Pi 2.6mmol/L. On day 6, calcium deposition was significantly suppressed by osteoprotegerin in a concentration-dependent manner. 3. On day 6, Von Kossa's staining showed that RVSMCs calcification did not exist in normal phosphate levels, In contrast, Pi 2.6mmol/L induced dramatic RVSMCs calcification, and Osteoprotegerin dramaticly inhibited calcification. 4. RVSMCs had expression of OPG in normal phosphate levels, in high phosphate levels, OPG expression increased. 5. On day 6, RVSMCs apoptosis were increased by Pi 2.6mmol/L compared with Pi 1.4mmol/L. Osteoprotegerin reduced apoptosis compared with Pi 2.6mmol/L.Conclusion: 1. Pi induces calcium deposition in RVSMCs. 2. Pi increase expression of OPGmRNA in RVSMCs. 2. Osteoprotegerin inhibits Pi-induced RVSMCs calcification. 3. Inhibitory effect of osteoprotegerin on RVSMCs calcification is association with preventing apoptosis.