Inflammation In Diabetic Retinopathy Research Articles

Modulation of diabetes-related retinal pathophysiology by PTX3

Diabetic retinopathy (DR) is a common complication of diabetes characterized by vascular pathology and neuroinflammation. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule that functions at the crossroads between innate immunity, inflammation, and tissue remodeling. DR is known to involve inflammatory pathways, although the potential relevance of PTX3 has not been explored. We found that PTX3 protein levels increased in the retina of diabetic mice. Similarly, evaluation of a publicly available transcriptomic human dataset revealed increased PTX3 expression in DR with diabetic macular edema and proliferative retinopathy, when compared to nondiabetic retinas or diabetic retinas without complications. To further understand the role of PTX3 within DR, we employed the streptozotocin-induced diabetes model in PTX3 knockout mice (PTX3KO), which were followed up for 9 mo to evaluate hallmarks of disease progression. In diabetic PTX3KO mice, we observed decreased reactive gliosis, diminished microglia activation, and reduced vasodegeneration, when compared to diabetic PTX3 wild-type littermates. The decrease in DR-associated pathological features in PTX3KO retinas translated into preserved visual function, as evidenced by improved optokinetic response, restored b-wave amplitude in electroretinograms, and attenuated neurodegeneration. We showed that PTX3 induced an inflammatory phenotype in human retinal macroglia, characterized by GFAP upregulation and increased secretion of IL6 and PAI-1. We confirmed that PTX3 was required for TNF-α-induced reactive gliosis, as PTX3KO retinal explants did not up-regulate GFAP in response to TNF-α. This study reveals a unique role for PTX3 as an enhancer of sterile inflammation in DR, which drives pathogenesis and ultimately visual impairment.

Bidirectional link between periodontitis and systemic inflammation in diabetic retinopathy.

Periodontitis is independently associated with numerous lifestyle diseases. Diabetic patients have approximately threefold increased odds of periodontitis, which in turn increases the risk of systemic inflammation. The study by Thazhe Poyil et al is an effort to establish the inflammatory link between diabetic re-tinopathy (DR) and periodontitis based on the periodontal inflamed surface area in diabetic patients with and without DR. To further advance the study, we suggest refining the eligibility criteria to explicitly state the clinical correlates of periodontitis and DR, larger sample size and improved sampling methodology, matching of baseline characteristics of the two groups, as well as improved statistical approach and interpretation of the study findings. Measurement of hemoglobin A1c (HbA1c) in studies comparing type 2 diabetes mellitus patients with DR of matched severity with and without periodontitis could provide a clearer picture of whether HbA1c level is indeed influenced by periodontitis.

Nerolidol Alleviates Oxidative Stress and Inflammation in Diabetic Retinopathy Mediated by High Glucose through Ameliorating Nrf2/HO-1 Pathway

Background Diabetic retinopathy (DR) is the foremost microvascular problem that causes drastic visual impairment in diabetes patients. Hyperglycemia-triggered reaction cascade of inflammation and oxidative stress constitute the DR pathogenesis. The existing treatment options are not completely satisfactory. Materials and Methods We investigated the cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, inflammatory mediators, lactate dehydrogenase (LDH), superoxide dismutase, glutathione, and malonaldehyde (MDA) levels by ELISA and qRT-PCR assay, protein expression of Nrf2 and heme oxygenase-1 (HO-1) by western blotting assay were analyzed. Results According to our research, nerolidol (NRD) increases the proliferation and antioxidant activity of human retinal endothelial cells (HRECs) by inducing Nrf2/HO-1 signaling, while attenuating MDA, an oxidative stress marker, LDH, and inflammatory mediators. These outcomes suggest that a substantial reaction of inflammation and oxidative stress injury happened in DR, which might be correlated to the instigation of the signaling Nrf2/HO-1. Conclusion NRD effectively suppresses oxidative stress and inflammation in HG-induced HRECs. The primary mechanism of NRD on DR may be linked to the activation of the Nrf2/HO-1 pathway and may give a useful medicine for DR treatment.

The role of inflammation in diabetic retinopathy in patients with type II diabetes; potential therapeutic perspectives

The role of inflammation in diabetic retinopathy in patients with type II diabetes; potential therapeutic perspectives

Mm9_circ_014683 regulates microglia polarization through canonical NFκB signaling pathway in diabetic retinopathy

Diabetic retinopathy (DR) is still the major cause of visual loss in working-aged people, one of the critical pathological processes are retinal microglia-mediated inflammation. Our previous study demonstrated that enhanced M1 microglial polarization was involved in retinal inflammation in DR, but the detailed mechanism needs further investigation. Circular RNAs (circRNAs) are important kind of noncoding RNAs involved in the regulation of various cell biological processes. Herein, the circRNA expression profiles of BV2 mouse microglia treated with or without glucose were detected, and a total of 347 differentially expressed circRNAs were identified in glucose-treated BV2 cells. The key circRNA mm9_circ_014683 increased after glucose stimulation. Inhibiting or overexpressing mm9_circ_014683 showed no effect on the proliferation and apoptosis of microglia. Inhibiting mm9_circ_014683 impeded M1 polarization and promoted M2 polarization, and overexpressing mm9_circ_014683 showed the opposite effect. A total of 216 differentially expressed genes were identified in mm9_circ_014683-knockdown BV2 cells, which were enriched in several signaling pathways, including the NFκB signaling pathway. Moreover, mm9_circ_014683 positively regulated the canonical, NFκB signaling pathway. Besides, mm9_circ_014683 was highly expressed in the retinal microglia of diabetic mice, and intraocular injection of Lv-circRNA inhibited M1 but enhanced M2 retinal microglial polarization. In conclusion, mm9_circ_014683 regulates microglial polarization through the canonical NFκB signaling pathway in diabetic retinopathy. This study may provide insight into the pathogenesis and treatment of DR.

MiR-26a-5p Attenuates Oxidative Stress and Inflammation in Diabetic Retinopathy through the USP14/NF-κB Signaling Pathway.

Diabetic retinopathy (DR) is an ocular disease caused by diabetes and may lead to vision impairment and even blindness. Oxidative stress and inflammation are two key pathogenic factors of DR. Recently, regulatory roles of different microRNAs (miRNAs) in DR have been widely verified. miR-26a-5p has been confirmed to be a potential biomarker of DR. Nevertheless, the specific functions of miR-26a-5p in DR are still unclear. Primary cultured mouse retinal Müller cells in exposure to high glucose (HG) were used to establish an in vitro DR model. Müller cells were identified via morphology observation under phase contrast microscope and fluorescence staining for glutamine synthetase. The in vivo animal models for DR were constructed using streptozotocin-induced diabetic C57BL/6 mice. Western blotting was performed to quantify cytochrome c protein level in the cytoplasm and mitochondria of Müller cells and to measure protein levels of glial fibrillary acidic protein (GFAP), ubiquitin-specific peptidase 14 (USP14), as well as factors associated with NF-κB signaling (p-IκBα, IκBα, p-p65, and p65) in Müller cells or murine retinal tissues. ROS production was detected by CM-H2DCFDA staining, and the concentration of oxidative stress markers (MDA, SOD, and CAT) was estimated by using corresponding commercial kits. Quantification of mRNA expression was conducted by RT-qPCR analysis. The concentration of proinflammatory factors (TNF-α, IL-1β, and IL-6) was evaluated by ELISA. Hematoxylin-eosin staining for murine retinal tissues was performed for histopathological analysis. Immunofluorescence staining was conducted to determine NF-κB p65 nuclear translocation in Müller cells. Furthermore, the interaction between miR-26a-5p and USP14 was verified via the luciferase reporter assays. HG stimulation contributed to Müller cell dysfunction by inducing inflammation, oxidative injury, and mitochondrial damage to Müller cells. miR-26a-5p was downregulated in Müller cells under HG condition, and overexpression of miR-26a-5p relieved HG-induced Müller cell dysfunction. Moreover, miR-26a-5p targeted USP14 and inversely regulated USP14 expression. Additionally, HG-evoked activation of NF-κB signaling was suppressed by USP14 knockdown or miR-26a-5p upregulation. Rescue assays showed that the protective impact of miR-26a-5p upregulation against HG-induced Müller cell dysfunction was reversed by USP14 overexpression. Furthermore, USP14 upregulation and activation of NF-κB signaling in the retinas of DR mice were detected in animal experiments. Injection with miR-26a-5p agomir improved retinal histopathological injury and weakened the concentration of proinflammatory cytokines and oxidative stress markers in the retinas of DR mice. miR-26a-5p inhibits oxidative stress and inflammation in DR progression by targeting USP14 and inactivating the NF-κB signaling pathway.

Fetuin-B Overexpression Promotes Inflammation in Diabetic Retinopathy Through Activating Microglia and the NF-κB Signaling Pathway

Purpose To investigate the expression, source, role, and mechanism of Fetuin-B (FETUB) in diabetic retinopathy (DR). Methods ELISA and immunofluorescence were used to analyze the concentration of FETUB in plasma, aqueous fluid, and tissue specimens of patients with DR and healthy controls. Immunofluorescence, q-PCR, and western blotting were used to examine the expression of FETUB in DR mice and cells cultured with different concentrations of glucose. BV2 microglia cell line and DR mice were treated using FETUB recombination protein and FETUB shRNA to explore the function of FETUB in DR by q-PCR, western blotting, and immunofluorescence. Results FETUB concentrations in plasma, aqueous fluid, and tissue specimens were significantly increased in DR patients. The mice in DR group had a higher concentration of FETUB in the retina and liver tissues than those in the control group, and the expression of FETUB was increased in both ARPE19 and BV2 cells under a high-glucose environment. The ratio of p-P65 (Phospho-P65)/P65 and the expression levels of TNF-α, VEGF, and ionized calcium binding adaptor molecule (IBA)-1 were increased in BV2 cells cultured with FETUB recombinant protein, while they were decreased in BV2 cells transfected with FETUB shRNA. Immunofluorescence staining showed that there were more IBA-1+ activated microglia in the retinas of the FETUB recombination protein group than in the retinas of the DR group, and there were fewer IBA-1+ activated microglia in the retinas of the FETUB shRNA group than in the retinas of the DR group. Conclusions FETUB sourced from endocrine, autocrine, and paracrine pathways could promote inflammation in DR by activating the NF-κB pathway and microglia.

Systemic Inflammasome Biomarkers as Predictors of Diabetic Retinopathy Progression: Evidence from a Pilot Study

The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) protein 3 (NLRP3) inflammasome pathway is believed to mediate chronic inflammation in diabetic retinopathy (DR); however, its impact on the progression of DR remains to be elucidated. Therefore, the primary aim of this pilot study was to determine whether systemic inflammasome biomarkers interleukin (IL)-1β and IL-18 can be used to predict DR progression. DR screening results were analyzed against weight, level of glycated hemoglobin (HbA1c), and plasma levels of inflammasome biomarkers (IL-1β and IL-18), as well as general inflammation markers (C-reactive protein (CRP), IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF)) in patients with type 2 diabetes at baseline and 1 year post-bariatric surgery. Cross-sectional analysis demonstrated that weight, HbA1c, CRP, and IL-18 did not correlate with DR severity. The progressed group showed a higher relative change in IL-18 and CRP levels compared to the stable and regressed groups. Furthermore, relative changes in plasma CRP levels correlated with those of IL-18. Although further validation with larger cohorts is necessary, this pilot study supports the hypothesis that systemic inflammasome activation is associated with DR progression.

Evaluation of changes in epivascular glia before and after intravitreal dexamethasone implant: An OCT pilot study

PurposeTo detect changes in epivascular glia (EVG) in diabetic retinopathy after intravitreal dexamethasone implant using en face optical coherence tomography (enface OCT) and OCT angiography (OCTA) and to correlate improvements in functional and structural features. MethodsA total of 38 eyes of 38 patients were enroled in this prospective study. They were divided into two different study groups: the first group including 20 eyes with diabetic retinopathy type 1 complicated by macular oedema and the control group including 18 eyes from 18 healthy age-matched patients. The main outcome measures were: (i) differences at baseline in the foveal avascuar zone (FAZ) area in the study group versus control; (ii) the presence of epivascular glia in the study group versus control, (iii) differences at baseline between foveal macular thickness versus control; (iv) changes in foveal macular thickness, FAZ and epivascular glia in the study group before and after intravitreal dexamethasone implant. ResultsAt baseline FAZ area detected at OCTA was larger in the study group than in the control group, and epivascular glia was only detected in the study group. Three months after intravitreal injection of dexamethasone implant in the study group the best corrected visual acuity (BCVA) improved and central macular tickness reduced (P<0.0001). No significant differences were found in FAZ area while epivascular glia disappeared in 80% of the patients after treatment. ConclusionsGlia activation due to retinal inflammation in diabetic retinopathy (DR) can be detected on en face-OCT as epivascular glia. Intravitreal dexamethasone (DEX) implant improves both the anatomical and functional condition in the presence of these signs.

325-OR: Glucagon-Like Peptide 1 Receptor Agonists Preserved Vascular Endothelial Junction through Inhibition of STING Signaling–Elicited Inflammation in Diabetic Retinopathy

Background: Glucagon like-peptide 1 receptor agonists (GLP-1 RAs) are protective to macrovascular diseases and nephropathy in diabetic patients; however, their role in diabetic retinopathy (DR) are still debatable. This study aimed to address the effect of GLP-1 RAs on endothelial junction and inflammation in DR. Methods: GLP-1 RAs were injected to db/db mice, then retinal inflammation and vascular changes were determined by RNA-sequencing analysis, immunostaining, fluorescein fundus imaging and Optical coherence tomography. Cross-sectional study of 1626 type 2 diabetic patients was performed. Findings: GLP-1R was majorly detected in retinal vessels, which was down-regulated by diabetes. GLP-1 RAs significantly rescued diabetes-induced retinal thinning and vascular tortuosity, likely through altered the expression profiles of junction- and mitochondria-related genes, which was confirmed by exedin-4 treatment in vitro. Stimulator of interferon genes (STING) was most abundant in the endothelial cells from fibrovascular membranes, which was positively correlated with the levels of angiogenic factors and inflammatory factors in endothelial cells from retinas with retinopathy and fibrovascular membranes. Further analysis showed prominent inhibition by GLP-1 RAs on STING activation in db/db retinas. Cross-sectional study further showed decreased DR incidence by GLP-1 RAs. Conclusions: Activation of the STING pathway induced inflammatory factors while suppressed junctional proteins in endothelial cells of DR. GLP-1 RAs ameliorated diabetes-elicited vascular damages through inhibition of the STING pathway. Disclosure X.He: None. G.Shi: None. Y.Chen: None. S.Wen: None. X.Tang: None. Z.Wen: None. R.Zhang: None. S.Li: None. R.Gao: None. Y.Zhu: None. Y.Lu: None. Funding National Natural Science Foundation of China (82000782, 82270886, 82070811); Third Affiliated Hospital of Sun Yat-sen University (2020G2RPYQN11); China International Medical Foundation (2018-N-01); Science and Technology Plan Project of Guangzhou City (202007040003)

Inflammation: The Link between Neural and Vascular Impairment in the Diabetic Retina and Therapeutic Implications.

The etiology of diabetic retinopathy (DR) is complex, multifactorial and compromises all the elements of the retinal neurovascular unit (NVU). This diabetic complication has a chronic low-grade inflammatory component involving multiple inflammatory mediators and adhesion molecules. The diabetic milieu promotes reactive gliosis, pro-inflammatory cytokine production and leukocyte recruitment, which contribute to the disruption of the blood retinal barrier. The understanding and the continuous research of the mechanisms behind the strong inflammatory component of the disease allows the design of new therapeutic strategies to address this unmet medical need. In this context, the aim of this review article is to recapitulate the latest research on the role of inflammation in DR and to discuss the efficacy of currently administered anti-inflammatory treatments and those still under development.

The 0.19-mg Fluocinolone Acetonide Implant for the Treatment of Diabetic Macular Edema: An Expert Consensus.

To better understand the level of agreement among retina specialists on the role of inflammation in diabetic retinopathy (DR) and diabetic macular edema (DME), and the use of 0.19-mg fluocinolone acetonide (FAc) implant in DME treatment, a consensus survey was drafted and disseminated to retina specialists across the United States. Using the modified Delphi method, a list of 12 consensus statements were generated by the coauthors based on short-answer responses to an initial survey. In total, 56 retina specialists completed the entire consensus survey. Except for two multiple-choice questions, there were 10 consensus statements that used a modified Likert scale to indicate their level of agreement to the statement: Agree = 3, Mostly Agree = 2, Mostly Disagree = 1, Disagree = 0. Percentage agreement and 95% confidence intervals (CIs) were calculated, and a consensus threshold was set at > 80% agreement for each statement. Seven of 10 consensus statements using the modified Likert scale reached consensus, including those on the role of inflammation in pathophysiology of DR/DME, injection burden and patient adherence, and efficacy and safety of the FAc implant. The remaining three statements displayed high agreement with average scores > 80%, but the 95% CIs were below threshold. These included the impact of the FAc implant on DR progression, FAc as baseline therapy for DME, and the effectiveness of the steroid challenge to mitigate intraocular pressure risk after FAc use. Two multiple-choice questions focused on clinical situations in which corticosteroids would be used as baseline therapy for DME (pseudophakic eye [73%], recent stroke/myocardial infarction [66%], and pregnancy/breastfeeding [66%]) and which delivery route satisfies the steroid challenge for the FAc implant (intravitreal [100%], sub-tenon/periocular [73%], and topical [57%]). Physicians highly agreed on the role of inflammation in pathophysiology of DR/DME, injection burden and patient adherence, and efficacy and safety of the FAc implant. However, full consensus was not found on the impact of the FAc implant on DR progression, FAc as baseline therapy for DME, and the effectiveness of the steroid challenge to mitigate intraocular pressure risk after FAc use. [Ophthalmic Surg Lasers Imaging Retina. 2023;54(3):166-173.].

Proinflammatory Cytokines Trigger the Onset of Retinal Abnormalities and Metabolic Dysregulation in a Hyperglycemic Mouse Model.

Recent evidence has shown that retinal inflammation is a key player in diabetic retinopathy (DR) pathogenesis. To further understand and validate the metabolic biomarkers of DR, we investigated the effect of intravitreal proinflammatory cytokines on the retinal structure, function, and metabolism in an in vivo hyperglycemic mouse model. C57Bl/6 mice were rendered hyperglycemic within one week of administration of a single high-dose intraperitoneal injection of streptozotocin, while control mice received vehicle injection. After confirming hyperglycemia, the mice received an intravitreal injection of either proinflammatory cytokines (TNF-α and IL-1β) or vehicle. Similarly, control mice received an intravitreal injection of either proinflammatory cytokines or vehicle. The retinal structure was evaluated using fundus imaging and optical coherence tomography, and retinal function was assessed using a focal electroretinogram (ERG), two days after cytokine injection. Retinas were collected for biochemical analysis to determine key metabolite levels and enzymatic activities. Hyperglycemic mice intraocularly injected with cytokines developed visible retinal vascular damage and intravitreal and intraretinal hyper-reflective spots two days after the cytokines injection. These mice also developed a significant functional deficit with reduced a-wave and b-wave amplitudes of the ERG at high light intensities compared to control mice. Furthermore, metabolic disruption was evident in these mice, with significantly higher retinal glucose, lactate, ATP, and glutamine levels and a significant reduction in glutamate levels compared with control mice. Minimal or no metabolic changes were observed in hyperglycemic mice without intraocular cytokines or in control mice with intraocular cytokines at 2 days post hyperglycemia. Proinflammatory cytokines accelerated the development of vascular damage in the eyes of hyperglycemic mice. Significant changes were observed in retinal structure, function, and metabolic homeostasis. These findings support the idea that with the onset of inflammation in DR, there is a deficit in metabolism. Therefore, early intervention to prevent inflammation-induced retinal changes in diabetic patients may improve the disease outcome.

Inflammation in diabetic retinopathy: possible roles in pathogenesis and potential implications for therapy.

Diabetic retinopathy, characterized as a microangiopathy and neurodegenerative disease, is the leading cause of visual impairment in diabetic patients. Many clinical features observed in diabetic retinopathy, such as capillary occlusion, acellular capillaries and retinal non-perfusion, aggregate retinal ischemia and represent relatively late events in diabetic retinopathy. In fact, retinal microvascular injury is an early event in diabetic retinopathy involving multiple biochemical alterations, and is manifested by changes to the retinal neurovascular unit and its cellular components. Currently, intravitreal anti-vascular endothelial growth factor therapy is the first-line treatment for diabetic macular edema, and benefits the patient by decreasing the edema and improving visual acuity. However, a significant proportion of patients respond poorly to anti-vascular endothelial growth factor treatments, indicating that factors other than vascular endothelial growth factor are involved in the pathogenesis of diabetic macular edema. Accumulating evidence confirms that low-grade inflammation plays a critical role in the pathogenesis and development of diabetic retinopathy as multiple inflammatory factors, such as interleukin-1β, monocyte chemotactic protein-1 and tumor necrosis factor -α, are increased in the vitreous and retina of diabetic retinopathy patients. These inflammatory factors, together with growth factors such as vascular endothelial growth factor, contribute to blood-retinal barrier breakdown, vascular damage and neuroinflammation, as well as pathological angiogenesis in diabetic retinopathy, complicated by diabetic macular edema and proliferative diabetic retinopathy. In addition, retinal cell types including microglia, Müller glia, astrocytes, retinal pigment epithelial cells, and others are activated, to secrete inflammatory mediators, aggravating cell apoptosis and subsequent vascular leakage. New therapies, targeting these inflammatory molecules or related signaling pathways, have the potential to inhibit retinal inflammation and prevent diabetic retinopathy progression. Here, we review the relevant literature to date, summarize the inflammatory mechanisms underlying the pathogenesis of diabetic retinopathy, and propose inflammation-based treatments for diabetic retinopathy and diabetic macular edema.

Sustained therapeutic effect of an anti-inflammatory peptide encapsulated in nanoparticles on ocular vascular leakage in diabetic retinopathy.

Pigment epithelium-derived factor (PEDF), an endogenous Wnt signaling inhibitor in the serine proteinase inhibitors (SERPIN) super family, is present in multiple organs, including the vitreous. Significantly low levels of PEDF in the vitreous are found to associate with pathological retinal vascular leakage and inflammation in diabetic retinopathy (DR). Intravitreal delivery of PEDF represents a promising therapeutic approach for DR. However, PEDF has a short half-life after intravitreal injection, which represents a major hurdle for the long-term treatment. Here we report the prolonged therapeutic effects of a 34-mer peptide of the PEDF N-terminus, encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (PEDF34-NP), on DR. PEDF34-NP inhibited hypoxia-induced expression of vascular endothelial growth factor and reduced levels of intercellular adhesion molecule 1 (ICAM-1) in cultured retinal cells. In addition, PEDF34-NP significantly ameliorated ischemia-induced retinal neovascularization in the oxygen-induced retinopathy rat model, and significantly reduced retinal vascular leakage and inflammation in streptozotocin-induced diabetic rats up to 4weeks after intravitreal injection, as compared to PLGA-NP control. Intravitreal injection of PEDF34-NP did not display any detectable toxicities to retinal structure and function. Our findings suggest that PEDF34-NP can confer sustained therapeutic effects on retinal inflammation and vascular leakage, having considerable potential to provide long-term treatment options for DR.

The role of inflammation in immune system of diabetic retinopathy: Molecular mechanisms, pathogenetic role and therapeutic implications.

Diabetic retinopathy is one of the most common complications of diabetes mellitus and the leading cause of low vision and blindness worldwide. Mounting evidence demonstrates that inflammation is a key mechanism driving diabetes-associated retinal disturbance, yet the pathophysiological process and molecular mechanisms of inflammation underlying diabetic retinopathy are not fully understood. Cytokines, chemokines, and adhesion molecules interact with each other to form a complex molecular network that propagates the inflammatory and pathological cascade of diabetic retinopathy. Therefore, it is important to understand and elucidate inflammation-related mechanisms behind diabetic retinopathy progression. Here, we review the current understanding of the pathology and pathogenesis of inflammation in diabetic retinopathy. In addition, we also summarize the relevant clinical trials to further suggest inflammation-targeted therapeutics for prevention and management of diabetic retinopathy.

Molecular investigation of candidate genes for pyroptosis-induced inflammation in diabetic retinopathy.

BackgroundDiabetic retinopathy is a diabetic microvascular complication. Pyroptosis, as a way of inflammatory death, plays an important role in the occurrence and development of diabetic retinopathy, but its underlying mechanism has not been fully elucidated. The purpose of this study is to identify the potential pyroptosis-related genes in diabetic retinopathy by bioinformatics analysis and validation in a diabetic retinopathy model and predict the microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) interacting with them. Subsequently, the competing endogenous RNA (ceRNA) regulatory network is structured to explore their potential molecular mechanism.MethodsWe obtained mRNA expression profile dataset GSE60436 from the Gene Expression Omnibus (GEO) database and collected 51 pyroptosis-related genes from the PubMmed database. The differentially expressed pyroptosis-related genes were obtained by bioinformatics analysis with R software, and then eight key genes of interest were identified by correlation analysis, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein–protein interaction (PPI) network analysis. Then, the expression levels of these key pyroptosis-related genes were validated with quantitative real-time polymerase chain reaction (qRT-PCR) in human retinal endothelial cells with high glucose incubation, which was used as an in vitro model of diabetic retinopathy. Western blot was performed to measure the protein levels of gasdermin D (GSDMD), dasdermin E (GSDME) and cleaved caspase-3 in the cells. Moreover, the aforementioned genes were further confirmed with the validation set. Finally, the ceRNA regulatory network was structured, and the miRNAs and lncRNAs which interacted with CASP3, TLR4, and GBP2 were predicted.ResultsA total of 13 differentially expressed pyroptosis-related genes were screened from six proliferative diabetic retinopathy patients and three RNA samples from human retinas, including one downregulated gene and 12 upregulated genes. A correlation analysis showed that there was a correlation among these genes. Then, KEGG pathway and GO enrichment analyses were performed to explore the functional roles of these genes. The results showed that the mRNA of these genes was mainly related to inflammasome complex, interleukin-1 beta production, and NOD-like receptor signaling pathway. In addition, eight hub genes—CASP3, TLR4, NLRP3, GBP2, CASP1, CASP4, PYCARD, and GBP1—were identified by PPI network analysis using Cytoscape software. High glucose increased the protein level of GSDMD and GSDME, as critical effectors of pyroptosis, in retinal vascular endothelial cells. Verified by qRT-PCR, the expression of all these eight hub genes in the in vitro model of diabetic retinopathy was consistent with the results of the bioinformatics analysis of mRNA chip. Among them, CASP4, GBP1, CASP3, TLR4, and GBP2 were further validated in the GSE179568 dataset. Finally, 20 miRNAs were predicted to target three key genes—CASP3, GBP2, and TLR4, and 22 lncRNAs were predicted to potentially bind to these 20 miRNAs. Then, we constructed a key ceRNA network that is expected to mediate cellular pyroptosis in diabetic retinopathy.ConclusionThrough the data analysis of the GEO database by R software and verification by qRT-PCR and validation set, we successfully identified potential pyroptosis-related genes involved in the occurrence of diabetic retinopathy. The key ceRNA regulatory network associated with these genes was structured. These findings might improve the understanding of molecular mechanisms underlying pyroptosis in diabetic retinopathy.

44-OR: NFAT Inhibition Reduces Retinal Vascular Leakage and Inflammation in Diabetic Retinopathy

Purpose: Macular edema resulting from retinal vascular leakage is the leading cause of vision loss in diabetic retinopathy (DR) . The nuclear factor of activated T-cells (NFAT) family of transcription factors is known to upregulate inflammatory cytokines in response to high glucose. This study explored the effect of NFAT inhibition on retinal vascular leakage and inflammation in DR in vivo and in vitro. Methods: The streptozocin-induced (STZ) model was used as the mouse model of diabetic retinopathy. Hyperglycemia was induced at 4 weeks in C57BL/6J mice and confirmed when fasting plasma glucose became persistently elevated (&amp;gt;300 mg/dL) . Experiments were performed at weeks of hyperglycemia. STZ mice were injected intravitreally with the NFAT signaling inhibitor INCA-6 (25 uM) or vehicle (0.1% DMSO in PBS) . Uninjected eyes of both STZ and control mice served as additional controls. Evans blue dye (EBD) was used to measure retinal vascular leakage. Primary mouse retinal microvascular endothelial cells (MRMECs) were used for in vitro experiments. MRMEC monolayers were treated with INCA-6 (2.5 uM) or with vehicle and cell permeability was induced with mouse VEGF165. The transendothelial electrical resistance (TEER) was measured with the electrical cell-substrate impedance sensing method. MRMEC IL-6 production was investigated by ELISA. Results: In the STZ model of diabetic retinopathy, EBD vascular leakage was significantly reduced in INCA-6-treated eyes (INCA-6: 0.85 ± 0.57, n=5; vehicle: 1.78 ± 0.38, n= 7; P=0.007) , and in INCA-6 treated eyes compared to untreated eyes (untreated: 2.83 ± 1.63, n=4, P=0.04) . In the VEGF-induced MRMEC monolayer permeability model, INCA-6 significantly preserved TEER compared to vehicle (n=12, P&amp;lt;0.05) and decreased the VEGF-induced production of IL-6 (n=5, P&amp;lt;0.05) . Conclusions: NFAT inhibition has therapeutic potential to prevent and treat retinal vascular leakage and inflammation in DR. Disclosure I.De la huerta: None. J.L.Nunez: None. J.M.Poloway: None. V.Reddy: None. J.Penn: None. Funding National Institutes of Health (EY032620) International Retinal Research Foundation Grant Knights Templar Foundation Career Starter Grant

Investigating Atypical Inflammatory Signaling in Vascular Pericytes as Potential Target for Controlling Blood‐Retinal‐Barrier (BRB) Inflammation in Diabetic Retinopathy

Pericytes are master regulators of vascular homeostasis and play key roles in the progression of many vasculopathies, including neurovascular inflammation and diabetic retinopathy. During inflammation, pericytes can be activated by many signals, leading to pericyte induced proinflammatory signaling and pericyte migration away from the vasculature leaving endothelial tubules exposed and fragile. Despite a clear role for G protein‐coupled receptors (GPCR) in the regulation of vascular endothelial inflammation, GPCR control of pericytes has been understudied. Furthermore, pericytes represent a promising candidate for blocking inflammatory responses that could mitigate the onset and progression of neurovascular disease. Our prior studies revealed an atypical inflammatory pathway in endothelial cells, regulated by GPCR induced autoactivation of the mitogen‐activated protein kinase (MAPK) p38 pathway. This Atypical inflammatory pathway has not been studied in pericytes and is a promising therapeutic target. Our central hypothesis is that neurovascular inflammation in the brain and eye is controlled by atypical p38 signaling in vascular pericytes. To investigate this we are initially using primary human brain pericytes. We show that that thrombin, histamine and prostaglandin E2 (PGE2) can activate p38 via the atypical p38 signaling pathway in primary human pericytes and that a peptide inhibitor of atypical p38 signaling, blocks PGE2 dependent p38 activation, suppressing proinflammatory cytokine expression. Furthermore we are currently using single cell morphometric analysis to characterize heterogeneous pericyte responses to proinflammatory stimuli. This will enable the correlation of morphological variations to proinflammatory roles in T‐cell modulation, cytokine regulation, and migration. These studies are revealing novel insights into GPCR dependent control of pericytes and will be extrapolated into studies investigating pericyte‐endothelial cell interactions and the dysregulation of the blood‐retinal‐barrier during diabetic retinopathy. Our current studies focus on defining this role and in understanding how the neurovascular unit in the eye is regulated through atypical signaling, and whether inhibition of atypical signaling can protect the retina from damage. Thus, these studies represent a novel approach to regulate vascular p38 activity and pro‐inflammatory signaling in retinal disease.

Nature against Diabetic Retinopathy: A Review on Antiangiogenic, Antioxidant, and Anti-Inflammatory Phytochemicals.

Background and Purpose. Diabetes mellitus (DM), hyperglycemia, and hypertension can result in diabetic retinopathy (DR), which is a major cause of blindness on a global scale. Development of DR is associated with decreased endothelial cells, increased basal membrane thickness, permeation of the retinal blood barrier, and neovascularization in patients. The purpose of the present review is to provide an overview of the findings regarding applications of phytochemicals for DR treatment and could be a beneficial resource for further clinical studies and also a basis for pharmaceutical purposes for drug design. Materials and Methods. A narrative literature review was performed from electronic databases including Web of Science, PubMed, and Scopus to analyze the effects of different phytochemicals to prevent or treat oxidation, angiogenesis, and inflammation in diabetic retinopathy. The inclusion criteria were original studies, which included the effects of different phytochemicals on diabetic retinopathy. The exclusion criteria included studies other than original articles, studies which assessed the effects of phytochemicals on nondiabetic retinopathy, and studies which used phytochemical-rich extracts. Results and Conclusions. Studies have shown that increased levels of inflammatory cytokines, angiogenic, and oxidative stress factors are involved in the progression and pathogenesis of DR. Therefore, phytochemicals with their anti-inflammatory, antiangiogenic, and antioxidant properties can prevent DR progression and retinal damage through various cellular mechanisms. It is also shown that some phytochemicals can simultaneously affect the inflammation, oxidation, and angiogenesis in DR.