Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC

Dark Matters in AMD Genetics: Epigenetics and Stochasticity

Missing heritability in the study of complex disease has been called dark matter. This review enlarges the scope of this term to include epigenetics and stochasticity, nongenetic sources of risk in complex diseases such as age-related macular degeneration

The use of hyperoxia to induce chronic mild oxidative stress

The retinal pigment epithelial (RPE) cell is exposed to chronic oxidative stress in part from exposure to high partial pressures of oxygen and in part, from its high level of oxygen metabolism which generates large amounts of reactive oxygen intermediates (ROI) An alternative and perhaps more applicable approach to studying the effects of oxidative stress on RPE cells would apply chronic and lower levels of oxidative stress than the acute levels resulting from chemical oxidants. Recently, von Zglinicki et al. used chronic hyperoxia treatment to induce long term oxidative stress on lung fibroblasts METHODS Cell culture: The RPE340 cell line from one globe of a 1-year-old trauma victim was propagated as previously described Methods: RPE340 cells and WI38 lung fibroblasts were grown in normal oxygen (20% O 2 ) and hyperoxia (40% O 2 or 60% O 2 ). After cell viability was examined, the levels of reactive oxygen intermediates (ROI) by flow cytometry and heme oxygenase-1 (HO-1) mRNA by northern analysis were measured as markers of oxidative stress in both cell types. Proliferative ability and gene expression pattern of growth factors were studied to demonstrate the phenotypic changes induced by mild oxidative stress upon these cells. Results: While decreased by 60% O 2 , 40% O 2 did not affect viability in both cell types, ROI production and HO-1 mRNA expression were elevated in hyperoxia compared to controls, but were inhibited with the antioxidant dehydro-ascorbic acid (DHA). The proliferation of cells by hyperoxia was inhibited in both cell types. The expression of growth factors induced by hyperoxia was cell type dependent. Fibroblast growth factor-2 mRNA was unchanged in RPE cells, but was increased in fibroblasts. Transforming growth factor-β2 was decreased in RPE cells, but unchanged in fibroblasts. Vascular endothelial growth factor was downregulated in RPE cells, while upregulated in fibroblasts. Connective tissue growth factor was decreased in RPE cells, but was unchanged in fibroblasts. Conclusions: The results demonstrate that hyperoxia induces mild oxidative stress which alters the phenotype of cells in a cell type specific manner

Topographic and age-dependent expression of heme oxygenase-1 and catalase in the human retinal pigment epithelium. Invest Ophthalmol Vis Sci.

PURPOSE. To investigate the hypothesis that there are topographic and age-related changes in the expression of heme oxygenase (HO)-1 and catalase in the RPE. METHODS. Cryosections of the macula and periphery of human eyes (n ϭ 18; aged 27-87 years) were subjected to a highsensitivity digoxigenin (DIG)-labeled cRNA in situ hybridization protocol to determine the expression of HO-1 and catalase. The immunoreactivity of HO-1 and catalase were also investigated in the same sample set. Specimens were examined by light microscopy, and images were captured with a digital camera. The total number of RPE cells and HO-1-and catalaselabeled RPE cells was counted in each section, and the ratio of labeled RPE cells to total RPE cells was calculated in both the macular and the peripheral regions of each donor eye. RESULTS. There was a mosaic pattern of mRNA and protein expression of HO-1 and catalase in macular and peripheral RPE. Topographical differences in the expression of HO-1 at the mRNA level and catalase at both the mRNA and protein levels was also observed. The topographical differences between the expression of HO-1 in the macula and periphery protein were not statistically significant but showed similar trends. For HO-1, the only significant age-related decline in expression was observed in the macula and periphery. Expression of HO-1 at the protein level and that of catalase at both the mRNA and protein levels showed no significant decline with age. CONCLUSIONS. There is a possible age-related decline in HO-1 expression, whereas catalase expression remains unchanged with aging. Both exhibit mosaic patterns in the RPE monolayer

Comparison of In Vivo

Purpose. To identify retinal pigment epithelium (RPE)/choroid genes and their relevant expression pathways affected by intravitreal injections of dexamethasone and triamcinolone acetonide in mice at clinically relevant time points for patient care. Methods. Differential gene expression of over 34,000 well-characterized mouse genes in the RPE/choroid of 6-week-old C57BL/6J mice was analyzed after intravitreal steroid injections at 1 week and 1 month postinjection, using Affymetrix Mouse Genome 430 2.0 microarrays. The data were analyzed using GeneSpring GX 12.5 and Ingenuity Pathway Analysis (IPA) microarray analysis software for biologically relevant changes. Results. Both triamcinolone and dexamethasone caused differential activation of genes involved in “Circadian Rhythm Signaling” pathway at both time points tested. Triamcinolone (TAA) uniquely induced significant changes in gene expression in “Calcium Signaling” (1 week) and “Glutamate Receptor Signaling” pathways (1 month). In contrast, dexamethasone (Dex) affected the “GABA Receptor Signaling” (1 week) and “Serotonin Receptor Signaling” (1 month) pathways. Understanding how intraocular steroids affect the gene expression of RPE/choroid is clinically relevant. Conclusions. This in vivo study has elucidated several genes and pathways that are potentially altering the circadian rhythms and several other neurotransmitter pathways in RPE/choroid during intravitreal steroid injections, which likely has consequences in the dysregulation of RPE function and neurodegeneration of the retina

Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

PurposeTo determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.MethodsQuantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.ResultsCfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.ConclusionsOnly Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC

GSTM1 and GSTM5 Genetic Polymorphisms and Expression in Age-Related Macular Degeneration*

PurposePreviously, two cytosolic antioxidant enzymes, Glutathione S-transferase Mu 1 (GSTM1) and Mu 5 (GSTM5), were reduced in retinas with age-related macular degeneration (AMD). This study compared genomic copy number variations (gCNV) of these two antioxidant enzymes in AMD versus controls.MethodsGenomic copy number (gCN) assays were performed using Taqman Gene Copy Number Assays (Applied Biosystems, Darmstadt, Germany) in technical quadruplicate for both GSTM1 and GSTM5. Peripheral leukocyte RNA levels were compared with controls in technical triplicates. Statistical comparisons were performed in SAS v9.2 (SAS Institute Inc., Cary, NC).ResultsA large percentage of patients in both AMD and age-matched control groups had no copies of GSTM1 (0/0). The mean gCN of GSTM1 was 1.40 (range 0-4) and 1.61 (range 0-5) for AMD and control, respectively (p = 0.29). A greater percentage of control patients had > 3 gCNs of GSTM1 compared with AMD, respectively (15.3% versus 3.0%, p = 0.004). The gCN of GSTM5 was 2 in all samples except one control sample. The relative quantification of GSTM1 and GSTM5 mRNA from peripheral blood leukocytes in patients showed significant differences in relative expression in AMD versus control (p < 0.05). Peripheral blood leukocyte mRNA and gCN were not significantly correlated (p = 0.27).ConclusionSince high copy numbers of GSTM1 are found more frequently in controls than in AMD, it is possible that high copy number leads to increased retinal antioxidant defense. Genomic polymorphisms of GSTM1 and GSTM5 do not significantly affect the peripheral blood leukocyte mRNA levels