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

Abstract

Background and Purpose: Epidemiological data suggests an excess risk of cardiovascular disease (CVD) at low doses (0.05 and 0.1 Gy) of ionizing radiation (IR). Furthermore, the underlying biological and molecular mechanisms of radiation-induced CVD are still unclear. Because damage to the endothelium could be critical in IR-related CVD, this study aimed to identify the effects of radiation on immortalized endothelial cells in the context of atherosclerosis. Material and Methods: Microarrays and RT-qPCR were used to compare the response of endothelial cells irradiated with a single X-ray dose (0.05, 0.1, 0.5, 2 Gy) measured after various post-irradiation (repair) times (1 day, 7 days, 14 days). To consolidate and mechanistically support the endothelial cell response to X-ray exposure identified via microarray analysis, DNA repair signaling (γH2AX/TP53BP1-foci quantification), cell cycle progression (BrdU/7AAD flow cytometric analysis), cellular senescence (β-galactosidase assay with CPRG and IGFBP7 quantification) and pro-inflammatory status (IL6 and CCL2) was assessed. Results: Microarray results indicated persistent changes in cell cycle progression and inflammation. Cells underwent G1 arrest in a dose-dependent manner after high doses (0.5 and 2 Gy), which was compensated by increased proliferation after 1 week and almost normalized after 2 weeks. However, at this point irradiated cells showed an increased β-Gal activity and IGFBP7 secretion, indicative of premature senescence. The production of pro-inflammatory cytokines IL6 and CCL2 was increased at early time points. Conclusions: IR induces pro-atherosclerotic processes in endothelial cells in a dose-dependent manner. These findings give an incentive for further research on the shape of the dose-response curve, as we show that even low doses of IR can induce premature endothelial senescence at later time points. Furthermore, our findings on the time- and dose-dependent response regarding differentially expressed genes, cell cycle progression, inflammation and senescence bring novel insights into the underlying molecular mechanisms of the endothelial response to X-ray radiation. This may in turn lead to the development of risk-reducing strategies to prevent IR-induced CVD, such as the use of cell cycle modulators and anti-inflammatory drugs as radioprotectors and/or radiation mitigators.

Keywords: X-ray; atherosclerosis; cardiovascular disease; cell cycle; endothelium.

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., ; 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.