THE EFFECT OF HIF-1Α/ANGPTL4 EXPRESSION CHANGES ON ARPE-19 CELL MIGRATION AND MONOLAYER CELL PERMEABILITY AND ITS POSSIBLE MECHANISM

Juan Li¹, Fei Sun¹, Xiaobo Li¹, Yunfeng Wang^2*

¹Department of Ophthalmology, Shenzhen Sami Medical Center, Shenzhen 518000, Guangdong Province, China - ²Department of Endocrinology, Shenzhen Sami Medical Center, Shenzhen 518000, Guangdong Province, China

Objective: To analyze the effect of changes in the expression of hypoxia-inducible factor-1α/angiogenin-like protein 4 (HIF-1α/ANGPTL4) on human retinal pigment epithelial cell (ARPE-19) migration and monolayer cell permeability, and their possible pathological mechanism. 

Methods: 30 clean-grade, healthy male Wistar rats were randomly selected to establish a diabetic rat model. The rats were randomly divided into a control group (n=15) and a diabetic group (n=15). The control group and diabetic group were both randomly divided into 4w (n=5), 8w (n=5) and 12w groups (n=5). Rat retinal tissue samples were taken, and changes in both retinal structure and retinal expression levels of HIF-1α and ANGPTL4 for each group were measured. Human retinal pigment epithelial cells (ARPE-19) were taken and cultured. The cells were divided into normal, hypoxia 8 hour, hypoxia 16 hour, and hypoxia 24 hour groups. Anaerobic culture bags were used in the hypoxia groups to provide a hypoxic culture, and 5 complex groups were set in each group. The expression levels of HIF-1α and ANGPTL4 in the cells of each group were measured in hypoxic environments. HIF-1α and ANGPTL4 inhibitors were transfected into the cells, placed in an anaerobic culture bag and cultured under hypoxia for 16 hours. The cells were divided into normal, hypoxia, HIF-1α low expression, and ANGPTL4 low expression groups. Tests measuring cell migration ability, cell permeability, and the expression level of tight junction proteins ZO-1 and occludin were conducted for each group. The cells were also divided into normal, hypoxia, ANGPTL4 low expression, and WP1066 groups, in which the expression levels of VEGF, p-STAT3, ZO-1 and occludin in each group was measured. 

Results: The retina of rats in the control group had normal morphology, clear structure, and orderly arrangement. The 4w group began to display cell disorders, and as the course of the disease progressed in the 8w and 12w groups, significant increases in retinal damage were observed. A small amount of HIF-1α and ANGPTL4 were expressed in the retina of the control group. As the disease progressed, the expression levels of HIF-1α and ANGPTL4 increased significantly (P<0.05). Compared to the normal group, the expression levels of HIF-1α and ANGPTL4 in each hypoxic group increased significantly as the time spent in hypoxia increased (P<0.05). Compared to the normal group, the cell migration ability, monolayer cell permeability, and expression levels of ZO-1 and occludin in the hypoxia group were significantly enhanced (P<0.05). Compared to the hypoxia group, the cell migration ability, monolayer cell permeability, and ZO-1 and occludin expression levels were significantly reduced in both the HIF-1α low expression and ANGPTL4 low expression groups (P<0.05). There were no significant differences in the cell migration ability, monolayer cell permeability, or expression levels of ZO-1 and occludin between the HIF-1α low expression and the ANGPTL4 low expression groups (P>0.05). Compared to the normal group, the expression levels of ZO-1 and occludin in the hypoxia group were significantly reduced, while the expression levels of VEGF and p-STAT3 were significantly increased (P<0.05). Compared to the hypoxia group, in the ANGPTL4 low expression and WP1066 groups the expression level of ZO-1 and occludin increased significantly, while the expression level of VEGF and p-STAT3 decreased significantly (P<0.05). 

Conclusion: Inhibition of HIF-1α/ANGPTL4 expression can reduce ARPE-19 cell migration ability and monolayer cell permeability and may be achieved by activating STAT3 phosphorylation.