Inhibitory effects of apolipoprotein A-I on biological behavior and VEGF expression of human retinal epithelial cells in high glucose environment

Abstract

Background As a main cellular component of retinal microvascular, retinal vascular endothelial cells (RVECs) play critical roles in the occurrence and development of diabetic retinopathy (DR) by proliferating, migrating and angiogenesis.Apolipoprotein A-Ⅰ (ApoA-Ⅰ) is the major apolipoprotein of high density lipoprotein.ApoA-Ⅰ is overexpressed in the retina of diabetic patients and plays different effects on RVECs upon different microenvironments, but its relationship with RVECs in high glucose environment is still not elucidated. Objective This study was to investigate the effects of ApoA-Ⅰ on proliferation, migration, tubulogenesis and vascular endothelial growth factor (VEGF) expression in human RVECs (hRVECs) in high glucose environment. Methods hRVECs were cultured with DMEM containing 10% fetal bovine serum and passaged, and the cells at generation 3 to 6 were used in the study.The cells were divided into low-glucose group, low-glucose+ ApoA-Ⅰ group, high-glucose group and high-glucose+ ApoA-Ⅰ group, and the low concentration glucose (5 mmol/L), high contration glucose (25 mmol/L) and ApoA-Ⅰ (30 μg/ml) was added separately according to grouping.The proliferation and migration rate of the cells were evaluated by cell counting kit-8 (CCK-8) assay and scratch wound test respectively.The tubulogenesis of the cells was examined by tube formation test.The mRNA and protein expression of VEGF in the cells was detected by using real-time fluorescence quantitative PCR and Western blot. Results The prolifative value (absorbancy) and migration rate of the cells in the high-glucose group were significantly higher than those in the low-glucose group, and those in the high-glucose+ ApoA-Ⅰ group were significantly reduced in comparion with the high-glucose group (A value: P=0.001, 0.033; migration rate: P=0.001, 0.010). The number of tubes in the low-glucose group, low-glucose+ ApoA-Ⅰ group, high-glucose group and high-glucose+ ApoA-Ⅰ group was 7.250±2.217, 9.250±2.630, 19.000±3.916 and 11.500±3.697, showing a significant difference among the groups (F=10.335, P=0.001). The number of tubes in the high-glucose group was more than that in the low-glucose group, and the number of tubes in the high-glucose+ ApoA-Ⅰ group was less that that in the high-glucose group (P=0.001, 0.037). The relative expression levels of VEGF mRNA were 0.944±0.083, 1.117±0.204, 1.768±0.164 and 1.301±0.077, and those of VEGF protein were 1.000±0.130, 1.217±0.152, 1.871±0.101 and 1.609±0.087 in the low-glucose group, low-glucose+ ApoA-Ⅰ group, high-glucose group and high-glucose+ ApoA-Ⅰ group, respectively, with significant differences among the groups (mRNA: F=18.640, P=0.001; protein: F=10.335, P=0.001), and the expressions of VEGF mRNA and protein in the high-glucose group were significantly higher than those in the low-glucose group and high glucose+ ApoA-Ⅰ group (mRNA: P=0.000, 0.004; protein: P=0.000, 0.029). Conclusions ApoA-Ⅰ plays inhibitory effects on the proliferation, migration and tubulogenesis of hRVECs in high glucose environment, which may be associated with the downregulation of VEGF expression. Key words: Apolipoprotein A-Ⅰ; Diabetic retinopathy; Vascular endothelial cells; Humans; Cells, cultured; Glucose/administration & dosage; Vascular endothelial growth factor