Diabetic Retina Research Articles

Erythropoietin maintains VE-cadherin expression and barrier function in experimental diabetic retinopathy via inhibiting VEGF/VEGFR2/Src signaling pathway

AimsTo explore the mechanisms of erythropoietin (EPO)'s protection on inner blood-retinal barrier (iBRB) in experimental diabetic retinopathy. Material and methodsMale SD rats were rendered diabetic with streptozotocin, followed by intravitreal injection of EPO. The permeability of iBRB was examined with fluorescein isothiocyanate (FITC)-dextran. Human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs) were treated with glyoxal and studied for cell viability and barrier function. The expressions of vascular endothelial (VE)-cadherin, Src kinase, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) were analyzed with Western blot, ELISA, qPCR, or immunofluorescence. Key findingsVE-cadherin in rat retinas was down-regulated with diabetes progression. EPO treatment could increase VE-cadherin expression at week 8 and week 16. The expressions of p-Src and p-VE-cadherin were increased at week 2, while decreased at week 8 of diabetes; which were prevented by EPO. The leakage of FITC-dextran in 8-week diabetic rat retinas was ameliorated by EPO. In vitro results showed the expressions of VEGF, p-Src and p-VE-cadherin were increased significantly, accompanied with the decreased barrier function, which were prevented by EPO. Ranibizumab and CGP77675 also inhibited the glyoxal-induced phosphorylation of Src and VE-cadherin. Cellular fractionation showed EPO mitigated the VE-cadherin internalization in glyoxal-treated cells. SignificanceEPO maintained the expression of VE-cadherin in experimental diabetic retinopathy by inhibiting its phosphorylation and internalization through VEGF/VEGFR2/Src pathway, thus improved the integrity of iBRB.

Effect of Topical Administration of Somatostatin on Retinal Inflammation and Neurodegeneration in an Experimental Model of Diabetes

Somatostatin (SST) is a neuroprotective peptide but little is known regarding the potential role of its anti-inflammatory effects on retinal neuroprotection. In a previous study, we provided the first evidence that topical (eye drops) administration of SST prevents retinal neurodegeneration in streptozotocin (STZ)-induced diabetic rats. However, STZ by itself could cause neurotoxicity, thus acting as a confounding factor. The aims of the present study were: (1) to test the effect of topical administration of SST in the db/db mouse model, a spontaneous model of type 2 diabetes, thus avoiding the confounding effect of STZ on neurodegeneration; (2) to further explore the anti-inflammatory mechanisms of SST in glial cells. This task was performed by using mouse retinal explants and cell cultures. In summary, we confirm that SST topically administered was able to prevent retinal neurodysfunction and neurodegeneration in db/db mice. Furthermore, we found that SST prevented the activation of the classical M1 response of Bv.2 microglial cells upon Lipopolysaccharide (LPS) stimulation as a potent pro-inflammatory trigger. The anti-inflammatory effect of SST in Bv.2 cells was also observed in response to hypoxia. In conclusion, we provide evidence that the neuroprotective effect of SST in diabetic retinas can be largely attributed to anti-inflammatory mechanisms.

Arrangement of the photoreceptor mosaic in a diabetic rat model imaged with multiphoton microscopy.

Diabetic retinopathy (DR) is defined as a microvascular pathology. However, some data have suggested that the retinal photoreceptors (PRs) might be important in the pathogenesis of this ocular disease. In this study the organization of the PRs in control and diabetic-induced rats was compared using multiphoton microscopy. The PR mosaic was imaged at different locations in non-stained retinas. The density of PRs was directly quantified from cell counting. The spatially resolved density presents a double-slope pattern (from the central retina towards the periphery) in both healthy and pathological samples, although the values for the latter were significantly lower all across the retina. Moreover, Voronoi analysis was performed to explore changes in PR topography. In control specimens a hexagonally packed structure was dominant. However, despite the non-controlled effects of the disease in retinal structures, this PR regularity was fairly maintained in diabetic retinas.

Hypoglycemia Induces Mitochondrial Reactive Oxygen Species Production Through Increased Fatty Acid Oxidation and Promotes Retinal Vascular Permeability in Diabetic Mice.

Aims: Hypoglycemia is associated with increased reactive oxygen species (ROS) production and vascular events. We have previously reported that low-glucose (LG) conditions induce mitochondrial ROS (mtROS) production in aortic endothelial cells (ECs). However, the mechanism by which hypoglycemia promotes diabetic retinopathy (DR) is unclear. Blood-retinal barrier (BRB) disruption occurs in the early stages of DR. We hypothesized that the mechanisms underlying hypoglycemia-induced DR are associated with BRB breakdown due to mtROS generation during hypoglycemia. Here, we aimed to determine whether hypoglycemia exacerbated mtROS production and induced BRB disruption. Results: We observed that hypoglycemia induced mtROS production by increasing fatty acid oxidation (FAO), which was suppressed by overexpression of mitochondrial-specific manganese superoxide dismutase (MnSOD) in retinal ECs. Furthermore, FAO blockade decreased the hypoglycemia-induced mtROS production. Recurrent hypoglycemia increased albumin leak in diabetic mice retina, which was suppressed in diabetic vascular endothelial cell-specific MnSOD transgenic (eMnSOD-Tg) mice. Pharmacological FAO blockade also reduced mtROS production, reduced vascular endothelial growth factor (VEGF) production during hypoglycemia, and prevented retinal vascular permeability in diabetic mice. MnSOD overexpression or carnitine palmitoyltransferase I (CPT1) blockade suppressed vascular endothelial-cadherin phosphorylation under LG in retinal ECs. Innovation and Conclusion: Reduction of mtROS and VEGF production via pharmacological FAO and/or CPT1 blockade may prevent hypoglycemia-induced worsening of DR.

Effects of Diabetes on Mitochondrial Morphology and Its Implications in Diabetic Retinopathy.

PurposeTo determine whether high glucose (HG) or diabetes alters mitochondrial morphology and promotes mitochondrial fragmentation in retinal vascular cells and thereby triggers apoptosis associated with diabetic retinopathy.MethodsTo assess whether diabetes promotes mitochondrial fragmentation and thereby triggers apoptosis, retinas from nondiabetic and diabetic rats were analyzed using electron microscopy (EM) and in parallel, wild-type, diabetic, and OPA1+/− mice were analyzed for optic atrophy gene 1 (OPA1) and cytochrome c levels using Western blot (WB) analysis. To assess the relationship between mitochondrial fragmentation and OPA1 levels, rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mmol/L) medium, HG (30 mmol/L) medium, or in N medium transfected with OPA1 siRNA for seven days. Cells were examined for OPA1 expression and cytochrome c release by WB. In parallel, cells were stained with MitoTracker Red and assessed for mitochondrial fragmentation in live cells using confocal microscopy.ResultsEM images revealed significant mitochondrial fragmentation in vascular cells of retinal capillaries of diabetic rats compared with that of nondiabetic rats. WB analysis showed significant OPA1 downregulation concomitant with increased levels of proapoptotic cytochrome c levels in cells grown in HG and in cells transfected with OPA1 siRNA alone. Similarly, OPA1 level was significantly reduced in diabetic retinas compared with that of nondiabetic retinas. Interestingly, OPA1+/− animals exhibited elevated cytochrome c release similar to those of diabetic mice.ConclusionsFindings indicate that diabetes promotes mitochondrial fragmentation in retinal vascular cells, which are driven, at least in part, by decreased OPA1 levels leading to apoptosis in diabetic retinopathy.

Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy.

Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by systematically mapping the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina. Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (Ins2Akita×Vegfa+/-) mice, which are known to replicate features of clinical diabetic retinopathy. This resulted in transcriptome data for 9474 retinal cells, which could be annotated to eight distinct retinal cell types. Using STRING analysis, we studied differentially expressed gene networks in neuronal, glial and immune cell compartments to create a comprehensive view on the pathological changes that occur in the Akimba retina. Using subclustering analysis, we further characterised macroglial and inflammatory cell subpopulations. Prominent findings were confirmed at the protein level using immunohistochemistry, western blotting and ELISA. At 12weeks, the Akimba retina was found to display degeneration of rod photoreceptors and presence of inflammatory cells, identified by subclustering analysis as monocyte, macrophage and microglial populations. Analysis of differentially expressed genes in the rod, cone, bipolar cell and macroglial compartments indicated changes in cell metabolism and ribosomal gene expression, gliosis, activation of immune system pathways and redox and metal ion dyshomeostasis. Experiments at the protein level supported a metabolic shift from glycolysis to oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase), activation of microglia/macrophages (isolectin-B4), metal ion and oxidative stress response (metallothionein and haem oxygenase-1) and reactive macroglia (glial fibrillary acidic protein and S100) in the Akimba retina, compared with wild-type mice. Our single-cell approach also indicates macroglial subpopulations with distinct fibrotic, inflammatory and gliotic profiles. Our study identifies molecular pathways underlying inflammatory, metabolic and oxidative stress-mediated changes in the Akimba mouse model of diabetic retinopathy and distinguishes distinct functional subtypes of inflammatory and macroglial cells. RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI ( www.ebi.ac.uk/arrayexpress ) under accession number E-MTAB-9061. Graphical abstract.

Human Induced Pluripotent Stem Cell Derived Mesoderm Subset Ameliorates Diabetic Retinopathy by Reestablishing Retinal Function and Restoring Protective Signaling Cascades in Type 2 Diabetes

Human induced pluripotent stem cells (hiPSC) differentiated into a specific mesoderm subset expressing vascular endothelial growth factor receptor 2, neural cell adhesion molecule 1, and apelin G protein-coupled receptor APJ (called KNA+), possessed all of the phenotypic and functional endothelial colony forming cell potential that resides in differentiating hiPSC. Thus, we postulated KNA+ mesoderm treatment would correct diabetic retinal capillary vasodegeneration. KNA+ cells derived from diabetic (D) and non-diabetic (N) patient-derived hiPSC displayed comparable phenotypic and functional properties. N-KNA+ or D-KNA+ intravitreal injections into type II diabetic ( db/db ) murine eyes led to retinal vascular engraftment, showed no toxicity, visual acuity was significantly improved, and diabetes-induced scotopic and photopic ERG defects were corrected. Proteomic arrays revealed that hiPSC-derived KNA+ cell administration restored several aberrant cell signaling pathways in the neural retina. These results support the efficacy of both hiPSC-derived D-KNA+ and N-KNA+ cells in correcting vasodegeneration in the diabetic murine retina.

The Complex Relationship between Diabetic Retinopathy and High-Mobility Group Box: A Review of Molecular Pathways and Therapeutic Strategies.

High-mobility group box 1 (HMGB1) is a protein that is part of a larger family of non-histone nuclear proteins. HMGB1 is a ubiquitary protein with different isoforms, linked to numerous physiological and pathological pathways. HMGB1 is involved in cytokine and chemokine release, leukocyte activation and migration, tumorigenesis, neoangiogenesis, and the activation of several inflammatory pathways. HMGB1 is, in fact, responsible for the trigger, among others, of nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), toll-like receptor-4 (TLR-4), and vascular endothelial growth factor (VEGF) pathways. Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM) that is rapidly growing in number. DR is an inflammatory disease caused by hyperglycemia, which determines the accumulation of oxidative stress and cell damage, which ultimately leads to hypoxia and neovascularization. Recent evidence has shown that hyperglycemia is responsible for the hyperexpression of HMGB1. This protein activates numerous pathways that cause the development of DR, and HMGB1 levels are constantly increased in diabetic retinas in both proliferative and non-proliferative stages of the disease. Several molecules, such as glycyrrhizin (GA), have proven effective in reducing diabetic damage to the retina through the inhibition of HMGB1. The main focus of this review is the growing amount of evidence linking HMGB1 and DR as well as the new therapeutic strategies involving this protein.

Silencing circ_0001879 inhibits the proliferation and migration of human retinal microvascular endothelial cells under high-glucose conditions via modulating miR-30-3p.

Diabetic retinopathy is a severe diabetic complication and a major cause of blindness. In this study, we explored the role of circ_0001879 in retinal vascular dysfunction under diabetic conditions. Human retinal microvascular endothelial cells (HRMECs) were divided into normal glucose group (NG, 5.5mmol/L d-glucose), high glucose group (HG, 25mmol/L d-glucose), and osmotic control group (5.5mmol/L d-glucose+19.5mmol/L mannitol). The expression of circ_0001879 and miR-30-3p was assessed via qRT-PCR. The circ_0001879/miR-30-3p roles in retinal vascular dysfunction were investigated through Cell Counting Kit-8 and Transwell assay. Bioinformatics analysis and luciferase reporter assays were applied to examine interactions between circ_0001879 and miR-30-3p in HRMECs. The relative circ_0001879 expression was remarkably increased in diabetic retinas group than that in the control group. Silencing circ_0001879 suppressed the proliferation and migration of HRMECs under high-glucose conditions. In addition, circ_0001879 acted as a binding platform and miRNA sponge for miR-30-3p. Circ_0001879 modulated the function of HRMECs via targeting miR-30-3p. Silencing circ_0001879 inhibited the proliferation and migration of HRMECs under high-glucose conditions via modulating miR-30-3p, which might shed new light on a novel potentially marker and molecular therapeutic target for diabetic retinopathy.

Inhibition of HDAC6 Attenuates Diabetes-Induced Retinal Redox Imbalance and Microangiopathy.

We investigated the contributing role of the histone deacetylase 6 (HDAC6) to the early stages of diabetic retinopathy (DR). Furthermore, we examined the mechanism of action of HDAC6 in human retinal endothelial cells (HuREC) exposed to glucidic stress. Streptozotocin-induced diabetic rats (STZ-rats), a rat model of type 1 diabetes, were used as model of DR. HDAC6 expression and activity were increased in human diabetic postmortem donors and STZ-rat retinas and were augmented in HuREC exposed to glucidic stress (25 mM glucose). Administration of the HDAC6 specific inhibitor Tubastatin A (TS) (10 mg/kg) prevented retinal microvascular hyperpermeability and up-regulation of inflammatory markers. Furthermore, in STZ-rats, TS decreased the levels of senescence markers and rescued the expression and activity of the histone deacetylase sirtuin 1 (SIRT1), while downregulating the levels of free radicals and of the redox stress markers 4-hydroxynonenal (4-HNE) and nitrotyrosine (NT). The antioxidant effects of TS, consequent to HDAC6 inhibition, were associated with preservation of Nrf2-dependent gene expression and up-regulation of thioredoxin-1 activity. In vitro data, obtained from HuREC, exposed to glucidic stress, largely replicated the in vivo results further confirming the antioxidant effects of HDAC6 inhibition by TS in the diabetic rat retina. In summary, our data implicate HDAC6 activation in mediating hyperglycemia-induced retinal oxidative/nitrative stress leading to retinal microangiopathy and, potentially, DR.

PPAR\u03b1 activation directly upregulates thrombomodulin in the diabetic retina

Two large clinical studies showed that fenofibrate, a commonly used peroxisome proliferator-activated receptor α (PPARα) agonist, has protective effects against diabetic retinopathy. However, the underlying mechanism has not been clarified. We performed genome-wide analyses of gene expression and PPARα binding sites in vascular endothelial cells treated with the selective PPARα modulator pemafibrate and identified 221 target genes of PPARα including THBD, which encodes thrombomodulin (TM). ChIP-qPCR and luciferase reporter analyses showed that PPARα directly regulated THBD expression via binding to the promoter. In the rat diabetic retina, treatment with pemafibrate inhibited the expression of inflammatory molecules such as VCAM-1 and MCP1, and these effects were attenuated by intravitreal injection of small interfering RNA targeted to THBD. Furthermore, pemafibrate treatment inhibited diabetes-induced vascular leukostasis and leakage through the upregulation of THBD. Our results indicate that PPARα activation inhibits inflammatory and vasopermeable responses in the diabetic retina through the upregulation of TM.

Rethinking the Hydroxychloroquine Dosing and Retinopathy Screening Guidelines

To describe the rationale for revising the hydroxychloroquine (HCQ) dosing and screening guidelines and to identify the barriers to more effective guidelines in the future. Literature review. A PubMed query of studies on HCQ dosing and HCQ retinopathy (HCQR) screening was conducted with a selective review of the English language literature. Three iterations of the American Academy of Ophthalmology HCQ dosing and HCQR screening guidelines have been published without including prescribing physicians on the writing committees. This may contribute to prescribing physicians' low adherence to the guidelines. As ancillary tests have improved, asymptomatic HCQR is being detected earlier, leading to a higher reported prevalence of HCQR and a drop in the ceiling for safe dosing. These trends put stricter constraints on prescribers and their patients, who may have had well-controlled autoimmune disease on HCQ doses that were previously considered to be below the high-risk threshold for HCQR. Indeed, stopping HCQ at the earliest sign of HCQR should be reconsidered; for cases of early HCQR, dose reduction and more intensive monitoring for retinopathy may strike a more appropriate balance between HCQ risk and benefits. A prospective study using the Diabetic Retinopathy Clinical Research Retina Network with standardized collection of data, HCQ blood levels, centralized grading of ancillary tests, and community and academic ophthalmologists would provide a stronger evidence base for future HCQ guidelines. The HCQ dosing and screening guidelines should be updated and a prospective study of HCQ dosing and HCQR should be initiated with the joint efforts of ophthalmologists and prescribing physicians.

Increased Ephrin-B2 expression in pericytes contributes to retinal vascular death in rodents

AimsDiabetes-induced retinal vascular cell death aggravates diabetic retinopathy (DR) to the proliferative stage and blindness. Pericytes play a crucial role in retinal capillaries survival, stability, and angiogenesis. Ephrin-B2 is a tyrosine kinase that regulates pericytes/endothelial cells communication during angiogenesis; yet, its role in DR is still unclear. We hypothesize that diabetes increases Ephrin-B2 signaling in pericytes, which contributes to inflammation and retinal vascular cell death. MethodsSelective inhibition of the Ephrin-B2 expression in the retinal pericytes was achieved using an intraocular injection of adeno-associated virus (AAV) with a specific pericyte promotor. Vascular death was determined by retinal trypsin digest. Pathological angiogenesis was assessed using the oxygen-induced retinopathy model in pericyte-Ephrin-B2 knockout mice, wild type, and wild type injected with AAV. Angiogenic properties, inflammatory, and apoptotic markers were measured in human retinal pericytes (HRP) grown under diabetic conditions. Key findingDiabetes significantly increased the expression of Ephrin-B2, inflammatory, and apoptotic markers in the diabetic retinas and HRP. These effects were prevented by silencing Ephrin-B2 in HRP. Moreover, Ephrin-B2 silencing in retinal pericytes decreased pathological angiogenesis and acellular capillaries formation in diabetic retinas. SignificanceIncreased Ephrin-B2 expression in the pericytes contributed to diabetes-induced retinal inflammation and vascular death. These results identify pericytes-Ephrin-B2 as a therapeutic target for DR.

Papillary Trunk Bifurcation Angles as Major Determinants of Ocular Perfusion: A Cross-Sectional Study

Background: Notable work has been done by Cecile and Murray in as early as 1926, on the structural and topological aspects of vasculature based on the concept that blood vessel size and arrangement is such that it provides blood flow with minimum energy loss. In the theoretical analysis of arterial networks, the existence of geometric optimisation for enabling functional adequacy has long been suggested, but observational studies have not yet fully corroborated these theories. Data on branching or bifurcation angles of retinal microvasculature is not only insufficient but also inconclusive. Aim: Attending to this gap in information, we compared the central retinal artery (CRA) branching angles of known diabetics with healthy subjects and explored their possible role in determining the circulatory adequacy of human retina. Methods: This study was done on North Indian subjects attending retina clinics of prominent tertiary centers of north India during 2019 through 2020. Fundus images were acquired through convenience sampling from 860 consecutive eyes of 430 subjects. Fluorescein fundus angiography was followed up for diabetics. CRA branching angles from digitalized fundus photographs of known diabetics and healthy subjects was analyzed through semi-automated digital image management tool and the readings were clinically correlated to note the occurrence and severity of retinopathic perfusion defects was Results: We found marked differences between CRA branching angles among diabetics and healthy subjects and a significant correlation between branching anless and the occurrence and severity of retinopathic perfusion defects. Discussion and Conclusion: The orientation of retinal blood vessels on the fundus plane is not merely ‘a matter of chance’ in the anatomic chronicle of the human body; rather, it has biological heralds and functional consequences. Direction, branching, angles; all determine the efficacy and abundance of blood flow in the human retina that hold indispensible relevance for optimal vision and might be subtle indicators of micro vascular damage in disease states. This study has elucidated retinal vascular geometrics of healthy and diseased Indians, a knowledge that can improve our understanding of ‘abnormal features’ and ‘natural variants’ in retinal vascular architecture. In this study, we quantified the ‘structural factors’ behind perfusion defects of the Diabetic retina.

566-P: Sulodexide Suppresses NLRP3 Inflammasome Activation via NOX4/ROS Signaling in the Retinal Microvasculature

Aims: Sulodexide (SDX) protects endothelial cells via restoring endothelial glycocalyx, but the effect of SDX on endothelial inflammation remains unknown. We showed that SDX potently inhibited the activation of NLRP3 inflammasome in diabetic retinas, but the underlying mechanism was not clear. As reactive oxygen species (ROS) activates NLRP3 inflammasome, and NADPH oxidase 4 (NOX4) is a key generator of ROS in endothelial cells, this study thus investigated if NOX4/ROS signaling mediates the effect of SDX on NLRP3 inflammasome, hoping to provide novel insights into SDX in treating diabetic retinopathy (DR). Methods: Diabetes was induced in mice by high fat diet and streptozotocin, followed by i.p. injection of SDX (10mg/kg) every other day for 3 months. Human retinal microvascular endothelial cells were exposed to high glucose, accompanied with overexpression or knocking down of NOX4 and a serial concentration of SDX. The retinal levels of NOX4, NLRP3 inflammasome, inflammatory factors, junction components, vascular leakage and ROS levels were determined. Results: SDX treatment dramatically suppressed the hyperglycemia-induced overexpression of NOX4 and activation of NLRP3 inflammasome in retinas compared to controls. Moreover, SDX preserved the levels of ZO-1, VE-cadherin and ameliorated the retinal vascular permeability. In addition, SDX significantly ameliorated high glucose-induced NOX4 up-regulation, ROS production and NLRP3 inflammasome activation, and restored the reduction of ZO-1 and VE-cadherin in endothelial cells. Conversely, overexpression of NOX4 exacerbated endothelial inflammation through over production of ROS and subsequent activation of NLRP3 inflammasome, which was rescued by SDX treatment. Conclusion: Our study suggests that SDX protects the retinal microvasculature by inhibiting NOX4/ROS-induced NLRP3 inflammasome activation, thus provides novel evidence of utilizing SDX for the treatment of DR. Disclosure T. Li: None. X. He: None. R. Peng: None. X. Fu: None. G. Shi: None. Y. Chen: None. Funding Research and Development Plans in Key Areas of Guangdong Province (2019B020227003); National Natural Science Foundation of China (81770826); National Key Research and Development Program of China (2017YFA0105803)

Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes.

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood–retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1β, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 μM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Effect of sustained insulin-releasing device made of poly(ethylene glycol) dimethacrylates on retinal function in streptozotocin-induced diabetic rats.

In this study, we developed a subcutaneous insulin-releasing device consisting of a disk-shaped capsule and drug formulation comprised of poly(ethylene glycol) dimethacrylates, then evaluated its efficacy on retinal function in streptozotocin (STZ)-induced diabetic rats. In vitro release studies showed that recombinant human insulin was released with a constant rate for more than 30 days. The device was able to maintain a basal level of blood glucose in diabetic rats for a prolonged period of more than 30 days, simultaneously preventing a decrease in body weight. For assessing the pharmacological effect of the device on retinal function in diabetic rats, electroretinograms were conducted for 12 weeks. The reduction in amplitude and delay in implicit time were attenuated by the device during the initial 4 weeks of application. The increase in gene expression of protein kinase C (PKC)-γ and caspase-3 in the diabetic retina was also attenuated by the device. Immunohistochemistry showed that the increase in glial fibrillary acidic protein expression in the diabetic retina was attenuated by the device. Histological evaluation of subcutaneous tissue around the device showed the biocompatibility of the device. In conclusion, the insulin-releasing device attenuated the reduction of retinal function in STZ-induced diabetic conditions for 4 weeks and the efficacy of the device might be partially related to PKC signaling in the retina. The long-term ability to control the blood glucose level might help to reduce the daily frequency of insulin injections.

Interleukin-6 trans-signaling inhibition prevents oxidative stress in a mouse model of early diabetic retinopathy

PurposeDiabetic retinopathy (DR), a microvascular complication of diabetes, is the leading cause of visual disability and blindness in diabetic patients. Chronic hyperglycemia leads to increased oxidative stress and inflammation in the retina, resulting in microvascular damage. Our recent in vitro studies have demonstrated that inhibition of interleukin-6 (IL-6) trans-signaling significantly reduces oxidative stress in retinal endothelial cells. The purpose of this study was to further explore the relationship between IL-6 trans-signaling and oxidative stress using a streptozotocin (STZ) induced mouse model of early diabetic retinopathy. MethodsDiabetes was induced in eight week-old male C57BL/6J mice using STZ injections. sgp130Fc (mouse sgp130Fc protein) treatment was used for inhibition of IL-6 trans-signaling. Studies were conducted to evaluate the effects of IL-6 trans-signaling on oxidative balance at the systemic and retinal level. ResultsDecreased antioxidant capacity and increased oxidative stress was observed in diabetic mice, which returned to near-normal levels with sgp130Fc treatment. Similarly, superoxide levels, lipid peroxidation, and markers of oxidative DNA damage were increased in the diabetic retina, and these effects were abrogated by sgp130Fc treatment. Inhibition of IL-6 trans-signaling also restored normal expression of catalase and endothelial nitric oxide synthase in mouse retinas. ConclusionsInhibition of IL-6 trans-signaling significantly reduces diabetes-induced oxidative damage at the systemic level and in the retina. These findings provide further evidence for the role of IL-6 trans-signaling in diabetes-mediated oxidative stress.

Transcriptomics analysis of pericytes from retinas of diabetic animals reveals novel genes and molecular pathways relevant to blood-retinal barrier alterations in diabetic retinopathy

Selective pericyte loss, the histological hallmark of early diabetic retinopathy (DR), enhances the breakdown of the blood-retinal barrier (BRB) in diabetes. However, the role of pericytes on BRB alteration in diabetes and the signaling pathways involved in their effects are currently unknown. To understand the role of diabetes-induced molecular alteration of pericytes, we performed transcriptomic analysis of sorted retinal pericytes from mice model of diabetes. Retinal tissue from non-diabetic and diabetic (duration 3 months) mouse eyes (n = 10 in each group) were used to isolate pericytes through fluorescent activated cell sorting (FACS) using pericyte specific fluorescent antibodies, PDGFRb-APC. For RNA sequencing and qPCR analysis, a cDNA library was generated using template switching oligo and the resulting libraries were sequenced using paired-end Illumina sequencing. Molecular functional pathways were analyzed using differentially expressed genes (DEGs). Differential expression analysis revealed 217 genes significantly upregulated and 495 genes downregulated, in pericytes isolated from diabetic animals. These analyses revealed a core set of differentially expressed genes that could potentially contribute to the pericyte dysfunction in diabetes and highlighted the pattern of functional connectivity between key candidate genes and blood retinal barrier alteration mechanisms. The top up-regulated gene list included: Ext2, B3gat3, Gpc6, Pip5k1c and Pten and down-regulated genes included: Notch3, Xbp1, Gpc4, Atp1a2 and AKT3. Out of these genes, we further validated one of the down regulated genes, Notch 3 and its role in BRB alteration in diabetic retinopathy. We confirmed the downregulation of Notch3 expression in human retinal pericytes exposed to Advanced Glycation End-products (AGEs) treatment mimicking the chronic hyperglycemia effect. Exploration of pericyte-conditioned media demonstrated that loss of NOTCH3 in pericyte led to increased permeability of endothelial cell monolayers. Collectively, we identify a role for NOTCH3 in pericyte dysfunction in diabetes. Further validation of other DEGs to identify cell specific molecular change through whole transcriptomic approach in diabetic retina will provide novel insight into the pathogenesis of DR and novel therapeutic targets.

Long Noncoding RNA Hotair Facilitates Retinal Endothelial Cell Dysfunction in Diabetic Retinopathy

Background: Retinal endothelial cell (REC) dysfunction induced by diabetes mellitus (DM) is an important pathological step of diabetic retinopathy (DR). Long noncoding RNAs (lncRNAs) have emerged as novel modulators in DR. This study aimed to investigate the role and mechanism of lncRNA Hotair in regulating DM-induced REC dysfunction. Methods: The retinal vascular preparations and immunohistochemical staining assays were conducted to assess the role of Hotair in retinal vessel impairment in vivo. The EdU, transwell, cell permeability, CHIP, luciferase activity, RIP, RNA pull-down, and Co-IP assays were employed to investigate the underlying mechanism of Hotair-mediated REC dysfunction in vitro. Results: Hotair expression was significantly increased in diabetic retinas and high glucose (HG)-stimulated REC. Hotair knockdown inhibited the proliferation, migration, and permeability of HG-stimulated REC in vitro and reduced the retinal acellular capillaries and vascular leakage in vivo. Mechanistically, Hotair bound to LSD1 to inhibit VE-cadherin transcription by reducing the H3K4me3 level on its promoter or to facilitate transcription factor HIF1α-mediated transcriptional activation of VEGFA. Furthermore, LSD1 mediated the effects of Hotair on REC function under HG condition. Conclusion: The Hotair exerts its role in DR by binding to LSD1, decreasing VE-cadherin transcription and increasing VEGFA transcription, leading to REC dysfunction. These findings revealed that Hotair is a potential therapeutic target of DR. Funding Statement: This study was supported by the funding of the National science foundation for youth (81700729). Declaration of Interests: All authors declare that they have no conflicts of interest in this work. Ethics Approval Statement: All animal experiments were approved by the Ethics Committees of the First Affiliated Hospital of Zhengzhou University.