Effects of High Glucose on the Expression of LAMA1 and Biological Behavior of Choroid Retinal Endothelial Cells

Abstract

Hyperglycemia is one of the main causes of proliferative diabetic retinopathy (PDR) characterized by thickening of the vascular basement membrane. Laminin alpha 1 (LAMA1) is a primary component of laminin, a major protein constituent of the basement membrane. In this study, we investigated the role of LAMA1 in the development of PDR. Retinal choroidal vascular endothelial cells (RF/6A line) were exposed to glucose at different concentrations (5 mM, 15 mM, 25 mM, and 35 mM) and analyzed for cell growth, migration, proliferation, and adhesion. LAMA1 expression was examined 24 and 48 h following glucose treatment using Western blotting, RT-PCR, and immunofluorescence. The results showed that the proliferation, migration, and adhesion of RF/6A cells were increased by high glucose, whereas LAMA1 expression was slightly higher at 15 mM but decreased at 25 mM and 35 mM glucose compared to control. Thus, the changes in the biological behavior of high glucose-exposed retinal vascular endothelial cells correspond to variations in LAMA1 expression, indicating a possibility for LAMA1 involvement in PDR development. Our findings suggest that LAMA1 may play a role in PDR and, thus, may serve as a potential target for DR diagnosis and/or treatment.

Figure 1

Effects of high glucose on the growth of RF/6A cells. RF/6A cells were grown in medium supplemented with different concentrations of glucose for the indicated times.

Figure 2

Immunocytochemistry analysis of LAMA1 expression in RF/6A cells treated with different concentrations of glucose for 24 and 48 h.

Figure 3

Expression of LAMA1 protein in RF/6A cells. RF/6A cells were treated with different concentrations of glucose for 24 and 48 h and LAMA1 expression was analyzed by Western blotting; GAPDH was used as loading control. (a) Representative gel images. Left, 24 h; right, 48 h. (b) Quantitative analysis of LAMA1 expression performed using the ImageJ software. The data are presented as the mean ± SD of three independent experiments; ^∗P < 0.05.

Figure 4

LAMA1 mRNA expression. RF/6A cells were treated with different concentrations of glucose for 24 and 48 h and analyzed for LAMA1 mRNA levels by real-time PCR. There was a significant decrease of LAMA1 mRNA expression in cells exposed to 35 mM glucose both at 24 and 48 h. The data are presented as the mean ± SD of three independent experiments; ^∗P < 0.05.

Figure 5

Cell migration ability. RF/6A cells grown at different concentrations of glucose for 24, 48, and 72 h were analyzed by the wound healing assay. (a) Representative microscopic images of wound healing. (b) Quantitative analysis of the gap size measured using the Image-Pro Plus software; ^∗P < 0.05.

Figure 6

Proliferation of RF/6A cells exposed to high glucose concentrations. RF/6A cells grown at different concentrations of glucose for 96 h were analyzed for cell proliferation every 24 h using the CCK-8 assay. The data are presented as the mean ± SD of three independent experiments; ^∗P < 0.05.

Figure 7

Adhesion ability of RF/6A cells exposed to high glucose. RF/6A cells suspended in medium containing different concentrations of glucose were seeded in 96-well plates and analyzed for adhesion after indicated times using the modified CCK-8 method. The data are presented as the mean ± SD of three independent experiments; ^∗P < 0.05.