Regulatory effect of Gpr124 on proliferation, migration and angiogenesis of retinal microvascular endothelial cells under high-glucose condition

Abstract

Objective To investigate the changes in G protein-coupled receptor 124 (Gpr124) expression in human retinal microvascular endothelial cells (HRMEC) under high-glucose condition and the regulatory effect of Gpr124 interference on HRMEC. Methods Immunofluorescence assay was used to observe the expression of Gpr124 in HRMEC. The cells were divided into a blank group and a high glucose group. CCK-8 assay and EdU cell proliferation assay were used to detect cell viability and proliferation, respectively. The Gpr124 expression was knocked down by transducting lentiviral virus carrying shRNA targeting Gpr124. Thus, the cells were divided into a blank control group, a high glucose control group, a high glucose+shRNA group, and a high glucose+Gpr124 shRNA group. Cell scratch test was performed to evaluate cell migration, and Matrigel plug assay was employed to assess the cell tubule formation. Western blotting was applied to detect the protein levels of Gpr124, VEGFA, MMP9, and β-catenin. Results The HRMEC from the high glucose group showed enhanced cell viability and increased number of proliferating cells compared to the cells of the blank group (P<0.01). High glucose treatment also induced increased expression levels of Gpr124, VEGFA and MMP9 (P<0.01), and larger migratory area and in vitro angiogenic area as well as longer tube diameter at all time points when compared with the conditions in the blank control group (P<0.01). However, knockdown of Gpr124 resulted in a significant decrease in HRMEC migration and in vitro angiogenic capacity, accompanied by decreases in protein expression of VEGFA, MMP9 and β-catenin (P<0.01). Conclusion Gpr124 can regulate the proliferation, migration, and tube formation ability of HRMEC under high-glucose condition, and also inhibit the overexpression of VEGFA and MMP9, which may be through regulating the classical Wnt/β-catenin pathway.

Keywords

• diabetic retinopathy
• g protein-coupled receptor 124
• vascular endothelial cells
• cell migration assay
• angiogenesis