Advanced glycation end product (AGE) accumulation on Bruch's membrane: links to age-related RPE dysfunction.

PURPOSE: Advanced glycation end products (AGEs) accumulate during aging and have been observed in postmortem eyes within the retinal pigment epithelium (RPE), Bruch's membrane, and subcellular deposits (drusen). AGEs have been associated with age-related dysfunction of the RPE-in particular with development and progression to age-related macular degeneration (AMD). In the present study the impact of AGEs at the RPE-Bruch's membrane interface was evaluated, to establish how these modifications may contribute to age-related disease. METHODS: AGEs on Bruch's membrane were evaluated using immunohistochemistry. A clinically relevant in vitro model of substrate AGE accumulation was established to mimic Bruch's membrane ageing. Responses of ARPE-19 growing on AGE-modified basement membrane (AGE-BM) for 1 month were investigated by using a microarray approach and validated by quantitative (q)RT-PCR. In addition to identified AGE-related mRNA alterations, lysosomal enzyme activity and lipofuscin accumulation were also studied in ARPE-19 grown on AGE-BM. RESULTS: Autofluorescent and glycolaldehyde-derived AGEs were observed in clinical specimens on Bruch's membrane and choroidal extracellular matrix. In vitro analysis identified a range of dysregulated mRNAs in ARPE-19 exposed to AGE-BM. Altered ARPE-19 degradative enzyme mRNA expression was observed on exposure to AGE-BM. AGE-BM caused a significant reduction in cathepsin-D activity in ARPE-19 (P

Homocysteine, Methylenetetrahydrofolate Reductase C677T Polymorphism, and Risk of Retnal Vein Occlusion: A Meta-analysis

Objective: To assess the role of plasma total homocysteine (tHcy) concentrations and homozygosity for the thermolabile variant of the methylenetetrahydrofolate reductase (MTHFR) C677T gene as risk factors for retinal vascular occlusive disease.

Design: Retinal vein occlusion (RVO) is an important cause of vision loss. Early meta-analyses showed that tHcy was associated with an increased risk of RVO, but a significant number of new studies have been published. Participants and/or Controls: RVO patients and controls.

Methods: Data sources included MEDLINE, Web of Science, and PubMed searches and searching reference lists of relevant articles and reviews. Reviewers searched the databases, selected the studies, and then extracted data. Results were pooled quantitatively using meta-analytic methods.

Main Outcome Measures: tHcy concentrations and MTHFR genotype.

Results: There were 25 case-control studies for tHcy (1533 cases and 1708 controls) and 18 case-control studies for MTHFR (1082 cases and 4706 controls). The mean tHcy was on average 2.8 mol/L (95% confidence
interval [CI], 1.8 –3.7) greater in the RVO cases compared with controls, but there was evidence of between-study heterogeneity (P0.001, I2 93%). There was funnel plot asymmetry suggesting publication bias. There was no evidence of association between homozygosity for the MTHFR C677T genotype and RVO (odds ratio [OR] 1.20; 95% CI, 0.84–1.71), but again marked heterogeneity (P 0.004, I2 53%) was observed.

Conclusions: There was some evidence that elevated tHcy was associated with RVO, but not homozygosity for the MTHFR C677T genotype. Both analyses should be interpreted cautiously because of marked heterogeneity between the study estimates and possible effect of publication bias on the tHcy findings.

Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07.

PURPOSE. A spontaneously arising retinal pigment epithelial (RPE) cell line (B6-RPE07) was cloned from a primary culture of mouse RPE cells and maintained in culture for more than 18 months. Morphologic and functional properties of this cell line have been characterized.

METHODS. The morphology of the B6-RPE07 cells was examined by phase-contrast light microscopy, electron microscopy, and confocal microscopy. Barrier properties were measured by the flux of fluorescence from the apical to the basolateral compartment of culture chambers. The abilities of the cells to bind/phagocytose photoreceptor outer segments (POS) were determined by confocal microscopy, electron microscopy, and flow cytometry. Cytokine/chemokine secretion was measured by cytometric bead array. The expression of visual cycle proteins was determined by RT-PCR and Western blotting.

RESULTS. In standard culture conditions, B6-RPE07 cells display cobblestone morphology. When cultured on three-dimensional (3D) collagen gel–coated membranes, B6-RPE07 cells exhibit a monolayer epithelial polarization with apical surface microvilli. Immunohistochemistry of B6-RPE07 cultures revealed a high expression of pan-cytokeratin. B6-RPE07 cells also expressed the retinal pigment epithelium-specific marker CRALBP, but not RPE65. Cell junction proteins ZO-1 and ß-catenin, but not claudin-1/3 or occludin-1, were observed in B6-RPE07 cells. B6-RPE07 cells are able to bind, phagocytose, and digest POS. Finally, B6-RPE07 cells produce high levels of IL-6 and CCL2.

CONCLUSIONS. This is the first report of a mouse RPE cell line with morphology, phenotype, and function similar to those of in vivo mouse RPE cells. This cell line will be a valuable resource for future RPE studies, in particular for in vivo gene modification and transplantation studies.