Heparanase-mediated histone 3 acetylation regulates VEGF gene transcription in the hyperglycemia and hypoxia human retinal endothelial cells.

Heparanase (HPA) is believed that might mediate histone 3 lysine 9 acetylation (H3K9ac) to regulate vascular endothelial growth factor (VEGF) gene expressions in the hyperglycemia and hypoxia human retinal endothelial cells (HRECs). Cultured human retinal endothelial cells (HRECs) in hyperglycemia, hypoxia, siRNA, and normal medium, respectively. Distributions of H3K9ac and HPA in HRECs were analyzed by immunofluorescence. Western blot and real-time PCR were respectively used to evaluate the expression of HPA, H3K9ac, and VEGF. The differences in occupancies of H3K9ac and RNA polymerase II at VEGF gene promoter among three groups were studied by Chromatin immunoprecipitation (ChIP) combined with real-time PCR. Co-immunoprecipitation (Co-IP) was used to measure the status of HPA and H3K9ac. Re-ChIP was used to verify whether HPA and H3K9ac associate to the transcription of VEGF gene. HPA was consistent with that of H3K9ac in the hyperglycemia and hypoxia groups. And the fluorescent lights of H3K9ac and HPA in siRNA groups were similar to the control group, fainter than that of hyperglycemia, hypoxia, and non-silencing groups. Western blot results showed that the expressions of HPA, H3K9ac, and VEGF in hyperglycemia and hypoxia HRECs were statistically higher than that of the control. HPA, H3K9ac, and VEGF expressions in siRNA groups were statistically lower than hyperglycemia and hypoxia HRECs. The same trends also were found in real-time PCR. ChIP exhibited the occupancies of H3K9ac and RNA Pol II at VEGF gene promoter in hyperglycemia and hypoxia groups were significantly more increased than in the control group. Co-IP revealed that HPA combined with H3K9ac in hyperglycemia and hypoxia groups; while it was not discovered in the control group. Re-ChIP showed that HPA combined with H3K9ac at VEGF gene promoter in the hyperglycemia and hypoxia HRECs nuclear. In our study HPA can influence expressions of H3K9ac and VEGF in the hyperglycemia and hypoxia HRECs. HPA can probably combine with H3K9ac and regulate the transcription of the VEGF gene in the hyperglycemia and hypoxia HRECs.

Metformin inhibiting the activation of NLRP3 inflammasome and pyroptosis in diabetic retinal vascular endothelial cells / 中华眼底病杂志

Objective:To observe the effect of metformin (Met) on inflammatory bodies and focal death in human retinal microvascular endothelial cells (hRMEC) in diabetes mellitus (DM) microenvironment.Methods:Experimental research was divided into in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 9 healthy C57BL/6J male mice were randomly divided into DM group, normal control group, and DM+Met group, with 3 mice in each group. DM group and DM+Met group mice were induced by streptozotocin to establish DM model, and DM+Met group was given Met 400 mg/ (kg · d) intervention. Eight weeks after modeling, the expression of NLRP3, cleaved-membrane perforating protein D (GSDMD) and cleaved-Caspase-1 in the retina of mice in the normal control group, DM group and DM+Met group were observed by immunohistochemical staining. In vitro cell experiments: hRMEC was divided into conventional culture cell group (N group), advanced glycation end products (AGE) group, and AGE+Met group. Joining the AGE, AGE+Met groups cells were induced by 150 μg/ml of glycation end products, and 2.0 mmol/L Met was added to the AGE+Met group. Pyroptosis was detected by flow cytometry; 2' ,7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was used to detect the expression of reactive oxygen species (ROS) in cells of each group. Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the relative mRNA and protein expression levels of NLRP3, cleaved-GSDMD, cleaved-Caspase-1 in each group of cells. Single factor analysis of variance was used for comparison among the three groups.Results:In vivo animal experiments: compared with the DM group, the expression of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in the retina of normal control group and DM+Met group mice was significantly reduced, with significant difference among the 3 groups ( F=43.478, 36.643, 24.464; P<0.01). In vitro cell experiment and flow cytometry showed that the pyroptosis rate of AGE group was significantly higher than that of N group and AGE+Met group ( F=32.598, P<0.01). The DCFH-DA detection results showed that the intracellular ROS levels in the N group and AGE+Met group were significantly lower than those in the AGE group, with the significant difference ( F=47.267, P<0.01). The mRNA ( F=51.563, 32.192, 44.473; P<0.01) and protein levels ( F=63.372, 54.463, 48.412; P<0.01) of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in hRMEC of the AGE+Met group were significantly reduced compared to the N group. Conclusion:Met can down regulate the expression of NLRP3 inflammatory body related factors in hRMEC and inhibit pyroptosis.

Up-regulation of p21 activated kinase 4 expression in the retina of diabetes mice and its effects on the behavior and mitochondrial function in retinal vascular endothelial cells / 中华眼底病杂志

Objective:To observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells.Methods:The experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. Results:In vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance ( t=25.372, 22.419, 25.372; P<0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance ( F=36.821, 38.692, 29.421; P<0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant ( F=39.649, P<0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance ( F=27.472, 22.315, 31.147, 27.472; P<0.05). Conclusion:Over-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.

Effect of high expression of polypyrimidine tract-binding protein-associated splicing factor on retinal microvascular endothelial cells / 中华眼底病杂志

Objective:To observe the effect of high expression of polypyrimidine tract-binding protein-associated splicing factor (PSF) on low concentration of 4-hydroxynonenal (4-HNE) induced human retinal microvascular endothelial cells (HRMECs), and explore the possible mechanism.Methods:The HRMECs cultured in vitro were divided into 4-HNE treated group, PSF overexpression group combined with 4-HNE group (PSF+4-HNE group), PSF overexpression+ML385 treatment combined with 4-HNE group (PSF+ML385+4-HNE group), and 4-HNE induced PSF overexpression group with LY294002 pretreatment (LY294002+4-HNE+PSF group). Cell culture medium containing 10 μmmol/L 4-HNE was added into 4-HNE treatment group, PSF+4-HNE group, PSF+ML385+4-HNE group for 12 hours to stimulate oxidative stress. 1.0 μg of pcDNA-PSF eukaryotic expression plasmid were transfected into PSF+4-HNE group and PSF+ML385+4-HNE group to achieve the overexpression of PSF. Also cells were pretreated with ML385 (5 μmol/L) for 48 hours in the PSF+ML385+4-HNE group, meanwhile within the LY294002+4-HNE+PSF group, after pretreatment with LY294002, cells were treated with plasmid transfection and 4-HNE induction. Transwell detects the migration ability of PSF to HRMECs. The effect of PSF on the lumen formation of HRMECs was detected by using Matrigel in vitro three-dimensional molding method. Flow cytometer was used to detect the effect of PSF overexpression on reactive oxygen (ROS) level in HRMECs. Protein immunoblotting was used to detect the relative expression of PSF, nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein, and phosphoserine threonine protein kinase (pAkt) protein. The comparison between the two groups was performed using a t-test. Results:The number of live cells, migrating cells, and intact lumen formation in the 4-HNE treatment group and the PSF+4-HNE group were 1.70±0.06, 0.80±0.13, 24.00±0.58, 10.00±0.67, and 725.00±5.77, 318.7±12.13, respectively. There were significant differences in the number of live cells, migrating cells, and intact lumen formation between the two groups ( t=12.311, 15.643, 17.346; P<0.001). The results of flow cytometry showed that the ROS levels in the 4-HNE treatment group, PSF+4-HNE group, and PSF+ML385+4-HNE group were 816.70±16.67, 416.70±15.44, and 783.30±17.41, respectively. There were statistically significant differences between the two groups ( t=16.311, 14.833, 18.442; P<0.001). Western blot analysis showed that the relative expression levels of pAkt, Nrf2, and HO-1 proteins in HRMECs in the 4-HNE treatment group, PSF+4-HNE group and LY294002+4-HNE+PSF group were 0.08±0.01, 0.57±0.04, 0.35±0.09, 0.17±0.03, 1.10±0.06, 0.08±0.11 and 0.80±0.14, 2.50±0.07, 0.50±0.05, respectively. Compared with the PSF+4-HNE group, the relative expression of pAkt, Nrf2, and HO-1 proteins in the LY294002+4-HNE+PSF group decreased significantly, with significant differences ( t=17.342, 16.813, 18.794; P<0.001). Conclusion:PSF upregulates the expression of HO-1 by activating the phosphatidylinositol 3 kinase/Akt pathway and inhibits cell proliferation, migration, and lumen formation induced by low concentrations of 4-HNE.

Inhibitory effect of GALNT2 gene knockdown on apoptosis of human retinal vascular endothelial cells in high glucose culture and its mechanism / 中华实验眼科杂志

Objective:To investigate the effect of polypeptide N-acetylgalactosaminaminyltransferase 2 (GALNT2) on the proliferation and apoptosis of human retinal vascular endothelial cells (HRCECs) cultured in high glucose and its possible mechanism.Methods:The small hairpin RNA (shRNA) targeting GALNT2 gene was constructed to interfere with the lentiviral vector and infect HRCECs.HRCECs were divided into blank control group, model group, NC-shGALNT2 group and shGALNT2 group, which were cultured in medium containing 5.5 mmol/L glucose, 25 mmol/L glucose, shGALNT2 negative control virus 25 mmol/L glucose and shGALNT2 knockdown virus 25 mmol/L glucose for 24 hours, respectively.The relative expression of GALNT2 mRNA in the four groups was detected by real-time fluorescence quantitative PCR.The relative expression levels of GALNT2, epidermal growth factor (EGF), EGF receptor (EGFR) and phosphorylated EGFR (p-EGFR) were detected by Western blot.The proliferative values of HRCECs were detected by cell counting kit-8 method.The apoptosis rate of different groups was detected by flow cytometry. Results:The relative expression levels of GALNT2 mRNA and protein were significantly higher in model group than in blank control group, and were significantly lower in shGALNT2 group than in blank control group (all at P<0.05). The cell proliferation value was significantly lower in model group than in blank control group, and was significantly higher in shGALNT2 than in model group and NC-shGALNT2 group (all at P<0.05). The apoptosis rates of blank control group, model group, NC-shGALNT2 group and shGALNT2 group were (4.73±0.26)%, (8.66±0.25)%, (9.26±1.12)% and (5.47±0.18)%, respectively, with a significant overall difference ( F=342.921, P<0.001). The apoptosis rate was significantly higher in model group than in blank control group, and was significantly lower in shGALNT2 group than in model group and NC-shGALNT2 group (all at P<0.05). The relative expression level of EGFR protein was significantly higher and the relative expression level of p-EGFR protein was significantly lower in model group than in blank control group (all at P<0.05). The relative expression of p-EGFR protein was significantly higher in shGALNT2 group than in model group (all at P<0.05). Conclusions:Knocking down GALNT2 can improve the proliferative ability of HRCECs under high glucose culture and reduce apoptosis, which may be related to the activation of EGFR signaling pathway.

Ultrasound-targeted cationic microbubble-mediated gene transfection and inhibition of retinal neovascularization.

AIM: To investigate whether ultrasound-targeted cationic microbubbles (CMBs) destruction could deliver endostatin-green fluorescent protein (GFP) plasmids efficiently to the human retinal endothelial cells (HRECs) and inhibit retinal neovascularization in mice. METHODS: CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy. Experiments assessing HRECs migration and vascular formation were performed to evaluate gene therapy's efficiency in vitro. A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl, Bcl-2, vascular endothelial growth factor (VEGF) and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction (qPCR). The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5, 14, and 28d after treatment. RESULTS: The gene expression of endostatin was the highest in the group of the CMBs. Besides, the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro. In vivo, retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups. CONCLUSION: The research illustrates ultrasound-targeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.

Down-regulation of LCN2 attenuates retinal vascular dysfunction and caspase-1-mediated pyroptosis in diabetes mellitus.

Background: Diabetic retinopathy (DR) is a diabetic microangiopathy with increasing incidence, which seriously threatens the quality of life of patients. This study investigated the molecular regulation mechanism of lipocalin-2 (LCN2) in DR by targeting the function of human retinal vascular endothelial cells (HRVECs). Methods: The expression of LCN2 in the retinal tissue of diabetic and high glucose (HG)-induced HRVECs was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis and western blotting assay. After intravitreal injection of adeno-associated virus (AAV)-NC or AAV-sh-LCN2, in vivo experiments, hematoxylin and eosin (H&E) staining, and retinal trypsin digestion experiments were performed to analyze the effect of LCN2 silencing on DR retinal tissue. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining was used to evaluate apoptosis and immunohistochemical (IHC) staining was performed to detect the expressions of caspase-1. Western blot was used to detect the expressions of pyroptosis-associated proteins. After transfection of sh-NC and sh-LCN2, the function of HRVECs cells induced by HG was evaluated by wound healing assay, Transwell assay, and tube formation assay. Results: The expression of LCN2 was significantly up-regulated in diabetic retinal tissue and HG-induced HRVECs. In vivo experiments showed that LCN2 silencing can significantly reduce diabetic retinal injury. Cell function experiments also revealed that LCN2 silencing inhibited cell migration, invasion, and angiogenesis. Flow cytometry and immunofluorescence staining showed that downregulation of LCN2 could inhibit caspase-1 mediated pyroptosis in HG-induced HRVECs. Conclusions: Down-regulation of LCN2 can significantly inhibit cell migration, invasion, and angiopoiesis, and pyroptosis regulated by caspase-1, thus attenuating the progression of DR.

Three-dimensional spheroids of choroid-retinal vascular endothelial cells as an in-vitro model for diabetic retinopathy: Proof-of-concept investigation.

Diabetic retinopathy (DR) is a primary microvascular complication of diabetes mellitus and a vision-threatening condition. Vascular endothelial growth factor (VEGF) induces neovascularization and causes metabolic damage to the retinal and choroidal vasculature in diabetic patients. Existing drug screening models and treatment strategies for DR need to be refined through the establishment of relevant pre-clinical models, which may enable development of effective and safe therapies. The present study discusses the development of an in-vitro three-dimensional (3D) spheroid model, using RF/6A choroid-retinal vascular endothelial cells, to closely mimic the in-vivo disease condition. Compact, reproducibly-sized, viable and proliferating RF/6A spheroids were fabricated, as confirmed by microscopy, live/dead assay, cell proliferation assay and histological staining. In-vitro angiogenesis was studied by evaluating individual effects of VEGF and an anti-VEGF monoclonal antibody, Bevacizumab, and their combination on cellular proliferation and 3D endothelial sprout formation. VEGF stimulated angiogenic sprouting while Bevacizumab demonstrated a dose-dependent anti-angiogenic effect, as determined from the cellular proliferation observed and extent and length of sprouting. These investigations validated the potential of RF/6A spheroids in providing an alternative-to-animal, pathophysiologically-relevant model to facilitate pre-clinical and biomedical research related to DR.

Hydroxychloroquine Alleviates EAU by Inhibiting Uveitogenic T Cells and Ameliorating Retinal Vascular Endothelial Cells Dysfunction.

Purpose: Inflammation triggers the activation of CD4+T cells and the breakdown of blood-retinal barrier, thus contributing to the pathology of experimental autoimmune uveitis (EAU). We explored the anti-inflammatory effect of hydroxychloroquine (HCQ) on EAU and the potential mechanisms active in T cells and retinal vascular endothelial cells (RVECs). Methods: C57BL/6J mice were immunized with interphotoreceptor retinoid binding protein 1-20 (IRBP1-20) to induce EAU and then treated with the vehicle or HCQ (100 mg/kg/day). On day 7, 14, 21, 30 and 60 after immunization, clinical scores were evaluated. On day 14, histopathological scores were assessed, and retinas, spleens, and lymph nodes were collected for quantitative polymerase chain reaction or flow cytometry analysis. RVEC dysfunction was induced by tumor necrosis factor α (TNF-α) stimulation. The expression of cytokines, chemokines, adhesion molecules, and lectin-like oxidized LDL receptor-1 (LOX-1)/nuclear factor κB (NF-κB) was measured in RVECs with or without HCQ. Results: HCQ treatment protected mice from uveitis, evidenced by reduced expression of inflammatory factors, chemokines, and adhesion molecules in the retina. In systemic immune response, HCQ inhibited the activation of naïve CD4+T cells and frequencies of T effector cells, and promoted T regulatory cells. HCQ decreased IRBP1-20-specific T cell responses and proliferation of CD4+T cells in vitro. Further studies established that TNF-α induced RVECs to express inflammatory cytokines, chemokines, and adhesion molecules, whereas HCQ alleviated the alterations via the LOX-1/NF-κB pathways. Conclusions: HCQ alleviates EAU by regulating the Teff/Treg balance and ameliorating RVECs dysfunction via the LOX-1/NF-κB axis. HCQ may be a promising therapeutic candidate for uveitis.

Key Role of 12-Lipoxygenase and Its Metabolite 12-Hydroxyeicosatetraenoic Acid (12-HETE) in Diabetic Retinopathy.

PURPOSE: Abnormal lipid metabolism has been proved to be implicated in the complex pathogenesis of diabetic retinopathy (DR). 12-lipoxygenase (12-LOX) is a member of lipoxygenase family responsible for the oxygenation of cellular polyunsaturated fatty acids to produce lipid mediators which modulate cell inflammation. This review explores the role of 12-lipoxygenase and its products in the pathogenesis of DR. METHODS: A comprehensive medical literature search was conducted on PubMed till September 2021. RESULTS: Emerging evidence has demonstrated that 12-LOX and its main product 12- hydroxyeicosatetraenoic acid (12-HETE) activate retinal cells, especially retinal vascular endothelial cells, through the activation of NADPH oxidase and the subsequent generation of reactive oxygen species (ROS), mediating multiple pathological changes during DR. Genetic deletion or pharmacological inhibition models of 12-LOX in mice show protection from DR. CONCLUSION: 12-LOX and its product 12-HETE take important part in DR pathogenesis and show their potential as future therapeutic targets for DR. Further studies are needed on the specific mechanism including 12-LOX pathway related molecules, 12-HETE receptors and downstream signaling pathways.

VEGF gene polymorphisms regulate human retinal vascular endothelial cell proliferation and apoptosis through ASF/SF2-associated alternative splicing.

This study investigated the effects of single nucleotide polymorphisms (SNPs) of the VEGF （vascular endothelial growth factor） gene, which are associated with susceptibility to age-related macular degeneration (AMD), on the expression of VEGF proteins (VEGF165 and VEGF165b) and their role in cell proliferation and apoptosis in human retinal vascular endothelial cells (hRVECs). Cell viability and VEGF165 and VEGF165b expressions were evaluated in hRVECs transfected with VEGF genes containing different SNPs (rs3025039, rs3025033, and rs10434). The Cell Counting Kit 8 assay, quantitative real-time PCR, western blotting, TUNEL assay, and enzyme-linked immunosorbent assay were used to examine the effects of VEGF gene SNPs on cell viability, VEGF165 and VEGF165b expressions, and cell apoptosis in hRVECs. The interaction and localization of the RNA-binding protein alternative splicing factor/splicing factor 2 (ASF/SF2) were assessed using RNA pull-down. Although VEGF165 expression decreased, VEGF165b levels increased significantly in hRVECs transfected with rs3025039, which decreased cell viability and induced apoptosis. The SNPs rs3025033 and rs10434 had no significant effects on VEGF165b protein production and apoptosis; however, they promoted cell proliferation. SNPs affected the interaction between RNA and ASF/SF2, a splicing factor for intron retention. Insulin-like growth factor-1 treatment induced the expression of VEGF165, but not VEGF165b, whereas SRPIN340 treatment, an inhibitor of ASF/SF2, increased VEGF165b protein levels. VEGF gene sequence variations affected hRVEC proliferation and apoptosis via alternative gene splicing. Thus, the regulation of splicing via ASF/SF2 could be a potential strategy in treating pathological neovascularization in patients with AMD.

Effect of Dapagliflozin on high glucose-induced apoptosis and oxidative stress in human retinal vascular endothelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To explore the effect of dapagliflozin on the apoptosis and oxidative stress of high glucose-induced human retinal vascular endothelial cells and its regulatory effect on forkhead FOXO4. <p>METHODS: High glucose-induced human retinal vascular endothelial cells(HRVECs)were used to establish a cell injury model(high glucose group). Experimental groups include high glucose+dapagliflozin low-dose group(1ng/L dapagliflozin), high glucose+dapagliflozin medium-dose group(5ng/L dapagliflozin), high glucose+dapagliflozin high-dose group(10ng/L dapagliflozin), high glucose+dapagliflozin high-dose+pcDNA group, high glucose+dapagliflozin high-dose+pcDNA-FOXO4 group, and normal sugar group(5.5mmol/L D-glucose). Flow cytometry was used to detect the apoptosis rate. The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were tested with corresponding kits. Western blot assay was used to detect the protein level of FOXO4. <p>RESULTS: Compared with the normal sugar group, the apoptosis rate(<i>P</i><0.05), the level of MDA(<i>P</i><0.05)and FOXO4(<i>P</i><0.05)were increased, but the level of SOD was decreased(<i>P</i><0.05)in high-glucose group. Compared with the high glucose group, cell apoptosis rate(<i>P</i><0.05), the level of MDA(<i>P</i><0.05)and the protein level of FOXO4 were decreased(<i>P</i><0.05), but the level of SOD was increased(<i>P</i><0.05)in high glucose+medium-dose dapagliflozin group and high glucose+high-dose dapagliflozin group. Compared with high glucose+dapagliflozin high-dose+pcDNA group, the apoptosis rate(<i>P</i><0.05)and the level of MDA(<i>P</i><0.05)were increased, but the level of SOD was decreased(<i>P</i><0.05)in high glucose+dapagliflozin high-dose+pcDNA-FOXO4 group(<i>P</i><0.05). <p>CONCLUSION: Dapagliflozin could inhibit oxidative stress and cell apoptosis in high glucose-induced HRVECs by down-regulating FOXO4, thereby reducing cell damage.

Mesenchymal stem cell-derived exosomes inhibit the VEGF-A expression in human retinal vascular endothelial cells induced by high glucose.

AIM: To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor A (VEGF-A) in human retinal vascular endothelial cells (HRECs). METHODS: Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy, Western blotting and nanoparticle tracking analysis. HRECs were randomly divided into a normal control group (group A), a high glucose model group (group B), a high glucose group with 25 µg/mL (group C), 50 µg/mL (group D), and 100 µg/mL exosomes (group E). Twenty-four hours after coculture, the cell proliferation rate was detected using flow cytometry, and the VEGF-A level was detected using immunofluorescence. After coculture 8, 16, and 24h, the expression levels of VEGF-A in each group were detected using PCR and Western blots. RESULTS: The characteristic morphology (membrane structured vesicles) and size (diameter between 50 and 200 nm) were observed under transmission electron microscopy. The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis (NTA). The exosomal markers CD9, CD63, and HSP70 were strongly detected. The proliferation rate of the cells in group B increased after 24h of coculture. Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes (F=39.03, P<0.01). The upregulation of VEGF-A protein (group C: F=7.96; group D: F=17.29; group E: F=11.89; 8h: F=9.45; 16h: F=12.86; 24h: F=42.28, P<0.05) and mRNA (group C: F=4.137; group D: F=13.64; group E: F=22.19; 8h: F=7.253; 16h: F=16.98; 24h: F=22.62, P<0.05) in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes (P<0.05). CONCLUSION: hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.

Cytochrome P450 2J2 inhibits the proliferation and angiogenesis of retinal vascular endothelial cells by regulating the Notch signaling pathway in a hypoxia-induced retinopathy model.

Retinopathy of Prematurity (ROP), a type of retinal neovascularization in premature infants, has become a serious problem that drastically affects the quality of life of premature infants. ROP is associated with angiogenesis and neovascularization. Here, we aimed to explain the function and latent roles of Cytochrome P450 2J2 (CYP2J2) in hypoxia-induced retinopathy in retinal vascular endothelial cells (HRVECs). HRVECs were stimulated with hypoxia for 24 h to establish an in vitro retinopathy model. Cell viability and migration were evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Transwell assays, respectively. Protein and gene expression was determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analysis. We observed that pcDNA3.1(+)-CYP2J2 promoted CYP2J2 and Jagged1 expression, while Dll4 was down-regulated in hypoxia-stimulated HRVECs. Additionally, pcDNA3.1(+)-CYP2J2 inhibited HRVEC viability, reduced PCNA expression, and inhibited the migration of HRVECs. Further, the Notch pathway was inhibited in the Hypoxia+pcDNA3.1(+)-CYP2J2 group. Opposite results were observed upon Terfenadone treatment in hypoxia induced HRVECs. Finally, our findings further verified that DAPT promotes the effects of CYP2J2 on cell viability, migration, and Notch signaling in hypoxia-induced HRVECs, while EDTA reversed the inhibitory effects of CYP2J2 on hypoxia-induced HRVECs. In conclusions, CYP2J2 was found to inhibit the viability and angiogenesis of HRVECs by inhibiting Notch signaling in a hypoxia-induced retinopathy model.

Scutellaria barbata D. Don Polysaccharides Inhibit High Glucose-Induced Proliferation and Angiogenesis of Retinal Vascular Endothelial Cells.

PURPOSE: The traditional Chinese medicine Scutellaria barbata D. Don (S. barbata) has been reported to exhibit anti-cancer and anti-inflammation activities. The ethanol extract of S. barbata has been confirmed to attenuate diabetic retinopathy (DR). This study aimed to investigate the effects and underlying mechanisms of the polysaccharides isolated from S. barbata (PSB) on the proliferation and angiogenesis of retinal vascular endothelial cells (RVECs) in DR. METHODS: Human RVECs (HRVECs) were cultured in normal glucose (NG, 5.5 mM), mannitol (MA, 30 mM), high glucose (HG, 30 mM) and HG plus 40 µg/mL PSB, respectively. Then, cell proliferation, migration and angiogenesis were evaluated. The cell proliferation was also estimated in the presence of SLIGKV, which was used to induce the phosphorylation of ERK (p-ERK). RESULTS: PSB reduced normal and HG-induced HRVECs cell viability in a concentration-dependent manner. The protein expression of proliferating cell nuclear antigen (PCNA) and proliferating antigen KI67 (Ki67), the migration rate and tube formation ability, which were increased by HG treatment, were significantly decreased by PSB. PSB also inhibited the phosphorylation of Raf, MEK and ERK in HG-stimulated HRVECs. Moreover, the application of SLIGKV recovered cell viability and the expression of p-ERK, PCNA and Ki67, in HG plus PSB-treated cells. Finally, the HG-enhanced expression of VE-cadherin, Frizzed, ß-catenin, MMP-2 and MMP-9 was all reversed by PSB. CONCLUSION: PSB could inhibit HG-induced HRVECs proliferation, migration and neovascularization, and these effects might work through blocking the activation of MEK/ERK pathway and VEGF/VE-cadherin axis.

STEAP4 Inhibits HIF-1α/PKM2 Signaling and Reduces High Glucose-Induced Apoptosis of Retinal Vascular Endothelial Cells.

BACKGROUND: Diabetic retinopathy (DR) is a vascular lesion induced by high glucose. STEAP4 is an indispensable membrane protein, which is closely related to hyperglycemic-induced cell inflammation and injury, while STEPT4 has not been studied in hyperglycemic-induced retinal vascular endothelial cell injury. METHODS: The expression of STEAP4 was detected by RT-qPCR and Western blot. CCK-8 was used to detect cell survival. STEAP4 was overexpressed by cell transfection. The expressions of cytokines TNF-α, IL-1, IL-6, ICAM-1, MDA, SOD and ROS were detected by ELISA. Cell apoptosis was detected by flow cytometry. The expressions of proteins associated with cell damage VEGF, KLF2, eNOS and apoptosis-related proteins Bax, cleaved caspase3 and Bcl2 were detected by Western blot. Finally, the expressions of HIFα and PKM2 were detected by immunofluorescence and Western blot. RESULTS: The expression of STEAP4 in hyperglycemic-induced retinal vascular endothelial cells (HRCECs) decreased gradually. Overexpression of STEAP4 reduced inflammation and apoptosis of HRCECs and improved dysfunction of them. Meanwhile, overexpression of steap4 inhibited the expression of HIF-1α/PKM2 signal. CONCLUSION: STEAP4 can be a potential therapeutic target for diabetic retinopathy by inhibiting HIF1/PKM2 signaling to reduce hyperglycemic-induced retinal cell apoptosis.

Knockdown of MALAT1 attenuates high-glucose-induced angiogenesis and inflammation via endoplasmic reticulum stress in human retinal vascular endothelial cells.

Diabetic retinopathy (DR) is one of the most severe complications of diabetes mellitus, and retinal endoplasmic reticulum stress (ERS) plays an important role in the pathogenesis of DR. However, the exact mechanisms by which ERS mediates DR remain unclear. In this study, human retinal vascular endothelial cells (RVECs) were cultured in high-glucose (HG) medium to mimic the environment of DR. The expression of long non-coding RNA (lncRNA)-metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was determined by quantitative real time PCR. ERS markers (glucose-regulated protein 78 [GRP78] and C/EBP homologous protein [CHOP]) were measured by immunofluorescence and western blotting. Cell viability was analyzed by the CCK-8 assay. The angiogenesis of RVECs was evaluated by tube formation assays. The levels of pro-inflammation cytokines TNF-α and IL-6 in RVECs were determined by ELISA assays. We found that exposure to HG levels upregulated MALAT1 and GRP78 expression in RVECs. While, GRP78 overexpression strengthened CHOP expression, cell proliferation suppression, capillary morphogenesis and inflammation in HG-treated RVECs. Importantly, knockdown of MALAT1 reversed HG-induced cell proliferation suppression, inhibited capillary morphogenesis, and inflammation in RVECs, and those effects were reversed by GRP78 overexpression. These results suggest that MALAT1 promotes HG-induced angiogenesis and inflammation in RVECs by upregulating ER stress, and might be target for treating DR.

Research status and progress of miR-15a in ophthalmological diseases / 国际眼科杂志(Guoji Yanke Zazhi)

@#miRNA-15a(miR-15a)is a non-coding small molecule RNA located on 13q14 gene. It affects the growth, development, differentiation and apoptosis of all organs and cells of the whole body. As the study progressively deepened, it was found that the role of miR-15a in different tissues and cells was not entirely consistent. Sometimes it plays a role in suppressing cancer, and sometimes it promotes cancer. The signal pathways it affects are complex and diverse. With the deepening of biological research into cell signaling pathways, miRNA-15a has become a miRNA more extensively studied. But in the ophthalmology, the corresponding research is not much. In this article, we mainly focus on the mechanism of miR-15a and its current research situation in ophthalmic diseases, so as to provide a reference for further study and their treatment.

Effect of miR-96-5p targeting FOXO4 on proliferation and apoptosis of rat retinal vascular endothelial cells induced by high glucose / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effect of microRNA-96-5p(miR-96-5p)on proliferation and apoptosis of rat retinal vascular endothelial cells induced by high glucose and to explore its mechanism. <p>METHODS: SD rat retinal vascular endothelial cells(RRVEC)were cultured and the RRVEC was divided into control group(NG)and high glucose group(HG). The high glucose-induced RRVECs were harvested separately or co-transfected with miR-96-5p mimic, miR-NC, si-FOXO4, si-NC. The expression of miR-96-5p and FOXO4 was detected by qRT-PCR and Western blotting, respectively. MTT assay was used to detect the proliferation activity. Flow cytometry was used to detect the apoptosis rate. The dual luciferase reporter assay validated the target gene of miR-96-5p. Western blotting was used to detect the expression of CyclinD1, p21, p27, Bcl-2, Bax and cleaved-caspased-3. <p>RESULTS:The expression levels of miR-96-5p, CyclinD1 and Bcl-2 in RRVEC were significantly decreased after high glucose treatment, and the expression levels of FOXO4, p21, p27, Bax and cleaved-caspased-3 were significantly increased, inhibiting cell proliferation activity, but promoting apoptosis. Overexpression of miR-96-5p and inhibition of FOXO4 expression increased the expression levels of CyclinD1 and Bcl-2, inhibited the expression of p21, p27, Bax, cleaved-caspased-3, enhanced cell proliferation and inhibited apoptosis. Dual luciferase reporter assay demonstrated that FOXO4 was a target gene for miR-96-5p. Overexpression of FOXO4 reversed the effect of miR-96-5p overexpression on high glucose-induced proliferation and apoptosis of RRVEC. <p>CONCLUSION:miR-96-5p inhibits high glucose-induced apoptosis of rat retinal vascular endothelial cells and promotes cell proliferation by targeting FOXO4.

Research status and progress of miR-15a in ophthalmological diseases / 国际眼科杂志(Guoji Yanke Zazhi)

@#miRNA-15a(miR-15a)is a non-coding small molecule RNA located on 13q14 gene. It affects the growth, development, differentiation and apoptosis of all organs and cells of the whole body. As the study progressively deepened, it was found that the role of miR-15a in different tissues and cells was not entirely consistent. Sometimes it plays a role in suppressing cancer, and sometimes it promotes cancer. The signal pathways it affects are complex and diverse. With the deepening of biological research into cell signaling pathways, miRNA-15a has become a miRNA more extensively studied. But in the ophthalmology, the corresponding research is not much. In this article, we mainly focus on the mechanism of miR-15a and its current research situation in ophthalmic diseases, so as to provide a reference for further study and their treatment.