| There is a growing body of evidence that the vascular adventitia could act as a critical regulator modulating (directly or indirectly) the structure and function of the vascular wall, thus play a role in the process of atherosclerosis. As the principal cell type in the adventitia, fibroblasts were shown to correlate with vascular dysfunction, vascular restenosis and atherosclerosis. Advanced glycation end products (AGEs) and vascular adventitial fibroblasts (AFs) are involved in diabetes-related vascular complications. However, the effect of AGEs on AFs remains unclear. The aim of this study was to observe the impact of AGEs on cell migration capacity,oxidative stress and associated inflammatory responses of AFs. It is made up of four parts:Part I Effect of advanced glycation end products on the expression of receptor for advanced glycation end products in vascular adventitial fibroblastsObjective: To investigate the effect of advanced glycation end products (AGEs) on the expression of receptor for advanced glycation end products (RAGE) in vascular adventitial fibroblasts (AFs).Methods: Isolated vascular adventitial fibroblast of Sprague-Dawley (SD) rats were cultured. The expressions of specific antigens on cell surface were analyzed by fluorescence immunocytochemistry. After 24 hours synchronization, AFs were exposed to AGE-HSA (concentration of 0, HSA200, 50, 100, 150, 200, 300μg/ml) for 24 hours. Expressions of RAGE were measured by RT-PCR and Western-blot.Results: AGEs (concentration range from 0 to 300μg/ml) up-regulated the expression of RAGE at mRNA and protein levels, peaked at a concentration of 200μg/mL(P<0.05), this effect could be significantly inhibited by p38, ERK1/2 and JNK MAPK inhibitors as well as Candesartan.Conclusion: AGEs can up-regulate the expression of RAGE in AFs. Candesartan can inhibit this effectPart II Advanced glycation end products enhance migration of vascular adventitial fibroblasts and effect of Candesartan Objective: To investigate the effects, mechanism on the migration of rat adventitial fibroblasts induced by AGEs and to study the intervention effects of Candesartan.Methods: Isolated vascular adventitial fibroblast of Sprague-Dawley (SD) rats were cultured. Migratory potential was estimated by transwell chamber in vitro. Expressions of phosphorylated mitogen-activated protein kinase (MAPK) of AFs were measured by RT-PCR and Western-blot.Results: AGEs increased expression of phosphorylated forms of p38, ERK1/2 and JNK mitogen-activated protein kinases in AFs, JNK peaked at 20min and p38, ERK1/2 peaked at 30min(P<0.05). Phosphorylation of p38, ERK1/2 and JNK was suppressed by p38, ERK1/2 and JNK MAPK inhibitor,respectively.This effect also could be significantly inhibited by Candesartan and anti-RAGE neutralizing antibody. The migration of AFs enhanced by AGEs (concentration ranging from 0 to 300μg/ml) was markedly increased in a dose-dependent manner, peaked at a concentration of 200μg/mL(P<0.05). Anti-RAGE neutralizing antibody, p38, ERK1/2 MAPK inhibitors and Candesartan suppressed AGEs-induced migration of AFs(P<0.01).Conclusion: This study indicated that AGEs promote AFs migration via RAGE and MAPK pathway.Candesartan can effectively inhibit the migration of AFs. It might be a novel mechanism on vascular protection of Candesartan.Part III Mechanism on activation of oxidative stress in vascular adventitial fibroblasts induced by advanced glycation end productsObjective: To investigate the effect and mechanism of high advanced glycation end products (AGEs) on the oxidative stress in vascular adventitial fibroblasts (AFs).Methods: Isolated vascular adventitial fibroblast of Sprague-Dawley (SD) rats were cultured. 2,7-dichlorofluorescein-diacetate(DCFH-DA) was used as a reactive oxygen species (ROS) capture agent.The fluorescent intensity of 2,7-dichlorofluorescein (DCF), which was the product of cellular oxidation of DCFH-DA, was detected, and the level of ROS was thus measured . The expressions of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22phox and p47phox were measured by RT-PCR and Western-blot.Results: (1) The increase in protein and mRNA expressions of p22phox and p47phox induced by AGE-HSA was significantly inhibited by co-treatment with NADPH oxidase inhibitor APO, DPI, anti-RAGE neutralizing antibody and Candesartan (P<0.05). (2)The production of ROS in AFs induced by AGE-HSA significantly increased. The effect can be inhibited by NADPH oxidase inhibitor DPI, APO, anti-RAGE neutralizing antibody and Candesartan.Conclusion: The production of ROS in AFs is correlated with the interaction of AGEs and RAGE which can upregulate p22phox mRNA expression. NADPH oxidase inhibitors and Candesartan can reduce ROS by downregulating p22phox and p47phox expression.Part IV Advanced Glycation End Products Enhance inflammatory response of Vascular Adventitial Fibroblasts and Effect of CandesartanObjective: To investigate the effects, mechanism on the inflammation of vascular adventitial fibroblasts induced by AGEs and the intervention effects of Candesartan.Methods: Isolated vascular adventitial fibroblast of Sprague-Dawley (SD) rats were cultured. Expression of MCP-1, IL-6, VCAM-1 mRNA was analyzed by RT-PCR.MCP-1, IL-6, VCAM-1 in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA). NF-κB and I-κB-αwas analyzed by electrophoretic mobility shift assay (EMSA) and Western-blot..Results: NF-κB transcriptional activity was increased after treatment with AGE-HSA (200μg/ml) for 0.5h, 1 h and 2 h. Incubating AFs with AGE-HSA (200μg/ml) for 0.5 h and for 1 h caused phosphorylation of I-κB-α. AGE-HSA (200μg/ml) treatment increased nuclear NF-κB, Candesartan co-treatment inhibited this translocation. I-κB-αphosphorylation in response to AGE-HSA (200μg/ml) was also suppressed by Candesartan. AGEs (concentration range from 0 to 300μg/ml) elevated the mRNA expression of MCP-1, IL-6, and VCAM-1. Compared with control, the expression of IL-6,VCAM-1,MCP-1 mRNA increased significantly in AGE-HSA 50,100μg/ml groups(P<0.05) and AGE-HSA 200,300μg/ml groups(P<0.01). Pretreatment of the cells with anti-RAGE neutralizing antibody , MG-132, an inhibitor of NF-κB, and Candesartan decreased the AGE-HSA induced expression of IL-6,VCAM-1, and MCP-1 and concentration of them in the supernatants(P<0.05). Conclusion: This study indicated that AGEs increase the expression of MCP-1, IL-6, and VCAM-1 in AFs via RAGE and NF-κB pathways. Candesartan can effectively inhibit phosphorylation of I-κB-α, nuclear translocation of NF-κB and expression of inflammatory factors, suggesting its treatment function of vascular complication in the diabetes.Part V Expression of Galectin-3 in Vascular Adventitial Fibroblasts and RNA Interference of Galectin-3 GeneObjective: To observe the expression of galectin-3 in AFs, to construct a lentivirus vector of RNA interference (RNAi) of galectin-3 gene and to observe effects of AGEs receptor galectin-3 on vascular fibroblasts.Methods: The expression of galectin-3 in AFs was detected by RT-PCR. DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized.Short hairpin RNA targeting to galectin-3 was cloned to lentivirus work vector.The recombined vector was identified by restriction enzyme analysis and DNA sequencing . Work vector and package plasmids were cotransfected to 293T cells.Lentivirus was collected after 72 h and was added to cultured AFs.The expression of galectin-3 was detected by Western blot.Results: AGEs (concentration range from 0 to 300μg/ml) up-regulated the expression of RAGE at mRNA and protein levels. The effective target of galectin-3 gene for RNAi was screened out. The galectin-3 siRNA lentivirus vector was established successfully. This vector can infected vascular adventitial fibroblasts and knocked down expression of galectin-3. Expression level of galectin-3 in vascular adventitial fibroblasts decreased.Conclusion: The constructed lentiviral vector can effectively inhibit the expression of galectin-3 in the adventitial fibroblasts. |
PDF Full Text Request