Effect of age on vascular calcification induced by vitamin D3 and nicotine

Objective: To explore the effect of age on vascular calcification induced by vitamine D3 and nicotine.Methods: Vascular calcification in rats was induced by adminstration of vitamine D3 plus nicotine(VDN treatment).After six weeks,Von Kossa staining,calcium content,alkaline phosphatase activity,phosphorus and calcium content in plasma were assayed.Carotid blood pressue,cardiac function and the relative amounts of osteopontin(OPN)、osteoprotegerin(OPG)、matrix Gla protein(MGP)、bone morphogenetic protein-2(BMP2) mRNA level and smooth musule actin-α(α-SMA)protein level were measured.Results: Compared with control group,the systolic blood pressure(SBP)of the rats of 2,8 and 16 months with vascular calcification respectively increased by 20.7%,29.4% and 22.2%(P0.05);the left ventricular systolic pressure(LVSP) respectively increased by 13.6%,21.1% and 16.2%(P0.05);+ LVdP/dtmax respectively increased by 49.1%(P0.01),21.4% and 13.1%(P0.05);-LVdP/dtmax respectively increased by 56.3%(P0.01),24.4% and 11.3%(P0.05).Aortic calcium contents of the 2-,8-and 16-month calcified rats were respectively 2.62-fold(P0.05),24.87-fold(P0.01) and 10.01-fold(P0.05)of the age-matched control group.As compared with the aortic calcium contents of calcified groups at different ages,the calcification group of 8 months had higher aortic calcium content than those of 2 and 16 months,which were respectively,5.28-fold and 2.63-fold(P0.05).Compared with the control groups,alkaline phosphatases activity(ALP) of calcification groups increased respectively by 126.6%,115.2% and 227.9%(P0.01)in the 2-,8-and 16-month rats.As compared with the ALP activity of calcified groups at different ages,ALP acti-vity of aortic calcification group of the 8-month-old rats was higher than that of the 2-month-old and 16-month-old rats,which increased by 176% and 75% respectively(all P0.01).Von kossa staining for calcification showed positive staining as black /brown areas within the main,large,nodular structures as shown in extracellular matrix and cytoplasma in VDN groups at different ages,especially in the 8-month-old VDN group,with the most dispersed calcific nodules deposited and a few of the elastic fibers of the medial layer collapse.The mRNA expressions of OPN,OPG,MGP,BMP2 were up-regulated(P0.01 or P0.05) and protein levels of α-SMA were down-regulated in different calcification groups(P0.05).The mRNA levels of OPN in 8-month-old calcification group increased by 3.41-fold(P0.01) and 1.34-fold(P0.05) respectively compared with the 2-month-old and 16-month-old calcification groups.And the α-SMA protein expression levels were lower at calcification groups in different ages,which were respectively equivalent to 17.6% of the 2-month-old control gruop(P0.01),11% of the 8-month-old control group(P0.05) and 41.7% of 16-month-old control group(P0.01).Conclusion: SD rats of 2,8 and 16 months can all be used to duplicate vascular calcification model induced by vitamine D3 plus nicotine and the 8-month-old rat has the most sensitivity to the calcification treatment,which means that the 8-month-old rat may be the most appropriate age for the study of vascular calcification.