Procyanidin Alleviates Diabetic Retinopathy by Activating the Expression of Activating Transcription Factor 1 Expression to Upregulate Synoviolin 1 and Inhibit the High Mobility Group Box1/Toll-Like Receptor 4 Pathway.

Diabetic retinopathy (DR) is a severe diabetes-induced eye disease, in which its pathological phenomena basically include abnormal proliferation, migration, and angiogenesis of microvascular endothelial cells in the retina. Long non-coding RNAs (lncRNAs) have been proven to be important regulators in various biological processes, but their participation in DR remains largely undiscovered. In the present study, we aimed to unveil the role of lncRNA small nucleolar RNA host gene 16 (SNHG16) in regulating the functions of human retinal microvascular endothelial cells (hRMECs) under a high-glucose (HG) condition. We found that SNHG16 expression was significantly upregulated in hRMECs treated with HG. Functionally, SNHG16 could facilitate hRMEC proliferation, migration, and angiogenesis. Moreover, SNHG16 was associated with nuclear factor κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Mechanistically, SNHG16 could promote hRMEC dysfunction by sequestering microRNA (miR)-146a-5p and miR-7-5p to act as a competing endogenous RNA (ceRNA) with interleukin-1 receptor-associated kinase 1 (IRAK1) and insulin receptor substrate 1 (IRS1). In conclusion, our results illustrated the potential role of SNHG16 in facilitating hRMEC dysfunction under HG treatment, providing a novel approach for DR therapy.

To investigate the mechanisms underlying 15(S)-HETE-induced angiogenesis, we have studied the role of the small GTPase, Rac1. We find that 15(S)-HETE activated Rac1 in human retinal microvascular endothelial cells (HRMVEC) in a time-dependent manner. Blockade of Rac1 by adenovirus-mediated expression of its dominant negative mutant suppressed HRMVEC migration as well as tube formation and Matrigel plug angiogenesis. 15(S)-HETE stimulated Src in HRMVEC in a time-dependent manner and blockade of its activation inhibited 15(S)-HETE-induced Rac1 stimulation in HRMVEC and the migration and tube formation of these cells as well as Matrigel plug angiogenesis. 15(S)-HETE stimulated JNK1 in Src-Rac1-dependent manner in HRMVEC and adenovirus-mediated expression of its dominant negative mutant suppressed the migration and tube formation of these cells and Matrigel plug angiogenesis. 15(S)-HETE activated ATF-2 in HRMVEC in Src-Rac1-JNK1-dependent manner and interference with its activation via adenovirus-mediated expression of its dominant negative mutant abrogated migration and tube formation of HRMVEC and Matrigel plug angiogenesis. In addition, 15(S)-HETE-induced MEK1 stimulation was found to be dependent on Src-Rac1 activation. Blockade of MEK1 activation inhibited 15(S)-HETE-induced JNK1 activity and ATF-2 phosphorylation. Together, these findings show that 15(S)-HETE activates ATF-2 via the Src-Rac1-MEK1-JNK1 signaling axis in HRMVEC leading to their angiogenic differentiation.

The present study is aimed to determine the mechanism of the protective effect of mailuoning and its main component luteolin on diabetic retinopathy. Human retinal microvascular endothelial cells were obtained and cultured in high glucose medium. Different concentrations of mailuoning and luteolin were added to the cells. MTT, flow cytometry and TUNEL staining assays were performed to investigate the effects of mailuoning on high glucose-induced proliferation and apoptosis in human retinal microvascular endothelial cells. Enzyme-linked immunosorbent assay was used to detect changes in inflammatory factors, and qPCR was used to detect the expression of apoptosis-related protein Bax and antiapoptotic protein Bcl-2. Western blot was used to detect changes of VEGFR2 and its phosphorylated p-VEGFR2. After adding mailuoning, cell viability of human retinal microvascular endothelial cells was significantly decreased in a concentration-dependent manner while 200 μg/ml mailuoning was the highest concentration without cytotoxic effects. Under high glucose conditions, apoptosis of human retinal microvascular endothelial cells was remarkably increased in a concentration-dependent manner and 100 μg/ml mailuoning was the optimum concentration on human retinal microvascular endothelial cells. Further studies indicated that mailuoning and luteolin inhibited the expression of apoptotic protein Bax and upregulated the expression of antiapoptotic protein Bcl-2. At the same time, inhibited the expression of inflammatory factors IL-6, TNFα and IL-1β. Mechanism studies have shown that mailuoning and luteolin could inhibit p-VEGFR2. Mailuoning and luteolin could prevent high glucose-induced human retinal microvascular endothelial cell apoptosis in diabetic retinopathy, suggesting that mailuoning and luteolin could be useful in the clinical treatment of diabetic retinopathy.

Diabetic retinopathy (DR) is a common complication of diabetes and the main cause of vision loss in the middle‐aged and elderly people. miRNAs play vital roles in the development of DR. This study aimed to explore the effects of miR‐16‐5p on high glucose (HG)–stimulated human retinal microvascular endothelial cells (HRECs) by modulating vascular endothelial growth factor A (VEGFA) and transforming growth factor beta receptor 1 (TGFBR1). HRECs were treated with 5 mM, 10 mM, 20 mM, and 30 mM of HG to induce the DR cell model. Real‐time quantitative polymerase chain reaction (RT‐qPCR) was used to detect the expression of miR‐16‐5p and mRNAs of VEGFA and TGFBR1. Western blot was used to examine VEGFA and TGFBR1 protein levels. The 3‐(4, 5‐dimethyl‐2‐thiazolyl)‐2, 5‐diphenyl‐2‐H‐tetrazolium bromide assay was conducted to test cell proliferation. Flow cytometry with Annexin V‐FITC/PI double staining was carried out to assess cell apoptosis ratio. Dual‐luciferase assay was used to identify the target relationship between miR‐16‐5p and VEGFA and TGFBR1. Results found that the expression of miR‐16‐5p in HG‐treated HRECs was reduced, and VEGFA and TGFBR1 expressions were upregulated. Knockdown of miR‐16‐5p increased VEGFA and TGFBR1 mRNA and protein levels, promoted cell proliferation, and inhibited apoptosis in HG‐treated HRECs. VEGFA and TGFBR1 inhibition reversed the effect of knocking down miR‐16‐5p on HRECs. Dual‐luciferase reporter assay revealed that VEGFA and TGFBR1 were the target of miR‐16‐5p. Overall, knockdown of miR‐16‐5p enhances proliferation and inhibits apoptosis of HRECs by upregulating VEGFA and TGFBR1 expression.

Knockdown of ChREBP ameliorates retinal microvascular endothelial cell injury and angiogenic responses in diabetic retinopathy

Diabetic retinopathy (DR), the leading cause of adult blindness, has its risk increased by excessive endoplasmic reticulum stress (ERS). Nuclear receptor subfamily 2 group f member 2 (NR2F2) is an orphan nuclear receptor with essential roles in angiogenesis. However, its roles in DR remain unknown. Notably, NR2F2 protein expression was upregulated in streptozotocin-induced diabetic mice retina and the retinal endothelial cells characterized by endothelial marker CD31. Retinal NR2F2 expression was knocked down in diabetic mice using adeno-associated virus serotype 2. NR2F2 knockdown increased retinal thickness, decreased acellular capillary formation and FITC-labeled dextran leakage, and increased tight junction (TJ) protein ZO-1 expression invivo. Additionally, NR2F2 knockdown inhibited reactive oxygen species generation and the ERS marker CHOP expression invivo. In high glucose (HG)-induced human retinal microvascular endothelial cell (HRMEC) monolayers, NR2F2 knockdown inhibited FITC-dextran flux, increased transendothelial electrical resistance, reduced VEGFA secretion and restored expression and continuous distribution of TJ proteins. NR2F2 knockdown also reduced ERS markers expression and inhibited ERS-related signaling pathways invitro. mRNA-seq analysis in NR2F2-knockdown HRMECs exposed to HG showed that NR2F2 inhibition upregulated CYB5R2 mRNA level. NR2F2 was bound directly to the CYB5R2 promoter for transcriptional repression in HRMECs. CYB5R2 overexpression inhibited ERS-related protein expressions and barrier dysfunction in HRMEC monolayers, and CYB5R2 knockdown reversed the endothelial protective effect of NR2F2 inhibition. In conclusion, NR2F2 knockdown attenuates vascular dysfunction by alleviating ERS in retinal microvascular endothelial cells and alleviates disease progression in DR mice. This may be achieved by reducing CYB5R2 transcriptional repression.

Introduction: It is known that the metabolic disorder caused by high glucose is one of pathogenesis in diabetic retinopathy (DR), the leading cause of blindness, due to the main pathological change of apoptosis of endothelial cells (ECs). In previous studies, the potential impact of sodium glucose cotransporter-2 (SGLT-2), whose inhibitors slow the progression of DR, has not been elucidated. The purpose of the presented study was to explore the effect of SGLT-2 inhibitors dapagliflozin (DAPA) on apoptosis of diabetic mice retina and human retinal microvascular endothelial cells (HRMECs), examine the effects of dapagliflozin on HRMECs metabolism, and explore the molecular processes that affect DR. Methods and Results: The eyeballs of male streptozotocin (STZ)-induced diabetic C57BL/6N mice were evaluated. C57BL/6N mice were divided into control group (CON), diabetic untreated group (DM), diabetic dapagliflozin treatment group (DM + DAPA) and diabetic insulin treatment group (DM + INS). Hematoxylin-Eosin (HE) staining was performed to observe the pathological structure of the mice retina, and TUNEL staining to detect apoptosis of mice retinal cells. In vitro, DCFH-DA and western blot (WB) were used to evaluate ROS, Bcl-2, BAX, cleaved-caspase 3 in HRMECs and metabolomics detected the effect of dapagliflozin on the metabolism of HRMECs. And then, we performed correlation analysis and verification functions for significantly different metabolites. In vivo, dapagliflozin reduced the apoptosis of diabetic mice retina independently of hypoglycemic. In vitro, SGLT-2 protein was expressed on HRMECs. Dapagliflozin reduced the level of ROS caused by high glucose, decreased the expression of cleaved-caspase3 and the ratio of BAX/Bcl-2. Metabolomics results showed that dapagliflozin did not affect the intracellular glucose level. Compared with the high glucose group, dapagliflozin reduced the production of arachidonic acid (AA) and inhibited the phosphorylation of ERK1/2, therefore, reducing the phosphorylation of cPLA2, which is a key enzyme for arachidonic acid release. Conclusion: Collectively, results unearthed for the first time that dapagliflozin reduced apoptosis of retina induced by DM whether in vivo or in vitro. Dapagliflozin did not affect the glucose uptake while mitigated intracellular arachidonic acid in HRMECs. Dapagliflozin alleviated HRMECs apoptosis induced by high glucose through ERK/1/2/cPLA2/AA/ROS pathway.

Diabetic retinopathy (DR) is a leading cause of vision loss and is primarily driven by chronic hyperglycemia, which induces retinal vascular damage through mechanisms involving vascular endothelial growth factor (VEGF) and the renin-angiotensin system (RAS). This study investigated the effects of hyperglycemia and different insulin formulations-regular, glulisine, and aspart-on VEGF-A and angiotensinogen (AGT) gene expression in two human retinal cell types: retinal pigment epithelial (RPE) cells and human retinal microvascular endothelial cells (HRECs). Cells were cultured from donor tissue and exposed to physiologic and hyperglycemic glucose concentrations, with or without insulin treatment. Gene expression levels were quantified using real-time PCR. Hyperglycemia significantly upregulated VEGF-A and AGT in both RPE and HREC cells (e.g., VEGF-A in RPE: 2.62-fold, P = 0.001; AGT in RPE: 3.32-fold, P = 0.093), supporting a role for both osmotic and glucose-specific pathways. Among insulin treatments, regular insulin significantly reduced VEGF-A expression in both RPE (0.72-fold, P = 0.033) and HRECs (0.57-fold, P = 0.009). In contrast, aspart and glulisine had modest effects on VEGF-A in HRECs (0.82-fold each; P = 0.035 and P = 0.060, respectively) and no significant impact in RPE cells. Regarding AGT, aspart insulin showed the most consistent suppressive effect, reducing expression in both RPE (0.15-fold, P < 0.001) and HRECs (0.22-fold, P = 0.004). Glulisine significantly increased AGT in RPE (1.56-fold, P = 0.009) but reduced it in HRECs (0.58-fold, P = 0.074). Regular insulin showed no effect on AGT in RPE (P = 0.680) and a non-significant increase in HRECs (1.36-fold, P = 0.097). These findings highlight the differential biological effects of insulin analogues and suggest that aspart insulin, in particular, may offer therapeutic benefits beyond glycemic control by modulating both VEGF-A and RAS-related pathways. Tailored insulin therapies could represent innovative strategies for managing or slowing the progression of diabetic retinopathy.

GPR120 has been reported to ameliorate inflammation in diabetes and diabetic complications. In this study, GW9508, the GPR120 agonist, was utilized in human retinal microvascular endothelial cells (HRMECs) exposed to high glucose (HG) to investigate the involvement of GPR120 in cellular viability and apoptosis as well as the association with the NLRP3 inflammasome. The expression of GPR120 in HRMECs cultured under HG was firstly detected by Western blotting. HRMECs were then assigned to the normal control, GW9508, HG, and HG + GW9508 groups. The expression of the NLRP3 inflammasome consists of NLRP3, ASC, and caspase-1 and was detected by Western blotting and the downstream IL-1β and IL-18 by ELISA. The cellular viability and apoptosis of HRMECs were detected by CCK-8 and flow cytometry, respectively. The expressions of apoptosis-related proteins Bax and Bcl-2 were detected by Western blotting. Finally, nonspecific siRNA (NS) or GPR120 siRNA (siGPR120) was transfected to the cells, followed by stimulation with or without GW9508 or HG, and the expression of NLRP3, ASC, and caspase-1 were detected by Western blotting in these groups. GPR120 is expressed in HRMECs, and HG can reduce its expression in a time-dependent manner. GW9508 can attenuate inflammation by reducing the expression of NLRP3, ASC, caspase-1, IL-1β, and IL-18 under HG. GW9508 rescues the viability of HRMCs and reduces cell apoptosis by preventing an increase in Bax expression and the reduction in Bcl-2 expression. Additionally, knockdown of GPR120 by siRNA weakened the effects of GW9508 on NLRP3 inflammasome expression. Activation of GPR120 protects retinal vascular endothelial cells from HG through inhibiting NLRP3 inflammasome. Thus, GPR120 might be a potential therapeutic target to reduce retinal endothelial damage in diabetic retinopathy.

Objective To explore the impact of Hedgehog protein on human retinal microvascular endothelial cell(HRMEC)and its signaling pathway. Methods The cultured HRMECs were divided into normal control group, 0.5 μmol/L agonist group and 1.0 μmol/L agonist group, and were cultured in medium with final concentration of 0, 0.5 and 1.0 μmol/L Hedgehog agonist, respectively; HRMECs cultured in high glucose medium were divided into high glucose control group, 1.5 μmol/L inhibitor group and 2.5 μmol/L inhibitor group.Erismodegib, the Smoothed inhibitor with final concentration of 0, 1.5 and 2.5 μmol/L was added into corresponding group, respectively.MTS method and Transwell cell migration method were used to detect the proliferation(A490 value)and relative mobility of HRMEC.The phosphorylation of PLCγ1, Akt and Erk proteins were detected by Western blot. Results The relative expression of Hedgehog protein in the high glucose control group was 6.24±0.11, which was significantly higher than 1.00±0.00 in the normal control group(t=667.573, P<0.001). The A490 value was 1.349±0.050 and 1.422±0.053, and the relative mobility rate was 2.34±0.14 and 3.59±0.32 in the0.5 μmol/L agonist group and the 1.0 μmol/L agonist group, respectively, which were significantly higher than 1.203±0.101 and 1.00±0.00 in the normal control group(all at P<0.01). The A490 value was 0.849±0.010 and 0.737±0.030, and the relative mobility rate was 0.43±0.02 and 0.27 ±0.01 in the 1.5 μmol/L inhibitor group and the 2.5 μmol/L inhibitor group, respectively, which were significantly lower than 1.000±0.040 and 1.00±0.00 in the high glucose control group(all at P<0.01). The phosphorylation ratios of PLCγ1, Akt and Erk in the 0.5 μmol/L agonist group and the 1.0 μmol/L agonist group were significantly higher than those in the normal control group(all at P<0.01). The phosphorylation ratios of PLCγ1, Akt and Erk in the 1.5 μmol/L inhibitor group and the 2.5 μmol/L inhibitor group were significantly lower than those in the high glucose control group(all at P<0.01). Conclusions High glucose induces the expression of Hedgehog protein in HRMEC.Hedgehog protein may regulate the function of HRMEC by regulating the phosphorylation of PLCγ1, Akt and Erk in G Protein-coupled receptors pathway. Key words: Diabetic retinopathy; Hedgehog protein; G Protein-coupled receptors signaling pathway; Human retinal microvascular endothelial cell

Diabetes mellitus (DM) is a chronic metabolic disorder affecting millions of people worldwide, characterized by dysregulated glucose homeostasis and hyperglycemia. Diabetic retinopathy (DR) is one of the serious multisystemic complications. Aging is an important risk factor for DR. Endothelial sirtuin 1 (SIRT1) plays an important role in regulating the pathophysiology of glucose metabolism, cellular senescence, and aging. Liraglutide, an analog of Glucagon-like peptide 1 (GLP-1), has been widely used in the treatment of DM. However, the effects of Liraglutide on DR are less reported. Here, we investigated whether treatment with Liraglutide has beneficial effects on high glucose (HG)-induced injury in human retinal microvascular endothelial cells (HRECs). First, we found that exposure to HG reduced the expression of glucagon-like peptide 1 receptor 1 (GLP-1R). Additionally, Liraglutide ameliorated HG-induced increase in the expression of vascular endothelial growth factor-A (VEGF-A) and interleukin 6 (IL-6). Importantly, Liraglutide ameliorated cellular senescence and increased telomerase activity in HG-challenged HRECs. Liraglutide also reduced the levels of p53 and p21. Mechanistically, Liraglutide restored the expression of SIRT1 against HG. In contrast, the knockdown of SIRT1 abolished the protective effects of Liraglutide in cellular senescence of HRECs. Our findings suggest that Liraglutide might possess a benefit on DR mediated by SIRT1.

Association between Insulin and Nitric Oxide in Human Retinal Microvascular Endothelial Cells in vitro

The damage of hyperglycemia to the retinal pigment epithelial (RPE) cells is a critical event in diabetic retinopathy (DR). Procyanidin (PC), a kind of polyphenol compounds, has shown to be effective in preventing and treating diabetes as well as its complications, in which autophagy disorder is involved in the pathological mechanism. However, it remains unclear whether PC can play a protective role in DR by regulating the autophagy of RPE. Here, the effect of PC on RPE under high glucose conditions and the role of autophagy were investigated. The cell viability of ARPE-19, a human RPE cell line, was detected by cell counting kit-8 (CCK-8) and the apoptosis rate was measured by flow cytometry. The protein expressions of apoptosis markers, including Bax, Bcl-2, and Caspase-3, as well as autophagy markers including LC3, p62, p53, and mTOR were detected by Western blotting. Autophagic flux in ARPE-19 cells was detected by transfection with Ad-mCherry-GFP-LC3B. Under high glucose conditions, the viability of ARPE-19 was decreased and the apoptosis rate increased, the protein expressions of Bax, Caspase-3, LC3-II/LC3-I, and p-p53 were all increased and the expressions of Bcl-2, p62, and p-mTOR decreased, and autophagic flux was increased compared with that of the controls. Treatment with PC weakened all these changes caused by high glucose. When rapamycin (RPM), an autophagy agonist was added, the cell viability of ARPE-19 by PC treatment was decreased while the apoptosis was increased. Our findings indicate that through the p53/mTOR autophagy pathway, PC may protect RPE cells from high glucose-induced injury.

MicroRNAs (miRNAs) have been reported to be involved in the progression of various diseases. Studying the regulatory mechanisms of miRNAs can help clinical treatment. Diabetic retinopathy (DR) is one of the complications of diabetes. The objective of this study was to elucidate the underlying molecular mechanisms by which miR-200c-3p regulates the pyroptosis of DR cell. Human retinal microvascular endothelial cells (HRMECs) and high glucose (HG) cultures established DR cell model in vitro. RT-qPCR is used to detect the expression level of miRNAs. CCK-8 assays and flow cytometry are used to detect apoptosis of HRMECs cell. Western blotting is used to detect cleaved caspase-3, cleaved caspase-1, and N-GSDMD proteins levels in HRMECs. The ELISA assay is used to detect the expression of IL-1β and IL-18. Predict and validate potential binding sites between miR-200c-3p and SLC30A7 by dual luciferase reporter gene analysis. The results showed that HG caused damage to HRMECs through the pyroptosis pathway rather than the apoptosis pathway. MiR-200c-3p is highly expressed in HG induced-HRMECs, and knockdown of miR-200c-3p mitigates HG-induced HRMECs pyroptosis. MiR-200c-3p negatively targets SLC30A7 in HRMECs, and miR-200c-3p regulates pyroptosis of HG-induced HRMECs by targeting SLC30A7. The results suggest that miR-200c-3p might be a promising interference target for DR prevention and treatment. The results of current study may provide new insights into development of therapeutic strategies for DR.

Glucose-Regulated Protein 78, via Releasing β-Catenin from Adherens Junctions, Facilitates Its Interaction with STAT3 in Mediating Retinal Neovascularization