Authors: Sun Jiaxing, Dou Guorui, Chang Tianfang, Li Manhong, Yang Ziyan, Yan Xianchun, Liu Yuan, Han Hua, Wang Yusheng
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To investigate whether vascular endothelial cells in choroidal neovascularization whether hypoxia condition can up-regulate SNAI1 and activate matrix metalloproteinase (MMP)2 and MMP9 therefore to participate in choroidal neovascularization(CNV).
Sixteen SPF male C57 mice aged 6-8 weeks were divided into control group and model group.CNV models were induced by retinal laser photocoagulation, and flat-mount and frozen sections of retinal pigment epithelium (RPE)-choroid-sclera compound were prepared at 7 days after modeling.The CNV in flat-mount was examined by Isolectin B4 staining, and the location of SNAI1, MMP2 and MMP9 in frozen sections was determined by immunofluorescence technology.The expression of SNAI1, MMP2 and MMP9 at mRNA level in CNV was detected by real-time fluorescence quantitative PCR (real-time PCR). The use and care of experimental animals complied with Statement for the Use of Animals in Ophthalmic and Visual Research.The RF/6A cells were divided into normoxia group and hypoxia group and cultured for 24 hours in 5%CO2 condition and mix condition of 94%N2, 5%CO2 and 1%O2, respectively.The expression of SNAI1, MMP2 and MMP9 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay, respectively.Small interfering RNA of SNAI1 (siSNAI1) was transfected into the cells, and then the expression of MMP2 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay, respectively, and the migrating number of the cells was assayed by Transwell chamber assay.
CD31 and SNAI1 positive-response cells were seen in RPE-choroid-sclera flat-mounts under the laser scanning confocal microscope.The relative expression levels of SNAI1 mRNA and MMP2 mRNA in RPE-choroid-sclera tissues were higher in the model group than those in the control group (SNAI1 mRNA: 1.291±0.060 vs.0.759±0.074, P=0.001; MMP2 mRNA: 1.610±0.424 vs.0.772±0.080, P=0.044). The expression of MMP9 mRNA was not significantly elevated between model group and control group (P>0.05). The relative expression level of MMP2 mRNA was higher in comparison with MMP9 mRNA in the model group (P<0.01). The relative expressions of hypoxic induced factor 1α (HIF-1α), SNAI1 and MMP2 at mRNA level and protein level in RF/6A cells were significantly higher in the hypoxia group than those in the normoxia group (all at P<0.05) and no considerable difference was seen in MMP9 mRNA expression between the two groups (P>0.05). The relative expressions of MMP2 mRNA in the cells were 0.217±0.036 and 0.818±0.105, and those of MMP2 protein in the cells were 0.236±0.009 and 1.043±0.120 in the hypoxia+ siSNAI1 group and only hypoxia group, respectively, with significant differences between them (P=0.002, 0.003). The migrating number of the cells was (254.60±71.31)/field in the hypoxia+ siSNAI1 group, which was significantly less than (534.10±96.21) /field in the control group (P=0.029).
The hypoxic environment at CNV can activate MMP2 by up-regulating the expression of SNAI1, which promotes the migration of vascular endothelial cells and therefore participates in CNV formation, and the intervention of SNAI1 activation under the hypoxic condition can inhibit this process.