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.

Multimodal Assessment of the Prognostic Role of Ectopic Inner Foveal Layers on Epiretinal Membrane Surgery.

BACKGROUND: To perform a multimodal assessment of the ectopic inner foveal layers' (EIFL) prognostic role on idiopathic epiretinal membrane (ERM) surgery. METHODS: We retrospectively followed-up for 12 months 27 patients who underwent ERM surgery and stratified them based on EIFL presence (group 1) or absence (group 2) at baseline. Central Retinal Thickness (CRT) and best-corrected visual acuity (BCVA) were compared pre- and post-operatively at 1, 4 and 12 months, whereas fixation stability (FS), macular sensitivity (MS) and multifocal electroretinogram (mfERG) responses were confronted at baseline and 12 months. RESULTS: In group 1, BCVA improved at 4 and 12 months (MD = 0.14 (SE = 0.04); MD = 0.13 (SE = 0.05), respectively) as well as in group 2 (MD = 0.31 (SE = 0.07); MD = 0.41 (SE = 0.08), respectively). CRT did not change in group 1, whereas it decreased in group 2 at 4 and 12 months (MD = -73.13; SE = 23.56; MD = -76.20; SE = 23.56). MS showed no changes in both groups after surgery. FS did not change in group 1, whereas group 2 improved FS 2° (+8.91 ± 13.97) and FS 4° (+4.33 ± 3.84). MfERG P1 wave did not change in group 1, while in group 2 αP1-2, αP1-3 and αP1-4 improved postoperatively (27.97 ± 27.62; 12.51 ± 17.36; 10.49 ± 17.19, respectively). CONCLUSIONS: Multimodal assessment confirmed that EIFL negatively affected ERM surgery outcomes.

Oral Administration of Vitamin D3 Prevents Corneal Damage in a Knock-Out Mouse Model of Sjögren's Syndrome.

BACKGROUND: Vitamin D deficiency has been associated with dry eye development during Sjögren's syndrome (SS). Here, we investigated whether repeated oral vitamin D3 supplementation could prevent the corneal epithelium damage in an SS mouse model. METHODS: 30 female mouse knock-out for the thrombospondin 1 gene were randomized (six per group) in untreated mice euthanized at 6 weeks as negative control (C-) or at 12 weeks as the positive control for dry eye (C+). Other mice were sacrificed after 6 weeks of oral vitamin D3 supplementation in the drinking water (1000, 8000, and 20,000 IU/kg/week, respectively). RESULTS: The C+ mice showed alterations in their corneal epithelial morphologies and thicknesses (p < 0.01 vs. C-), while the mice receiving 8000 (M) and 20,000 (H) IU/kg/week of vitamin D3 showed preservation of the corneal epithelium morphology and thickness (p < 0.01 vs. C+). Moreover, while the C+ mice exhibited high levels and activity of corneal tumor necrosis factor alpha converting enzyme (TACE), neovascularization and fibrosis markers; these were all reduced in the M and H mice. CONCLUSIONS: Oral vitamin D3 supplementation appeared to counteract the negative effect of TACE on corneal epithelium in a mouse model of SS-associated dry eye.

Effects of the Calix[4]arene Derivative Compound OTX008 on High Glucose-Stimulated ARPE-19 Cells: Focus on Galectin-1/TGF-ß/EMT Pathway.

Diabetic retinopathy (DR) is a neurovascular disease characterized by the reduction of retina integrity and functionality, as a consequence of retinal pigment epithelial cell fibrosis. Although galectin-1 (a glycan-binding protein) has been associated with dysregulated retinal angiogenesis, no evidence has been reported about galectin-1 roles in DR-induced fibrosis. ARPE-19 cells were cultured in normal (5 mM) or high glucose (35 mM) for 3 days, then exposed to the selective galectin-1 inhibitor OTX008 (2.5-5-10 µM) for 6 days. The determination of cell viability and ROS content along with the analysis of specific proteins (by immunocytochemistry, Western blotting, and ELISA) or mRNAs (by real time-PCR) were performed. OTX008 5 µM and 10 µM improved cell viability and markedly reduced galectin-1 protein expression in cells exposed to high glucose. This was paralleled by a down-regulation of the TGF-ß/, NF-kB p65 levels, and ROS content. Moreover, epithelial-mesenchymal transition markers were reduced by OTX008 5 µM and 10 µM. The inhibition of galectin-1 by OTX008 in DR may preserve retinal pigment epithelial cell integrity and functionality by reducing their pro-fibrotic phenotype and epithelial-mesenchymal transition phenomenon induced by diabetes.

Sex and Age-Related Differences in Neuroinflammation and Apoptosis in Balb/c Mice Retina Involve Resolvin D1.

(1) Background: The pro-resolving lipid mediator Resolvin D1 (RvD1) has already shown protective effects in animal models of diabetic retinopathy. This study aimed to investigate the retinal levels of RvD1 in aged (24 months) and younger (3 months) Balb/c mice, along with the activation of macro- and microglia, apoptosis, and neuroinflammation. (2) Methods: Retinas from male and female mice were used for immunohistochemistry, immunofluorescence, transmission electron microscopy, Western blotting, and enzyme-linked immunosorbent assays. (3) Results: Endogenous retinal levels of RvD1 were reduced in aged mice. While RvD1 levels were similar in younger males and females, they were markedly decreased in aged males but less reduced in aged females. Both aged males and females showed a significant increase in retinal microglia activation compared to younger mice, with a more marked reactivity in aged males than in aged females. The same trend was shown by astrocyte activation, neuroinflammation, apoptosis, and nitrosative stress, in line with the microglia and Müller cell hypertrophy evidenced in aged retinas by electron microscopy. (4) Conclusions: Aged mice had sex-related differences in neuroinflammation and apoptosis and low retinal levels of endogenous RvD1.

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.

Two-year macular volume assessment in multiple sclerosis patients treated with fingolimod.

BACKGROUND: Fingolimod (FNG) is associated with the development of symptomatic macular edema (ME) in a small subset of multiple sclerosis (MS) patients. By using spectral domain optical coherence tomography (SD-OCT), an increase in the total macular volume (TMV) was rarely detected during the first months of treatment. OBJECTIVES: The objective of this study is to assess whether FNG treatment leads to long-term macular changes in a real-life setting. METHODS: Sixty RRMS patients starting FNG, according to therapeutic indication, were enrolled at three Italian MS centers and followed for 2 years. RESULTS: The mean TMV did not change between baseline and the follow-up. No patients experienced visual acuity drop during the follow-up. CONCLUSIONS: Initiation of FNG in MS is associated with a modest, not significant, increase in macular volume followed by no further significant changes over 2 years, highlighting the good safety profile of such treatment in MS.

Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina.

Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

Retinal and circulating miRNA expression patterns in diabetic retinopathy: An in silico and in vivo approach.

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.

Targeting and silencing of rhodopsin by ectopic expression of the transcription factor KLF15.

The genome-wide activity of transcription factors (TFs) on multiple regulatory elements precludes their use as gene-specific regulators. Here we show that ectopic expression of a TF in a cell-specific context can be used to silence the expression of a specific gene as a therapeutic approach to regulate gene expression in human disease. We selected the TF Krüppel-like factor 15 (KLF15) based on its putative ability to recognize a specific DNA sequence motif present in the rhodopsin (RHO) promoter and its lack of expression in terminally differentiated rod photoreceptors (the RHO-expressing cells). Adeno-associated virus (AAV) vector-mediated ectopic expression of KLF15 in rod photoreceptors of pigs enables Rho silencing with limited genome-wide transcriptional perturbations. Suppression of a RHO mutant allele by KLF15 corrects the phenotype of a mouse model of retinitis pigmentosa with no observed toxicity. Cell-specific-context conditioning of TF activity may prove a novel mode for somatic gene-targeted manipulation.

Macular hole closure patterns associated with different internal limiting membrane flap techniques.

PURPOSE: To compare the anatomic and functional outcome of two variants of the inverted internal limiting membrane (I-ILM) flap technique for idiopathic macular holes (IMH) larger than 400 µm. METHODS: Twenty-seven consecutive patients undergoing PPV for IMH were randomly assigned to different variants of I-ILM technique: the Cover group included 14 patients in which the I-ILM was folded upside-down over the MH as a single layer while the Fill group enrolled 13 patients in which the I-ILM was folded within the MH in multiple layers. RESULTS: MH closed in 12/14 Cover and in 13/13 Fill eyes (84.6 vs. 100%, p = 0.14; n.s.). Vision at 1 month was Snellen 0.44 ± 0.17 vs. 0.28 ± 0.21 (p = 0.05) and 0.48 ± 0.20 vs. 0.37 ± 0.25 (n.s.) at 3 months. IS/OS line interruption width was 463 ± 385 vs. 602 ± 210 µm, respectively, at 1 month (n.s.) and 602 ± 210 vs. 563 ± 209 µm at 3 months (n.s.). The Cover group showed outer retina cystic changes more often (p < 0.01). MH over 700 µm closed in 0/2 and in 2/2 cases, respectively, in the Cover and Fill groups (0.045). CONCLUSIONS: Cover and Fill I-ILM techniques allowed similar closure rates and post-operative vision at 3 months. The Cover group showed better anatomical restoration and vision at 1 month while the Fill technique might be more efficient in closing larger MHs.

Rhodopsin targeted transcriptional silencing by DNA-binding.

Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.