Inhibitory effect of recombinant canstatin protein on alkali burn-induced corneal neovascularization in mouse

Abstract

Background Alkali burn-induced corneal neovascularization(CNV) usually leads to blindness.Recently,study determined that vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) were the main regulating factors inducing angiogenesis,and canstatin proteins can inhibit the growth of VEGF and bFGF and thereby inhibit CNV growth.Objective The present study was to investigate the effect of recombinant canstatin protein on mouse CNV induced by alkali burn and its mechanism.Methods CNV models were induced in the right eyes of 40 female BALB/c mice by sticking the 2.0 mm filtering paper with 1 mol/L NaOH at the central cornea for 10 seconds.The animals then were randomized into two groups.Recombinant canstatin protein drops(5 mg/L) was topically administered 4 times daily in the mice of the experimental group,and normal saline solution was used at the same way in the control group.Corneas of the mice were examined under the slit lamp to calculate the CNV area 1 day,3,7,14 days after modeling.The mice were sacrificed at above time points and corneas were obtained.The expressions of VEGF protein and bFGF protein in cornea were detected by Western blot,and the results were analyzed by enhanced chemiluminescence(ECL).The use of the experimental animals complied with the Instructive Notions with Respect to Caring for Laboratory Animals by State Ministry of Science and Technology.Results In 3,7 and 14 days after establishment of models,the area of CNV was (1.98-0.31) mm2,(6.21 ±0.44) mm2 and (9.83±0.72) mm2 in the experimental group,and that in the control group was (2.92 ± 0.41) mm2,(8.04 ± 0.56) mm2 and (11.78 ±0.84) mm2,showing significant reduce in the mice treated with recombinant canstatin protein(t3d =4.332,P=0.005 ;t7 d =11.729,P =0.000 ;t14 d =14.562,P =0.000).Western blot analysis showed that there was significant increase in the gray scale of VEGF protein 1 day,3,7,14 days following alkali burn in the experimental group compared with the control group(t1 d =-3.980,P＜0.001 ;t3d =-10.020,P＜0.001 ;t7d =-4.355,P＜0.001 ;t14 d =-8.156,P＜0.001),and the gray scale of bFGF was significantly ascent at various time points in the the experimental in comparison with the control groups (t1 d =-3.488,P＜0.001 ; t3 d =-2.124,P =0.013; t7 d =-1.977,P =0.028; t14 d =-4.542,P＜0.001).Conclusions Topical application of recombinant canstatin protein drops inhibits CNV growth induced by alkali burn by down-regulating the expressions of VEGF and bFGF proteins. Key words: Corneal neovascularization; Recombinant canstatin protein; Alkali burn; Vascular endothelial growth factor; Basic fibroblast growth factor