Expression of anti-angiogenic vascular endothelial growth factor isoforms VEGFxxxb in human retinal pigment epithelium cells / 中华实验眼科杂志

ABSTRACT

Background Hypoxia can increase the secretion of vascular endothelial growth factor-A (VEGF A) by retinal pigment epithelium (RPE) cells and initiate choroidal neovascularization (CNV) consequently.Two VEGF-A isoforms,the angiogenic VEGF-A family (VEGFxxx) and the anti-angiogenic VEGF-A family (VEGFxxxb),are found and formed by alternative splicing.However,the expressing changes and its effect in human RPE cells under the normoxia and hypoxia are unclear.Objective This study was to investigate the expression of VEGFxxx b in human RPE cells under hypoxia.Methods ARPE-19,a human RPE cell line,were cultured.CoCl2 150 μmol/L was added into the medium to mimic the hypoxia environment for 0 hour (normoxia group),3,12 and 24 hours to induce the hypoxia cells.The cells or suspension was harvested at various time points.The expressions of VEGFxxx mRNA and VEGFxxxb mRNA in the cells were detected,and the expressions of the total VEGF-A protein and VEGFxxxb protein in the cells were assayed by Western blot.ELISA was used to determine the contents of total VEGF-A and VEGFxxxb proteins in suspension.Results VEGFxxx mRNA and VEGFxxxb mRNA were expressed in the ARPE-19 cells in both normoxia and hypoxia,and multiple VEGFxxx mRNA isoforms appeared,including VEGF121 mRNA,VEGF165 mRNA and extremely faint VEGF189 mRNA,but only VEGF165b band was seen in VEGFxxxb mRNA.With the prolong of hypoxia culture,the expression of VEGFxxx mRNA showed gradual increase,while VEGFxxx b mRNA appeared gradual decrease.Western blot exhibited that VEGFxxxb was expressed in the cells cultured by normoxia and hypoxia with the strongest expression in VEGF165b.The VEGFxxxb content in suspension of medium was (166.82± 2.55) pg/ml under the normoxia environment and (125.35 ±2.10) pg/ml in 24 hours under the hypoxia,and the total VEGF-A protein elevated from (294.27 ± 11.97) pg/ml under the normoxia environment to (582.26 ± 12.98) pg/ml in 24 hours under the hypoxia.In addition,the proportion of VEGFxxxb to total VEGF-A in the cells lowed from (56.71 ± 1.02) ％ under the normoxia environment to (21.53 ±0.08) ％ in 24 hours under the hypoxia.Conclusions VEGFxxxb isoforms are expressed in both normoxia and hypoxia ARPE-19 cells.VEGFxxxb isoform is a predominant isoform in normoxia ARPE-19 cells.In hypoxia ARPE-19 cells,however,the expression of VEGF A is significantly stronger than that of VEGFxxxb.