ABSTRACT
·AIM: To investigate the effect and mechanism of Caspase- 1 on microglia in oxygen - induced retinal neovascularization in mice. ·METHODS: Twelve 7-day-old (P7) C57BL/6J mice were randomly divided into normal group,OIR group and OIR+VX-765 group. OIR models were established in OIR group and OIR+VX-765 group. Caspase-1 inhibitor VX-765(4mg/kg, dissolved in 0.4% polyethylene glycol) or 0. 4% polyethylene glycol, were intraperitoneally injected from P12 to P16 into the mice of OIR+VX-765 and OIR groups,respectively. Whole retinal flatmounts of P17 mice were prepared, and Lectin staining was performed to calculate the ratio of avascular and neovascular area to retina area. The frozen sections of the posterior ocular segment were prepared,and the distribution of Caspase-1 and activated microglial cells were detected by immunofluorescence technique. Cultured BV-2 cells were divided into control group, hypoxia group and inhibitor group. The cells of inhibitor and hypoxia groups were pre-treated with VX-765 or 0.4% polyethylene glycol for 3h, and then hypoxic incubated for 24h. The expression levels of Caspase-1, p20 (active form of Caspase-1), IL-1β and VEGF were detected by Western blotting. The angiogenesis and migration capacity of cultured RF/6A cells were assessed by endothelial cell tube formation assay and migration assay,after they were incubated with supernatant of those different BV-2 groups. ·RESULTS:The distribution and morphology of retinal blood vessels were normal in P17 mice of the normal group,and avascular and new blood vessel cluster were found in the mice of OIR group and OIR+VX-765 group. The ratio of avascular area was 12.23% ±1.02% and that of the new blood vessel area was 2.16% ±0.52% in the OIR+VX- 765 group, which decreased in comparison with 16.58% ± 1.14% and 4.00% ± 0.41% of the OIR group(P<0.01 ). Caspase - 1 was rarely detected by immunofluorescence staining in the normal retina of the mice, whereas it was mainly co-located with activated microglial cells in the ganglion cell layer and the inner plexiform layer in the mice of OIR group. The expression of Caspase-1, p20, IL-1β and VEGF increased in BV-2 cells of the hypoxia group, which were down-regulated by VX-765(P<0.05), except Caspase-1. The tube length was 271±12,and the number of migrated cells was 347±34 in RF/6A cells cultured with supernatant of BV-2 cells in the hypoxia group, which significantly decreased to 171 ± 22 and 212±27 with inhibitor of Caspase-1 (P<0.05). · CONCLUSION: Caspase - 1 promotes retinal neovascularization in the mice with OIR, probably by activating the downstream inflammatory factor IL-1β in microglial cells and accelerating the release of VEGF.
ABSTRACT
Objective To explore the role of integrin αvβ3 in the promotion of the development of choroidal neovascularization (CNV) by SDF-1/CXCR4.Methods This study was divided into two parts in vitro and in vivo.As for the in vivo study,a CNV model was induced by laser on C57BL/6J mice,and then assigned into 4 groups:mice with solely CNV modeling as control group,with intravitreal injection of SDF-1 after immediate CNV modeling as SDF-1 group,with intravitreal injection of SDF-1 + CXCR4 inhibitor (AMD3100) after CNV modeling as SDF-1 + AMD3100 group,and mice with intravitreal injection of SDF-1 + αvβ3 inhibitor (SB273005) after modeling as SDF-1 + SB273005 group.CXCR4 and αvβ3 expression levels in laser-induced eyes were quantified by qRT-PCR at time points of day 1,3,5,7,10 and 14 after modeling,and immunofluorescence staining was applied to detect αvβ3 expression in regional CNV and its endothelial cells in the four groups.Finally,OCT was used to observe the height of retinal pigment epithelial (RPE) layers in CNV after treatment in the four groups.Moreover,in the experiment in vitro,Western blot was used to measure the expression of CXCR4 protein of RF/6A cells in normal control group,Si-CXCR4 knockdown group and Si-NC knockdown model group.Meanwhile,the expression of integrin subunit β3 protein was determined in the normal control group,SDF-1 group,SDF-1 + AMD3100group,SDF-1 + Si-NC group and SDF-1 + Si-CXCR4 group.Transwell assay was conducted to detect the migration ability of RF/6A cells in the normal control group,SDF-1group,SDF-1 +AMD3100 group,SDF-1 + SB273005 group.Results On the one hand,the study in vivo,qRT-PCR showed that the expression of CXCR4 and integrin subunit β3 mRNA was up-regulated at first,and then down-regulated with time passed after CNV induction,with the highest expression level of CXCR4 mRNA (4.263 ± 0.464) on day 3,and the peak expression of β3 mRNA (3.678 ±0.364) on day 7 after CNV modeling.The results of immunofluorescence staining showed that the β3 fluorescence intensity of SDF-1 group was significantly enhanced,and the ratio of β3/CD31 was also significantly increased,which both were significantly higher than those of the control group (P ＜ 0.01).However,the β3 fluorescence intensity and β3/CD31 ratio of SDF-1 +AMD3100 group and SDF-1 + SB273005 group were significantly weakened and decreased,respectively (P ＜0.05).OCT showed that the elevation level of RPE layer inSDF-1 group was significantly higher than that in the control group [(135.503 ± 10.301) μm vs.(94.443 ± 12.156) μm](P＜0.05).The height of RPE uplift in SDF-1 + AMD3100 group [(95.283 ±20.062) μm] and SDF-1 + SB273005 group [(99.807 ± 10.403) μm] was significantly decreased (P ＜ 0.05).On the other hand,in experiment in vitro,Western blot showed that the expression levels of integrin β3 in SDF-1 group and SDF-1 + Si-NC group were significantly higher than those in the control group [(1.301 ± 0.043) and (1.273 ± 0.077) vs.(0.244 ± 0.069)] (P ＜ 0.01).The levels of integrin subunit β3 protein in SDF-1 + si-CXCR4 group (0.322 ± 0.042) and SDF-1 + AMD3100 group (0.336 ± 0.077) were significantly down-regulated (P ＜ 0.01).Transwell assay showed that the amount of migrating cells in SDF-1 group increased,which was significantly higher than that of the control group (P ＜ 0.01),while the number of migrating cells in SDF-1 +AMD3100 group and SDF-1 + SB273005 group was significantly decreased.Conclusion Integrin αvβ3 can promote the development of CNV by mediating SDF-1/CXCR4 signaling in endothelial cells.