Authors: Qi Yun, Bai Yujing, Li Xiaoxin, Shi Xuan
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Choroidal neovascularization (CNV) is the primary pathogenic cause of many fundus diseases.Oxidative stress injury of retinal pigment epithelial (RPE) cells plays important role in angiogenesis of choroid new blood vessels.Oxidative stress injury can active p75NTR receptor, a member of tumor necrosis factors family, resulting in the proliferation of vascular endothelial cells.However, the mechanisms of vascular endothelial cell proliferation remain unclear.
This study was conducted to investigate the effect of p75NTR overexpression on CNV and the relative mechanism.
The ARPE-19 cell line was used in this study.RPE cells were transfected with p75 NTR receptor overexpressed plasmid, and untransfected cells served as the control group.The transfected results were verified by reverse transcription-PCR and Western blot assay.Viability of the cells over time was determined in the p75 NTRreceptor plasmid transfected group by using BrdU assay.The percentage of apoptotic cells was detected by flow cytometry using Annexin V-FITC/PI fluorescence staining.The percentage of reactive oxygen species (ROS) expression in the cells was detected by using H2DCFDA fluorescence and flow cytometry.Mitochondrial membrane potential and cytochrome C expression were examined under the confocal microscope.The protein expressions of cleaved caspase-3, Fas and VEGF were determined by Western blot assay.
The relative expression level of p75NTR receptor mRNA was (6.11±0.77) times higher than that of the control group, and relative expression level of p75NTR receptor protein in the cells in the p75 NTR receptor plasmid transfected group was (7.42±0.48) times higher than that in the control group (t=11.49 and 23.17, both at P<0.01). The absorbency values of the p75NTR receptor plasmid transfected group were (93.12±0.56)%, (86.30±0.66)%, (72.53±0.86)% and (60.77±2.81)% in 12, 24, 36 and 48 hours after plasmid transfection, which were significantly lower than 100% in the control group, and the apoptotic percentages were evidently higher than that in the control group (all at P<0.05). The relative fluorescence intensity of ROS fluorescence in the p75NTRreceptor plasmid transfected group was 2.4 times higher than that in the control group, showing significant difference (t=16.45, P<0.01). The positive expressing rate of mitomarker (mitochondrial membrane potentials) was 100% in the control group and (37.30±2.06)% in the p75NTR receptor plasmid transfected group, with significant difference between them (t=57.71, P<0.01). The fluorescence intensity of cytochrome C expression was elevated in the p75NTR receptor plasmid transfected group compared with the control group.Compared with the control group, the expressing levels of cleaved caspase-3, Fas and VEGF165 proteins in the cells were significantly raised in the p75NTR receptor plasmid transfected group (all at P<0.01).
Overexpression of p75 NTR receptors in RPE cells leads to mitochondrial damage and cellular apoptosis and the secretion of VEGF protein, which sequentially promote CNV.P75 NTR receptor may be another important regulation pathway in RPE oxygen damage.