Animal Models Of Diabetic Retinopathy Research Articles

Manifestation of Pathology in Animal Models of Diabetic Retinopathy Is Delayed from the Onset of Diabetes.

Diabetic retinopathy (DR) is the most common complication that develops in patients with diabetes mellitus (DM) and is the leading cause of blindness worldwide. Fortunately, sight-threatening forms of DR develop only after several decades of DM. This well-documented resilience to DR suggests that the retina is capable of protecting itself from DM-related damage and also that accumulation of such damage occurs only after deterioration of this resilience. Despite the enormous translational significance of this phenomenon, very little is known regarding the nature of resilience to DR. Rodent models of DR have been used extensively to study the nature of the DM-induced damage, i.e., cardinal features of DR. Many of these same animal models can be used to investigate resilience because DR is delayed from the onset of DM by several weeks or months. The purpose of this review is to provide a comprehensive overview of the literature describing the use of rodent models of DR in type-1 and type-2 diabetic animals, which most clearly document the delay between the onset of DM and the appearance of DR. These readily available experimental settings can be used to advance our current understanding of resilience to DR and thereby identify biomarkers and targets for novel, prevention-based approaches to manage patients at risk for developing DR.

Network pharmacology mechanism of Scutellarin to inhibit RGC pyroptosis in diabetic retinopathy

To investigate the effect of scutellarin (SCU) in diabetic retinopathy (DR) and explore the associated molecular network mechanism. The animal model of DR was established from diabetic mellitus (DM) rats by intraperitoneally injected streptozotocin (STZ) at dosage 55 mg/kg. Meanwhile, SCU was intraperitoneally administrated to protect retina from cell pyroptosis induced by DM, and cell pyroptosis was detected by using HE, Nissl staining, and immunofluorescence recognition. Moreover, the hub gene involving in pyroptosis in DR was screened by bioinformatics and network pharmacology, designated as Venny intersection screen, GO and KEGG analysis, PPI protein interaction, and molecular docking. Lastly, the expressional change of hub genes were validated with experimental detection. Cell pyroptosis of the DR, specifically in retina ganglion cells (RGC), was induced in DM rats; SCU administration results in significant inhibition in the cell pyroptosis in DR. Mechanically, 4084 genes related to DR were screened from GeneCards and OMIM databases, and 120 SCU therapeutic targets were obtained, by using GeneCards, TCMSP with Swiss Target Prediction databases. Moreover, 357 targets related to pyroptosis were found using GenenCards database, and Drug, disease and phenotypic targets were analyzed online using the Draw Venn Diagram website, and 12 cross targets were obtained. Through GO function and KEGG pathway enrichment analysis, 659 BP related items, 7 CC related items, 30 MF related items, and 70 signal pathways were screened out; Of these, eleven proteins screened from cross-target PPI network were subsequently docked with the SCU, and their expressions including caspase-1, IL-1β, IL-18, GSDMD and NLRP3 in RGC indicated by immunofluorescence, and the mRNA expression for caspase-1 in DR indicated by quantitative PCR, were successfully validated. SCU can effectively protect RGC pyroptosis in DR, and underlying mechanisms are involved in the inhibition of caspase-1, GSDMD, NLRP3, IL-1β and IL-18. Our findings therefore provide crucial evidence to support the clinic practice of SCU for the treatment of DR, and explained the underlying molecular network mechanism.

PS-BPB06-8: THE NON-STEROIDAL MINERALOCORTICOID RECEPTOR ANTAGONIST, FINERENONE, REDUCES VASCULAR PATHOLOGY AND INFLAMMATION IN PRE-CLINICAL DIABETIC RETINOPATHY

Objective: Diabetic retinopathy is the leading cause of vision loss in the working population across the globe. Diabetes has devastating effects on the retinal vasculature, and can lead to vision-threatening vascular leakage, odema, and neovascularization. Current treatments such as anti-vascular endothelial growth factor (VEGF) are administered directly into the eye late in the disease process but are ineffective in about 50% of patients. Hypertension is a risk factor for the development of diabetic retinopathy and the renin-angiotensin aldosterone system contributes to disease pathogenesis. Here we aimed to determine if finerenone, a non-steroidal mineralocorticoid receptor (MR) antagonist, reduced vascular pathology and inflammation in animal models of diabetic retinopathy. Methods: To study diabetic retinopathy, streptozotocin diabetes was induced in female hypertensive transgenic Ren-2 rats overexpressing renin in extra-renal tissues. Diabetic Ren-2 rats were randomised to receive finerenone (10 mg/kg/day) by oral gavage or the angiotensin converting enzyme inhibitor perindopril (10 mg/kg/day) in drinking water for 12 weeks. As retinal neovascularization does not develop in diabetic rodents, the oxygen-induced retinopathy (OIR) model was studied. Litters of C57BL/6J mice were exposed to high oxygen (75%) between postnatal days 7 to 12 and then room air until postnatal day 18. Mouse pups were randomised to be OIR controls, or treated with finerenone (5 mg/kg/day) by intraperitoneal injection between postnatal days 7 to 18. Comparisons were made to mice exposed to room air. Damage to the blood-retinal barrier was evaluated by measuring the gliosis of macroglial Müller cells which confer barrier properties, vascular leakage (albumin ELISA), neovascularization, and the expression of VEGF. Inflammation was evaluated by the quantitation of retinal microglia and the expression of pro-inflammatory mediators. Results: Systolic blood pressure was similar between untreated non-diabetic (174.2 ± 6.3 mmHG) and diabetic (184.2 ± 8.5 mmHG) Ren-2 rats. Finerenone (138.5 ± 5.01 mmHG, P < 0.0001) and perindopril (106 ± 2.9 mmHG, P < 0.0001) reduced systolic blood pressure compared to untreated diabetic rats. In diabetic rats, finerenone reduced the gliosis of retinal Müller cells, vascular leakage, microglial density and the expression of VEGF, ICAM-1 and IL-1β;. In diabetic rats, perindopril did not reduce retinal VEGF levels, microglial density and the expression of pro-inflammatory mediators. In OIR mice, finerenone reduced retinal neovascularization, vascular leakage and microglial density. Conclusion: Our findings placed in the context of the FIDELIO-DKD trial reporting the benefits of finerenone on renal and cardiovascular outcomes in diabetic kidney disease, indicate the potential of finerenone to be an effective orally administered treatment for diabetic retinopathy.

Pdx1 Knockout Leads to a Diabetic Nephropathy- Like Phenotype in Zebrafish and Identifies Phosphatidylethanolamine as Metabolite Promoting Early Diabetic Kidney Damage.

The pdx1-/- zebrafish mutant was recently established as a novel animal model of diabetic retinopathy. In this study, we investigate whether knockout of pdx1 also leads to diabetic kidney disease (DKD). pdx1-/- larvae exhibit several signs of early DKD, such as glomerular hypertrophy, impairments in the filtration barrier corresponding to microalbuminuria, and glomerular basement membrane (GBM) thickening. Adult pdx1-/- mutants show progressive GBM thickening in comparison with the larval state. Heterozygous pdx1 knockout also leads to glomerular hypertrophy as initial establishment of DKD similar to the pdx1-/- larvae. RNA sequencing of adult pdx1+/- kidneys uncovered regulations in multiple expected diabetic pathways related to podocyte disruption and hinting at early vascular dysregulation without obvious morphological alterations. Metabolome analysis and pharmacological intervention experiments revealed the contribution of phosphatidylethanolamine in the early establishment of kidney damage. In conclusion, this study identified the pdx1 mutant as a novel model for the study of DKD, showing signs of the early disease progression already in the larval stage and several selective features of later DKD in adult mutants.

Diabetic Retinopathy: From Animal Models to Cellular Signaling.

Diabetic retinopathy (DR) is an ocular complication of diabetes mellitus (DM), a metabolic disorder characterized by elevation in blood glucose level. The pathogenesis of DR includes vascular, neuronal, and inflammatory components leading to activation of complex cellular molecular signaling. If untreated, the disease can culminate in vision loss that eventually leads to blindness. Animal models mimicking different aspects of DM complications have been developed to study the development and progression of DR. Despite the significant contribution of the developed DR models to discovering the mechanisms of DR and the recent achievements in the research field, the sequence of cellular events in diabetic retinas is still under investigation. Partially, this is due to the complexity of molecular mechanisms, although the lack of availability of models that adequately mimic all the neurovascular pathobiological features observed in patients has also contributed to the delay in determining a precise molecular trigger. In this review, we provide an update on the status of animal models of DR to help investigators choose an appropriate system to validate their hypothesis. We also discuss the key cellular and physiological events of DR in these models.

Superoxide dismutase 3 prevents early stage diabetic retinopathy in streptozotocin-induced diabetic rat model.

PurposeTo identify the effects of superoxide dismutase (SOD)3 on diabetes mellitus (DM)-induced retinal changes in a diabetic rat model.MethodsDiabetic models were established by a single intraperitoneal injection of streptozotocin (STZ) in Sprague-Dawley rats. After purification of the recombinant SOD3, intravitreal injection of SOD3 was performed at the time of STZ injection, and 1 and 2 weeks following STZ injection. Scotopic and photopic electroretinography (ERG) were recorded. Immunofluorescence staining with ɑ-smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), pigment epithelium-derived factor (PEDF), Flt1, recoverin, parvalbumin, extracellular superoxide dismutase (SOD3), 8-Hydroxy-2’deoxyguanosine (8-OHdG) and tumor necrosis factor-ɑ (TNF-ɑ) were evaluated.ResultsIn the scotopic ERG, the diabetic group showed reduced a- and b-wave amplitudes compared with the control group. In the photopic ERG, b-wave amplitude showed significant (p < 0.0005) reduction at 8 weeks following DM induction. However, the trend of a- and b-wave reduction was not evident in the SOD3 treated group. GFAP, Flt1, 8-OHdG and TNF-ɑ immunoreactivity were increased, and ɑ-SMA, PEDF and SOD3 immunoreactivity were decreased in the diabetic retina. The immunoreactivity of these markers was partially recovered in the SOD3 treated group. Parvalbumin expression was not decreased in the SOD3 treated group. In the diabetic retinas, the immunoreactivity of recoverin was weakly detected in both of the inner nuclear layer and inner plexiform layer compared to the control group but not in the SOD3 treated group.ConclusionsSOD3 treatment attenuated the loss of a/b-wave amplitudes in the diabetic rats, which was consistent with the immunohistochemical evaluation. We also suggest that in rod-dominant rodents, the use of blue on green photopic negative response (PhNR) is effective in measuring the inner retinal function in animal models of diabetic retinopathy. SOD3 treatment ameliorated the retinal Müller cell activation in diabetic rats and pericyte dysfunction. These results suggested that SOD3 exerted protective effects on the development of diabetic retinopathy.

Evidence for a dysfunction and disease-promoting role of the circadian clock in the diabetic retina

Diabetic retinopathy is a major complication of chronic hyperglycemia and a leading cause of blindness in developed countries. In the present study the interaction between diabetes and retinal clocks was investigated in mice. It was seen that in the db/db mouse – a widely used animal model of diabetic retinopathy – clock function and circadian regulation of gene expression was disturbed in the retina. Remarkably, elimination of clock function by Bmal1-deficiency mitigates the progression of pathophysiology of the diabetic retina. Thus high-fat diet was seen to induce histopathology and molecular markers associated with diabetic retinopathy in wild type but not in Bmal1-deficient mice. The data of the present study suggest that Bmal1/the retinal clock system is both, a target and an effector of diabetes mellitus in the retina and hence represents a putative therapeutic target in the pathogenesis of diabetic retinopathy.

Animal models of diabetic retinopathy.

The retina is the posterior neuro-integrated layer of the eye that conducts impulses induced by light to the optic nerve for human vision. Diseases of the retina often leads to diminished vision and in some cases blindness. Diabetes mellitus (DM) is a worldwide public health issue and globally, there is an estimated 463 million people that are affected by DM and its consequences. Diabetic retinopathy (DR) is a blinding complication of chronic uncontrolled DM and is the most common cause of blindness in the United States between the ages 24–75. It is estimated that the global prevalence of DR will increase to 191.0 million by 2030, of those 56.3 million possessing vision-threatening diabetic retinopathy (VTDR). For the most part, current treatment modalities control the complications of DR without addressing the underlying pathophysiology of the disease. Therefore, there is an unmet need for new therapeutics that not only repair the damaged retinal tissue, but also reverse the course of DR. The key element in developing these treatments is expanding our basic knowledge by studying DR pathogenesis in animal models of proliferative and non-proliferative DR (PDR and NPDR). There are numerous models available for the research of both PDR and NPDR with substantial overlap. Animal models available include those with genetic backgrounds prone to hyperglycemic states, immunologic etiologies, or environmentally induced disease. In this review we aimed to comprehensively summarize the available animal models for DR while also providing insight to each model’s ocular therapeutic potential for drug discovery.

Sex and Age-Related Differences in Neuroinflammation and Apoptosis in Balb/c Mice Retina Involve Resolvin D1.

(1) Background: The pro-resolving lipid mediator Resolvin D1 (RvD1) has already shown protective effects in animal models of diabetic retinopathy. This study aimed to investigate the retinal levels of RvD1 in aged (24 months) and younger (3 months) Balb/c mice, along with the activation of macro- and microglia, apoptosis, and neuroinflammation. (2) Methods: Retinas from male and female mice were used for immunohistochemistry, immunofluorescence, transmission electron microscopy, Western blotting, and enzyme-linked immunosorbent assays. (3) Results: Endogenous retinal levels of RvD1 were reduced in aged mice. While RvD1 levels were similar in younger males and females, they were markedly decreased in aged males but less reduced in aged females. Both aged males and females showed a significant increase in retinal microglia activation compared to younger mice, with a more marked reactivity in aged males than in aged females. The same trend was shown by astrocyte activation, neuroinflammation, apoptosis, and nitrosative stress, in line with the microglia and Müller cell hypertrophy evidenced in aged retinas by electron microscopy. (4) Conclusions: Aged mice had sex-related differences in neuroinflammation and apoptosis and low retinal levels of endogenous RvD1.

ASK1/p38‑mediated NLRP3 inflammasome signaling pathway contributes to aberrant retinal angiogenesis in diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness among the working-age population in several countries. Despite the available treatments, some patients are diagnosed at the late stages of the disease when treatment is more difficult. Hence, it is crucial that novel targets are identified in order to improve the clinical therapy of DR. In the present study, an animal model of DR and a cell model using primary human retinal microvascular endothelial cells exposed to high glucose were constructed to examine the association between apoptosis signal-regulating kinase 1 (ASK1)/p38 and NLR family pyrin domain containing 3 (NLRP3) in DR. The results revealed that DR induced inflammatory response and micro-vascular cell proliferation. NLRP3 contributed to DR-mediated inflammatory development and progression, which promoted the expression of inflammatory-related cytokines. In addition, NLRP3 promoted the tube formation of retinal microvascular endothelial cells and angiogenesis. Moreover, further research indicated that the NLRP3-mediated aberrant retinal angiogenesis in DR was regulated by ASK1 and p38. It was thus suggested that ASK1/p38 may be novel target for the treatment of DR.

A potential novel therapeutic target in diabetic retinopathy: a chemokine receptor (CCR2/CCR5) inhibitor reduces retinal vascular leakage in an animal model.

We have previously shown that the chemokine CCL2 plays an important role in monocyte trafficking into the retina and alteration of the BRB in an animal model of diabetic retinopathy. In this study, we examined the effect of pharmacologically targeting the chemokine pathway to reduce the increased retinal vascular permeability in this model. C57BL/6J mice were made diabetic using streptozotocin. After 4months of diabetes, mice (n = 10) were treated by intraperitoneal injections of TAK-779 (dual CCR2/CCR5 inhibitor) (30mg/kg) daily for 2weeks. Retinal vascular permeability and protein expression were done using western blot. The SDF-1 levels were measured by ELISA. Immune cell infiltration in the retinas was measured using flow cytometry. The dual inhibitor significantly decreased retinal vascular permeability in diabetic animals. There was a significant reduction in macrophage/microglia infiltration in the retinas of treated animals. Levels of SDF-1 and ICAM-1 were significantly reduced and the tight junction protein ZO-1 level was increased, and phospho-VE-Cad was significantly reduced with drug treatment. A chemokine receptor inhibitor (CCR2/CCR5) can reduce retinal vascular permeability in diabetic animals. Targeting the chemokine pathway pharmacologically may be used as a novel therapeutic strategy in management of diabetic macular edema.

A Robust Model System for Retinal Hypoxia: Live Imaging of Calcium Dynamics and Gene Expression Studies in Primary Human Mixed Retinal Culture.

The detailed mechanisms underlying oxidative stress that leads to neuroinflammation and neurodegeneration in retinal vascular conditions, including diabetic retinopathy, retinopathy of prematurity etc., remain largely unexplored mainly due to a lack of suitable disease models that can simulate the inherent neuron–glia interactions in human retina. Specifically, establishment of a mixed retinal culture (MRC) containing both neuron and glial cell types remains a challenge due to different conditions required for their optimal growth and differentiation. Here, we establish a novel primary MRC model system containing neurons, astrocytes, Müller glia, and microglia from human donor retina that can be used to study the neuromodulatory effects of glial cells under the stress. The cell characterization based on immunostaining with individual cell type–specific markers and their presence in close vicinity to each other further underscores their utility for studying their cross talk. To the best of our knowledge, this is the first instance of an in vitro model obtained from human donor retina containing four major cell types. Next, we induce hypoxic stress to MRC to investigate if hypoxia activated neuroglia modulates altered gene expression for inflammatory, apoptotic, and angiogenic markers and Ca2+ transients by live cell imaging. Further, we performed k-means clustering of the Ca2+ responses to identify the modification of clustering pattern in stressed condition. Finally, we provide the evidence that the altered Ca2+ transient correlates to differential expression of genes shown to be involved in neuroinflammation, angiogenesis, and neurodegeneration under the hypoxic conditions as seen earlier in human cell lines and animal models of diabetic retinopathy. The major features of the hypoxic conditions in the proposed human MRC model included: increase in microglia activity, chemokine and cytokine expression, and percentage of cells having higher amplitude and frequency of Ca2+ transients. Thus, the proposed experimental system can potentially serve as an ideal in vitro model for studying the neuroinflammatory and neurodegenerative changes in the retina and identifying newer drug targets.

Blood flow distribution and the endothelial surface layer in the diabetic retina.

Diabetic retinopathy is known as a microvascular complication of hyperglycemia, with a breakdown of the blood-retinal barrier, loss of pericytes, formation of microhemorrhages, early decreases in perfusion and areas of ischemia, with the latter speculated to induce the eventual proliferative, angiogenic phase of the disease. Our animal models of diabetic retinopathy demonstrate similar decreases in retinal blood flow as seen in the early stages of diabetes in humans. Our studies also show an alteration in the retinal distribution of red blood cells, with the deep capillary layer receiving a reduced fraction, and with flow being diverted more towards the superficial vascular layer. Normal red blood cell distribution is dependent on the presence of the endothelial surface layer, specifically the glycocalyx, which has been reported to be partially lost in the diabetic retina of both humans and animals. This review addresses these two phenomena in diabetes: altered perfusion patterns and loss of the glycocalyx, with a possible connection between the two.

Intravitreal pro-inflammatory cytokines in non-obese diabetic mice: Modelling signs of diabetic retinopathy.

Diabetic retinopathy is a vascular disease of the retina characterised by hyperglycaemic and inflammatory processes. Most animal models of diabetic retinopathy are hyperglycaemia-only models that do not account for the significant role that inflammation plays in the development of the disease. In the present study, we present data on the establishment of a new animal model of diabetic retinopathy that incorporates both hyperglycaemia and inflammation. We hypothesized that inflammation may trigger and worsen the development of diabetic retinopathy in a hyperglycaemic environment. Pro-inflammatory cytokines, IL-1β and TNF-α, were therefore injected into the vitreous of non-obese diabetic (NOD) mice. CD1 mice were used as same genetic background controls. Fundus and optical coherence tomography images were obtained before (day 0) as well as on days 2 and 7 after intravitreal cytokine injection to assess vessel dilation and beading, retinal and vitreous hyper-reflective foci and retinal thickness. Astrogliosis and microgliosis were assessed using immunohistochemistry. Results showed that intravitreal cytokines induced vessel dilation, beading, severe vitreous hyper-reflective foci, retinal oedema, increased astrogliosis and microglia upregulation in diabetic NOD mice. Intravitreal injection of inflammatory cytokines into the eyes of diabetic mice therefore appears to provide a new model of diabetic retinopathy that could be used for the study of disease progression and treatment strategies.

Deep Phenotyping of a Spontaneous Nonhuman Primate Model of Diabetic Macular Edema

Diabetic retinopathy (DR) is a diabetic complication involving damages to blood vessels in the retina that, if left untreated, can lead to blindness. It is estimated that over 93 million people suffer from DR worldwide and in fact the disease accounts for 12% of all new cases of blindness each year in the United States. Diabetic macular edema (DME) is one of the manifestations of DR, in which fluids leak from the blood vessels into the centre of the macula, causing pain, swelling, and blurred vision. However, the underlying pathogenesis of DR/DME is complex and not well understood, thus impeding the development of effective treatment. Consequently, the use of animal models of DR is of critical importance for better understanding of the underlying pathogenesis of DME, as well as developing new drugs. Non-human primate (NHP) models of diabetes are known to be the most clinically translatable animal models, where the animals develop major clinical phenotypes along the disease progression, including DR. As such, in this study we phenotyped a cohort of type 2 prediabetes/diabetes NHPs (n=123, weight: 11.5±2.0kg, age: 15.5±2.9years) using clinically relevant circulating and imaging biomarkers. Specifically, spectral domain optical coherence tomography (OCT) was first used to identify a subgroup of the animals with significantly thickened retina (329 ± 6.77µm) with an accumulation of hard exudates and inter-retinal cysts (n= 59). Fundus fluorescein angiography (FA) imaging further confirmed the presence of retinal vascular leakage into the cystoid spaces during the late-phase FA. Levels of vascular endothelial growth factor (VEGF) in both plasma and vitreous fluid were also measured and the results appear to correlate with retina thickness (vs. aged-matched controls). Taken together, our findings demonstrate a clinically translatable NHP model for DR/DME that allows characterization of disease progression in vivo, investigation of co-morbidities, and evaluation of novel therapeutics. Disclosure S. Chia: Employee; Self; Merck Sharp &amp; Dohme Corp.. L. Gong: None. S. Tiu: Employee; Self; Merck &amp; Co., Inc. S. Annamalai: Employee; Self; Merck Sharp &amp; Dohme Corp. N.X. Li: Employee; Self; Merck &amp; Co., Inc.. L. Pan: None. L. Hong: Employee; Self; Merck Sharp &amp; Dohme Corp.. Employee; Spouse/Partner; Illumina, Inc. A. Abu Bakar Ali: Employee; Self; Merck &amp; Co., Inc. C. Chin: Employee; Self; Merck &amp; Co., Inc.. W. Zeng: None.

Paying attention to the differences and applications of animal models of diabetic retinopathy

Diabetic retinopathy (DR) is a common complication of diabetes, and remains the leading cause of blindness among working-age individuals all over the world.Current treatments for DR mainly focus on the advanced stages of the disease and are associated with significant adverse effects.As the pathogenesis of DR is not thoroughly revealed, the development of more effective therapy of DR is challenging, in which a good DR animal model is essential.At present, these animal models involve mammals, non-mammals and other species.The modeling methods also include chemical injection, high glucose diet, genetic engineering and ex vivo retinal explant cultures.Although most of the models discussed, especially the rodent models, have demonstrated the basic features of NPDR, the key feature of human PDR (pre-retinal neovascularization secondary to diabetes per se) is not recapitulated in any diabetic animal models.Therefore, choosing the appropriate DR animal model according to the purpose of research will be very important in understanding development of the disease and new approaches that prevent or delay the onset of DR. Key words: Diabetes; Diabetic retinopathy; Animal model

Sustained inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown.

In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

Mesenchymal marker expression is elevated in Müller cells exposed to high glucose and in animal models of diabetic retinopathy.

Müller cells are retinal glial cells and exhibit a fibroblast-like phenotype and ability to migrate in diabetic retinopathy (DR). However, expression of mesenchymal markers, which promote fibrosis in various organs, has not been characterized in the diabetic retina. We examined changes in the expression of these markers in Müller cells exposed to high glucose and in animal models of diabetic retinopathy. High glucose conditions increased mesenchymal maker expression and migration in Müller cells. Snail, N-cadherin, Vimentin, β-catenin, and α-smooth muscle actin (α-SMA) levels were all dramatically increased in retinas from humans with diabetic retinopathy (DR) and from DR mouse models. In addition, Snail overexpression increased the expression of connective tissue growth factor (CTGF) and fibronectin, while Snail knockdown attenuated high glucose-induced increases in fibronectin and CTGF expression. These results demonstrate for the first time that mesenchymal markers are upregulated in retinas from a diabetic mouse model, and that Snail and N-cadherin levels are also increased in Müller cells exposed to high glucose. This suggests mesenchymal proteins may play a crucial role in the development of DR.

High glucose-induced changes in hyaloid-retinal vessels during early ocular development of zebrafish: a short-term animal model of diabetic retinopathy.

Although a variety of animal models have been used to test drug candidates and examine the pathogenesis of diabetic retinopathy, time-saving and inexpensive models are still needed to evaluate the increasing number of therapeutic approaches. We developed a model for diabetic retinopathy using the early stage of transgenic zebrafish (flk:EGFP) by treating embryos with 130 mM glucose, from 3-6 days post fertilisation (high-glucose model). On day 6, lenses from zebrafish larvae were isolated and treated with 3% trypsin, and changes in hyaloid-retinal vessels were analysed using fluorescent stereomicroscopy. In addition, expression of tight junction proteins (such as zonula occludens-1), effects of hyperosmolar solutions and of hypoxia, and Vegf expression were assessed by RT -PCR. NO production was assessed with a fluorescent substrate. Effects of inhibitors of the VEGF receptor, NO synthesis and a VEGF antibody (ranibizumab) were also measured. In this high-glucose model, dilation of hyaloid-retinal vessels, on day 6, was accompanied by morphological lesions with disruption of tight junction proteins, overproduction of Vegf mRNA and increased NO production. Treatment of this high-glucose model with an inhibitor of VEGF receptor tyrosine kinase or an inhibitor of NO synthase or ranibizumab decreased dilation of hyaloid-retinal vessels. These findings suggest that short-term exposure of zebrafish larvae to high-glucose conditions could be used for screening and drug discovery for diabetic retinopathy and particularly for disorders of retinal vessels related to disruption of tight junction proteins and excessive VEGF and NO production.

High Glucose-induced Retinal Pericyte Apoptosis Depends on Association of GAPDH and Siah1

Diabetic retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive, and have limited effectiveness. Retinal pericyte death is an early pathologic feature of DR. Although it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified in ocular tissues. In this article we examined the involvement of the GAPDH/Siah1 interaction in human retinal pericyte (hRP) apoptosis. HRP were cultured in 5 mm normal glucose, 25 mm l- or d-glucose for 48 h (osmotic control and high glucose treatments, respectively). Siah1 siRNA was used to down-regulate Siah1 expression. TAT-FLAG GAPDH and/or Siah1-directed peptides were used to block GAPDH and Siah1 interaction. Co-immunoprecipitation assays were conducted to analyze the effect of high glucose on the association of GAPDH and Siah1. Apoptosis was measured by Annexin V staining and caspase-3 enzymatic activity assay. High glucose increased Siah1 total protein levels, induced the association between GAPDH and Siah1, and led to GAPDH nuclear translocation. Our findings demonstrate that dissociation of the GAPDH/Siah1 pro-apoptotic complex can block high glucose-induced pericyte apoptosis, widely considered a hallmark feature of DR. Thus, the work presented in this article can provide a foundation to identify novel targets for early treatment of DR.