Epithelial-mesenchymal transition of human retinal pigment epithelial cells under the high glucose condition in vitro

Background

Several types of cells participate in the formation of proliferative membrane in proliferative retinopathy (PVR), and the proliferation, migration and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells play an important role.Many studies have confirmed high blood glucose is the basic pathogenesis of diabetic retinopathy (DR).However, whether EMT could be induced in RPE cells under the high glucose condition has not been reported.

Objective

This study was to investigate the effects of high glucose on the migration and EMT of RPE cells in high glucose culture model in vitro.

Methods

Human RPE cell line D407 were cultured and passaged in DMEM/F12 medium with 10% fetal bovine serum, and 6-8 generations of cells were used in experiment.The cells were divided into 3 groups based on different glucose concentrations in medium.The glucose at the final concentration 5.5 mmol/L or 60.0 mmol/L was respectively used in the normal control group or high glucose group, and the DMEM with 5.5 mmol/L glucose and mannitol was used in the hypertonic control group.The migration rate of the cells were detected 0, 24, 48 and 72 hours after scratching by wound-scratch test.Real-time PCR was used to detect the relative expressions of zonula occludens-1 (ZO-1) and α-smooth muscle actin (α-SMA) in the cells.

Results

Cultured cells showed a polygon shape with the clear nucleolus and dense arrangement in the normal control group and the hypertonic control group, but the cells were larger and elongated with the lapse of culture time with the indistinct structure and loose arrangement.At 48 hours after scratching, migrating cells were seen in the scratching area, and the scratching area disappeared at 72 hours after scratching in the high glucose group, but the scratching area still was existed in the normal control group or hypertonic control group.The migrating rate of the cells was higher in the high glucose group than that in the normal control group or hypertonic control group, showing total differences among 3 groups and various time points (F_group =328.600, P=0.000;F_time=773.270, P=0.000).Compared with the normal control group, the expression level of ZO-1 mRNA was significantly lower, and α-SMA mRNA level was higher 48 hours and 72 hours in the high glucose group than those in the normal control group (all at P<0.05).

Conclusions

High glucose induce the migration and EMT of RPE cells in vitro, which may be associated with the pathogenesis of proliferative diabetic retinopathy.

Human; Retinal pigment epithelium cell; High glucose; Epithelial-mesenchymal transition; Diabetes mellitus/complication, retinopathy; Cell culture