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.

Integrating network pharmacology: The next-generation approach in ocular drug discovery.

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.

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.

Safety Profile of Lutein- Versus Triamcinolone Acetonide-Based Vitreous Staining.

Purpose: To assess the safety profile of a new lutein-based vitreous dye (LB-VD) formulation compared with various triamcinolone acetonide (TA) formulations with and without subsequent exposure to perfluorodecalin (PFD) in vitro. Methods: Human adult retinal pigment epithelial cells (ARPE-19) were treated with the following formulations: undiluted preserved TA (TA-BA), diluted preserved TA (D-TA-BA), preservative-free TA (TA-PF), and LB-VD. First, cell tolerability was evaluated with MTT, LDH, and ATPlite assays after 1, 5, and 30 minutes of exposure to each tested formulation. Then, cells were sequentially exposed to formulations and PFD. After 24 hours of exposure to PFD, cell tolerability was evaluated through MTT and ATPlite assays. Results: Among the formulations tested, LB-VD showed the highest levels of cell viability, cell metabolism, and cell proliferation and induced the lowest release of LDH, whereas the TA-based formulations demonstrated a cytotoxic effect on ARPE-19 cells in vitro. After subsequent 24-hour exposure to PFD, a greater reduction of cell viability was noted for all the formulations; however, this reduction was not significant only for the combination LB-VD-PFD, which was the best tolerated condition. Conclusions: LB-VD showed a better safety profile compared with all TA-based formulations, even when used in combination with PFD. Translational Relevance: In surgical practice, LB-VD may be preferred to TA-based formulations for vitreous staining in the light of its more favorable safety profile.

Vitamin D3 preserves blood retinal barrier integrity in an in vitro model of diabetic retinopathy.

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 D3 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 D3 attenuates cell damage elicited by HG, maintaining cell viability and reducing the expression of inflammatory cytokines such as IL-1ß and ICAM-1. Furthermore, we showed that vitamin D3 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 D3, particularly as regard as the early phase of diabetic retinopathy, characterized by BRB breakdown and inflammation.

Effects of Vitamin D3 and Meso-Zeaxanthin on Human Retinal Pigmented Epithelial Cells in Three Integrated in vitro Paradigms of Age-Related Macular Degeneration.

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, Aß (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) 2D3 (vitamin D3) 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 Aß (1-42) oligomers, H2O2, LPS, and TNF-α, respectively. In the present study, we demonstrated that the combination of 1,25(OH)2D3 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)2D3 and meso-zeaxathin could be a useful approach to contrast pathological features of AMD, such as retinal inflammation and oxidative stress.

1α,25-dihydroxyvitamin D3 protects retinal ganglion cells in glaucomatous mice.

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)2D3 (1α,25-dihydroxyvitamin D3, calcitriol), in a genetic model of age-related glaucomatous neurodegeneration (DBA/2J mice). METHODS: DBA/2J mice were randomized to 1,25(OH)2D3 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)2D3 for 5 weeks showed improved PERG and FERG amplitudes and reduced RGCs death, compared to vehicle-treated age-matched controls. 1,25(OH)2D3 treatment decreased microglial and astrocyte activation, as well as expression of inflammatory cytokines and pNF-κB-p65 (p < 0.05). Moreover, 1,25(OH)2D3-treated DBA/2J mice displayed increased mRNA levels of neuroprotective factors (p < 0.05), such as BDNF. CONCLUSIONS: 1,25(OH)2D3 protected RGCs preserving retinal function, reducing inflammatory cytokines, and increasing expression of neuroprotective factors. Therefore, 1,25(OH)2D3 could attenuate the retinal damage in glaucomatous patients and warrants further clinical evaluation for the treatment of optic neuropathies.

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.

Diabetes Exacerbates the Intraocular Pressure-Independent Retinal Ganglion Cells Degeneration in the DBA/2J Model of Glaucoma.

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

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.

TGF-ß Serum Levels in Diabetic Retinopathy Patients and the Role of Anti-VEGF Therapy.

Transforming growth factor ß1 (TGFß1) 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 TGFß1 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 TGFß1 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, TGFß1 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, TGFß1, and diabetes duration. In conclusion, our data have strengthened the hypothesis that TGFß1 would be a biomarker and pharmacological target of diabetic retinopathy.

Stabilization of HIF-1α in Human Retinal Endothelial Cells Modulates Expression of miRNAs and Proangiogenic Growth Factors.

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 (CoCl2) 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 TGFß 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 TGFß1.

P2X7 receptor antagonism preserves retinal ganglion cells in glaucomatous mice.

To investigate the role of P2X7 receptor to preserve retinal ganglion cells (RGCs) structure and function in a genetic mouse model (DBA/2J mouse) of age-related glaucomatous neurodegeneration. Chronic treatment with P2X7 receptor antagonist eye drops was carried out in order to assess RGCs function and density by pattern electroretinogram (PERG) and RBPMS immunostaining, respectively. Further, microglia activation was assessed in flat-mounted retina by using Iba-1 immunostaining. Untreated glaucomatous eyes displayed significant microglia activation, alteration of PERG signal, and RGCs loss. In the P2X7 receptor antagonist-treated eyes, the PERG signal was significantly (p < 0.05) improved compared to controls, along with a significant (p < 0.05) reduction in terms of retinal microglial activation, and remarkable preservation of RGCs density. Altogether, these findings demonstrated that topical treatment with a P2X7 receptor antagonist has a neuroprotective effect on RGCs in glaucomatous mice, suggesting an appealing pharmacological approach to prevent retinal degenerative damage in optic neuropathy.

Effects of protein-protein interface disruptors at the ligand of the glucocorticoid-induced tumor necrosis factor receptor-related gene (GITR).

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.

Novel indole derivatives targeting HuR-mRNA complex to counteract high glucose damage in retinal endothelial cells.

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.

Ocular Pharmacological Profile of Hydrocortisone in Dry Eye Disease.

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-1ß, 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.

Blood-retinal barrier protection against high glucose damage: The role of P2X7 receptor.

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-1ß 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-1ß 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.

Aflibercept regulates retinal inflammation elicited by high glucose via the PlGF/ERK pathway.

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.

Novel ophthalmic formulation of myriocin: implications in retinitis pigmentosa.

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.

NAP modulates hyperglycemic-inflammatory event of diabetic retina by counteracting outer blood retinal barrier damage.

Diabetic retinopathy (DR) is a common microvascular complication of diabetes. Prolonged hyperglycemia stimulates inflammatory pathway characterized by the release of some cytokines leading to the impairment of blood retinal barrier (BRB). NAP exerts a protective effect in various eye diseases, including DR. So far, the role of NAP in the modulation of inflammatory event during early phase of this pathology has not been investigated yet. In the current study, we have studied the retinal protective effect of NAP, injected into the eye, in diabetic rats. NAP treatment exerts a dual effect downregulating interleukin (IL)-1ß and its related receptors and upregulating IL-1Ra expression. We have also tested the role of this peptide in human retinal epithelial cells (ARPE19) cultured on a semipermeable support and exposed to hyperglycemic-inflammatory insult, representing a in vitro model of diabetic macular edema, a clinical manifestation of DR. The results have shown that NAP prevents outer BRB impairment by upregulating the tight junctions. In conclusion, deepened characterization of NAP action mechanism on hyperglycemic-inflammatory damage may be useful to develop a new strategy to prevent retinal damage during DR.