Showing papers in "Molecular Vision in 2013"

Astaxanthin protects ARPE-19 cells from oxidative stress via upregulation of Nrf2-regulated phase II enzymes through activation of PI3K/Akt

Abstract: Purpose: Oxidative stress on retinal pigment epithelial (RPE) cells is thought to play a crucial role in the development and progression of age-related macular degeneration. Astaxanthin (AST) is a carotenoid that shows significant antioxi dant properties. This study was designed to investigate the protective effect of AST on ARPE-19 cells against oxidative stress and the possible underlying mechanism. Methods: ARPE-19 cells exposed to different doses of H 2 O 2 were incubated with various concentrations of AST and cell viability subsequently detected with the (4-[3-[4-iodophenyl]-2–4(4-nitrophenyl)-2H-5- tetrazolio-1,3-benzene disulfonate]; WST-1) assay. The apoptosis rate and intracellular levels of reactive oxygen species (ROS) were measured with flow cytometry. NAD(P)H quinine oxidoreductase 1 (NQO1), hemeoxygenase-1 (HO-1), glutamate-cysteine ligase modifi er subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC) expression were examined with realtime PCR and western blotting. The nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein and the expression levels of cleaved caspase-3 and protein kinase B proteins were evaluated with western blotting. Results: AST clearly reduced H 2 O 2 -induced cell viability loss, cell apoptosis, and intracellular generation of ROS. Furthermore, treatment with AST activated the Nrf2-ARE pathway by inducing Nrf2 nuclear localization. Consequently, Phase II enzymes NQO1, HO-1, GCLM, and GCLC mRNA and proteins were increased. AST inhibited expression of H 2 O 2 -induced cleaved caspase-3 protein. Activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway was involved in the protective effect of AST on the ARPE-19 cells. Conclusions: AST protected ARPE-19 cells against H 2 O 2 -induced oxidative stress via Nrf2-mediated upregulation of the expression of Phase II enzymes involving the PI3K/Akt pathway.

Study of 27 aqueous humor cytokines in patients with type 2 diabetes with or without retinopathy

Abstract: Purpose To compare the changes in the levels of 27 aqueous humor cytokines between nondiabetic controls and patients with type 2 diabetes and to ascertain the association of these cytokines with diabetic retinopathy (DR).

Evaluation of the percentage of ganglion cells in the ganglion cell layer of the rodent retina

Abstract: Purpose: Retinal ganglion cells comprise a percentage of the neurons actually residing in the ganglion cell layer (GCL) of the rodent retina. This estimate is useful to extrapolate ganglion cell loss in models of optic nerve disease, but the values reported in the literature are highly variable depending on the methods used to obtain them. Methods: We tested three retrograde labeling methods and two immunostaining methods to calculate ganglion cell number in the mouse retina (C57BL/6). Additionally, a double-stain retrograde staining method was used to label rats (Long-Evans). The number of total neurons was estimated using a nuclear stain and selecting for nuclei that met specific criteria. Cholinergic amacrine cells were identified using transgenic mice expressing Tomato fluorescent protein. Total neurons and total ganglion cell numbers were measured in microscopic fields of 10 4 µm 2 to determine the percentage of neurons comprising ganglion cells in each field. Results: Historical estimates of the percentage of ganglion cells in the mouse GCL range from 36.1% to 67.5% depending on the method used. Experimentally, retrograde labeling methods yielded a combined estimate of 50.3% in mice. A retrograde method also yielded a value of 50.21% for rat retinas. Immunolabeling estimates were higher at 64.8%. Immunolabeling may introduce overestimates, however, with non-specific labeling effects, or ectopic expression of antigens in neurons other than ganglion cells. Conclusions: Since immunolabeling methods may overestimate ganglion cell numbers, we conclude that 50%, which is consistently derived from retrograde labeling methods, is a reliable estimate of the ganglion cells in the neuronal population of the GCL.

Differential regulation of microRNA-146a and microRNA-146b-5p in human retinal pigment epithelial cells by interleukin-1β, tumor necrosis factor-α, and interferon-γ.

Abstract: Purpose The inflammatory response of the retinal pigment epithelium (RPE) is implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB pathway. The aim of the present study is to investigate the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines.

Status of serum VEGF and ICAM-1 and its association with external limiting membrane and inner segment-outer segment junction disruption in type 2 diabetes mellitus

Abstract: Purpose To correlate the serum levels of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) with the severity of retinopathy and disruption of the external limiting membrane (ELM) and inner segment-outer segment (IS-OS) junction in type 2 diabetes mellitus (DM).

MicroRNAs in vitreus humor from patients with ocular diseases

Abstract: Purpose: Based on evidence that microRNAs (miRNAs) are found in many biologic fluids (e.g., urine, saliva, pleural fluid), we sought to detect the presence of miRNAs and analyze their profile in vitreous humor (VH) from patients af fected by various ocular diseases. Methods: MiRNAs were purified from VH samples taken during vitrectomy, by using the Qiagen miRNeasy Mini Kit. The expression profile on 745 miRNAs was performed by using TaqMan Low Density Array. Single TaqMan expres sion assays were performed on 18 VH samples (six each from patients with choroidal melanomas, retinal detachment, or macular hole) for miRNAs commonly expressed in serum or retinal cells: let-7b, miR-21, miR-26a, miR-146a, miR199-3p, miR-210, miR-374a*, miR-532-5p. RNA extracted from serum of six healthy donors or from formalin-fixed, paraffin-embedded samples of choroidal melanocytes from four uveal melanomas (epithelioid cells) and from three unaffected eyes were used as controls. Results: We identified the presence of 94 circulating small RNAs in the vitreous, some of which (miR-9, miR-9*, miR125a-3p, miR-184, miR-211, miR-214, miR-302c, miR-452, miR-628, miR-639) are particularly abundant in the VH but downrepresented or not detectable in serum. MiR-146a and miR-26a were overexpressed more than threefold in VH from patients with uveal melanomas compared to the other pathological groups (Wilcoxon signed-rank test, p value <0.05). Conclusions: Our experimental data suggest that a specific set of circulating miRNAs is secreted in the vitreous, which is quite different from the miRNA pattern in serum, and that the quantity of vitreal miRNAs could change, depending on the pathologies of the eye.

Increased malondialdehyde concentration and reduced total antioxidant capacity in aqueous humor and blood samples from patients with glaucoma.

Abstract: Purpose: To evaluate the levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) in the blood and aqueous humor of glaucomatous and nonglaucomatous patients. To measure the adenosine triphosphate/adenosine diphosphate/adenosine monophosphate (ATP/ADP/AMP) concentration as a biomarker of the blood energy charge potential. Methods: We examined 40 consecutive patients with primary open-angle glaucoma scheduled for cataract surgery. Twenty-six age-matched subjects scheduled for cataract surgery were enrolled as a control group. Blood and aqueous humor samples were collected at the time of surgery. MDA concentrations and blood nucleotides were measured with high-performance liquid chromatography. The TAC of the samples was estimated with the oxygen-radical absorbance capacity method. Results: Blood and aqueous humor MDA levels in glaucoma patients (respectively, 0.976±0.370 and 0.145±0.065 μmol/ ml) were significantly increased (p<0.001 for both) over those of the control group (respectively, 0.454±0.395 and 0.060±0.039 μmol/ml). In contrast, the control group presented significantly higher TACs than did the glaucoma group in both the blood (control: 2.681±1.101 and glaucoma: 1.617±0.674 μmol Trolox Equi/g; p<0.001) and aqueous humor (control: 0.963±0.302 and glaucoma: 0.788±0.346 μmol Trolox Equi/g; p=0.039). The control group (0.869±0.037) exhibited statistically significant (p<0.001) higher values of blood adenosine triphosphate/adenosine diphosphate (ATP-ADP) levels than did the glaucoma group (0.791±0.037). Conclusions: Our data further support the hypothesis that oxidative stress and decreased antioxidant defenses are involved in glaucoma. High-performance liquid chromatography appears to be an effective and sensitive method to detect altered levels of oxidative stress markers in glaucoma patients.

Review: corneal epithelial stem cells, their niche and wound healing.

Abstract: Stem cells emerged as a concept during the second half of 19(th) century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet's membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing.

Global microRNA expression profiling: Curcumin (diferuloylmethane) alters oxidative stress-responsive microRNAs in human ARPE-19 cells

Abstract: Purpose: In recent years, microRNAs (miRNAs) have been reported to play important roles in a broad range of biologic processes, including oxidative stress-mediated ocular diseases. In addition, the polyphenolic compound curcumin has been shown to possess anti-inflammatory, antioxidant, anticancer, antiproliferative, and proapoptotic activities. The aim of this study was to investigate the impact of curcumin on the expression profiles of miRNAs in ARPE-19 cells exposed to oxidative stress. Methods: MiRNA expression profiles were measured in ARPE-19 cells treated with 20 μM curcumin and 200 μM H 2 O 2 . PCR array analysis was performed using web-based software from SABiosciences. The cytotoxicity of ARPE-19 cells was determined with the CellTiter-Blue cell viability assay. The effects of curcumin on potential miRNA targets were analyzed with quantitative real-time PCR and western blotting. Results: Curcumin treatment alone for 6 h had no effect on ARPE-19 cell viability. Incubation with H 2 O 2 (200 µM) alone for 18 h decreased cell viability by 12.5%. Curcumin alone downregulated 20 miRNAs and upregulated nine miRNAs compared with controls. H 2 O 2 downregulated 18 miRNAs and upregulated 29 miRNAs. Furthermore, curcumin pretreat- ment in cells exposed to H 2 O 2 significantly reduced the H 2 O 2 -induced expression of 17 miRNAs. As determined with quantitative real-time PCR and western blotting, curcumin increased the expression of antioxidant genes and reduced angiotensin II type 1 receptor, nuclear factor-kappa B, and vascular endothelial growth factor expression at the mes- senger RNA and protein levels. Conclusions: The results demonstrated that curcumin alters the expression of H 2 O 2 -modulated miRNAs that are puta- tive regulators of antioxidant defense and renin-angiotensin systems, which have been reported to be linked to ocular diseases.

Diagnostic performance of a tear protein panel in early dry eye

Abstract: Purpose: To evaluate the tear protein pattern in patients with recent subjective symptoms of dry eye (DE) and with poor distinctive DE clinical signs. Methods: One hundred sixty patients suspected of suffering from mild to moderate DE according to the Dry Eye Workshop (DEWS report 2007) severity grade and 45 matched normal volunteers were included in the study. Subjective symptom score (Ocular Surface Disease index score), Schirmer test I, tear film break-up time, cornea and conjunctiva staining (National Eye Institute score); and tear protein analysis were performed. Statistical evaluation of data was performed with Mann–Whitney unpaired and Student t tests, (significance p<0.05). Correlations between variables were evaluated by using Pearson’s (r) or Spearman’s (ρ) correlation coefficients. Thresholds were selected from receiver operating curves; sensitivity, specificity, likelihood ratio (LR+), and positive predictive values were calculated for each protein. The combination of variables was carried out by univariate analysis, representing the best combination of tests for early DE diagnosis. Results: Total protein content (TP) and the following proteins were recognized in all samples: lysozyme-C (LYSC), lactoferrin (LACTO), tear lipocalin 1 (LIPOC-1), zinc-alpha-2-glycoprotein (ZAG-2), transferrin (TRANSF), and exudated serum albumin (ALB). A statistically significant decrease was demonstrated between normal subjects and patients with DE (mg/ml, mean±SD) for TP (9.89±2.28 versus 6.44±2.1), LYS-C (3.06±1.07 versus 2.15±0.78), LIPOC-1 (1.71±0.52 versus 0.98±0.5), ZAG-2 (0.43±0.24 versus 0.25±0.2), TRANSF (0.9±0.6 versus 0.33±0.3), and LACTO (2.11±0.74 versus 1.47±0.76), while an increase was found for ALB (0.21±0.5 versus 0.94±1.28). LIPOC-1 and ZAG-2 were strongly correlated to tear film break-up time. The proteins were related to the DEWS severity grade. Changes in each protein were a better predictor of early DE than were clinical variables; TP, LIPOC-1, and ALB exhibited the highest diagnostic performance either alone (LR+ 16.7, 12.3, 4.7, respectively) or when combined in a univariate analysis (LR+: 41.8, positive predictive value: 99.9). Conclusions: Our results demonstrated in tears from patients with early DE a significant reduction in tear protein content as a whole, associated with a decrease in proteins with antibacterial and protective functions. A decrease in proteins with lipid binding properties and an increase in inflammatory-related proteins were also shown. Changes in the abundance of a panel of tear proteins with divergent functions was found to better diagnose early DE than did conventional clinical tests.

Scleral structural alterations associated with chronic experimental intraocular pressure elevation in mice.

Abstract: PURPOSE To study changes in scleral structure induced by chronic experimental intraocular pressure elevation in mice. METHODS We studied the effect of chronic bead-induced glaucoma on scleral thickness, collagen lamellar structure, and collagen fibril diameter distribution in C57BL/6 (B6) and CD1 mice, and in collagen 8α2 mutant mice (Aca23) and their wild-type littermates (Aca23-WT) using electron and confocal microscopy. RESULTS In unfixed tissue, the control B6 peripapillary sclera was thicker than in CD1 mice (p<0.001). After 6 weeks of glaucoma, the unfixed CD1 and B6 sclera thinned by 9% and 12%, respectively (p<0.001). The fixed sclera, measured by electron microscopy, was significantly thicker in control Aca23 than in B6 or CD1 mice (p<0.05). The difference between fresh and fixed scleral thickness was nearly 68% in untreated control B6 and CD1 mice, but differed by only 10% or less in fresh/fixed glaucoma scleral comparisons. There were 39.3±9.6 lamellae (mean, standard deviation) in control sclera, categorized as 41% cross-section, 24% cellular, 20% oblique, and 15% longitudinal. After glaucoma, mean peripapillary thickness significantly increased in fixed specimens of all mouse strains by 10.3 ±4.8 µm (p=0.001) and the total number of lamellae increased by 18% (p=0.01). The number of cellular and cross-section lamellae increased in glaucoma eyes. After glaucoma, there were more small and fewer large collagen fibrils (p<0.0001). Second harmonic generation imaging showed that the normal circumferential pattern of collagen fibrils in the peripapillary sclera was altered in significantly damaged glaucomatous eyes. CONCLUSIONS Dynamic responses of the sclera to experimental mouse glaucoma may be more important than baseline anatomic features in explaining susceptibility to damage. These include decreases in nonfibrillar elements, alterations in lamellar orientation, an increased number of smaller collagen fibrils and fewer larger fibrils, and relative increase in the number of scleral fibroblast layers.

Regulation of constitutive vascular endothelial growth factor secretion in retinal pigment epithelium/choroid organ cultures: p38, nuclear factor kappaB, and the vascular endothelial growth factor receptor-2/phosphatidylinositol 3 kinase pathway

Abstract: Purpose: The retinal pigment epithelium (RPE) is a major source of vascular endothelial growth factor (VEGF) in the eye. Despite the role of VEGF in ocular pathology, VEGF is an important factor in maintaining the choroid and the RPE. Accordingly, the VEGF is constitutively expressed in RPE. In this study, the regulation of constitutive VEGF expression was investigated in an RPE/choroid organ culture. Methods: To investigate VEGF regulation, RPE/choroid of porcine origin were used. VEGF content was evaluated with enzyme-linked immunosorbent assay. The influence of several molecular factors was assessed with commercially available inhibitors (SU1498, bisindolylmaleimide, LY294002, nuclear factor kappaB [NFkB] activation inhibitor, mithramycin, YC-1, Stattic, SB203580). For toxicity measurements of inhibitors, primary RPE cells of porcine origin were used, and toxicity was evaluated with methyl thiazolyl tetrazolium assay. Results: VEGF secretion as measured in the RPE/choroid organ culture was diminished after long-term (48 h) inhibition of vascular endothelial growth factor receptor-2 by VEGFR-2-antagonist SU1498. VEGF secretion was also diminished after phosphatidylinositol 3 kinase was inhibited by LY294002 for 48 h. Coapplication of the substances did not show an additive effect, suggesting that they use the same pathway in an autocrine-positive VEGF regulation loop. Inhibition of protein kinase C by bisindolylmaleimide, on the other hand, did not influence VEGF secretion in organ culture. Inhibition of the transcription factor SP-1 by mithramycin displayed effects after 24 h and 48 h. Inhibiting hypoxia-inducible factor-1 (HIF-1) and Stat3 did not show any influence on constitutive VEGF secretion. Inhibition of the transcription factor NFkB diminished VEGF secretion after 6 h (earliest measured time point) and remained diminished at all measured time points (24 h, 48 h). The same pattern was found when the inhibitor of mitogen-activated kinase p38 was applied. A combination of NFkB and p38 inhibitors displayed an additive effect, completely abolishing VEGF secretion. Conclusions: Constitutive VEGF secretion in the RPE/choroid seems to be regulated by the transcription factor NFkB and the mitogen-activated kinase p38 in an independent manner. Constitutive VEGF secretion may be regulated to a lesser extent by the transcription factor SP-1, while Stat3 and hypoxia-inducible factor-1 do not seem to be involved. Additionally, VEGF secretion seems to be regulated long-term by an autocrine positive loop via vascular endothelial growth factor receptor-2 and phosphatidylinositol 3 kinase.

Review: Epigenetic mechanisms in ocular disease.

Abstract: Epigenetics has become an increasingly important area of biomedical research. Increasing evidence shows that epigenetic alterations influence common pathologic responses including inflammation, ischemia, neoplasia, aging, and neurodegeneration. Importantly, epigenetic mechanisms may have a pathogenic role in many complex eye diseases such as corneal dystrophy, cataract, glaucoma, diabetic retinopathy, ocular neoplasia, uveitis, and age-related macular degeneration. The emerging emphasis on epigenetic mechanisms in studies of eye disease may provide new insights into the pathogenesis of complex eye diseases and aid in the development of novel treatments for these diseases.

Role of N-ε- carboxy methyl lysine, advanced glycation end products and reactive oxygen species for the development of nonproliferative and proliferative retinopathy in type 2 diabetes mellitus.

Abstract: e-CML), advanced glycation end-products (AGEs), and reactive oxygen species (ROS) for the development of retinopathy among type 2 diabetic subjects. Methods: Seventy type 2 diabetic subjects with nonproliferative diabetic retinopathy (NPDR), 105 subjects with proliferative diabetic retinopathy (PDR), and 102 patients with diabetes but without retinopathy (DNR) were enrolled in this study. In addition, 95 normal individuals without diabetes were enrolled as healthy controls in this study. Serum and vitreous N e -CML and AGEs were measured by enzyme-linked immunosorbent assay. The peripheral blood mononuclear cell (PBMC) ROS level was measured by flow cytometric analysis. Serum and PBMC total thiols were measured by spectrophotometry. Results: Serum AGEs and N e -CML levels were significantly elevated in subjects with PDR (p<0.0001) and NPDR (p=0.0297 and p<0.0001, respectively) compared to DNR subjects. Further vitreous AGEs and N e -CML levels were found to be significantly high among PDR subjects compared to the control group (p<0.0001). PBMC ROS production was found to be strikingly high among NPDR (p<0.0001) and PDR (p<0.0001) subjects as compared to the DNR group. Serum and PBMC total thiol levels were remarkably decreased in NPDR (p<0.0001 and p=0.0043, respectively) and PDR (p=0.0108 and p=0.0332 respectively) subjects than those were considered as DNR. Conclusions: Our findings suggest that N e -CML and ROS are the key modulators for the development of nonproliferative retinopathy among poorly controlled type 2 diabetic subjects. Furthermore, AGEs under persistent oxidative stress and the deprived antioxidant state might instigate the pathogenic process of retinopathy from the nonproliferative to the proliferative state.

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3β inhibitor, SB216763.

Abstract: Purpose Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3β (GSK-3β) in regulating mMPT. Using direct inhibition of GSK-3β with the GSK-3β inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential.

Altered gene expression in dry age-related macular degeneration suggests early loss of choroidal endothelial cells.

Abstract: Purpose Age-related macular degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE) and choroid from early AMD and control maculas with exon-based arrays.

Retinal O-linked N-acetylglucosamine protein modifications: implications for postnatal retinal vascularization and the pathogenesis of diabetic retinopathy

Abstract: Purpose Hyperglycemia activates several metabolic pathways, including the hexosamine biosynthetic pathway. Uridine diphosphate N-acetylglucosamine (GlcNAc) is the product of the hexosamine biosynthetic pathway and the substrate for O-linked GlcNAc (O-GlcNAc) modification. This modification affects a wide range of proteins by altering their activity, cellular localization, and/or protein interactions. However, the role O-GlcNAcylation may play in normal postnatal retinal vascular development and in the ocular complications of diabetes, including diabetic retinopathy, requires further investigation.

Microglial ramification and redistribution concomitant with the attenuation of choroidal neovascularization by neuroprotectin D1.

Abstract: Purpose Neuroprotectin D1 (NPD1) attenuates laser-induced choroidal neovascularization (CNV) when administered intraperitoneally. Due to its lipophilicity and low molecular weight, NPD1 is well suited for topical delivery; thus, we investigated the efficacy of topically applied NPD1 in attenuating CNV. We also examined the effect of NPD1 on the recruitment and activation of microglia surrounding CNV lesions. Methods Mice were given laser-induced CNV and treated with NPD1 eye drops. CNV was evaluated by fluorescein leakage using a novel image analysis method and by isolectin B4 immunofluorescence of neovasculature. Microglia; recruitment was assessed by quantification. Using form factor, solidity, convexity, and fractal dimension, microglial activation was quantitatively assessed by two-dimensional, and for the first time, three-dimensional morphology. An ImageJ plugin, 3D Shape, was developed to enable this analysis. Results NPD1 attenuated leakage and neovascularization. The proximity of microglia to CNV lesions was significantly closer with NPD1. Consistent with the cellular ramification, microglia in NPD1-treated eyes were larger and exhibited a lower form factor and higher fractal dimension. Conclusions Our data show that NPD1 signaling induces a ramified, non-injury-inducing microglial phenotype coincident with attenuation of CNV. Since microglia are crucial participants in neurodegenerative diseases, the discovery that microglia are potential targets of NPD1 signaling warrants further investigation.

Topical administration of interleukin-1 receptor antagonist as a therapy for aqueous-deficient dry eye in autoimmune disease.

Abstract: Purpose: Dry eye is commonly associated with autoimmune diseases such as Sjogren’s syndrome (SS), in which exocrinopathy of the lacrimal gland leads to aqueous tear deficiency and keratoconjunctivitis sicca (KCS). KCS is among the most common and debilitating clinical manifestations of SS that is often recalcitrant to therapy. We established mice deficient in the autoimmune regulator ( Aire) gene as a model for autoimmune-mediated aqueous-deficient dry eye. In Aire-deficient mice, CD4+ T cells represent the main effector cells and local signaling via the interleukin-1 (IL-1/IL-1R1) pathway provides an essential link between autoreactive CD4+ T cells and ocular surface disease. In the current study, we evaluated the efficacy of topical administration of IL-1R1 antagonist (IL-1RA) anakinra in alleviating ocular surface damage resulting from aqueous-deficient dry eye in the setting of autoimmune disease. Methods: We compared the effect of commercially available IL-1R1 antagonist, anakinra (50 μg/mL concentration) to that of carboxymethylcellulose (CMC) vehicle control as a treatment for dry eye. Age-matched, Aire-deficient mice were treated three times daily with anakinra or CMC vehicle for 14 days using side-by-side (n=4 mice/group) and paired-eye (n=5) comparisons. We assessed (1) ocular surface damage with lissamine green staining; (2) tear secretion with wetting of phenol-red threads; (3) goblet cell (GC) mucin glycosylation with lectin histochemistry; (4) immune cell infiltration using anti-F4/80, CD11c, and CD4 T cell antibodies; and (5) gene expression of cornified envelope protein, Small ProlineRich Protein-1B (SPRR1B) with real-time quantitative polymerase chain reaction. Results: Aire-deficient mice treated with anakinra experienced significant improvements in ocular surface integrity and tear secretion. After 7 days of treatment, lissamine green staining decreased in eyes treated with anakinra compared to an equivalent increase in staining following treatment with CMC vehicle alone. By day 14, lissamine green staining in anakinra-treated eyes remained stable while eyes treated with CMC vehicle continued to worsen. Accordingly, there was a progressive decline in tear secretion in eyes treated with the CMC vehicle compared to a progressive increase in the anakinra-treated eyes over the 2-week treatment period. Aberrant acidification of GC mucins and pathological keratinization of the ocular surface were significantly reduced in anakinra-treated eyes. Significantly fewer Maackia amurensis leukoagglutinin positive goblet cells were noted in the conjunctiva of anakinra-treated eyes with a corresponding decrease in the expression of the pathological keratinization marker, SPRR1B. Finally, there was a downward trend in the infiltration of each immune cell type following anakinra treatment, but the cell counts compared to eyes treated with the vehicle alone were not significantly different. Conclusions: IL-1R antagonist, anakinra, demonstrates therapeutic benefits as a topical treatment for aqueous-deficient dry eye in a spontaneous mouse model of autoimmune KCS that mimics the clinical characteristics of SS. Targeting the IL-1/IL-1R1 signaling pathway through topical administration of IL-1RA may provide a novel option to improve ocular surface integrity, increase tear secretion, and restore the normal glycosylation pattern of GC mucins in patients with SS.

Review: mesenchymal stem cells and corneal reconstruction.

Abstract: Corneal reconstruction is among the most effective methods for curing corneal injury due to various clinical disorders. Mesenchymal stem cells (MSCs) are a type of multipotent cells distributed in various tissues, which can be easily isolated and expanded in vitro. MSCs are self-renewable and have the potential to transdifferentiate into other type of cells under certain conditions. More recently, the modulating angiogenesis, anti-inflammatory, and immunomodulatory properties of MSCs have been confirmed in animal models. The potential roles of MSCs are valuable for corneal reconstruction. Thus, in this review, we summarized the current understanding of the possible roles of MSCs in corneal reconstruction.

Altered cytokine profiles of human retinal pigment epithelium: oxidant injury and replicative senescence.

Abstract: PURPOSE Age-related macular degeneration (AMD) is a local, chronic inflammatory disease of the eye that is influenced by oxidative stress and dysregulation of the retinal pigment epithelium (RPE) associated with aging. The purpose of this study is to characterize the effects of oxidative stress and replicative senescence on the secreted cytokine profiles of RPE in vitro. METHODS We used multiple, serial passages of human RPE cells from primary culture as an in vitro model of aging. Responses of early passage 5 (P5) and late passage 21 (P21) RPE cells were compared. Oxidative stress was induced in RPE cells (P5) by exposure to 75 μM hydroquinone (HQ) for 24 h. The secretome profiles of the RPE cells were measured with a multiplex suspension assay that assayed human cytokine, chemokine, and growth factors. Immunohistochemistry on younger (≤55 years old) and older (≥70 years old) human post-mortem donor eyes was used to verify selected cytokines. RESULTS Supernatant of HQ-treated RPE cultures exhibited increased secreted levels of vascular endothelial growth factor (VEGF), interleukin (IL)-12, and IL-10 that reached statistical significance (p<0.05). Supernatant of late passage P21 RPE cultures exhibited decreased secreted levels of stromal cell-derived factor (SDF)-1α, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-8, IL-15, IL-6, and an increased level of IL-1ra compared to early passage P5 RPE cultures that reached statistical significance (p<0.05). Immunohistochemical analysis demonstrated increased expression of IL-1ra in RPE cells from older post-mortem donor eyes (≥70 years old) versus younger eyes (≤55 years old). CONCLUSIONS Our data demonstrate a unique cytokine secretion profile of primary culture RPE cells at early and late passage. Our in vitro data suggest an age-specific modulation of cytokine secretion in RPE and is consistent with immunohistochemical analysis on post-mortem eyes. The secretion profile associated with RPE under conditions that mimic oxidative stress, another factor associated with the pathogenesis of AMD, emphasizes upregulation of the angiogenic growth factor, vascular endothelial growth factor. Together, these data support the role of advanced age and oxidative stress in inflammatory cytokine modulation in RPE cells.

Proapoptotic and survival signaling in the neuroretina at early stages of diabetic retinopathy

Abstract: Purpose Diabetic retinopathy (DR) has been classically considered a microcirculatory disease of the retina. However, before any microcirculatory abnormalities can be detected in ophthalmoscopic examination, retinal neurodegeneration is already present. The aim of the study was to analyze proapoptotic and survival signaling in the neuroretinas of diabetic patients at early stages of DR.

Review: the history and role of naturally occurring mouse models with Pde6b mutations.

Abstract: Mouse models are useful tools for developing potential therapies for human inherited retinal diseases, such as retinitis pigmentosa (RP), since more strains are being identified with the same mutant genes and phenotypes as humans with corresponding retinal degenerative diseases. Mutations in the beta subunit of the human rod phosphodiesterase (PDE6B) gene are a common cause of autosomal recessive RP (arRP). This article focuses on two well-established naturally occurring mouse models of arRP caused by spontaneous mutations in Pde6b, their discovery, phenotype, mechanism of degeneration, strengths and limitations, and therapeutic approaches to restore vision and delay disease progression. Viral vector, especially adeno-associated viral vector (AAV) -mediated gene replacement therapy, pharmacological treatment, cell-based therapy and other approaches that extend the therapeutic window of treatment, is a potentially promising strategy for improving photoreceptor function and significantly slowing the process of retinal degeneration.

Association of MTHFR gene C677T mutation with diabetic peripheral neuropathy and diabetic retinopathy

Abstract: Diabetes is a common disorder with various systemic complications including diabetic peripheral neuropathy (DPN), which affects most diabetic patients and causes sensory, motor, and/or autonomic dysfunctions [ 1,2]. Diabetic retinopathy is the most common complication of diabetes mellitus [3] and is rapidly emerging as a global health issue that may threaten patients’ visual acuity and visual func-tioning. However, the underlying pathogenesis of DPN and diabetic retinopathy has not been well defined. It is widely accepted that vascular complication is the most common cause of diabetes death and disability and has been linked to diabetic neuropathy [4]. A risk factor for vasculopathy in diabetes is elevated homocysteine levels [5]. It has been reported that hyperhomocysteinemia in diabetic patients was associated with the prevalence of diabetic nephropathy and diabetic retinopathy [6,7]. Hyperhomocysteinemia was also independently related to the prevalence of diabetic neuropathy in diabetic patients [8-10].Methylenetetrahydrofolate reductase (MTHFR) is the enzyme that catalyzes the transformation of homocysteine to methionine via the remethylation pathway (gene located in 1p36) [11]. Hyperhomocysteinemia is the consequence of decreased activity of MTHFR [ 12]. Mutations of the

IGF-1 protects retinal ganglion cells from hypoxia-induced apoptosis by activating the Erk-1/2 and Akt pathways

Abstract: Purpose Hypoxia-induced retinal ganglion cell (RGC) apoptosis has been implicated in many optic neuropathies. Insulin-like growth factor-1 (IGF-1) is important in maintaining neuronal survival, proliferation, and differentiation. The purpose of this study is to explore whether IGF-1 can protect RGCs from hypoxia-induced apoptosis and to determine the precise mechanisms that regulate this process.

Homozygosity mapping in autosomal recessive retinitis pigmentosa families detects novel mutations

Abstract: Purpose Autosomal recessive retinitis pigmentosa (arRP) is a genetically heterogeneous disease resulting in progressive loss of photoreceptors that leads to blindness. To date, 36 genes are known to cause arRP, rendering the molecular diagnosis a challenge. The aim of this study was to use homozygosity mapping to identify the causative mutation in a series of inbred families with arRP.

Estrogen pathway polymorphisms in relation to primary open angle glaucoma: An analysis accounting for gender from the United States

Abstract: Purpose Circulating estrogen levels are relevant in glaucoma phenotypic traits. We assessed the association between an estrogen metabolism single nucleotide polymorphism (SNP) panel in relation to primary open angle glaucoma (POAG), accounting for gender.

Genetic variants of FZD4 and LRP5 genes in patients with advanced retinopathy of prematurity.

Abstract: Purpose Retinopathy of prematurity (ROP) is a complex disease with a genetic predisposition, but little is known about its genetic background. It has a clinical resemblance to familial exudative vitreoretinopathy (FEVR), a hereditary disease characterized by defects in the development of retinal vessels. Several studies have suggested that mutations in the causative genes for FEVR may account for a proportion of advanced ROP, but conflicting data have also been reported for some variants. To address the possibility of genetic involvement of FEVR genes in ROP, we performed comprehensive sequence analyses of 53 Japanese patients with advanced ROP for the FEVR-causing genes. Methods Peripheral blood DNA was obtained from 53 patients referred to our hospitals for retinal surgery. Polymerase chain reaction followed by direct sequencing of the coding regions of the known FEVR-causing genes (FZD4, LRP5, TSPAN12, and NDP) and a noncoding exon of the NDP gene was performed. Possible pathogenicity of the sequence changes were analyzed by orthologous protein sequence alignment and by computational predictions. Results We identified six different nonsynonymous DNA variants in the coding region of either the FZD4 gene (p.H69Y, p.R127H, and p.Y211H) or the LRP5 gene (p.R1219H, p.H1383P, and p.T1540M) in seven patients. The corresponding codons of these changes were highly conserved among species, and these changes were predicted to be pathogenic by at least two of four computational prediction programs. No such changes were found in the TSPAN12 and NDP genes. Conclusions Six possibly pathogenic variants of FZD4 or LRP5 were found in seven advanced ROP patients. Although these variants do not yet provide definitive evidence that they are causal, the results imply a role of the FZD4 and LRP5 genes in the development of advanced ROP.

Serum levels of matrix metalloproteinase 2 and matrix metalloproteinase 9 elevated in polypoidal choroidal vasculopathy but not in age-related macular degeneration

Abstract: Purpose Age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) are the leading causes of vision loss in the elderly Asian population. Previous studies have confirmed that abnormal extracellular matrix (ECM) metabolism plays an important role in the pathogenesis of AMD and PCV. However, the dynamic metabolism of the ECM is closely regulated by matrix metalloproteinases (MMPs) and tissue metalloproteinase inhibitors (TIMPs). Whether MMPs and TIMPs participate in the pathogenesis of AMD and PCV remains unclear. The aim of this study was to investigate the correlation between circulating MMP and TIMP levels and AMD and PCV. Methods The serum levels of MMPs (MMP1, MMP2, MMP3, and MMP9) and TIMPs (TIMP1 and TIMP3) were quantified using enzyme-linked immunosorbent assays in four groups of subjects (n=342): early AMD (group 1, n=75), neovascular AMD (group 2, n=89), PCV (group 3, n=98), and age- and gender-matched controls (group 4, n=80). Results The mean concentrations of the two gelatinases, MMP2 and MMP9, in the PCV group were significantly higher than that of the control (p=0.001, p 0.05). The mean serum levels of MMP1, MMP3, TIMP1, and TIMP3 were not significantly different among the four groups. Conclusions This pilot study first reveals a link between increased levels of circulating gelatinases (MMP2 and MMP9) and PCV but not AMD, which may provide a biologically relevant marker of ECM metabolism in patients with PCV. This finding suggests that the two disorders may have different molecular mechanisms.

Effects of polymorphisms in vitamin E-, vitamin C-, and glutathione peroxidase-related genes on serum biomarkers and associations with glaucoma.

Abstract: +-dependent L-ascorbic acid transporter 2 (SLC23A2) gene, rs6994076 in the tocopherol alpha transfer protein (TTPA) gene, rs737723 in the tocopherol-associated protein (SEC14L2/TAP) gene, and rs757228 in the glutathione peroxidase 4 (GPX4) gene. We also analyzed expression of the SLC23A2 gene in a subsample. Results: We found a novel association between the rs737723 polymorphism and POAG risk. Homozygous subjects for the C allele had a higher POAG risk than carriers of the ancestral G allele (adjusted odds ratio 1.73, 95% confidence interval 1.13–2.65, p=0.011). This association remained statistically significant after adjustment for multiple comparisons. We also confirmed the association between the rs1279683 polymorphism and a higher POAG risk in GG homozygous subjects and detected statistically significant differences in SLC23A2 gene expression between POAG cases and controls, even after adjustment for multiple testing. We observed a nominally significant (p<0.05) gene–gene interaction between the SEC14L2/TAP and SLC23A2 polymorphisms in determining POAG risk, increasing POAG risk in those subjects who had both risk genotypes at the same time (p<0.01). This increase was statistically significant even after adjustment for multiple comparisons. We did not detect any association with POAG risk for the rs6994076 or rs757228 polymorphisms. We also found that POAG patients had statistically significant (after correction for multiple testing) lower plasma vitamin E (p<0.001) and vitamin C (p<0.001) concentrations than control subjects. However, we detected a higher plasma GPx activity in POAG cases than in controls (p<0.001). The rs6994076 and rs1279683 polymorphisms were significantly (p<0.001) associated with plasma vitamin E and vitamin C, respectively. However, the rs757228 polymorphism in the GPX4 gene was not associated with plasma GPx activity. Conclusions: We have described a novel association between the rs737723 polymorphism (SEC14L2/TAP) and higher POAG risk and confirmed the association between rs1279683 ( SLC23A2) and POAG. Our results also suggested a gene– gene interaction between both polymorphisms that increases POAG risk.