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.

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.

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.

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.

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.

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.

Low survival rate and muscle fiber-dependent aging effects in the McArdle disease mouse model.

McArdle disease is an autosomal recessive disorder caused by the absence of the muscle glycogen phosphorylase, which leads to impairment of glycogen breakdown. The McArdle mouse, a model heavily affected by glycogen accumulation and exercise intolerance, was used to characterize disease progression at three different ages. The molecular and histopathological consequences of the disease were analyzed in five different hind-limb muscles (soleus, extensor digitorum longus, tibialis anterior, gastrocnemius and quadriceps) of young (8-week-old), adult (35-week-old) and old (70-week-old) mice. We found that McArdle mice have a high perinatal and post-weaning mortality. We also observed a progressive muscle degeneration, fibrosis and inflammation process that was not associated with an increase in muscle glycogen content during aging. Additionally, this progressive degeneration varied among muscle and fiber types. Finally, the lack of glycogen content increase was associated with the inactivation of glycogen synthase and not with compensatory expression of the Pygl and/or Pygb genes in mature muscle.