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Diabetic retinopathy is a secondary microvascular complication of diabetes mellitus. This disease progresses from two stages, non-proliferative and proliferative diabetic retinopathy, the latter characterized by retinal abnormal angiogenesis. Pharmacological management of retinal angiogenesis employs expensive and invasive intravitreal injections of biologic drugs (anti-vascular endothelial growth factor agents). To search small molecules able to act as anti-angiogenic agents, we focused our study on axitinib, which is a tyrosine kinase inhibitor and represents the second line treatment for renal cell carcinoma. Axitinib is an inhibitor of vascular endothelial growth factor receptors, and among the others tyrosine kinase inhibitors (sunitinib and sorafenib) is the most selective towards vascular endothelial growth factor receptors 1 and 2. Besides the well-known anti-angiogenic and immune-modulatory functions, we hereby explored the polypharmacological profile of axitinib, through a bioinformatic/molecular modeling approach and in vitro models of diabetic retinopathy. We showed the anti-angiogenic activity of axitinib in two different in vitro models of diabetic retinopathy, by challenging retinal endothelial cells with high glucose concentration (fluctuating and non-fluctuating). We found that axitinib, along with inhibition of vascular endothelial growth factor receptors 1 (1.82 ± 0.10; 0.54 ± 0.13, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) and vascular endothelial growth factor receptors 2 (2.38 ± 0.21; 0.98 ± 0.20, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively), was able to significantly reduce ( p < 0.05) the expression of Nrf2 (1.43 ± 0.04; 0.85 ± 0.01, protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) in retinal endothelial cells exposed to high glucose, through predicted Keap1 interaction and activation of melanocortin receptor 1. Furthermore, axitinib treatment significantly ( p < 0.05) decreased reactive oxygen species production (0.90 ± 0.10; 0.44 ± 0.06, fluorescence units in high glucose vs . axitinib 1 µM, respectively) and inhibited ERK pathway (1.64 ± 0.09; 0.73 ± 0.06, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) in HRECs exposed to high glucose. The obtained results about the emerging polypharmacological profile support the hypothesis that axitinib could be a valid candidate to handle diabetic retinopathy, with ancillary mechanisms of action., Competing Interests: Authors PS and SA were employed by Dr. Reddy’s Laboratories Ltd. 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 author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Lazzara, Conti, Sasmal, Alikunju, Rossi, Drago, Platania and Bucolo.)

Introduction: The purine analog 6-thioguanine (6TG), an old drug approved in the 60s to treat acute myeloid leukemia (AML), was tested in the diabetic retinopathy (DR) experimental in vivo setting along with a molecular modeling approach., Methods: A computational analysis was performed to investigate the interaction of 6TG with MC1R and MC5R. This was confirmed in human umbilical vein endothelial cells (HUVECs) exposed to high glucose (25 mM) for 24 h. Cell viability in HUVECs exposed to high glucose and treated with 6TG (0.05-0.5-5 µM) was performed. To assess tube formation, HUVECs were treated for 24 h with 6TG 5 µM and AGRP (0.5-1-5 µM) or PG20N (0.5-1-5-10 µM), which are MC1R and MC5R antagonists, respectively. For the in vivo DR setting, diabetes was induced in C57BL/6J mice through a single streptozotocin (STZ) injection. After 2, 6, and 10 weeks, diabetic and control mice received 6TG intravitreally (0.5-1-2.5 mg/kg) alone or in combination with AGRP or PG20N. Fluorescein angiography (FA) was performed after 4 and 14 weeks after the onset of diabetes. After 14 weeks, mice were euthanized, and immunohistochemical analysis was performed to assess retinal levels of CD34, a marker of endothelial progenitor cell formation during neo-angiogenesis., Results: The computational analysis evidenced a more stable binding of 6TG binding at MC5R than MC1R. This was confirmed by the tube formation assay in HUVECs exposed to high glucose. Indeed, the anti-angiogenic activity of 6TG was eradicated by a higher dose of the MC5R antagonist PG20N (10 µM) compared to the MC1R antagonist AGRP (5 µM). The retinal anti-angiogenic effect of 6TG was evident also in diabetic mice, showing a reduction in retinal vascular alterations by FA analysis. This effect was not observed in diabetic mice receiving 6TG in combination with AGRP or PG20N. Accordingly, retinal CD34 staining was reduced in diabetic mice treated with 6TG. Conversely, it was not decreased in diabetic mice receiving 6TG combined with AGRP or PG20N., Conclusion: 6TG evidenced a marked anti-angiogenic activity in HUVECs exposed to high glucose and in mice with DR. This seems to be mediated by MC1R and MC5R retinal receptors., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Trotta, Gesualdo, Lepre, Russo, Ferraraccio, Panarese, Marano, Grieco, Petrillo, Hermenean, Simonelli, D’Amico, Bucolo, Lazzara, De Nigris, Rossi and Platania.)

With the spread of the "omics" sciences, the approaches of systems biology can be considered as new paradigms of pharmacological research for discovery of novel targets and/or treatments for complex multifactorial diseases. Data from omics sciences can be used for the design of biologic networks, that in turn can be quantitatively analyzed to identify new pharmacological targets. In this review, we will introduce the concept of network pharmacology, particularly the application of this innovative approach in the field of ocular pharmacology, with a focus on retinal diseases such as diabetic retinopathy (DR), age-related macular degeneration (AMD) and glaucoma., 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 article., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The impairment of the blood retinal barrier (BRB) represents one of the main features of diabetic retinopathy, a secondary microvascular complication of diabetes. Hyperglycemia is a triggering factor of vascular cells damage in diabetic retinopathy. The aim of this study was to assess the effects of vitamin D 3 on BRB protection, and to investigate its regulatory role on inflammatory pathways. We challenged human retinal endothelial cells with high glucose (HG) levels. We found that vitamin D 3 attenuates cell damage elicited by HG, maintaining cell viability and reducing the expression of inflammatory cytokines such as IL-1β and ICAM-1. Furthermore, we showed that vitamin D 3 preserved the BRB integrity as demonstrated by trans-endothelial electrical resistance, permeability assay, and cell junction morphology and quantification (ZO-1 and VE-cadherin). In conclusion this in vitro study provided new insights on the retinal protective role of vitamin D 3 , particularly as regard as the early phase of diabetic retinopathy, characterized by BRB breakdown and inflammation., 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 Lazzara, Longo, Giurdanella, Lupo, Platania, Rossi, Drago, Anfuso and Bucolo.)

Purinergic ionotropic receptors, such as the P2X7 receptor, are activated by extracellular adenosine triphosphate (ATP). The P2X7 receptor is a trimeric ATP-gated cation channel and its activation results in several downstream events, including the release of proinflammatory mediators and cell damage. The P2X7 receptor has been studied as a pharmacological target for inflammatory and neuroinflammatory diseases, and preclinical studies have recently provided evidence that P2X7 receptor activation is implicated in pathophysiology of several retinal age-related diseases. These diseases are devastating conditions that have an deep impact on the quality of life of patients and on the health systems of all countries. In this review, we discuss the role of the P2X7 receptor in retinal age-related conditions such as glaucoma, age-related macular degeneration, and diabetic retinopathy. Furthermore, we focus on the pharmacological modulation of the P2X7 receptor that could have a relevant clinical impact to prevent retinal diseases., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Caffeine, one of the most consumed central nervous system (CNS) stimulants, is an antagonist of A 1 and A 2A adenosine receptors. In this study, we investigated the potential protective effects of this methylxanthine in the retinal tissue. We tested caffeine by using in vitro and in vivo paradigms of retinal inflammation. Human retinal pigment epithelial cells (ARPE-19) were exposed to lipopolysaccharide (LPS) with or without caffeine. This latter was able to reduce the inflammatory response in ARPE-19 cells exposed to LPS, attenuating the release of IL-1β, IL-6, and TNF-α and the nuclear translocation of p-NFκB. Additionally, caffeine treatment restored the integrity of the ARPE-19 monolayer assessed by transepithelial electrical resistance (TEER) and the sodium fluorescein permeability test. Finally, the ischemia reperfusion (I/R) injury model was used in C57BL/6J mice to induce retinal inflammation and investigate the effects of caffeine treatment. Mouse eyes were treated topically with caffeine, and a pattern electroretinogram (PERG) was used to assess the retinal ganglion cell (RGC) function; furthermore, we evaluated the levels of IL-6 and BDNF in the retina. Retinal BDNF dropped significantly ( p < 0.05) in the I/R group compared to the control group (normal mice); on the contrary, caffeine treatment maintained physiological levels of BDNF in the retina of I/R eyes. Caffeine was also able to reduce IL-6 mRNA levels in the retina of I/R eyes. In conclusion, these findings suggest that caffeine is a good candidate to counteract inflammation in retinal diseases., 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 Conti, Lazzara, Romano, Platania, Drago and Bucolo.)

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults, showing a high mortality due to metastasis. Although it is considered a rare disease, a growing number of papers have reported altered levels of RNAs (i.e., coding and non-coding RNAs) in cancerous tissues and biological fluids from UM patients. The presence of circulating RNAs, whose dysregulation is associated with UM, paved the way to the possibility of exploiting it for diagnostic and prognostic purposes. However, the biological meaning and the origin of such RNAs in blood and ocular fluids of UM patients remain unexplored. In this review, we report the state of the art of circulating RNAs in UM and debate whether the amount and types of RNAs measured in bodily fluids mirror the RNA alterations from source cancer cells. Based on literature data, extracellular RNAs in UM patients do not represent, with rare exceptions, a snapshot of RNA dysregulations occurring in cancerous tissues, but rather the complex and heterogeneous outcome of a systemic dysfunction, including immune system activity, that modifies the mechanisms of RNA delivery from several cell types.

Age-related macular degeneration (AMD) is a degenerative retinal disease and one of major causes of irreversible vision loss. AMD has been linked to several pathological factors, such as oxidative stress and inflammation. Moreover, Aβ (1-42) oligomers have been found in drusen, the extracellular deposits that accumulate beneath the retinal pigmented epithelium in AMD patients. Hereby, we investigated the hypothesis that treatment with 1,25(OH) 2 D 3 (vitamin D 3 ) and meso-zeaxathin, physiologically present in the eye, would counteract the toxic effects of three different insults on immortalized human retinal pigmented epithelial cells (ARPE-19). Specifically, ARPE-19 cells have been challenged with Aβ (1-42) oligomers, H 2 O 2 , LPS, and TNF-α, respectively. In the present study, we demonstrated that the combination of 1,25(OH) 2 D 3 and meso-zeaxanthin significantly counteracted the cell damage induced by the three insults, at least in these in vitro integrated paradigms of AMD. These results suggest that combination of 1,25(OH) 2 D 3 and meso-zeaxathin could be a useful approach to contrast pathological features of AMD, such as retinal inflammation and oxidative stress., 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 Lazzara, Conti, Platania, Eandi, Drago and Bucolo.)

Age-related disorders, such as Alzheimer's disease (AD) and age-related macular degeneration (AMD) share common features such as amyloid-β (Aβ) protein accumulation. Retinal deposition of Aβ aggregates in AMD patients has suggested a potential link between AMD and AD. In the present study, we analyzed the expression pattern of a focused set of miRNAs, previously found to be involved in both AD and AMD, in the retina of a triple transgenic mouse model of AD (3xTg-AD) at different time-points. Several miRNAs were differentially expressed in the retina of 3xTg-AD mice, compared to the retina of age-matched wild-type (WT) mice. In particular, bioinformatic analysis revealed that miR-155 had a central role in miRNA-gene network stability, regulating several pathways, including apoptotic and inflammatory signaling pathways modulated by TNF-related apoptosis-inducing ligand (TNFSF10). We showed that chronic treatment of 3xTg-AD mice with an anti-TNFSF10 monoclonal antibody was able to inhibit the retinal expression of miR-155, which inversely correlated with the expression of its molecular target SOCS-1. Moreover, the fine-tuned mechanism related to TNFSF10 immunoneutralization was tightly linked to modulation of TNFSF10 itself and its death receptor TNFRSF10B, along with cytokine production by microglia, reactive gliosis, and specific AD-related neuropathological hallmarks (i.e., Aβ deposition and Tau phosphorylation) in the retina of 3xTg-AD mice. In conclusion, immunoneutralization of TNFSF10 significantly preserved the retinal tissue in 3xTg-AD mice, suggesting its potential therapeutic application in retinal degenerative disorders., (© 2021. The Author(s).)

This study aimed to investigate the interactions between fingolimod, a sphingosine 1-phosphate receptor (S1PR) agonist, and melanocortin receptors 1 and 5 (MCR1, MCR5). In particular, we investigated the effects of fingolimod, a drug approved to treat relapsing-remitting multiple sclerosis, on retinal angiogenesis in a mouse model of diabetic retinopathy (DR). We showed, by a molecular modeling approach, that fingolimod can bind with good-predicted affinity to MC1R and MC5R. Thereafter, we investigated the fingolimod actions on retinal MC1Rs/MC5Rs in C57BL/6J mice. Diabetes was induced in C57BL/6J mice through streptozotocin injection. Diabetic and control C57BL/6J mice received fingolimod, by oral route, for 12 weeks and a monthly intravitreally injection of MC1R antagonist (AGRP), MC5R antagonist (PG20N), and the selective S1PR1 antagonist (Ex 26). Diabetic animals treated with fingolimod showed a decrease of retinal vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2), compared to diabetic control group. Fingolimod co-treatment with MC1R and MC5R selective antagonists significantly ( p < 0.05) increased retinal VEGFR1, VEGFR2, and VEGFA levels compared to mice treated with fingolimod alone. Diabetic animals treated with fingolimod plus Ex 26 (S1PR1 selective blocker) had VEGFR1, VEGFR2, and VEGFA levels between diabetic mice group and the group of diabetic mice treated with fingolimod alone. This vascular protective effect of fingolimod, through activation of MC1R and MC5R, was evidenced also by fluorescein angiography in mice. Finally, molecular dynamic simulations showed a strong similarity between fingolimod and the MC1R agonist BMS-470539. In conclusion, the anti-angiogenic activity exerted by fingolimod in DR seems to be mediated not only through S1P1R, but also by melanocortin receptors., 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 Gesualdo, Balta, Platania, Trotta, Herman, Gharbia, Rosu, Petrillo, Giunta, Della Corte, Grieco, Bellavita, Simonelli, D’Amico, Hermenean, Rossi and Bucolo.)

Background: Glaucoma is an optic neuropathy characterized by loss of function and death of retinal ganglion cells (RGCs), leading to irreversible vision loss. Neuroinflammation is recognized as one of the causes of glaucoma, and currently no treatment is addressing this mechanism. We aimed to investigate the anti-inflammatory and neuroprotective effects of 1,25(OH) 2 D 3 (1α,25-dihydroxyvitamin D 3 , calcitriol), in a genetic model of age-related glaucomatous neurodegeneration (DBA/2J mice)., Methods: DBA/2J mice were randomized to 1,25(OH) 2 D 3 or vehicle treatment groups. Pattern electroretinogram, flash electroretinogram, and intraocular pressure were recorded weekly. Immunostaining for RBPMS, Iba-1, and GFAP was carried out on retinal flat mounts to assess retinal ganglion cell density and quantify microglial and astrocyte activation, respectively. Molecular biology analyses were carried out to evaluate retinal expression of pro-inflammatory cytokines, pNFκB-p65, and neuroprotective factors. Investigators that analysed the data were blind to experimental groups, which were unveiled after graph design and statistical analysis, that were carried out with GraphPad Prism. Several statistical tests and approaches were used: the generalized estimated equations (GEE) analysis, t-test, and one-way ANOVA., Results: DBA/2J mice treated with 1,25(OH) 2 D 3 for 5 weeks showed improved PERG and FERG amplitudes and reduced RGCs death, compared to vehicle-treated age-matched controls. 1,25(OH) 2 D 3 treatment decreased microglial and astrocyte activation, as well as expression of inflammatory cytokines and pNF-κB-p65 (p < 0.05). Moreover, 1,25(OH) 2 D 3 -treated DBA/2J mice displayed increased mRNA levels of neuroprotective factors (p < 0.05), such as BDNF., Conclusions: 1,25(OH) 2 D 3 protected RGCs preserving retinal function, reducing inflammatory cytokines, and increasing expression of neuroprotective factors. Therefore, 1,25(OH) 2 D 3 could attenuate the retinal damage in glaucomatous patients and warrants further clinical evaluation for the treatment of optic neuropathies., (© 2021. The Author(s).)

Glucose induces corneal epithelial dysfunctions characterized by delayed wound repair. Nuclear erythroid 2-related factor 2 (Nrf2) mediates cell protection mechanisms even through the Heme oxygenase-1 (HO-1) up-regulation. Here, we synthesized new HO-1 inducers by modifying dimethyl fumarate (DMF) and used docking studies to select VP13/126 as a promising compound with the best binding energy to Kelch-like ECH-associated protein 1 (keap1), which is the the regulator of Nrf2 nuclear translocation. We verified if VP13/126 protects SIRC cells from hyperglycemia compared to DMF. SIRC were cultured in normal (5 mM) or high glucose (25 mM, HG) in presence of DMF (1-25 μM) or VP13/126 (0.1-5 μM) with or without ERK1/2 inhibitor PD98059 (15 μM). VP13/126 was more effective than DMF in the prevention of HG-induced reduction of cell viability and proliferation. Reduction of wound closure induced by HG was similarly counteracted by 1 μM VP13/126 and 10 μM DMF. VP13/126 strongly increased phospho/total ERK1/2 and restored HO-1 protein in HG-treated SIRC; these effects are completely counteracted by PD98059. Moreover, high-content screening analysis showed a higher rate of Nrf2 nuclear translocation induced by VP13/126 than DMF in HG-stimulated SIRC. These data indicate that VP13/126 exerts remarkable pro-survival properties in HG-stimulated SIRC, promoting the Nrf2/HO-1 axis.

Eye drop formulations allowing topical treatment of retinal pathologies have long been sought as alternatives to intravitreal administration. This study aimed to assess whether a novel nanostructured microemulsions system (NaMESys) could be usefully employed to deliver sorafenib to the retina following topical instillation. NaMESys carrying 0.3% sorafenib (NaMESys-SOR) proved to be cytocompatible in vitro on rabbit corneal cells, and well-tolerated following b.i.d. ocular administration to rabbits during a 3-month study. In rats subject to retinal ischemia-reperfusion, NaMESys-SOR significantly inhibited retinal expression of tumor necrosis factor-alpha (TNFα, 20.7%) and inducible nitric oxide synthase (iNos, 87.3%) mRNAs in comparison to controls. Similarly, in streptozotocin-induced diabetic rats, NaMESys-SOR inhibited retinal expression of nuclear factor kappa B (NFκB), TNFα, insulin like growth factor 1 (IGF1), IGF1 receptor (IGF1R), vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2) mRNAs by three-fold on average compared to controls. Furthermore, a reduction in TNFα, VEGFR1 and VEGFR2 protein expression was observed by western blot. Moreover, in mice subject to laser-induced choroidal neovascularization, NaMESys-SOR significantly inhibited neovascular lesions by 54%. In conclusion, NaMESys-SOR was shown to be a well-tolerated ophthalmic formulation able to deliver effective amounts of sorafenib to the retina, reducing proinflammatory and pro-angiogenic mediators in reliable models of proliferative retinopathies. These findings warrant further investigations on the full therapeutic potential of NaMESys-SOR eye drops, aiming to address unmet needs in the pharmacotherapy of retinal neovascular diseases.

In this study we analyzed the expression of circulating miRNAs, in the serum of diabetic retinopathy (DR) patients. Five miRNAs (hsa-miR-195-5p, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-27b-3p and hsa-miR-451a) were validated as biomarkers for stratification of DR stages, from the early non-proliferative (NPDR) to the late proliferative (PDR) phase. Furthermore, circulating levels of these miRNAs correlated with retinal hyper-reflective spots (HRS), assessed by optical coherence tomography (OCT). The number of HRS increased with worsening of DR stages. On the contrary, no significant vascular density differences between NPDR and PDR patients were detected by angio-OCT (OCTA). A post-hoc bioinformatics analysis associated these five miRNAs to target genes belonging to the "Tumor Necrosis Factor alfa signaling" pathway, and several molecules were predicted to modify miRNAs expression. In conclusion, correlation between specific circulating miRNAs and intraretinal hyper-reflective spots was demonstrated, confirming that these miRNAs were validated as prognostic biomarkers, and also as potential pharmacological targets, warranting further clinical evaluation to explore novel therapeutics for diabetic retinopathy., (Copyright © 2021. Published by Elsevier Inc.)

Allosteric drugs are ligands that when bound to an allosteric site modify the conformational state of the pharmacological target, leading then to a modification of functional response upon binding of the endogenous ligand. Pharmacological targets are defined as biological entities, to which a ligand/drug binds and leads to a functional effect. Pharmacological targets can be proteins or nucleic acids. Computational approaches such as molecular dynamics (MD) sped up discovery and identification of allosteric binding sites and allosteric ligands. Classical all-atom and hybrid classical/quantum MD simulations can be generalized as simulation techniques aimed at analysis of atoms and molecular motion. Main limitations of MD simulations are related to high computational costs, that in turn limit the conformational sampling of biological systems. Indeed, other techniques have been developed to overcome limitations of MD, such as enhanced sampling MD simulations. In this chapter, classical MD and enhanced sampling MD simulations will be described, along with their application to drug discovery, with a focus on allosteric drugs.

Novel heme oxygenase-1 (HO-1) inducers based on dimethyl fumarate (DMF) structure are reported in this paper. These compounds are obtained by modification of the DMF backbone. Particularly, maintaining the α, β-unsaturated dicarbonyl function as the central chain crucial for HO-1 induction, different substituted or unsubstituted phenyl rings are introduced by means of an ester or amide linkage. Symmetric and asymmetric derivatives are synthesized. All compounds are tested on a human hepatic stellate cell line LX-2 to assay their capacity for modifying HO-1 expression. Compounds 1b , 1l and 1m stand out for their potency as HO-1 inducers, being 2-3 fold more active than DMF, and for their ability to reverse reactive oxygen species (ROS) production mediated using palmitic acid (PA). These properties, coupled with a low toxicity toward LX-2 cell lines, make these compounds potentially useful for treatment of diseases in which HO-1 overexpression may counteract inflammation, such as hepatic fibrosis. Docking studies show a correlation between predicted binding free energy and experimental HO-1 expression data. These preliminary results may support the development of new approaches in the management of liver fibrosis.

Transforming growth factor β1 (TGFβ1) is a proinflammatory cytokine that has been implicated in the pathogenesis of diabetic retinopathy (DR), particularly in the late phase of disease. The aim of the present study was to validate serum TGFβ1 as a diagnostic and prognostic biomarker of DR stages. Thirty-eight subjects were enrolled and, after diagnosis and evaluation of inclusion and exclusion criteria, were assigned to six groups: (1) healthy age-matched control, (2) diabetic without DR, (3) non-proliferative diabetic retinopathy (NPDR) naïve to treatment, (4) NPDR treated with intravitreal (IVT) aflibercept, (5) proliferative diabetic retinopathy (PDR) naïve to treatment and (6) PDR treated with IVT aflibercept. Serum levels of vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF) and TGFβ1 were measured by means of enzyme-linked immunosorbent assay (ELISA). Foveal macular thickness (FMT) in enrolled subjects was evaluated by means of structural-optical coherence tomography (S-OCT). VEGF-A serum levels decreased in NPDR and PDR patients treated with aflibercept, compared to naïve DR patients. PlGF serum levels were modulated only in aflibercept-treated NPDR patients. Particularly, TGFβ1 serum levels were predictive of disease progression from NPDR to PDR. A Multivariate ANOVA analysis (M-ANOVA) was also carried out to assess the effects of fixed factors on glycated hemoglobin (HbA1c) levels, TGFβ1, and diabetes duration. In conclusion, our data have strengthened the hypothesis that TGFβ1 would be a biomarker and pharmacological target of diabetic retinopathy.

Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1β, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.

The tumor necrosis factor (TNF) superfamily (TNFSF) includes about thirty structurally related receptors (TNFSFRs) and about twenty protein ligands that bind to one or more of these receptors. Receptors of the tumor necrosis factor (TNF) superfamily (TNFSFRs) are pharmacological targets for treatment of inflammatory and autoimmune diseases. Currently, drugs targeting TNFSFR signaling are biological drugs (monoclonal antibodies, decoy receptors) aimed at binding and sequestering TNFSFR ligands. The glucocorticoid-induced tumor necrosis factor receptor-related gene (GITR) signaling is involved in a series of inflammatory and autoimmune diseases, such as rheumatoid arthritis and Crohn's disease. Our study aimed at repurposing FDA approved small molecules as protein-protein disruptors at the GITR ligand (GITRL) trimer, in order to inhibit the binding of GITRL to its receptor (GITR). A structure based molecular modeling approach was carried out to identify, through high throughput virtual screening, GITRL monomer-monomer disruptors. We used a database of ~8,000 FDA approved drugs, and after virtual screening, we focused on two hit compounds, minocycline and oxytetracycline. These two compounds were tested for their capability to modulate IL-17, IL-21 and RORγT expression in T lymphocytes, isolated from wild-type and GITR knock-out (GITR -/- ) mice. Minocycline showed immunomodulatory effects specific to GITR activation and could represent a novel pharmacological tool to treat inflammatory diseases., 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 © 2020 Elsevier Inc. All rights reserved.)

Retinal hypoxia is one of the causative factors of diabetic retinopathy and is also one of the triggers of VEGF release. We hypothesized that specific dysregulated miRNAs in diabetic retinopathy could be linked to hypoxia-induced damage in human retinal endothelial cells (HRECs). We investigated in HRECs the effects of chemical (CoCl 2 ) hypoxia on the expression of HIF-1α, VEGF, PlGF, and of a focused set of miRNAs. We found that miR-20a-5p, miR-20b-5p, miR-27a-3p, miR-27b-3p, miR-206-3p, miR-381-3p correlated also with expression of TGFβ signaling pathway genes in HRECs, challenged with chemical hypoxic stimuli. In conclusion, our data suggest that retinal angiogenesis would be promoted, at least under HIF-1α activation, by upregulation of PlGF and other factors such as miRNAs, VEGFA, and TGFβ1., (Copyright © 2020 Lazzara, Trotta, Platania, D’Amico, Petrillo, Galdiero, Gesualdo, Rossi, Drago and Bucolo.)

Gestational methylazoxymethanol acetate (MAM) treatment produces offspring with adult phenotype relevant to schizophrenia, including positive- and negative-like symptoms, cognitive deficits, dopaminergic dysfunction, structural and functional abnormalities. Here we show that adult rats prenatally treated with MAM at gestational day 17 display significant increase in dopamine D3 receptor (D3) mRNA expression in prefrontal cortex (PFC), hippocampus and nucleus accumbens, accompanied by increased expression of dopamine D2 receptor (D2) mRNA exclusively in the PFC. Furthermore, a significant change in the blood perfusion at the level of the circle of Willis and hippocampus, paralleled by the enlargement of lateral ventricles, was also detected by magnetic resonance imaging (MRI) techniques. Peripubertal treatment with the non-euphoric phytocannabinoid cannabidiol (30 mg/kg) from postnatal day (PND) 19 to PND 39 was able to reverse in MAM exposed rats: i) the up-regulation of the dopamine D3 receptor mRNA (only partially prevented by haloperidol 0.6 mg/kg/day); and ii) the regional blood flow changes in MAM exposed rats. Molecular modelling predicted that cannabidiol could bind preferentially to dopamine D3 receptor, where it may act as a partial agonist according to conformation of ionic-lock, which is highly conserved in GPCRs. In summary, our results demonstrate that the mRNA expression of both dopamine D2 and D3 receptors is altered in the MAM model; however only the transcript levels of D3 are affected by cannabidiol treatment, likely suggesting that this gene might not only contribute to the schizophrenia symptoms but also represent an unexplored target for the antipsychotic activity of cannabidiol., Competing Interests: Declaration of Competing Interest VD is a consultant for GW Pharmaceuticals, UK, and FAI, FP and VD receive funding from GW Pharmaceuticals, UK. CTW is employed by Boehringer Ingelheime Pharma & Co KG which did not influence design, analysis and interpretation of the study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Our study was aimed at assessing the retinal binding of a new synthetic Brilliant Blue G (BBG) derivative (pure benzyl-Brilliant Blue G; PBB) ophthalmic formulation, to improve vitreoretinal surgery procedure. Protein affinity of the new molecule was evaluated in vitro (cell-free assay) and in silico . Furthermore, an ex vivo model of vitreoretinal surgery was developed by using porcine eyes to assess the pharmacological profile of PBB, compared to commercial formulations based on BBG and methyl-BBG (Me-BBG). PBB showed a higher affinity for proteins (p < 0.05), compared to BBG and Me-BBG. In vitro and in silico studies demonstrated that the high selectivity of PBB could be related to high lipophilicity and binding affinity to fibronectin, the main component of the retinal internal limiting membrane (ILM). The PBB staining capabilities were evaluated in porcine eyes in comparison with BBG and Me-BBG. Forty microliters of each formulation were slowly placed over the retinal surface and removed after 30 s. After that, ILM peeling was carried out, and the retina collected. BBG, Me-BBG, and PBB quantification in ILM and retina tissues was carried out by HPLC analysis. PBB levels in the ILM were significantly (p < 0.05) higher compared to BBG and Me-BBG formulations. On the contrary, PBB showed a much lower (p < 0.05) distribution in retina (52 ng/mg tissue) compared to BBG and Me-BBG, in particular PBB levels were significantly (p < 0.05) lower. Therefore, the new synthetic Brilliant Blue derivative (PBB) showed a great ILM selectivity in comparison to underneath retinal layers. In conclusion, these findings had high translational impact with a tangible improving in ex vivo model of retinal surgery, suggesting a future use during surgical practice., (Copyright © 2020 Spadaro, Rao, Lorenti, Romano, Augello, Eandi, Platania, Drago and Bucolo.)

The ELAVL1 (or human antigen R - HuR) RNA binding protein stabilizes the mRNA, with an AU-rich element, of several genes such as growth factors (i.e. VEGF) and inflammatory cytokines (i.e. TNFα). We hereby carried out a virtual screening campaign in order to identify and test novel HuR-mRNA disruptors. Best-scored compounds were tested in an in-vitro model of diabetic retinopathy, namely human retinal endothelial cells (HRECs) challenged with high-glucose levels (25 mM). HuR, VEGF and TNFα protein contents were evaluated by western-blot analysis in total cell lysates. VEGF and TNFα released from HRECs were measured in cell medium by ELISA. We found that two derivatives bearing indole moiety, VP12/14 and VP12/110, modulated HuR expression and decreased VEGF and TNF-α release by HREC exposed to high glucose (HG) levels. VP12/14 and VP12/110 inhibited VEGF and TNF-α release in HRECs challenged with high glucose levels, similarly to dihydrotanshinone (DHTS), a small molecule known to interfere with HuR- TNFα mRNA binding. The present findings demonstrated that VP12/14 and VP12/110 are innovative molecules with anti-inflammatory and anti-angiogenic properties, suggesting their potential use as novel candidates for treatment of diabetic retinopathy., 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 © 2020 Elsevier Inc. All rights reserved.)

Stimulation of retinal photoreceptors with elevated glucose concentration (30 mM) for 96 h, served as diabetic retinopathy in vitro model to study Resolvin D1 (50 nM) effects on neovascularization. VEGF and anti-angiogenic miR-20a-3p, miR-20a-5p, miR-106a-5p, and miR-20b expression was assessed either in photoreceptors exposed to HG or in exosomes released by those cells. High glucose increased VEGF levels and concurrently decreased anti-angiogenic miRNAs content in photoreceptors and exosomes. RvD1 reverted the effects of glucose damage in photoreceptors and exosomal pro-angiogenic potential, tested with the HUVEC angiogenesis assay. By activating FPR2 receptor, RvD1 modulated both the expression of anti-angiogenic miRNA, which decrease VEGF, and the pro-angiogenic potential of exosomes released by primary retinal cells. HUVEC transfection with miR-20a-3p, miR-20a-5p, miR-106a-5p, and miR-20b antagomirs, followed by exposure to exosomes from photoreceptors, confirmed the VEGF-related miRNAs mechanism and the anti-angiogenic effects of RvD1., (Copyright © 2020 Maisto, Trotta, Petrillo, Izzo, Cuomo, Alfano, Hermenean, Barcia, Galdiero, Platania, Bucolo and D’Amico.)

Deletion of dystrobrevin binding protein 1 has been linked to Hermansky-Pudlak syndrome type 7 (HPS-7), a rare disease characterized by oculocutaneous albinism and retinal dysfunction. We studied dysbindin-1 null mutant mice (Dys -/- ) to shed light on retinal neurodevelopment defects in HPS-7. We analyzed the expression of a focused set of miRNAs in retina of wild type (WT), Dys +/- and Dys -/- mice. We also investigated the retinal function of these mice through electroretinography (ERG). We found that miR-101-3p, miR-137, miR-186-5p, miR-326, miR-382-5p and miR-876-5p were up-regulated in Dys -/- mice retina. Dys -/- mice showed significant increased b-wave in ERG, compared to WT mice. Bioinformatic analysis highlighted that dysregulated miRNAs target synaptic plasticity and dopaminergic signaling pathways, affecting retinal functions of Dys -/- mice. Overall, the data indicate potential mechanisms in retinal neurodevelopment of Dys -/- mice, which may have translational significance in HSP-7 patients, both in terms of diagnostic/prognostic biomarkers and novel pharmacological targets.

Myriocin is an antibiotic derived from Mycelia sterilia, and is a potent inhibitor of serine palmitoyltransferase, the enzyme involved in the first step of sphingosine synthesis. Myriocin, inhibiting ceramide synthesis, has a great potential for treatment of diseases characterized by high ceramide levels in affected tissues, such as retinitis pigmentosa (RP). Drug delivery to the retina is a challenging task, which is generally by-passed through intravitreal injection, that represents a risky invasive procedure. We, therefore, developed and characterized an ophthalmic topical nanotechnological formulation based on a nanostructured lipid carrier (NLC) and containing myriocin. The ocular distribution of myriocin in the back of the eye was assessed both in rabbits and mice using LC-MS/MS. Moreover, rabbit retinal sphingolipid and ceramides levels, after myriocin-NLC (Myr-NLC) eye drops treatment, were assessed. The results demonstrated that Myr-NLC formulation is well tolerated and provided effective levels of myriocin in the back of the eye both in rabbits and mice. We found that Myr-NLC eye drops treatment was able to significantly decrease retinal sphingolipid levels. In conclusion, these data suggest that the Myr-NLC ophthalmic formulation is suitable for pharmaceutical development and warrants further clinical evaluation of this eye drops for the treatment of RP.

To investigate the ocular pharmacological profile of hydrocortisone (HC) using in vitro and in vivo models of dry eye disease. Rabbit corneal epithelial cells (SIRCs) were used to assess the effect of HC in two paradigms of corneal damage: hyperosmotic stress and scratch-wound assay. Dry eye was induced in albino rabbits by topical administration of atropine sulfate or by injection of concanavalin A (ConA) into the lacrimal gland. TNFα, TNF-related apoptosis-inducing ligand (TRAIL), IL-1β, and IL-8 were determined by ELISA or western blot in a corneal damage hyperosmotic in vitro model, with or without HC treatment. Inflammatory biomarkers, such as TNFα, IL-8, and MMP-9, were evaluated in tears of rabbit eye injected with ConA and treated with HC. Tear volume and tear film integrity, in both in vivo models, were evaluated by the Schirmer test and tear break-up time (TBUT). Ocular distribution of four formulations containing HC (0.001%, 0.003%, 0.005%, and 0.33%) was performed in the rabbit eye. Aqueous humor samples were collected after 15, 30, 60, and 90 min from instillation and then detected by LC-MS/MS. Hyperosmotic insult significantly activated protein expression of inflammatory biomarkers, which were significantly modulated by HC treatment. HC significantly enhanced the re-epithelialization of scratched SIRCs. Treatment with HC eye drops significantly reduced the tear concentrations of TNF-α, IL-8, and MMP-9 vs. vehicle in the ConA dry eye model. Moreover, HC significantly restored the tear volume and tear film integrity to levels of the control eyes, both in ConA- and atropine-induced dry eye paradigms. Finally, we demonstrated that HC crossed, in a dose-dependent manner, the corneal barrier when the eyes were topically treated with HC formulations (dose range 0.003-0.33%). No trace of HC was detected in the aqueous humor after ocular administration of eye drops containing the lowest dose of the drug (0.001%), indicating that, at this very low concentration, the drug did not pass the corneal barrier avoiding potential side effects such as intraocular pressure rise. Altogether, these data suggest that HC, at very low concentrations, has an important anti-inflammatory effect both in vitro and in vivo dry eye paradigms and a good safety profile., (Copyright © 2019 Bucolo, Fidilio, Fresta, Lazzara, Platania, Cantarella, Di Benedetto, Burgaletto, Bernardini, Piazza, Barabino and Drago.)

Blood retinal barrier (BRB) breakdown is a hallmark of diabetic retinopathy, whose occurrence in early or later phases of the disease has not yet been completely clarified. Recent evidence suggests that hyperglycemia induces activation of the P2X7 receptor (P2X7R) leading to pericyte cell death. We herein investigated the role of P2X7R on retinal endothelial cells viability and expression of tight- and adherens-junctions following high glucose (HG) exposure. We found that HG elicited P2X7R activation and expression and release of the pro-inflammatory cytokine IL-1β in human retinal endothelial cells (HRECs). Furthermore, HG exposure caused a decrease in HRECs viability and a damage of the BRB. JNJ47965567, a P2X7R antagonist, protected HRECs from HG-induced damage (LDH release) and preserved the BRB, as shown by transendothelial electrical resistance and cell junction morphology (ZO-1, claudin-5 and VE-cadherin). Moreover, JNJ47965567 treatment significantly decreased IL-1β expression and release, elicited by HG. These data indicate that P2X7R plays an important role to regulate BRB integrity, in particular the block of this receptor was useful to counteract the damage elicited by HG in HRECs, and warranting further clinical evaluation of P2X7R antagonists for the treatment of diabetic macular edema., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Diabetic retinopathy (DR) is a secondary complication of diabetes. DR can cause irreversible blindness, and its pathogenesis is considered multifactorial. DR can progress from non-proliferative DR to proliferative DR, characterized by retinal neovascularization. The main cause of vision loss in diabetic patients is diabetic macular edema, caused by vessel leakage and blood retinal barrier breakdown. Currently, aflibercept is an anti-VEGF approved for diabetic macular edema. Aflibercept can bind several members of vascular permeability factors, namely VEGF-A, B, and PlGF. We analyzed the aflibercept-PlGF complex at molecular level, through an in silico approach. In order to explore the role of PlGF in DR, we treated primary human retinal endothelial cells (HRECs) and mouse retinal epithelial cells (RPEs) with aflibercept and an anti-PlGF antibody. We explored the hypothesis that aflibercept has anti-inflammatory action through blocking of PlGF signaling and the ERK axis in an in vitro and in vivo model of DR. Both aflibercept and the anti-PlGF antibody exerted protective effects on retinal cells, by inhibition of the ERK pathway. Moreover, aflibercept significantly decreased (p < 0.05) the expression of TNF-α in an in vitro and in vivo model of DR. Therefore, our data suggest that inhibition of PlGF signaling, or a selective blocking, may be useful in the management of early phases of DR when the inflammatory process is largely involved., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background and Purpose: Diabetic retinopathy, a secondary complication of diabetes mellitus, can lead to irreversible vision loss. Currently, no treatment is approved for early phases of diabetic retinopathy. Modifications of the expression pattern of miRNAs could be involved in the early retinal damage of diabetic subjects. Therefore, we aimed at identification of dysregulated miRNAs-mRNA interactions that might be biomarkers and pharmacological targets for diagnosis and treatment of early diabetic retinopathy., Methods: A focused set of miRNAs was predicted through a bioinformatic analysis accessing to Gene Expression Omnibus dataset and enrichment of information approach (GENEMANIA-Cytoscape). Identification of miRNAs-mRNA interactions was carried out with miRNET analysis. Diabetes was induced in C57BL6J mice by streptozotocin and samples analysed at 5 and 10 weeks after diabetes induction. Retinal ultrastructure of diabetic mice was analysed through electron microscopy. We used Real-time PCR, western blot analysis, elisa, and immunohistochemistry to study expression of miRNAs and possible targets of dysregulated miRNAs., Key Results: We found that miR-20a-5p, miR-20a-3p, miR-20b, miR-106a-5p, miR-27a-5p, miR-27b-3p, miR-206-3p, and miR-381-3p were dysregulated in the retina and serum of diabetic mice. VEGF, brain-derived neurotrophic factor (BDNF), PPAR-α, and cAMP response element-binding protein 1 (CREB1) are targets of dysregulated miRNAs, which then modulated protein expression in diabetic retina. We found structural modifications in retinas from diabetic mice., Conclusions and Implications: Serum and retina of diabetic mice express eight dysregulated miRNAs, which modified the expression of VEGF, BDNF, PPAR-α, and CREB1, before vasculopathy in diabetic retinas., (© 2019 The British Pharmacological Society.)

Diabetic retinopathy was included by the World Health Organization in the eye disease priority list. Up to now, only proliferative diabetic retinopathy can be treated with approved drugs, such as intravitreal anti-vascular endothelial growth factor (VEGF) agents or steroids. In this perspective, there is the urgent need to explore novel pharmacological targets for treatment of diabetic retinopathy. Drug discovery todays exploits the noticeable ability of computational systems biology methods to identify novel drug targets in complex pathologies bearing multifactorial etiology and wide and varying symptomatology. This is especially true for diseases, where the identification of specific molecular mechanisms, and thus drug targets, is a challenging, when not impossible, task. Within this framework, we applied a systems biology approach to identify novel drug targets for diabetic retinopathy. The complexity of diabetic retinopathy was investigated through the analysis of transcriptomics data, retrieved from Gene Expression Omnibus Dataset repository (GEO) datasets. Analysis of GEO datasets was carried out with an enrichment-information approach, which gave as output a series of complex gene-pathway and drug-gene networks. Analysis of these networks identified genes and biological pathways related with inflammation, fibrosis and G protein-coupled receptors that are potentially involved in development of the disease. This analysis provided new clues on novel pharmacological targets, useful to treat diabetic retinopathy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Diabetic retinopathy (DR), a secondary complication of diabetes, is a leading cause of irreversible blindness accounting for 5% of world blindness cases in working age. Oxidative stress and inflammation are considered causes of DR. Curcumin, a product with anti-oxidant and anti-inflammatory properties, is currently proposed as oral supplementation therapy for retinal degenerative diseases, including DR. In this study we predicted the pharmacodynamic profile of curcumin through an in silico approach. Furthermore, we tested the anti-oxidant and anti-inflammatory activity of curcumin on human retinal pigmented epithelial cells exposed to oxidative stress, human retinal endothelial and human retinal pericytes (HRPCs) cultured with high glucose. Because currently marketed curcumin nutraceutical products have not been so far evaluated for their ocular bioavailability; we assessed retinal distribution of curcumin, following oral administration, in rabbit eye. Curcumin (10 μM) decreased significantly ( p < 0.01) ROS concentration and TNF-α release in retinal pigmented epithelial cells and retinal endothelial cells, respectively. The same curcumin concentration significantly ( p < 0.01) protected retinal pericytes from high glucose damage as assessed by cell viability and LDH release. Among the tested formulations, only that containing a hydrophilic carrier provided therapeutic levels of curcumin in rabbit retina. In conclusion, our data suggest that curcumin, when properly formulated, may be of value in clinical practice to manage retinal diseases.

Nanostructured lipid carriers (NLCs) loaded with palmitoylethanolamide (PEA) were formulated with the aim to enhance ocular bioavailability of PEA, particularly to the back of the eye. Technological characterization (e.g., size, charge) of NLC loaded with PEA formulation (PEA-NLC) was performed, and NLC morphology was characterized by electron microscopy. Ocular pharmacokinetic study, after topical administration of the formulation, was carried out in rabbit eye. Ultra-high performance liquid chromatography tandem mass spectrometry analysis was carried out to detect PEA levels in ocular tissues. Finally, the ocular tolerability of PEA-NLC formulation was assessed in rabbit eye. The novel formulation significantly increased PEA levels in ocular tissues compared to PEA suspension. Vitreous and retinal levels of PEA were significantly higher in the group treated with PEA-NLC formulation versus PEA suspension (PEA-NLC C max 5919 ± 541 pmol/g and 315 ± 70 pmol/g in vitreous and retina, respectively). The PEA-NLC formulation was characterized by high stability and robust ocular bioavailability. Therefore, this innovative formulation may be useful in clinical practice to manage retinal diseases.

Purpose: To assess the protective effects of ophthalmic formulations based on taurine (TAU) and sodium hyaluronate (SH) in ocular surface., Methods: Rabbit corneal epithelial cells [Statens Seruminstitut Rabbit Cornea (SIRC)] were subjected to oxidative stress (1 mM H 2 O 2 ) and treated with the following formulations: 0.2% SH, 0.4% SH, 0.4% SH +0.5% TAU. Reactive oxygen species (ROS) were evaluated by commercial kit. Dry eye was induced by atropine sulfate and topical treatment was carried out with the following formulations: 0.2% SH, 0.4% SH, 0.4% SH +0.5% TAU. Schirmer's test, tear breakup time (TBUT), and tear osmolarity were evaluated. Furthermore, tear matrix metalloproteinase 9 (MMP-9) expression was assessed by Western blot., Results: TAU significantly (P < 0.05) decreased ROS production in SIRC after oxidative stress. Topical administration of atropine in the rabbit eye significantly (P < 0.05) reduced tear volume and TBUT. Tear osmolarity was also significantly (P < 0.05) modified by atropine treatment. All the altered parameters were significantly (P < 0.05) reversed by 0.5% TAU +0.4% SH treatment; furthermore, this formulation was more effective than SH alone. Moreover, tear levels of MMP-9 were significantly (P < 0.05) lower in the group treated with 0.5% TAU +0.4% SH., Conclusions: Altogether these data suggest that TAU has a relevant antioxidant effect in corneal epithelial cells and prevents the ocular surface damage elicited by atropine. Therefore, our findings suggest that TAU in combination with SH may be useful in clinical practice to manage ocular surface diseases.

Background: Glaucoma is a progressive optic neuropathy characterized by retinal ganglion cell death and alterations of visual field. Elevated intraocular pressure (IOP) is considered the main risk factor of glaucoma, even though other factors cannot be ruled out, such as epigenetic mechanisms., Objective: An overview of the ultimate promising experimental drugs to manage glaucoma has been provided., Results: In particular, we have focused on purinergic ligands, KATP channel activators, gases (nitric oxide, carbon monoxide and hydrogen sulfide), non-glucocorticoid steroidal compounds, neurotrophic factors, PI3K/Akt activators, citicoline, histone deacetylase inhibitors, cannabinoids, dopamine and serotonin receptors ligands, small interference RNA, and Rho kinase inhibitors., Conclusions: The review has been also endowed of a brief chapter on last reports about potential neuroprotective benefits of anti-glaucoma drugs already present in the market., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Dysregulation of the transforming growth factor-β1 (TGF-β1)/selected small mother against decapentaplegic (SMAD) pathway can be implicated in development of age-related macular degeneration (AMD), and the delivery of TGF-β1 could be beneficial for AMD. We developed a new ophthalmic formulation of TGF-β1 assessing the ocular pharmacokinetic profile of TGF-β1 in the rabbit eye. Small unilamellar vesicles (SUV) loaded with TGF-β1 were complemented with Annexin V and Ca 2+ , and the vitreous bioavailability of TGF-β1 was assessed after topical ocular administration by a commercial ELISA kit. We detected high levels of TGF-β1 (C max 114.7 ± 12.40 pg/mL) in the vitreous after 60 min (T max ) from the topical application of the liposomal suspension. Ocular tolerability was also assessed by a modified Draize's test. The new formulation was well tolerated. In conclusion, we demonstrated that the novel formulation was able to deliver remarkable levels of TGF-β1 into the back of the eye after topical administration. Indeed, this TGF-β1 delivery system may be useful in clinical practice to manage ophthalmic conditions such as age-related macular degeneration, skipping invasive intraocular injections., Competing Interests: The authors declare no conflict of interest.

Diabetic retinopathy (DR) is the most frequent complication of diabetes and one of leading causes of blindness worldwide. Early phases of DR are characterized by retinal pericyte loss mainly related to concurrent inflammatory process. Recently, an important link between P2X7 receptor (P2X7R) and inflammation has been demonstrated indicating this receptor as potential pharmacological target in DR. Here we first carried out an in silico molecular modeling study in order to characterize the allosteric pocket in P2X7R, and identify a suitable P2X7R antagonist through molecular docking. JNJ47965567 was identified as the hit compound in docking calculations, as well as for its absorption, distribution, metabolism and excretion (ADME) profile. As an in vitro model of early diabetic retinopathy, human retinal pericytes were exposed to high glucose (25mM, 48h) that caused a significant (p<0.05) release of IL-1β and LDH. The block of P2X7R by JNJ47965567 significantly (p<0.05) reverted the damage elicited by high glucose, detected as IL-1β and LDH release. Overall, our findings suggest that the P2X7R represents an attractive pharmacological target to manage the early phase of diabetic retinopathy, and the compound JNJ47965567 is a good template to discover other P2X7R selective antagonists., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Age related macular degeneration (AMD) is the leading cause of blindness among people aged 50 and over. Retinal deposition of amyloid-β (Aβ) aggregates in AMD patients has suggested a potential link between AMD and Alzheimer's disease (AD). We have evaluated the differential retinal expression profile of miRNAs in a rat model of AMD elicited by Aβ. A serum profile of miRNAs in AMD patients has been also assessed using single TaqMan assay. Analysis of retina from rats intravitreally injected with Aβ revealed that miR-27a, miR-146a, and miR-155 were up-regulated in comparison to control rats. Seven miRNA (miR-9, miR-23a, miR-27a, miR-34a, miR-126, miR-146a, and miR-155) have been found to be dysregulated in serum of AMD patients in comparison to control group. Analysis of pathways has revealed that dysregulated miRNAs, both in the AMD animal model and in AMD patients, can target genes regulating pathways linked to neurodegeneration and inflammation, reinforcing the hypothesis that AMD is a protein misfolding disease similar to AD. In fact, miR-9, miR-23a, miR-27a, miR-34a, miR-146a, miR-155 have been found to be dysregulated both in AMD and AD. In conclusion, we suggest that miR-9, miR-23a, miR-27a, miR-34a, miR-146a, miR-155 represent potential biomarkers and new pharmacological targets for AMD.

Oxidative stress is a hallmark of retinal degenerations such as age-related macular degeneration and diabetic retinopathy. Enhancement of heme oxygenase-1 (HO-1) activity in the retina would exert beneficial effects by protecting cells from oxidative stress, therefore promoting cell survival. Because a crosstalk exists between nitric oxide (NO) and HO-1 in promotion of cell survival under oxidative stress, we designed novel NO-releasing molecules also capable to induce HO-1. Starting from curcumin and caffeic acid phenethyl ester (CAPE), two known HO-1 inducers, the molecules were chemically modified by acylation with 4-bromo-butanoyl chloride and 2-chloro-propanoyl chloride, respectively, and then treated in the dark with AgNO 3 to obtain the nitrate derivatives VP10/12 and VP10/39. Human retinal pigment epithelial cells (ARPE-19) subjected to H 2 O 2 -mediated oxidative stress were treated with the described NO-releasing compounds. VP10/39 showed significant ( p < 0.05) antioxidant and protecting activity against oxidative damage, in comparison to VP10/12, which in turn showed at 100  μ M concentration a slight but significant cell toxicity. Only VP10/39 significantly ( p < 0.05) induced HO-1 in ARPE-19, most likely through covalent bond formation at Cys151 of the Keap1-BTB domain, as revealed from molecular docking analysis. In conclusion, the present data indicate VP10/39 as a promising candidate to protect ARPE-19 cells against oxidative stress.