Epigenomic and Transcriptomic Changes During Human RPE EMT in a Stem Cell Model of Epiretinal Membrane Pathogenesis and Prevention by Nicotinamide

Abstract

Epithelial to mesenchymal transition (EMT) is a biological process involved in tissue morphogenesis and disease that causes dramatic changes in cell morphology, migration, proliferation, and gene expression. The retinal pigment epithelium (RPE), which supports the neural retina, can undergo EMT, producing fibrous epiretinal membranes (ERMs) associated with vision-impairing clinical conditions, such as macular pucker and proliferative vitreoretinopathy (PVR). We found that co-treatment with TGF-β and TNF-α (TNT) accelerates EMT in adult human RPE stem cell-derived RPE cell cultures. We captured the global epigenomic and transcriptional changes elicited by TNT treatment of RPE and identified putative active enhancers associated with actively transcribed genes, including a set of upregulated transcription factors that are candidate regulators. We found that the vitamin B derivative nicotinamide downregulates these key transcriptional changes, and inhibits and partially reverses RPE EMT, revealing potential therapeutic routes to benefit patients with ERM, macular pucker and PVR.

Keywords: contractility; epigenetics; epithelial to mesenchymal transition; mesenchymal-to-epithelial transition; nicotinamide; proliferative vitreoretinopathy; retinal pigment epithelium; whole transcriptome.

Graphical abstract

Figure 1

TGF-β1 + TNF-α Co-treatment (TNT) of RPESC-RPE Cells Induces EMT and Formation of In Vitro Membranes Similar to ERMs (A) Schematic of RPE isolation and in vitro ERM model generation. (B) Phase images showing RPE cells in control conditions (cobblestone morphology) or 5 days after treatment with 10 ng/mL of TGF-β1, TNF-α, or both TGF-β1 + TNF-α (TNT). (C) Time course (0, 1, 3, and 5 days) of SNAI1, SNAI2, and TWIST1 expression in control RPESC-RPE cells (vehicle treated) and with 10 ng/mL of TGF-β1, TNF-α, or both (TNT), n = 3 biological replicates. (D) Immunofluorescence images of RPE cultures stained with anti-SNAI1 antibody show upregulation in TNT conditions. (E) Fundus image of a patient with ERM (indicated by dotted line). (F) RNA isolated from patient ERMs compared with in vitro RPE in control and TNT conditions assessed by qPCR for expression of RPE and EMT genes. n = 4 biological replicates. Scale bars, 50 μm. ^∗∗p ≤ 0.01.

Figure 2

TGF-β1 + TNF-α (TNT) Co-treatment of RPESC-RPE Cells Induces Global Epigenetic Changes at Enhancer Elements (A) TNT co-treatment induced changes in H3K27 acetylation: abscissa—normalized read density of H3K27ac ChIP-seq at chromatin features (putative enhancers and promoters) in cobblestone RPE cells; ordinate—normalized read density of H3K27ac ChIP-seq in TNT-treated RPE cells. Regions in red have significantly upregulated ChIP signal upon TNT treatment (FDR < 0.01), in blue downregulated (FDR < 0.01), and in orange no change (FDR < 0.1). (B) Changes in the H3K4me1 ChIP signal are correlated with H3K27ac changes, regions color coded according to the H3K27ac classes defined in (A). (C) TGF-β1 + TNF-α treatment results in relatively few changes in promoter signatures. Negative values indicate a promoter-like chromatin signature at interrogated sites. Color coding as in previous panels. (D) Most changes in H3K27 acetylation occur at sites distal from annotated transcription start sites. Plotted is the absolute distance to the closest annotated TSS versus the log2 fold change in the H3K27ac ChIP signal: genes associated with distal elements with upregulated H3K27ac (red), downregulated H3K27ac (blue), or unchanged (orange). The differences are significant with p values indicated (Mann-Whitney-Wilcoxon test). n = 2 biological replicates.

Figure 3

RNA-Seq of RPE Undergoing TNT-Stimulated EMT Correlated with Epigenomic Changes (A and B) KEGG pathway enrichment analysis using the genes significantly upregulated (A) or downregulated (B) after TNT treatment (C and D) Gene ontology category analysis with goseq in combination with cateGOizer to classify and summarize the enrichment analysis revealed pathways enriched TNT-induced EMT RPE (C) and pathways enriched in cobblestone RPE (D). N = 2 biological replicates.

Figure 4

Integration of Transcriptional Changes and Epigenetic Changes during TNT-Induced EMT (A) Scatterplot of expression changes after TNT treatment with selected transcription factors highlighted. (B) Venn diagram showing the intersection between the differentially expressed genes and genes with either enhancer only or enhancer and promoter peaks. (C) Venn diagrams of genes with significantly changing peaks in the cobblestone RPE (yellow) or with TNT-induced EMT RPE (green) overlapping with genes exhibiting increased (red) or decreased (blue) expression during EMT. (D and E) Homer motif analysis of the subset of peaks and genes identified in (C) by ^∗ (D) or $ (E) identified key TFs potentially regulating the genes in each subset. Directed graphs based on the motif analysis were constructed and the minimum connecting dominating sets were identified. Shown are the subgraphs of the TFs for the RPE network (D) and the TNT network (E). Complete graphs and the motif analysis can be found at http://neuralsci.org/computing.

Figure 5

Genes Changing Expression during TNT-Induced EMT versus Impact of NAM Treatment on Human RPE (A) RPE genes changing after NAM treatment and after TNT treatment. (B) Scatterplot of the 226 genes significantly changed in RPE cells common to both NAM and TNT treatments. x axis: log2 fold change after TNT treatment; y axis: log2 fold change after NAM treatment. Lower right and upper left quadrants (blue boxes) contain genes whose expression in TNT is opposed by NAM treatment. (C and D) Gene ontology enrichment analysis of gene categories aggregated based on semantic similarity using REVIGO and plotted as treemaps with top level descriptions, (C) genes upregulated during TNT-induced EMT, (D) genes from the lower right quadrant of (B). Comparison of the treemaps reveals similar strand enrichment for gene ontology categories related to blood vessel development, cell movement and adhesion, and apoptosis. Minor categories have been removed from treemap display to increase readability.

Figure 6

NAM Protects RPESC-RPE Cells from Undergoing EMT (A) Schematic of RPESC-RPE culture procedure and analysis. (B) Phase images of adult human RPESC-RPE cultures without (CONTROL) and with NAM. Scale bars, 100 μm. (C) TER measurements of adult human RPE cultured without (CONT) and with NAM. N = 3 biological replicates. (D) Immunofluorescence images of ZO-1 and CLDN19 (red) after 1 month in culture with NAM (nuclear stain, DAPI, blue). Scale bars, 100 μm. (E) qPCR for RPE identity genes MITF, OTX2, and RPE65. N = 12 biological replicates. (F) Western blot analysis for E- and N-cadherin in three adult human RPE lines; vinculin as loading control. (G) qPCR analysis of EMT master regulator genes (SNAI1 and SNAI2) and EMT marker (ACTA2). All values are relative to control without NAM (−). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; n = 3 biological replicates.

Figure 7

NAM Promotes RPESC-RPE MET and Inhibits 3D ERM-like Membrane Formation (A) Schematic of experiment to determine if NAM can stimulate MET. (B) Phase images of RPE cultures showing NAM treatment restores cobblestone morphology, scale bars, 100 μm. (C and D) qPCR analysis for (C) RPE identity genes MITF and RPE65, and (D) EMT master genes SNAI1 and SNAI2. N = 5 biological replicates. (E) Schematic of experiment to determine if NAM can prevent TNT-induced changes in adult RPESC-RPE cultures. (F) Phase images of cultured adult human RPE cells after 5 days of culture in CONTROL (Vehicle), TNT, or TNT + NAM. Scale bars, 100 μm. (G) qPCR analysis of EMT master genes SNAI1 and SNAI2. N = 3 biological replicates. (H) Phase images of primary adult human RPE after 5 days in culture in CONTROL (Vehicle), TNT, or TNT + NAM. Scale bars, 100 μm. (I) Quantification of number of 3D masses produced primary RPE with vehicle control (pCON), primary RPE treated with TNT (pTNT), RPESC-RPE (sTNT), primary RPE treated with TNT + NAM (pTNT + NAM). N = 3 biological replicates. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.