Global Transcriptome Analysis Reveals Distinct Phases of the Endothelial Response to TNF

Abstract

The vascular endothelium acts as a dynamic interface between blood and tissue. TNF-α, a major regulator of inflammation, induces endothelial cell (EC) transcriptional changes, the overall response dynamics of which have not been fully elucidated. In the present study, we conducted an extended time-course analysis of the human EC response to TNF, from 30 min to 72 h. We identified regulated genes and used weighted gene network correlation analysis to decipher coexpression profiles, uncovering two distinct temporal phases: an acute response (between 1 and 4 h) and a later phase (between 12 and 24 h). Sex-based subset analysis revealed that the response was comparable between female and male cells. Several previously uncharacterized genes were strongly regulated during the acute phase, whereas the majority in the later phase were IFN-stimulated genes. A lack of IFN transcription indicated that this IFN-stimulated gene expression was independent of de novo IFN production. We also observed two groups of genes whose transcription was inhibited by TNF: those that resolved toward baseline levels and those that did not. Our study provides insights into the global dynamics of the EC transcriptional response to TNF, highlighting distinct gene expression patterns during the acute and later phases. Data for all coding and noncoding genes is provided on the Web site (http://www.endothelial-response.org/). These findings may be useful in understanding the role of ECs in inflammation and in developing TNF signaling-targeted therapies.

FIGURE 1.

Overview of TNF-regulated genes in human endothelial cells. HUVECs (ECs, n = 5) were treated with or without TNF-α and harvested at 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, or 72 h before RNA-sequencing analysis. Genes were classified as (A) upregulated or (B) downregulated by TNF (FC versus untreated control >2 or <0.5, respectively). Plots show (i) total TNF-regulated genes (in both sexes) and corresponding biotype and the number of (ii) protein coding, (iii) lncRNA, or (iv) pseudogenes regulated at each time point, by sex. (C) GO analysis showing overrepresented terms for all TNF-regulated transcripts. (D and E) The number of genes classified as either upregulated or downregulated, respectively, in females or males only or in both sexes. Heatmaps show relative expression in control or TNF-treated female (left) or male (right) samples for genes reaching the threshold for classification as TNF regulated in (i) males or (ii) females only.

FIGURE 2.

WGNCA reveals temporal relationships between TNF upregulated genes: Group 1 “early induced.” HUVECs (ECs, n = 5) were treated with or without TNF-α and harvested at 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, or 72 h before RNA-sequencing analysis. WGNCA was used to cluster genes into modules, based on expression pattern similarity across sample sets. (A) (i) Dendrogram showing WGNCA modules and (ii) corresponding distribution of genes previously classified as TNF upregulated. For modules (B) orange, (C) purple, (D) black, (E) and royal blue, (i) relative gene expression plots displaying the module eigengenes and (ii) heatmaps showing the temporal expression profile for all genes in the module with eigengene >0.8 (p < 0.05). (iii) Circle plots for the top 50 genes classified as TNF upregulated within each module, showing expression values in control and stimulated ECs, the peak FC, the biotype and number of PubMed hits for “gene name” + “TNF,” and (iv) overrepresented terms by GO analysis (biological processes).

FIGURE 3.

WGNCA reveals temporal relationships between TNF upregulated genes: Group 2 “delayed induced.” HUVECs (ECs, n = 5) were treated with or without TNF-α and harvested at 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, or 72 h before RNA-sequencing analysis. WGNCA was used to cluster genes into modules, based on expression pattern similarity across sample sets. (A) (i) Dendrogram showing WGNCA modules and (ii) corresponding distribution of genes previously classified as TNF downregulated. For modules (B) tan and (C) turquoise, (i) relative gene expression plots displaying the module eigengenes and (ii) heatmaps showing the temporal expression profile for all genes in the module with correlation to the eigengene >0.8 (p < 0.05). (iii) Circle plots for the top 50 genes classified as TNF upregulated within each module, showing expression values in control and stimulated ECs, the peak FC, the biotype and number of PubMed hits for “gene name” + “TNF,” and (iv) overrepresented terms by GO analysis (biological processes). (D and E) The number of hits returned for TNF upregulated genes in the tan or turquoise modules, respectively, in a PubMed search for “gene name” + “IFN” and “gene name” + “IFN” + “endothelial,” with temporal expression plots for selected examples (created using the Web site tool provided as part of this study). (F) Temporal distribution of IFN-related genes upregulated by TNF across orange, royal blue, tan, and turquoise modules.

FIGURE 4.

TNF upregulation of IFN-stimulated and pattern recognition receptor gene expression is independent of de novo IFN production. (A) Temporal expression profiles in unstimulated control or TNF-α–stimulated ECs (n = 6) for IFNB1 and (i) ISG20, (ii) IFIT3, (iii) IFI35, (iv) CXCL10, (v) CXCL11, (vi) MX1, (vii) DDX58, (viii) TRL2, (ix) NOD2, and (x) IFIH1 (sample set F). (B) Summary of key genes and pathways linked to IFN-stimulated gene expression: (i) NFKB-IRF1 signaling (94), (ii) RIG-I-like receptor signaling (95), (iii) JAK-STAT signaling (96), (iv) TLR signaling (97), and (v) cGAS-STING signaling (95). Heatmaps show the differential gene expression between unstimulated control and TNF-stimulated ECs for the adjacent gene. Gray squares in the heatmap are the result of zero TPM values; thus, differential expression is not calculated. Bold gene symbols denote those that were classified as TNF upregulated. Heatmaps were created using the Web site tool provided as part of this study.

FIGURE 5.

WGNCA reveals temporal relationships between TNF-downregulated genes. HUVECs (ECs, n = 5) were treated with or without TNF-α and harvested at 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, or 72 h before RNA-sequencing analysis. WGNCA was used to cluster genes into modules, based on expression pattern similarity across sample sets. (A) (i) Dendrogram showing WGNCA modules and (ii) corresponding distribution of genes previously classified as TNF downregulated. For modules (B) dark orange, (C) saddle brown, (D) green, and (E) light yellow: (i) relative gene expression plots displaying the module eigengenes and (ii) heatmaps showing the temporal expression profile for all genes in the module with correlation to the eigengene >0.8 (p < 0.05). (iii) Circle plots for genes classified as TNF downregulated within each module, showing expression values in control and stimulated ECs, the peak FC, and the biotype and number of PubMed hits for “gene name” + “TNF.”