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Purpose Age-related macular degeneration (AMD) is a leading cause of irreversible blindness. We wished to elaborate mechanisms that regulate RPE-choroidal microenvironment in AMD. We hypothesize that retinal pigment epithelial cells (RPE) produce interleukin 33 (IL-33) and regulate choroidal stromal fibroblasts and mast cell activation and angiogenesis in an ST2-dependent manner. Through such mechanisms, change in choroidal architecture may contribute to AMD phenotypes observed clinically. Methods Upon treatment, RPE cells, human choroidal fibroblasts and bone-marrow-derived mast cells (BMMC) were assayed by RT- PCR, Western Blot and ELISA. Choroidal sprouting assay and laser-induced choroidal neovascularization (CNV) were used as models of ocular angiogenesis. Results TLR-stimulation of RPE significantly up-regulated IL-33 expression. ST2+ BMMC generated a spectrum of inflammatory cytokines when cultured with IL-33 rich RPE supernatant. Pretreatment with IL-33 antagonist markedly inhibited the ability of BMMC to produce inflammatory mediators. Importantly, activation of inflammatory cascade upon RPE supernatant treatment was abrogated in ST2-/- BMMC. In a wound-healing assay, recombinant IL-33 treatment of human choroidal fibroblasts impaired their ability to migrate and contract collagen gel. Furthermore, IL-33 treatment promoted vascular choroidal sprouting in WT and IL33-/- explants. CNV was also regulated by exogenous and endogenous IL-33 in WT and IL33-/- mice respectively. Conclusions Our data illuminate an endogenous IL-33/ ST2 pathway between RPE function and choroidal stroma, influencing tissue remodeling and regulating angiogenesis. Our findings support IL-33/ ST2 axis as a therapeutic target in AMD. [ABSTRACT FROM AUTHOR]

Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor-associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants of IRAK3 , which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD. Irak3 -knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)-expressing human IRAK3 rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in Irak3 -knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.

The present study concerns the preparation of hybrid nanostructures composed of carbon dots (CDs) synthesized in our lab and a double-hydrophilic poly(2-dimethylaminoethyl methacrylate- co -oligo(ethylene glycol) methyl ether methacrylate) (P(DMAEMA- co -OEGMA)) random co polymer through electrostatic interactions between the negatively charged CDs and the positively charged DMAEMA segments of the co polymer. The synthesis of P(DMAEMA- co -OEGMA) co polymer was conducted through RAFT polymerization. Furthermore, the co polymer was converted into a strong cationic random polyelectrolyte through quaternization of the amine groups of DMAEMA segments with methyl iodide (CH 3 I), and it was subsequently utilized for the complexation with the carbon dots. The molecular, physicochemical, and photophysical characterization of the aqueous solution of the co polymers and their hybrid nanoparticles was conducted using dynamic and electrophoretic light scattering (DLS, ELS) and spectroscopic techniques, such as UV-Vis, fluorescence (FS), and FT-IR spectroscopy. In addition, studies of their aqueous solution using DLS and ELS showed their responsiveness to external stimuli (pH, temperature, ionic strength). Finally, the interaction of selected hybrid nanoparticles with iron (III) ions was confirmed through FS spectroscopy, demonstrating their potential application for heavy metal ions sensing.

Background/objectives: Timely assessment and treatment of patients with neovascular AMD (nAMD) are crucial to preservation of vision. Loss to follow up (LTFU) in these patients is a problem but this has not been systematically investigated., Subjects/methods: A retrospective review of electronic medical records of patients with nAMD first treated with anti-VEGF therapy from 1st Jan 2014 to 31st Dec 2018, was conducted in January 2021. Any patient not seen for more than 12 months was classed as no longer attending., Results: Of the 1328 patients who attended between 2014 and 2018, 348 had failed to attend and were eligible for inclusion in this study. Reasons noted for discontinuation of care: discharged by clinician (33.3%), died (20.7%), moved to another unit outside of area (17.5%), stopped attending due to ill-health (13.5%), discharged due to failure to attend (5.6%) and patient choice to no longer attend (4.6%). There were 16 (4.6%) who did not receive any further appointments despite clinician request for follow-up. After 5 years, 50.5% of patients were no longer attending for treatment. Age was a factor in failure to attend, with 7 out of 12 patients aged >100 years no longer being followed up, compared to 1 out of 11 of 50-59 year-olds., Conclusions: When analysing visual outcomes in an AMD service it is important to characterise the patients who are lost to follow up. The outcomes for this group may be avoidably poor and understanding the factors influencing LTFU rate is crucial to addressing shortcomings in a hospital AMD service., (© 2023. The Author(s).)

Age-related macular degeneration (AMD), a degenerative disease affecting the retinal pigment epithelium (RPE) and photoreceptors in the macula, is the leading cause of central blindness in the elderly. AMD progresses to advanced stages of the disease, atrophic AMD (aAMD), or in 15% of cases "wet" or neovascular AMD (nAMD), associated with substantial vision loss. Whilst there has been advancement in therapies treating nAMD, to date, there are no licenced effective treatments for the 85% affected by aAMD, with disease managed by changes to diet, vitamin supplements, and regular monitoring. AMD has a complex pathogenesis, involving highly integrated and common age-related disease pathways, including dysregulated complement/inflammation, impaired autophagy, and oxidative stress. The intricacy of AMD pathogenesis makes therapeutic development challenging and identifying a target that combats the converging disease pathways is essential to provide a globally effective treatment. Interleukin-33 is a cytokine, classically known for the proinflammatory role it plays in allergic disease. Recent evidence across degenerative and inflammatory disease conditions reveals a diverse immune-modulatory role for IL-33, with promising therapeutic potential. Here, we will review IL-33 function in disease and discuss the future potential for this homeostatic cytokine in treating AMD., (© 2021. The Author(s).)

Purpose: Studies examining association of estrogen receptor alpha (ERα) polymorphisms with early puberty are scarce and results are controversial; data in Caucasian girls are lacking. Main objective was to determine association of Xbal and Pvull polymorphisms of ERα gene in Greek girls with precocious/early puberty METHODS: We studied 107 girls with idiopathic precocious/early puberty and 81 young women with pubertal maturation within normal age (controls). Pubertal stage, height SDS (HSDS), and BMI z-score were determined in patients. In controls, height was measured and menarcheal age was self-reported. All participants in the study were genotyped for XbaI and PvuII polymorphisms of the ERα gene., Results: There was no significant difference in XbaI and PvuII polymorphisms between patients and controls. Homozygous, xx and pp, girls had an earlier onset of puberty, although non-significant, than heterozygous or with no polymorphisms p = 0.9; in girls with pubertal onset <7 years, the association tended to become significant, p = 0.09. Girls with xxpp genotype were significantly taller, HSDS 1.63, p = 0.014. In controls, homozygosity for Xbal (xx) and PvuII (pp) was associated with significantly earlier menarche than in women with no polymorphism, p = 0.013 and p = 0.026, respectively, and xxpp genotype was associated with taller adult height, p = 0.017., Conclusion: XbaI and PvuII polymorphisms are not related to idiopathic precocious/early puberty. Early pubertal girls homozygous for both polymorphisms presented earlier onset of puberty, although statistically non-significant, and taller height than girls heterozygous or without these polymorphisms. Homozygosity for both polymorphisms is associated with earlier menarche and taller adult height.

It remains unresolved how retinal pigment epithelial cell metabolism is regulated following immune activation to maintain retinal homeostasis and retinal function. We exposed retinal pigment epithelium (RPE) to several stress signals, particularly Toll-like receptor stimulation, and uncovered an ability of RPE to adapt their metabolic preference on aerobic glycolysis or oxidative glucose metabolism in response to different immune stimuli. We have identified interleukin-33 (IL-33) as a key metabolic checkpoint that antagonizes the Warburg effect to ensure the functional stability of the RPE. The identification of IL-33 as a key regulator of mitochondrial metabolism suggests roles for the cytokine that go beyond its extracellular "alarmin" activities. IL-33 exerts control over mitochondrial respiration in RPE by facilitating oxidative pyruvate catabolism. We have also revealed that in the absence of IL-33, mitochondrial function declined and resultant bioenergetic switching was aligned with altered mitochondrial morphology. Our data not only shed new light on the molecular pathway of activation of mitochondrial respiration in RPE in response to immune stressors but also uncover a potentially novel role of nuclear intrinsic IL-33 as a metabolic checkpoint regulator.

The leading cause of central vision loss, age-related macular degeneration (AMD), is a degenerative disorder characterized by atrophy of retinal pigment epithelium (RPE) and photoreceptors. For 15% of cases, neovascularization occurs, leading to acute vision loss if left untreated. For the remaining patients, there are currently no treatment options and preventing progressive RPE atrophy remains the main therapeutic goal. Previously, we have shown treatment with interleukin-33 can reduce choroidal neovascularization and attenuate tissue remodelling. Here, we investigate IL-33 delivery in aged, high-fat diet (HFD) fed mice on a wildtype and complement factor H heterozygous knockout background. We characterize the non-toxic effect following intravitreal injection of IL-33 and further demonstrate protective effects against RPE cell death with evidence of maintaining metabolic retinal homeostasis of Cfh+/-~HFD mice. Our results further support the potential utility of IL-33 to prevent AMD progression., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Diabetic retinopathy (DR) is one of the most severe clinical manifestations of diabetes mellitus and a major cause of blindness. DR is principally a microvascular disease, although the pathogenesis also involves metabolic reactive intermediates which induce neuronal and glial activation resulting in disruption of the neurovascular unit and regulation of the microvasculature. However, the impact of neural/glial activation in DR remains controversial, notwithstanding our understanding as to when neural/glial activation occurs in the course of disease. The objective of this study was to determine a potential protective role of neuropeptide Y (NPY) using an established model of DR permissive to N-methyl-D-aspartate (NMDA)-induced excitotoxic apoptosis of retinal ganglion cells (RGC) and vascular endothelial growth factor (VEGF)-induced vascular leakage. In vitro evaluation using primary retinal endothelial cells demonstrates that NPY promotes vascular integrity, demonstrated by maintained tight junction protein expression and reduced permeability in response to VEGF treatment. Furthermore, ex vivo assessment of retinal tissue explants shows that NPY can protect RGC from excitotoxic-induced apoptosis. In vivo clinical imaging and ex vivo tissue analysis in the diabetic model permitted assessment of NPY treatment in relation to neural and endothelial changes. The neuroprotective effects of NPY were confirmed by attenuating NMDA-induced retinal neural apoptosis and able to maintain inner retinal vascular integrity. These findings could have important clinical implications and offer novel therapeutic approaches for the treatment in the early stages of DR., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Age-related macular degeneration (AMD) is the leading cause of central retinal vision loss worldwide, with an estimated 1 in 10 people over the age of 55 showing early signs of the condition. There are currently no forms of therapy available for the end stage of dry AMD, geographic atrophy (GA). Here, we show that the inner blood-retina barrier (iBRB) is highly dynamic and may play a contributory role in GA development. We have discovered that the gene CLDN5, which encodes claudin-5, a tight junction protein abundantly expressed at the iBRB, is regulated by BMAL1 and the circadian clock. Persistent suppression of claudin-5 expression in mice exposed to a cholesterol-enriched diet induced striking retinal pigment epithelium (RPE) cell atrophy, and persistent targeted suppression of claudin-5 in the macular region of nonhuman primates induced RPE cell atrophy. Moreover, fundus fluorescein angiography in human and nonhuman primate subjects showed increased retinal vascular permeability in the evening compared with the morning. These findings implicate an inner retina-derived component in the early pathophysiological changes observed in AMD, and we suggest that restoring the integrity of the iBRB may represent a novel therapeutic target for the prevention and treatment of GA secondary to dry AMD.

Regulation of vascular permeability plays a major role in the pathophysiology of visually threatening conditions such as retinal vein occlusion and diabetic retinopathy. Principally, several factors such as vascular endothelial growth factor (VEGF), are up-regulated or induced in response to hypoxia thus adversely affecting the blood-retinal barrier (BRB), resulting in retinal edema and neovascularisation. Furthermore, current evidence supports a dysregulation of the inner retinal neural-vascular integrity as a critical factor driving retinal ganglion cell (RGC) death and visual loss. The principal objective of this study was to interrogate whether Substance P (SP), a constitutive neurotransmitter of amacrine and ganglion cells, may protect against N-methyl-d-aspartate (NMDA)-induced excitotoxic apoptosis of ganglion cells and VEGF-induced vessel leakage in the retina. Tight junctional protein expression and a Vascular Permeability Image Assay were used to determine vascular integrity in vitro. The protective effect of SP on RGC was established in ex vivo retinal explants and in vivo murine models. After NMDA administration, a reduction in TUNEL+ cells and a maintained number of Brn-3a+ cells were found, indicating an inhibition of RGC apoptosis mediated by SP. Additionally, SP maintained endothelial tight junctions and decreased VEGF-induced vascular permeability. In conclusion, administration of SP protects against NMDA apoptosis of RGC and VEGF-induced endothelial barrier breakdown., (Copyright © 2019. Published by Elsevier Inc.)

Brain is an organ with the highest lipid concentration in the body. Cellular membrane lipids can affect both the positioning and the functioning of membrane proteins, thus regulating several cell actions. Changes in the lipid composition of cell membrane can modulate the microenvironment and consequently the function of its proteins, e.g. neurotransmission. Some of the first studies on the subject have shown a negative correlation between serum cholesterol levels and depression, suicidality and behavioral disorders. Several studies -but not all- have found decreased concentrations of ω-3 polyunsaturated fatty acids (ω-3-PUFA) in plasma or erythrocyte membranes of patients with depression, bipolar disorder or after a suicide attempt. In some cases, positive results after their administration have been reported. The effect of ω-3-PUFA in affective disorders is attributed to their action on neurotransmission, neuroplasticity as well as to their anti-inflammatory and anti-oxidative properties. Besides, decreased levels of ω-3-PUFA have been found in erythrocytes or platelets of schizophrenic patients. Some studies have shown in schizophrenics an increased rate of membrane phospholipids breakdown and a decreased ratio of ω-3-PUFA incorporation in phospholipids, possibly because of increased activity of phospholipase A2, an enzyme with crucial role in signaling transduction. Deficient dietetic ingestion of ω-3-PUFA may increase the risk for development of schizophrenia, while a diet rich in ω-3-PUFA may have a preventive role for the disease or improve its course. Although there is no evidence for their action as a monotherapy, they may be useful as an add-on therapy to drug treatment. Some authors suggest that abnormal sphingolipid metabolism, leading to accumulation of ceramides, may be responsible for the development of mood and anxiety disorders, as well as for induction of inflammation or oxidative stress, mechanisms possibly responsible for the physical symptoms of depression. Some drugs seem to combine inhibition of sphingomyelinase (an enzyme catalyzing the production of ceramides) and antidepressant effect. Despite the multitude of related studies, many aspects of the subject remain obscure. Current research focuses on the validity of preventive (especially in the perinatal period) or therapeutic administration of ω-3-PUFA as well as to the pharmacological manipulation of enzymes involved in lipid metabolism (e.g. sphingomyelinase) for the treatment of psychiatric disorders.

Background/aims: Hypoxia of the retina is a common pathogenic drive leading to vision loss as a result of tissue ischemia, increased vascular permeability and ultimately retinal neovascularisation. Here we tested the hypothesis that Müller cells stabilize the neurovascular unit, microvasculature by suppression of HIF-1α activation as a result of hypoxic preconditioning., Methods: Tube Formation Assay and In vitro Vascular Permeability Image Assay were used to analyze angiogenesis and vascular integrity. Seahorse XF Cell Mito Stress Test was used to measure mitochondrial respiration. Gene and protein expression were examined by qRTPCR, ELISA and western blot., Results: Hypoxic insult induces a significant induction of proangiogenic factors including vascular endothelial growth factor (VEGF) and angiopoietinlike 4 (ANGPTL-4) resulting in angiogenesis and increased vascular permeability of vascular endothelial cells. Hypoxic preconditioning of a human retinal Müller glia cell line significantly attenuates HIF-1α activation through the inhibition of mTOR and concomitant induction of aerobic glycolysis, stabilizing endothelial cells., Conclusion: Hypoxic preconditioning of Müller cells confers a robust protection to endothelial cells, through the suppression of HIF1α activation and its downstream regulation of VEGF and ANGPTL-4., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Despite strong genetic associations, compelling human histological data and numerous hypotheses generated with supportive animal data, the mechanisms of inflammation or inflammatory control of cell health during progression of age-related macular degeneration arguably remain elusive. This perspective delivers a view that maintaining tissue health requires active immune cellular and tissue pathways, but when responses are perturbed or exaggerated, chronic inflammation is destructive. There are potential pathways and processes to enable understanding and determine how potential causative factors including altered cellular metabolism, senescence, oxidative stress disrupt tissue homeostasis are engaged. Establishing differences in the immune phenotype between normal aging and AMD, and how the inter-relatedness of these triggers contribute to pathobiology is integral for future therapeutic success.

Purpose: We have observed an increasing incidence of myelodysplastic syndromes (MDS) in the geographic area of Western Greece during the past two decades. The objective of this study was to investigate potential risk factors for the manifestation of MDS in this area of Greece., Methods: A hospital-based case-control study was conducted in the public hospitals of the region. Participants were interviewed based on a questionnaire regarding demographics, occupational exposures, smoking, alcohol consumption, dietary, and domestic factors., Results: A total of 228 individuals (126 cases, 102 controls) were recruited in this study. Univariate analysis showed that risk of MDS was associated with a family history of hematologic malignancy or solid tumor, exposure to pesticides, insecticides, herbicides, increased weekly intake of meat and eggs, and increased alcohol intake, whereas fruit intake had a protective effect. Analysis by pesticide ingredient showed a weak association of exposure to paraquat and glyphosate with the occurrence of MDS. Multivariate analysis showed that independent risk factors for the manifestation of MDS were family history of solid tumor (OR 2.47, 95% CI 1.32-4.65), meat intake for ≥5 days/week (OR 2.67, 95% CI 1.05-6.80) and exposure to pesticides (OR 3.25, 95% CI 1.73-6.11)., Conclusions: Exposure to pesticides is a major risk factor of MDS in Western Greece. Family history of solid tumor and increased meat intake also appear to play a role in the pathogenesis of MDS. Public health authorities should implement policies to advise and protect farmers from the harmful effects of agrochemicals. Emphasis should also be given to health promotion advice including healthy eating.

Purpose: To report the short-term safety of dexamethasone implants to treat macular edema due to retinal vein occlusion (RVO), in eyes with treated glaucoma or ocular hypertension at baseline using an as-needed re-treatment regimen., Methods: Retrospective clinical database study from two centers using the same electronic medical record system. Extracted data included: intraocular pressure (IOP), visual acuity (VA), central 1 mm retinal thickness (CRT) by optical coherence tomography, phakic status, number of injections, glaucoma treatment, and peri-operative complications., Results: Thirty-three eyes of 33 patients on IOP-lowering treatment for glaucoma or ocular hypertension (OHT) at baseline and mean IOP of 16 mmHg at baseline received one to four (mean, 1.8; median, 1) dexamethasone implants over 18 months for RVO-related macular edema. Fourteen eyes (42 %) had IOP of ≥21 mmHg, and three eyes (9 %) had IOP of ≥35 mmHg at one or more visits during the study period. Nine of 14 eyes (64 %) with raised IOP required additional topical treatment only for a mean (SE) period of 8.5 months (3.2), while the remaining five eyes (36 %) required long-term additional IOP-lowering treatment for a mean (SE) of 16 months (1.44). Surgery for IOP lowering was not required in any eye. Mean VA (SE) improved from 44 (3) ETDRS letters at baseline to 47 letters (5) at 2 months (p = 0.049), 48 (8) letters at 6 months and 46 (4) letters at 12 months. Mean CRT (SE) improved from 530 (25) μm at baseline to 323 (27) μm at 2 months (p < 0.001), 498 (76) μm at 6 months, and 359 (25) μm at 12 months (p < 0.001)., Conclusion: The short-term IOP rise after intravitreal dexamethasone implant in eyes with glaucoma or ocular hypertension at baseline was acceptable and consistent with previous reports in patients without preexisting glaucoma. Treated OHT or glaucoma may not be a strict contraindication against the use of dexamethasone implant, but close monitoring of IOP is required.

Age-related macular degeneration (AMD) is the leading cause of central vision loss worldwide. Loss of retinal pigment epithelium (RPE) is a major pathological hallmark in AMD with or without pathological neovascularization. Although activation of the immune system is implicated in disease progression, pathological pathways remain diverse and unclear. Here, we report an unexpected protective role of a pro-inflammatory cytokine, interleukin-33 (IL-33), in ocular angiogenesis. IL-33 and its receptor (ST2) are expressed constitutively in human and murine retina and choroid. When RPE was activated, IL-33 expression was markedly elevated in vitro. We found that IL-33 regulated tissue remodelling by attenuating wound-healing responses, including reduction in the migration of choroidal fibroblasts and retinal microvascular endothelial cells, and inhibition of collagen gel contraction. In vivo, local administration of recombinant IL-33 inhibited murine choroidal neovascularization (CNV) formation, a surrogate of human neovascular AMD, and this effect was ST2-dependent. Collectively, these data demonstrate IL-33 as a potential immunotherapy and distinguishes pathways for subverting AMD pathology. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

The ophthalmic manifestations of dengue fever include a visually impairing maculopathy, where patients are left with a central or paracentral relative scotoma. The authors present a case of a 26-year-old female patient returning from Thailand with unilateral reduction in visual acuity and a central scotoma associated with dengue fever. The authors report the use of the optical coherence tomography angiography (OCTA) as a noninvasive imaging platform to demonstrate its value in showing the persistent changes corresponding to the functional central scotoma in dengue-related maculopathy, which often cannot be visualized clinically or by standard OCT and fundus fluorescein angiography. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:1057-1060.]., (Copyright 2016, SLACK Incorporated.)

Age-related decreases in autophagy contribute to the progression of age-related macular degeneration (AMD). We have now studied the interaction between autophagy impaired in retinal pigment epithelium (RPE) and the responses of macrophages. We find that dying RPE cells can activate the macrophage inflammasome and promote angiogenesis. In vitro, inhibiting rotenone-induced autophagy in RPE cells elicits caspase-3 mediated cell death. Co-culture of damaged RPE with macrophages leads to the secretion of IL-1β, IL-6 and nitrite oxide. Exogenous IL-6 protects the dysfunctional RPE but IL-1β causes enhanced cell death. Furthermore, IL-1β toxicity is more pronounced in dysfunctional RPE cells showing reduced IRAK3 gene expression. Co-culture of macrophages with damaged RPE also elicits elevated levels of pro-angiogenic proteins that promote ex vivo choroidal vessel sprouting. In vivo, impaired autophagy in the eye promotes photoreceptor and RPE degeneration and recruitment of inflammasome-activated macrophages. The degenerative tissue environment drives an enhanced pro-angiogenic response, demonstrated by increased size of laser-induced choroidal neovascularization (CNV) lesions. The contribution of macrophages was confirmed by depletion of CCR2(+) monocytes, which attenuates CNV in the presence of RPE degeneration. Our results suggest that the interplay between perturbed RPE homeostasis and activated macrophages influences key features of AMD development.

Background: Age-related macular degeneration is a leading cause of irreversible blindness. Altered immune responses drive degeneration, in response to oxidative stress and hypoxia-induced regulation of metabolism. We tested the hypothesis that toll-like receptor activation of retinal pigment epithelium and cellular metabolic switch upregulate interleukin 33, which acts through its receptor ST2 to activate both choroidal stromal fibroblasts and mast cells. By such mechanisms, the fibrosis and insidious degeneration, which we observe clinically, is accentuated., Methods: Retinal pigment epithelial cells (ARPE-19 and B6-RPE07) were stimulated with toll-like receptor ligands, and energetic pathways were assessed through lactate production and the expression of glycolytic enzymes. Expression profile and secretion of interleukin 33 were determined by RT-PCR and western blots. Function and expression profile of bone-marrow-derived mast cells and human choroidal fibroblasts were also assessed., Findings: The production of lactate, determining aerobic glycolysis, increased after stimulation of retinal pigment epithelial cells with LPS or poly(I:C), indicating an increase in the glycolytic activity after toll-like receptor stimulation. Increased levels of GLUT1 transcripts, and upregulation of GAPDH expression corroborated this finding. Furthermore, increased expression of interleukin 33 was dependent on a glycolytic metabolic switch and was enhanced under hypoxic conditions. ST2 was highly expressed in retinal pigment epithelium, choroidal mast cells, and choroidal fibroblasts in mouse and man. ST2+ bone-marrow-derived mast cells generated a spectrum of inflammatory cytokines and PGS2 when cultured with interleukin-33-rich retinal pigment epithelium supernatant. Interleukin-33 treatment impaired fibroblast migration and gel contraction alongside suppression of MMP-2 and MMP-9 expression., Interpretation: Our data highlight an unrecognised link between retinal pigment epithelium bioenergetic status and tissue remodelling of choroidal stroma. Our findings suggest that the interleukin 33/ST2 axis and changing bioenergetic sources are potential therapeutic targets to inhibit progression of age-related macular degeneration., Funding: National Institute for Health Research, National Eye Research Centre., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Recent studies suggest that the anti-diabetic drug metformin may reduce the risk of cancer and have anti-proliferative effects for some but not all cancers. In this study, we examined the effects of metformin on human retinoblastoma cell proliferation in vitro and in vivo. Two different human retinoblastoma cell lines (Y79, WERI) were treated with metformin in vitro and xenografts of Y79 cells were established in nu/nu immune-deficient mice and used to assess the effects of pharmacological levels of metformin in vivo. Metformin inhibited proliferation of the retinoblastoma cells in vitro. Similar to other studies, high concentrations of metformin (mM) blocked the cell cycle in G0‑G1, indicated by a strong decrease of G1 cyclins, especially cyclin D, cyclin-dependent kinases (4 and 6), and flow cytometry assessment of the cell cycle. This was associated with activation of AMPK, inhibition of the mTOR pathways and autophagy marker LC3B. However, metformin failed to suppress growth of xenografted tumors of Y79 human retinoblastoma cells in nu/nu mice, even when treated with a maximally tolerated dose level achieved in human patients. In conclusion, suprapharmacological levels (mM) of metformin, well above those tolerated in vivo, inhibited the proliferation of retinoblastoma cells in vitro. However, physiological levels of metformin, such as seen in the clinical setting, did not affect the growth of retinoblastoma cells in vitro or in vivo. This suggests that the potential beneficial effects of metformin seen in epidemiological studies may be limited to specific tumor types or be related to indirect effects/mechanisms not observed under acute laboratory conditions.