Abstract
BackgroundAge-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of neovascular fibrosis and/or cell death. Here, we investigate how repeated wound stimulus leads to seminal changes in gene expression and the onset of a perpetual state of stimulus-independent wound response in retinal pigmented epithelial (RPE) cells, a cell-type central to the etiology of AMD.MethodsTranscriptome wide expression profiles of human fetal RPE cell cultures as a function of passage and time post-plating were determined using Agilent 44 K whole genome microarrays and RNA-Seq. Using a systems level analysis, differentially expressed genes and pathways of interest were identified and their role in the establishment of a persistent mesenchymal state was assessed using pharmacological-based experiments.ResultsUsing a human fetal RPE cell culture model that considers monolayer disruption and subconfluent culture as a proxy for wound stimulus, we show that prolonged wound stimulus leads to terminal acquisition of a mesenchymal phenotype post-confluence and altered expression of more than 40 % of the transcriptome. In contrast, at subconfluence fewer than 5 % of expressed transcripts have two-fold or greater expression differences after repeated passage. Protein-protein and pathway interaction analysis of the genes with passage-dependent expression levels in subconfluent cultures reveals a 158-node interactome comprised of two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGFβ pathway activators: TGFB1, TGFB2, INHBA, INHBB, GDF6, CTGF, and THBS1. Significantly, inhibition of TGFBR1/ACVR1B mediated signaling using receptor kinase inhibitors both forestalls and largely reverses the passage-dependent loss of epithelial potential; thus extending the effective lifespan by at least four passages. Moreover, a disproportionate number of RPE wound response genes have altered expression in neovascular and geographic AMD, including key members of the TGFβ pathway.ConclusionsIn RPE cells the switch to a persistent mesenchymal state following prolonged wound stimulus is driven by lasting activation of the TGFβ pathway. Targeted inhibition of TGFβ signaling may be an effective approach towards retarding AMD progression and producing RPE cells in quantity for research and cell-based therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0183-x) contains supplementary material, which is available to authorized users.
Highlights
Age-related macular degeneration (AMD) is a leading cause of blindness
In this study we investigated the molecular basis of intrinsic retinal pigmented epithelial (RPE) wound repair, how extended periods of wound stimulus lead to a chronic state of wound response that is independent of further stimulus, and how these processes might relate to the progression of AMD
Using a cell culture model system that views disruption of cell monolayers during routine passage as a wound stimulus, we showed that when repeatedly passaged RPE reach confluence they fail to achieve a normal cell density, they fail to inactivate genes associated with successful wound repair, they fail to reactivate genes associated with terminally differentiated RPE, and they aberrantly upregulate genes that are unique to a terminal mesenchymal phenotype
Summary
Age-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of neovascular fibrosis and/or cell death. We investigate how repeated wound stimulus leads to seminal changes in gene expression and the onset of a perpetual state of stimulus-independent wound response in retinal pigmented epithelial (RPE) cells, a cell-type central to the etiology of AMD. Wound responses encompass a wide range of processes directed at repairing tissue and cell damage. Wound responses are often secondary or bystander responses that occur as a result of damage to neighboring cells or tissue, they can occur at the singlecell level [1]. Wound response inactivation requires cessation of damage, wound closure, resolution of the inflammatory response, and tissue remodeling or regeneration. The core processes of wound repair are common to all tissues, there can be substantial differences depending on the extent and nature of the insult, as well as the affected tissues and cell types
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