Small-molecule agonist AdipoRon alleviates diabetic retinopathy through the AdipoR1/AMPK/EGR4 pathway.

BACKGROUND: Diabetes mellitus (DM) is a progressive disease that involves multiple organs due to increased blood glucose, and diabetic retinopathy (DR) is the main complication of DM in the eyes and causes irreversible vision loss. In the pathogenesis of diabetic vascular disease, oxidative stress caused by hyperglycemia plays an important role in Müller cell impairment. In recent years, AdipoRon, an adiponectin analog that demonstrated important physiological functions in obesity, diabetes, inflammation, and cardiovascular diseases, demonstrated cellular protection from apoptosis and reduced inflammatory damage through a receptor-dependent mechanism. Here, we investigated how AdipoRon reduced oxidative stress and apoptosis in Müller glia in a high glucose environment. RESULTS: By binding to adiponectin receptor 1 on Müller glia, AdipoRon activated 5' adenosine monophosphate-activated protein kinase (AMPK)/acetyl-CoA carboxylase phosphorylation downstream, thereby alleviating oxidative stress and eventual apoptosis of cells and tissues. Transcriptome sequencing revealed that AdipoRon promoted the synthesis and expression of early growth response factor 4 (EGR4) and inhibited the cellular protective effects of AdipoRon in a high-glucose environment by reducing the expression of EGR4. This indicated that AdipoRon played a protective role through the EGR4 and classical AMPK pathways. CONCLUSIONS: This provides a new target for the early treatment of DR.

Integration of Vitreous Lipidomics and Metabolomics for Comprehensive Understanding of the Pathogenesis of Proliferative Diabetic Retinopathy.

As a vision-threatening complication of diabetes mellitus (DM), proliferative diabetic retinopathy (PDR) is associated with sustained metabolic disorders. Herein, we collected the vitreous cavity fluid of 49 patients with PDR and 23 control subjects without DM for metabolomics and lipidomics analyses. Multivariate statistical methods were performed to explore relationships between samples. For each group of metabolites, gene set variation analysis scores were generated, and we constructed a lipid network by using weighted gene co-expression network analysis. The association between lipid co-expression modules and metabolite set scores was investigated using the two-way orthogonal partial least squares (O2PLS) model. A total of 390 lipids and 314 metabolites were identified. Multivariate statistical analysis revealed significant vitreous metabolic and lipid differences between PDR and controls. Pathway analysis showed that 8 metabolic processes might be associated with the development of PDR, and 14 lipid species were found to be altered in PDR patients. Combining metabolomics and lipidomics, we identified fatty acid desaturase 2 (FADS2) as an important potential contributor to the pathogenesis of PDR. Collectively, this study integrates vitreous metabolomics and lipidomics to comprehensively unravel metabolic dysregulation and identifies genetic variants associated with altered lipid species in the mechanistic pathways for PDR.

MEK1-dependent MondoA phosphorylation regulates glucose uptake in response to ketone bodies in colorectal cancer cells.

The Mondo family transcription factor MondoA plays a pivotal role in sensing metabolites, such as glucose, glutamine, and lactic acid, to regulate glucose metabolism and cell proliferation. Ketone bodies are important signals for reducing glucose uptake. However, it is unclear whether MondoA functions in ketone body-regulated glucose transport. Here we reported that ketone bodies promoted MondoA nuclear translocation and binding to the promoter of its target gene TXNIP. Ketone bodies reduced glucose uptake, increased apoptosis and decreased proliferation of colorectal cancer cells, which was impeded by MondoA knockdown. Moreover, we identified MEK1 as a novel component of the MondoA protein complex using a proteomic approach. Mechanistically, MEK1 interacted with MondoA and enhanced tyrosine 222, but not serine or threonine, phosphorylation of MondoA, inhibiting MondoA nuclear translocation and transcriptional activity. Ketone bodies decreased MEK1-dependent MondoA phosphorylation by blocking MondoA and MEK1 interaction, leading to MondoA nuclear translocation, TXNIP transcription, and inhibition of glucose uptake. Therefore, our study not only demonstrated that ketone bodies reduce glucose uptake, promote apoptosis, and inhibit cell proliferation in colorectal cancer cells by regulating MondoA phosphorylation but also identified MEK1-dependent phosphorylation as a new mechanism to manipulate MondoA activity.

Serum Ang-1/Ang-2 ratio may be a promising biomarker for evaluating severity of diabetic retinopathy.

PURPOSE: To investigate the predictive role of serum angiopoietin-1 and angiopoietin-2 (Ang-1/Ang-2) in evaluating the severity of diabetic retinopathy (DR). METHODS: A total of 101 outpatients with type 2 diabetes mellitus (T2DM) were recruited and were further divided into the following five groups: T2DM without DR (non-DR), mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR and proliferative DR (PDR) in accordance with the International Clinical Diabetic Retinopathy Guidelines. Furthermore, 101 serum samples were included in the further analysis using enzyme-linked immunosorbent assays. A receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of each index. RESULTS: The expression of Ang-1 in the PDR group was significantly lower than that in the non-DR group, while Ang-2 showed an opposite upward trend (p < 0.05). The Ang-1/Ang-2 ratio of the non-DR group was significantly lower than that of the moderate NPDR, severe NPDR and PDR (p < 0.05, p < 0.01 and p < 0.01, respectively). Differences in the Ang-1/Ang-2 ratio were observed earlier than those in the individual Ang-1 and Ang-2 measurements. The maximal Youden index was 0.512 with a calculated area under the curve (AUC) value of 0.734 (p < 0.01). CONCLUSIONS: The Ang-1/Ang-2 ratio was helpful in assessing the severity of DR and may provide potential clinical benefits as a biomarker and early warning signs for DR diagnosis.

Protective effect of pentraxin 3 on pathological retinal angiogenesis in an in vitro model of diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is the most common retinal microvascular disease caused by diabetes. Previous studies indicated that Pentraxin 3 (PTX3), an acute phase reactant, was closely related to the development of DR. But the exact effect of PTX3 in diabetic retinopathy needs more investigations. METHODS: Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) analysis and western blot (WB) were used to detect the expression of PTX3 in vitro. The Ki67 immunofluorescent staining, scratch-wound migration assay, and tube formation experiments were performed to detect the effect of PTX3 knockdown and overexpression on the fibroblast growth factor (FGF)-induced proliferation, migration and tube-forming ability of human retinal microvascular endothelial cells (HRMECs). The phosphorylation levels of extracellular regulated protein kinases (ERK) and fibroblast growth factor receptor (FGFR) in HRMECs were detected by WB. RESULTS: In vitro, the mRNA and protein expressions of PTX3 in the high-concentration glucose condition group were upregulated compared with the normal group (p < 0.05). The proliferation, migration and tube-forming abilities of HRMECs exposed to high-concentration glucose were enhanced (p < 0.01, p < 0.01, p < 0.05 respectively), and the phosphorylation of FGFR and ERK1/2 were increased (p < 0.01, p < 0.05 respectively) compared with the normal condition group. Compared with the high glucose condition group, the proliferation, migration and tube-forming abilities of HRMECs in the high glucose + PTX3 siRNA condition group were further strengthened (p < 0.001, p < 0.0001, p < 0.05 respectively), and the phosphorylation of FGFR and ERK1/2 were increased (p < 0.001, p < 0.01 respectively). Compared with the high glucose condition group, the proliferation, migration and tube-forming abilities of HRMECs in the high glucose + PTX3 overexpression condition group were compromised (p < 0.001, p < 0.05, p < 0.01 respectively), and the phosphorylation of FGFR and ERK1/2 were inhibited (p < 0.001, p < 0.0001 respectively). Neither the scramble siRNA condition group nor the blank plasmid condition group showed significant difference on the proliferation, migration and tube-forming abilities of HRMECs compared with the high glucose condition group (p > 0.05). CONCLUSIONS: The upregulated expression of PTX3 may play a protective role on pathological angiogenesis in DR. PTX3 may serve as a new target for the treatment of DR.

Endomucin restores depleted endothelial glycocalyx in the retinas of streptozotocin-induced diabetic rats.

Endothelial glycocalyx plays a significant role in the development and progression of diabetic complications. Endomucin (EMCN) is an anti-inflammatory membrane glycoprotein that is mainly expressed in venous and capillary endothelial cells. However, the function of EMCN in diabetic retinopathy (DR) progression is still completely unknown. We first investigated the change of EMCN expression in the retina and human retinal microvascular endothelial cells. We then overexpressed EMCN in the retina with adeno-associated virus and induced DR with streptozotocin (STZ). We analyzed EMCN's effect on the integrity of endothelial glycocalyx under conditions of DR. Furthermore, we investigated EMCN's protective effect against inflammation and blood-retinal barrier (BRB) destruction. We found that EMCN is specifically expressed in retinal endothelial cells and that its levels are decreased during hyperglycemia in vitro and in vivo. Overexpression of EMCN can restore the retinal endothelial glycocalyx of STZ-induced diabetic rats. Furthermore, EMCN overexpression can decrease leukocyte-endothelial adhesion to ameliorate inflammation and stabilize the BRB to inhibit vessel leakage in rats with DR. EMCN may protect patients with diabetes from retinal vascular degeneration by restoring the endothelial glycocalyx. EMCN may thus represent a novel therapeutic strategy for DR because it targets endothelial glycocalyx degradation associated with this disease.-Niu, T., Zhao, M., Jiang, Y., Xing, X., Shi, X., Cheng, L., Jin, H., Liu, K. Endomucin restores depleted endothelial glycocalyx in the retinas of streptozotocin-induced diabetic rats.

O- glycosylation can regulate the proliferation and migration of human retinal microvascular endothelial cells through ZFR in high glucose condition.

PURPOSE: Angiogenesis is an essential part of the diabetes retinopathy (DR) process, and Zinc Finger RNA Binding Protein (ZFR) is important for vascularization to occur. However, the function and regulation of ZFR in DR development are not well understood. We hypothesized that high glucose condition could result in ZFR up-regulation in human retinal microvascular endothelial cells (HRMECs), thus contributing to disease progression, and O-glycosylation may be participated in this regulation. METHODS: Retinas were harvested from streptozotocin (STZ)-induced rat model of diabetes. Human umbilical vein endothelial cells (HUVECs) and HRMECs cultured in high glucose concentration, and retinal tissues were detected for ZFR expression. We examined the role of ZFR on vasculogenic processes including proliferation and migration in the cell model of DR. The effect of O-glycosylation modification on ZFR was further assessed in HRMECs. RESULTS: Expression of ZFR was up-regulated in high glucose condition both in vitro and in vivo. ZFR induced proliferation and migration of HRMECs. Inhibition of O-glycosylation modification attenuated the expression of ZFR. CONCLUSION: ZFR plays an important role in the pathogenesis of DR, and its mechanism may through the modification of O-glycosylation. Our research suggests that ZFR may be used as a potential prognostic marker or potential therapeutic target for DR.

RUNX1 can mediate the glucose and O-GlcNAc-driven proliferation and migration of human retinal microvascular endothelial cells.

INTRODUCTION: This study aims to determine whether high glucose condition and dynamic O-linked N-acetylglucosamine (O-GlcNAc) modification can promote the proliferation and migration of human retinal microvascular endothelial cells (HRMECs) and whether Runt-related transcription factor 1 (RUNX1) could mediate the glucose and O-GlcNAc-driven proliferation and migration of HRMECs. RESEARCH DESIGN AND METHODS: Western blot analysis was used to detect the O-GlcNAc modification level and RUNX1 level in cells and retina tissues, cell growth was studied by cell counting kit-8 assay, cell proliferation was detected by immunofluorescence staining. Then, cell migration and tube formation were investigated by scratch-wound assay, Transwell assay, and tube-forming assay. The changes of retinal structure were detected by H&E staining. The O-GlcNAc modification of RUNX1 was detected by immunoprecipitation. RESULTS: High glucose increases pan-cellular O-GlcNAc modification and the proliferation and migration of HRMECs. Hence, O-GlcNAc modification is critical for the proliferation and migration of HRMECs. RUNX1 mediates the glucose and O-GlcNAc-driven proliferation and migration in HRMECs. RUNX1 can be modified by O-GlcNAc, and that the modification is enhanced in a high glucose environment. CONCLUSIONS: The present study reveals that high glucose condition directly affects retinal endothelial cells (EC) function, and O-GlcNAc modification is critical for the proliferation and migration of HRMECs, RUNX1 may take part in this mechanism, and maybe the function of RUNX1 is related to its O-GlcNAc modification level, which provides a new perspective for studying the mechanism of RUNX1 in diabetic retinopathy.

Pathogenesis Study Based on High-Throughput Single-Cell Sequencing Analysis Reveals Novel Transcriptional Landscape and Heterogeneity of Retinal Cells in Type 2 Diabetic Mice.

Diabetic retinopathy (DR) is the leading cause of acquired blindness in middle-aged people. The complex pathology of DR is difficult to dissect, given the convoluted cytoarchitecture of the retina. Here, we performed single-cell RNA sequencing (scRNA-seq) of retina from a model of type 2 diabetes, induced in leptin receptor-deficient (db/db) and control db/m mice, with the aim of elucidating the factors mediating the pathogenesis of DR. We identified 11 cell types and determined cell-type-specific expression of DR-associated loci via genome-wide association study (GWAS)-based enrichment analysis. DR also impacted cell-type-specific genes and altered cell-cell communication. Based on the scRNA-seq results, retinaldehyde-binding protein 1 (RLBP1) was investigated as a promising therapeutic target for DR. Retinal RLBP1 expression was decreased in diabetes, and its overexpression in Müller glia mitigated DR-associated neurovascular degeneration. These data provide a detailed analysis of the retina under diabetic and normal conditions, revealing new insights into pathogenic factors that may be targeted to treat DR and related dysfunctions.

Identification of novel differentially expressed genes in retinas of STZ-induced long-term diabetic rats through RNA sequencing.

BACKGROUND: The aim of this research was to investigate the retinal transcriptome changes in long-term streptozotocin (STZ)-induced rats' retinas using RNA sequencing (RNA-seq), to explore the molecular mechanisms of diabetic retinopathy (DR), and to identify novel targets for the treatment of DR by comparing the gene expression profile we obtained. METHODS: In this study, 6 healthy male SD rats were randomly divided into wild-type (WT) group and streptozotocin (STZ)-induced group, 3 rats each group. After 6 months, 3 normal retina samples and 3 DM retina samples (2 retinas from the same rat were considered as 1 sample) were tested and differentially expressed genes (DEGs) were measured by RNA-seq technology. Then, we did Gene Ontology (GO) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and validated the results of RNA-seq through qRT-PCR. RESULTS: A total of 118 DEGs were identified, of which 72 were up-regulated and 46 were down-regulated. The enriched GO terms showed that 3 most significant enrichment terms were binding (molecular function), cell part (cellular component), and biological regulation (biological process). The results of the KEGG pathway analysis revealed a significant enrichment in cell adhesion molecules, PI3K-Akt signaling pathway, and allograft rejection, etc. CONCLUSION: Our research has identified specific DEGs and also speculated their potential functions, which will provide novel targets to explore the molecular mechanisms of DR.

The Anti-Inflammatory Effect of KS23, A Novel Peptide Derived From Globular Adiponectin, on Endotoxin-Induced Uveitis in Rats.

Purpose: Adiponectin has been shown to exert potent anti-inflammatory activities in a range of systemic inflammatory diseases. This study aimed to investigate the potential therapeutic effects of KS23, a globular adiponectin-derived peptide, on endotoxin-induced uveitis (EIU) in rats and lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW 264.7 cells. Methods: EIU was induced in Lewis rats by subcutaneous injection of LPS into a single footpad. KS23 or phosphate-buffered saline (PBS) was administered immediately after LPS induction via intravitreal injection. Twenty-four hours later, clinical and histopathological scores were evaluated, and the aqueous humor (AqH) was collected to determine the infiltrating cells, protein concentration, and levels of inflammatory cytokines. In vitro, cultured RAW 264.7 cells were stimulated with LPS in the presence or absence of KS23, inflammatory cytokine levels in the supernatant, nuclear translocation of nuclear factor kappa B (NF-κB) subunit p65, and the expression of NF-kB signaling pathway components were analyzed. Results: KS23 treatment significantly ameliorated the clinical and histopathological scores of EIU rats and reduced the levels of infiltration cells, protein, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the aqueous humor. Consistently, KS23 decreased the expression of TNF-α and IL-6 in the supernatant of LPS-stimulated RAW 264.7 cells and inhibited the LPS-induced nuclear translocation of NF-κB p65 and the phosphorylation of IKKα/ß/IκBα/NF-κB. Conclusion: The in vivo and in vitro results demonstrated the anti-inflammatory effects of the peptide KS23 and suggested that KS23 is a compelling, novel therapeutic candidate for the treatment of ocular inflammation.