Effect of captopril on radiation-induced TGF-β1 secretion in EA.Hy926 human umbilical vein endothelial cells

Abstract

The pathophysiological mechanism involved in the sustained endothelial secretion of cytokines that leads to fibrosis 6-16 months after radiotherapy remains unclear. Angiotensin II (Ang II) is produced by the endothelium in response to stressing stimuli, like radiation, and may induce the synthesis of TGF-β, a profibrotic cytokine. In this study we tested the hypothesis that captopril, an angiotensin-converting enzyme (ACE) inhibitor, inhibits or attenuates radiation-induced endothelial TGF-β1 secretion. The human endothelial hybrid cell line EA.HY926 was irradiated with split doses of x-rays (28 Gy delivered in 14 fractions of 2 Gy). TGF-β1 mRNA, TNF-α mRNA and TGF-β1 protein levels were evaluated by RT-PCR and western blotting each month until the fifth month post radiation. Ang II was detected using radioimmunoassays, NF-κB activity was examined using EMSA, and western blotting was used to detect the expression of Iκ-Bα. To explore the role of Ang II on radiation-induced TGF-β1 release and Iκ-Bα expression, captopril was added to cultured cells before, during, or after irradiation. Sustained strong expression of TGF-β1 was observed after conventional fractionated irradiation. TNF-α, Ang II, and NF-κB activity were also increased in EA.Hy926 cells after radiation. Captopril decreased Ang II expression, inhibited the NF-κB pathway and reduced TGF-β1 expression. These data suggest that captopril might protect the endothelium from radiation-induced injury.

Keywords: EA.Hy926; NF-κB; TGF-β1; captopril; irradiation.

Figure 1. Sustained secretion of TGF-β1 in irradiated EA.Hy926 cells

(A) After exposure to ionizing radiation, no sustained expression of TGF-β1 mRNA was detected in A549 cells. (B) From the first month to the fifth month following 28 Gy radiation, the expression of TGF-β1 mRNA in EA.Hy926 cells increased significantly compared with the control group (P < 0.05). (C and D) TGF-β1 protein levels varied consistently with the mRNA changes (P < 0.05). *P < 0.05 compared to the control group; **P < 0.01 compared to the control group.

Figure 2. Effects of radiation on TNF-α and Ang II mRNA expression

(A) Significantly higher TNF-α mRNA expression was observed three months after irradiation in A549 cells. (B) TNF-α mRNA levels increased significantly at one and four months after irradiation in EA.Hy926 cells. (C) No significant Ang II mRNA expression changes were observed after radiation exposure in A549 cells. (D) Compared with control cells, Ang II expression increased significantly in EA.Hy926 cells one to four months after radiation exposure. *P < 0.05 compared to the control group; **P < 0.01 compared to the control group.

Figure 3. Radiation activates the NF-κB pathway in EA.Hy926 cells

(A and B) Compared with control cells, Iκ-Bα protein levels were significantly elevated after irradiation. (C) EMSA assay showing NF-κB activation in irradiated EA.Hy926 cells. Lane 1: EA.Hy926 no radiation Negative control; Lane 2: EA.Hy926 no radiation Experimental group; Lane 3: EA.Hy926 no radiation Cold competition group; Lane 4: EA.Hy926 no radiation Competition mutation group; Lane 5: EA.Hy926 2 Gy × 14 Negative control; Lane 6: EA.Hy926 2 Gy × 14 Experimental group; Lane 7: EA.Hy926 2 Gy × 14 Cold competition group; Lane 8: EA.Hy926 2 Gy × 14 Competition mutation group. *P < 0.05 compared to the control group; **P < 0.01 compared to the control group.

Figure 4. Captopril blocks angiotensin II expression in EA.Hy926 cells

(A) After the last irradiation, quantitative real-time RT-PCR was used to detect TNF-α. Cap treatment had no effect on radiation-induced TNF-α mRNA levels. (B) After the last irradiation, Ang II expression in the cell supernatants was measured by radioimmunoassay. While Cap alone had no effect, Cap treatment before IR, after IR, or in-between IR all inhibited Ang II expression induced by IR. *P < 0.05 compared to the control group; **P < 0.01 compared to the control group.

Figure 5. Captopril reduces TGF-β1 expression by inhibiting the transcriptional activity of NF-κB

(A) Cap treatment before IR, after IR, or in-between IR significantly decreased TGF-β1 mRNA expression induced by IR (B). TGF-β1 and Iκ-Bα protein expression assessed by Western blot. After irradiation, TGF-β1 protein changes were coincident with the changes observed in TGF-β1 mRNA. Densitometry analysis showed that while Cap treatment alone did not affect the expression of either protein, it decreased IR-induced TGF-β1 (C) and Iκ-Bα (D) protein levels.