| BackgroundIt is well known that cataract is a leading cause of blindness in the world. The growing percentage of the aging people in the world leads to increasing the incidence of age-related cataract (ARC) insistently. The occurrence of ARC is correlated with a lot of factors and oxidative stress is the most importance. Oxidative stress can induce cells dysmetabolism and generate reaction oxygen species. It can damage the structure and the function of lens epithelial cells, destroy the epithelium barrier and then induce cataract. However, the mechanism of the process is still not clear. A multifunctional cytokine, vascular endothelial growth factor (VEGF) plays an important role in regulating cell proliferation, differentiation, migration and permeability in some cells. VEGF is correlated with angiogenesis and vasopermeability, but also express in avascular lens cells. The protein level of VEGF in the eyes was upregulated in hypoxia. However, whether VEGF involved in ARC formation in mature lenses is still unclear. It has been known that Src-family kinase (SFK), a nonreceptor tyrosine kinase, is involved in the activation of some signal transduction induced by oxidative stress, UV, osmotic stress. In our earlier research, abnormal activation of SFK has been proved involving in development of cortical cataract. It has been found that VEGF may change cell-cell junction for regulating the cell permeability together with VEGFR and c-Src. Whether these signal transduction pathway involved in cataract formation in human lens epithelial cells (HLECs) by affecting the integrality and permeability of HLECs is the issue we focused on in the study.ObjectiveTo observe the influence of oxidative stress on VEGF expression in HLECs of cataract lenses and investigate the effects of oxidative damage and SFK/VEGF on cell-cell junction in lens epithelium and on the formation of cataract.Methods(1) The undiluted aqueous humor and the anterior capsule sample of ARC patients were collected in cataract surgery. Correlated clinical factors of ARC patients were collected. VEGF levels in undiluted aqueous humor were tested by ELISA assay and VEGF expression in anterior capsule were assayed by indirect immunofluorescence stain. The correlation of VEGF expression and related factors was analyzed by SPSS 13.0 soft. (2) Undiluted supernatant samples from cultured HLECs (SRA01/04) incubated with different concentrations of H2O2 (10 nM~1 mM) were collected for VEGF assay. The level of VEGF was determined with ELISA assay.(3) SRA01/04 cells were treated with 100?M H2O2 and/or 300 U/ml CAT. Expression of VEGF and Src were determined by RT-PCR and Western-Blot assay.(4) SRA01/04 cells were incubated with 300 U/ml CAT, 10?M PP1 (specific inhibiter of SFK) and/or 0.25?g/ml Avastin (specific antibody of VEGF) together with 100?M H2O2. Expression of VEGF, p-Src, ZO-1 and N-cadherin were determined with Western-Blot. Expression of F-actin, ZO-1 and N-cadherin were determined with immunofluorescence stain and laser confocal microscopy. The change of cell cycle was determined with flow cytometry and cell migration with scarification experiment respectively.(5) Cultured murine lens in vitro were treated with 100?M H2O2 and/or 300 U/ml CAT. Lens opacity was observed by stereomicroscope. Expression of VEGF and Src in lens was determined by RT-PCR and Western-Blot assay in mRNA and protein level.Results(1) The VEGF concentration in aqueous humor from 59 ARC patients (range 41y-90y) was 151.28±2.1 (mean±SD)?g/ml. It was positively correlated to age significantly (rs = 0.347; P = 0.008) by spearman rank-order correlation. The mean fluorescence intensity of VEGF were 42.91±4.99,50.84±4.54,60.86±3.55,57.59±6.70 in capsules of 54y, 65y, 75y and 83y patients by immunofluorescence stain respectively. By students'-t test, the fluorescence intensity in 65y, 75y and 83y patients were significantly higher than in 54y patients (t = 2.63, 6.56, 3.93; P = 0.030, 0.000, 0.004).(2) The VEGF level in the supernatant of cells incubated with different concentrations of H2O2 (10 nM~100?M) were significantly higher than control (F = 14.68, 26.49 and 35.69; P = 0.00, 0.00 and 0.00). However, VEGF levels in supernatant of cells incubated with 100?M H2O2 at 6 h, 12 h and 24 h were 201.78±8.78, 323.21±15.81 and 737.32±78.29?g/ml respectively.. They were 1.57, 1.58 and 2.64 times than in control, respectively. However, VEGF levels in cell supernatant incubated with 100?M H2O2 was higher than with 1mM H2O2 (169.82±10.70, 314.61±13.33 and 709.85±62.68, P =0.00, 0.44, 0.47) significantly at 6 h, 12 h and 24 h.(3) The expressions of VEGF and active Src (p-Src) in SRA01/04 incubated with 100?M H2O2 increased with the cultured times in mRNA and protein level. And this was inhibited by incubated with 300 U/ml of CAT together.(4) In SRA01/04 incubated with 100?M H2O2, the expression of VEGF and p-Src was up-regulated and the normal distribution of ZO-1, N-cadherin and F-actin were destroyed. Furthermore, the (G2+S) % in the cell cycle was raise up and the cell migration was decreased. These changes can be inhibited by 300 U/ml CAT totally and by 10?M PP1 and/or 0.25?g/ml Avastin partially.(5) The formation of cataract in cultured murine lens treated with 100?M H2O2 in vitro was accelerated and severer than in controls. The expression of VEGF and p-Src in lens incubated with 100?M H2O2 up-regulated. This effect could be inhibited by 300 U/ml of CAT.ConclusionVEGF levels in aqueous humor and the anterior capsules were closely correlated with age of ARC patients. HLECs could express and secrete VEGF by being incubated with H2O2. In cultured HLECs and murine lens in vitro, the expression of VEGF and p-Src were up-regulated and cataract formation accelerated by being incubated with H2O2. H2O2 destroyed intercellular junctions, affected the cell cycle and cell migration through activate SFK/VEGF pathway. |
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