Blocking Hemopexin With Specific Antibodies: A New Strategy for Treating Diabetic Retinopathy.

Hemopexin (HPX) is overexpressed in the retina of patients with diabetes and induces the breakdown of the blood-retinal barrier in vitro. The aim of this study was to evaluate whether HPX blockade by specific antibodies (aHPX) could avoid vascular leakage in vivo and microvascular angiogenesis in vitro and ex vivo. For this purpose, the effect of intravitreal (IVT) injections of aHPX on vascular leakage was evaluated in db/db mice and rats with streptozotocin-induced diabetes using the Evans Blue method. Retinal neurodegeneration and inflammation were also evaluated. The antiangiogenic effect of aHPX on human retinal endothelial cells (HRECs) was tested by scratch wound healing and tube formation using standardized methods, as well as by choroidal sprouting assays from retinal explants obtained in rats. We found that IVT injection of aHPX significantly reduced vascular leakage, retinal neurodegeneration, and inflammation. In addition, treatment with aHPX significantly reduced HREC migration and tube formation induced by high glucose concentration and suppressed choroidal sprouting even after vascular endothelial growth factor stimulation, with this effect being higher than obtained with bevacizumab. The antipermeability and antiangiogenic effects of IVT injection of aHPX suggest the blockade or inhibition of HPX as a new strategy for the treatment of advanced stages of diabetic retinopathy. ARTICLE HIGHLIGHTS: Hemopexin (HPX) is the best-characterized permeability factor in steroid-sensitive nephrotic syndrome. We have previously reported that HPX is overexpressed in the retina of patients with diabetes and induces the breakdown of the blood-retinal barrier in vitro. Here, we report that intravitreal injection of anti-HPX antibodies significantly reduces vascular leakage, retinal neurodegeneration, and inflammation in diabetic murine models and that the immunoneutralization of HPX exerts a significant antiangiogenic effect in vitro and in retinal explants. The blockade of HPX can be considered as a new therapy for advanced stages of diabetic retinopathy.

Caffeine and the Risk of Diabetic Retinopathy in Type 2 Diabetes Mellitus: Findings from Clinical and Experimental Studies.

The aim of this study was to assess the potential benefits of caffeine intake in protecting against the development of diabetic retinopathy (DR) in subjects with type 2 diabetes (T2D). Furthermore, we tested the effect of topical administration of caffeine on the early stages of DR in an experimental model of DR. In the cross-sectional study, a total of 144 subjects with DR and 147 individuals without DR were assessed. DR was assessed by an experienced ophthalmologist. A validated food frequency questionnaire (FFQ) was administered. In the experimental model, a total of 20 mice were included. One drop (5 µL) of caffeine (5 mg/mL) (n = 10) or vehicle (5 µL PBS, pH 7.4) (n = 10) was randomly administered directly onto the superior corneal surface twice daily for two weeks in each eye. Glial activation and retinal vascular permeability were assessed using standard methods. In the cross-sectional study in humans, the adjusted-multivariable model showed that a moderate and high (Q2 and Q4) caffeine intake had a protective effect of DR (odds ratio (95% confidence interval) = 0.35 (0.16-0.78); p = 0.011 and 0.35 (0.16-0.77); p = 0.010, respectively). In the experimental model, the administration of caffeine did not improve either reactive gliosis or retinal vascular permeability. Our results suggest a dose-dependent protective effect of caffeine in the development of DR, while the potential benefits of antioxidants in coffee and tea should also be considered. Further research is needed to establish the benefits and mechanisms of caffeinated beverages in the development of DR.

Antioxidant Effects of DPP-4 Inhibitors in Early Stages of Experimental Diabetic Retinopathy.

Hyperglycemia-induced oxidative stress plays a key role in the impairment of the retinal neurovascular unit, an early event in the pathogenesis of DR. The aim of this study was to assess the antioxidant properties of topical administration (eye drops) of sitagliptin in the diabetic retina. For this purpose, db/db mice received sitagliptin or vehicle eye drops twice per day for two weeks. Age-matched db/+ mice were used as the control group. We evaluated retinal mRNA (RT-PCR) and protein levels (Western blotting and immunohistochemistry) of different components from both the antioxidant system (NRF2, CAT, GPX, GR, CuZnSOD, and MnSOD) and the prooxidant machinery (PKC and TXNIP). We also studied superoxide levels (dihydroethidium staining) and oxidative damage to DNA/RNA (8-hydroxyguanosine immunostaining) and proteins (nitrotyrosine immunostaining). Finally, NF-кB translocation and IL-1ß production were assessed through Western blotting and/or immunohistochemistry. We found that sitagliptin protected against diabetes-induced oxidative stress by reducing superoxide, TXNIP, PKC, and DNA/RNA/protein oxidative damage, and it prevented the downregulation of NRF2 and antioxidant enzymes, with the exception of catalase. Sitagliptin also exerted anti-inflammatory effects, avoiding both NF-кB translocation and IL-1ß production. Sitagliptin prevents the diabetes-induced imbalance between ROS production and antioxidant defenses that occurs in diabetic retinas.

Diabetic Retinopathy: Role of Neurodegeneration and Therapeutic Perspectives.

ABSTRACT: Retinal neurodegeneration plays a significant role in the pathogenesis of diabetic retinopathy, the leading cause of preventable blindness. The hallmarks of diabetes-induced neurodegeneration are neural cell apoptosis and glial activation, which seem even before vascular lesions can be detected by ophthalmoscopic examination. The molecular mediators of retinal neurodegeneration include proinflamma- tory cytokines, oxidative stress, mitochondrial dysfunction, and the molecular pathways closely related to chronic hyperglycemia. In this article, an overview of the main components of neurodegeneration, its key underlying mechanisms, and the more useful experimental models for investigative purposes will be given. In addition, the results of most relevant treatments based on neuroprotection, and the research gaps that should be filled will be critically reviewed.

Minimum Effective Dose of DPP-4 Inhibitors for Treating Early Stages of Diabetic Retinopathy in an Experimental Model.

The neurovascular unit (NVU) plays an essential role in the development of diabetic retinopathy (DR). We previously reported that the topical administration (eye drops) of sitagliptin and saxagliptin, two dipeptidyl peptidase-4 inhibitors (DPP-4i), prevents retinal neurodegeneration and vascular leakage in db/db mice. The aim of the present study is to evaluate the minimum effective dose of the topical administration of these DPP-4i. For this purpose, sitagliptin and saxagliptin were tested at different concentrations (sitagliptin: 1 mg/mL, 5 and 10 mg/mL, twice per day; saxagliptin: 1 and 10 mg/mL, once or twice per day) in db/db mice. As end points of efficacy, the hallmarks of NVU impairment were evaluated: reactive gliosis, neural apoptosis, and vascular leakage. These parameters were assessed by immunohistochemistry, cell counting, and the Evans blue method, respectively. Our results demonstrated that the minimum effective dose is 5 mg/mL twice per day for sitagliptin, and 10 mg/mL twice per day for saxagliptin. In conclusion, this study provides useful results for the design of future preclinical regulatory studies and for planning clinical trials.

Transcriptomic Analysis Reveals That Retinal Neuromodulation Is a Relevant Mechanism in the Neuroprotective Effect of Sitagliptin in an Experimental Model of Diabetic Retinopathy.

Synaptic dysfunction and neuronal damage have been extensively associated with diabetic retinopathy (DR). Our group evidenced that chronic hyperglycemia reduces the retinal expression of presynaptic proteins, which are crucial for proper synaptic function. The aim of the study was to explore the effect of topically administered sitagliptin, an inhibitor of the enzyme dipeptidyl peptidase-4, on the retinal expression patterns of an experimental model of DR. Transcriptome analysis was performed, comparing the retinas of 10 diabetic (db/db) mice randomly treated with sitagliptin eye drops (10 mg/mL) twice daily and the retinas of 10 additional db/db mice that received vehicle eye drops. Ten non-diabetic mice (db/+) were used as a control group. The Gene Ontology (GO) and Reactome databases were used to perform the gene set enrichment analysis (GSEA) in order to explore the most enriched biological pathways among the groups. The most differentiated genes of these pathways were validated through quantitative RT-PCR. Transcriptome analysis revealed that sitagliptin eye drops have a significant effect on retinal expression patterns and that neurotransmission is the most enriched biological process. Our study evidenced enriched pathways that contain genes involved in membrane trafficking, transmission across chemical synapses, vesicle-mediated transport, neurotransmitter receptors and postsynaptic signal transmission with negative regulation of signaling as a consequence of neuroprotector treatment with sitagliptin. This improves the modulation of the macromolecule biosynthetic process with positive regulation of cell communication, which provides beneficial effects for the neuronal metabolism. This study suggests that topical administration of sitagliptin ameliorates the abnormalities on presynaptic and postsynaptic signal transmission during experimental DR and that this improvement is one of the main mechanisms behind the previously demonstrated beneficial effects.

ERM Complex, A Therapeutic Target for Vascular Leakage Induced by Diabetes.

BACKGROUND: Ezrin, radixin, and moesin (the ERM complex) interact directly with membrane proteins regulating their attachment to actin filaments. ERM protein activation modifies cytoskeleton organization and alters the endothelial barrier function, thus favoring vascular leakage. However, little is known regarding the role of ERM proteins in diabetic retinopathy (DR). OBJECTIVE: This study aimed to examine whether overexpression of the ERM complex exists in db/db mice and its main regulating factors. METHODS: 9 male db/db mice and 9 male db/+ aged 14 weeks were analyzed. ERM proteins were assessed by western blot and by immunohistochemistry. Vascular leakage was determined by the Evans blue method. To assess ERM regulation, HRECs were cultured in a medium containing 5.5 mM D-glucose (mimicking physiological conditions) and 25 mM D-glucose (mimicking hyperglycemia that occurs in diabetic patients). Moreover, treatment with TNF-α, IL-1ß, or VEGF was added to a high glucose condition. The expression of ERM proteins was quantified by RT-PCR. Cell permeability was evaluated by measuring movements of FITC-dextran. RESULTS: A significant increase of ERM in diabetic mice in comparison with non-diabetic mice was observed. A high glucose condition alone did not have any effect on ERM expression. However, TNF-α and IL-1ß induced a significant increase in ERM proteins. CONCLUSION: The increase of ERM proteins induced by diabetes could be one of the mechanisms involved in vascular leakage and could be considered as a therapeutic target. Moreover, the upregulation of the ERM complex by diabetes is induced by inflammatory mediators rather than by high glucose itself.

Neuromodulation Induced by Sitagliptin: A New Strategy for Treating Diabetic Retinopathy.

Diabetic retinopathy (DR) involves progressive neurovascular degeneration of the retina. Reduction in synaptic protein expression has been observed in retinas from several diabetic animal models and human retinas. We previously reported that the topical administration (eye drops) of sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, prevented retinal neurodegeneration induced by diabetes in db/db mice. The aim of the present study is to examine whether the modulation of presynaptic proteins is a mechanism involved in the neuroprotective effect of sitagliptin. For this purpose, 12 db/db mice, aged 12 weeks, received a topical administration of sitagliptin (5 µL; concentration: 10 mg/mL) twice per day for 2 weeks, while other 12 db/db mice were treated with vehicle (5 µL). Twelve non-diabetic mice (db/+) were used as a control group. Protein levels were assessed by western blot and immunohistochemistry (IHC), and mRNA levels were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Our results revealed a downregulation (protein and mRNA levels) of several presynaptic proteins such as synapsin I (Syn1), synaptophysin (Syp), synaptotagmin (Syt1), syntaxin 1A (Stx1a), vesicle-associated membrane protein 2 (Vamp2), and synaptosomal-associated protein of 25 kDa (Snap25) in diabetic mice treated with vehicle in comparison with non-diabetic mice. These proteins are involved in vesicle biogenesis, mobilization and docking, membrane fusion and recycling, and synaptic neurotransmission. Sitagliptin was able to significantly prevent the downregulation of all these proteins. We conclude that sitagliptin exerts beneficial effects in the retinas of db/db mice by preventing the downregulation of crucial presynaptic proteins. These neuroprotective effects open a new avenue for treating DR as well other retinal diseases in which neurodegeneration/synaptic abnormalities play a relevant role.

Faim knockout leads to gliosis and late-onset neurodegeneration of photoreceptors in the mouse retina.

Fas Apoptotic Inhibitory Molecule protein (FAIM) is a death receptor antagonist and an apoptosis regulator. It encodes two isoforms, namely FAIM-S (short) and FAIM-L (long), both with significant neuronal functions. FAIM-S, which is ubiquitously expressed, is involved in neurite outgrowth. In contrast, FAIM-L is expressed only in neurons and it protects them from cell death. Interestingly, FAIM-L is downregulated in patients and mouse models of Alzheimer's disease before the onset of neurodegeneration, and Faim transcript levels are decreased in mouse models of retinal degeneration. However, few studies have addressed the role of FAIM in the central nervous system, yet alone the retina. The retina is a highly specialized tissue, and its degeneration has proved to precede pathological mechanisms of neurodegenerative diseases. Here we describe that Faim depletion in mice damages the retina persistently and leads to late-onset photoreceptor death in older mice. Immunohistochemical analyses showed that Faim knockout (Faim-/- ) mice present ubiquitinated aggregates throughout the retina from early ages. Moreover, retinal cells released stress signals that can signal to Müller cells, as shown by immunofluorescence and qRT-PCR. Müller cells monitor retinal homeostasis and trigger a gliotic response in Faim-/- mice that becomes pathogenic when sustained. In this regard, we observed pronounced vascular leakage at later ages, which may be caused by persistent inflammation. These results suggest that FAIM is an important player in the maintenance of retinal homeostasis, and they support the premise that FAIM is a plausible early marker for late photoreceptor and neuronal degeneration.

Neurovascular Unit: A New Target for Treating Early Stages of Diabetic Retinopathy.

The concept of diabetic retinopathy as a microvascular disease has evolved and is now considered a more complex diabetic complication in which neurovascular unit impairment plays an essential role and, therefore, can be considered as a main therapeutic target in the early stages of the disease. However, neurodegeneration is not always the apparent primary event in the natural story of diabetic retinopathy, and a phenotyping characterization is recommendable to identify those patients in whom neuroprotective treatment might be of benefit. In recent years, a myriad of treatments based on neuroprotection have been tested in experimental models, but more interestingly, there are drugs with a dual activity (neuroprotective and vasculotropic). In this review, the recent evidence concerning the therapeutic approaches targeting neurovascular unit impairment will be presented, along with a critical review of the scientific gaps and problems which remain to be overcome before our knowledge can be transferred to clinical practice.

Effects of the Topical Administration of Semaglutide on Retinal Neuroinflammation and Vascular Leakage in Experimental Diabetes.

BACKGROUND: An unexpected increase in the rate of severe diabetic retinopathy was observed in the Semaglutide in Subjects with Type 2 Diabetes (SUSTAIN)-6 clinical trial. Although this effect was attributed to a rapid decrease in blood glucose levels, a direct deleterious effect of semaglutide on the retina could not be ruled out. In order to shed light on this issue, we have performed a study aimed at testing the direct effect of semaglutide administered by eye drops on retinal neuroinflammation and microvascular abnormalities using the db/db mouse model. METHODS: Eye drops containing semaglutide (0.33 mg/mL; 5 µL once/daily) or vehicle (PBS; 5 µL once daily) were administered for 15 days. RESULTS: We found that semaglutide significantly reduced glial activation, as well as the retinal expression of Nuclear factor kB (NF-κB), proinflammatory cytokines (IL-1ß, IL-6, IL-18) and Intercellular Adhesion Molecule (ICAM)-1. In addition, semaglutide prevented the apoptosis of cells from the retinal ganglion layer and activated the protein kinase B (AKT) pathway. Finally, a dramatic decrease in vascular leakage was observed in db/db mice treated with semaglutide. All these findings were observed without any change in blood glucose levels and, therefore, can be directly attributed to semaglutide. CONCLUSIONS: These experimental findings point to a beneficial rather than a deleterious effect of semaglutide on the retina of subjects with diabetes.

Beneficial Effects of Glucagon-Like Peptide-1 (GLP-1) in Diabetes-Induced Retinal Abnormalities: Involvement of Oxidative Stress.

BACKGROUND: Hyperglycemia-induced oxidative stress plays a key role in diabetic complications, including diabetic retinopathy. The main goal of this study was to assess whether the topical administration (eye drops) of glucagon-like peptide-1 (GLP-1) has any effect on oxidative stress in the retina. METHODS: db/db mice were treated with eye drops of GLP-1 or vehicle for three weeks, with db/+ mice being used as control. Studies included the assessment by western blot of the antioxidant defense markers CuZnSOD, MnSOD, glutathione peroxidase and reductase; immunofluorescence measurements of DNA/RNA damage, nitro tyrosine and Ki67 and Babam2 proteins. RESULTS: GLP-1 eye drops protected from oxidative stress by increasing the protein levels of glutathione reductase, glutathione peroxidase and CuZnSOD and MnSOD in diabetic retinas. This was associated with a significant reduction of DNA/RNA damage and the activation of proteins involved in DNA repair in the retina (Babam2) and Ki67 (a biomarker of cell proliferation). CONCLUSIONS: GLP-1 modulates the antioxidant defense system in the diabetic retina and has a neuroprotective action favoring DNA repair and neuron cells proliferation.

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.

SOCS1-Derived Peptide Administered by Eye Drops Prevents Retinal Neuroinflammation and Vascular Leakage in Experimental Diabetes.

Current treatments for diabetic retinopathy (DR) target late stages when vision has already been significantly affected. Accumulating evidence suggests that neuroinflammation plays a major role in the pathogenesis of DR, resulting in the disruption of the blood-retinal barrier. Suppressors of cytokine signaling (SOCS) are cytokine-inducible proteins that function as a negative feedback loop regulating cytokine responses. On this basis, the aim of the present study was to evaluate the effect of a SOCS1-derived peptide administered by eye drops (2 weeks) on retinal neuroinflammation and early microvascular abnormalities in a db/db mouse model. In brief, we found that SOCS1-derived peptide significantly reduced glial activation and neural apoptosis induced by diabetes, as well as retinal levels of proinflammatory cytokines. Moreover, a significant improvement of electroretinogram parameters was observed, thus revealing a clear impact of the histological findings on global retinal function. Finally, SOCS1-derived peptide prevented the disruption of the blood-retinal barrier. Overall, our results suggest that topical administration of SOCS1-derived peptide is effective in preventing retinal neuroinflammation and early microvascular impairment. These findings could open up a new strategy for the treatment of early stages of DR.

New Insights into the Mechanisms of Action of Topical Administration of GLP-1 in an Experimental Model of Diabetic Retinopathy.

The main goals of this work were to assess whether the topical administration of glucagon-like peptide-1 (GLP-1) could revert the impairment of the neurovascular unit induced by long-term diabetes (24 weeks) in diabetic mice and to look into the underlying mechanisms. For that reason, db/db mice were treated with eye drops of GLP-1 or vehicle for 3 weeks. Moreover, db/+ mice were used as control. Studies performed in vivo included electroretinogramand the assessment of vascular leakage by using Evans Blue. NF-κB, GFAP and Ki67 proteins were analyzed by immunofluorescence (IF). Additionally, caspase 9, AMPK, IKBα, NF-κB, AKT, GSK3, ß-catenin, Bcl-xl, and VEGF were analyzed by WB. Finally, VEGF, IL-1ß, IL-6, TNF-α, IL-18, and NLRP3 were studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. We found that topical administration of GLP-1 reverted reactive gliosis and albumin extravasation, and protected against apoptosis and retinal dysfunction. Regarding the involved mechanisms, GLP-1 exerted an anti-inflammatory action by decreasing NF-κB, inflammosome, and pro-inflammatory factors. In addition, it also decreased VEGF expression. Furthermore, GLP-1 promoted cell survival by increasing the anti-apoptotic protein Bcl-xl and the signaling pathway Akt/GSK3b/ß-catenin. Finally, Ki67 results revealed that GLP-1 treatment could induce neurogenesis. In conclusion, the topical administration of GLP-1 reverts the impairment of the neurovascular unit by modulating essential pathways involved in the development of diabetic retinopathy (DR). These beneficial effects on the neurovascular unit could pave the way for clinical trials addressed to confirm the effectiveness of GLP-1 in early stages of DR.

Topical Administration of Bosentan Prevents Retinal Neurodegeneration in Experimental Diabetes.

Experimental evidence suggests that endothelin 1 (ET-1) is involved in the development of retinal microvascular abnormalities induced by diabetes. The effects of ET-1 are mediated by endothelin A- and B-receptors (ETA and ETB). Endothelin B-receptors activation mediates retinal neurodegeneration but there are no data regarding the effectiveness of ETB receptor blockage in arresting retinal neurodegeneration induced by diabetes. The main aim of the present study was to assess the usefulness of topical administration of bosentan (a dual endothelin receptor antagonist) in preventing retinal neurodegeneration in diabetic (db/db) mice. For this purpose, db/db mice aged 10 weeks were treated with one drop of bosentan (5 mg/mL, n = 6) or vehicle (n = 6) administered twice daily for 14 days. Six non-diabetic (db/+) mice matched by age were included as the control group. Glial activation was evaluated by immunofluorescence using specific antibodies against glial fibrillary acidic protein (GFAP). Apoptosis was assessed by TUNEL method. A pharmacokinetic study was performed in rabbits. We found that topical administration of bosentan resulted in a significant decrease of reactive gliosis and apoptosis. The results of the pharmacokinetic study suggested that bosentan reached the retina through the trans-scleral route. We conclude that topical administration of bosentan was effective in preventing neurodegeneration in the diabetic retina and, therefore, could be a good candidate to be tested in clinical trials.

Effects of Liposomal Formulation of Citicoline in Experimental Diabetes-Induced Retinal Neurodegeneration.

Diabetic retinopathy (DR) has been classically considered a microcirculatory disease of the retina. However, there is growing evidence to suggest that retinal neurodegeneration is also an early event in the pathogenesis of DR. Citicoline has been successfully used as a neuroprotective agent in the treatment of glaucoma but their effects on DR remain to be elucidated. On this basis, the main aim of the present study was to evaluate the effect of topical administration of citicoline in liposomal formulation on retinal neurodegeneration in db/db mouse and to investigate the underlying mechanisms of action. The treatment (citicoline or vehicle) was topically administered twice daily for 15 days. Retinal analyses were performed in vivo by electroretinography and ex vivo by using Western blot and immunofluorescence measurements. We found that the liposomal formulation of citicoline prevented glial activation and neural apoptosis in the diabetic retina. The main mechanism implicated in these beneficial effects were the inhibition of the downregulation of synaptophysin and its anti-inflammatory properties by means of preventing the upregulation of NF-κB and TNF-α (Tumor Necrosis Factor α) induced by diabetes. Overall, these results suggest that topical administration of citicoline in liposomal formulation could be considered as a new strategy for treating the early stages of DR.

Calcium dobesilate prevents the oxidative stress and inflammation induced by diabetes in the retina of db/db mice.

AIM: Calcium dobesilate (CaD) is beneficial in early stages of diabetic retinopathy (DR), but its mechanisms of action remains to be elucidated. The aim was to investigate the effect of CaD on proinflammatory cytokines and oxidative stress. METHODS: db/db mice were randomly assigned to daily oral treatment with CaD (200mg/kg/day) or vehicle for 15days. Biomarkers of oxidative stress (dihydroethidium, malondialdehyde), NF-κB, and proinflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α, MCP-1) were examined in the retina by immunohistochemical analysis. Cultures of human retinal endothelial cells (HRECs) were used for complementary experiments. RESULTS: CaD significantly reduced the biomarkers of oxidative stress in the retina of db/db mice. In addition, CaD prevented the increase of NF-κB, IL-6, IL-8, TNF-α and MCP-1 induced by diabetes. CaD inhibited the activation of NF-kß induced by IL-1ß by preventing IKKB-α phosphorylation in HRECs and reduced the upregulation of IL-6 and IL-18 induced by TNF-α in a dose-dependent manner. CONCLUSION: Our results suggest that antioxidant and antiinflammatory effects are crucial in accounting for the effectiveness of CaD for treating DR.

Topical administration of DPP-IV inhibitors prevents retinal neurodegeneration in experimental diabetes.

AIMS/HYPOTHESIS: The main aims of the present study were: (1) to assess the expression and content of dipeptidyl peptidase IV (DPP-IV) in human and db/db mouse retinas, and in human vitreous fluid; and (2) to determine whether the topical administration of the DPP-IV inhibitors (DPP-IVi) would prevent retinal neurodegeneration and vascular leakage in db/db mice by reducing endogenous glucagon-like peptide 1 (GLP-1) degradation. METHODS: To assess the expression and content of DPP-IV, human samples of vitreous fluid and retinas were obtained from participants with type 2 diabetes (n = 8) and age-matched non-diabetic individuals (n = 8), as well as from db/db (n = 72) and db/+ (n = 28) mice. The interventional study, which included 72 db/db mice, consisted of the topical administration (eye drops) of saxagliptin, sitagliptin or vehicle for 14 days. DPP-IV mRNA levels were assessed by RT-PCR, and protein content was measured by ELISA or western blotting. GLP-1 was assessed by immunofluorescence, and its downstream effector exchange protein activated by cAMP-1 (EPAC-1) was used as a measure of GLP-1 receptor activation. Retinal analyses were performed in vivo by electroretinography and ex vivo by RT-PCR (Epac-1, Iba-1 [also known as Aif1]), western blotting (EPAC-1, glial fibrillar acidic protein [GFAP], glutamate-aspartate transporter [GLAST]) and immunofluorescence measurements (GLP-1, GFAP, ionised calcium binding adaptor molecule 1 [IBA-1], TUNEL, GLAST, albumin and collagen IV). Glutamate was quantified by HPLC. In addition, vascular leakage was examined by the Evans Blue method. RESULTS: DPP-IV was present in human vitreous fluid but in a range 100-fold less than in plasma. Both mRNA levels and protein content were much lower in the retina than in the liver or bowel, but were significantly higher in retinal pigment epithelium (RPE) from diabetic donors in comparison to non-diabetic donors (p < 0.05). Topical treatment with DPP-IVi prevented glial activation, apoptosis and vascular leakage induced by diabetes in db/db mice (p < 0.05). Moreover, it also significantly prevented diabetes-induced functional abnormalities in the electroretinogram. A significant increase of both GLP-1 and EPAC-1 was found after treatment with DPP-IVi (p < 0.05). Furthermore, GLAST downregulation induced by diabetes was prevented, resulting in a significant reduction of extracellular glutamate concentrations. All these effects were observed without any changes in blood glucose levels. CONCLUSIONS/INTERPRETATION: The topical administration of DPP-IVi is effective in preventing neurodegeneration and vascular leakage in the diabetic retina. These effects can be attributed to an enhancement of GLP-1, but other mechanisms unrelated to the prevention of GLP-1 degradation cannot be ruled out.