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Vitamin A dimers trigger the protracted death of retinal pigment epithelium cells.

ABSTRACT: Cellular events responsible for the initiation of major neurodegenerative disorders of the eye leading to blindness, including age-related macular degeneration, Stargardt and Best diseases, are poorly understood. Accumulation of vitamin A dimers, such as N-retinylidene-N-retinylethanolamine (A2E) in the retinal pigment epithelium (RPE), is one of the earliest measurable events preceding retinal degeneration. However, the extent to which these dimers contribute to tissue degeneration is not clear. To determine if A2E could trigger morphological changes associated with the degenerating RPE and subsequent cell death, we evaluated its toxicity to cultured human RPE cells (ARPE-19). We show that A2E triggered the accumulation of debris followed by a protracted death. A2E was up to ? 14-fold more toxic than its precursor, retinaldehyde. Measurements reveal that the concentration of A2E in the aged human eye could exceed the concentration of all other retinoids, opening the possibility of A2E-triggered cell death by several reported mechanisms. Findings suggest that accumulation of vitamin A dimers such as A2E in the human eye might be responsible for the formation of ubiquitous RPE debris, an early indication of retinal degeneration, and that preventing or reducing the accumulation of vitamin A dimers is a prudent strategy to prevent blindness.

SUBMITTER: Mihai DM

PROVIDER: S-EPMC4123103 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Vitamin A dimers trigger the protracted death of retinal pigment epithelium cells.

Mihai D M DM Washington I I

Cell death & disease 20140724

Cellular events responsible for the initiation of major neurodegenerative disorders of the eye leading to blindness, including age-related macular degeneration, Stargardt and Best diseases, are poorly understood. Accumulation of vitamin A dimers, such as N-retinylidene-N-retinylethanolamine (A2E) in the retinal pigment epithelium (RPE), is one of the earliest measurable events preceding retinal degeneration. However, the extent to which these dimers contribute to tissue degeneration is not clear ...[more]

PMID: 25058422

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Project description:The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell-derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE-derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting "immature" gene regulatory networks.

Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

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Vitamin A dimers trigger the protracted death of retinal pigment epithelium cells.

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Vitamin A dimers trigger the protracted death of retinal pigment epithelium cells.

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