Efficacy of fatty acids dietary supplement in polyethylene glycol-induced mouse model of retinal degeneration

Current knowledge of the benefits of nutrition supplements for eye pathologies is based largely on the use of appropriate animal models, together with defined dietary supplementation. Here, C57BL6 mice were subretinally injected with polyethylene glycol (PEG)-400, an established model of retinal degeneration with a dry age-related macular degeneration (AMD)-like phenotype, an eye pathology that lacks treatment. In response to PEG-400, markers of the complement system, angiogenesis,inflammation,gliosis,andmacrophageinfiltrationwereupregulatedinbothretinasand retinal pigment epithelium (RPE)/choroids, whereas dietary supplementation with a mixture based on fatty acids counteracted their upregulation. Major effects include a reduction of inflammation, in both retinas and RPE/choroids, and an inhibition of macrophage infiltration in the choroid, yet not in the retina, suggesting a targeted action through the choroidal vasculature. Histological analysis revealed a thinning of the outer nuclear layer (ONL), together with dysregulation of the epithelium layer in response to PEG-400. In addition, immunohistofluorescence demonstrated Müller cell gliosis and macrophage infiltration into subretinal tissues supporting the molecular findings. Reduced ONL thickness,gliosis,andmacrophageinfiltrationwerecounteractedbythedietsupplement. The present data suggest that fatty acids may represent a useful form of diet supplementation to prevent or limit the progression of dry AMD

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen-induced retinopathy: beneficial effect of the absence of AQP4

Hypoxia-dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin-4 (AQP4) is involved in the hypoxia-dependent VEGF upregulation in the retina of a mouse model of oxygen-induced retinopathy (OIR). The expression levels of VEGF, the hypoxia-inducible factor-1a (HIF-1a) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF-1 binding site (HBS) in the VEGF gene promoter, the binding of HIF-1a to the HBS, the retinal vascularization and function have been determined in the retina of wild-type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF-1a, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF-1a. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1a to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF-1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF-induced retinal damage in response to hypoxia

Gaining insight on mitigation of rubeosis iridis by UPARANT in a mouse model associated with proliferative retinopathy

Proliferative retinopathies (PR) lead to an increase in neovascularization and inflammation factors, at times culminating in pathologic rubeosis iridis (RI). In mice, uveal puncture combined with injection of hypoxia-conditioned media mimics RI associated with proliferative retinopathies. Here, we investigated the effects of the urokinase plasminogen activator receptor (uPAR) antagonist-UPARANT-on the angiogenic and inflammatory processes that are dysregulated in this model. In addition, the effects of UPARANT were compared with those of anti-vascular endothelial growth factor (VEGF) therapies. Administration of UPARANT promptly decreased iris vasculature, while anti-VEGF effects were slower and less pronounced. Immunoblot and qPCR analysis suggested that UPARANT acts predominantly by reducing the upregulated inflammatory and extracellular matrix degradation responses. UPARANT appears to be more effective in comparison to anti-VEGF in the treatment of RI associated with PR in the murine model, by modulating multiple uPAR-associated signaling pathways. Furthermore, UPARANT effectiveness was maintained when systemically administered, which could open to novel improved therapies for proliferative ocular diseases, particularly those associated with PR. KEY MESSAGES: • Further evidence of UPARANT effectiveness in normalizing pathological iris neovascularization. • Both systemic and local administration of UPARANT reduce iris neovascularization in a model associated with proliferative retinopathies. • In the mouse models of rubeosis iridis associated with proliferative retinopathy, UPARANT displays stronger effects when compared with anti-vascular endothelial growth factor regimen

the urokinase receptor derived peptide uparant recovers dysfunctional electroretinogram and blood retinal barrier leakage in a rat model of diabetes

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UPARANT, a novel multitarget drug for neovascular and inflammatory ocular diseases

Pharmacologic control of neovascularization and inflammation is a promising approach for the treatment of sight threatening ocular disease such as the retinopathy of prematurity (ROP), age-related macular degeneration (AMD) and diabetic retinopathy (DR). Currently, the mainstay treatment for these diseases is based on surgical interventions and on pharmacological approaches targeting the main angiogenic factor, the vascular endothelial growth factor (VEGF). However, anti-VEGF therapies require intravitreal injections of anti- VEGF drugs and are associated with potential risks. Indeed, the high frequency of intravireal injections may lead to adverse side effects such as ocular inflammation and endophthalmitis. The urokinase receptor (uPAR) and its ligand, the urokinase (uPA) are major players in VEGF induced angiogenesis. Indeed, the uPAR-uPA system is involved in the first phase of VEGFmediated angiogenesis by triggering extracellular matrix remodeling and thus promoting endothelial cell migration. uPAR is capable of inducing cell migration by interacting with G protein-coupled receptors (GPCRs) of the formyl peptide receptor (FPR) family, which are involved in chemotaxis of the immune system. For these reasons, the uPAR-uPA system has been actively targeted for development of novel therapeutics against diseases in which angiogenesis and inflammation play a crucial role. In search for novel therapies against inflammatory/neovascular ocular pathologies, peptide analogues targeting uPAR have been developed. uPAR is an important component of the angiogenic-inflammatory response in the retina Among the series of newly synthesized analogues, the tetrapeptide Ac-L-Arg-Aib-L-Arg-LCα( Me)Phe-NH2, named UPARANT, displays strong inhibition of endothelial cell migration, longtime resistance to enzymatic digestion and high stability in blood and plasma. Using angiogenic assays both in vitro and in vivo, it has been demonstrated that UPARANT inhibits VEGF-driven angiogenesis by preventing FPR activation. Indeed, a possible clinical application of UPARANT in human ocular diseases would find a great benefit from the demonstration of its effectiveness in in vivomodels characterized by neovascularization and/or inflammation. If the antiangiogenic/anti-inflammatory activity of UPARANT will be also demonstrated in models of ROP, DR and AMD, then, the proven effectiveness in animal models will constitute a starting point, which will bring closer the likelihood of commencing preliminary studies aimed at assessing safety first and thereafter effectiveness in humans. 6 With this aim in mind, we tested the efficacy of UPARANT in different in vivo models of ocular diseases. We firstly assayed the efficacy of UPARANT in counteracting neovascularization and inflammatory in a model of oxygen induced retinopathy (OIR), an acknowledged model of ROP, in which UPARANT was found effective in counteracting retinal neovascularization. The efficacy of UPARANT was further addressed in a mouse model of laser-induced choroidal neovascularization, a model that closely mimics neovascular AMD. Moreover, in a model of early DR, mimicking type 1 diabetes, UPARANT promoted visual function recovery by restoring the blood–retinal barrier integrity and by inhibiting inflammatory and angiogenic responses. These results were also confirmed in a model of type 2 diabetes. Lastly, UPARANT was effective in mitigating rubeosis iridis in a model where neovascularization is associated with mechanisms of wound healing which are independent on VEGF signalling

Il sistema β-adrenergico nelle retinopatie proliferative: uno studio funzionale

Le retinopatie proliferative (PRs) costituiscono la causa più comune di cecità nei paesi sviluppati e in quelli in via di sviluppo. In particolare, la retinopatia diabetica (DR), e la retinopatia del prematuro (ROP) presentano costi sociali molto elevati. Nelle PRs, il danno retinico è causato da una condizione di ischemia che promuove il rilascio di fattori pro-angiogenici come il vascular endothelial growth factor (VEGF), che causano neo-angiogenesi. Le PRs sono caratterizzate da neovascolarizzazione retinica, ovvero dalla crescita di nuovi vasi da quelli preesistenti. Questi nuovi vasi esprimono bassi livelli di proteine delle giunzioni strette permettendo la fuoriuscita di plasma nel parenchima retinico. Negli ultimi anni, tra i possibili bersagli dei farmaci antiangiogenici, un intensa attività di ricerca si è focalizzata sul sistema β- adrenergico. Vi sono diverse evidenze che i recettori β adrenergici (β-ARs) possano regolare l’angiogenesi patologica in modelli murini di retinopatia indotta da ossigeno (OIR), un modello che mima la patogenesi di ROP e DR. In particolare, è stato osservato che i β-ARs sono espressi nella retina, i β1- e β2-ARs sono localizzati principalmente nelle cellule di Muller, le quali sono le principali fonti di VEGF, mentre i β3-ARs sono localizzati nei capillari retinici. Il fatto che l’OIR sia caratterizzato da elevati livelli retinici di noriepinefrina (NE) suggerisce la possibilità che essi possano over stimolare i β-ARs attivando meccanismi che causano un aumento di fattori pro-angiogenici. A questo proposito, è stato dimostrato che molecole che ripristinano l’omeostasi simpatetica possono migliorare la disfunzione vascolare. Ad esempio il blocco dei β1/2-AR con propranololo o il blocco del β2-AR con antagonisti selettivi riduce la neovascolarizzazione patologica e migliora l’integrità della barriera emato-retinica (BRB) riducendo anche i fattori pro-angiogenici. Inoltre, in un recente lavoro è stato osservato che il propranololo è in grado di contrastare la neovascolarizzazione retinica in neonati pretermine affetti da ROP. La maggiorparte degli studi recenti sull’uso dei farmaci anti angiogenici nelle PRs si sono focalizzati soprattutto sui meccanismi e sui fattori che causano la neovascolarizzazione, tralasciando spesso tuttavia gli effetti sulla funzione visiva. Lo scopo del presente studio è stato quello di valutare il ruolo dei distinti β-ARs sulla neovascolarizzazione retinica e sulla disfunzione visiva che caratterizza l’OIR. Per questo scopo, sono stati utilizzati diversi agonosti ed antagonisti dei distinti β-ARs, cosi come topi con delezione genica dei recettori β1/2-AR. In particolare abbiamo analizzato gli effetti degli antagonisti/agonisti dei β-ARs e della delezione genica sui livelli di VEGF, sulla neovascolarizzazione retinica, sull’integrità della BRB. Inoltre sono stati investigati gli effetti sull’espressione di marker apotptotici, poichè la morte cellulare è una carattersitica critica dell’OIR. Infine, mediante elettroretinogramma (ERG), è stato analizzato se, le diverse molecole o la delezione genica, hanno un effetto sulla funzione visiva. In accordo con studi precedenti, i nostri risultati dimostrano che le retine dei topi OIR sono caratterizzate da elevati livelli di VEGF, neovascolarizzazione, perdità di integrità della BRB, morte cellulare e disfunzione visiva. In una prima serie di esperimenti, abbiamo valutato gli effetti del blocco e/o attivazione dei β2-ARs. I nostri risultati dimostrano che sia l’antagonismo sia l’agonismo, causa una riduzione del processo neovascolare, dei livelli di VEGF e di citocromo c, migliora l’integrità della BRB e previene la disfunzione visiva. In un secondo set di espermimenti sono stati utilizzati topi knockout (KO) per valutare il ruolo dei β1- e β2-ARs nella risposta angiogenica caratteristica dell’OIR. I topi KO sonoi stati utilizzati per comprendere il ruolo funzionale dei β3-Ars. Al contrario di ciò che si è osservato nei topi wild type (WT), i topi KO non mostrano neovascolarizzazione retinica indicando un effetto migliorativo della delezione dei β1/2-AR sulla neovascolarizzazione retinica. Non sono state osservate differenze significative tra i WT ed i KO nei livelli di VEGF e sull’integrità della BRB, mentre i livelli di citocromo c sono più elevati nel KO che nel WT. Nonostante la migliore vascolarizzazione dei KO la funzionalità visiva non migliora, rimanendo simile a quella dei WT OIR. L’attivazione dei β3-ARs causa un aumento della formazione di nuovi vasi sia nei WT che nei KO. Gli effetti vascolari dell’agonismo del β3-AR sono accompagnati da aumentati livelli di VEGF senza differenze tra i due ceppi. Inoltre in ambo i ceppi, l’attivazione del β3-AR non influenza le risposte ERG. Il blocco del β3-AR, in ambo i ceppi, non influenza la formazione di nuovi vasi, ma causa un decremento dei livelli di VEGF e citocromo c senza influenzare l’ ERG disfunzionale. In conclusione, il blocco del β2-AR può effettivamente contrastare i processi neoangiogenici e prevenire la disfunzione visiva. Il fatto che, effetti comparabili possono essere indotti dall’agonismo del β2-AR può essere spiegato da risultati preliminari che dimostrano che i β2-AR sono desensitizzati da esposizioni prolungate ad agonisti, quindi risultando in una ridotta overstimolazione dei β2-ARs e ridotto swtich pro-angiogenico. Tuttavia, è necessario molto lavoro per comprendere completamente il ruolo di molecole agoniste o antagoniste dei β2-ARs. A questo proposito, il risultato che la neovascolarizzazione indotta da ipossia è quasi del tutto prevenuta dalla delezione dei β1/2-AR è in accordo con i risultati del blocco o della desensitizzazione in seguito ad agonista. Il fatto che il ripristino della vascolarizzazione retinica non sia sempre seguita da miglioramenti della funzione visiva necessita di ulteriori studi

Protective Efficacy of a Dietary Supplement Based on Forskolin, Homotaurine, Spearmint Extract, and Group B Vitamins in a Mouse Model of Optic Nerve Injury

Glaucoma is a multifactorial blinding disease with a major inflammatory component ultimately leading to apoptotic retinal ganglion cell (RGC) death. Pharmacological treatments lowering intraocular pressure can help slow or prevent vision loss although the damage caused by glaucoma cannot be reversed. Recently, nutritional approaches have been evaluated for their efficacy in preventing degenerative events in the retina although mechanisms underlying their effectiveness remain to be elucidated. Here, we evaluated the efficacy of a diet supplement consisting of forskolin, homotaurine, spearmint extract, and vitamins of the B group in counteracting retinal dysfunction in a mouse model of optic nerve crush (ONC) used as an in vivo model of glaucoma. After demonstrating that ONC did not affect retinal vasculature by fluorescein angiography, we determined the effect of the diet supplement on the photopic negative response (PhNR) whose amplitude is strictly related to RGC integrity and is therefore drastically reduced in concomitance with RGC death. We found that the diet supplementation prevents the reduction of PhNR amplitude (p < 0.001) and concomitantly counteracts RGC death, as in supplemented mice, RGC number assessed immunohistochemically is significantly higher than that in non-supplemented animals (p < 0.01). Major determinants of the protective efficacy of the compound are due to a reduction of ONC-associated cytokine secretion leading to decreased levels of apoptotic markers that in supplemented mice are significantly lower than in non-supplemented animals (p < 0.001), ultimately causing RGC survival and ameliorated visual dysfunction. Overall, our data suggest that the above association of compounds plays a neuroprotective role in this mouse model of glaucoma thus offering a new perspective in inflammation-associated neurodegenerative diseases of the inner retina

Protective Efficacy of a Dietary Supplement Based on Forskolin, Homotaurine, Spearmint Extract, and Group B Vitamins in a Mouse Model of Optic Nerve Injury

Glaucoma is a multifactorial blinding disease with a major inflammatory component ultimately leading to apoptotic retinal ganglion cell (RGC) death. Pharmacological treatments lowering intraocular pressure can help slow or prevent vision loss although the damage caused by glaucoma cannot be reversed. Recently, nutritional approaches have been evaluated for their efficacy in preventing degenerative events in the retina although mechanisms underlying their effectiveness remain to be elucidated. Here, we evaluated the efficacy of a diet supplement consisting of forskolin, homotaurine, spearmint extract, and vitamins of the B group in counteracting retinal dysfunction in a mouse model of optic nerve crush (ONC) used as an in vivo model of glaucoma. After demonstrating that ONC did not affect retinal vasculature by fluorescein angiography, we determined the effect of the diet supplement on the photopic negative response (PhNR) whose amplitude is strictly related to RGC integrity and is therefore drastically reduced in concomitance with RGC death. We found that the diet supplementation prevents the reduction of PhNR amplitude (p < 0.001) and concomitantly counteracts RGC death, as in supplemented mice, RGC number assessed immunohistochemically is significantly higher than that in non-supplemented animals (p < 0.01). Major determinants of the protective efficacy of the compound are due to a reduction of ONC-associated cytokine secretion leading to decreased levels of apoptotic markers that in supplemented mice are significantly lower than in non-supplemented animals (p < 0.001), ultimately causing RGC survival and ameliorated visual dysfunction. Overall, our data suggest that the above association of compounds plays a neuroprotective role in this mouse model of glaucoma thus offering a new perspective in inflammation-associated neurodegenerative diseases of the inner retina

Fatty Acids Dietary Supplements Exert Anti-Inflammatory Action and Limit Ganglion Cell Degeneration in the Retina of the EAE Mouse Model of Multiple Sclerosis

Optic neuritis is an acute inflammatory demyelinating disorder of the optic nerve (ON) and is an initial symptom of multiple sclerosis (MS). Optic neuritis is characterized by ON degeneration and retinal ganglion cell (RGC) loss that contributes to permanent visual disability and lacks a reliable treatment. Here, we used the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, a well-established model also for optic neuritis. In this model, C57BL6 mice, intraperitoneally injected with a fragment of the myelin oligodendrocyte glycoprotein (MOG), were found to develop inflammation, Müller cell gliosis, and infiltration of macrophages with increased production of oncomodulin (OCM), a calcium binding protein that acts as an atypical trophic factor for neurons enabling RGC axon regeneration. Immunolabeling of retinal whole mounts with a Brn3a antibody demonstrated drastic RGC loss. Dietary supplementation with Neuro-FAG (nFAG®), a balanced mixture of fatty acids (FAs), counteracted inflammatory and gliotic processes in the retina. In contrast, infiltration of macrophages and their production of OCM remained at elevated levels thus eventually preserving OCM trophic activity. In addition, the diet supplement with nFAG exerted a neuroprotective effect preventing MOG-induced RGC death. In conclusion, these data suggest that the balanced mixture of FAs may represent a useful form of diet supplementation to limit inflammatory events and death of RGCs associated to optic neuritis. This would occur without affecting macrophage infiltration and the release of OCM thus favoring the maintenance of OCM neuroprotective role