The Identification of New Pharmacological Targets for the Treatment of Glaucoma: A Network Pharmacology Approach.

Background: Glaucoma is a progressive optic neuropathy characterized by the neurodegeneration and death of retinal ganglion cells (RGCs), leading to blindness. Current glaucoma interventions reduce intraocular pressure but do not address retinal neurodegeneration. In this effort, to identify new pharmacological targets for glaucoma management, we employed a network pharmacology approach. Methods: We first retrieved transcriptomic data from GEO, an NCBI database, and carried out GEO2R (an interactive web tool aimed at comparing two or more groups of samples in a GEO dataset). The GEO2R statistical analysis aimed at identifying the top differentially expressed genes (DEGs) and used these as input of STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) app within Cytoscape software, which builds networks of proteins starting from input DEGs. Analyses of centrality metrics using Cytoscape were carried out to identify nodes (genes or proteins) involved in network stability. We also employed the web-server software MIRNET 2.0 to build miRNA-target interaction networks for a re-analysis of the GSE105269 dataset, which reports analyses of microRNA expressions. Results: The pharmacological targets, identified in silico through analyses of the centrality metrics carried out with Cytoscape, were rescored based on correlations with entries in the PubMed and clinicaltrials.gov databases. When there was no match (82 out of 135 identified central nodes, in 8 analyzed networks), targets were considered "potential innovative" targets for the treatment of glaucoma, after further validation studies. Conclusions: Several druggable targets, such as GPCRs (e.g., 5-hydroxytryptamine 5A (5-HT5A) and adenosine A2B receptors) and enzymes (e.g., lactate dehydrogenase A or monoamine oxidase B), were found to be rescored as "potential innovative" pharmacological targets for glaucoma treatment.

Anti-angiogenic and antioxidant effects of axitinib in human retinal endothelial cells: implications in diabetic retinopathy.

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.

Ocular pharmacological and biochemical profiles of 6-thioguanine: a drug repurposing study.

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.

From Molecular Biology to Novel Immunotherapies and Nanomedicine in Uveal Melanoma.

Molecular biology studies of uveal melanoma have resulted in the development of novel immunotherapy approaches including tebentafusp-a T cell-redirecting bispecific fusion protein. More biomarkers are currently being studied. As a result, combined immunotherapy is being developed as well as immunotherapy with bifunctional checkpoint inhibitory T cell engagers and natural killer cells. Current trials cover tumor-infiltrating lymphocytes (TIL), vaccination with IKKb-matured dendritic cells, or autologous dendritic cells loaded with autologous tumor RNA. Another potential approach to treat UM could be based on T cell receptor engineering rather than antibody modification. Immune-mobilizing monoclonal T cell receptors (TCR) against cancer, called ImmTAC TM molecules, represent such an approach. Moreover, nanomedicine, especially miRNA approaches, are promising for future trials. Finally, theranostic radiopharmaceuticals enabling diagnosis and therapy with the same molecule bring hope to this research.

Protective effect of pituitary adenylate cyclase activating polypeptide in diabetic keratopathy.

Diabetic keratopathy (DK) is the major complication of the cornea characterizing diabetes-affected patients. This ocular pathology is correlated with the hyperglycemic state leading to delayed corneal wound healing and recurrent corneal ulcers. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread distribution throughout the body, and exerting cytoprotective effects in the neural and non-neuronal parts of the eye, including the cornea. The purpose of the present study was to investigate whether changes in PACAP expression can concur for delayed epithelial wound healing in diabetic cornea and whether the protective effect of the peptide could be mediated through the activation of the EGFR signaling pathway, which has been reported to be impaired in DK. Expression and distribution of PACAP, PAC1R, and EGFR were investigated through immunohistochemistry analysis in the cornea of normal and diabetic rats. The role of the peptide on wound healing during DK was evaluated in an in vitro model represented by rabbit corneal epithelial cells grown in high glucose conditions. Western blotting and immunofluorescence analysis were used to examine the ability of PACAP to induce the activation of the EGFR/ERK1/2 signaling pathway. Our results showed that in diabetic cornea the expression of PACAP, PAC1R, and EGFR is drastically reduced. The treatment with PACAP via PAC1R activation enhanced cell viability and corneal epithelium wound healing in cells grown under high glucose conditions. Furthermore, both EGFR and ERK1/2 signaling was induced upon the peptide treatment. Overall, our results showed the trophic efficiency of PACAP for enhancing the corneal epithelium re-epithelialization suggesting that the peptide could be beneficially valuable as a treatment for DK.

PACAP-ADNP axis prevents outer retinal barrier breakdown and choroidal neovascularization by interfering with VEGF secreted from retinal pigmented epitelium cells.

During diabetic retinopathy (DR) progression, the retina undergoes various metabolic changes, including hypoxia-signalling cascade induction in the cells of retinal pigmented epithelium (RPE). The overexpression of hypoxic inducible factors causes transcription of many target genes including vascular endothelial growth factor (VEGF). The RPE cells form the outer blood retinal barrier (oBRB), a specialized structure that regulates ions and metabolites flux into the retina to maintain a suitable quality of its extracellular microenvironment. VEGF worsens retinal condition since its secretion from the basolateral compartment of RPE cells compromises the barrier's integrity and induces choroidal neovascularization. In this work, we hypothesized that PACAP prevents the damage to oBRB and controls choroidal neovascularization through the induction of ADNP. Firstly, we demonstrated that ADNP is expressed in Streptozotocin (STZ)-induced diabetic animals. To validate our hypothesis, we cultured endothelial cells (H5V) forming vessels-like structures, in a conditioned medium (CM) derived from ARPE-19 cells exposed to hyperglycaemic/hypoxic insult, containing a known VEGF concentration. The involvement of PACAP-ADNP axis on oBRB integrity was evaluated through the measurement of trans-epithelial-electrical resistance and permeability assay performed on ARPE cell monolayer cultured in CM and by analysing the expression of two tight junction forming proteins, ZO1 and occludin. By culturing H5V in CM, we demonstrated that PACAP-ADNP axis counteracted vessels-like structures formation promoted by VEGF. In conclusion, the results suggested a primary role of PACAP/ADNP axis in preventing oBRB damage and in controlling aberrant choroidal neovascularization induced by VEGF secreted from RPE cells exposed to hyperglycaemia/hypoxic insult in DR.

Carnosine Counteracts the Molecular Alterations Aß Oligomers-Induced in Human Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) has been described as a progressive eye disease characterized by irreversible impairment of central vision, and unfortunately, an effective treatment is still not available. It is well-known that amyloid-beta (Aß) peptide is one of the major culprits in causing neurodegeneration in Alzheimer's disease (AD). The extracellular accumulation of this peptide has also been found in drusen which lies under the retinal pigment epithelium (RPE) and represents one of the early signs of AMD pathology. Aß aggregates, especially in the form of oligomers, are able to induce pro-oxidant (oxidative stress) and pro-inflammatory phenomena in RPE cells. ARPE-19 is a spontaneously arising human RPE cell line validated for drug discovery processes in AMD. In the present study, we employed ARPE-19 treated with Aß oligomers, representing an in vitro model of AMD. We used a combination of methods, including ATPlite, quantitative real-time PCR, immunocytochemistry, as well as a fluorescent probe for reactive oxygen species to investigate the molecular alterations induced by Aß oligomers. In particular, we found that Aß exposure decreased the cell viability of ARPE-19 cells which was paralleled by increased inflammation (increased expression of pro-inflammatory mediators) and oxidative stress (increased expression of NADPH oxidase and ROS production) along with the destruction of ZO-1 tight junction protein. Once the damage was clarified, we investigated the therapeutic potential of carnosine, an endogenous dipeptide that is known to be reduced in AMD patients. Our findings demonstrate that carnosine was able to counteract most of the molecular alterations induced by the challenge of ARPE-19 with Aß oligomers. These new findings obtained with ARPE-19 cells challenged with Aß1-42 oligomers, along with the well-demonstrated multimodal mechanism of action of carnosine both in vitro and in vivo, able to prevent and/or counteract the dysfunctions elicited by Aß oligomers, substantiate the neuroprotective potential of this dipeptide in the context of AMD pathology.

Topically Administered NOX4 Inhibitor, GLX7013114, Is Efficacious in Treating the Early Pathological Events of Diabetic Retinopathy.

NADPH oxidases (NOXs) are major players in generating reactive oxygen species (ROS) and are implicated in various neurodegenerative ocular pathologies. The aim of this study was to investigate the role of a NOX4 inhibitor (GLX7013114) in two in vivo, experimental streptozotocin (STZ) paradigms depicting the early events of diabetic retinopathy (DR). Animals in the diabetic treated group received GLX7013114 topically (20 µL/eye, 10 mg/mL, once daily) for 14 days (paradigm A: preventive) and 7 days (paradigm B: treated) at 48 h and 4 weeks after STZ injection, respectively. Several methodologies were used (immunohistochemistry, Western blot, real-time PCR, ELISA, pattern electroretinography [PERG]) to assess the diabetes-induced early events of DR, namely oxidative stress, neurodegeneration, and neuroinflammation, and the effect of GLX7013114 on the diabetic insults. GLX7013114, administered as eye drops (paradigms A and B), was beneficial in treating the oxidative nitrative stress, activation of caspase-3 and micro- and macroglia, and attenuation of neuronal markers. It also attenuated the diabetes-induced increase in vascular endothelial growth factor, Evans blue dye leakage, and proinflammatory cytokine (TNF-α protein, IL-1ß/IL-6 mRNA) levels. PERG amplitude values suggested that GLX7013114 protected retinal ganglion cell function (paradigm B). This study provides new findings regarding the pharmacological profile of the novel NOX4 inhibitor GLX7013114 as a promising therapeutic candidate for the treatment of the early stage of DR. ARTICLE HIGHLIGHTS: NADPH oxidases (NOXs) are implicated in the early pathological events of diabetic retinopathy (DR). The NOX4 inhibitor GLX7013114, topically administered, reduced oxidative damage and apoptosis in the rat streptozotocin model of DR. GLX7013114 protected retinal neurons and retinal ganglion cell function and reduced the expression of pro-inflammatory cytokines in the diabetic retina. GLX7013114 diminished the diabetes-induced increase in vascular endothelial growth factor levels and Evans blue dye leakage in retinal tissue. GLX7013114 exhibits neuroprotective, anti-inflammatory, and vasculoprotective properties that suggest it may have a role as a putative therapeutic for the early events of DR.

Purinergic ionotropic P2X7 and metabotropic glutamate mGlu5 receptors crosstalk influences pro-inflammatory conditions in microglia.

Microglia represent the resident immune system in the brain. They mediate neuroinflammatory processes and have been described as important regulators of homeostasis in the central nervous system (CNS). Among several players and mechanisms contributing to microglial function in inflammation, ATP and glutamate have been shown to be involved in microgliosis. In this study, we focused on receptor subtypes that respond to these neurotransmitters, purinergic ionotropic P2X7 receptor and metabotropic glutamate mGlu5 receptor. We found that both receptors are functionally expressed in a murine microglia cell line, BV2 cells, and we performed patch-clamp experiments to measure purinergic ionotropic P2X7 receptor ion flux in control condition and after metabotropic glutamate mGlu5 receptor activation. The selective purinergic ionotropic P2X7 receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP, 100 µM), elicited a robust current that was prevented by the selective purinergic ionotropic P2X7 receptor antagonist A438079 (10 µM). When BV2 cells were acutely stimulated with the selective metabotropic glutamate mGlu5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 200 µM), purinergic ionotropic P2X7 receptor current was increased. This positive modulation was prevented by the selective metabotropic glutamate mGlu5 receptor antagonist 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP, 1 µM). Moreover, nitric oxide synthesis elicited by purinergic ionotropic P2X7 receptor activation was enhanced by metabotropic glutamate mGlu5 receptor co-stimulation. Taken together, our results suggest an important crosstalk between ATP and glutamate in inflammation. Pro-inflammatory effects mediated by purinergic ionotropic P2X7 receptor might be exacerbated by simultaneous exposure of microglia to ATP and glutamate, suggesting new pharmacological targets to modulate neuroinflammation.

The P2X7 receptor as a new pharmacological target for retinal diseases.

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.

Activity-Dependent Neuroprotective Protein (ADNP)-Derived Peptide (NAP) Counteracts UV-B Radiation-Induced ROS Formation in Corneal Epithelium.

The corneal epithelium, the outermost layer of the cornea, acts as a dynamic barrier preventing access to harmful agents into the intraocular space. It is subjected daily to different insults, and ultraviolet B (UV-B) irradiation represents one of the main causes of injury. In our previous study, we demonstrated the beneficial effects of pituitary adenylate cyclase-activating polypeptide (PACAP) against UV-B radiation damage in the human corneal endothelium. Some of its effects are mediated through the activation of the intracellular factor, known as the activity-dependent protein (ADNP). In the present paper, we have investigated the role of ADNP and the small peptide derived from ADNP, known as NAP, in the corneal epithelium. Here, we have demonstrated, for the first time, ADNP expression in human and rabbit corneal epithelium as well as its protective effect by treating the corneal epithelial cells exposed to UV-B radiations with NAP. Our results showed that NAP treatment prevents ROS formation by reducing UV-B-irradiation-induced apoptotic cell death and JNK signalling pathway activation. Further investigations are needed to deeply investigate the possible therapeutic use of NAP to counteract corneal UV-B damage.

Do Extracellular RNAs Provide Insight into Uveal Melanoma Biology?

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.

Targeting the miRNA-155/TNFSF10 network restrains inflammatory response in the retina in a mouse model of Alzheimer's disease.

Age-related disorders, such as Alzheimer's disease (AD) and age-related macular degeneration (AMD) share common features such as amyloid-ß (Aß) protein accumulation. Retinal deposition of Aß 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., Aß 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.

New Therapeutic Perspectives in the Treatment of Uveal Melanoma: A Systematic Review.

Uveal melanoma (UM) is a rare disease, but the most common primary intraocular cancer, mostly localized in the choroid. Currently, the first-line treatment options for UM are radiation therapy, resection, and enucleation. However, although these treatments could potentially be curative, half of all patients will develop metastatic disease, whose prognosis is still poor. Indeed, effective therapeutic options for patients with advanced or metastatic disease are still lacking. Recently, the development of new treatment modalities with a lower incidence of adverse events, a better disease control rate, and new therapeutic approaches, have merged as new potential and promising therapeutic strategies. Additionally, several clinical trials are ongoing to find new therapeutic options, mainly for those with metastatic disease. Many interventions are still in the preliminary phases of clinical development, being investigated in phase I trial or phase I/II. The success of these trials could be crucial for changing the prognosis of patients with advanced/metastatic UM. In this systematic review, we analyzed all emerging and available literature on the new perspectives in the treatment of UM and patient outcomes; furthermore, their current limitations and more common adverse events are summarized.

Brimonidine is Neuroprotective in Animal Paradigm of Retinal Ganglion Cell Damage.

To investigate the neuroprotective effect of brimonidine after retinal ischemia damage on mouse eye. Glaucoma is an optic neuropathy characterized by retinal ganglion cells (RGCs) death, irreversible peripheral and central visual field loss, and high intraocular pressure. Ischemia reperfusion (I/R) injury model was used in C57BL/6J mice to mimic conditions of glaucomatous neurodegeneration. Mouse eyes were treated topically with brimonidine and pattern electroretinogram were used to assess the retinal ganglion cells (RGCs) function. A wide range of inflammatory markers, as well as anti-inflammatory and neurotrophic molecules, were investigated to figure out the potential protective effects of brimonidine in mouse retina. In particular, brain-derived neurotrophic factor (BDNF), IL-6, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor DR-5, TNF-α, GFAP, Iba-1, NOS, IL-1ß and IL-10 were assessed in mouse retina that underwent to I/R insult with or without brimonidine treatment. Brimonidine provided remarkable RGCs protection in our paradigm. PERG amplitude values were significantly (p < 0.05) higher in brimonidine-treated eyes in comparison to I/R retinas. Retinal BDNF mRNA levels in the I/R group dropped significantly (p < 0.05) compared to the control group (normal mice); brimonidine treatment counteracted the downregulation of retinal BDNF mRNA in I/R eyes. Retinal inflammatory markers increased significantly (p < 0.05) in the I/R group and brimonidine treatment was able to revert that. The anti-inflammatory IL-10 decreased significantly (p < 0.05) after retinal I/R insult and increased significantly (p < 0.05) in the group treated with brimonidine. In conclusion, brimonidine was effective in preventing loss of function of RGCs and in regulating inflammatory biomarkers elicited by retinal I/R injury.

Epiretinal Membrane Vitrectomy With and Without Intraoperative Intravitreal Dexamethasone Implant: A Systematic Review With Meta-Analysis.

Purpose: To evaluate the efficacy of vitrectomy combined with intravitreal dexamethasone implant vs. vitrectomy without the implant in patients with epiretinal membrane (ERM) by conducting a systematic review and meta-analysis. Methods: Studies that compared ERM vitrectomy with and without intraoperative dexamethasone implant with a follow-up ≥3 months were included. The primary outcome was mean best corrected visual acuity (BCVA) change between eyes undergoing ERM vitrectomy combined with dexamethasone implant (DEX group) and eyes undergoing ERM vitrectomy alone (control group) at 3 months. Secondary outcomes included mean BCVA change at 6 months and mean optical coherence tomography central macular thickness (CMT) change at both 3-months and 6-months follow-up. Mean differences (MDs) with their 95% confidence interval (95%CI) were calculated. Meta-analyses were based either on random effect model or fixed effect model according to heterogeneity. Results: Four studies were included. At 3 months, ERM vitrectomy combined with dexamethasone implant yielded a greater visual gain compared to vitrectomy alone (MD = 9.7; 95%CI = 2.6-16.8; p = 0.01). However, significant heterogeneity was found. A sensitivity analysis excluding the only retrospective non-randomized study confirmed a greater visual gain in the DEX group (MD = 7.1; 95%CI = 2.7-11.6; p < 0.01), with no heterogeneity. At 6 months, a non-significant but borderline difference in visual gain was shown between in the two groups (MD = 5.1; 95%CI = -0.3-10.5; p = 0.06), with no heterogeneity. Three-month analysis of CMT revealed a greater reduction in the DEX group (MD = -80.2; 95%CI =-149.1-11.2; p = 0.02), but with significant heterogeneity. A sensitivity analysis excluding the only retrospective non-randomized study allowed to reduce heterogeneity, but no difference in 3-months CMT change was found between the two groups (MD = -50.0; 95%CI = -106.2-6.2; p = 0.08). At 6 months, no difference in CMT change was shown between the two groups (MD = -48.5; 95%CI = -120.5-23.5; p = 0.19), with significant heterogeneity. Conclusions: Intraoperative dexamethasone implant in eyes undergoing vitrectomy for ERM provided a better visual outcome at 3 months compared to ERM vitrectomy without the implant, with limited evidence of better anatomic outcome as well. Further studies are needed to ascertain whether dexamethasone implant would ensure a significant long-term visual benefit as a result of a faster reduction of macular thickening.

Assessment of a New Nanostructured Microemulsion System for Ocular Delivery of Sorafenib to Posterior Segment of the Eye.

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.

Circulating miRNAs in diabetic retinopathy patients: Prognostic markers or pharmacological targets?

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.

Caffeine Protects Against Retinal Inflammation.

Caffeine, one of the most consumed central nervous system (CNS) stimulants, is an antagonist of A1 and A2A 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-1ß, 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.