Synergistic effect of chitosan and ß-carotene in inhibiting MNU-induced retinitis pigmentosa.

In this study, N-Methyl-N-nitrosourea (MNU) was intraperitoneally injected to construct a mouse retinitis pigmentosa (RP) model to evaluate the protective effect of chitosan and ß-carotene on RP. The results demonstrated that chitosan synergized with ß-carotene significantly reduced retinal histopathological structural damage in RP mice. The co-treatment group of ß-carotene and chitosan restored the retinal thickness and outer nuclear layer thickness better than the group treated with the two alone, and the thickness reached the normal level. The content of ß-carotene and retinoids in the liver of chitosan and ß-carotene co-treated group increased by 46.75 % and 20.69 %, respectively, compared to the ß-carotene group. Chitosan and ß-carotene supplement suppressed the expressions of Bax, Calpain2, Caspase3, NF-κB, TNF-α, IL-6, and IL-1ß, and promoted the up-regulation of Bcl2. Chitosan and ß-carotene interventions remarkably contributed to the content of SCFAs and enhanced the abundance of Ruminococcaceae, Rikenellaceae, Odoribacteraceae and Helicobacteraceae. Correlation analysis demonstrated a strong association between gut microbiota and improvement in retinitis pigmentosa. This study will provide a reference for the study of the gut-eye axis.

Long-term intake of Lactobacillus helveticus enhances bioavailability of omega-3 fatty acids in the mouse retina.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA), are required for the structure and function of the retina. Several observational studies indicate that consumption of a diet with relatively high levels of n-3 PUFAs, such as those provided by fish oils, has a protective effect against the development of age-related macular degeneration. Given the accumulating evidence showing the role of gut microbiota in regulating retinal physiology and host lipid metabolism, we evaluated the potential of long-term dietary supplementation with the Gram-positive bacterium Lactobacillus helveticus strain VEL12193 to modulate the retinal n-3 PUFA content. A set of complementary approaches was used to study the impact of such a supplementation on the gut microbiota and host lipid/fatty acid (FA) metabolism. L. helveticus-supplementation was associated with a decrease in retinal saturated FAs (SFAs) and monounsaturated FAs (MUFAs) as well as an increase in retinal n-3 and omega-6 (n-6) PUFAs. Interestingly, supplementation with L. helveticus enriched the retina in C22:5n-3 (docosapentaenoic acid, DPA), C22:6n-3 (DHA), C18:2n-6 (linoleic acid, LA) and C20:3n-6 (dihomo gamma-linolenic acid, DGLA). Long-term consumption of L. helveticus also modulated gut microbiota composition and some changes in OTUs abundance correlated with the retinal FA content. This study provides a proof of concept that targeting the gut microbiota could be an effective strategy to modulate the retinal FA content, including that of protective n-3 PUFAs, thus opening paths for the design of novel preventive and/or therapeutical strategies for retinopathies.

Extracellular vesicles in degenerative retinal diseases: A new therapeutic paradigm.

Nanoscale extracellular vesicles (EVs), consisting of exomers, exosomes and microvesicles/ectosomes, have been extensively investigated in the last 20 years, although their biological role is still something of a mystery. EVs are involved in the transfer of lipids, nucleic acids and proteins from donor to recipient cells or distant organs as well as regulating cell-cell communication and signaling. Thus, EVs are important in intercellular communication and this is not limited to sister cells, but may also mediate the crosstalk between different cell types even over long distances. EVs play crucial functions in both cellular homeostasis and the pathogenesis of diseases, and since their contents reflect the status of the donor cell, they represent an additional valuable source of information for characterizing complex biological processes. Recent advances in isolation and analytical methods have led to substantial improvements in both characterizing and engineering EVs, leading to their use either as novel biomarkers for disease diagnosis/prognosis or even as novel therapies. Due to their capacity to carry biomolecules, various EV-based therapeutic applications have been devised for several pathological conditions, including eye diseases. In the eye, EVs have been detected in the retina, aqueous humor, vitreous body and also in tears. Experiences with other forms of intraocular drug applications have opened new ways to use EVs in the treatment of retinal diseases. We here provide a comprehensive summary of the main in vitro, in vivo, and ex vivo literature-based studies on EVs' role in ocular physiological and pathological conditions. We have focused on age-related macular degeneration, diabetic retinopathy, glaucoma, which are common eye diseases leading to permanent blindness, if not treated properly. In addition, the putative use of EVs in retinitis pigmentosa and other retinopathies is discussed. Finally, we have reviewed the potential of EVs as therapeutic tools and/or biomarkers in the above-mentioned retinal disorders. Evidence emerging from experimental disease models and human material strongly suggests future diagnostic and/or therapeutic exploitation of these biological agents in various ocular disorders with a good possibility to improve the patient's quality of life.

Exploring the Potential of Wild Olive (Acebuche) Oil as a Pharm-Food to Prevent Ocular Hypertension and Fibrotic Events in the Retina of Hypertensive Mice.

SCOPE: Our laboratory has previously described the antioxidant and anti-inflammatory potential of a wild olive (acebuche, ACE) oil against hypertension-associated vascular retinopathies. The current study aims to analyze the antifibrotic effect of ACE oil on the retina of hypertensive mice. METHODS AND RESULTS: Mice are rendered hypertensive by administration of NG-nitro-L-arginine-methyl-ester (L-NAME) and simultaneously subjected to dietary supplementation with ACE oil or a reference extra virgin olive oil (EVOO). Intraocular pressure (IOP) is measured by rebound tonometry, and retinal vasculature/layers are analyzed by fundus fluorescein angiography and optical coherence tomography. Different fibrosis-related parameters are analyzed in the retina and choroid of normotensive and hypertensive mice with or without oil supplementation. Besides preventing the alterations found in hypertensive animals, including increased IOP, reduced fluorescein signal, and altered retinal layer thickness, the ACE oil-enriched diet improves collagen metabolism by regulating the expression of major fibrotic process modulators (matrix metalloproteinases, tissue inhibitors of metalloproteinases, connective tissue growth factor, and transforming growth factor beta family). CONCLUSION: Regular consumption of EVOO and ACE oil (with better outcomes in the latter) might help reduce abnormally high IOP values in the context of hypertension-related retinal damage, with significant reduction in the surrounding fibrotic process.

Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina.

Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca2+. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases.

Repeated Low-Level Red-Light Therapy for Controlling Onset and Progression of Myopia-a Review.

Repeated low-level red-light (RLRL), characterized by increased energy supply and cellular metabolism, thus enhancing metabolic repair processes, has gained persistent worldwide attention in recent years as a new novel scientific approach for therapeutic application in myopia. This therapeutic revolution led by RLRL therapy is due to significant advances in bioenergetics and photobiology, for instance, enormous progresses in photobiomodulation regulated by cytochrome c oxidase, the primary photoreceptor of the light in the red to near infrared regions of the electromagnetic spectrum, as the primary mechanism of action in RLRL therapy. This oxidase is also a key mitochondrial enzyme for cellular bioenergetics, especially for the nerve cells in the retina and brain. In addition, dopamine (DA)-enhanced release of nitric oxide may also be involved in controlling myopia by activation of nitric oxide synthase, enhancing cGMP signaling. Recent evidence has also suggested that RLRL may inhibit myopia progression by inhibiting spherical equivalent refraction (SER) progression and axial elongation without adverse effects. In this review, we provide scientific evidence for RLRL therapy as a unique paradigm to control myopia and support the theory that targeting neuronal energy metabolism may constitute a major target for the neurotherapeutics of myopia, with emphasis on its molecular, cellular, and nervous tissue levels, and the potential benefits of RLRL therapy for myopia.

Epigallocatechin-3-Gallate, an Active Green Tea Component to Support Anti-VEGFA Therapy in Wet Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a largely incurable disease and an emerging problem in aging societies. It occurs in two forms, dry and wet (exudative, neovascular), which may cause legal blindness and sight loss. Currently, there is not any effective treatment for dry AMD. Meanwhile, repeated intravitreal injections with antibodies effective against vascular endothelial growth factor A (VEGFA) slow down wet AMD progression but are not free from complications. (-)-Epigallocatechin-3-gallate (EGCG) is an active compound of green tea, which exerts many beneficial effects in the retinal pigment epithelium and the neural retina. It has been reported to downregulate the VEGFA gene by suppressing its activators. The inhibition of mitogen-activated protein kinases 1 and 3 (MAPK1 and MAPK3) may lie behind the antiangiogenic action of EGCG mediated by VEGFA. EGCG exerts protective effects against UV-induced damage to retinal cells and improves dysfunctional autophagy. EGCG may also interact with the mechanistic target rapamycin (MTOR) and unc-51-like autophagy activating kinase (ULK1) to modulate the interplay between autophagy and apoptosis. Several other studies report beneficial effects of EGCG on the retina that may be related to wet AMD. Therefore, controlled clinical trials are needed to verify whether diet supplementation with EGCG or green tea consumption may improve the results of anti-VEGFA therapy in wet AMD.

Cell-Based Therapies for Glaucoma.

Glaucomatous optic neuropathy (GON) is the major cause of irreversible visual loss worldwide and can result from a range of disease etiologies. The defining features of GON are retinal ganglion cell (RGC) degeneration and characteristic cupping of the optic nerve head (ONH) due to tissue remodeling, while intraocular pressure remains the only modifiable GON risk factor currently targeted by approved clinical treatment strategies. Efforts to understand the mechanisms that allow species such as the zebrafish to regenerate their retinal cells have greatly increased our understanding of regenerative signaling pathways. However, proper integration within the retina and projection to the brain by the newly regenerated neuronal cells remain major hurdles. Meanwhile, a range of methods for in vitro differentiation have been developed to derive retinal cells from a variety of cell sources, including embryonic and induced pluripotent stem cells. More recently, there has been growing interest in the implantation of glial cells as well as cell-derived products, including neurotrophins, microRNA, and extracellular vesicles, to provide functional support to vulnerable structures such as RGC axons and the ONH. These approaches offer the advantage of not relying upon the replacement of degenerated cells and potentially targeting earlier stages of disease pathogenesis. In order to translate these techniques into clinical practice, appropriate cell sourcing, robust differentiation protocols, and accurate implantation methods are crucial to the success of cell-based therapy in glaucoma. Translational Relevance: Cell-based therapies for glaucoma currently under active development include the induction of endogenous regeneration, implantation of exogenously derived retinal cells, and utilization of cell-derived products to provide functional support.

Salvianolic acid B ameliorates retinal deficits in an early-stage Alzheimer's disease mouse model through downregulating BACE1 and Aß generation.

Alzheimer's disease (AD) is a neurodegenerative disease with subtle onset, early diagnosis remains challenging. Accumulating evidence suggests that the emergence of retinal damage in AD precedes cognitive impairment, and may serve as a critical indicator for early diagnosis and disease progression. Salvianolic acid B (Sal B), a bioactive compound isolated from the traditional Chinese medicinal herb Salvia miltiorrhiza, has been shown promise in treating neurodegenerative diseases, such as AD and Parkinson's disease. In this study we investigated the therapeutic effects of Sal B on retinopathy in early-stage AD. One-month-old transgenic mice carrying five familial AD mutations (5×FAD) were treated with Sal B (20 mg·kg-1·d-1, i.g.) for 3 months. At the end of treatment, retinal function and structure were assessed, cognitive function was evaluated in Morris water maze test. We showed that 4-month-old 5×FAD mice displayed distinct structural and functional deficits in the retinas, which were significantly ameliorated by Sal B treatment. In contrast, untreated, 4-month-old 5×FAD mice did not exhibit cognitive impairment compared to wild-type mice. In SH-SY5Y-APP751 cells, we demonstrated that Sal B (10 µM) significantly decreased BACE1 expression and sorting into the Golgi apparatus, thereby reducing Aß generation by inhibiting the ß-cleavage of APP. Moreover, we found that Sal B effectively attenuated microglial activation and the associated inflammatory cytokine release induced by Aß plaque deposition in the retinas of 5×FAD mice. Taken together, our results demonstrate that functional impairments in the retina occur before cognitive decline, suggesting that the retina is a valuable reference for early diagnosis of AD. Sal B ameliorates retinal deficits by regulating APP processing and Aß generation in early AD, which is a potential therapeutic intervention for early AD treatment.

Akimba Proliferative Diabetic Retinopathy Model: Understanding Molecular Mechanism and Drug Screening for the Progression of Diabetic Retinopathy.

As the prevalence of diabetes has reached epidemic proportions worldwide, diabetic retinopathy incidence is increasing rapidly. An advanced diabetic retinopathy (DR) stage can lead to a sight-threatening form. There is growing evidence showing diabetes causes a range of metabolic changes that subsequently lead to pathological modifications in the retina and retinal blood vessels. To understand the complex mechanism of the pathophysiology of DR, a precise model is not readily available. By crossbreeding the Akita and Kimba strains, a suitable proliferative DR model was acquired. This new Akimba strain manifests marked hyperglycemia and vascular changes, which resemble the early and advanced stage of DR.Here, we describe the breeding method, colony screening for experiments, and imaging techniques widely used to investigate the DR progression in this model. We elaborate step-by-step protocols to set up and perform fundus, fluorescein angiography, optical coherence tomography, and optical coherence tomography-angiogram to study retinal structural changes and vascular abnormalities. In addition, we show a method to label the leukocytes with fluorescence and laser speckle flowgraphy to examine the inflammation in the retina and retinal vessel blood flow speed, respectively. Lastly, we describe electroretinogram to evaluate the functional aspect of the DR transformations.

The essential role of docosahexaenoic acid and its derivatives for retinal integrity.

The fatty acid composition of photoreceptor outer segment (POS) phospholipids diverges from other membranes, being highly enriched in polyunsaturated fatty acids (PUFAs). The most abundant PUFA is docosahexaenoic acid (DHA, C22:6n-3), an omega-3 PUFA that amounts to over 50% of the POS phospholipid fatty acid side chains. Interestingly, DHA is the precursor of other bioactive lipids such as elongated PUFAs and oxygenated derivatives. In this review, we present the current view on metabolism, trafficking and function of DHA and very long chain polyunsaturated fatty acids (VLC-PUFAs) in the retina. New insights on pathological features generated from PUFA deficient mouse models with enzyme or transporter defects and corresponding patients are discussed. Not only the neural retina, but also abnormalities in the retinal pigment epithelium are considered. Furthermore, the potential involvement of PUFAs in more common retinal degeneration diseases such as diabetic retinopathy, retinitis pigmentosa and age-related macular degeneration are evaluated. Supplementation treatment strategies and their outcome are summarized.

Three Major Causes of Metabolic Retinal Degenerations and Three Ways to Avoid Them.

An imbalance of homeostasis in the retina leads to neuron loss and this eventually results in a deterioration of vision. If the stress threshold is exceeded, different protective/survival mechanisms are activated. Numerous key molecular actors contribute to prevalent metabolically induced retinal diseases-the three major challenges are age-related alterations, diabetic retinopathy and glaucoma. These diseases have complex dysregulation of glucose-, lipid-, amino acid or purine metabolism. In this review, we summarize current knowledge on possible ways of preventing or circumventing retinal degeneration by available methods. We intend to provide a unified background, common prevention and treatment rationale for these disorders and identify the mechanisms through which these actions protect the retina. We suggest a role for herbal medicines, internal neuroprotective substances and synthetic drugs targeting four processes: parainflammation and/or glial cell activation, ischemia and related reactive oxygen species and vascular endothelial growth factor accumulation, apoptosis and/or autophagy of nerve cells and an elevation of ocular perfusion pressure and/or intraocular pressure. We conclude that in order to achieve substantial preventive or therapeutic effects, at least two of the mentioned pathways should be targeted synergistically. A repositioning of some drugs is considered to use them for the cure of the other related conditions.

Genetic deletion of Bco2 and Isx establishes a golden mouse model for carotenoid research.

OBJECTIVE: Low plasma levels of carotenoids are associated with mortality and chronic disease states. Genetic studies in animals revealed that the tissue accumulation of these dietary pigments is associated with the genes encoding ß-carotene oxygenase 2 (BCO2) and the scavenger receptor class B type 1 (SR-B1). Here we examined in mice how BCO2 and SR-B1 affect the metabolism of the model carotenoid zeaxanthin that serves as a macular pigment in the human retina. METHODS: We used mice with a lacZ reporter gene knock-in to determine Bco2 expression patterns in the small intestine. By genetic dissection, we studied the contribution of BCO2 and SR-B1 to zeaxanthin uptake homeostasis and tissue accumulation under different supply conditions (50 mg/kg and 250 mg/kg). We determined the metabolic profiles of zeaxanthin and its metabolites in different tissues by LC-MS using standard and chiral columns. An albino Isx-/-/Bco2-/- mouse homozygous for Tyrc-2J was generated to study the effect of light on ocular zeaxanthin metabolites. RESULTS: We demonstrate that BCO2 is highly expressed in enterocytes of the small intestine. Genetic deletion of Bco2 led to enhanced accumulation of zeaxanthin, indicating that the enzyme serves as a gatekeeper of zeaxanthin bioavailability. Relaxing the regulation of SR-B1 expression in enterocytes by genetic deletion of the transcription factor ISX further enhanced zeaxanthin accumulation in tissues. We observed that the absorption of zeaxanthin was dose-dependent and identified the jejunum as the major zeaxanthin-absorbing intestinal region. We further showed that zeaxanthin underwent oxidation to ε,ε-3,3'-carotene-dione in mouse tissues. We detected all three enantiomers of the zeaxanthin oxidation product whereas the parent zeaxanthin only existed as (3R, 3'R)-enantiomer in the diet. The ratio of oxidized to parent zeaxanthin varied between tissues and was dependent on the supplementation dose. We further showed in an albino Isx-/-/Bco2-/- mouse that supra-physiological supplementation doses (250 mg/kg) with zeaxanthin rapidly induced hypercarotenemia with a golden skin phenotype and that light stress increased the concentration of oxidized zeaxanthin in the eyes. CONCLUSIONS: We established the biochemical basis of zeaxanthin metabolism in mice and showed that tissue factors and abiotic stress affect the metabolism and homeostasis of this dietary lipid.

Photobiomodulation therapy activates YAP and triggers proliferation and dedifferentiation of Müller glia in mammalian retina.

Photobiomodulation therapy has been proposed as a promising therapeutic approach for retinal degenerative diseases. However, its effect on the regenerative capacity in mammalian retina and its intracellular signalling mechanisms remain unknown. Here, we show that photobiomodulation with 670 nm light stimulates Müller glia cell cycle re-entry and dedifferentiation into a progenitor-like state in both the uninjured and injured retina. We also find that 670 nm light treatment inhibits the Hippo pathway, which is activated in Müller glia following NaIO3-induced retinal injury. YAP, a major downstream effector of the Hippo signalling pathway was translocated into the nucleus of Müller glia along with YAP dephosphorylation in retina treated with 670 nm light. Deficiency of YAP attenuated Müller glia cell cycle re-entry and dedifferentiation. Our data reveal that the Hippo-YAP signalling pathway is associated with the photostimulatory effect on regenerative response in mammalian retina, and suggest a potential therapeutic strategy for retinal degenerative diseases. [BMB Reports 2023; 56(9): 502-507].

Wolfberry water extract attenuates blue light-emitting diode damage to ARPE-19 cells and mouse retina by activating the NRF2 signaling pathway.

The wolfberry is believed to improve eyesight in traditional Chinese medicine. Soaking wolfberry in thermos cups has become a common health-preserving practice. The object of this paper was to research the protective effects of wolfberry water extract (WWE) on oxidative injury induced by blue light-emitting diodes (LEDs) in ARPE-19 cells and C57BL/6J mice. Wolfberry water extract significantly increased cell viability, reduced ROS production, stabilized mitochondrial membrane potential, and inhibited apoptosis in blue LED-induced cells (P < 0.05). The protective effects of WWE against blue LED-induced cytotoxicity and ROS accumulation in cells were abolished by transfection with Nrf2 siRNA. In blue LED-exposed C57BL/6J mice, WWE treatment markedly increased the amplitudes of electroretinogram (ERG) waves a and b, increased the thickness of retinal outer nuclear layer (ONL), activated endogenous antioxidant enzymes, and decreased MDA levels in the retina and lens. WWE also promoted NRF2 translocation and the expression of the downstream genes Ho-1, Nqo1, Gclc, and Gclm in the retina. The protection of WWE in ERG a and b wave amplitudes and ROS levels were abrogated in Nrf2 knockout mice. These results suggested that WWE has beneficial effects on retinal injury induced by blue LED, and mechanisms of action at least partly via the NRF2 signaling pathway.

Systemic C-peptide supplementation ameliorates retinal neurodegeneration by inhibiting VEGF-induced pathological events in diabetes.

Diabetic retinopathy (DR) is caused by retinal vascular dysfunction and neurodegeneration. Intraocular delivery of C-peptide has been shown to be beneficial against hyperglycemia-induced microvascular leakage in the retina of diabetes; however, the effect of C-peptide on diabetes-induced retinal neurodegeneration remains unknown. Moreover, extraocular C-peptide replacement therapy against DR to avoid various adverse effects caused by intravitreal injections has not been studied. Here, we demonstrate that systemic C-peptide supplementation using osmotic pumps or biopolymer-conjugated C-peptide hydrogels ameliorates neurodegeneration by inhibiting vascular endothelial growth factor-induced pathological events, but not hyperglycemia-induced vascular endothelial growth factor expression, in the retinas of diabetic mice. C-peptide inhibited hyperglycemia-induced activation of macroglial and microglial cells, downregulation of glutamate aspartate transporter 1 expression, neuronal apoptosis, and histopathological changes by a mechanism involving reactive oxygen species generation in the retinas of diabetic mice, but transglutaminase 2, which is involved in retinal vascular leakage, is not associated with these pathological events. Overall, our findings suggest that systemic C-peptide supplementation alleviates hyperglycemia-induced retinal neurodegeneration by inhibiting a pathological mechanism, involving reactive oxygen species, but not transglutaminase 2, in diabetes.

MicroRNA-376b-3p Suppresses Choroidal Neovascularization by Regulating Glutaminolysis in Endothelial Cells.

Purpose: Choroidal neovascularization (CNV) is a common pathological change of various ocular diseases that causes serious damage to central vision. Accumulated evidence shows that microRNAs (miRNAs) are closely related with the regulation of endothelial metabolism, which plays crucial roles in angiogenesis. Here, we investigate the molecular mechanism underlying the regulation of endothelial glutamine metabolism by miR-376b-3p in the progression of CNV. Methods: Human retinal microvascular endothelial cells (HRMECs) were transfected with control or miR-376b-3p mimics, and the expression of glutaminase 1 (GLS1), a rate-limiting enzyme in glutaminolysis, was detected by real-time PCR or Western blotting. The biological function and glutamine metabolism of transfected HRMECs were measured by related kits. Luciferase reporter assays were used to validate the CCAAT/enhancer-binding protein beta (CEBPB) was a target of miR-376b-3p. Chromatin immunoprecipitation and RNA immunoprecipitation assays were performed to verify the binding of CEBPB on the promoter region of GLS1. Fundus fluorescein angiography and immunofluorescence detected the effect of miR-376b-3p agomir on rat laser-induced CNV. Results: The expression of miR-376b-3p was decreased, whereas GLS1 expression was increased in the retinal pigment epithelial-choroidal complexes of rats with CNV. HRMECs transfected with miR-376b-3p mimic showed inhibition of CEBPB, resulting in the inactivation of GLS1 transcription and glutaminolysis. Moreover, the miR-376b-3p mimic inhibited proliferation, migration and tube formation but promoted apoptosis in HRMECs, whereas these effects counteracted by α-ketoglutarate supplementation or transfection with CEBPB overexpression plasmid. Finally, the intravitreal administration of the miR-376b-3p agomir restrained CNV formation. Conclusions: Collectively, miR-376b-3p is a suppressor of glutamine metabolism in endothelial cells that could be expected to become a therapeutic target for the treatment of CNV-related diseases.

Saffron extract attenuates Sofosbuvir-induced retinal neurodegeneration in albino rat.

Sofosbuvir is a novel drug candidate for the treatment of hepatitis C viral infection; however, vision loss is one of its growing adverse effects. Saffron is a natural biomolecule with a high antioxidant potential that has been efficiently used in some diseases caused by oxidative stress. This study evaluated Sofosbuvir's neurodegenerative effect on the retina of albino rat and examined the potential protective role of saffron aqueous extract. Twenty-one adult male albino rats were randomly divided into three groups: Control, Sofosbuvir-treated (41.1 mg/kg /day for 6 weeks), and Sofosbuvir + Saffron co-treated groups. Retinal specimens were biochemically analyzed for malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels. In addition, light and transmission electron microscopic examination, as well as immunohistochemical staining for Caspase-3, COX-2, and GFAP were performed. Sofosbuvir treatment caused a significant increase in retinal MDA, IL-6, and TNF-α levels coupling with a significant decrease in retinal total antioxidant capacity level. Histopathological findings revealed disturbed retinal architecture, detached pigment epithelium, vacuolated photoreceptors, in addition to a significant decrease in the thicknesses of both outer and inner nuclear layers, and the number of ganglionic cells. Ultrastructural examination revealed extensive degenerative changes in all retinal layers. Caspase-3, COX-2, and GFAP immunohistochemical expressions were significantly increased. Meanwhile, concomitant treatment with Saffron significantly improved retinal redox status, inflammation, histological, and ultrastructural parameters. Saffron may protect the retina from the hazardous effects of Sofosbuvir. Saffron could be used as an adjuvant therapy to protect patients receiving Sofosbuvir from retinal damage.

AMP kinase activation by Omega-3 polyunsaturated fatty acid protects the retina against ischemic insult: An in vitro and in vivo study.

PURPOSE: To investigate the possible beneficial effects of omega-3 polyunsaturated fatty acids (ω3-PUFAs) in ischemic retinal angiogenesis and whether AMP-activated protein kinase (AMPK) is involved. METHODS: Human retinal microvascular endothelial cells (hRMECs) were exposed to dimethyloxalylglycine (DMOG), a hypoxia-inducible factor hydroxylase inhibitor, in the presence or absence of docosahexaenoic acid (DHA) and small interfering RNA (siRNA) for AMPKα for 24 h. Ischemic factors, endothelial mesenchymal transition marker, endothelial barrier integrity, cell migration, and tube formation were evaluated. Neonatal AMPKα2-/- and control wild-type (WT) mice were submitted to an oxygen-induced retinopathy (OIR) protocol; their nursing mother mice were either fed ω3-PUFAs or not. In the end, ischemic markers and endothelial cell proliferation were evaluated in neonatal mouse retinal tissue through immunohistochemical or immunofluorescent assays among all studied groups. RESULTS: Cells exposed to DMOG displayed increased expressions of hypoxic and endothelial mesenchymal transition (vimentin) markers and barrier disarrangement of Zonula Occludens-1 compared to the control, accompanied by increased cellular migration and tube formation (p < 0.05). AMPK activity was significantly decreased. Supplementation with DHA restored the mentioned alterations compared to DMOG (p<0.05). In siRNAAMPKα-treated cells, the beneficial effects observed with DHA were abolished. DHA upregulated G-protein receptor-120 (GPR120), which promptly increased intracellular levels of calcium (p ≤ 0.001), which consequently increased Calcium/calmodulin-dependent protein kinase kinase ß expression (CaMKKß) thus phosphorylating AMPKThr172. AMPKα2-/- and wild-type (WT) OIR mice exhibited similar retinal ischemic changes, and the oral supplementation with ω3-PUFA efficiently prevented the noticed ischemic alterations only in WT mice, suggesting that AMPKα2 is pivotal in the protective effects of ω3-PUFA. CONCLUSIONS: ω3-PUFAs protect the retina from the effects of ischemic conditions, and this effect occurs via the GPR120-CaMKKß-AMPK axis. A better understanding of this mechanism might improve the control of pathological angiogenesis in retinal ischemic diseases.

Resvega, a Nutraceutical Preparation, Affects NFκB Pathway and Prolongs the Anti-VEGF Effect of Bevacizumab in Undifferentiated ARPE-19 Retina Cells.

Age-related macular degeneration (AMD) is an irreversible chronic degenerative pathology that affects the retina. Despite therapeutic advances thanks to the use of anti-vascular endothelial growth factor (VEGF) agents, resistance mechanisms have been found to accentuate the visual deficit. In the present study, we explored whether a nutraceutical formulation composed of omega-3 fatty acids and resveratrol, called Resvega®, was able to disrupt VEGF-A secretion in human ARPE-19 retina cells. We found that Resvega® inhibits VEGF-A secretion through decreases in both the PI3K-AKT-mTOR and NFκB signaling pathways. In NFκB signaling pathways, Resvega® inhibits the phosphorylation of the inhibitor of NFκB, IκB, which can bind NFκB dimers and sequester them in the cytoplasm. Thus, the NFκB subunits cannot migrate to the nucleus where they normally bind and stimulate the transcription of target genes such as VEGF-A. The IκB kinase complex (IKK) is also affected by Resvega® since the nutraceutical formulation decreases both IKKα and IKKß subunits and the IKKγ subunit which is required for the stimulation of IKK. Very interestingly, we highlight that Resvega® could prolong the anti-angiogenic effect of Avastin®, which is an anti-VEGF agent typically used in clinical practice. Our results suggest that Resvega® may have potential interest as nutritional supplementation against AMD.