Functional Gene Analysis Reveals Cell Cycle Changes and Inflammation in Endothelial Cells Irradiated with a Single X-ray Dose

Figure 1. Irradiated TICAE cells differentially express genes controlling cell cycling and inflammation. Gene ontology and ChIP enrichment analysis (ChEA) was performed on differentially expressed genes in irradiated vs. sham-irradiated TICAE cells. (n = 3). (A) Top 10 GO enrichment terms among upregulated differentially expressed genes 1 day after irradiation with a single X-ray dose of 2 Gy. (B) As in (A) but showing downregulated differentially expressed genes. (C) Top 10 enriched predicted upregulated transcriptional regulators in TICAE cells 1 day after irradiation with a single X-ray dose of 2 Gy. (D) As in (C) but showing downregulated transcriptional regulators. (E) As in (A) but at 7 days post-irradiation. (F) As in (B) but at 7 days post-irradiation. (G) As in (C) but at 7 days post-irradiation. (H) As in (D) but at 7 days post-irradiation. PubMed ID numbers are included in the ChIP enrichment analysis graphs. Enrichment was scored using FDR-corrected P-value. Full gene ontology and ChIP enrichment analysis results are listed in the Supplementary data files.

Figure 2. Irradiation induces dose- and time-dependent repression of the expression of genes controlling mitotic endothelial cell proliferation. Graphs show changes in gene expression of (A) BUB1, (B) MKI67 and (C) FAM111B. Left graphs show data obtained using microarrays (n = 3) and right graphs using RT-qPCR (n = 6). Of note, FAM111B expression was below detection threshold by RT-qPCR on day 14 post-irradiation. *P < 0.05, †P < 0.005, ‡P < 0.001 compared to sham, using 2-way ANOVA with Bonferroni post-hoc test.

Figure 3. Irradiation induces endothelial DNA damage signaling. Graphs represent the amount of γH2AX (A), TP53BP1 (B) and colocalized γH2AX/TP53BP1 (C) foci 30 min, 1, 4, and 24 h after irradiation with a single X-ray dose of 0.05, 0.1, 0.5 and 2 Gy (n = 6). Data show means ± SEM. *P < 0.05, †P < 0.005, ‡P < 0.001 compared to sham on the same day, using 2-way ANOVA with Bonferroni post-hoc test. (D) Representative images showing γH2AX (green), TP53BP1 (red) and γH2AX+TP53BP1 (yellow) foci in DAPI stained nuclei (blue) of TICAE cells 30 min after irradiation with a single X-ray dose of 2 Gy.

Figure 4. Irradiation reversibly inhibits cell cycle progression, induces premature endothelial senescence and promotes endothelial cell inflammation. (A) The histogram represents the % of TICAE cells in the different phases of the cell cycle at the indicated times after irradiation with the indicated doses (n = 6). (B) The histogram represents the activity of senescence-associated β-galactosidase (SA-β-gal) 14 days after irradiation at the indicated doses (n = 16). Data are normalized to cell numbers and control values. (C,D,E) Histograms represent the amount of IGFBP7 (C), IL6 (D) and CCL2 (E) secreted by TICAE cells at the indicated times after irradiation at the indicated doses (n = 8). Data are normalized to cell numbers alone (C) or to cell numbers and control values (D,E). Data show means ± SEM. *P < 0.05, †P < 0.005, ‡P < 0.001 compared to sham on the same day, using 2-way ANOVA with Bonferroni post-hoc test.

Figure 5. Schematic overview based on our experimental findings and literature that explains the possible role of p53 and NF-κB signaling in radiation-induced premature senescence. Irradiation of endothelial cells with a single X-ray dose leads to the formation of DNA damage, resulting in both p53 and NF-κB signaling, well-known signaling responses in irradiated cells (Yu, 2012). At day 0–2 post-irradiation, induced p53 signaling can result in a G1/S cell cycle block (Agarwal et al., 1995), which is removed at day 7 post-irradiation. Although not conclusive (question mark symbol), we also suggest the presence of a persistent p53 signaling at this time-point. NF-κB signaling on day 0–2 post-irradiation, on another hand, could induce endothelial inflammation (Kempe et al., 2005) that persists until day 7 post-irradiation. Both the persistent p53 signaling (Rufini et al., 2013) and inflammatory state (Freund et al., 2010; Kojima et al., 2013) may lead to premature senescence observed at day 14 after irradiation with a single X-ray dose. Symbols indicate the irradiation dose at which the described effects were observed: ‡ = 2 Gy, † = 0.5 Gy, * = 0.1 Gy, and $ = 0.05 Gy.