Alzheimer's disease as a causal risk factor for diabetic retinopathy: a Mendelian randomization study.

Objectives: This study aims to investigate the causal relationship between Alzheimer's Disease (AD) and Diabetic Retinopathy (DR). Methods: Employing Mendelian Randomization (MR), Generalized Summary-data-based Mendelian Randomization (GSMR), and the MR-Steiger test, this study scrutinizes the genetic underpinnings of the hypothesized causal association between AD and DR, as well as its Proliferative DR (PDR) and Non-Proliferative DR (NPDR) subtypes. Comprehensive data from Genome-Wide Association Studies (GWAS) were analyzed, specifically AD data from the Psychiatric Genomics Consortium (71,880 cases/383,378 controls), and DR, PDR, and NPDR data from both the FinnGen consortium (FinnGen release R8, DR: 5,988 cases/314,042 controls; PDR: 8,383 cases/329,756 controls; NPDR: 3,446 cases/314,042 controls) and the IEU OpenGWAS (DR: 14,584 cases/176,010 controls; PDR: 8,681 cases/204,208 controls; NPDR: 2,026 cases/204,208 controls). The study also incorporated Functional Mapping and Annotation (FUMA) for an in-depth analysis of the GWAS results. Results: The MR analyses revealed that genetic susceptibility to AD significantly increases the risk of DR, as evidenced by GWAS data from the FinnGen consortium (OR: 2.5090; 95% confidence interval (CI):1.2102-5.2018, false discovery rate P-value (PFDR)=0.0201; GSMR: bxy=0.8936, bxy_se=0.3759, P=0.0174), NPDR (OR: 2.7455; 95% CI: 1.3178-5.7197, PFDR=0.0166; GSMR: bxy=0.9682, bxy_se=0.3802, P=0.0126), and PDR (OR: 2.3098; 95% CI: 1.2411-4.2986, PFDR=0.0164; GSMR: bxy=0.7962, bxy_se=0.3205, P=0.0129) using DR GWAS from FinnGen consortium. These results were corroborated by DR GWAS datasets from IEU OpenGWAS. The MR-Steiger test confirmed a significant association of all identified instrumental variables (IVs) with AD. While a potential causal effect of DR and its subtypes on AD was identified, the robustness of these results was constrained by a low power value. FUMA analysis identified OARD1, NFYA, TREM1 as shared risk genes between DR and AD, suggesting a potential genetic overlap between these complex diseases. Discussion: This study underscores the contribution of AD to an increased risk of DR, as well as NPDR and PDR subtypes, underscoring the necessity of a holistic approach in the management of patients affected by these conditions.

Deciphering Müller cell heterogeneity signatures in diabetic retinopathy across species: an integrative single-cell analysis.

Diabetic retinopathy (DR), a leading cause of visual impairment, demands a profound comprehension of its cellular mechanisms to formulate effective therapeutic strategies. Our study presentes a comprehensive single-cell analysis elucidating the intricate landscape of Müller cells within DR, emphasizing their nuanced involvement. Utilizing scRNA-seq data from both Sprague-Dawley rat models and human patients, we delineated distinct Müller cell clusters and their corresponding gene expression profiles. These findings were further validated through differential gene expression analysis utilizing human transcriptomic data. Notably, certain Müller cell clusters displayed upregulation of the Rho gene, implying a phagocytic response to damaged photoreceptors within the DR microenvironment. This phenomenon was consistently observed across species. Additionally, the co-expression patterns of RHO and PDE6G within Müller cell clusters provided compelling evidence supporting their potential role in maintaining retinal integrity during DR. Our results offer novel insights into the cellular dynamics of DR and underscore Müller cells as promising therapeutic targets for preserving vision in retinal disorders induced by diabetes.

LncRNA XIST promotes neovascularization in diabetic retinopathy by regulating miR-101-3p/VEGFA.

Objective: This study sought to investigate the regulation of long noncoding RNA (lncRNA) XIST on the microRNA (miR)-101-3p/vascular endothelial growth factor A (VEGFA) axis in neovascularization in diabetic retinopathy (DR). Materials and methods: Serum of patients with DR was extracted for the analysis of XIST, miR-101-3p, and VEGFA expression levels. High glucose (HG)-insulted HRMECs and DR model rats were treated with lentiviral vectors. MTT, transwell, and tube formation assays were performed to evaluate cell viability, migration, and angiogenesis, and ELISA was conducted to detect the levels of inflammatory cytokines. Dual-luciferase reporter, RIP, and RNA pull-down experiments were used to validate the relationships among XIST, miR-101-3p, and VEGFA. Results: XIST and VEGFA were upregulated and miR-101-3p was downregulated in serum from patients with DR. XIST knockdown inhibited proliferation, migration, vessel tube formation, and inflammatory responsein HG-treated HRMECs, whereas the above effects were nullified by miR-101-3p inhibition or VEGFA overexpression. miR-101-3p could bind to XIST and VEGFA. XIST promoted DR development in rats by regulating the miR-101-3p/VEGFA axis. Conclusion: LncRNA XIST promotes VEGFA expression by downregulating miR-101-3p, thereby stimulating angiogenesis and inflammatory response in DR.

Causal relationships between blood metabolites and diabetic retinopathy: a two-sample Mendelian randomization study.

Background: Diabetic retinopathy (DR) is a microvascular complication of diabetes, severely affecting patients' vision and even leading to blindness. The development of DR is influenced by metabolic disturbance and genetic factors, including gene polymorphisms. The research aimed to uncover the causal relationships between blood metabolites and DR. Methods: The two-sample mendelian randomization (MR) analysis was employed to estimate the causality of blood metabolites on DR. The genetic variables for exposure were obtained from the genome-wide association study (GWAS) dataset of 486 blood metabolites, while the genetic predictors for outcomes including all-stage DR (All DR), non-proliferative DR (NPDR) and proliferative DR (PDR) were derived from the FinnGen database. The primary analysis employed inverse variance weighted (IVW) method, and supplementary analyses were performed using MR-Egger, weighted median (WM), simple mode and weighted mode methods. Additionally, MR-Egger intercept test, Cochran's Q test, and leave-one-out analysis were also conducted to guarantee the accuracy and robustness of the results. Subsequently, we replicated the MR analysis using three additional datasets from the FinnGen database and conducted a meta-analysis to determine blood metabolites associated with DR. Finally, reverse MR analysis and metabolic pathway analysis were performed. Results: The study identified 13 blood metabolites associated with All DR, 9 blood metabolites associated with NPDR and 12 blood metabolites associated with PDR. In summary, a total of 21 blood metabolites were identified as having potential causal relationships with DR. Additionally, we identified 4 metabolic pathways that are related to DR. Conclusion: The research revealed a number of blood metabolites and metabolic pathways that are causally associated with DR, which holds significant importance for screening and prevention of DR. However, it is noteworthy that these causal relationships should be validated in larger cohorts and experiments.

Cellular communication network factor 1 promotes retinal leakage in diabetic retinopathy via inducing neutrophil stasis and neutrophil extracellular traps extrusion.

BACKGROUND: Diabetic retinopathy (DR) is a major cause of blindness and is characterized by dysfunction of the retinal microvasculature. Neutrophil stasis, resulting in retinal inflammation and the occlusion of retinal microvessels, is a key mechanism driving DR. These plugging neutrophils subsequently release neutrophil extracellular traps (NETs), which further disrupts the retinal vasculature. Nevertheless, the primary catalyst for NETs extrusion in the retinal microenvironment under diabetic conditions remains unidentified. In recent studies, cellular communication network factor 1 (CCN1) has emerged as a central molecule modulating inflammation in pathological settings. Additionally, our previous research has shed light on the pathogenic role of CCN1 in maintaining endothelial integrity. However, the precise role of CCN1 in microvascular occlusion and its potential interaction with neutrophils in diabetic retinopathy have not yet been investigated. METHODS: We first examined the circulating level of CCN1 and NETs in our study cohort and analyzed related clinical parameters. To further evaluate the effects of CCN1 in vivo, we used recombinant CCN1 protein and CCN1 overexpression for gain-of-function, and CCN1 knockdown for loss-of-function by intravitreal injection in diabetic mice. The underlying mechanisms were further validated on human and mouse primary neutrophils and dHL60 cells. RESULTS: We detected increases in CCN1 and neutrophil elastase in the plasma of DR patients and the retinas of diabetic mice. CCN1 gain-of-function in the retina resulted in neutrophil stasis, NETs extrusion, capillary degeneration, and retinal leakage. Pre-treatment with DNase I to reduce NETs effectively eliminated CCN1-induced retinal leakage. Notably, both CCN1 knockdown and DNase I treatment rescued the retinal leakage in the context of diabetes. In vitro, CCN1 promoted adherence, migration, and NETs extrusion of neutrophils. CONCLUSION: In this study, we uncover that CCN1 contributed to retinal inflammation, vessel occlusion and leakage by recruiting neutrophils and triggering NETs extrusion under diabetic conditions. Notably, manipulating CCN1 was able to hold therapeutic promise for the treatment of diabetic retinopathy.

Effects of Nox4 upregulation on PECAM-1 expression in a mouse model of diabetic retinopathy.

Diabetic Retinopathy (DR) is the leading cause of vision loss in working-age adults. The hallmark features of DR include vascular leakage, capillary loss, retinal ischemia, and aberrant neovascularization. Although the pathophysiology is not fully understood, accumulating evidence supports elevated reactive oxygen species associated with increased activity of NADPH oxidase 4 (Nox4) as major drivers of disease progression. Previously, we have shown that Nox4 upregulation in retinal endothelial cells by diabetes leads to increased vascular leakage by an unknown mechanism. Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell surface molecule that is highly expressed in endothelial cells and regulates endothelial barrier function. In the present study, using endothelial cell-specific human Nox4 transgenic (TG) mice and endothelial cell-specific Nox4 conditional knockout (cKO) mice, we investigated the impact of Nox4 upregulation on PECAM-1 expression in mouse retinas and brain microvascular endothelial cells (BMECs). Additionally, cultured human retinal endothelial cells (HRECs) transduced with adenovirus overexpressing human Nox4 were used in the study. We found that overexpression of Nox4 increases PECAM-1 mRNA but has no effect on its protein expression in the mouse retina, BMECs, or HRECs. Furthermore, PECAM-1 mRNA and protein expression was unchanged in BMECs isolated from cKO mice compared to wild type (WT) mice with or without 2 months of diabetes. Together, these findings do not support a significant role of Nox4 in the regulation of PECAM-1 expression in the diabetic retina and endothelial cells. Further studies are warranted to elucidate the mechanism of Nox4-induced vascular leakage by investigating other intercellular junctional proteins in endothelial cells and their implications in the pathophysiology of diabetic retinopathy.

Exploration of potential novel drug targets for diabetic retinopathy by plasma proteome screening.

The aim of this study is to identify novel potential drug targets for diabetic retinopathy (DR). A bidirectional two-sample Mendelian randomization (MR) analysis was performed using protein quantitative trait loci (pQTL) of 734 plasma proteins as the exposures and clinically diagnosed DR as the outcome. Genetic instruments for 734 plasma proteins were obtained from recently published genome-wide association studies (GWAS), and external plasma proteome data was retrieved from the Icelandic Decoding Genetics Study and UK Biobank Pharma Proteomics Project. Summary-level data of GWAS for DR were obtained from the Finngen Consortium, comprising 14,584 cases and 202,082 population controls. Steiger filtering, Bayesian co-localization, and phenotype scanning were used to further verify the causal relationships calculated by MR. Three significant (p < 6.81 × 10-5) plasma protein-DR pairs were identified during the primary MR analysis, including CFH (OR = 0.8; 95% CI 0.75-0.86; p = 1.29 × 10-9), B3GNT8 (OR = 1.09; 95% CI 1.05-1.12; p = 5.9 × 10-6) and CFHR4 (OR = 1.11; 95% CI 1.06-1.16; p = 1.95 × 10-6). None of the three proteins showed reverse causation. According to Bayesian colocalization analysis, CFH (coloc.abf-PPH4 = 0.534) and B3GNT8 (coloc.abf-PPH4 = 0.638) in plasma shared the same variant with DR. All three identified proteins were validated in external replication cohorts. Our research shows a cause-and-effect connection between genetically determined levels of CFH, B3GNT8 and CFHR4 plasma proteins and DR. The discovery implies that these proteins hold potential as drug target in the process of developing drugs to treat DR.

A novel fusion of genetic grey wolf optimization and kernel extreme learning machines for precise diabetic eye disease classification.

In response to the growing number of diabetes cases worldwide, Our study addresses the escalating issue of diabetic eye disease (DED), a significant contributor to vision loss globally, through a pioneering approach. We propose a novel integration of a Genetic Grey Wolf Optimization (G-GWO) algorithm with a Fully Convolutional Encoder-Decoder Network (FCEDN), further enhanced by a Kernel Extreme Learning Machine (KELM) for refined image segmentation and disease classification. This innovative combination leverages the genetic algorithm and grey wolf optimization to boost the FCEDN's efficiency, enabling precise detection of DED stages and differentiation among disease types. Tested across diverse datasets, including IDRiD, DR-HAGIS, and ODIR, our model showcased superior performance, achieving classification accuracies between 98.5% to 98.8%, surpassing existing methods. This advancement sets a new standard in DED detection and offers significant potential for automating fundus image analysis, reducing reliance on manual examination, and improving patient care efficiency. Our findings are crucial to enhancing diagnostic accuracy and patient outcomes in DED management.

Upregulation of HMOX1 associated with M2 macrophage infiltration and ferroptosis in proliferative diabetic retinopathy.

The roles of immune cell infiltration and ferroptosis in the progression of proliferative diabetic retinopathy (PDR) remain unclear. To identify upregulated molecules associated with immune infiltration and ferroptosis in PDR, GSE60436 and GSE102485 datasets were downloaded from the Gene Expression Omnibus (GEO). Genes associated with immune cell infiltration were examined through Weighted Gene Co-expression Network Analysis (WGCNA) and CIBERSORT algorithm. Common differentially expressed genes (DEGs) were intersected with ferroptosis-associated and immune cell infiltration-related genes. Localization of cellular expression was confirmed by single-cell analysis of GSE165784 dataset. Findings were validated by qRT-PCR, ELISA, Western blotting, and immunofluorescence staining. As a result, the infiltration of M2 macrophages was significantly elevated in fibrovascular membrane samples from PDR patients than the retinas of control subjects. Analysis of DEGs, M2 macrophage-related genes and ferroptosis-related genes identified three hub intersecting genes, TP53, HMOX1 and PPARA. qRT-PCR showed that HMOX1 was significantly higher in the oxygen-induced retinopathy (OIR) mouse model retinas than in controls. Single-cell analysis confirmed that HMOX1 was located in M2 macrophages. ELISA and western blotting revealed elevated levels of HMOX1 in the vitreous humor of PDR patients and OIR retinas, and immunofluorescence staining showed that HMOX1 co-localized with M2 macrophages in the retinas of OIR mice. This study offers novel insights into the mechanisms associated with immune cell infiltration and ferroptosis in PDR. HMOX1 expression correlated with M2 macrophage infiltration and ferroptosis, which may play a crucial role in PDR pathogenesis.

Apolipoprotein-CIII O-Glycosylation Is Associated with Micro- and Macrovascular Complications of Type 2 Diabetes.

Apolipoprotein-CIII (apo-CIII) inhibits the clearance of triglycerides from circulation and is associated with an increased risk of diabetes complications. It exists in four main proteoforms: O-glycosylated variants containing either zero, one, or two sialic acids and a non-glycosylated variant. O-glycosylation may affect the metabolic functions of apo-CIII. We investigated the associations of apo-CIII glycosylation in blood plasma, measured by mass spectrometry of the intact protein, and genetic variants with micro- and macrovascular complications (retinopathy, nephropathy, neuropathy, cardiovascular disease) of type 2 diabetes in a DiaGene study (n = 1571) and the Hoorn DCS cohort (n = 5409). Mono-sialylated apolipoprotein-CIII (apo-CIII1) was associated with a reduced risk of retinopathy (ß = -7.215, 95% CI -11.137 to -3.294) whereas disialylated apolipoprotein-CIII (apo-CIII2) was associated with an increased risk (ß = 5.309, 95% CI 2.279 to 8.339). A variant of the GALNT2-gene (rs4846913), previously linked to lower apo-CIII0a, was associated with a decreased prevalence of retinopathy (OR = 0.739, 95% CI 0.575 to 0.951). Higher apo-CIII1 levels were associated with neuropathy (ß = 7.706, 95% CI 2.317 to 13.095) and lower apo-CIII0a with macrovascular complications (ß = -9.195, 95% CI -15.847 to -2.543). In conclusion, apo-CIII glycosylation was associated with the prevalence of micro- and macrovascular complications of diabetes. Moreover, a variant in the GALNT2-gene was associated with apo-CIII glycosylation and retinopathy, suggesting a causal effect. The findings facilitate a molecular understanding of the pathophysiology of diabetes complications and warrant consideration of apo-CIII glycosylation as a potential target in the prevention of diabetes complications.

Exploring the Associated Genetic Causes of Diabetic Retinopathy as a Model of Inflammation in Retinal Diseases.

To investigate potential biomarkers and biological processes associated with diabetic retinopathy (DR) using transcriptomic and proteomic data. The OmicsPred PheWAS application was interrogated to identify genes and proteins associated with DR and diabetes mellitus (DM) at a false discovery rate (FDR)-adjusted p-value of <0.05 and also <0.005. Gene Ontology PANTHER analysis and STRING database analysis were conducted to explore the biological processes and protein interactions related to the identified biomarkers. The interrogation identified 49 genes and 22 proteins associated with DR and/or DM; these were divided into those uniquely associated with diabetic retinopathy, uniquely associated with diabetes mellitus, and the ones seen in both conditions. The Gene Ontology PANTHER and STRING database analyses highlighted associations of several genes and proteins associated with diabetic retinopathy with adaptive immune response, valyl-TRNA aminoacylation, complement activation, and immune system processes. Our analyses highlight potential transcriptomic and proteomic biomarkers for DR and emphasize the association of known aspects of immune response, the complement system, advanced glycosylation end-product formation, and specific receptor and mitochondrial function with DR pathophysiology. These findings may suggest pathways for future research into novel diagnostic and therapeutic strategies for DR.

Diabetic Nephropathy, Retinopathy, and Functional Hypogonadism in a Patient with MODY10: A Case Report.

(1) Background and objectives: Maturity-onset diabetes of the young (MODY) is a group of diabetes caused by gene defects related to insulin secretion. MODY1, MODY2, and MODY3 are the most common and account for approximately 80% of all cases. Other types are relatively rare. This study describes the clinical, analytical, and genetic characteristics of a patient with MODY10, and diabetic nephropathy, retinopathy, and functional hypogonadism diagnosis. (2) Materials and methods: A clinical case was analyzed and whole exome generation sequencing (WES) was used to detect mutations related to a monogenic variant. (3) Results: A seventeen-year-old male patient, who was diagnosed with apparent type 1 diabetes at the age of eight was started with insulin therapy. He came to the emergency room with glycemic decompensation, facial, and lower limb edema. During his evaluation, he had near-nephrotic range proteinuria of 2902 mg/24 h, a kidney ultrasound showing mild pyelocalyceal dilation, proliferative diabetic retinopathy, and was also diagnosed with functional hypogonadotropic hypogonadism. These comorbidities improved with adequate glycemic control. WES showed missense variant c.94G>A (p.Gly32Ser) in the INS gene, according to Clinvar corresponding to MODY10. It was a "de novo" variant not reported in his parents. (4) Conclusions: Monogenic diabetes (MD) is rare and MODY10 is among the less frequent types. MODY should be suspected in patients with type 1 phenotype with negative autoimmunity even in the absence of a family history of diabetes. To the best of our knowledge, we present here the first patient with these phenotypic traits of MODY10 reported in Latin America.

NSUN2 affects diabetic retinopathy progression by regulating MUC1 expression through RNA m5C methylation.

BACKGROUND: Diabetic retinopathy (DR) is the leading cause of blinding eye disease among working adults and is primarily attributed to the excessive proliferation of microvessels, which leads to vitreous hemorrhage and retinal traction, thereby significantly impairing patient vision. NSUN2-mediated RNA m5C methylation is implicated in various diseases, and in this investigation, we focused on elucidating the impact of NSUN2 on the regulation of the expression of the downstream gene MUC1, specifically through RNA m5C methylation, on the progression of DR. METHOD: Utilizing Microarray analysis, we examined patient vitreous fluid to pinpoint potential therapeutic targets for DR. Differential expression of NSUN2 was validated through qRT-PCR, Western blot, and immunofluorescence in human tissue, animal tissue, and cell model of DR. The relationship between NSUN2 and DR was explored in vitro and in vivo through gene knockdown and overexpression. Various techniques, such as MeRIP-qPCR and dot blot, were applied to reveal the downstream targets and mechanism of action of NSUN2. RESULTS: The levels of both NSUN2 and RNA m5C methylation were significantly elevated in the DR model. Knockdown of NSUN2 mitigated DR lesion formation both in vitro and in vivo. Mechanistically, NSUN2 promoted MUC1 expression by binding to the RNA m5C reader ALYREF. Knockdown of ALYREF resulted in DR lesion alterations similar to those observed with NSUN2 knockdown. Moreover, MUC1 overexpression successfully reversed a series of DR alterations induced by NSUN2 silencing. CONCLUSIONS: NSUN2 regulates the expression of MUC1 through ALYREF-mediated RNA m5C methylation, thereby regulating the progression of DR and providing a new option for the treatment of DR in the future.

Causal association of circulating metabolites with diabetic retinopathy: a bidirectional Mendelian randomization analysis.

Introduction: The retina is a highly metabolically active tissue, and there is a lack of clarity about the relationship between metabolites and diabetic retinopathy (DR). This study used two-sample bidirectional Mendelian randomization (MR) analyses to identify causal relationships between metabolites and DR. Methods: Genetic variants were selected from the open-access Genome-Wide Association Studies (GWAS) summary database as proxies for the 1400 most recently published metabolites. MR analysis was performed to examine associations between these metabolite traits and DR. Single nucleotide polymorphism (SNP) data that were significantly associated with exposure were screened through association analysis. Validated instrumental variables (IVs) were obtained by removing SNPs with linkage disequilibrium (LD) and F-statistic values below 10. MR analyses were performed using the inverse variance weighted (IVW) method as the primary approach. The robustness of the results was verified by sensitivity analyses, including assessments of heterogeneity, horizontal pleiotropy, and the leave-one-out method. Results: In the IVW approach and in the primary analysis of several sensitivity analyses, genetically determined glycolithocholate sulfate levels, androstenediol (3 beta, 17 beta) monosulfate (1) levels, 1-stearoyl-2-arachidonoyl-GPE (18:0/20:4) levels, 1-oleoyl-2-arachidonoyl-GPE (18:1/20:4) levels, 1-oleoyl-2-linoleoyl-GPE (18:1/18:2) levels, X-26109 levels, N6-methyllysine levels, (N6,N6-dimethyllysine levels), and (N2-acetyl,N6,N6-dimethyllysine levels) were negatively associated with the risk of DR. 5-hydroxymethyl-2-furoylcarnitine levels and the glutamate-to-alanine ratio were positively associated with the risk of DR. No reverse causal association was found between DR and metabolites. Discussion: This MR study suggests that nine metabolites may have a protective effect in DR, while two metabolites may be associated with an increased risk of DR. However, further research is needed to confirm these findings. Supplementation with beneficial metabolites may reduce DR risk and could potentially be a novel therapeutic approach to DR treatment.

Unveiling the molecular complexity of proliferative diabetic retinopathy through scRNA-seq, AlphaFold 2, and machine learning.

Background: Proliferative diabetic retinopathy (PDR), a major cause of blindness, is characterized by complex pathogenesis. This study integrates single-cell RNA sequencing (scRNA-seq), Non-negative Matrix Factorization (NMF), machine learning, and AlphaFold 2 methods to explore the molecular level of PDR. Methods: We analyzed scRNA-seq data from PDR patients and healthy controls to identify distinct cellular subtypes and gene expression patterns. NMF was used to define specific transcriptional programs in PDR. The oxidative stress-related genes (ORGs) identified within Meta-Program 1 were utilized to construct a predictive model using twelve machine learning algorithms. Furthermore, we employed AlphaFold 2 for the prediction of protein structures, complementing this with molecular docking to validate the structural foundation of potential therapeutic targets. We also analyzed protein-protein interaction (PPI) networks and the interplay among key ORGs. Results: Our scRNA-seq analysis revealed five major cell types and 14 subcell types in PDR patients, with significant differences in gene expression compared to those in controls. We identified three key meta-programs underscoring the role of microglia in the pathogenesis of PDR. Three critical ORGs (ALKBH1, PSIP1, and ATP13A2) were identified, with the best-performing predictive model demonstrating high accuracy (AUC of 0.989 in the training cohort and 0.833 in the validation cohort). Moreover, AlphaFold 2 predictions combined with molecular docking revealed that resveratrol has a strong affinity for ALKBH1, indicating its potential as a targeted therapeutic agent. PPI network analysis, revealed a complex network of interactions among the hub ORGs and other genes, suggesting a collective role in PDR pathogenesis. Conclusion: This study provides insights into the cellular and molecular aspects of PDR, identifying potential biomarkers and therapeutic targets using advanced technological approaches.

Role of Hsa_circ_0000880 in the Regulation of High Glucose-Induced Apoptosis of Retinal Microvascular Endothelial Cells.

Purpose: Circular RNAs (circRNAs) have been verified to participate in multiple biological processes and disease progression. Yet, the role of circRNAs in the pathogenesis of diabetic retinopathy (DR) is still poorly understood and deserves further study. This study aimed to investigate the role of circRNAs in the regulation of high glucose (HG)-induced apoptosis of retinal microvascular endothelial cells (RMECs). Methods: Epiretinal membranes from patients with DR and nondiabetic patients with idiopathic macular epiretinal membrane were collected for this study. The circRNA microarrays were performed using high-throughput sequencing. Hierarchical clustering, functional enrichment, and network regulation analyses were used to analyze the data generated by high-throughput sequencing. Next, RMECs were subjected to HG (25 mM) conditions to induce RMECs apoptosis in vitro. A series of experiments, such as Transwell, the Scratch wound, and tube formation, were conducted to explore the regulatory effect of circRNA on RMECs. Fluorescence in situ hybridization (FISH), immunofluorescence staining, and Western blot were used to study the mechanism underlying circRNA-mediated regulation. Results: A total of 53 differentially expressed circRNAs were found in patients with DR. Among these, hsa_circ_0000880 was significantly upregulated in both the diabetic epiretinal membranes and in an in vitro DR model of HG-treated RMECs. Hsa_circ_0000880 knockout facilitated RMECs vitality and decreased the paracellular permeability of RMECs under hyperglycemia. More importantly, silencing of hsa_circ_0000880 significantly inhibited HG-induced ROS production and RMECs apoptosis. Hsa_circ_0000880 acted as an endogenous sponge for eukaryotic initiation factor 4A-III (EIF4A3). Knockout of hsa_circ_0000880 reversed HG-induced decrease in EIF4A3 protein level. Conclusions: Our findings suggest that hsa_circ_0000880 is a novel circRNA can induce RMECs apoptosis in response to HG conditions by sponging EIF4A3, offering an innovative treatment approach against DR. Translational Relevance: The circRNAs participate in the dysregulation of microvascular endothelial function induced by HG conditions, indicating a promising therapeutic target for DR.

EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells.

Purpose: To examine whether increased ephrin type-B receptor 1 (EphB1) leads to inflammatory mediators in retinal Müller cells. Methods: Diabetic human and mouse retinal samples were examined for EphB1 protein levels. Rat Müller cells (rMC-1) were grown in culture and treated with EphB1 siRNA or ephrin B1-Fc to explore inflammatory mediators in cells grown in high glucose. An EphB1 overexpression adeno-associated virus (AAV) was used to increase EphB1 in Müller cells in vivo. Ischemia/reperfusion (I/R) was performed on mice treated with the EphB1 overexpression AAV to explore the actions of EphB1 on retinal neuronal changes in vivo. Results: EphB1 protein levels were increased in diabetic human and mouse retinal samples. Knockdown of EphB1 reduced inflammatory mediator levels in Müller cells grown in high glucose. Ephrin B1-Fc increased inflammatory proteins in rMC-1 cells grown in normal and high glucose. Treatment of mice with I/R caused retinal thinning and loss of cell numbers in the ganglion cell layer. This was increased in mice exposed to I/R and treated with the EphB1 overexpressing AAVs. Conclusions: EphB1 is increased in the retinas of diabetic humans and mice and in high glucose-treated Müller cells. This increase leads to inflammatory proteins. EphB1 also enhanced retinal damage in response to I/R. Taken together, inhibition of EphB1 may offer a new therapeutic option for diabetic retinopathy.

Transcriptional patterns of human retinal pigment epithelial cells under protracted high glucose.

BACKGROUND: The retinal pigment epithelium (RPE) is essential for retinal homeostasis. Comprehensively exploring the transcriptional patterns of diabetic human RPE promotes the understanding of diabetic retinopathy (DR). METHODS AND RESULTS: A total of 4125 differentially expressed genes (DEGs) were screened out from the human primary RPE cells subjected to prolonged high glucose (HG). The subsequent bioinformatics analysis is divided into 3 steps. In Step 1, 21 genes were revealed by intersecting the enriched genes from the KEGG, WIKI, and Reactome databases. In Step 2, WGCNA was applied and intersected with the DEGs. Further intersection based on the enrichments with the GO biological processes, GO cellular components, and GO molecular functions databases screened out 12 candidate genes. In Step 3, 13 genes were found to be simultaneously up-regulated in the DEGs and a GEO dataset involving human diabetic retinal tissues. VEGFA and ERN1 were the 2 starred genes finally screened out by overlapping the 3 Steps. CONCLUSION: In this study, multiple genes were identified as crucial in the pathological process of RPE under protracted HG, providing potential candidates for future researches on DR. The current study highlights the importance of RPE in DR pathogenesis.

Assessing the possible association between MTHFR (rs1801133) and GPx-1 (rs1050450) polymorphisms with the risk of type 2 diabetes, diabetic neuropathy, and diabetic retinopathy.

PURPOSE: Oxidative stress in chronic hyperglycemia could injure the tissues and onset of diabetes-related complications like retinopathy and neuropathy. This study investigates the association between methylenetetrahydrofolate reductase (MTHFR) and glutathione peroxidase (GPx) genetic variants with these complications. METHODS: In this case-control study, 400 individuals, including 100 healthy subjects and 300 patients with type 2 diabetes mellitus (T2DM) in three subgroups: with retinopathy(n = 100), with neuropathy(n = 100), and without complication (n = 100) from West Iran, were studied. MTHFR (rs1801133) and GPx-1 (rs1050450) variants were identified by the PCR-RFLP method. The plasma levels of GPx activity, glutathione, malondialdehyde (MDA), total antioxidant capacity (TAC), and total oxidative stress (TOS) were measured by chemical methods. RESULTS: Higher BMI, TOS and MDA levels were observed in patients with neuropathy compared to other patients and controls. Diabetic patients with neuropathy had lower levels of glutathione (7.8 ± 4.5; P < 0.001), GPx activity (39.5 ± 8.5; P < 0.001), and TAC (703.1 ± 129.1; P = 0.0001) in comparison with other groups. The patients without complication and retinopathic patients had higher plasma levels of glutathione (12.2 ± 2.4; p = 0.02) and TAC (793.4 ± 124.6; P < 0.001), respectively. MTHFR TT genotype significantly correlated with lower levels of TOS (3.5 ± 1.1; P < 0.001) and OSI (0.0050 ± 0.001; P < 0.001). Subjects with the GPx-1 TT genotype had higher levels of MDA (6.8 ± 2.5; P = 0.02) and lower levels of TOS (3.7 ± 1.6; P < 0.001), which is statistically significant. TT genotype of MTHFR was associated with 3.9 fold (95% CI 1.04-4.76; P = 0.0436) increased risk of neuropathy. Also, GPx-1 CT genotype increased the risk of retinopathy [OR = 2.7 (95% CI = 1.38-5.44; P = 0.0039)]. CONCLUSION: The MTHFR TT genotype increased the risk of neuropathy in diabetic patients significantly. The GPx-1 CT genotype is related to increased retinopathy risk among diabetic patients. Both MTHFR and Gpx-1 TT genotypes were associated with higher BMI levels.

MSC-derived small extracellular vesicles mitigate diabetic retinopathy by stabilizing Nrf2 through miR-143-3p-mediated inhibition of neddylation.

Diabetic retinopathy (DR) is a highly hazardous and widespread complication of diabetes mellitus (DM). The accumulated reactive oxygen species (ROS) play a central role in DR development. The aim of this research was to examine the impact and mechanisms of mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEV) on regulating ROS and retinal damage in DR. Intravitreal injection of sEV inhibited Cullin3 neddylation, stabilized Nrf2, decreased ROS, reduced retinal inflammation, suppressed Müller gliosis, and mitigated DR. Based on MSC-sEV miRNA sequencing, bioinformatics software, and dual-luciferase reporter assay, miR-143-3p was identified to be the key effector for MSC-sEV's role in regulating neural precursor cell expressed developmentally down-regulated 8 (NEDD8)-mediated neddylation. sEV were able to be internalized by Müller cells. Compared to advanced glycation end-products (AGEs)-induced Müller cells, sEV coculture decreased Cullin3 neddylation, activated Nrf2 signal pathway to combat ROS-induced inflammation. The barrier function of endothelial cells was impaired when endothelial cells were treated with the supernatant of AGEs-induced Müller cells, but was restored when treated with supernatant of AGEs-induced Müller cells cocultured with sEV. The protective effect of sEV was, however, compromised when miR-143-3p was inhibited in sEV. Moreover, the protective efficacy of sEV was diminished when NEDD8 was overexpressed in Müller cells. These findings showed MSC-sEV delivered miR-143-3p to inhibit Cullin3 neddylation, stabilizing Nrf2 to counteract ROS-induced inflammation and reducing vascular leakage. Our findings suggest that MSC-sEV may be a potential nanotherapeutic agent for DR, and that Cullin3 neddylation could be a new target for DR therapy.