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Retinal pigment epithelial expression of complement regulator CD46 is altered early in the course of geographic atrophy.

ABSTRACT: In geographic atrophy (GA), the non-neovascular end stage of age-related macular degeneration (AMD), the macular retinal pigment epithelium (RPE) progressively degenerates. Membrane cofactor protein (MCP, CD46) is the only membrane-bound regulator of complement expressed on the human RPE basolateral surface. Based on evidence of the role of complement in AMD, we hypothesized that altered CD46 expression on the RPE would be associated with GA development and/or progression. Here we report the timeline of CD46 protein expression changes across the GA transition zone, relative to control eyes, and relative to events in other chorioretinal layers. Eleven donor eyes (mean age 87.0 ± 4.1 yr) with GA and 5 control eyes (mean age 84.0 ± 8.9 yr) without GA were evaluated. Macular cryosections were stained with PASH for basal deposits, von Kossa for calcium, and for CD46 immunoreactivity. Internal controls for protein expression were provided by an independent basolateral protein, monocarboxylate transporter 3 (MCT3) and an apical protein, ezrin. Within zones defined by 8 different semi-quantitative grades of RPE morphology, we determined the location and intensity of immunoreactivity, outer segment length, and Bruch's membrane calcification. Differences between GA and control eyes and between milder and more severe RPE stages in GA eyes were assessed statistically. Increasing grades of RPE degeneration were associated with progressive loss of polarity and loss of intensity of staining of CD46, beginning with the stages that are considered normal aging (grades 0-1). Those GA stages with affected CD46 immunoreactivity exhibited basal laminar deposit, still-normal photoreceptors, and concomitant changes in control protein expression. Activated or anteriorly migrated RPE (grades 2-3) exhibited greatly diminished CD46. Changes in RPE CD46 expression thus occur early in GA, before there is evidence of morphological RPE change. At later stages of degeneration, CD46 alterations occur within a context of altered RPE polarity. These changes precede degeneration of the overlying retina and suggest that therapeutic interventions be targeted to the RPE.

SUBMITTER: Vogt SD

PROVIDER: S-EPMC3202648 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Retinal pigment epithelial expression of complement regulator CD46 is altered early in the course of geographic atrophy.

Vogt Susan D SD Curcio Christine A CA Wang Lan L Li Chuan-Ming CM McGwin Gerald G Medeiros Nancy E NE Philp Nancy J NJ Kimble James A JA Read Russell W RW

Experimental eye research 20110612 4

In geographic atrophy (GA), the non-neovascular end stage of age-related macular degeneration (AMD), the macular retinal pigment epithelium (RPE) progressively degenerates. Membrane cofactor protein (MCP, CD46) is the only membrane-bound regulator of complement expressed on the human RPE basolateral surface. Based on evidence of the role of complement in AMD, we hypothesized that altered CD46 expression on the RPE would be associated with GA development and/or progression. Here we report the tim ...[more]

PMID: 21684273

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Project description:PurposeLipofuscin contained in the retinal pigment epithelium (RPE) is the main source of fundus autofluorescence (FAF), the target of an imaging method useful for estimating the progression of geographic atrophy (GA) in clinical trials. To establish a cellular basis for hyperfluorescent GA border zones, histologic autofluorescence (HAF) was measured at defined stages of RPE pathologic progression.DesignExperimental study.Participants and controlsTen GA donor eyes (mean age ± standard deviation, 87.1 ± 4.0 years) and 3 age-matched control eyes (mean age ± standard deviation, 84.0 ± 7.2 years) without GA.MethodsThe 10-micrometer-thick sections were divided into zones of RPE morphologic features according to an 8-point scale. Any HAF excited by 488 nm light was imaged by laser confocal microscopy. The HAF intensity summed along vertical lines perpendicular to Bruch's membrane at 0.2-?m intervals served as a surrogate for FAF. Intensity profiles in 151 zones were normalized to grade 0 at a standard reference location in each eye. Cross-sectional area, mean, and sum autofluorescence for individual RPE cells were measured (cellular autofluorescence [CAF]).Main outcome measuresStatistically significant differences in intensity and localization of HAF and CAF at defined stages of RPE morphologic progression for GA and control eyes.ResultsThe RPE morphologic features were most abnormal (cell rounding, sloughing, and layering; grade 2) and HAF intensity profiles were highest and most variable immediately adjacent to atrophic areas. Peaks in HAF intensity frequently were associated with vertically superimposed cells. The HAF value that optimally separated reactive RPE was 0.66 standard deviations more than the mean for uninvolved RPE and was associated with a sensitivity of 75.8% and a specificity of 76.3%. When variable cell area was accounted for, neither mean nor sum CAF differed significantly among the RPE pathologic grades.ConclusionsAreas with advanced RPE alterations are most likely to exhibit clinically recognizable patterns of elevated FAF around GA, but may not predict cells about to die, because of vertically superimposed cells and cellular fragments. These data do not support a role for lipofuscin-related cell death and call into question the rationale of treatments targeting lipofuscin.

Project description:Purpose: The morphology of the RPE shows minimal change as the neural retina and choriocapillaris differentiate. Nonetheless, initial studies of barrier-related proteins suggest extensive remodeling of the RPE in response to this changing environment. A genomic approach was used to investigate the extent of this remodeling. Methods: RPE was isolated from E7, E10, E14 and E18 chick embryos and total RNA extracted for probing the entire genome on Affymetrix microarray chips. Statistical parameters using ANOVA were adjusted to yield a theoretical false discovery rate of 5%. STEM software was used to cluster genes into statistically related patterns of expression. Gene ontology clustering, using Affymetrix software was used for functional clustering. The proteinlounge.com database was used as a source of known biological pathways. Results: Of the 37,694 probe sets on the microarray, 17,199 were not absent. Of the 20,495 expressed probes, the expression of 8,889 was developmentally regulated. 4814 of these could be clustered into 12 patterns of expression that were statistically significant. The developmental patterns of expression for 22 tight and adherens junction proteins have been reported. Only two showed small variations from the patterns revealed by the microarray. Further, as would be predicted, expression of genes that promote progression through the cell cycle decreased and genes that inhibit progression increased with age. The data indicate extensive remodeling of the extracellular matrix, cell surface receptors, cell-cell junctions, transcellular ion transport and signal transduction pathways throughout development. Notably, the appearance of claudin 20, ZO-3 and cadherins 13 and 20 very late in development suggest barrier properties continue to change after functional junctions are formed. Conclusions: The data reveal a far more dynamic view of the RPE and its interactions with its environment than would be expected from morphological examination. The remodeling of junctional complexes, extracellular matrix interactions and transcellular transport capabilities indicates a continuous remodeling of the blood-retinal barrier. These data provide a standard whereby culture models of RPE function and regulation may be judged. Keywords: time course

Project description:RationaleAge-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in the developed world. Aging, inflammation and complement dysregulation affecting the retinal pigment epithelium (RPE), are considered significant contributors in its pathogenesis and several evidences have linked tumor necrosis factor alpha (TNF-α) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown to have cytoprotective effects in human clinical trials as well as having anti-inflammatory and anti-vascular exudative effects in animals. The purpose of this study was to evaluate if acadesine is able to suppress TNF-α induced C3 in RPE cells.MethodsARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-α was used for C3 induction in the presence or absence of acadesine. Small molecule inhibitors and siRNA were used to determine if acadesine exerts its effect via the extracellular or intracellular pathway and to evaluate the importance of AMPK for these effects. The expression level of C3 was determined by immunoblot analysis.ResultsAcadesine suppresses TNF-α induced C3 in a dose dependent manner. When we utilized the adenosine receptor inhibitor dipyridamole (DPY) along with acadesine, acadesine's effects were abolished, indicating the necessity of acadesine to enter the cell in order to exert it's action. However, pretreatment with 5-iodotubericidin (5-Iodo), an adenosine kinase (AK) inhibitor, didn't prevent acadesine from decreasing TNF-α induced C3 expression suggesting that acadesine does not exert its effect through AMP conversion and subsequent activation of AMPK. Consistent with this, knockdown of AMPK α catalytic subunit did not affect the inhibitory effect of acadesine on TNF-α upregulation of C3.ConclusionsOur results suggest that acadesine suppresses TNF-α induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.

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Retinal pigment epithelial expression of complement regulator CD46 is altered early in the course of geographic atrophy.

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Retinal pigment epithelial expression of complement regulator CD46 is altered early in the course of geographic atrophy.

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