Amelioration of ultraviolet-induced photokeratitis in mice treated with astaxanthin eye drops

Purpose: Ultraviolet (UV) acts as low-dose ionizing radiation. Acute UVB exposure causes photokeratitis and induces apoptosis in corneal cells. Astaxanthin (AST) is a carotenoid, present in seafood, that has potential clinical applications due to its high antioxidant activity. In the present study, we examined whether topical administration of AST has preventive and therapeutic effects on UV-photokeratitis in mice. Methods: C57BL/6 mice were administered with AST diluted in polyethylene glycol (PEG) in instillation form (15 μl) to the right eye. Left eyes were given vehicle alone as controls. Immediately after the instillation, the mice, under anesthesia, were irradiated with UVB at a dose of 400 mJ/cm2. Eyeballs were collected 24 h after irradiation and stained with H&E and TUNEL. In an in vitro study, mouse corneal epithelial (TKE2) cells were cultured with AST before UV exposure to quantify the UV-derived cytotoxicity. Results: UVB exposure induced cell death and thinning of the corneal epithelium. However, the epithelium was morphologically well preserved after irradiation in AST-treated corneas. Irradiated corneal epithelium was significantly thicker in eyes treated with AST eye drops, compared to those treated with vehicles (p<0.01), in a doses dependent manner. Significantly fewer apoptotic cells were observed in AST-treated eyes than controls after irradiation (p<0.01). AST also reduced oxidative stress in irradiated corneas. The in vitro study showed less cytotoxicity of TKE2 cells in AST-treated cultures after UVB-irradiation (p<0.01). The cytoprotective effect increased with the dose of AST. Conclusions: Topical AST administration may be a candidate treatment to limit the damages by UV irradiation with wide clinical applications

Bioinformatic Analysis Reveals Central Role for Tumor-Infiltrating Immune Cells in Uveal Melanoma Progression

Tumor-infiltrating immune cells are capable of effective cancer surveillance, and their abundance is linked to better prognosis in numerous tumor types. However, in uveal melanoma (UM), extensive immune infiltrate is associated with poor survival. This study aims to decipher the role of different tumor-infiltrating cell subsets in UM in order to identify potential targets for future immunotherapeutic treatment. We have chosen the TCGA-UVM cohort as a training dataset and GSE22138 as a testing dataset by mining publicly available databases. The abundance of 22 immune cell types was estimated using CIBERSORTx. Then, to determine the significance of tumor-infiltrating cell subsets in UM, we built a multicell type prognostic signature, which was validated in the testing cohort. The created signature was built upon the negative prognostic role of CD8+ T cells and M0 macrophages and the positive role of neutrophils. Based on the created signature score, we divided the patients into low- and high-risk groups. Kaplan-Meier, Cox, and ROC analyses demonstrated superior performance of our risk score compared to either clinical or pathologic characteristics of both cohorts. Further, we found the molecular pathways associated with cancer immunoevasion and metastasis to be enriched in the high-risk group, explaining both the lack of adequate immune surveillance despite increased infiltration of CD8+ T cells as well as the higher metastatic potential. Genes associated with tryptophan metabolism (IDO1 and KYNU) and metalloproteinases were among the most differentially expressed between the high- and low-risk groups. Our correlation analyses interpreted in context of published in vitro data strongly suggest the central role of CD8+ T cells in shifting the UM tumor microenvironment towards suppressive and metastasis-promoting. Therefore, we propose further investigations of IDO1 and metalloproteinases as novel targets for immunotherapy in lymphocyte-rich metastatic UM patients

Transcriptome-Wide Analysis of CXCR5 Deficient Retinal Pigment Epithelial (RPE) Cells Reveals Molecular Signatures of RPE Homeostasis

Age-related macular degeneration (AMD) is the most common cause of irreversible blindness in the elderly population. In our previous studies, we found that deficiency of CXCR5 causes AMD-like pathological phenotypes in mice, characterized by abnormalities and dysfunction of the retinal pigment epithelium (RPE) cells. The abnormalities included abnormal cellular shape and impaired barrier function. In the present study, primary RPE cells were derived separately from CXCR5 knockout (KO) mice and from C57BL6 wild type (WT). The isolated primary cells were cultured for several days, and then total RNA was isolated and used for library preparation, sequencing, and the resultant raw data analyzed. Relative to the WT, a total of 1392 differentially expressed genes (DEG) were identified. Gene ontology analysis showed various biological processes, cellular components, and molecular functions were enriched. Pathway enrichment analysis revealed several pathways, including the PI3K-Akt signaling, mTOR signaling, FoxO, focal adhesion, endocytosis, ubiquitin-mediated proteolysis, TNF alpha-NF-kappa B Signaling, adipogenesis genes, p53 signaling, Ras, autophagy, epithelial-mesenchymal transition (EMT), and mitochondrial pathway. This study explores molecular signatures associated with deficiency of CXCR5 in RPE cells. Many of these signatures are important for homeostasis of this tissue. The identified pathways and genes require further evaluation to better understand the pathophysiology of AMD.Funding Agencies|University of Missouri; National Eye Institute (NEI), Bethesda, MD, USA [EY027824]</p

Autoimmune-Mediated Retinopathy in CXCR5-Deficient Mice as the Result of Age-Related Macular Degeneration Associated Proteins Accumulation

Previous research has shown that CXCR5(-/-) mice develop retinal degeneration (RD) with age, a characteristic related to age macular degeneration (AMD). RD in these mice is not well-understood, and in this study, we sought to characterize further the RD phenotype and to gain mechanistic insights into the function of CXCR5 in the retina. CXCR5(-/-) and WT control mice were used. Fundus images demonstrated a significant (p amp;lt; 0.001) increase of hypo-pigmented spots in the retina of aged CXCR5(-/-) mice compared with WT control mice. PAS staining indicated localization of deposits in the sub-retinal pigment epithelia (RPE) layer. AMD-associated proteins Cryab, amyloid beta, and C3d were detected within the RPE/sub-RPE tissues by immunofluorescence (IF). In addition, western blot analysis of COX-2, Arg1, and VEGF-a revealed an increase in the signaling of these molecules within the RPE/choroid complex. Transmission electron microscopy (TEM) indicated a drusen-like structure of sub-RPE deposits with an accumulation of vacuolated cellular debris. Loss of photoreceptors was detected by peanut lectin staining and was corroborated by a reduction in MAP2 signaling. Loss of blood-retinal barrier integrity was demonstrated by a reduction of ZO-1 expression. Inflammatory cells were detected in the sub-RPE space, with an increase in IBA-1 positive microglia cells on the surface of the RPE. Mass spectrometry analysis of CXCR5(-/-) mouse RPE/choroid proteins extracts, separated by SDS-page and incubated with autologous serum, identified autoantibodies against AMD-associated proteins: Cryaa, Cryab, and Anxa2. In vitro evaluations in BV-2 cell culture indicated a significant increase in production of Arg-1 (p amp;lt; 0.001) and COX-2 (p amp;lt; 0.01) in the presence of anti-CXCR5 antibody when compared with Igg-treated control BV-2 cells stimulated with IL-4 and TNF alpha/IFN gamma, respectively. Anti-CXCR5 antibody treatment without stimulating agents did not affect Arg-1 and COX-2 expression; this suggests that CXCR5 may have a regulatory role in microglia cells activation. These results indicate that with age, CXCR5(-/-) mice develop RD characterized by microglia dysfunction, increased production of CXCL13 in the RPE progressive photoreceptor, neuronal loss, and sub-RPE deposition of cellular debris, resulting in the production of immunogenic proteins and autoimmune-mediated RD.Funding Agencies|BrightFocus Foundation [M2014124]; Missouri University start-up fund (Hu Huang research group); NSFC fund [81870677]</p

Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea

Angiogenesis as a pathological process in the eye can lead to blindness. In the cornea, suppression of angiogenesis by anti-VEGF treatment is only partially effective while steroids, although effective in treating inflammation and angiogenesis, have broad activity leading to undesirable side effects. In this study, genome-wide expression was investigated in a suture-induced corneal neovascularization model in rats, to investigate factors differentially targeted by dexamethasone and anti-Vegf. Topical treatment with either rat-specific anti-Vegf, dexamethasone, or normal goat IgG (sham) was given to sutured corneas for 48 hours, after which in vivo imaging, tissue processing for RNA microarray, and immunofluorescence were performed. Dexamethasone suppressed limbal vasodilation (P amp;lt; 0.01) and genes in PI3K-Akt, focal adhesion, and chemokine signaling pathways more effectively than anti-Vegf. The most differentially expressed genes were confirmed by immunofluorescence, qRTPCR and Western blot. Strong suppression of Reg3g and the inflammatory chemokines Ccl2 and Cxcl5 and activation of classical complement pathway factors C1r, C1s, C2, and C3 occurred with dexamethasone treatment, effects absent with anti-Vegf treatment. The genome-wide results obtained in this study provide numerous potential targets for specific blockade of inflammation and angiogenesis in the cornea not addressed by anti-Vegf treatment, as possible alternatives to broad-acting immunosuppressive therapy.Funding Agencies|Swedish Research Council [2012-2472]; Swedish Foundation Stiftelsen Synframjandets Forskningsfond/Ogonfonden</p

Transcriptome-wide analysis of differentially expressed chemokine receptors, SNPs, and SSRs in the age-related macular degeneration

Abstract Background Age-related macular degeneration (AMD) is the most common, progressive, and polygenic cause of irreversible visual impairment in the world. The molecular pathogenesis of the primary events of AMD is poorly understood. We have investigated a transcriptome-wide analysis of differential gene expression, single-nucleotide polymorphisms (SNPs), indels, and simple sequence repeats (SSRs) in datasets of the human peripheral retina and RPE-choroid-sclera control and AMD. Methods and results Adaptors and unbiased components were removed and checked to ensure the quality of the data sets. Molecular function, biological process, cellular component, and pathway analyses were performed on differentially expressed genes. Analysis of the gene expression datasets identified 5011 upregulated genes, 11,800 downregulated genes, 42,016 SNPs, 1141 indels, and 6668 SRRs between healthy controls and AMD donor material. Enrichment categories for gene ontology included chemokine activity, cytokine activity, cytokine receptor binding, immune system process, and signal transduction respectively. A functional pathways analysis identified that chemokine receptors bind chemokines, complement cascade genes, and create cytokine signaling in immune system pathway genes (p value < 0.001). Finally, allele-specific expression was found to be significant for Chemokine (C-C motif) ligand (CCL) 2, 3, 4, 13, 19, 21; C-C chemokine receptor (CCR) 1, 5; chemokine (C-X-C motif) ligand (CXCL) 9, 10, 16; C-X-C chemokine receptor type (CXCR) 6; as well as atypical chemokine receptor (ACKR) 3,4 and pro-platelet basic protein (PPBP). Conclusions Our results improve our overall understanding of the chemokine receptors’ signaling pathway in AMD conditions, which may lead to potential new diagnostic and therapeutic targets

Amelioration of endotoxin-induced uveitis treated with the sea urchin pigment echinochrome in rats

Purpose: Echinochrome is a pigment present in the shells and spines of sea urchins. It has been reported to have several biologic protective effects, including in experimental models of myocardial ischemia/reperfusion injury, for which the proposed mechanisms are scavenging reactive oxygen species (ROS) and chelating iron. Endotoxin-induced uveitis (EIU) is an animal model of acute anterior segment intraocular inflammation that is induced by the injection of lipopolysaccharide (LPS). In this study, the therapeutic effect of echinochrome was examined in uveitis using the EIU model. Methods: EIU was induced in Lewis rats via 200 mu g subcutaneous injections of LPS from Escherichia coli. Echinochrome was administered intravenously in 10, 1, or 0.1 mg/kg doses suspended in PBS (controls were injected with PBS only). Twenty-four hours after LPS injection, the number of infiltrating cells and the protein concentration in aqueous humor were determined. Aqueous tumor necrosis factor alpha (TNF-alpha) concentration was quantified with enzyme-linked immunosorbent assay, eyes were stained with nuclear factor (NF) kappa B antibodies, and ROS production was determined by dihydroethidium staining in fresh frozen samples. Results: The number of inflammatory aqueous cells and protein levels were lower in the groups treated with 10 and 1 mg/kg of echinochrome than in the untreated LPS group (p<0.01). Treatment with 10 and 1 mg/kg of echinochrome significantly reduced TNF-alpha concentrations in aqueous humor (p<0.01). The numbers of NF kappa B-positive cells and ROS signals were also reduced by echinochrome administration (p<0.05). Conclusions: Echinochrome ameliorated intraocular inflammation caused by EIU by reducing ROS production, thereby also decreasing the expression of NF kappa B and TNF-alpha. As a natural pigment, echinochrome may therefore be a promising candidate for the safe treatment of intraocular inflammation. The use of sea urchin shells and spines in health foods and medical products is thus both economically and environmentally meaningful

Genome-wide expression datasets of anti-VEGF and dexamethasone treatment of angiogenesis in the rat cornea

Therapeutics against pathologic new blood vessel growth, particularly those targeting vascular endothelial growth factor (VEGF) are of enormous clinical interest. In the eye, where anti-VEGF agents are in widespread clinical use for treating retinal and corneal blindness, only partial or transient efficacy and resistance to anti-VEGF agents are among the major drawbacks. Conversely, corticosteroids have long been used in ophthalmology for their potency in suppressing inflammation and angiogenesis, but their broad biological activity can give rise to side effects such as glaucoma and cataract. To aid in the search for more targeted and effective anti-angiogenic therapies in the eye, we present here a dataset comparing gene expression changes in dexamethasone versus anti-Vegfa treatment of inflammation leading to angiogenesis in the rat cornea. Global gene expression analysis with GeneChip Rat 230 2.0 microarrays was conducted and the metadata submitted to Expression Omnibus repository. Here, we present a high-quality validated dataset enabling genome-wide comparison of genes differentially targeted by dexamethasone and anti-Vegf treatments, to identify potential alternative therapeutic targets for evaluation.Funding Agencies|Swedish Research Council [2012- 2472]; Swedish Ophthalmological Society Stiftelsen Synframjandets Forskningsfond/Ogonfonden</p

Repeat Corneal Neovascularization is Characterized by More Aggressive Inflammation and Vessel Invasion Than in the Initial Phase

Purpose: Treatment of corneal neovascularization can lead to vessel regression and recovery of corneal transparency. Here, we examined the response of the cornea to a repeated stimulus after initial vessel regression comparing the second wave of neovascularization with the first. Methods: Corneal neovascularization was induced by surgical suture placement in the rat cornea for 7 days, followed by suture removal and a 30-day regression period. Corneas were then re-sutured and examined for an additional 4 days. Longitudinal slit-lamp imaging, in vivo confocal microscopy, and microarray analysis of global gene expression was conducted to assess the inflammatory and neovascularization response. Inhibitory effect of topical dexamethasone for repeat neovascularization was assessed. Results: After initial robust neovascularization, 30 days of regression resulted in the recovery of corneal transparency; however, a population of barely functional persistent vessels remained at the microscopic level. Upon re-stimulation, inflammatory cell invasion, persistent vessel dilation, vascular invasion, and gene expression of Vegfa, Il1β, Il6, Ccl2, Ccl3, and Cxcl2 all doubled relative to initial neovascularization. Repeat neovascularization occurred twice as rapidly as initially, with activation of nitric oxide and reactive oxygen species, matrix metalloproteinase, and leukocyte extravasation signaling pathways, and suppression of anti-inflammatory LXR/RXR signaling. While inhibiting initial neovascularization, a similar treatment course of dexamethasone did not suppress repeat neovascularization. Conclusions: Persistent vessels remaining after the initial resolution of neovascularization can rapidly reactivate to facilitate more aggressive inflammation and repeat neovascularization, highlighting the importance of achieving and confirming complete vessel regression after an initial episode of corneal neovascularization.Funding Agencies|Swedish Research Council (Stockholm, Sweden) [2012-2472]</p