Human Retinal Capillary Endothelial Cells Research Articles

Pipecolic acid mitigates ferroptosis in diabetic retinopathy by regulating GPX4-YAP signaling

Diabetic retinopathy (DR) is currently recognized as the leading cause of end-stage eye disease. Pipecolic acid, a metabolite, has a significant regulatory effect on several pathological processes. However, the exact mechanism by which it causes damage in diabetic retinopathy is unknown. Between September 2021 and December 2022, 40 patients were retrospectively examined and divided into two groups: the healthy group (n = 20) and the DR group (n = 20). Metabolomic analysis found that pipecolic acid plays an important role in this process. Streptozotocin-induced diabetic mice and high-glucose cultured human retinal capillary endothelial cells (HRCECs) were then treated with pipecolic acid. Several oxidative stress measurements and RNA sequencing of retinal cells were tested. A gene interaction study was conducted using bioinformatics. Comparison of serological metabolites between healthy volunteers and DR patients showed that pipecolic acid was significantly lower in DR patients, and there was a negative correlation between the level of pipecolic acid with blood glucose and glycated hemoglobin. Yes-associated protein (YAP) mRNA, Malondialdehyde (MDA), and reactive oxygen species (ROS) levels were significantly higher in diabetic mice, but glutathione peroxidase (GSH-Px) levels were significantly lower. Pipecolic acid significantly alleviated oxidative stress and YAP expression. The number of vascular tubes was significantly higher in the DR group, and pipecolic acid treatment significantly reduced tube formation. RNA-Sequencing analysis revealed that YAP and glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) expression was reduced, and functional enrichment analysis revealed that ferroptosis and Hippo signaling pathways play an important role in this process. Additionally, pipecolic acid's ability to improve DR is diminished after YAP and GPX4 ablation. This study found that pipecolic acid, as a metabolite, may impede the progression of DR by inhibiting the YAP-GPX4 signaling pathway.

TFEB attenuates hyperglycemia-induced retinal capillary endothelial cells injury via autophagy regulation.

Diabetic retinopathy is a common microvascular complication of diabetes mellitus. The maintenance of retinal capillary endothelial cell homeostasis requires a complete and unobtrusive flow of autophagy because it may help combat the inflammatory response, apoptosis, and oxidative stress damage of cells in diabetes mellitus. The transcription factor EB is a master regulator of autophagy and lysosomal biogenesis, but its role in diabetic retinopathy remains unknown. This study aimed to confirm the involvement of transcription factor EB in diabetic retinopathy and explore the role of transcription factor EB in hyperglycemia-linked endothelial injury in vitro. First, the expression levels, including the nuclear location of transcription factor EB and autophagy, were reduced in diabetic retinal tissues and high glucose-treated human retinal capillary endothelial cells. Subsequently, autophagy was mediated by transcription factor EB in vitro. Moreover, transcription factor EB overexpression reversed high glucose-induced autophagy inhibition and lysosomal dysfunction and protected human retinal capillary endothelial cells from inflammation, apoptosis, and oxidative stress damage caused by high glucose treatment. Additionally, under high-glucose stimulation, the autophagy inhibitor chloroquine attenuated transcription factor EB overexpression-mediated protection, and the autophagy agonist Torin1 rescued transcription factor EB knockdown-induced damage effects. Taken together, these results suggest that transcription factor EB is involved in the development of diabetic retinopathy. In addition, transcription factor EB protects human retinal capillary endothelial cells from high glucose-induced endothelial damage via autophagy.

Identification of potential ferroptosis-related biomarkers and a pharmacological compound in diabetic retinopathy based on machine learning and molecular docking.

Diabetic retinopathy (DR), a neurovascular disease, is a leading cause of visual loss worldwide and severely affects quality of life. Several studies have shown that ferroptosis plays an important role in the pathogenesis of DR; however, its molecule mechanism remains incompletely elucidated. Hence, this study aimed to investigate the pathogenesis of ferroptosis and explore potential ferroptosis-related gene biomarkers and a pharmacological compound for treating DR. Ferroptosis-related differentially expressed genes (DEGs) were identified in the GSE102485 dataset. Functional enrichment analyses were then performed and a protein-protein interaction (PPI) network was constructed to screen candidates of ferroptosis-related hub genes (FRHGs). FRHGs were further screened based on least absolute shrinkage and selection operator (LASSO) regression and random forest algorithms, and were then validated with the GSE60436 dataset and previous studies. A receiver operating characteristic (ROC) curve monofactor analysis was conducted to evaluate the diagnostic performance of the FRHGs, and immune infiltration analysis was performed. Moreover, the pharmacological compound targeting the FRHGs were verified by molecular docking. Finally, the FRHGs were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The 40 ferroptosis-related DEGs were extracted, and functional enrichment analyses mainly implicated apoptotic signaling, response to oxidative stress, ferroptosis, and lipid and atherosclerosis pathways. By integrating the PPI, LASSO regression, and random forest analyses to screen the FRHGs, and through validation, we identified five FRHGs that performed well in the diagnosis (CAV1, CD44, NOX4, TLR4, and TP53). Immune infiltration analysis revealed that immune microenvironment changes in DR patients may be related to these five FRHGs. Molecular docking also showed that glutathione strongly bound the CAV1 and TLR4 proteins. Finally, the upregulated expression of FRHGs (CD44, NOX4, TLR4, and TP53) was validated by qRT-PCR analysis in human retinal capillary endothelial cells cultured under high-glucose environment. CAV1, CD44, NOX4, TLR4, and TP53 are potential biomarkers for DR and may be involved in its occurrence and progression by regulating ferroptosis and the immune microenvironment. Further, glutathione exhibits potential therapeutic efficacy on DR by targeting ferroptosis. Our study provides new insights into the ferroptosis-related pathogenesis of DR, as well as its diagnosis and treatment.

TRIM46 aggravated high glucose-induced hyper permeability and inflammatory response in human retinal capillary endothelial cells by promoting IκBα ubiquitination

BackgroundDiabetic retinopathy (DR) as a severe diabetic complication contributes to blindness. The increased permeability of retinal capillary endothelial cells (RCECs) as well as the production of inflammatory markers are closely related to DR occurrence. We recently revealed that TRIM46 promotes high glucose (HG)-caused ferroptosis in human RCECs (HRCECs). The current study aims to explore the molecular mechanism of how TRIM46 plays its role in DR progression.MethodsWestern blot was utilized to determine protein expression. The cell counting kit-8 assay was used to observe cell viability. The permeability of the cell layer was determined by measuring the transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran leak. Enzyme-linked immunosorbent assay was used to quantify the protein level of pro-inflammatory cytokines and co-immunoprecipitation was employed to verify the relationship between TRIM46 and IκBα.ResultsHG dramatically upregulated TRIM46 protein expression in a dose-dependent way. Silencing TRIM46 effectively reversed HG-induced cell growth inhibition, cell cycle arrest, hyper permeability and pro-inflammatory cytokines secretion in HRCECs, while overexpression of TRIM46 exhibited an opposite effect. Furthermore, TRIM46 was able to interact with IκBα and promote the ubiquitination and degradation of IκBα. IκBα overexpression recovered the effects of TRIM46 overexpression in HRCECs. Furthermore, inhibiting the activation of NF-κB partially recovered HG-induced HRCEC injury, whereas TRIM46 overexpression reversed these effects.ConclusionThis study demonstrates that TRIM46 interacts with IκBα to activate the NF-κB signaling pathway, thereby enhancing cell proliferation inhibition, hyper permeability and the inflammatory response of HRCECs in a HG state.

Bie-Jia-Ruan-Mai-Tang, a Chinese Medicine Formula, Inhibits Retinal Neovascularization in Diabetic Mice Through Inducing the Apoptosis of Retinal Vascular Endothelial Cells.

Proliferative diabetic retinopathy (PDR) is one of the main complications of diabetes, mainly caused by the aberrant proliferation of retinal vascular endothelial cells and the formation of new blood vessels. Traditional Chinese medicines possess great potential in the prevention and treatment of PDR. Bie-Jia-Ruan-Mai-Tang (BJ), a Chinese medicine formula, has a good therapeutic effect on PDR clinically; however, the mechanism of action involved remains unclear. Therefore, we investigated the effect of BJ on PDR through in vitro and in vivo experiments. A diabetic mouse model with PDR was established by feeding a high-fat–high-glucose diet combined with an intraperitoneal injection of streptozotocin (STZ), while high-glucose-exposed human retinal capillary endothelial cells (HRCECs) were employed to mimic PDR in vitro. The in vivo experiments indicated that BJ inhibited the formation of acellular capillaries, decreased the expression of VEGF, and increased the level of ZO-1 in diabetic mice retina. In vitro experiments showed that high glucose significantly promoted cell viability and proliferation. However, BJ inhibited cell proliferation by cycle arrest in the S phase, thus leading to apoptosis; it also increased the production of ROS, decreased the mitochondrial membrane potential, reduced the ATP production, and also reduced the expressions of p-PI3K, p-AKT, and Bcl-xL, but increased the expressions of Bax and p-NF-κB. These results suggest that BJ induces the apoptosis of HRCECs exposed to high glucose through activating the mitochondrial death pathway by decreasing the PI3K/AKT signaling and increasing the NF-κB signaling to inhibit the formation of acellular capillaries in the retina, thus impeding the development of PDR.

Calycosin Alleviates Oxidative Stress and Pyroptosis Induced by High Glucose in Human Retinal Capillary Endothelial Cells Induced by High Glucose

Calycosin is an isoflavone isolated from the roots of Astragalus membranaceus. Due to its antioxidant and anti-inflammatory activities, it has been extensively used in traditional Chinese medicine formulations. Herein, we have evaluated the mechanism of action of calycosin using high glucose induced HRCEC cells as a model. The observed effects of calycosin include alleviation of oxidative stress induced by high glucose through promotion of SOD, GSH-Px and inhibition of ROS expression. Moreover, calycosin relieved HG-induced inflammation and pyroptosis by upregulating IL-1β, IL-18 and downregulating NLRP3, ASC, and cleaving caspase-1/procaspase-1. Calycosin also inhibited HMGB1/NF-κB pathway activation by promoting p-P65/P65, p-IκBα, HMGB1 and decreasing IκBα. All of these results support the notion that calycosin alleviates oxidative stress and pyroptosis of HRCEC cells induced by high glucose by inhibiting activation of the HMGB1/NF-κB pathway.

Secreted Protein Acidic and Rich in Cysteine Mediates the Development and Progression of Diabetic Retinopathy.

BackgroundsDiabetic retinopathy (DR) is one of the most severe microvascular complications of diabetes mellitus (DM). Secreted protein acidic and rich in cysteine (SPARC) has been found to play an important role in many diseases, but its role and mechanism in DR remain unknown.MethodsWe studied the role of SPARC and integrin β1 in vascular pathophysiology and identified potential therapeutic translation. The SPARC levels were tested in human serum and vitreous by ELISA assay, and then the Gene Expression Omnibus (GEO) dataset was used to understand the key role of the target gene in DR. In human retinal capillary endothelial cells (HRCECs), we analyzed the mRNA and protein level by RT-PCR, immunohistochemistry, and Western blotting. The cell apoptosis, cell viability, and angiogenesis were analyzed by flow cytometry, CCK-8, and tube formation.ResultsIn this study, we investigated the role of SPARC in the development and progression of human DR and high glucose-induced HRCEC cells and found that the SPARC-ITGB1 signaling pathway mimics early molecular and advanced neurovascular pathophysiology complications of DR. The result revealed that DR patients have a high-level SPARC expression in serum and vitreous. Knockdown of SPARC could decrease the expressions of inflammatory factors and VEGFR, inhibit cell apoptosis and angiogenesis, and increase cell viability by regulating integrin β1 in HRCECs.ConclusionSPARC promotes diabetic retinopathy via the regulation of integrin β1. The results of this study can provide a potential therapeutic application for the treatment of DR.

Prohibitin inhibits high glucose-induced apoptosis via maintaining mitochondrial function in human retinal capillary endothelial cells.

Mitochondrial dysfunction and excessive apoptosis of vascular endothelial cells play a critical role in the development of diabetic retinopathy (DR). Prohibitin (PHB), a significant regulator, maintains mitochondrial function and protects vascular endothelial cells against apoptosis. However, the mechanism underlying the protective effect of PHB on DR remains unclear. Since mitochondria are key regulators of vascular homeostasis, the present study aimed to investigate the molecular mechanism of PHB on maintaining mitochondrial function in human retinal capillary endothelial cells (HRCECs). To evaluate the role of PHB in cell apoptosis, HRCECs, transfected with or without PHB overexpression plasmid or small interfering RNA clones targeting PHB, were cultured in the presence of 5.5 mmol/l normal glucose (NG) or 30 mmol/l high glucose (HG). Subsequently, the apoptosis rate of HRCECs was determined using flow cytometry. The results showed that PHB was upregulated in HRCECs, while PHB knockdown promoted the generation of reactive oxygen species from mitochondria via inhibition of the activation of complex I. Additionally, the apoptosis rate of HRCECs in the HG group was notably enhanced compared with that in the NG group. Interestingly, PHB overexpression attenuated the increase in HG-mediated HRCEC apoptosis. Furthermore, treatment with HG upregulated expression of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase in vitro. The present study indicated that PHB could be a key modulator of mitochondrial homeostasis and could protect HRCECs against HG-induced apoptosis. Overall, the aforementioned findings provided experimental evidence supporting the potential protective effects of PHB on DR.

Paeoniflorin alleviates the progression of retinal vein occlusion via inhibiting hypoxia inducible factor-1α/vascular endothelial growth factor/STAT3 pathway

ABSTRACT Retinal vein occlusion (RVO) is a severe retinal vascular disease involving several complications, leading to weakening of vision and even blindness. Globally, over 16 million patients with RVO were found in the middle-aged population. Paeoniflorin (PF), a monomer of Taohong Siwu decoction, was reported to exhibit many pharmacological activities including anti-inflammatory, antioxidant, cardioprotective, and neuroprotective effects. However, the effect of PF on the progression of RVO remains unclear. In the current study, CCK8 assay was performed to investigate the cell viability. In addition, transwell assay and western blot were used to measure cell invasion and protein expression, respectively. Moreover, a mouse model of oxygen-induced dischemic retinopathy (OIR) was established. We found PF was able to inhibit the migration and angiogenesis of human retinal capillary endothelial cells under normoxia. Additionally, PF notably prevented hypoxia-induced angiogenesis of human retinal capillary endothelial cells via inhibiting hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF)/STAT3 pathway. Eventually, PF significantly alleviated the retinal lesions in the mouse with OIR. All in all, PF was able to alleviate the progression of retinal vein occlusion via inhibiting HIF-1α/VEGF/STAT3 pathway. These findings might provide some theoretical knowledge for exploring novel effective treatment for patients with RVO.

Glucocorticoids Promote Extracellular Matrix Component Remodeling by Activating YAP in Human Retinal Capillary Endothelial Cells.

Remodeling of extracellular matrix (ECM) components of endothelial cells is the main cause of retinal vascular basement membrane (BM) thickening, which leads to the initiation and perpetuation of microvasculopathy of diabetic retinopathy (DR). Excessive amounts of glucocorticoids (GCs) are related to the presence and severity of DR, however transcriptional effects of GCs on the biology of human retinal capillary endothelial cells (HRCECs) and its impacts on DR are still unclear. Here, we showed that GC (hydrocortisone) treatment induced ECM component [fibronectin (FN) and type IV collagen (Col IV)] expression and morphological changes in HRCECs via the glucocorticoid receptor (GR), which depended on the nuclear translocation of YAP coactivator. Mechanistically, GCs induced stress fiber formation in HRCECs, while blocking stress fiber formation inhibited GC-induced YAP nuclear translocation. Overexpression of FN, but not Col IV, activated YAP through the promotion of stress fiber formation via ECM-integrin signaling. Thus, a feedforward loop is established to sustain YAP activity. Using mRNA sequencing of HRCECs with overexpressed YAP or GC treatment, we found a similarity in Gene Ontology (GO) terms, differentially expressed genes (DEGs) and transcription factors (TFs) between the two RNA-seq datasets. In vivo, YAP was activated in retina vascular ECs of STZ-induced diabetic mice, and TF prediction analysis of published RNA-seq data of dermal vascular ECs from T2DM patients showed that GR and TEAD (the main transcription factor for YAP) were enriched. Together, GCs activate YAP and promote ECM component (FN and Col IV) remodeling in retinal capillary endothelial cells, and the underlying regulatory mechanism may provide new insights into the vascular BM thickening of the retina in the early pathogenesis of DR.

LINC00963 silencing inhibits the proliferation and migration of high glucose-induced retinal endothelial cells via targeting miR-27b.

The association between long intergenic non-protein-coding RNA 963 (LINC00963) and diabetes has not been fully elucidated. Therefore, the present study aimed to investigate the effect of the long non-coding RNA LINC00963 on diabetic retinopathy (DR), in order to provide a new therapeutic target for this condition. Human retinal capillary endothelial cells (HRECs) were induced with high concentrations of glucose to establish a DR model. The expression levels of LINC00963, cell viability, the protein expression levels of proliferating cell nuclear antigen (PCNA) and Ki67, and the migratory capacity of HRECs were determined using reverse transcription-quantitative PCR (RT-qPCR), Cell Counting Kit-8 assay, western blot analysis, and wound healing and Transwell assays, respectively. Furthermore, the Encyclopedia of RNA Interactomes database was used to predict the binding targets of LINC00963, and luciferase reporter assay was used to verify the direct binding of microRNA (miR)-27b to LINC00963. RT-qPCR was also utilized to measure the expression levels of miR-27b, PCNA and Ki67. The results demonstrated that LINC00963 silencing inhibited glucose-induced HREC proliferation and migration, and downregulated PCNA and Ki67 expression. Following transfection with miR-27b inhibitor, cell proliferation and migration were notably enhanced, and the protein expression levels of PCNA and Ki67 were increased. Taken together, the results of the present study suggested that the LINC00963/miR-27b axis may regulate the proliferation and migration of glucose-induced HRECs. Therefore, LINC00963 may be considered as a potential therapeutic target for DR.

TRIM46 contributes to high glucose-induced ferroptosis and cell growth inhibition in human retinal capillary endothelial cells by facilitating GPX4 ubiquitination

PurposeIncreased permeability of retinal capillary endothelial cells is a key feature in the progression of diabetic retinopathy (DR). Precisely why and how diabetes causes dysfunction in retinal capillary endothelial cells is not well understood, making it challenging to explore more advanced therapeutics. MethodsCell proliferation was assessed by the Cell Counting Kit-8 assay. Ferroptosis was evaluated by measuring lipid reactive oxygen species levels by flow cytometry and determining malondialdehyde, superoxide dismutase, and glutathione peroxidase levels through biochemical assays. Western blot analysis and quantitative PCR were respectively used to check the expression of proteins and RNAs. Co-immunoprecipitation assays were used to confirm the interaction between TRIM46 and GPX4. ResultsHigh glucose (HG, 25 mM glucose) significantly suppressed cell growth, which could be reversed by the ferroptosis inhibitor, ferrostatin-1. HG treatment time-dependently induced ferroptosis in human retinal capillary endothelial cells (HRCECs) and induced TRIM46 expression. Lentiviral-mediated overexpression of TRIM46 decreased cell resistance against HG-induced ferroptosis, whereas knockdown showed the opposite effect. Administration of RSL3, a ferroptosis agonist, was able to reverse the protective effects of TRIM46 silencing. TRIM46 interacted with GPX4, an important enzyme that suppresses ferroptosis, and promoted GPX4 ubiquitination. Furthermore, lentiviral-mediated overexpression ofGPX4 ameliorated the effects of TRIM46 overexpression and conferred protection to cells against HG-induced ferroptosis. ConclusionTRIM46 and GPX4 form a regulatory pathway that controls HG-induced ferroptosis of HRCECs. Inhibiting this pathway or sustaining the expression of GPX4 enables cells to resist damage caused by HG. We provide new mechanistic insight into the pathology of DR and identified TRIM46 and GPX4 as two molecular targets for the development of effective drugs for DR treatment.

Hu-Zhang-Qing-Mai-Yin Inhibits Proliferation of Human Retinal Capillary Endothelial Cells Exposed to High Glucose

Background: Diabetic retinopathy (DR) is one of the serious complications of diabetes and an important cause of blindness. Despite much research on the pathogenesis of DR, there is still a lack of safe and effective treatment methods. Hu-zhang-qing-mai-yin (HZQMY), a Chinese medicine formula, has been clinically used in the safe and effective treatment of DR for many years. However, the systematic pharmacological research is lacking. The aim of this study was to evaluate the anti-DR effects of HZQMY and explore the possible mechanism involved. Methods: The constituents of HZQMY were analyzed by LC-MS/MS. DR model was established by high glucose simulation on human retinal capillary endothelial cells (HRCECs) in vitro. The cell viability, cell proliferation, cell apoptosis, and tube formation were assessed. Subsequently the related mechanisms were analyzed by assays for JC-1 mitochondrial membrane potential (MMP), intracellular ROS, ATP, western blot and proteomics. Results: 27 main chemical components contained in HZQMY were identified. HZQMY significantly inhibited the viability and proliferation of HRCECs exposed to high glucose, and promoted the apoptosis. In addition, HZQMY also boosted the release of ROS and suppressed tube formation of HRCECs under high glucose exposure. Meanwhile, HRCECs treated with high glucose released more ROS than normal cells, which could be markedly inhibited by HZQMY in a dose-dependent manner. Additionally, western blot assay indicated that HZQMY increased the expression of proteins related to the P38 signaling pathway and inhibited nuclear factor kappa-B (NF-κB) pathway. Proteomic analysis predicted that HSPA4, MAPK3, ENO1, EEF2 and ERPS may be the candidate targets of HZQMY in HRCECs. Conclusions: HZQMY inhibited the proliferation and promoted the Mitochondria related apoptosis of HRCECs exposed to high glucose possibly through regulating P38 and NF-κB signaling pathway.

Cytoskeleton-associated protein 2 (CKAP2) is regulated by vascular endothelial growth factor and p53 in retinal capillary endothelial cells under high-glucose conditions

PurposeWe previously found the mutation frequency of cytoskeleton-associated protein 2 (CKAP2) was significantly increased in proliferative diabetic retinopathy (PDR) patients through whole exome sequencing. The present study was conducted to explore the expression and possible mechanism of CKAP2 in PDR patients and human retinal capillary endothelial cells (HRCECs) under high-glucose (HG) conditions. MethodsExpression of CKAP2 and p53 in the vitreous fluid and fibrovascular membrane (FVM) of PDR patients and HRCECs under HG conditions was observed. Cell proliferation, migration and tubule formation were assessed. Ranibizumab and siRNA transfection were used in the inhibition assay. ResultsCKAP2 and p53 were significantly increased in the ocular tissues of PDR patients. HG combined with VEGF treatment significantly up-regulated expression of CKAP2 and p53 in HRCECs. Inhibition of CKAP2 attenuated the abilities of cell proliferation, migration and tube formation under HG conditions. Blocking VEGF or p53 significantly decreased CKAP2 expression, whereas inhibition of CKAP2 failed to alter the level of VEGF or p53. ConclusionsCKAP2 is involved in the pathogenesis of PDR and regulated by VEGF and p53 under HG conditions.

VDAC1 regulates mitophagy in NLRP3 inflammasome activation in retinal capillary endothelial cells under high-glucose conditions

Diabetic retinopathy (DR) has been considered to involve mitochondrial alterations and be related to the nucleotide-binding oligomerization domain-like receptors 3 (NLRP3) inflammasome activation. The voltage-dependent anion channel 1 (VDAC1) protein is one of the key proteins that regulates the metabolic and energetic functions of the mitochondria. To explore the involvement of VDAC1 in mitophagy regulation of NLRP3 inflammasome activation under high-glucose (HG) conditions, this study examined expressions of VDAC1, mitochondrial function and mitophagy-related proteins, and NLRP3 inflammasome-related proteins in human retinal capillary endothelial cells (HRCECs) cultured with 30 mM of glucose in the presence or absence of mitophagy inhibitor (Mdivi-1) using Western blot. Mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were detected using flow cytometry. GFP-tagged pAdTrack-VDAC1 adenovirus was used to overexpress VDAC1. Cell biological behaviors, including proliferation, migration, tubule formation, and apoptosis, were also observed. Our results showed that when compared to the normal glucose and high mannitol groups, increased amounts of mitochondrial fragments, reduced mitochondrial membrane potential, increased expression of mitochondrial fission protein Drp 1, decreased expression of mitochondrial fusion protein Mfn 2, accumulation of mtROS, and activation of the NLRP3 inflammasome were observed in the HG group. Meanwhile, HG markedly reduced the protein expressions of PINK1, Parkin and VDAC1. Inhibition of mitophagy reduced PINK1 expression, enhanced NLRP3 expression, but failed to alter VDAC1. VDAC1 overexpression promoted PINK1 expression, inhibited NLRP3 activation and changed the cell biological behaviors under HG conditions. These findings demonstrate that VDAC1-mediated mitophagy plays a crucial role in regulating NLRP3 inflammasome activation in retinal capillary endothelial cells under HG conditions, suggesting that VDAC1 may be a potential target for preventing or treating DR.

Salusin-β Mediates High Glucose-Induced Inflammation and Apoptosis in Retinal Capillary Endothelial Cells via a ROS-Dependent Pathway in Diabetic Retinopathy

BackgroundDiabetic retinopathy (DR) is characterized by retinal vascular endothelial cell death and vascular inflammation, which are microvascular complications of diabetes mellitus (DM). Salusin-β, a newly identified peptide, is closely associated with hypertension, atherosclerosis and diabetic cardiomyopathy. However, the exact role of salusin-β in high glucose (HG)-induced retinal capillary endothelial cell (REC) inflammation and apoptosis remains unclear.Patients and MethodsA total of 60 patients with type 2 diabetes and 20 healthy controls were included in this study. Based on fundus fluorescein angiography findings, the diabetic patients were divided into three subgroups: diabetes without retinopathy (DWR), non-proliferative DR (NPDR) and proliferative DR (PDR). Serum salusin-β levels were measured by enzyme-linked immunosorbent assay. Human RECs (HRECs) were cultured in normal glucose (NG) and HG medium with or without salusin-β. Salusin-β expression was analysed by Western blotting and immunofluorescence staining. Expression of the pro-inflammatory cytokines MCP-1, IL-1β, TNF-α, and VCAM-1 was analysed by Western blotting. Reactive oxygen species (ROS) production was measured with 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Cell apoptosis rates were determined by flow cytometry. The levels of p38, JNK, p-p38, and p-JNK and the apoptosis-related proteins cleaved caspase-3, Bax, and cl2 were analysed by Western blotting.ResultsSerum salusin-β levels were higher in diabetic patients than in healthy controls (p = 0.0027), especially in patients with NPDR and PDR (both p<0.01). HG upregulated salusin-β expression in HRECs in a time-dependent manner. Salusin-β exacerbated inflammation and apoptosis, upregulated intracellular ROS production in HG-induced HRECs, and activated ROS-dependent JNK and p38 MAPK signalling, while knockdown of salusin-β suppressed these effects.ConclusionOur findings indicate that salusin-β can promote inflammation and apoptosis via ROS-dependent JNK and p38 MAPK signalling in HG-induced HRECs and could be a therapeutic target for DR.

MicroRNA-431-5p encapsulated in serum extracellular vesicles as a biomarker for proliferative diabetic retinopathy.

Early diagnosis and precise monitoring of the development of proliferative diabetic retinopathy (PDR) can significantly improve therapeutic strategies and help decrease blindness caused by it. Extracellular vesicles (EVs) were recently found to be involved in intercellular communications and are a potential source for the discovery of novel biomarkers. The current study aims to investigate the effectiveness of microRNAs (miRNAs) encapsulated in small EVs (sEVs) as minimally invasive biomarkers for PDR. SEVs were extracted from plasma of healthy subjects, diabetic patients, nonPDR patients and PDR patients. Then, we performed microarray analysis to determine the miRNA expression profile. MiR-431-5p expression doubled in the PDR patients compared with the healthy controls and the diabetic patients. We further found that miR-431-5p expression was 2.3 times higher in 4-hydroxynonenal treated human retinal capillary endothelial cells (HRCECs) than the control. After transfection with miR-431-5p mimics, proliferation of HRCECs was promoted, while transfection with miR-431-5p inhibitor demonstrated the opposite effect. The present findings indicate that circulating sEVs showed a differential miRNA profile in PDR patients. MiR-431-5p was involved in the pathogenesis of PDR development and may function as a novel biomarker for PDR.

RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy.

Diabetic retinopathy (DR), currently considered as a neurovascular disease, has become the major cause of blindness. More and more scholars believe that DR is no longer just a kind of microvascular disease, but accompanied by retinal neurodegenerative changes. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is a classic treatment for DR; however, anti-VEGF drugs can exacerbate fibrosis and eventually lead to retinal detachment. The aim of this study was to explore the pathogenesis of DR and identified new treatments that can provide dual-target intervention for angiogenesis and fibrosis. We explored changes in gene expression in high glucose-induced vascular endothelial cells using RNA sequencing (RNA-seq) technology. We identified bone morphogenetic protein 4 (BMP4) and SMAD family member 9 (SMAD9) among 449 differentially expressed genes from RNA-seq data and confirmed the expression of these two genes in the blood of diabetes patients by RT-PCR and in streptozotocin-induced rat retinas by RT-PCR, immunofluorescence, and western blot. Moreover, considering that DR is a multifactorial and multicellular disease, we used hydrogen peroxide (H2O2), advanced glycation end products (AGEs), CoCl2, 4-hydroxynonenal (4-HNE), and hypoxia to induce three human retinal cell types (Müller, retinal pigment epithelium, and human retinal capillary endothelial cells) to simulate the pathogenesis of DR, and MTT experiment, scratch experiment, Transwell experiment, and lumen formation experiment were used to test whether the model was successfully established. Then, we verified the overexpression of these two genes in the cell models by RT-PCR, immunofluorescence, and western blot. We further tested the effects of BMP4 on retinal cells. We use BMP4 to stimulate retinal cells and observe the effect of BMP4 on retinal cells by MTT experiment, scratch experiment, and RT-PCR. The results demonstrated that BMP4 and SMAD9 were highly expressed in both in vivo and in vitro models, while BMP4 could significantly upregulate the expression of SMAD9 and promote the expression of VEGF and fibrosis factors. This study is the first to analyze the mechanism by which high glucose levels affect retinal vascular endothelial cells through RNA transcriptome sequencing and indicates that BMP4 may be a potential target for the dual-target treatment (anti-VEGF and anti-fibrosis) of DR. • High-glucose effect on vascular endothelial cell was analyzed by RNA-seq. • KEGG analysis revealed enrichment of TGF-beta signaling pathway. • SMAD9 and BMP4 expression was upregulated in all samples. • Dual-target therapy of PDR by antagonizing BMP4.

MicroRNA‑126 suppresses the proliferation and migration of endothelial cells in experimental diabetic retinopathy by targeting polo‑like kinase4.

As a specific microvascular complication of diabetes, diabetic retinopathy (DR) causes severe visual impairment in patients with diabetes. The expression of microRNA-126 (miRNA/miR-126) has previously been found to be significantly decreased in the serum of patients with DR. In the present study, the functions of miR-126 and its mechanisms of action in experimental diabetic retinopathy were examined in rats with streptozotocin (STZ)-induced diabetes and in high glucose (HG)-induced human retinal capillary endothelial cells (HRCECs). In vivo, diabetic rat models were established and the rats were intravitreally injected with lentivirus expressing rno-miR-126 (lenti-miR-126) or negative control (lenti-NC). RT-qPCR was used to determine the miR-126 level in the serum and retina. Paraffin sections and retinal vasculature were used to determine the extent of retinopathy. The protein content of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in the retina was used as an auxiliary measurement of retinopathy. Western blot analysis and immunofluorescence staining were used to measure the expression of polo-like kinase 4 (PLK4) in rat retinal tissue. In vitro, the cells were transfected with miR-126 inhibitor or mimic and treated with the PLK4 inhibitor, CFI-400945 fumarate. RT-qPCR and western blot analysis were used to detect the miR-126 level and PLK4 expression. Cell proliferation and migration were measured by EdU and Transwell assays. The diabetic rats were found to exhibit downregulated serum and retinal miR-126 levels compared with the non-diabetic rats. The intravitreal delivery of miR-126 alleviated retinopathy and reduced the diabetes-induced upregulation of PLK4 in retinal tissues. Luciferase reporter assays confirmed that PLK4 mRNA was the target of miR-126. In HG-induced HRCECs, transfection with miR-126 mimic increased the miR-126 level, whereas it downregulated that of its downstream target, PLK4, which was opposite to the effects exerted by the miR-126 inhibitor. Furthermore, miR-126 mimic and CFI-400945 fumarate reduced the HG-induced upregulation of PLK4 expression, as well as cell proliferation and migration. On the whole, the findings of the present study demonstrate that miR-126 reduces experimental diabetic retinopathy and suppresses endothelial cell proliferation and migration by targeting PLK4. Thus, miR-126 and CFI-400945 fumarate may be therapeutic targets for DR.

The expression and significance of mTORC1 in diabetic retinopathy

BackgroundTo investigate the expression and significance of mechanistic target of rapamycin complex 1(mTORC1) in diabetic retinopathy (DR), and to find new targets and new methods for the treatment of DR.MethodsA DR rat model was prepared by general feeding combined with intraperitoneal injection of 10% streptozotocin (60 mg/kg). The rats were randomly divided into a control group (NDM group) and a diabetes group (DM group). Three months later, the degrees of retinopathy was determined using hematoxylin and eosin staining, and the levels of p-S6, VEGF, and PEDF proteins were detected by immunohistochemistry and western blotting. Human retinal capillary endothelial cells (HRCECs) were cultured in high glucose (HG) conditions, then treated with rapamycin or transfected with siTSC1.The protein levels of p-S6 were assessed by western blotting. The 5-ethynyl-2′-deoxyuridine assay was used to detect cell proliferation, and the Transwell assay was used to detect cell migration.ResultsA DM rat model was successfully developed. The expressions of p-S6 and VEGF proteins were significantly increased in the DM group (p < 0.05), and the expression of PEDF protein was significantly decreased compared with the NDM group (p < 0.05). In vitro, the p-S6 protein, as well as cell proliferation and migration, in HG induced HRCECs were increased (p < 0.05) compared with the control (normal glucose) group (p < 0.05). After transfection with siTSC1 to activate mTORC1, the expression of p-S6, as well as cell proliferation and migration, were increased. In contrast, rapamycin decreased p-S6 expression, as well as proliferation and migration, in HG induced HRCECs compared to the control group (p < 0.05).ConclusionmTORC1 plays an important role in DR. After activation, mTORC1 induced expression of the p-S6 protein, regulated the expressions of VEGF and PEDF proteins, and changed the proliferation and migration of endothelial cells. The mTORC1 can therefore be used as a new target,as well as in the treatment of DR.