The Study On Mechanism Of Advanced Glycosylation End Products Inducing Atherosclerosis Via Dendritic Cells

The principal cause of morbidity and mortality is chronic vascular complications in diabetic patients. It has been found that advanced glycosylation end products (AGEs) play an important role in the pathogenesis and progression of atherosclerosis. Accumulating evidence suggests that atherosclerosis is a chronic inflammatory disease. Dendritic cells (DCs) play an important regulating function in immune process. The study on the effect of AGEs on DCs in atherosclerosis has not been seen in liturature. The aim of this research is to study the mechanism of AGEs in inducing atherosclerosis via DCs. It is made up of three parts:Part Ⅰ: Effect of advanced glycosylation end products on the expression of scavenger receptor A and receptor for advanced glycosylation end products in human monocyte-derived dendritic cellsObjective: To investigate the effect of advanced glycosylation end products (AGEs) on the expression of scavenger receptor A (SR-A) and receptor for advanced glycosylation end products (RAGE) in human monocyte -derived dendritic cells (DCs).Methods: Monocytes were purified (over 98%) using Anti-CD14+ microbeads, after eight days' culture in RPMI1640 medium containing recombinated human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (100ng/ml) and recombinated human interleukin-4 (rhIL-4) (50ng/ml), immature DCs were derived. At the eighth day, They were exposed to AGE-BSA (concentration of 0, 50, 100, 200, 300μg/ml ) for 24 hours, and exposed to AGE-BSA (protein concentration of 200μg/ml ) for 0, 6, 12, 24, 36 hours. Expression of SR-A was semi-quantified by RT-PCR and western-blot. In addition, immature DCs were treated by 200μg/mlAGE-BSA for 24 hours and then the expression of RAGE was semi-quantified by RT-PCR and western-blot.Results: (1) The mRNA of SR-A incubated by 50μg/ml AGE-BSA was higher than that of control and the protein of SR-A incubated by 100μg/ml AGE-BSA was higher than that of control at 24 h (P<0.05) . It reached the peak at 200μg/ml AGE-BSA (p<0.01). After intervention of AGE-BSA for periods of 12, 24 ,36 hours, the SR-A expression was increased markedly. It reached the peak at36 hours (P<0.01). (2) mRNA and protein of RAGE incubated by 200μg/ml AGE-BSA was higher than that of control at 24 h. Treatment of DCs with AGE-BSA resulted in about two-fold increase of the expression of RAGE (P<0.05) .Conclusion: AGEs can up-regulate the expression of SR-A and RAGE in DCs. It may be one of the mechanisms of AGEs inducing atherosclerosis via DCs.Part Ⅱ: Mechanism of advanced glycosylation end products on the expression of scavenger receptor A and receptor for advanced glycosylation end products in human monocyte-derivcd dendritic cellsObjective: To investigate the signal pathway of AGEs inducing the expression of SRA and RAGE.Methods: Monocytes were purified (over 98%) using Anti-CD14+ microbeads, after eight days' culture in RPMI1640 medium containing recombinated human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (100ng/ml) and recombinated human interleukin-4 (rhIL-4) (50ng/ml), immature DCs were derived. At the eighth day, they were exposed to 200μg/mL AGE-BSA for 0, 10, 20, 30 and 40 min, mitogen-activated protein kinase (MAPK), i.e. Phospho-Erk, phospho-Jnk and Phospho-p38MAPK were measured by Western-blot. In addition, DCs were treated by 200μg/mL AGE-BSA plus the inhibitor Erk, Jnk or p38MAPK and theexpressions of SRA and RAGE were measured by Western-blot.Results: (1) Increases in the phosphoylation levels of Erk, Jnk and P38 were visible at 10 minutes, peaked at 20 minutes and declined thereafter. (2) The increase in SR-A and RAGE expressions post AGE-BSA was significantly inhibited by co-treatment with Jnk inhibitor SP600125, but not by Erk inhibitor PD98059 and p38 MAPK inhibitor SB203580.Conclusion: AGEs can activate the MAPK signal pathway. The expressions of SRA and RAGE have a relation with Jnk signal pathway.Part Ⅲ: Effect of advanced glycosylation end products on the maturation and immune function of human monocyte-derived dendritic cellsObjective: To observe the effect of AGEs on the maturation and immune function of DCsMethods: Monocytes were purified (over 98%) using Anti-CD14+ microbeads, after eight days' culture in RPMI1640 medium containing recombinated human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (lOOng/ml) and recombinated human interleukin-4 (rhIL-4) (50ng/ml), immature DCs were derived. At the eighth day, they were exposed to 200μg/mL AGE-BSA, 200μg/mL AGE-BSA + 50μmol/L Jnk inhibitor SP600125, 200μg/mL AGE-BSA plus 50μg/mL anti-RAGE antibody or BSA for 24 hours. FACS was used to investigate the immunophenotypic expression (CDla, CD40, CD80, CD83, CD86, HLA-DR), allogeneic mixed T lymphocytes reaction and supernatants' cytokine (IL-12, IL-10, IFN-γ and IL-2) measurements were used for immune functional assays.Results: (1) Compared with the BSA and the untreated DCs, FACS analysis showed that the expressions of CDla, CD40, CD80, CD83, CD86 and HLA-DR in AGE-BSA-treated DCs were increased significantly. (2) Compared with the untreated control DCs, the AGE-BSA-treated DCs increased the proliferation of T...