Phenotypic and functional changes of rat retinal microvascular endothelial cells induced by advanced glycation end products

Abstract

Objective To investigate whether the advanced glycation end products (AGEs) can induce the apoptosis of rat retinal microvascular endothelial cells and the related mechanisms. Methods Twenty SPF male Wistar rats aged 7 to 8 weeks were used. Microvascular endothelial cells was isolated from rat retinal, and identified by immunofluorescence staining and flow cytometry for vWF, and the tube formation on Matrigel. MTT assay was used to analyze the effect of AGEs on the proliferation of rat retinal microvascular endothelial cells. Flow cytometry was used to detect AGEs-induced apoptosis. Reverse transcription PCR and flow cytometry were used to analyze the expression level of RAGE, PKC, ICAM-1 and iNOS with or without AGEs in culture medium. t-test were used for statistical analysis. Results The isolated and purified rat retinal microvascular endothelial cells express vWF, and can form capillary-like network structure in Matrigel. AGEs can inhibit proliferation of rat retinal endothelial cells in dose- and time-dependent ways: the proliferation of endothelial cells in 200 mg/L AGEs group was lower than control at 5-day(t=8.9, P<0.05), and more significant at 7- and 9-day (t=15.7 or 46.1, both P<0.01). The inhibition to proliferation of endothelial cells by 400 mg/L AGEs was significant at 3-day(t=12.5, P<0.05), and became greatly at 5-, 7- and 9-day(t=22.4, 41.5 or 77.7, all P<0.01). AGEs also induced apoptosis of retinal endothelial cells. Furthermore, AGEs can up-regulate the expression of RAGE, PKC, iNOS and ICAM-1 at mRNA level(t=91.8, 9.22, 16 and 42, both P<0.01) and protein level(t=20.2, 12.3, 7.7 and 13.9, all P<0.01). Conclusion AGEs might induced phenotypic and functional changes of rat retinal microvascular endothelial cells by up-regulating RAGE. Key words: Advanced glycation end products; Retinal endothelial cell; Apoptosis; Phenotype; Function