| Objective: To investigate the AGEs induced VSMCs to osteoblast-like cell phenotype transdifferentiation molecular mechanism and the effects and molecular mechanism of Rapamycin(RPM) on atherosclerosis(AS) induced by AGEs in renal transplantation recipients..Methods: Isolation and culture of primary SD rat thoracic aortic smooth muscle cells(VSMCs), for cell passage through 4-6 generations, α- smooth muscle actin was measured using an optical microscope to observe cell morphology and indirect immunofluorescence staining(α- SMA) expression to identify whether VSMCs cell characteristics. With 200 mg / L, respectively, the role of AGEs or BSA VSMCs cells at different times(0h ~ 12d); In addition, some cells 2.0ug of pc DNA3.1 or integrin streptokinase(ILK) plasmid 24 h, or be subject to anti-AGE body(RAGE) antibody pretreatment 12 h, which shall then 200 mg / L of AGEs or BSA treated 9d, the cells were collected on SD rat thoracic aorta VSMCs detected by Western blotting α- smooth muscle actin(α-SMA), osteopontin(OPN), the expression of ILK in, and armor-phenolphthalein complexone method for detecting changes in the calcium concentration in the cell. take the fifth VSMCs as research objects, the cells divided into four groups depending on treatment: Sirolimus group(sirolimus), AGEs group(AGEs treatment), sirolimus + AGEs group(sirolimus and AGEs co-treatment) and control group(solvent-treated). After SD rat thoracic aorta VSMCs by 10 nm or sirolimus and 200 mg / L AGEs treatment 9d, cells were collected and detected the expressions of α-Smooth Muscle Actin(α-SMA), Osteopontin(OPN) 、 Proliferating Cell Nuclear Antigen(PCNA) Alkaline phosphatase(ALP) and ILK by Western blot;The cellular calcium despoition was assessed by the o-cresolphthalein method.Results: Showing 4-6 generations SD rat thoracic aorta VSMCs cell morphology no significant difference in the primary cells under an optical microscope; indirect immunofluorescence staining results also show that the intensity of their expression of α-SMA and primary cells similar. In vitro experiments showed that: after AGEs SD ratthoracic aortic VSMCs role in the expression of α-SMA in a time-dependent decrease; at the same time, OPN expression in a time-dependent increase; and the calcium ion concentration also has AGEs concentration-dependent increase trend(P<0.05). Additionally, compared with the control group, treated with AGEs SD rat thoracic aorta VSMCs6 h, the cells significantly increased expression of ILK, and reached the peak at 12 h, 24 h start began to decrease(P<0.05); and transfected with empty plasmid(pc DNA3.1) of SD rat thoracic aorta VSMCs compared to wild-type ILK plasmid transfected cells upregulated ILK expression, the cells can express α-SMA decreased significantly; while OPN expression increased significantly, further after the effect of such cells to AGEs, then the expression of α-SMA to further reduce, OPN expression further increased. And with anti-RAGE neutralizing antibody pretreated cells, ILK inhibits expression induced by AGEs increased(P<0.05). we also found AGEs suppressed SD rat thoracic aorta VSMCs in a-SMA expression; induction of OPN, PCNA expression and upregulation of ILK, and cells expressing ALP and calcium ion concentrations were significantly increased(P<0.05); compared with AGEs group after pretreatment of cells with sirolimus inhibit these effects are mediated by AGEs(P<0.05).Conclusion: AGEs may--RAGE by binding to its receptor is activated ILK within the relevant intracellular signaling pathways thereby causing VSMCs to osteoblast phenotype transdifferentiation; but, Sirolimus may thereby inhibiting AGEs induced VSMCs happen to osteoblast phenotype transdifferentiation and inhibition of AGEs induced VSMCs proliferation, thereby inhibiting renal transplantation atherosclerosis occurrence / progression, and improved by suppressing the expression of ILK postoperative cardiovascular disease(CVD) incidence of kidney transplantation. |
PDF Full Text Request