The Mechanism Of Inhibition Effect Of DI-Ⅳon AGEs’ Inducing RF/6A Angiogenesis

ObjectiveDiabetic retinopathy (DR) is one of the major microvascular complications of diabetes. Proliferative diabetic retinopathy (PDR) with angiogenesis as its characteristic serves as a significant cause for blindness of diabetic patients. Some studies found that there was a positive correlation between advanced glycation end products level and PDR severity. Some results indicated that domain deletion mutant of β2-GPI---domain I-IV (DI-IV) inhibited angiogenesis of vascular endothelial cells. So rhesus retinal endothelial cells (RF/6A) were used in this study to investigate:1. the contribution of DI-IV to AGEs’ inducing angiogenesis effect on RF/6A.2. the mechanism of the contribution of DI-IV to AGEs’ inducing angiogenesis effect on RF/6A.Method1. RF/6A cells were cultivated and divided into five groups, a) NC group:without any interference factors. b) AGEs group:100ug/ml AGEs was added. c)10nM DI-IV group:100ug/ml AGEs was added with10nM DI-Ⅳ. d)100nM DI-IV group:100ug/ml AGEs was added with100nM DI-Ⅳ. e)400nM DI-Ⅳ group:100ug/ml AGEs was added with400nM DI-Ⅳ.2. Proliferition assay, wound healing assay and in vitro tubule formation on Matrigel were used to explore the contribution of different DI-IV concentration to AGEs’ inducing angiogenesis effect on RF/6A.3. Real time PCR was used to investigate the contribution of DI-IV to the mRNA expression of VEGFR-1(Flt-1) and VEGFR-2(KDR) of RF/6A intervened by AGEs.4. Western blot was used to determine the contribution of DI-IV to the expression of Akt in VEGF intracellular signal transduction pathway.Results 1. AGEs markedly induced proliferation of RF/6A (p<0.05) which was inhibited by DI-IV of different concentration, of which10nM and400nM DI-IV displayed more stronger inhibition (p<0.01)2. AGEs conspicuously increased migration of RF/6A by1.55times (p<0.01) which was restrained by DI-IV of different concentration, of which400nM DI-IV displayed the strongest inhibition, up to47.6%(p<0.05).3. AGEs observably induced tubule formation on Matrigel of RF/6A by70%(p<0.01) which was inhibited by DI-IV of different concentration, of which400nM DI-IV displayed the strongest inhibition, up to27.4%(p<0.05).4. AGEs markedly increased RF/6A mRNA expression of VEGFR-2(p<0.01) which was inhibited by DI-IV of different concentration, of which400nM DI-IV displayed the strongest inhibition (p<0.01). VEGFR-1expression among groups was not statistically different.5. AGEs markedly increased RF/6A p-Akt expression (p<0.05) which was inhibited by DI-IV of different concentration (p<0.01).Conclusion1. AGEs promoted proliferation, migration and tubule formation of RF/6A, might via increasing VEGFR-2expression and activating Akt with downstream signal transduction pathway.2. DI-IV antagonized AGEs’ inducing effect on proliferation, migration and tubule formation of RF/6A, might via inhibiting VEGF/VEGFR-2/Akt signal transduction pathway.3. VEGFR-1may not participated in proliferation, migration and tubule formation of RF/6A.