Degeneration modulates retinal response to transient exogenous oxidative injury.

PURPOSE: Oxidative injury is involved in retinal and macular degeneration. We aim to assess if retinal degeneration associated with genetic defect modulates the retinal threshold for encountering additional oxidative challenges. METHODS: Retinal oxidative injury was induced in degenerating retinas (rd10) and in control mice (WT) by intravitreal injections of paraquat (PQ). Retinal function and structure was evaluated by electroretinogram (ERG) and histology, respectively. Oxidative injury was assessed by immunohistochemistry for 4-Hydroxy-2-nonenal (HNE), and by Thiobarbituric Acid Reactive Substances (TBARS) and protein carbonyl content (PCC) assays. Anti-oxidant mechanism was assessed by quantitative real time PCR (QPCR) for mRNA of antioxidant genes and genes related to iron metabolism, and by catalase activity assay. RESULTS: Three days following PQ injections (1 µl of 0.25, 0.75, and 2 mM) the average ERG amplitudes decreased more in the WT mice compared with the rd10 mice. For example, following 2 mM PQ injection, ERG amplitudes reduced 1.84-fold more in WT compared with rd10 mice (p = 0.02). Injection of 4 mM PQ resulted in retinal destruction. Altered retina morphology associated with PQ was substantially more severe in WT eyes compared with rd10 eyes. Oxidative injury according to HNE staining and TBARS assay increased 1.3-fold and 2.1-fold more, respectively, in WT compared with rd10 mice. At baseline, prior to PQ injection, mRNA levels of antioxidant genes (Superoxide Dismutase1, Glutathione Peroxidase1, Catalase) and of Transferrin measured by quantitative PCR were 2.1-7.8-fold higher in rd10 compared with WT mice (p<0.01 each), and catalase activity was 1.7-fold higher in rd10 (p = 0.0006). CONCLUSIONS: This data suggests that degenerating rd10 retinas encounter a relatively lower degree of damage in response to oxidative injury compared with normal retinas. Constitutive up-regulation of the oxidative defense mechanism in degenerating retinas may confer such relative protection from oxidative injury.

Retinal function and structure in the hypotransferrinemic mouse.

PURPOSE: The iron carrier transferrin is expressed at remarkably high levels in normal retinas and is upregulated during retinal degeneration. The authors characterized the consequences of genetically reduced retinal transferrin production on retinal structure and function. METHODS: Hypotransferrinemic (HPX⁻/⁻) mice treated with weekly intraperitoneal salvage transferrin injections were examined at 1 and 2 months of age. HPX⁻/⁻, HPX⁺/⁻, and wild-type (WT) mice were evaluated by electroretinography, ophthalmoscopy, and histology. Retinal iron content and transferrin levels were measured. RNA levels of genes involved in iron homeostasis and antioxidative response were determined by quantitative PCR. Oxidative injury was assessed by immunostaining for 4-hydroxy-2-nonenal (HNE). RESULTS: At 2 months, dark-adapted, mixed rod-cone response b-wave amplitudes were significantly lower in HPX⁻/⁻ mice than in WT mice (340 ± 112 µV vs. 624 ± 134 µV [mean ± SEM]; P = 0.002). Oscillatory potentials were significantly suppressed in HPX mice, and ophthalmoscopy demonstrated marked retinal pallor. Quantitative immunostaining revealed a 39% reduction of transferrin content in HPX⁻/⁻ compared with WT retinas (P = 0.01). mRNA levels of Tf, Tf receptor, and ceruloplasmin were decreased, whereas mRNA for antioxidant genes were elevated in HPX⁻/⁻ retinas. HNE staining was reduced in mice carrying the mutant HPX allele. Histologic examination demonstrated preserved retinal structure, and retinal iron content was similar across the strains. CONCLUSIONS: Despite the lack of wild-type retinal transferrin production and low levels of retinal transferrin protein, the retinal morphology and retinal iron content in HPX⁻/⁻ mice treated by systemic salvage transferrin injections are normal until age 2 months. However, retinal function and gene expression of some of the iron-associated genes are significantly altered.

Zinc-desferrioxamine attenuates retinal degeneration in the rd10 mouse model of retinitis pigmentosa.

Iron-associated oxidative injury plays a role in retinal degeneration such as age-related macular degeneration and retinitis pigmentosa. The metallo-complex zinc-desferrioxamine (Zn/DFO) may ameliorate such injury by chelation of labile iron in combination with release of zinc. We explored whether Zn/DFO can affect the course of retinal degeneration in the rd10 mouse model of retinitis pigmentosa. Zn/DFO-treated animals showed significantly higher electroretinographic responses at 3 and 4.5 weeks of age compared with saline-injected controls. Corresponding retinal (photoreceptor) structural rescue was observed by quantitative histological and immunohistochemical techniques. When administered alone, the components of the complex, Zn and DFO, showed a lesser, partial effect. TBARS, a marker of lipid peroxidation, and levels of oxidative DNA damage as quantified by 8-OHdG immunostaining were significantly lower in Zn/DFO-treated retinas compared with saline-injected controls. Reduced levels of retinal ferritin as well as reduced iron content within ferritin molecules were measured in Zn/DFO-treated retinas. The data, taken together, suggest that the protective effects of the Zn/DFO complex are mediated through modulation of iron bioavailability, leading to attenuation of oxidative injury. Reducing iron-associated oxidative stress using complexes such as Zn/DFO may serve as a "common pathway" therapeutic approach to attenuate injury in retinal degeneration.

Characterizing the involvement of the nuclear factor-kappa B (NF kappa B) transcription factor in uveal melanoma.

Purpose. To examine the involvement of nuclear factor-kappa B (NFkappaB) pathways in uveal melanoma (UM) and to assess their potential as a therapeutic target for metastatic UM. Methods. Samples from primary (n = 7) and metastatic (n = 7) UM were evaluated for NFkappaB transcription factor family expression by quantitative PCR (QPCR), immunofluorescent staining, and Western blot analysis. The effect of two NFkappaB inhibitors, DHMEQ and BMS-345541, on two cell lines derived from UM liver metastases was assessed. Cell proliferation was examined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, methylene blue assay, and immunostaining for Ki-67. Apoptosis was assessed by immunostaining for activated caspase 3. Results. NFkappaB1, NFkappaB2, RelA, RelB, and NIK were expressed in primary UM and in its liver metastases. NFkappaB2, RelB, and NIK showed significantly higher mRNA levels in metastases from UM compared with primary tumors (3.4-fold, P = 0.03; 3.6-fold, P = 0.05; 3.5-fold, P = 0.03; respectively). NFkappaB2 protein activation was 3.9-fold higher in metastases (P = 0.03). NFkappaB inhibition reduced metastatic cell proliferation by 9.2-fold and 1.9-fold according to Ki67 staining (P = 0.04) and methylene blue assay (P = 6 x 10(-7)), respectively. Both NFkappaB inhibitors achieved dose-dependent reductions of UM cell proliferation in both cell lines (P < 0.001). NFkappaB inhibition resulted in a 6.3-fold increase of apoptosis (P = 7 x 10(-7)). Conclusions. These data indicate that the NFkappaB1 and NFkappaB2 pathways are active in both primary and metastatic UM and that these pathways regulate metastatic cell proliferation and apoptosis. The role of NFkappaB as a therapeutic target for UM should be further evaluated.

Association of neovascular age-related macular degeneration with specific gene expression patterns in peripheral white blood cells.

PURPOSE: Inflammation probably plays a major role in the pathogenesis of age-related macular degeneration (AMD). The authors evaluated whether AMD is associated with gene expression patterns in white blood cells (WBCs) and whether such a pattern may serve as a biomarker for the disease. METHODS: Microarray analysis of gene expression in peripheral WBCs was performed on patients with neovascular AMD (NVAMD; n = 16) and controls (n = 16). Results were validated using quantitative real-time RT-PCR (QPCR) on another set of patients (n = 14) and controls (n = 16), respectively. QPCR findings were evaluated using receiver operator characteristic (ROC) curves and correlated with genotyping for the major risk single nucleotide polymorphisms (SNPs) for AMD in the genes for complement factor H and LOC387715. RESULTS: NVAMD-associated expression was identified for eight sequences (false discovery rate [FDR] = 0%) and 167 sequences (FDR = 10%), respectively. There was an enrichment of genes involved in antigen presentation among the AMD-associated genes (P = 0.0029). QPCR confirmed increased expression (1.6- to 4.3-fold) of four genes (HSPA8, IGHG1, ANXA5, VKORC1) in association with NVAMD (P = 0.02-0.0002). Area under the curve for these genes according to ROC analysis ranged from 0.776 to 0.815. Gene expression was not associated with genotyping for risk SNPs or WBC counts. CONCLUSIONS: NVAMD is associated with altered gene expression in peripheral WBCs that is not underlined by the major risk SNPs for the disease. Such altered expression may potentially serve as a biomarker for the disease. These data support the involvement of systemic immune response in the pathogenesis of AMD.

Lack of association between the C2 allele of transferrin and age-related macular degeneration in the Israeli population.

BACKGROUND: Altered iron metabolism and transferrin expression were associated with neurodegenerations including age-related macular degeneration (AMD) and Alzheimer's disease (AD). Carriers of transferrin C2 allele alone or in combination with the hemochromatosis C282Y variant may have increased risk for developing AD. We aim to assess if these alleles also predispose to AMD. METHODS: DNA was collected from 290 AMD patients and 157 unaffected, age-matched, controls. Genotyping was performed for transferrin C1/C2 alleles and hemochromatosis C282Y allele, and association with AMD was evaluated. RESULTS: There was no association between the C1/C2 transferrin alleles and AMD. Hemochromatosis C282Y variant was identified in four individuals; one was an AMD patient and three were unaffected. CONCLUSION: Transferrin C2 and hemochromatosis C282Y alleles are not associated with increased risk for developing AMD in Israel.

Alteration in iron metabolism during retinal degeneration in rd10 mouse.

PURPOSE: Altered iron metabolism was implicated in retinal and macular degeneration. This study was designed to further elucidate iron homeostasis during the course of retinal degeneration in mice. METHODS: Retinal mRNA and protein expression of transferrin, transferrin receptor, and ceruloplasmin were evaluated during retinal degeneration in rd10 mice and chemokine receptor 2 (ccr2)-deficient mice. Retinal ferritin protein levels, ferritin-bound iron, and total iron were evaluated in rd10 mice. RESULTS: Transferrin and ceruloplasmin mRNA levels increased between 2- and 12-fold during the course of retinal degeneration in rd10 mice compared with same-age controls (P < 0.01), whereas transferrin receptor mRNA levels increased only at the late stages of degeneration in rd10 mice (2.7-fold; P = 0.005). Transferrin mRNA also increased in retinas of aged ccr2-deficient mice (1.5-fold; P = 0.05). Transferrin and ceruloplasmin protein levels corroborated with mRNA levels changes in rd10 mice albeit at a lower magnitude. Retinal ferritin protein levels increased between 1.5-fold and 2-fold (P < 0.03) in rd10 mice, and ferritin-bound iron levels increased 1.6-fold in 3-week-old rd10 mice (P = 0.03). Three-week-old rd10 mice also had a 1.4-fold increase in total retinal iron level (P = 0.05). CONCLUSIONS: Combined with previous reports, these data suggest that retinal degenerations are associated with altered iron homeostasis regardless of the primary insult. Given the potential of iron to generate oxidative injury, its role as a therapeutic target in retinal and macular degenerations should be evaluated.

Sequence variants in HTRA1 and LOC387715/ARMS2 and phenotype and response to photodynamic therapy in neovascular age-related macular degeneration in populations from Israel.

PURPOSE: Single nucleotide polymorphisms (SNPs) in the tightly linked LOC387715/ARMS2 and HTRA1 genes have been associated with age-related macular degeneration (AMD). We tested whether these SNPs are associated with AMD in Israeli populations, if they underlie variable phenotype and response to therapy in neovascular AMD (NVAMD), and if HTRA1 expression in vivo is associated with its promoter variant. METHODS: Genotyping for the rs10490924 SNP in LOC387715/ARMS2 and the rs11200638 SNP in HTRA1 was performed on 255 NVAMD patients and 119 unaffected controls from Ashkenazi and Sephardic Jewish, and from Arab origins which are the main ethnic groups composing the Israeli population. Genotyping was correlated with phenotype and response to therapy among 143 patients who underwent photodynamic therapy (PDT). HTRA1 mRNA levels in white blood cells (WBCs), measured by quantitative PCR, were correlated with genotype in 27 participants. RESULTS: Both SNPs were in almost complete linkage disequilibrium (D'=0.96-1). Homozygotes for the T allele of rs10490924 had an odds ratio (OR) of 8.6, with a 95% confidence interval (CI) of 3.5-20.8, and homozygotes for the A allele of rs11200638 had an OR of 10.7, with a 95% CI of 3.2-35.7, for having AMD (p<0.00001). There was no association among these SNPs and phenotype or response to PDT. HTRA1 mRNA levels in WBCs were not associated with rs11200638 genotypes. CONCLUSIONS: The rs10490924 SNP in LOC387715/ARMS2 and the rs11200638 SNP in HTRA1 are strongly associated with NVAMD in this Israeli population. These variants do not have a major contribution to the variable phenotype and response to PDT which characterize NVAMD.

Association of complement factor H Y402H polymorphism with phenotype of neovascular age related macular degeneration in Israel.

PURPOSE: The Tyr402His variant of complement factor H (CFH) is associated with age-related macular degeneration (AMD) in several populations. Our aim was to evaluate if this single nucleotide polymorphism (SNP) is associated with AMD in the Israeli population and see if it underlies heterogeneity in clinical manifestation and responses to photodynamic therapy (PDT), which characterize neovascular AMD (NVAMD). METHODS: Genotyping for the Tyr402His variant was performed in 240 NVAMD patients (78.1+/-7 age range) and 118 controls (70.8+/-8.2 age range). Genotyping was correlated with clinical characteristics and treatment parameters in sequential 131 NVAMD patients who underwent PDT. RESULTS: TheTyr402His coding allele was associated with NVAMD in the Israeli population: odds ratio (OR)=1.9; 95% confidence interval (CI)=1.3-2.6; p=0.0002. Homozygosity for this variant was associated with an OR of 3.4 (95% CI: 1.7-6.8) for having AMD. There was no association among this SNP and age of onset of NVAMD, gender, neovascular lesion size, initial or final visual acuity, and number of PDT sessions required. CONCLUSIONS: In accordance with findings from the majority of previous study populations, the Tyr402His variant of CFH is associated with NVAMD in Israel. However, heterogeneity in clinical manifestations of NVAMD and in its response to PDT is not underlined by this CFH variant and may be accounted for by other genetic and environmental factors.

Inhibitor of apoptosis proteins gene expression and its correlation with prognostic factors in primary and metastatic uveal melanoma.

PURPOSE: Members of the inhibitors of apoptosis proteins (IAPs) family are thought to promote tumor growth and interfere with response to therapy by suppressing apoptosis in several malignancies. We aimed to evaluate the expression of IAPs in uveal melanoma (UM) and its correlation with prognostic factors associated with death from metastatic UM. METHODS: Expression of eight IAP genes [Baculoviral IAP repeat-containing (BIRC) 1-8] was evaluated through reverse transcription (RT)-PCR. BIRC5 and BIRC7 expression was measured using quantitative PCR (QPCR). BIRC5 protein expression was assessed with immunohistochemistry. QPCR results were correlated with apoptosis rate and with prognostic factors in UM, including lesion dimensions, cell type, monosomy 3, and vascular mimicry patterns. RESULTS: IAP genes were expressed in the majority of primary and metastatic UM. BIRC5 and BIRC7 levels were 8.8-fold (p = 0.0003) and 7.0-fold (p = 0.003) higher in tumors (primary and metastatic tissue) vs. normal eye tissue, respectively. BIRC5 levels correlated with presence of monosomy 3 (p = 0.01) and higher levels of BIRC7 correlated with epithelioid cell type (p = 0.048). CONCLUSIONS: IAPs expression is up regulated in UM and is associated with some of its prognostic factors. Considering our findings together with previous reports on their role in a variety of malignancies and in UM cell lines, it is conceivable that IAPs contribute to the remarkable resistance of uveal melanoma to apoptosis-inducing chemotherapy.