The effects of nitric oxide synthase and its antagonist on alkali burn-induced corneal neovascularization

Abstract

Background Though nitric oxide (NO) and NO synthase (NOS) have a critical role in angiogenesis,their effects on corneal neovascularization (CNV) and mechanism need to be further explored.Objective The aim of this study was to explore the effects of NOS and its antagonist,Nw-nitro-L-arginine methyl ester (L-NAME) on experimental CNV in mice,and investigate the influence of NOS and L-NAME on the tube formation of human retinal endothelial cells (RECs) in vitro.Methods The CNV models were established in the left eyes of 36 male BALB/c mice aged 7-8 weeks by application of the filter paper with NaOH in the center of corneas.The mice were randomized into two groups.L-NAME of 10 g/L (0.5 ml) was intraperitoneally injected 1 week before induction of CNV three times a week for three weeks in the mice of the L-NAME injection,and PBS was used in the same way in the control group.CNV was examined under the slit lamp biomicroscope 2,4,7,14 days after NaOH burn.The expression of CD31 in the CNV was assayed to calculate the ratio of CNV area and total corneal area using whole mount technique.The expression of NOS mRNA in the corneal tissue was detected by reverse transcriptase polymerase chain reaction (PCR),and VEGF expression in the human RECs was assayed by Western blot.The vessel formation number of cultured human RECs with or without L-NAME was performed by matrigel in vitro.Grouped t test was used to compare the differences of the parameters between the two groups.Results CNV developed and peaked 2 weeks after the application of NaOH on the mice corneas,and the CNV was obviously less in the L-NAME group compared with the control group.The expression of NOS mRNA in the corneas (NOS mRNA/ GAPDH mRNA)was significantly lower in the L-NAME group than that of the control group 2,4,7 days after CNV induction (t =19.481,t=22.059,t=10.961,all at P＜0.01).The ratio of the CD31 positive area in whole corneal area was 0.59± 0.01 in the L-NAME group,and that of the control group was 0.78±0.10,showing a significant difference between the two groups (t =3.078,P＜0.05).Western blot assay showed that the relative expression of VEGF protein in human RECs was declined in the L-NAME group compared with the control group 0,2,4,7 days,with statistically significant differences in 4 days and 7 days after NaOH burn(t=7.696,t=17.953,both at P＜0.01).The number of vessel network was 46.33±1.86 in the L-NAME group and 64.00±4.51 in the control group,with a significant difference between them (t =3.623,P＜0.05).Conclusions NOS participated in the pathogenesis and aggravation of CNV induced by NaOH.L-NAME arrests CNV formation and human RECs tube formation through down regulating the VEGF expression and NOS activity. Key words: Cornea; Neovascularization; Nitric oxide synthase; Vascular endothelial growth factor; Alkali burn