Objective To observe the expression of C9 in laser-induced choroidal neovascularization (CNV) and the inhibitory effect of cobra venom factor (CVF) on CNV. Methods Thirty-six C57BL/6J mice were randomly divided into control group (n=12), laser photocoagulation group (n=12) and CVF group(n=12). The mice in control group had no interference treatment. CNV of the latter 2 groups were induced by photocoagulation. The mice of CVF group received intraperitoneal injection of CVF (0.1 mu;g/g) 2 days before and once a day after laser treatment. Six days after photocoagulation, the examination of fundus fluorescein angiography was used to confirm that CNV existed in the mice of laser the photocoagulation group. Then at 1, 3, 5, 7 days, the C9 expression was observed using SABCFITC histochemical stain and HE stain. The absorbance was measured by Image-Pro Plus 6.0 image software. Results Only in the laser group had formed CNV. At different time points, C9 expression was observed in the laser photocoagulation group but not in the CVF group. The A value showed a significant difference among 3 groups at different time points (F=146.045, P=0.000). The significant difference of A value exited in each group at different time points (F=9725, P=0.000). Conclusions There is C9 expression in laser-induced choroidal neovascularization. CVF can inhibit CNV formation by inducing complement consumption.
Intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) drugs, including monoclonal antibodies (such as bevacizumab and ranibizumab) and fusion protein agents (such as aflibercept and conbercept) have been proven to be effective in the treatment of wet age-related macular degeneration (wAMD). However, there are still some patients with poor efficacy, such as no response to initial treatment or poor response, and even relapse during the course of treatment. In view of the different targets and molecular characteristics of anti-VEGF drugs, the switch of anti-VEGF drugs and the adjustment of delivery pattern, dosages and intervals have been the strategies to cope with the poor efficacy in clinic. However, there are some differences in the results of current studies. Overall, the recovery of retinal anatomical outcome achieves more benefits, and it is relatively difficult to improve visual acuity. To determine which regimen would get the biggest benefits, a large number of randomized controlled clinical trials and long study period will be needed.
Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. MethodsHuman RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. ResultsCompared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). ConclusionsBMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.
Objective To observe the expression of miR-204 and 211 human embryonic stem cells (hESCs) differentiated into retinal pigment epithelial (RPE) cells. Methods RPE cells were derived from hESCs by natural differentiation method, and were identified. miRNA expression profiles and real-time polymerase chain reaction (RT-PCR) of miR-204 and 211 were generated from the following groups: hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and human fetal RPE (hfRPE) cells. Results miRNA-204 was continuously upregulated throughout the entire differentiation process of hESCs to RPE cells. It increased 5.026 times in hESCs-derived cells containing pigmented foci compared to hfRPE cells; it was increased 3.337 times in hESCs-derived RPE cells compared to hESCs-derived cells containing pigmented foci; it increased 13.574 times in hfRPE cells compared to hESCs-derived RPE cells. miR-211 does not change during differentiation from hESC to RPE, but it increased 44.333 times in hESCderived RPE cells compared to hfRPE cells. miR-211 was the biggest difference in the miRNA expression pattern. In four cell types of hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and hfRPE cells, RT-PCR showed the levels of miR-204 were 91.81plusmn;4.43, 2263.09plusmn;206.39, 5996.80plusmn;235.42, and 171676.45plusmn;999.82 respectively. miR-204 was significantly increased during the whole course (t=18.22, 20.66, 279.38;P＜0.001). The levels of miR-211 were 2.23plusmn;0.31, 129.33plusmn;3.75, 125.7592plusmn;4.78, and 16682.00plusmn;352.97 respectively. miR-211 was significantly increased from hESCs to cells containing pigmented foci and from hESCs-derived RPE cells to hfRPE (t=58.58, 81.24; P＜0.001). Conclusion There is a continuous change of miR-204 and 211 in differentiation of RPE cells from hESCs.
Objective To observe the effects of overexpression of polypyrimidine tract binding protein-associated splicing factor (PSF) on the endoplasmic reticulum (ER) oxidative stress damage of human retinal microvascular endothelial cells (hRMEC) under high concentration of 4-hydroxynonenal (4-HNE). MethodsThe logarithmic growth phase hRMEC cultured in vitro was divided into normal group, simple 4-HNE treatment group (simple 4-HNE group), empty plasmid combined with 4-HNE treatment group (Vec+4-HNE group), and PSF high expression combined with 4-HNE treatment group (PSF+4-HNE group). In 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group cell culture medium, 10 μmol/L 4-HNE was added and stimulated for 12 hours. Subsequently, the Vec+4-HNE group and PSF+4-HNE group were transfected with transfection reagent liposome 2000 into pcDNA empty bodies and pcDNA-PSF eukaryotic expression plasmids, respectively, for 24 hours. Flow cytometry was used to detect the effects of 4-HNE and PSF on cell apoptosis. The effect of PSF overexpression on the expression of reactive oxygen species (ROS) in hRMEC was detected by 2', 7'-dichlorodihydrofluorescein double Acetate probe. Western blot was used to detect ER oxide protein 1 (Ero-1), protein disulfide isomerase (PDI), C/EBP homologous transcription factor (CHOP), glucose regulatory protein (GRP) 78, protein kinase R-like ER kinase (PERK)/phosphorylated PERK (p-PERK), and Eukaryotic initiation factor (eIF) 2α/the relative expression levels of phosphorylated eIF (peIF) and activated transcription factor 4 (ATF4) proteins in hRMEC of normal group, 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group. Single factor analysis of variance was performed for inter group comparison. ResultsThe apoptosis rates of the simple 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group were (22.50±0.58)%, (26.93±0.55)%, and (11.70±0.17)%, respectively. The intracellular ROS expression levels were 0.23±0.03, 1.60±0.06, and 0.50±0.06, respectively. The difference in cell apoptosis rate among the three groups was statistically significant (F=24.531, P＜0.05). The expression level of ROS in the Vec+4-HNE group was significantly higher than that in the simple 4-HNE group and the PSF+4-HNE group, with a statistically significant difference (F=37.274, P＜0.05). The relative expression levels of ER Ero-1 and PDI proteins in the normal group, simple 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group were 1.25±0.03, 0.45±0.03, 0.63±0.03, 1.13±0.09, and 1.00±0.10, 0.27±0.10, 0.31±0.05, and 0.80±0.06, respectively. The relative expression levels of CHOP and GRP78 proteins were 0.55±0.06, 1.13±0.09, 0.90±0.06, 0.48±0.04 and 0.48±0.04, 1.25±0.03, 1.03±0.09, 0.50±0.06, respectively. The relative expression levels of Ero-1 (F=43.164), PDI (F=36.643), CHOP (F=42.855), and GRP78 (F=45.275) proteins in four groups were compared, and the differences were statistically significant (P＜0.05). Four groups of cells ER p-pERK/pERK (F=35.755), peIF2 α/ The relative expression levels of eIF (F=38.643) and ATF4 (F=31.275) proteins were compared, and the differences were statistically significant (P＜0.05). ConclusionPSF can inhibit cell apoptosis and ROS production induced by high concentration of 4-HNE, and its mechanism is closely related to restoring the homeostasis of ER and down-regulating the activation level of PERK/eIF2α/ATF4 pathway.
Objective To investigate the effect of Nodal protein on retinal neovascularization under hypoxia. MethodsIn vivo animal experiment: 48 healthy C57BL/6J mice were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+dimethyl sulfoxide (DMSO) group and OIR+SB431542 group, with 12 mice in each group. Retinal neovascularization was observed in mice at 17 days of age by retina flat mount. Counts exceeded the number of vascular endothelial nuclei in the retinal inner boundary membrane (ILM) by hematoxylin eosin staining. In vivo cell experiment: human retinal microvascular endothelial cells (hRMEC) were divided into normal group, hypoxia group, hypoxia+DMSO group and hypoxia +SB431542 group. The cell proliferation was detected by thiazolyl blue colorimetry (MTT). The effect of SB431542 on hRMEC lumen formation was detected by Matrigel three-dimensional in vitro molding method. Cell migration in hRMEC was detected by cell scratch assay. The Seahorse XFe96 Cell Energy Metabolism analyzer measured extracellular acidification rate (ECAR) of intracellular glycolysis, glycolysis reserve, and glycolysis capacity. One-way analysis of variance was used to compare groups. ResultsIn vivo animal experiment: compared with normal group, the neovascularization increased in OIR group (t=41.621, P＜0.001). Compared with OIR group, the number of vascular endothelial nuclei breaking through ILM in OIR+SB431542 group was significantly reduced, and the difference was statistically significant (F=36.183, P＜0.001). MTT test results showed that compared with normal group and hypoxia+SB431542 group, the cell proliferation of hypoxia group and hypoxia+DMSO group was significantly increased, and the difference was statistically significant (F=39.316, P＜0.01). The cell proliferation of hypoxia+SB431542 group was significantly lower than that of hypoxia+DMSO group, and the difference was statistically significant (t=26.182, P＜0.001). The number of intact lumen formation and migration cells in normal group, hypoxia group, hypoxia+DMSO group and hypoxia+SB431542 group were statistically significant (F=34.513, 41.862; P＜0.001, ＜0.01). Compared with the hypoxia+DMSO group, the number of intact lumen formation and migrating cells in the hypoxia+SB431542 group decreased significantly, and the differences were statistically significant (t=44.723, 31.178; P＜0.001, ＜0.01). The results of cell energy metabolism showed that compared with the hypoxia +DMSO group, the ECAR of intracellular glycolysis and glycolysis reserve in the hypoxia +SB431542 group was decreased, and the ECAR of glycolysis capacity was increased, with statistical significance (t=26.175, 33.623, 37.276; P＜0.05). ConclusionSB431542 can inhibit the proliferation, migration and the ability to form lumens, reduce the level of glycolysis of hRMECs cells induced by hypoxia.
Objective To observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) on hydrogen peroxide (H2O2) induced apoptosis of retinal pigment epithelial (RPE) cells in vitro. Methods RPE cells were cultured and divided into a normal group, normal+H2O2 group, Vec+H2O2group, PSF+H2O2 group according to the experimental design. Overexpression of PSF in RPE cells were achieved by pEGFP-PSF plasmid transient transfection into RPE cells, then RPE cells were exposed to H2O2. The morphological changes were observed by hematoxylin-eosin (HE) staining and Live/Dead staining while the survival rate of cells was detected by MTT assay. The effect of PSF on H2O2-induced RPE apoptosis was analyzed by Cell Death Detection ELISA kit. Meanwhile, intracellular reactive oxygen species (ROS) level was detected by using DCFH-DA method. Results Overexpression of PSF could effectively alleviate the morphological changes induced by H2O2 stimulation shown by HE staining, and effectively reduce dead cells number shown by Live/Dead staining. After H2O2 stimulation, the survival rate, apoptosis rate and ROS production level in PSF overexpression group were 0.68±0.12, 0.44±0.08 and 18 616±3 382.54 respectively, showing significant difference in comparison with the vector plasmid group and normal group (P＜0.05). Conclusion PSF overexpression plays a protective role in H2O2-induced apoptosis by inhibiting the production of ROS in RPE cells.
ObjectiveTo observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) towards advanced glycation end products (AGEs) induced the apoptosis of Müller cells in vitro.MethodsExperimental study. Müller cells were cultured and divided into groups according to the project design, plasmid enhanced green fluorescent protein-PSF were transfected into the cells to achieve the overexpression of PSF Müller cells in vitro, then cells were exposed to AGEs and the Morphological changes were observed by HE staining and Hoechst 33258 staining while the survival rate of cells were detected by MTT assay. The effects of PSF on AGEs-induced Müller apoptosis was measured by Cell Death Detection ELISA kit. Meanwhile, 2′,7′-dichlorofluorescin diacetate staining was performed to monitor the protective effects of PSF on AGEs-induced Müller cells ROS.ResultsThe morphology of cells in normal group was full and the cytoplasm staining was uniform. In N+AGEs group and Vec+AGEs group, cell volume decreased, cytoplasm was dense and concentrated, and eosinophilic staining was enhanced. The cell morphology of PSF+AGEs group was still full, with uniform cytoplasm staining and uniform nucleus staining. The viability of N+AGEs group, Vec+AGEs group and PSF+AGEs group were 0.42±0.11, 0.35±0.12 and 0.68±0.12. The apoptosis values were 1.08±0.16, 0.96±0.20 and 0.44±0.08. The intracellular ROS levels were 28 833.67±3 550.06, 28 356.67±4 854.81, 186 163.00±382.54. Compared with N+AGEs group and Vec+AGEs group, the cell viability of PSF+AGEs group was significantly improved (F=20.65, P=0.000), cell apoptosis value (F=43.43, P=0.000) and intracellular ROS level (F=18.86, P=0.000).ConclusionPSF overexpression play a protective role in AGEs-induced apoptosis by inhibiting the production of ROS in Müller cells.