Leukocyte chemotactic receptor Fpr1 protects against aging-related posterior subcapsular cataract formation.

Cataracts are a common consequence of aging; however, pathogenesis remains poorly understood. Here, we observed that after 3 months of age mice lacking the G protein-coupled leukocyte chemotactic receptor Fpr1 (N-formyl peptide receptor 1) began to develop bilateral posterior subcapsular cataracts that progressed to lens rupture and severe degeneration, without evidence of either systemic or local ocular infection or inflammation. Consistent with this, Fpr1 was detected in both mouse and human lens in primary lens epithelial cells (LECs), the only cell type present in the lens; however, expression was confined to subcapsular LECs located along the anterior hemispheric surface. To maximize translucency, LECs at the equator proliferate and migrate posteriorly, then differentiate into lens fiber cells by nonclassical apoptotic signaling, which results in loss of nuclei and other organelles, including mitochondria which are a rich source of endogenous N-formyl peptides. In this regard, denucleation and posterior migration of LECs were abnormal in lenses from Fpr1-/- mice, and direct stimulation of LECs with the prototypic N-formyl peptide agonist fMLF promoted apoptosis. Thus, Fpr1 is repurposed beyond its immunoregulatory role in leukocytes to protect against cataract formation and lens degeneration during aging.

Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration.

Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch's membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10 337 cases and 11 174 controls (OR = 1.10; P-value = 3.79 × 10(-5)). Thus, it appears that rare and common variants in a single gene--FBN2--can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch's membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.

Wnt signaling in age-related macular degeneration: human macular tissue and mouse model.

BACKGROUND: The wingless-type MMTV integration site (Wnt) signaling is a group of signal transduction pathways. In canonical Wnt pathway, Wnt ligands bind to low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6), resulting in phosphorylation and activation of the receptor. We hypothesize that canonical Wnt pathway plays a role in the retinal lesion of age-related macular degeneration (AMD), a leading cause of irreversible central visual loss in elderly. METHODS: We examined LRP6 phosphorylation and Wnt signaling cascade in human retinal sections and plasma kallistatin, an endogenous inhibitor of the Wnt pathway in AMD patients and non-AMD subjects. We also used the Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mouse models with AMD-like retinal degeneration to further explore the involvement of Wnt signaling activation in the retinal lesions in those models and to preclinically evaluate the role of Wnt signaling suppression as a potential therapeutic option for AMD. RESULTS: We found higher levels of LRP6 (a key Wnt signaling receptor) protein phosphorylation and transcripts of the Wnt pathway-targeted genes, as well as higher beta-catenin protein in AMD macula compared to controls. Kallistatin was decreased in the plasma of AMD patients. Retinal non-phosphorylated-ß-catenin and phosphorylated-LRP6 were higher in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice than that in wild type. Intravitreal administration of an anti-LRP6 antibody slowed the progression of retinal lesions in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mice. Electroretinography of treated eyes exhibited larger amplitudes compared to controls in both mouse models. A2E, a retinoid byproduct associated with AMD was lower in the treated eyes of Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice. Anti-LRP6 also suppressed the expression of Tnf-α and Icam-1 in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 retinas. CONCLUSIONS: Wnt signaling may be disturbed in AMD patients, which could contribute to the retinal inflammation and increased A2E levels found in AMD. Aberrant activation of canonical Wnt signaling might also contribute to the focal retinal degenerative lesions of mouse models with Ccl2 and Cx3cr1 deficiency, and intravitreal administration of anti-LRP6 antibody could be beneficial by deactivating the canonical Wnt pathway.

Platelet-derived growth factor (PDGF)-C inhibits neuroretinal apoptosis in a murine model of focal retinal degeneration.

Platelet-derived growth factor (PDGF)-C is a member of the PDGF family and is critical for neuronal survival in the central nervous system. We studied the possible survival and antiapoptotic effects of PDGF-C on focal retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) on C57BL/6N [Crb1(rd8)] (DKO rd8) background mice, a model for progressive and focal retinal degeneration. We found no difference in transcript and protein expression of PDGF-C in the retina between DKO rd8 mice and wild type (WT, C57BL/6N). Recombinant PDGF-CC protein (500 ng/eye) was injected intravitreally into the right eye of DKO rd8 mice with phosphate-buffered saline as controls into the left eye. The retinal effects of PDGF-C were assessed by fundoscopy, ocular histopathology, A2E levels, apoptotic molecule analysis, and direct flat mount retinal vascular labeling. We found that the PDGF-CC-treated eyes showed slower progression or attenuation of the focal retinal lesions, lesser photoreceptor and retinal pigment epithelial degeneration resulting in better-preserved photoreceptor structure. Lower expression of apoptotic molecules was detected in the PDGF-CC-treated eyes than in controls. In addition, no retinal neovascularization was observed after PDGF-CC treatment. Our results demonstrate that PDGF-C potently ameliorates photoreceptor degeneration via the suppression of apoptotic pathways without inducing retinal angiogenesis. The protective effects of PDGF-C suggest a novel alternative approach for potential age-related retinal degeneration treatment.

Nutrient supplementation with n3 polyunsaturated fatty acids, lutein, and zeaxanthin decrease A2E accumulation and VEGF expression in the retinas of Ccl2/Cx3cr1-deficient mice on Crb1rd8 background.

The Age-Related Eye Diseases Study 2 (AREDS2) clinical trial is assessing the effects of higher dietary xanthophyll (lutein and zeaxanthin) and long-chain n3 polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) intake on progression to advanced age-related macular degeneration (AMD). This study's purpose was to examine the retinal effects of the AREDS2 formulation on Chemokine (C-C motif) ligand 2 (Ccl2(-/-))/CX3C chemokine receptor 1 (Cx3cr1(-/-)) mice on Crumbs homolog 1 retinal degeneration phenotype 8 (Crb1(rd8)) background (DKO), which develop focal retinal lesions with certain features similar to AMD. DKO and C57BL/6N rd8 background mice (WT) were bred and randomized into 4 groups. Two groups, WT mice on AREDS2 diet (A-WT) and DKO mice on AREDS2 diet (A-DKO), were supplemented daily with 1.76 µmol of lutein, 35.1 µmol of zeaxanthin, 215 µmol EPA, and 107 µmol of DHA, and 2 control groups, WT mice on control diet (C-WT) and DKO mice on control diet (C-DKO), were fed an isocaloric diet. All mice had monthly fundus photos and were killed after 3 mo for biochemical and histologic analyses. After 3 mo, 81% of A-DKO mice had lesion regression compared with 25% of C-DKO mice (P < 0.05). Toxic retinal 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetra-enyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium (A2E) concentrations were significantly lower in A-DKO compared with C-DKO mice. The outer nuclear layer thickness in A-DKO mice was significantly greater than that in C-DKO mice. Retinal expression of inducible nitric oxide synthase (iNos) tumor necrosis factor-α (Tnf-α), Cyclooxygenase-2 (Cox-2), interleukin1beta (IL-1ß), and vascular endothelial growth factor (Vegf) was significantly lower in A-DKO compared with C-DKO mice. Xanthophylls and LCPUFAs have antiinflammatory, neuroprotective, and antiangiogenic properties. Our data provide potential mechanisms by which the AREDS2 formula has a protective effect on retinal lesions in DKO mice.

Genetic mechanisms and age-related macular degeneration: common variants, rare variants, copy number variations, epigenetics, and mitochondrial genetics.

Age-related macular degeneration (AMD) is a complex and multifaceted disease involving contributions from both genetic and environmental influences. Previous work exploring the genetic contributions of AMD has implicated numerous genomic regions and a variety of candidate genes as modulators of AMD susceptibility. Nevertheless, much of this work has revolved around single-nucleotide polymorphisms (SNPs), and it is apparent that a significant portion of the heritability of AMD cannot be explained through these mechanisms. In this review, we consider the role of common variants, rare variants, copy number variations, epigenetics, microRNAs, and mitochondrial genetics in AMD. Copy number variations in regulators of complement activation genes (CFHR1 and CFHR3) and glutathione S transferase genes (GSTM1 and GSTT1) have been associated with AMD, and several additional loci have been identified as regions of potential interest but require further evaluation. MicroRNA dysregulation has been linked to the retinal pigment epithelium degeneration in geographic atrophy, ocular neovascularization, and oxidative stress, all of which are hallmarks in the pathogenesis of AMD. Certain mitochondrial DNA haplogroups and SNPs in mitochondrially encoded NADH dehydrogenase genes have also been associated with AMD. The role of these additional mechanisms remains only partly understood, but the importance of their further investigation is clear to elucidate more completely the genetic basis of AMD.

Early degeneration of photoreceptor synapse in Ccl2/Cx3cr1-deficient mice on Crb1(rd8) background.

Photoreceptor ribbon synapse releases glutamate to postsynaptic targets. The synaptic ribbon may play multiple roles in ribbon synapse development, synaptic vesicle recycling, and synaptic transmission. Age-related macular degeneration (AMD) patients appear to have fewer or no detectable synaptic ribbons as well as abnormal swelling in the photoreceptor terminals in the macula. However, reports on changes of photoreceptor synapses in AMD are scarce and photoreceptor type and quantity affected in early AMD is still unclear. Here, we employed multiple anatomical techniques to investigate these questions in Ccl2⁻/⁻/Cx3cr1⁻/⁻ mouse on Crb1(rd8) background (DKO rd8) at one month of age. We found that approximately 17% of photoreceptors over the focal lesion were lost. Immunostaining for synapse-associated proteins (CtBP2, synaptophysin, and vesicular glutamate transporter 1) showed significantly reduced expression and ectopic localization. Cone opsins demonstrated dramatic reduction in expression (S-opsins) and extensive mislocalization (M-opsins). Quantitative ultrastructural analysis confirmed a significant decrease in the number of cone terminals and nuclei, numerous vacuoles in remaining cone terminals, reduction in the number of synaptic ribbons in photoreceptor terminals, and ectopic rod ribbon synapses. In addition, glutamate receptor immunoreactivity on aberrant sprouting of rod bipolar cells and horizontal cells were identified at the ectopic synapses. These results indicate that synaptic alterations occur at the early stages of disease and cones are likely more susceptible to damage caused by DKO rd8 mutation. They provide a new insight into potential mechanism of vision function lost due to synaptic degeneration before cell death in the early stages of AMD.

Epigenetics in ocular diseases.

Epigenetics pertains to heritable alterations in gene expression that do not involve modification of the underlying genomic DNA sequence. Historically, the study of epigenetic mechanisms has focused on DNA methylation and histone modifications, but the concept of epigenetics has been more recently extended to include microRNAs as well. Epigenetic patterning is modified by environmental exposures and may be a mechanistic link between environmental risk factors and the development of disease. Epigenetic dysregulation has been associated with a variety of human diseases, including cancer, neurological disorders, and autoimmune diseases. In this review, we consider the role of epigenetics in common ocular diseases, with a particular focus on DNA methylation and microRNAs. DNA methylation is a critical regulator of gene expression in the eye and is necessary for the proper development and postmitotic survival of retinal neurons. Aberrant methylation patterns have been associated with age-related macular degeneration, susceptibility to oxidative stress, cataract, pterygium, and retinoblastoma. Changes in histone modifications have also been observed in experimental models of diabetic retinopathy and glaucoma. The expression levels of specific microRNAs have also been found to be altered in the context of ocular inflammation, retinal degeneration, pathological angiogenesis, diabetic retinopathy, and ocular neoplasms. Although the complete spectrum of epigenetic modifications remains to be more fully explored, it is clear that epigenetic dysregulation is an important contributor to common ocular diseases and may be a relevant therapeutic target.

Subconjunctival Palomid 529 in the treatment of neovascular age-related macular degeneration.

BACKGROUND: Recent evidence suggests that neovascular age-related macular degeneration (AMD) may have an immune mediated component. Palomid 529, an investigational medication involving the immune Akt/mTOR pathway, is unique in dissociating both targets of rapamycin complexes TORC1 and TORC2. This small short-term pilot study assesses the safety of subconjunctival Palomid 529 in the treatment of neovascular AMD, with some limited efficacy information. METHODS: In this 12-week phase I open-label prospective pilot study, five participants with neovascular age-related macular degeneration that were refractory to intravitreal anti-vascular endothelial growth factor (VEGF) received three serial monthly subconjunctival doses of 1.9 mg Palomid 529. All participants were also offered concomitant monthly intravitreal anti-VEGF injections. Safety was monitored via adverse events recording. Additional outcome measures included visual acuity, optical coherence tomography, fluorescein angiography, indocyanine green angiography and fundus photography. RESULTS: The study drug was well-tolerated by all participants. There were no drug-related adverse events and no serious adverse events. A depot formed at the injection site, which persisted at the end of the study. In these anti-VEGF refractory patients, no clinically important changes in best-corrected visual acuity, fluorescein leakage pattern, choroidal neovascularization size on indocyanine green angiography, or autofluorescence pattern on fundus autofluorescence were observed compared to baseline. The fluid status, assessed with optical coherence tomography showed that central retinal thickness and macular volume remained stable in three participants, while the other two participants clinically progressed. CONCLUSIONS: Serial subconjunctival injections of Palomid 529 were well-tolerated and resulted in depot formation. There were no concerns for any ocular or systemic toxicity during this small short-term study. Larger randomized studies are required to determine efficacy.

Enhanced apoptosis in retinal pigment epithelium under inflammatory stimuli and oxidative stress.

Age-related macular degeneration (AMD) is a neurodegenerative disease that causes irreversible central vision loss in the elderly. Retinal pigment epithelium (RPE) has been found to be a key component in AMD pathogenesis. The Ccl2(-/-)/Cx3cr1(-/-) (DKO) mouse on Crb1(rd8) background is created as an AMD model, developing AMD-like retinal lesions. Our study aimed to examine RPE apoptosis in DKO mouse and human ARPE-19 cell line. DKO RPE expressed higher apoptotic proteins when compared with age-matched wild type (WT) RPE in physiological conditions. Apoptosis of primary cultured mouse RPE was evaluated under stimulation with lipopolysaccharide for inflammatory stimulation and 2,3,7,8-tetrachlorodibenzo-p-dioxin or H(2)O(2) for oxidative stress. Compared with WT RPE, DKO RPE was more susceptible to Fas ligand (FasL)-mediated apoptosis under both inflammatory and oxidative stress, with less cell viability and higher expression of apoptotic transcripts and proteins. Decreased cell viability was also observed in ARPE-19 cells under each stimulus. Furthermore, we also investigated the anti-apoptotic effects of decoy receptor 3 (DcR3), a decoy receptor for FasL, on ARPE-19 cells under inflammatory and oxidative stress. DcR3 pre-incubated ARPE-19 cells showed decreased apoptosis, with increased cell viability and decreased expression of apoptotic transcripts and proteins under the stimuli. On the contrary, knockdown of DcR3 in ARPE-19 cells showed totally opposite results. Our study demonstrates that FasL-mediated RPE apoptosis may play a pivotal role in AMD pathogenesis.

Anti-inflammatory recombinant TSG-6 stabilizes the progression of focal retinal degeneration in a murine model.

BACKGROUND: Inflammatory responses are detected in the retina of patients with age-related macular degeneration and Ccl2-/-/Cx3cr1-/- mice on rd8 background,(Ccl2-/-/Cx3cr1-/- mice) a model that develops progressive age-related macular degeneration-like retinal lesions including focal photoreceptor degeneration, abnormal retinal pigment epithelium and A2E accumulation. Tumor necrosis factor-inducible gene 6 protein is an anti-inflammatory protein and has been shown to improve myocardial infarction outcome and chemically injured cornea in mice by suppressing inflammation. In this study, we evaluated the effect of an intravitreous injection of recombinant TSG-6 on the retinal lesions of Ccl2-/-/Cx3cr1-/- mice. METHODS: Recombinant TSG-6 (400 ng) was administered by intravitreous injection into the right eye of six-week-old Ccl2-/-/Cx3cr1-/- mice. Their left eye was injected with phosphate-buffered saline as a control. Funduscopic pictures were taken before injection and sequentially once a month after injection. The mice were killed two months after injection and the ocular histology examined. Retinal A2E, a major component of lipofuscin, was measured by high performance liquid chromatography. The microarray of ocular mRNA of 92 immunological genes was performed. The genes showing differentiated expression in microarray were further compared between the injected right eye and the contralateral (control) eye by [real-time quantitative reverse transcription polymerase chain reaction] qRT-PCR. RESULTS: The continuous monitoring of the fundus for two months showed a slower progression or alleviation of retinal lesions in the treated right eyes as compared with the untreated left eyes. Among 23 pairs of eyes, the lesion levels improved in 78.3%, stayed the same in 8.7% and progressed in 13.0%. Histology confirmed the clinical observation. Even though there was no difference in the level of A2E between the treated and the untreated eyes, microarray analysis of 92 immune genes showed that IL-17a was substantially decreased after the treatment. Expression of TNF-α showed a similar pattern to IL-17a. The results were consistent in duplicated arrays and confirmed by qRT-PCR. CONCLUSIONS: We concluded that intravitreous administration of recombinant TSG-6 might stabilize retinal lesions in Ccl2-/-/Cx3cr1-/- mice on rd8 background. Modulation of ocular immunological gene expressions, especially IL-17a, could be one of the mechanisms.

Suggestive association between PLA2G12A single nucleotide polymorphism rs2285714 and response to anti-vascular endothelial growth factor therapy in patients with exudative age-related macular degeneration.

PURPOSE: The use of anti-vascular endothelial growth factor (anti-VEGF) therapy, with drugs such as ranibizumab and bevacizumab, to treat neovascular age-related macular degeneration (nAMD) produces an effective but widely variable response. Identifying markers that predict differentiated response could serve as a valuable assay in developing more personalized medicine. This study aimed to identify single nucleotide polymorphisms (SNPs) that influence the outcome of treatment with anti-VEGF therapy for AMD. METHODS: One hundred six patients with nAMD were treated with either ranibizumab or bevacizumab as needed over a period of 12 months. Visual acuity and the presence of macular fluid were measured with optical coherence tomography at baseline, six months, and 12 months. Patients were then classified as good or poor responders based on change in visual acuity and macular fluid on follow-up visits. DNA extracted from blood was genotyped with a TaqMan-based allelic discrimination SNP assay for 21 SNPs in six candidate genes (PLAG12A, IL23R, STAT3, VEGFA, KDR, and HIF1A). The SNPs were primarily selected based on previously reported associations with AMD and functional involvement in angiogenesis pathways. SNPs shown to be promising for association with anti-VEGF therapy were then assessed in an independent AMD case-control cohort. RESULTS: Of the 106 patients with nAMD, 77 were classified as good responders and 29 as poor responders. For rs2285714 (PLA2G12A), the frequency of minor allele T was 40.1% for good responders compared to 51.7% for poor responders (odds ratio: 1.60, 95% confidence interval of odds ratio: 0.87-2.94, p=0.13). Genetic model analysis of rs2285714 (PLA2G12A) demonstrated an association between rs2285714 (PLA2G12A) and therapy response in a dominant genotypic model. Patients carrying at least one T allele of rs2285714 were 2.79 times (95% confidence interval=1.02-7.69, p<0.05) more likely to be poor responders (79.3% of poor responders) than good responders (57.3% of good responders). However, after adjusting for multiple testing by the false discovery rate or Bonferroni correction, the initially observed association was no longer statistically significant. No association was identified between the remaining SNPs and response status. The SNP rs2285714 of PLA2G12A was not significantly associated with AMD in an independent AMD case-control cohort. CONCLUSIONS: Data suggest a possible weak association between rs2285714 (PLA2G12A) and response to anti-VEGF therapy, but the association must be confirmed in additional cohorts with larger patient samples. Identifying factors that predict the differentiated response could provide a valuable assay for developing approaches in personalized medicine.

Differential promoter activities of functional haplotypes in the 5'-flanking region of human sulfotransferase 1A1.

Previously, we reported five common single nucleotide polymorphisms (SNPs), -624G>C, -396G>A, -358A>C, -341C>G, and -294T>C, and six common haplotypes (CGACT, GAACT, GGAGC, GGACC, CAACT, and GAACC) in the 5'-flanking region of the SULT1A1 gene that were associated with altered enzymatic activity. In the present study, we performed in vitro assays to determine the functional impact of these genetic variations on the promoter activity. Dual luciferase reporter assays revealed that these SNPs are located in a negative regulatory fragment of the SULT1A1 gene. Further experiments demonstrated that these SNPs and haplotypes affected promoter activities of SULT1A1. Electrophoretic mobility shift assays showed distinctive binding patterns for the SNPs -396G>A and -294T>C, due to differential binding affinities of the G/A alleles and the T/C alleles to nuclear proteins extracted from the liver carcinoma cell lines, HepG2 and Huh7.

IL-10 -1082 SNP and IL-10 in primary CNS and vitreoretinal lymphomas.

OBJECTIVES: Most primary central nervous system lymphomas (PCNSLs) and primary vitreoretinal lymphomas (PVRLs) are B-cell lymphomas that produce high levels of interleukin (IL)-10, which is linked to rapid disease progression. The IL-10 (-1082) G → A polymorphism (IL-10 SNP) is associated with improved survival in certain non-CNS lymphoma patients. PDCD4 is a tumor suppressor gene and upstream regulator of IL-10. This study examined the correlation between the IL-10 SNP, PDCD4 mRNA expression, and IL-10 expression (at transcript and protein levels) in these lymphoma cells. MATERIALS AND METHODS: Single-nucleotide polymorphism (SNP)-typing at IL-10 (-1082) was performed after microdissecting cytospun PVRL cells from 26 specimens. Vitreal IL-10 and IL-6 levels were measured by ELISA. PCNSL cells from 52 paraffin-embedded sections were microdissected and SNP typed on genomic DNA. RT-PCR was performed to analyze expression of IL-10 and PDCD4 mRNA. IL-10 (-1082) SNP typing was performed on blood samples of 96 healthy controls. We measured IL-10 (-1082) SNP expression in 26 PVRLs and 52 PCNSLs and examined its relationship with IL-10 protein and gene expression, respectively. RESULTS: More PVRL patients expressed one copy of the IL-10 ( -1082 ) G → A SNP with the GA genotype compared to controls. The frequencies of the three genotypes (AA, AG, GG) significantly differed in PVRL versus controls and in PCNSL versus controls. In PVRLs, the vitreal IL-10/IL-6 ratio was higher in IL-10 (-1082) AG and IL-10 (-1082) AA patients, compared to IL-10 (-1082) GG patients. IL-10 mRNA expression was higher in IL-10 (-1082) AG and IL-10 (-1082) AA PCNSLs, compared to IL-10 (-1082) GG PCNSLs. No correlation was found between IL-10 and PDCD4 expression levels in 37 PCNSL samples. CONCLUSIONS: PVRL and PCNSL patients had similar IL-10 (-1082) A allele frequencies, but genotype distributions differed from healthy controls. The findings suggest that the IL-10 (-1082) A allele is a risk factor for higher IL-10 levels in PVRLs and PCNSLs. Higher IL-10 levels have been correlated with more aggressive disease in both PVRLs and PCNSLs, making this finding an important and potentially clinically significant observation.

Evidence for enhanced tissue factor expression in age-related macular degeneration.

Tissue factor (TF) is the primary initiator of blood coagulation. In addition to hemostasis, TF can initiate intracellular signaling and promote inflammation and angiogenesis, the key processes underlying the pathogenesis of age-related macular degeneration (AMD). AMD, the leading cause of irreversible blindness among the elderly, involves many genetic and environmental risk factors, including oxidative stress and inflammation. In this study, TF expression was examined in human AMD tissue and in the eyes of a model of AMD, the Ccl2(-/-)/Cx3cr1(-/-) (DKO) mouse, as well as in the ARPE-19 cell line after lipopolysaccharide (LPS) and H(2)O(2) stimulation. Total RNA was extracted from tissue samples and further analyzed by real-time RT-PCR. Immunohistochemistry was performed to evaluate TF protein expression. In the human retina, a 32-fold increase of TF mRNA expression was detected in AMD macular lesions compared with normal maculae. TF protein expression was also enhanced in human AMD maculae. Similarly, TF transcript and protein expression were moderately increased in retinal lesions, neuroretinal tissue, and cultured RPE cells of DKO mice compared with age-matched wild-type mice. TF expression level correlated with age in both wild-type and DKO mice. In order to better understand how AMD might lead to enhanced TF expression, 1, 5, and 10 µg/ml LPS as well as 100 and 200 µM H(2)O(2) were used to stimulate ARPE-19 cells for 24 and 2 h, respectively. LPS treatment consistently increased TF transcript and protein expression. H(2)O(2) alone or in combination with LPS also moderately enhanced TF expression. These results indicate that upregulated TF expression may be associated with AMD, and inflammatory and oxidative stress may contribute to TF expression in AMD eyes.

Complement component C5a promotes expression of IL-22 and IL-17 from human T cells and its implication in age-related macular degeneration.

BACKGROUND: Age related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Recent studies have demonstrated a strong genetic association between AMD and complement factor H (CFH), the down-regulatory factor of complement activation. Elevated levels of complement activating molecules including complement component 5a (C5a) have been found in the serum of AMD patients. Our aim is to study whether C5a can impact human T cells and its implication in AMD. METHODS: Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of exudative form of AMD patients using a Ficoll gradient centrifugation protocol. Intracellular staining and enzyme-linked immunosorbent assays were used to measure protein expression. Apoptotic cells were detected by staining of cells with the annexin-V and TUNEL technology and analyzed by a FACS Caliber flow cytometer. SNP genotyping was analyzed by TaqMan genotyping assay using the Real-time PCR system 7500. RESULTS: We show that C5a promotes interleukin (IL)-22 and IL-17 expression by human CD4+ T cells. This effect is dependent on B7, IL-1ß and IL-6 expression from monocytes. We have also found that C5a could protect human CD4+ cells from undergoing apoptosis. Importantly, consistent with a role of C5a in promoting IL-22 and IL-17 expression, significant elevation in IL-22 and IL-17 levels was found in AMD patients as compared to non-AMD controls. CONCLUSIONS: Our results support the notion that C5a may be one of the factors contributing to the elevated serum IL-22 and IL-17 levels in AMD patients. The possible involvement of IL-22 and IL-17 in the inflammation that contributes to AMD may herald a new approach to treat AMD.

Cellular and 3D optical coherence tomography assessment during the initiation and progression of retinal degeneration in the Ccl2/Cx3cr1-deficient mouse.

Retinal pathologies common to human eye diseases, including abnormal retinal pigment epithelial (RPE) cells, drusen-like accumulation, photoreceptor atrophy, and choroidal neovascularization, have been reported in the Ccl2/Cx3cr1-deficient mouse. The Ccl2 gene encodes the pro-inflammatory chemokine CCL2 (MCP-1), which is responsible for chemotactic recruitment of monocyte-derived macrophages to sites of inflammation. The Cx3cr1 gene encodes the fractalkine receptor, CX3CR1, and is required for accumulation of monocytes and microglia recruited via CCL2. Chemokine-mediated inflammation is implicated in retinal degenerative diseases such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, and uveoretinitis, and proper chemokine signaling from the RPE, Müller glia, and astrocytes is necessary to regulate leukocyte trafficking. Therefore, this mouse, possessing aberrant chemokine signaling coupled with retinal degenerative pathologies, presents an ideal opportunity to investigate the effect of altered signaling on retinal homeostasis and photoreceptor degeneration. Since this mouse is a recent development, more data covering the onset, location, and progression rate of pathologies is needed. In the present study we establish these parameters and show two photoreceptor cell death processes. Our observations of decreased glutamine synthetase and increased glial fibrillary acidic protein suggest that Müller cells respond very early within regions where lesions are forming. Finally, we suggest that retinal angiomatous proliferation contributes to pathological angiogenesis in this Ccl2/Cx3cr1-deficient mouse.

Macrophage polarization in the maculae of age-related macular degeneration: a pilot study.

Macrophages can be polarized to exhibit either pro-inflammatory M1 or pro-angiogenic M2 phenotypes, but have high phenotypic plasticity. This pilot study investigated macrophage polarization in the macular retina and choroid of age-related macular degeneration (AMD) and non-AMD subjects, as well as in AMD choroidal neovascular membranes (CNVM). All specimens were evaluated for routine histopathology. Quantitative real-time polymerase chain reaction for representative M1 (CXCL11) and M2 (CCL22) transcripts were performed on macular choroidal trephines (MCT) of 19 AMD and nine non-AMD eye bank eyes, on the microdissected macular retinal cells from the archived slides of five geographic atrophic AMD, five exudative/neovascular AMD, and eight normal autopsied eyes, and on microdissected inflammatory cells from two surgically removed CNVM that did not respond to anti-vascular endothelial growth factor (VEGF) therapy. High M2-chemokine transcript and a low ratio of M1 to M2 chemokine transcript were found in aging non-AMD MCT. Advanced AMD maculae had a higher M1 to M2 chemokine transcript ratio compared to normal autopsied eyes. Macrophages in the two CNVM of patients unresponsive to anti-VEGF therapy were polarized toward either M1 or M2 phenotypes. The number of M2 macrophages was increased compared to M1 macrophages in normal aging eyes. A pathological shift of macrophage polarization may play a potential role in AMD pathogenesis.

Effects of flickering light on refraction and changes in eye axial length of C57BL/6 mice.

AIMS: To investigate the effectiveness and feasibility of inducing myopia in mice by flickering-light (FL) stimulation. METHODS: Forty-five 28-day-old C57BL/6 (B6) mice were randomly assigned to three groups: control group, FL stimulation group and form deprivation (FD) group. Mice in the control group were raised under 250 lux illumination from 8:00 a.m. to 8:00 p.m. Mice in the FL group were raised under illumination with a duty cycle of 50% at a flash rate of 2 Hz from 8:00 a.m. to 8:00 p.m. for 6 weeks. Mice in the FD group were raised under the same conditions as the control group; the right eyes of the mice were covered with semitransparent hemispherical plastic shells serving as eye diffusers. The refractive state and axial length (AL) of the right eyes were measured by eccentric infrared photorefraction and A-scan ultrasonography, respectively, before treatment and after 2, 4, 6 or 8 weeks' treatment. RESULTS: After 6 weeks' exposure to FL, the refraction became more myopic compared with the control group as indicated by longer AL compared with the control group (p < 0.05); the FD eyes were more myopic than the FL eyes (p < 0.05). However, some mice lost their eye diffusers, and lens opacities were found. CONCLUSION: Myopia can be induced by FL in B6 mice. The myopic shift induced by FL is less than that induced by FD, but FL causes fewer side effects, and is safery and easier to manipulate.

Republished review: Gene therapy for ocular diseases.

The eye is an easily accessible, highly compartmentalised and immune-privileged organ that offers unique advantages as a gene therapy target. Significant advancements have been made in understanding the genetic pathogenesis of ocular diseases, and gene replacement and gene silencing have been implicated as potentially efficacious therapies. Recent improvements have been made in the safety and specificity of vector-based ocular gene transfer methods. Proof-of-concept for vector-based gene therapies has also been established in several experimental models of human ocular diseases. After nearly two decades of ocular gene therapy research, preliminary successes are now being reported in phase 1 clinical trials for the treatment of Leber congenital amaurosis. This review describes current developments and future prospects for ocular gene therapy. Novel methods are being developed to enhance the performance and regulation of recombinant adeno-associated virus- and lentivirus-mediated ocular gene transfer. Gene therapy prospects have advanced for a variety of retinal disorders, including retinitis pigmentosa, retinoschisis, Stargardt disease and age-related macular degeneration. Advances have also been made using experimental models for non-retinal diseases, such as uveitis and glaucoma. These methodological advancements are critical for the implementation of additional gene-based therapies for human ocular diseases in the near future.