The Effects Of Atorvastatin And Alendronate On Artery Calcification And Their Mechanisms

Artery calcification often occurs with advancing age, atherosclerosis, diabetes mellitus and chronic uremia. Artery calcification refers to the deposition of calcium phosphate mineral, most often hydroxyapatite in the artery wall. It may occur in intimal atherosclerotic plaques or in Monckeberg's sclerosis, which is defined as calcification restricted to the arterial medium, or in heart valve.Artery calcification is a common process. About 75%~95% of men and women at autopsy have coronary calcification regardless of the cause of their death. Coronary calcification is positively correlated with increased risk of myocardial infarction and increased risk of dissection after angioplasty, and is also correlated with adverse event in C ABG. The extent of coronary calcification ("calcium score") has been found to add incremental prognostic significance to conventional risk factors for coronary artery disease. Vascular calcification causes a reduction in elasticity of the artery wall and reduced compliance, which results in hypertension. Therefore, artery calcification contributes to both the morbidity and mortality associated with cardiovascular disease. It has clinical significance of studying the pathogenesis of artery calcification and its treatment.In the past, artery calcification was considered to be a passive process that occurred as a nonspecific response to tissue injury or necrosis, referred to calciumfrom bone transferred to artery wall. But with understanding deepens, now there is strong evidence to support the concept that artery calcification is not a passive process, but an active process similar to bone formation, and is regulated by the growth factors, matrix proteins, and other bone-related proteins.In this study, after successfully establishing the artery calcification model of rat, the effects of Atorvastatin and Alendronate on the induced artery calcification in vivo and on the aortic VSMC calcification in vitro were studied by the methods of histology, cytology and molecular biology. The study is aimed to offer experiment data to prevent cardiovascular disease, especially atherosclerosis in clinic.The study is composed of 5 parts.1. Establishment and characteristics of artery calcification induced by warfarin and vitamin D3 in rat.Artery calcification of rat was established by subcutaneous injection of vitamin D3 (300,000U/kg/d for 3 days) and Warfarin (15mg/100g/12h for 4 days) . Abdominal aortas were collected for paraffin section, stained by von Kossa staining to observe the area of calcification. Thoracic aortas were collected for detection of OPG and RANKL expression levels by RT-PCR. Aorta paraffin sections were used to detect the localization of OPG and RANKL by in situ hybridization. Von Kossa staining found black deep stained structure in the medium of aorta, which was calcified artery. In the normal artery of rat, there are the expressions of OPG and RANKL, which are mainly located in the medium smooth muscle layer. The expressions of OPG and RANKL in calcified artery are decreased.2. The protective role of Atorvastatin on the artery calcification induced by warfarin and vitamin D3 in rat.At 4 days before establishment of artery calcification, atorvastatin (100mg/kg/d) was treated by intragestric administration, and continued to administrate to the end. The effect of atorvastatin on the artery calcification wasobserved. Von Kossa staining found the black deep stained structure decreased with atorvastatin treatment. Compared to the untreated calcified artery, the expression level of OPG and RANKL had not significant difference in the atorvastatin treated arota. Right tibias were cut to measure the bone mineral density. Bone mineral density in the rat of artery calcification was decreased, and was slightly increased after treating with atorvastatin.3. The effects of Alendronate on the artery calcification induced by warfarin and vitamin D3.At 4 days before establishment of artery calcification, alendronate (lmg/kg/d) was administrated by subcutaneous injection, and continued to administrate to the end. The effect of alendronate on the artery calcification was observed. Von Kossa staining found the black deep stained structure decreased after alendronate treatment. The expressions of OPG and RANKL in the calcified arota had no significant difference between the alendronate treatment group and the untreated group. Bone mineral density in the rat of artery calcification was decreased, and was slightly increased after treating with alendronate.4. The effects of Atorvastatin on the calcification of rat aortic VSMC.Rat aortic VMSC was cultured in vitro by tissue explant. All cells were divided into 5 groups, which was normal, calcification group (control group), and atorvastatin 1UM, 5UM and 10UM group. Before inducing calcification, Atorvastatin 1, 5, 10UM group were individually pre-treated by final concentration of 1, 5, 10UM atorvastatin for 24 hours. ï¿¡ -glycerophosphate, were then added in calcification group and in all atorvastatin groups to induce VSMC calcification. All cells were cultured for 14 days. Cell crawling slice was applied to Alizarin red S staining to observe VSMC calcification. Colorimetric method was applied to measure contents of Ca2+, cell proteins, and ALP activity. The ratio of contents of Ca2+ and cell proteins was cell calcium deposits. Cell proliferation was measured by tetrazolium salt (MTT) method. VSMCs werecollected to detect the expression level of OPG and RANKL by RT-PCR. Compared with the control group, in all atorvastatin groups, the number of calcium nodulus, cell calcium depositions, cell ALP activity and cell proliferation decreased significantly and dose-dependently. Normal aorta VSMC has expression of OPG and RANKL. In calcified VSMC, OPG expressed increased, and RANKL expressed decreased. There was no difference in the expression of OPG and RANKL after treated with atorvastatin. 5. The effects of alendronate on the calcification of rat aortic VSMC.All cells were divided into 5 groups, which was normal, calcification group (control group), and alendronate 10"9M, 10'7M and 10'5M group. Before inducing calcification, Alendronate 10"9, 10~7, 10"5M group were individually pre-treated by final concentration of 10"9, 10"7, 10"5M Alendronate for 24 hours. P -glycerophosphate, were then added in calcification group and in all alendronate groups to induce VSMC calcification. All cells were cultured for 14 days. Compared with the control group, in all alendronate groups, calcium nodulus, cell calcium depositions, cell ALP activity and cell proliferation decreased significantly and dose-dependently. There was no difference in the expression of OPG and RANKL after treated with alendronate.Conclusion. OPG and RANKL are expressed in the normal rat aorta and locate mainly in the medium smooth muscle of arota. Expression level of OPG and RANKL decrease in the rat calcified artery induced by warfarin and vitamin D3.2. Atorvastatin can dramatically decrease the rat artery calcification induced by warfarin and vitamin D3.3. Atorvastatin does not affect the expression level of OPG and RANKL in calcified artery induced by warfarin and vitamin D34. Alendronate can dramatically decrease the rat artery calcification induced by warfarin and vitamin D3.