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Purpose: Retinal ganglion cells (RGCs) are highly susceptible to diabetes-induced metabolic stress. This study describes the early responses of RGCs to hyperglycemia and examines the effects of the neuroprotective somatostatin analog octreotide (OCT)., Methods: Thy1-green fluorescent protein (GFP)-M transgenic mice were used, which express GFP in a number of RGCs. OCT was intravitreally injected in mice with streptozotocin (STZ)-induced diabetes. A longitudinal electroretinography (ERG) analysis was performed up to 2 weeks from STZ treatment. RGC density was measured and extensive morphometric analyses were performed on identified RGC subtypes., Results: STZ treatment caused a decline of RGC function, which was counteracted by OCT. No differences in RGC density were recorded, indicating that impaired activity was unlikely to be related to RGC death. Different GFP-labeled RGC subtypes were identified and analyzed. Overall, large RGCs were mostly affected by diabetes and responded to OCT treatment, while those with smaller dendritic arborizations were less involved. Interestingly, depending on the complexity of the dendritic tree, OCT could completely rescue RGC morphometric parameters or increase the effects of hyperglycemia., Conclusions: There is an early response of RGCs to diabetes, which involves specific morpho-functional deficits but not overt cell death. OCT induces adaptive changes that, although different among RGC subtypes, contribute to RGC functionality in the presence of metabolic stress. These results highlight the importance of neuronal protection in the early phases of diabetic retinopathy, when cell loss has not yet started and RGC morphology can be preserved or adjusted to maintain RGC physiology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Age-related macular degeneration (AMD) is nowadays considered among the retinal diseases whose clinical management lacks established treatment approaches, mainly for its atrophic (dry) form. In this respect, the use of dietary patterns enriched in omega-3 and antioxidant xanthophylls has emerged as a promising approach to counteract dry AMD progression although the prophylactic potential of omega-3 of fish origin has been discussed. Whether enriched availability of omega-3 and xanthophylls may increase the effectiveness of diet supplementation in preventing dry AMD remains to be fully established. The present study aims at comparing the efficacy of an existing orally administered formulation based on lutein and fish oil, as a source of omega-3, with a novel formulation providing the combination of lutein and astaxanthin with Calanus oil (COil), which contains omega-3 together with their precursors policosanols. Using a mouse model of dry AMD based on subretinal injection of polyethylene glycol (PEG)-400, we assessed the comparative efficacy of both formulations on PEG-induced major hallmarks including oxidative stress, inflammation, glial reactivity and outer retinal thickness. Dietary supplementation with both mixtures has been found to exert a significant antioxidant and anti-inflammatory activity as reflected by the overall amelioration of the PEG-induced pathological hallmarks. Noteworthy, the formulation based on COil appeared to be more protective than the one based on fish oil, presumably because of the higher bioavailability of omega-3 in COil. These results support the use of dietary supplements combining omega-3 and xanthophylls in the prevention and treatment of AMD and suggest that the source of omega-3 might contribute to treatment efficacy., Competing Interests: DR was an employee of Fidia Farmaceutici SpA. Fidia Farmaceutici SpA had no direct role in the collection, analyses or intepretation of the data or in the decision to publish the results. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Melecchi, Amato, Lapi, Dal Monte, Rusciano, Bagnoli and Cammalleri.)

Retinopathy of prematurity (ROP) is one of the main blinding diseases affecting preterm newborns and is classically considered a vascular disorder. The premature exposure to the extrauterine environment, which is hyperoxic in respect to the intrauterine environment, triggers a cascade of events leading to retinal ischemia which, in turn, makes the retina hypoxic thus setting off angiogenic processes. However, many children with a history of ROP show persistent vision impairment, and there is evidence of an association between ROP and neurosensory disabilities. This is not surprising given the strict relationship between neuronal function and an adequate blood supply. In the present work, we revised literature data evidencing to what extent ROP can be considered a neurodegenerative disease, also taking advantage from data obtained in preclinical models of ROP. The involvement of different retinal cell populations in triggering the neuronal damage in ROP was described along with the neurological outcomes associated to ROP. The situation of ROP in Italy was assessed as well.

The role of the β-adrenoceptors (β-ARs) in hypoxia-driven diseases has gained visibility after the demonstration that propranolol promotes the regression of infantile hemangiomas and ameliorates the signs of retinopathy of prematurity (ROP). Besides the role of β2-ARs, preclinical studies in ROP have also revealed that β3-ARs are upregulated by hypoxia and that they are possibly involved in retinal angiogenesis. In a sort of figurative round trip, peculiarities typical of ROP, where hypoxia drives retinal neovascularization, have been then translated to cancer, a disease equally characterized by hypoxia-driven angiogenesis. In this step, investigating the role of β3-ARs has taken advantage of the assumption that cancer growth uses a set of strategies in common with embryo development. The possibility that hypoxic induction of β3-ARs may represent one of the mechanisms through which primarily embryo (and then cancer, as an astute imitator) adapts to grow in an otherwise hostile environment, has grown evidence. In both cancer and embryo, β3-ARs exert similar functions by exploiting a metabolic shift known as the Warburg effect, by acquiring resistance against xenobiotics, and by inducing a local immune tolerance. An additional potential role of β3-AR as a marker of stemness has been suggested by the finding that its antagonism induces cancer cell differentiation evoking that β3-ARs may help cancer to grow in a nonhospital environment, a strategy also exploited by embryos. From cancer, the round trip goes back to neonatal diseases for which new possible interpretative keys and potential pharmacological perspectives have been suggested., (© 2021 The Authors. Medicinal Research Reviews published by Wiley Periodicals LLC.)

A major player in the homeostatic response to hypoxia is the hypoxia-inducible factor (HIF)-1 that transactivates a number of genes involved in neovessel proliferation in response to low oxygen tension. In the retina, hypoxia overstimulates β-adrenoceptors (β-ARs) which play a key role in the formation of pathogenic blood vessels. Among β-ARs, β3-AR expression is increased in proliferating vessels in concomitance with increased levels of HIF-1α and vascular endothelial growth factor (VEGF). Whether, similarly to VEGF, hypoxia-induced β3-AR upregulation is driven by HIF-1 is still unknown. We used the mouse model of oxygen-induced retinopathy (OIR), an acknowledged model of retinal angiogenesis, to verify the hypothesis of β3-AR transcriptional regulation by HIF-1. Investigation of β3-AR regulation over OIR progression revealed that the expression profile of β3-AR depends on oxygen tension, similar to VEGF. The additional evidence that HIF-1α stabilization decouples β3-AR expression from oxygen levels further indicates that HIF-1 regulates the expression of the β3-AR gene in the retina. Bioinformatics predicted the presence of six HIF-1 binding sites (HBS #1-6) upstream and inside the mouse β3-AR gene. Among these, HBS #1 has been identified as the most suitable HBS for HIF-1 binding. Chromatin immunoprecipitation-qPCR demonstrated an effective binding of HIF-1 to HBS #1 indicating the existence of a physical interaction between HIF-1 and the β3-AR gene. The additional finding that β3-AR gene expression is concomitantly activated indicates the possibility that HIF-1 transactivates the β3-AR gene. Our results are indicative of β3-AR involvement in HIF-1-mediated response to hypoxia.

Retinopathy of prematurity (ROP) is an evolutive and potentially blinding eye disease that affects preterm newborns. Unfortunately, until now no conservative therapy of active ROP with proven efficacy is available. Although ROP is a multifactorial disease, premature exposition to oxygen concentrations higher than those intrauterine, represents the initial pathogenetic trigger. The increase of oxygenation in a retina still incompletely vascularized promotes the downregulation of proangiogenic factors and finally the interruption of vascularization (ischemic phase). However, the increasing metabolic requirement of the ischemic retina induces, over the following weeks, a progressive hypoxia that specularly increases the levels of proangiogenic factors finally leading to proliferative retinopathy (proliferative phase). Considering non-modifiable the coupling between oxygen levels and vascularization, so far, neonatologists and ophthalmologists have "played defense", meticulously searching the minimum necessary concentration of oxygen for individual newborns, refining their diagnostic ability, adopting a careful monitoring policy, ready to decisively intervene only in a very advanced stage of disease progression. However, recent advances have demonstrated the possibility to pharmacologically modulate the relationship between oxygen and vascularization, opening thus the perspective for new therapeutic or preventive opportunities. The perspective of a shift from a defensive towards an attack strategy is now at hand., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Filippi, Cammalleri, Amato, Ciantelli, Pini, Bagnoli and Dal Monte.)

In diabetic retinopathy (DR), high blood glucose drives chronic oxidative stress and inflammation that trigger alterations of the neurovascular balance finally resulting in vascular abnormalities and retinal cell death, which converge towards altered electroretinogram (ERG). In the last years, a growing body of preclinical evidence has suggested that nutrients with anti-inflammatory/antioxidant properties can be able to hamper DR progression since its very early stages. In the present study, we used a streptozotocin-induced rat model of DR, which mimics most aspects of the early stages of human DR, to test the preventive efficacy of a novel compound containing cyanidin-3-glucoside (C3G), verbascoside and zinc as nutrients with antioxidant and anti-inflammatory properties. Western blot, immunofluorescence and electroretinographic analyses demonstrated a dose-dependent inhibition of oxidative stress- and inflammation-related mechanisms, with a significant counterpart in preventing molecular mechanisms leading to DR-associated vasculopathy and its related retinal damage. Preventive efficacy of the compound on dysfunctional a- and b-waves was also demonstrated by electroretinography. The present demonstration that natural compounds, possibly as a consequence of vascular rescue following ameliorated oxidative stress and inflammation, may prevent the apoptotic cascade leading to ERG dysfunction, adds further relevance to the potential application of antioxidants as a preventive therapy to counteract DR progression., Competing Interests: MM received a study grant from Sooft Italia SpA. DR is an employee of Sooft Italia SpA. Sooft Italia SpA had no direct role in the collection, analyses or interpretation of data or in the decision to publish the results. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer ST declared a past collaboration with one of the authors MC to the handling editor., (Copyright © 2022 Canovai, Amato, Melecchi, Dal Monte, Rusciano, Bagnoli and Cammalleri.)

Renin-angiotensin systems produce angiotensin II (Ang II) and angiotensin 1-7 (Ang 1-7), which are able to induce opposite effects on circulation. This study in vivo assessed the effects induced by Ang II or Ang 1-7 on rat pial microcirculation during hypoperfusion-reperfusion, clarifying the mechanisms causing the imbalance between Ang II and Ang 1-7. The fluorescence microscopy was used to quantify the microvascular parameters. Hypoperfusion and reperfusion caused vasoconstriction, disruption of blood-brain barrier, reduction of capillary perfusion and an increase in reactive oxygen species production. Rats treated with Ang II showed exacerbated microvascular damage with stronger vasoconstriction compared to hypoperfused rats, a further increase in leakage, higher decrease in capillary perfusion and marker oxidative stress. Candesartan cilexetil (specific Ang II type 1 receptor (AT 1 R) antagonist) administration prior to Ang II prevented the effects induced by Ang II, blunting the hypoperfusion-reperfusion injury. Ang 1-7 or ACE2 activator administration, preserved the pial microcirculation from hypoperfusion-reperfusion damage. These effects of Ang 1-7 were blunted by a Mas (Mas oncogene-encoded protein) receptor antagonist, while Ang II type 2 receptor antagonists did not affect Ang 1-7-induced changes. In conclusion, Ang II and Ang 1-7 triggered different mechanisms through AT 1 R or MAS receptors able to affect cerebral microvascular injury.

The mobilization of endothelial progenitor cells (EPCs) into circulation from bone marrow is well known to be present in several clinical settings, including acute coronary syndrome, heart failure, diabetes and peripheral vascular disease. The aim of this review was to explore the current literature focusing on the great opportunity that EPCs can have in terms of regenerative medicine.

In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Light-induced retinal damage (LD) is characterized by the accumulation of reactive oxygen species leading to oxidative stress and photoreceptor cell death. The use of natural antioxidants has emerged as promising approach for the prevention of LD. Among them, lutein and cyanidin-3-glucoside (C3G) have been shown to be particularly effective due to their antioxidant and anti-inflammatory activity. However, less is known about the possible efficacy of combining them in a multicomponent mixture. In a rat model of LD, Western blot analysis, immunohistochemistry and electroretinography were used to demonstrate that lutein and C3G in combination or in a multicomponent mixture can prevent oxidative stress, inflammation, gliotic and apoptotic responses thus protecting photoreceptor cells from death with higher efficacy than each component alone. Combined efficacy on dysfunctional electroretinogram was also demonstrated by ameliorated rod and cone photoreceptor responses. These findings suggest the rationale to formulate multicomponent blends which may optimize the partnering compounds bioactivity and bioavailability.

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Milano, Carmosino, Gerbino, Saponara, Lapi, Dal Monte, Bagnoli, Svelto and Procino.)

Lisosan G (LG), a fermented powder obtained from whole grains, is a nutritional supplement containing a variety of metabolites with documented antioxidant properties. We have recently demonstrated that orally administered LG protects diabetic rodent retinas from oxidative stress, inflammation, apoptosis, blood-retinal barrier disruption, and functional damage. Here, we investigated whether LG may exert protective effects in a model of glaucoma and measured the amounts of selected LG components that reach the retina after oral LG administration. Six-month-old DBA/2J mice were given an aqueous LG solution in place of drinking water for 2 mo. During the 2 mo of treatment with LG, the intraocular pressure (IOP) was monitored and the retinal ganglion cell (RGC) functional activity was recorded with pattern-electroretinography (PERG). At the end of the 2-mo period, the expression of oxidative stress and inflammatory markers was measured with qPCR, and RGC survival or macroglial activation were assessed with immunofluorescence. Alternatively, LG was administered by gavage and the concentrations of four of the main LG components (nicotinamide, gallic acid, 4-hydroxybenzoic acid, and quercetin) were measured in the retinas in the following 24 h using mass spectrometry. LG treatment in DBA/2J mice did not influence IOP, but it affected RGC function since PERG amplitude was increased and PERG latency was decreased with respect to untreated DBA/2J mice. This improvement of RGC function was concomitant with a significant decrease of both oxidative stress and inflammation marker expression, of RGC loss, and of macroglial activation. All four LG metabolites were found in the retina, although with different proportions with respect to the amount in the dose of administered LG, and with different temporal profiles in the 24 h following administration. These findings are consistent with neuroenhancing and neuroprotective effects of LG in glaucoma that are likely to derive from its powerful antioxidant properties. The co-occurrence of different metabolites in LG may provide an added value to their beneficial effects and indicate LG as a basis for the potential treatment of a variety of retinal pathologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amato, Rossino, Cammalleri, Timperio, Fanelli, Dal Monte, Pucci and Casini.)

Purpose: Glaucoma is a multifactorial disease, causing retinal ganglion cells (RGCs) and optic nerve degeneration. The role of diabetes as a risk factor for glaucoma has been postulated but still not unequivocally demonstrated. The purpose of this study is to clarify the effect of diabetes in the early progression of glaucomatous RGC dysfunction preceding intraocular pressure (IOP) elevation, using the DBA/2J mouse (D2) model of glaucoma., Methods: D2 mice were injected with streptozotocin (STZ) obtaining a combined model of diabetes and glaucoma (D2 + STZ). D2 and D2 + STZ mice were monitored for weight, glycemia, and IOP from 3.5 to 6 months of age. In addition, the activity of RGC and outer retina were assessed using pattern electroretinogram (PERG) and flash electroretinogram (FERG), respectively. At the end point, RGC density and astrogliosis were evaluated in flat mounted retinas. In addition, Müller cell reactivity was evaluated in retinal cross-sections. Finally, the expression of inflammation and oxidative stress markers were analyzed., Results: IOP was not influenced by time or diabetes. In contrast, RGC activity resulted progressively decreased in the D2 group independently from IOP elevation and outer retinal dysfunction. Diabetes exacerbated RGC dysfunction, which resulted independent from variation in IOP and outer retinal activity. Diabetic retinas displayed decreased RGC density and increased glial reactivity given by an increment in oxidative stress and inflammation., Conclusions: Diabetes can act as an IOP-independent risk factor for the early progression of glaucoma promoting oxidative stress and inflammation-mediated RGC dysfunction, glial reactivity, and cellular death.

Neuropathic ocular pain is a frequent occurrence in medium to severe dry eye disease (DED). Only palliative treatments, such as lubricants and anti-inflammatory drugs, are available to alleviate patients' discomfort. Anesthetic drugs are not indicated, because they may interfere with the neural feedback between the cornea and the lacrimal gland, impairing tear production and lacrimation. Gabapentin (GBT) is a structural analog of gamma-amino butyric acid that has been used by systemic administration to provide pain relief in glaucomatous patients. We have already shown in a rabbit model system that its topic administration as eye drops has anti-inflammatory properties. We now present data on rabbits' eyes showing that indeed GBT given topically as eye drops has analgesic but not anesthetic effects. Therefore, opposite to an anesthetic drug such as oxybuprocaine, GBT does not decrease lacrimation, but-unexpectedly-even stimulates it, apparently through the upregulation of acetylcholine and norepinephrine, and by induction of aquaporin 5 (AQP5) expression in the lacrimal gland. Moreover, data obtained in vitro on a primary human corneal epithelial cell line also show direct induction of AQP5 by GBT. This suggests that corneal cells might also contribute to the lacrimal stimulation promoted by GBT and participate with lacrimal glands in the restoration of the tear film, thus reducing friction on the ocular surface, which is a known trigger of ocular pain. In conclusion, GBT is endowed with analgesic, anti-inflammatory and secretagogue properties, all useful to treat neuropathic pain of the ocular surface, especially in case of DED., Competing Interests: MDM received a study grant from Sooft Italia SpA. MO, SP, and DR are employees of Sooft Italia SpA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cammalleri, Amato, Olivieri, Pezzino, Bagnoli, Dal Monte and Rusciano.)

During retinal development, a physiologic hypoxia stimulates endothelial cell proliferation. The hypoxic milieu warrants retina vascularization and promotes the activation of several mechanisms aimed to ensure homeostasis and energy balance of both endothelial and retinal cells. Autophagy is an evolutionarily conserved catabolic system that contributes to cellular adaptation to a variety of environmental changes and stresses. In association with the physiologic hypoxia, autophagy plays a crucial role during development. Autophagy expression profile was evaluated in the developing retina from birth to post-natal day 18 of rat pups, using qPCR, western blotting and immunostaining methodologies. The rat post-partum developing retina displayed increased active autophagy during the first postnatal days, correlating to the hypoxic phase. In latter stages of development, rat retinal autophagy decreases, reaching a normalization between post-natal days 14-18, when the retina is fully vascularized and mature. Collectively, the present study elaborates on the link between hypoxia and autophagy, and contributes to further elucidate the role of autophagy during retinal development.

Melatonin is of great importance for regulating several eye processes, including pressure homeostasis. Melatonin in combination with agomelatine has been recently reported to reduce intraocular pressure (IOP) with higher efficacy than each compound alone. Here, we used the methylcellulose (MCE) rat model of hypertensive glaucoma, an optic neuropathy characterized by the apoptotic death of retinal ganglion cells (RGCs), to evaluate the hypotensive and neuroprotective efficacy of an eye drop nanomicellar formulation containing melatonin/agomelatine. Eye tissue distribution of melatonin/agomelatine in healthy rats was evaluated by HPLC/MS/MS. In the MCE model, we assessed by tonometry the hypotensive efficacy of melatonin/agomelatine. Neuroprotection was revealed by electroretinography; by levels of inflammatory and apoptotic markers; and by RGC density. The effects of melatonin/agomelatine were compared with those of timolol (a beta blocker with prevalent hypotensive activity) or brimonidine (an alpha 2 adrenergic agonist with potential neuroprotective efficacy), two drugs commonly used to treat glaucoma. Both melatonin and agomelatine penetrate the posterior segment of the eye. In the MCE model, IOP elevation was drastically reduced by melatonin/agomelatine with higher efficacy than that of timolol or brimonidine. Concomitantly, gliosis-related inflammation and the Bax-associated apoptosis were partially prevented, thus leading to RGC survival and recovered retinal dysfunction. We suggest that topical melatoninergic compounds might be beneficial for ocular health.

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.

Introduction: Retinopathy of Prematurity (ROP) is a sight-threatening disease representing one of the main disabling diseases affecting premature newborns. Presently, ROP is treated by surgical interventions and drug therapies are limited to the off-label use of a little amount of molecules approved for other pathologies., Areas Covered: Many drugs that may potentially be used in treating ROP are recently proposed, in many cases after the demonstration of their effectiveness in preclinical studies. In this review, the authors discuss safety and effectiveness of the main proposed approaches in the pharmacologic treatment of the disease, including approaches based on oxygen therapy and nutritional interventions., Expert Opinion: Surgical approaches to ROP are not without side effects. However, most of the proposed pharmacologic interventions can also raise specific concerns. In particular, these approaches follow a curative paradigm and are proposed in patients once the disease has progressed, with an effectiveness that is often smaller than expected. A goal in the treatment of ROP would be moving the paradigm toward a preventive approach that could be potentially effective in treating extremely low birth weight preterm infants before ROP becomes manifest.

Background : In retinitis pigmentosa (RP), inherited rod death is followed by cone loss and blindness. Why cones die is still a matter of consideration. Here, we investigate the pathogenic role of the sympathetic transmission in the rd10 mouse model of RP. Methods : Retinal levels of beta adrenergic receptor (BAR) 2 and norepinephrine (NE) were measured. After administration of the BAR1/2 blocker propranolol or the hypoxia-inducible factor (HIF)-1 activator dimethyloxalylglycine (DMOG), retinal levels of HIF-1α, BAR2 or proteins involved in BAR2 desensitization were also measured. In DMOG treated mice, expression and localization of BAR2, inflammatory markers and cone arrestin were determined. Finally, rd10 mice were subjected to electroretinogram (ERG) analysis to assess rod and cone function. Results : In the rd10 retina, BAR2 overexpression and NE accumulation were found, with BAR2 immunoreactivity localized to Müller cells. BAR2 overexpression was likely due to desensitization defects. Upregulated levels of BAR2 were drastically reduced by propranolol that also restored desensitization defects. Due to the low level of HIF-1 consequent to the hyperoxic environment in the rd10 retina, we hypothesized a link between HIF-1 and BAR2. HIF-1α stabilization with DMOG resulted in i. increased HIF-1α accumulation, ii. decreased BAR2 levels, iii. restored desensitization processes, iv. reduced expression of inflammatory markers and v. increased cone survival without improved retinal function. Conclusions : Our results support a pathogenic role of the sympathetic system in RP that might help to understand why rd10 mice show a positive response to BAR blockers.

There is indication that nutritional supplements protect retinal cells from degeneration. In a previous study, we demonstrated that dietary supplementation with an association of forskolin, homotaurine, spearmint extract and B vitamins efficiently counteracts retinal dysfunction associated with retinal ganglion cell (RGC) death caused by optic nerve crush. We extended our investigation on the efficacy of dietary supplementation with the use of a mouse model in which RGC degeneration depends as closely as possible on intraocular pressure (IOP) elevation. In this model, injecting the anterior chamber of the eye with methylcellulose (MCE) causes IOP elevation leading to RGC dysfunction. The MCE model was characterized in terms of IOP elevation, retinal dysfunction as determined by electrophysiological recordings, RGC loss as determined by brain-specific homeobox/POU domain protein 3A immunoreactivity and dysregulated levels of inflammatory and apoptotic markers. Except for IOP elevation, dysfunctional retinal parameters were all recovered by dietary supplementation indicating the involvement of non-IOP-related neuroprotective mechanisms of action. Our hypothesis is that the diet supplement may be used to counteract the inflammatory processes triggered by glial cell activation, thus leading to spared RGC loss and the preservation of visual dysfunction. In this respect, the present compound may be viewed as a potential remedy to be added to the currently approved drug therapies for improving RGC protection., Competing Interests: M.C. and M.D.M. received a study grant from Sooft Italia SpA. D.R. is an employee of Sooft Italia SpA. Sooft Italia SpA had no direct role in the collection, analyses, or interpretation of data and in the decision to publish the results. R.A and P.B. declare no conflict of interest.

Background: Melatoninergic agents are known to reduce intraocular pressure (IOP). The present study was performed to evaluate the effect of nanomicellar formulations of melatoninergic agents on IOP in the rat., Methods: Tonometry was used to measure IOP in eyes instilled with melatonin or agomelatine. Ocular hypertension was induced by the injection of methylcellulose in the anterior chamber., Results: Melatonin formulated in nanomicelles had a longer lasting hypotonizing effect on IOP with respect to melatonin in saline. Nanomicellar formulations of melatonin and agomelatine, either alone or in combination, had lowering effects that did not depend on their concentration or their combination, which, however, resulted in an increased duration of the hypotonizing effect. The duration of the lowering effect was further increased by the addition of lipoic acid., Conclusions: We demonstrated the effective hypotonizing activity of melatonin and agomelatine in combination with lipoic acid. Although results in animals cannot be directly translated to humans, the possibility of developing novel therapeutical approaches for patients suffering from hypertensive glaucoma should be considered., Competing Interests: M.D.M. and M.C. received grants from Sooft Italia SpA. S.P., R.C., and D.R. are employees of Sooft Italia SpA. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The somatostatin analog octreotide (OCT) displays important neuroprotective and anti-angiogenic properties that could make it an interesting candidate to treat diabetic retinopathy (DR). Unfortunately, systemic drug administration is hindered by severe side effects, therefore topical administration routes are preferable. However, drug delivery through eye drops may be difficult due to ocular barriers and, in the long term, could induce ocular damage. On the other hand, intraocular injections must be repeated to maintain drug concentration, and this may cause severe damage to the eye. To decrease injection frequency, long-term release and reduced biodegradation could be obtained by binding the drug to biodegradable polymeric nanoparticles. In the present study, we made a preparation of OCT bound to magnetic nanoparticles (MNP-OCT) and tested its possible use as an OCT delivery system to treat retinal pathologies such as DR. In particular, in vitro , ex vivo , and in vivo experimental models of the mammalian retina were used to investigate the possible toxicity of MNPs, possible effects of the binding to MNPs on OCT bioactivity, and the localization of MNP-OCT in the retina after intraocular injection. The results showed that, both in human retinal endothelial cells (HRECs) and in mouse retinal explants, MNPs were not toxic and the binding with MNPs did not influence OCT antiangiogenic or antiapoptotic activity. Rather, effects of MNP-OCT were observed at concentrations up to 100-fold (in HRECs) or 10-fold (in mouse retinal explants) lower compared to OCT, indicating that OCT bioactivity was enhanced in MNP-OCT. MNP-OCT in mouse retinas in vivo after intraocular delivery were initially localized mainly to the outer retina, at the level of the retinal pigment epithelium, while after 5 days they were observed throughout the retinal thickness. These observations demonstrate that MNP-OCT may be used as an OCT intraocular delivery system that may ensure OCT localization to the retina and enhanced OCT bioactivity. Further studies will be necessary to determine the OCT release rate in the retina and the persistence of drug effects in the long period., (Copyright © 2020 Amato, Giannaccini, Dal Monte, Cammalleri, Pini, Raffa, Lulli and Casini.)

β 3 -Adrenoceptors exhibit a restricted expression pattern, particularly in humans. However, they have been found to be upregulated in various cancers and under several conditions associated with hypoperfusion such as congestive heart failure and diabetes for instance in the heart and other tissues. These conditions are frequently associated with hypoxia. Furthermore, direct induction of hypoxia has consistently been reported to cause upregulation of β 3 -adrenoceptors across various tissues of multiple species including humans, rats, dogs, and fish. While a canonical hypoxia-response element in the promoter of the human β 3 -adrenoceptor gene may play a role in this, no such sequence was found in rodent homologs. Moreover, not all upregulation of β 3 -adrenoceptor protein is accompanied by increased expression of corresponding mRNA, indicating that the upregulation may involve factors other than transcriptional changes. We propose that upregulation of β 3 -adrenoceptors at the mRNA and/or protein level is a general marker of hypoxic conditions. Moreover, it may be an additional pathway whereby cells and tissues adapt to reduced oxygen levels.

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.

Background: Recent explorative studies suggest that propranolol reduces retinopathy of prematurity (ROP) progression, but the short-term effects of propranolol treatment at 1 year of corrected age have not been extensively evaluated. Methods: A multi-center retrospective observational cohort study was conducted to assess the physical development and the refractive outcome of infants with prior ROP treated with propranolol. Forty-nine infants treated with propranolol were compared with an equal number of patients who did not receive any propranolol therapy and represent the control group, with comparable anthropometrical characteristics and stages of ROP. Results: The weight, length, and head circumference at 1 year of corrected age were similar between infants who had been treated, or not, with propranolol, without any statistically significant differences. Refractive evaluation at 1 year showed spherical equivalent values decreasing with the progression of ROP toward more severe stages of the disease, together with an increasing number of infants with severe myopia. On the contrary, no differences were observed between infants who had been treated with propranolol and those who had not. Conclusion: This study confirms that the progression of ROP induces an increase of refractive errors and suggests that propranolol itself does not affect the refractive outcome. Therefore, if the efficacy of propranolol in counteracting ROP progression is confirmed by further clinical trials, the conclusion will be that propranolol might indirectly improve the visual outcome, reducing the progression of ROP., (Copyright © 2019 Filippi, Cavallaro, Perciasepe, Sandini, Araimo, Regiroli, Raffaeli, Bagnoli, Dal Monte, Calvani, Fortunato, Osnaghi, De Masi and Mosca.)

Diabetic retinopathy (DR) is a common complication of diabetes and constitutes a major cause of vision impairment and blindness in the world. DR has long been described exclusively as a microvascular disease of the eye. However, in recent years, a growing interest has been focused on the contribution of neuroretinal degeneration to the pathogenesis of the disease, and there are observations suggesting that neuronal death in the early phases of DR may favor the development of microvascular abnormalities, followed by the full manifestation of the disease. However, the mediators that are involved in the crosslink between neurodegeneration and vascular changes have not yet been identified. According to our hypothesis, vascular endothelial growth factor (VEGF) could probably be the most important connecting link between the death of retinal neurons and the occurrence of microvascular lesions. Indeed, VEGF is known to play important neuroprotective actions; therefore, in the early phases of DR, it may be released in response to neuronal suffering, and it would act as a double-edged weapon inducing both neuroprotective and vasoactive effects. If this hypothesis is correct, then any retinal stress causing neuronal damage should be accompanied by VEGF upregulation and by vascular changes. Similarly, any compound with neuroprotective properties should also induce VEGF downregulation and amelioration of the vascular lesions. In this review, we searched for a correlation between neurodegeneration and vasculopathy in animal models of retinal diseases, examining the effects of different neuroprotective substances, ranging from nutraceuticals to antioxidants to neuropeptides and others and showing that reducing neuronal suffering also prevents overexpression of VEGF and vascular complications. Taken together, the reviewed evidence highlights the crucial role played by mediators such as VEGF in the relationship between retinal neuronal damage and vascular alterations and suggests that the use of neuroprotective substances could be an efficient strategy to prevent the onset or to retard the development of DR., (Copyright © 2019 Rossino, Dal Monte and Casini.)

Puncture-induced iris neovascularization (rubeosis iridis; RI) in mice is associated with upregulation of extracellular matrix (ECM) degradation and inflammatory factors. The anti-angiogenic and anti-inflammatory efficacy of UPARANT in reducing RI was determined by noninvasive, in vivo iris vascular densitometry, and confirmed in vitro by quantitative vascular-specific immunostaining. Intravitreal administration of UPARANT successfully and rapidly reduced RI to non-induced control levels. Molecular analysis revealed that UPARANT inhibits formyl peptide receptors through a predominantly anti-inflammatory response, accompanied with a significant reduction in ECM degradation and inflammation markers. Similar results were observed with UPARANT administered systemically by subcutaneous injection. These data suggest that the tetrapeptide UPARANT is an effective anti-angiogenic agent for the treatment of RI, both by local and systemic administrations. The effectiveness of UPARANT in reducing RI in a model independent of the canonical vascular endothelial growth factor (VEGF) proposes an alternative for patients that do not respond to anti-VEGF treatments, which could improve treatment in proliferative ocular diseases. KEY MESSAGES: UPARANT is effective in the treatment of rubeosis iridis, both by local and systemic administrations. UPARANT can reduce VEGF-independent neovascularization.

Dysregulation of vascular networks is characteristic of eye diseases associated with retinal cell degeneration and visual loss. Visual impairment is also the consequence of photoreceptor degeneration in inherited eye diseases with a major inflammatory component, but without angiogenic profile. Among the pathways with high impact on vascular/degenerative diseases of the eye, a central role is played by a system formed by the ligand urokinase-type plasminogen activator (uPA) and its receptor uPAR. The uPAR system, although extensively investigated in tumors, still remains a key issue in vascular diseases of the eye and even less studied in inherited retinal pathologies such as retinitis pigmantosa (RP). Its spectrum of action has been extended far beyond a classical pro-angiogenic function and has emerged as a central actor in inflammation. Preclinical studies in more prevalent eye diseases characterized by neovascular formation, as in retinopathy of prematurity, wet macular degeneration and rubeosis iridis or vasopermeability excess as in diabetic retinopathy, suggest a critical role of increased uPAR signaling indicating the potentiality of its modulation to counteract neovessel formation and microvascular dysfunction. The additional observation that the uPAR system plays a major role in RP by limiting the inflammatory cascade triggered by rod degeneration rises further questions about its role in the diseased eye.

Retinitis pigmentosa (RP) is characterized by progressive loss of vision due to photoreceptor degeneration leading to secondary inflammation. The urokinase-type plasminogen activator (uPA) system contributes to retinal inflammation, but its role in RP is unknown. In the rd10 mouse model of RP, we addressed this question with the use of the peptide UPARANT designed to interact with the uPA system. UPARANT was systemically administered from post-natal day (PD) 10 to PD30 when its efficacy in RP rescue was investigated using electroretinographic recordings, Western blot and immunocytochemistry. Temporal profile of protein expression in the uPA system was also investigated. UPARANT reduced both Müller cell gliosis and up-regulated levels of inflammatory markers and exerted major anti-apoptotic effects without influencing the autophagy cascade. Rescue from retinal cell degeneration was accompanied by improved retinal function. No scotopic phototransduction was rescued in the UPARANT-treated animals as determined by the kinetic analysis of rod-mediated a-waves and confirmed by rod photoreceptor markers. In contrast, the cone photopic b-wave was recovered and its rescue was confirmed in the whole mounts using cone arrestin antibody. Investigation of the uPA system regulation over RP progression revealed extremely low levels of uPA and its receptor uPAR both of which were recovered by HIF-1α stabilization indicating that HIF-1 regulates the expression of the uPA/uPAR gene in the retina. Ameliorative effects of UPARANT were likely to occur through an inhibitory action on up-regulated activity of the αvβ3 integrin/Rac1 pathway that was suggested as a novel target for the development of therapeutic approaches against RP., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Stress plays a role in tumourigenesis through catecholamines acting at β-adrenoceptors including β 1 -, β 2 - and β 3 -adrenoceptors, and the use of β-adrenoceptor antagonists seems to counteract tumour growth and progression. Preclinical evidence and meta-analysis data demonstrate that melanoma shows a positive response to β-adrenoceptor blockers and in particular to propranolol acting mainly at β 1 - and β 2 -adrenoceptors. Although evidence suggesting that β 3 -adrenoceptors may play a role as a therapeutic target in infantile haemangiomas has been recently reviewed, a comprehensive analysis of the data available from preclinical studies supporting a possible role of β 3 -adrenoceptors in melanoma was not available. Here, we review data from the literature demonstrating that propranolol may be effective at counteracting melanoma growth, and we provide preclinical evidence that β 3 -adrenoceptors may also play a role in the pathophysiology of melanoma, thus opening the door for further clinical assays trying to explore β 3 -adrenoceptor blockers as novel alternatives for its treatment. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc., (© 2018 The British Pharmacological Society.)

Background and Purpose: Stress-related catecholamines have a role in cancer and β-adrenoceptors; specifically, β 2 -adrenoceptors have been identified as new targets in treating melanoma. Recently, β 3 -adrenoceptors have shown a pleiotropic effect on melanoma micro-environment leading to cancer progression. However, the mechanisms by which β 3 -adrenoceptors promote this progression remain poorly understood. Catecholamines affect the immune system by modulating several factors that can alter immune cell sub-population homeostasis. Understanding the mechanisms of cancer immune-tolerance is one of the most intriguing challenges in modern research. This study investigates the potential role of β 3 -adrenoceptors in immune-tolerance regulation., Experimental Approach: A mouse model of melanoma in which syngeneic B16-F10 cells were injected in C57BL-6 mice was used to evaluate the effect of β-adrenoceptor blockade on the number and activity of immune cell sub-populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with β-blockers (propranolol and SR59230A) and specific β-adrenoceptor siRNAs targeting β 2 - or β 3 -adrenoceptors were used., Key Results: Only β 3 -, but not β 2 -adrenoceptors, were up-regulated under hypoxia in peripheral blood mononuclear cells and selectively expressed in immune cell sub-populations including Treg, MDSC, and NK. SR59230A and β 3 -adrenoceptor siRNAs increased NK and CD8 number and cytotoxicity, while they attenuated Treg and MDSC sub-populations in the tumour mass, blood, and spleen. SR59230A and β 3 -adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes., Conclusions and Implications: Our data suggest that β 3 -adrenoceptors are involved in immune-tolerance, which opens the way for new strategic therapies to overcome melanoma growth., Linked Articles: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc., (© 2019 The British Pharmacological Society.)

Background: Oral propranolol reduces retinopathy of prematurity (ROP) progression, although not safely. Propranolol 0.1% eye micro-drops administered to newborns with stage 2 ROP are well-tolerated, but not sufficiently effective. Methods: A multi-center open-label trial was conducted to assess the safety and efficacy of propranolol 0.2% eye micro-drops in newborns with stage 1 ROP. The progression of the disease was evaluated with serial ophthalmologic examinations. Hemodynamic, respiratory, biochemical parameters, and propranolol plasma levels were monitored. Demographic and perinatal characteristics, co-morbidities and co-intervention incidences, together with ROP progression, were compared with a historical control group in the same centers participating in the trial. Results: Ninety-eight newborns were enrolled and compared with the historical control group. Populations were not perfectly homogeneous (as demonstrated by the differences in the Apgar score and the different incidence rate in surfactant administration and oxygen exposure). The progression to ROP stage 2 or 3 plus was significantly lower than the incidence expected on the basis of historical data (Risk Ratio 0.521, 95% CI 0.297- 0.916). No adverse effects related to propranolol were observed and the mean propranolol plasma level was significantly lower than the safety cut-off of 20 ng/mL. Unexpectedly, three newborns treated with oral propranolol before the appearance of ROP, showed a ROP that was unresponsive to propranolol eye micro-drops and required laser photocoagulation treatment. Conclusion: Propranolol 0.2% eye micro-drops were well-tolerated and appeared to reduce the ROP progression expected on the basis of a comparison with a historical control group. Propranolol administered too early appears to favor a more aggressive ROP, suggesting that a β-adrenoreceptor blockade is only useful during the proliferative phase. Further randomized placebo-controlled trials are required to confirm the current results. Clinical Trial Registration   The trial was registered at ClinicalTrials.gov with Identifier NCT02504944 and with EudraCT Number 2014-005472-29.

The urokinase-type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR -(s)uPAR- from circulation) is to regulate podocyte function through αvβ3 integrin/Rac-1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)-induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvβ3 integrin/Rac-1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ-induced up-regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvβ3 integrin/Rac-1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen-plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR-targeting approaches., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Lisosan G (LG), a fermented powder obtained from whole grains, is a recognized antioxidant compound that improves the bioactivity and survival of different cell types. The purpose of this study was to investigate whether LG ameliorates both the neural and the vascular damage characterizing early stages of diabetic retinopathy (DR). The effects of LG were studied in cultured explants of mouse retinas challenged with oxidative stress (OS) or in retinas of streptozotocin (STZ)-treated rats. Apoptosis, vascular endothelial growth factor (VEGF) expression, OS markers, blood-retinal barrier (BRB) integrity, and inflammation were assessed, while retinal function was evaluated with electroretinogram (ERG). LG extensively inhibited apoptosis, VEGF expression, and OS both in retinal explants and in STZ rats. In addition, STZ rats treated with LG displayed an almost total BRB integrity, reduced levels of inflammatory markers and a partially restored visual function as evaluated with ERG. In summary, we demonstrated that LG exhibits antioxidant and anti-inflammatory effects that exert powerful protective actions against neural and vascular defects characteristic of DR. Therefore, LG-containing foods or supplements may be considered to implement DR treatments.

The early phases of embryonic development and cancer share similar strategies to improve their survival in an inhospitable environment: both proliferate in a hypoxic and catecholamine-rich context, increasing aerobic glycolysis. Recent studies show that β 3-adrenergic receptor ( β 3-AR) is involved in tumor progression, playing an important role in metastasis. Among β -adrenergic receptors, β 3-AR is the last identified member of this family, and it is involved in cancer cell survival and induction of stromal reactivity in the tumor microenvironment. β 3-AR is well known as a strong activator of uncoupling protein 1 (UCP1) in brown fat tissue. Interestingly, β 3-AR is strongly expressed in early embryo development and in many cancer tissues. Induction of uncoupling protein 2 (UCP2) has been related to cancer metabolic switch, leading to accelerated glycolysis and reduced mitochondrial activity. In this study, for the first time, we demonstrate that β 3-AR is able to promote this metabolic shift in both cancer and embryonic stem cells, inducing specific glycolytic cytoplasmic enzymes and a sort of mitochondrial dormancy through the induction of UCP2. The β 3-AR/UCP2 axis induces a strong reduction of mitochondrial activity by reducing ATP synthesis and mitochondrial reactive oxygen species (mtROS) content. These effects are reverted by SR59230A, the specific β 3-AR antagonist, causing an increase in mtROS. The increased level of mtROS is neutralized by a strong antioxidant activity in embryonic stem cells, but not in cancer stem cells, where it causes a dramatic reduction in tumor cell viability. These results lead to the possibility of a selective antitumor therapeutic use of SR59230A. Notably, we demonstrate the presence of β 3-AR within the mitochondrial membrane in both cell lines, leading to the control of mitochondrial dormancy.

In the experimental autoimmune encephalomyelitis (EAE) mouse model of optic neuritis, we recently demonstrated that diet supplementation with a balanced mixture of fatty acids (FAs), including omega 3 and omega 6, efficiently limited inflammatory events in the retina and prevented retinal ganglion cell (RGC) death, although mechanisms underlying the efficacy of FAs were to be elucidated. Whether FAs effectiveness was accompanied by efficient rescue of demyelinating events in the optic nerve was also unresolved. Finally, the possibility that RGC rescue might result in ameliorated visual performance remained to be investigated. Here, the EAE model of optic neuritis was used to investigate mechanisms underlying the anti-inflammatory effects of FAs, including their potential efficacy on macrophage polarization. In addition, we determined how FAs-induced rescue of RGC degeneration was related to optic nerve histopathology by performing ultrastructural morphometric analysis with transmission electron microscopy. Finally, RGC rescue was correlated with visual performance by recording photopic electroretinogram, an efficient methodology to unravel the role of RGCs in the generation of electroretinographic waves. We conclude that the ameliorative effects of FAs were dependent on a predominant anti-inflammatory action including a role on promoting the shift of macrophages from the inflammatory M1 phenotype towards the anti-inflammatory M2 phenotype. This would finally result in restored optic nerve histopathology and ameliorated visual performance. These findings can now offer new perspectives for implementing our knowledge on the effectiveness of diet supplementation in counteracting optic neuritis and suggest the importance of FAs as possible adjuvants in therapies against inflammatory diseases of the eye.

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., Competing Interests: M.C. received a study grant from Sooft Italia SpA. D.R. is an employee of Sooft Italia SpA, which however had no direct role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Neuronal injury plays a major role in diabetic retinopathy (DR). Our hypothesis was that the balance between neuronal death and survival may depend on a similar equilibrium between apoptosis and autophagy and that a neuroprotectant may act by influencing this equilibrium. Ex vivo mouse retinal explants were treated with high glucose (HG) for 10days and the somatostatin analog octreotide (OCT) was used as a neuroprotectant. Chloroquine (CQ) was used as an autophagy inhibitor. Apoptotic and autophagic markers were evaluated using western blot and immunohistochemistry. HG-treated explants displayed a significant increase of apoptosis paralleled by a significant decrease of the autophagic flux, which was likely to be due to increased activity of the autophagy regulator mTOR (mammalian target of rapamycin). Treatment with OCT rescued HG-treated retinal explants from apoptosis and determined an increase of autophagic activity with concomitant mTOR inhibition. Blocking the autophagic flux with CQ completely abolished the anti-apoptotic effect of OCT. Immunohistochemical observations showed that OCT-induced autophagy is localized to populations of bipolar and amacrine cells and to ganglion cells. These observations revealed the antithetic role of apoptosis and autophagy, highlighting their equilibrium from which neuronal survival is likely to depend. These data suggest the crucial role covered by autophagy, which could be considered as a molecular target for DR neuroprotective treatment strategies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Diabetic retinopathy (DR) is a major complication of diabetes, characterized by extensive vascular pathology leading to vision loss. Neuronal suffering and death are also present in the diabetic retina as a result of different molecular mechanisms that are compromised or modified in response to high glucose. The aim of this paper is to highlight recent data indicating that neurodegeneration is likely to play a primary role in the development of DR and that strategies based on nanomedicine may be exploited to deliver neuroprotection to the retina., Methods: An extensive analysis of the publications dealing with the role of neuroprotection in DR and with nanoparticle-mediated drug delivery to the retina has been conducted using PubMed, with particular attention to the most recent papers., Results: There are important limitations related to possible systemic side effects of neuroprotective substances and to drug bioavailability in the retina such as, for instance, the amount of drug reaching the retina, the need of keeping to a minimum the number of administrations (especially, for example, in the case of intraocular injections) and the need of assuring a long-lasting, graded intraocular drug delivery. In recent years, a variety of investigations have been aimed at the exploitation of approaches of nanomedicine to enhance the pharmacokinetics and pharmacodynamic activity of intraocularly delivered drugs. In particular, we provide some preliminary results that we have obtained about the feasibility of delivering magnetic nanoparticles functionalized with a neuroprotectant to mouse eyes through intraocular injections., Conclusion: We propose that nanoparticles functionalized with neuroprotective substances may be used to protect the diabetic retina, thus causing an impact in the design of future pharmacologic treatments for DR., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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-1α (HIF-1α) 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-1α 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-1α, 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-1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1α 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., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

In a mouse model of oxygen induced retinopathy (OIR), beta adrenergic receptor (BAR) blockade has been shown to recover hypoxia-associated retinal damages. Although the adrenergic signaling is an important regulator of apoptotic and autophagic processes, the role of BARs in retinal cell death remains to be elucidated. The present study was aimed at investigating whether ameliorative effects of BAR blockers may occur through their coordinated action on apoptosis and autophagy. To this aim, retinas from control and OIR mice untreated or treated with propranolol, a non-selective BAR1/2 blocker, were characterized in terms of expression and localization of apoptosis and autophagy markers. The effects of propranolol on autophagy signaling were also evaluated and specific autophagy modulators were used to get functional information on the autophagic effects of BAR antagonism. Finally, propranolol effects on neurodegenerative processes were associated to an electrophysiological investigation of retinal function by recording electroretinogram (ERG). We found that retinas of OIR mice are characterized by increased apoptosis and decreased autophagy, while propranolol reduces apoptosis and stimulates autophagy. In particular, propranolol triggers autophagosome formation in bipolar, amacrine and ganglion cells that are committed to die by apoptosis in response to hypoxia. Also our data argue that propranolol, through the inhibition of the Akt-mammalian target of rapamycin pathway, activates autophagy which decreases retinal cell death. At the functional level, propranolol recovers dysfunctional ERG by recovering the amplitude of a- and b-waves, and oscillatory potentials, thus indicating an efficient restoring of retinal transduction. Overall, our results demonstrate that BAR1/2 are key regulators of retinal apoptosis/autophagy, and that BAR1/2 blockade leads to autophagy-mediated neuroprotection. Reinstating the balance between apoptotic and autophagic machines may therefore be viewed as a future goal in the treatment of retinopathies.

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, and macrophage infiltration were upregulated in both retinas and 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, and macrophage infiltration were counteracted by the diet supplement. The present data suggest that fatty acids may represent a useful form of diet supplementation to prevent or limit the progression of dry AMD., Competing Interests: M.C. received a study grant from Sooft Fidia Pharma. D.R. is an employee of Sooft Fidia Pharma. Sooft Fidia Pharma had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.

Background: Genetics plays an important role in the susceptibility to sporadic colorectal cancer (CRC). In the last 10 years genome-wide association studies (GWAS) have identified over 40 independent low penetrance polymorphic variants. However, these loci only explain around 1‑4% of CRC heritability, highlighting the dire need of identifying novel risk loci. In this study, we focused our attention on the genetic variability of the TAS2R16 gene, encoding for one of the bitter taste receptors that selectively binds to salicin, a natural antipyretic that resembles aspirin. Given the importance of inflammation in CRC, we tested whether polymorphic variants in this gene could affect the risk of developing this neoplasia hypothesizing a role of TAS2R16 in modulating chronic inflammation within the gut., Methods: We performed an association study using 6 tagging SNPs, (rs860170, rs978739, rs1357949, rs1525489, rs6466849, rs10268496) that cover all TAS2R16 genetic variability. The study was carried out on 1902 CRC cases and 1532 control individuals from four European countries., Results: We did not find any statistically significant association between risk of developing CRC and selected SNPs. However, after stratification by histology (colon vs. rectum) we found that rs1525489 was associated with increased risk of rectal cancer with a (P trend of = 0.0071)., Conclusions: Our data suggest that polymorphisms within TAS2R16 gene do not have a strong influence on colon cancer susceptibility, but a possible role in rectal cancer should be further evaluated in larger cohorts.

Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects. uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5'-CGGCGGGTGACCCATGTG-3' ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation. The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts. In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Retinopathy of prematurity (ROP) still represents one of the leading causes of visual impairment in childhood. Systemic propranolol has proven to be effective in reducing ROP progression in preterm newborns, although safety was not sufficiently guaranteed. On the contrary, topical treatment with propranolol eye micro-drops at a concentration of 0.1% had an optimal safety profile in preterm newborns with ROP, but was not sufficiently effective in reducing the disease progression if administered at an advanced stage (during stage 2). The aim of the present protocol is to evaluate the safety and efficacy of propranolol 0.2% eye micro-drops in preterm newborns at a more precocious stage of ROP (stage 1)., Methods: A multicenter, open-label, phase II, clinical trial, planned according to the Simon optimal two-stage design, will be performed to analyze the safety and efficacy of propranolol 0.2% eye micro-drops in preterm newborns with stage 1 ROP. Preterm newborns with a gestational age of 23-32 weeks, with a stage 1 ROP will receive propranolol 0.2% eye micro-drops treatment until retinal vascularization has been completed, but for no longer than 90 days. Hemodynamic and respiratory parameters will be continuously monitored. Blood samplings checking metabolic, renal and liver functions, as well as electrocardiogram and echocardiogram, will be periodically performed to investigate treatment safety. Additionally, propranolol plasma levels will be measured at the steady state, on the 10th day of treatment. To assess the efficacy of topical treatment, the ROP progression from stage 1 ROP to stage 2 or 3 with plus will be evaluated by serial ophthalmologic examinations., Discussion: Propranolol eye micro-drops could represent an ideal strategy in counteracting ROP, because it is definitely safer than oral administration, inexpensive and an easily affordable treatment. Establishing the optimal dosage and treatment schedule is to date a crucial issue., Trial Registration: ClinicalTrials.gov Identifier NCT02504944, registered on July 19, 2015, updated July 12, 2016. EudraCT Number 2014-005472-29.

Purpose: The activation of the urokinase-type plasminogen activator and its receptor system is associated with retinal diseases. Among peptide inhibitors of this system, UPARANT acts by preventing the onset of pathologic signs of neovascular ocular diseases. We investigated whether systemic UPARANT may act in a therapeutic regimen by suppressing the retinal damage that characterizes diabetic retinopathy using a rat model of streptozotocin-induced diabetes., Methods: In healthy rats, plasma, eye, and retina concentrations of UPARANT were evaluated by mass spectrometry. In rat models of streptozotocin-induced diabetes, the appearance of diabetic retinopathy was assessed by electroretinogram (ERG). UPARANT was then administered at different dosages and daily regimens. ERG recording, Evans blue perfusion, and real-time PCR were used to evaluate UPARANT efficacy. UPARANT safety was also determined., Results: UPARANT was found in plasma, eye, and retina soon after its administration and remained detectable after 24 hours. Between the 4th and the 5th week after diabetes onset, UPARANT at 8 mg/kg (daily for 5 days) was effective in recovering dysfunctional ERG. Three-day treatments at 8 mg/kg or a half dose for 5 days were ineffective. ERG recovery lasted approximately 2 weeks. ERG recovery was accompanied by restored blood-retinal barrier integrity and inhibition of inflammatory and angiogenic responses. UPARANT showed a safety profile., Conclusions: These data suggest that targeting the urokinase-type plasminogen activator and its receptor system by systemic UPARANT is a potential therapeutic approach for the treatment of early diabetic retinopathy, thus providing a potential alternative approach to delay disease progression in humans.

Background: Oral propranolol reduces retinopathy of prematurity (ROP) progression, although not safely. This study evaluated safety and efficacy of propranolol eye micro-drops in preterm newborns with ROP., Methods: A multicenter open-label trial, planned according to the Simon optimal two-stage design, was performed to analyze safety and efficacy of propranolol micro-drops in newborns with stage 2 ROP. To this end, hemodynamic and respiratory parameters were monitored, and blood samples were collected weekly, for 3 wk. Propranolol plasma levels were also monitored. The progression of the disease was evaluated with serial ophthalmologic examinations., Results: Twenty-three newborns were enrolled. Since the fourth of the first 19 newborns enrolled in the first stage of the study showed a progression to stage 2 or 3 with plus, the second stage was prematurely discontinued. Even though the objective to complete the second stage was not achieved, the percentage of ROP progression (26%) was similar to that obtained previously with oral propranolol administration. However, no adverse effects were observed and propranolol plasma levels were significantly lower than those measured after oral administration., Conclusion: Propranolol 0.1% eye micro-drops are well tolerated, but not sufficiently effective. Further studies are required to identify the optimal dose and administration schedule.

The spontaneously diabetic Torii (SDT) rat is of increasing preclinical interest because of its similarities to human type 2 diabetic retinopathy (DR). The system formed by urokinase-type plasminogen activator (uPA) and its receptor (uPAR) is a player in blood-retinal barrier (BRB) breakdown in DR. Here, we investigated whether in SDT rats, preventive administration of UPARANT, an inhibitor of the uPAR pathway, counteracts the retinal impairment in response to chronic hyperglycemia. Electroretinogram (ERG) monitoring was followed over time. Fluorescein-dextran microscopy, CD31 immunohistochemistry, quantitative PCR, ELISA, Evans blue perfusion, and Western blot were also used. UPARANT prevented ERG dysfunction, upregulation of vascular endothelial growth factor and fibroblast growth factor-2, BRB leakage, gliosis, and retinal cell death. The mechanisms underlying UPARANT benefits were studied comparing them with the acute streptozotocin (STZ) model in which UPARANT is known to inhibit DR signs. In SDT rats, but not in the STZ model, UPARANT downregulated the expression of uPAR and its membrane partners. In both models, UPARANT reduced the levels of transcription factors coupled to inflammation or inflammatory factors themselves. These findings may help to establish the uPAR system as putative target for the development of novel drugs that may prevent type 2 DR.

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β1- and β2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of β3-adrenergic receptor (β3-AR) in mouse kidney. The β3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that β3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the β3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of β3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of β3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of β3-AR stimulation in the kidney. Hence, β3-AR agonism might be useful to bypass AVPR2-inactivating mutations., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)