Gamma Radiation-Induced Disruption of Cellular Junctions in HUVECs Is Mediated through Affecting MAPK/NF- κ B Inflammatory Pathways - PubMed (original) (raw)

Gamma Radiation-Induced Disruption of Cellular Junctions in HUVECs Is Mediated through Affecting MAPK/NF- κ B Inflammatory Pathways

H Wang et al. Oxid Med Cell Longev. 2019.

Abstract

Ionizing radiation-induced cardiovascular diseases (CVDs) have been well documented. However, the mechanisms of CVD genesis are still not fully understood. In this study, human umbilical vein endothelial cells (HUVECs) were exposed to gamma irradiation at different doses ranging from 0.2 Gy to 5 Gy. Cell viability, migration ability, permeability, oxidative and nitrosative stresses, inflammation, and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-_κ_B) pathway activation were evaluated postirradiation. It was found that gamma irradiation at doses ranging from 0.5 Gy to 5 Gy inhibited the migration ability of HUVECs without any significant effects on cell viability at 6 h and 24 h postirradiation. The decreased transendothelial electrical resistance (TEER), increased permeability, and disruption of cellular junctions were observed in HUVECs after gamma irradiation accompanied by the lower levels of junction-related proteins such as ZO-1, occludin, vascular endothelial- (VE-) cadherin, and connexin 40. The enhanced oxidative and nitrosative stresses, e.g., ROS and NO2 - levels and inflammatory cytokines IL-6 and TNF-α were demonstrated in HUVECs after gamma irradiation. Western blot results showed that protein levels of mitogen-activated protein kinase (MAPK) pathway molecules p38, p53, p21, and p27 increased after gamma irradiation, which further induced the activation of the NF-_κ_B pathway. BAY 11-7085, an inhibitor of NF-_κ_B activation, was demonstrated to partially block the effects of gamma radiation in HUVECs examined by TEER and FITC-dextran permeability assay. We therefore concluded that the gamma irradiation-induced disruption of cellular junctions in HUVECs was through the inflammatory MAPK/NF-_κ_B signaling pathway.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1

Figure 1

Cell viability and migration ability in HUVECs after gamma irradiation of different doses. Gamma irradiation with doses ranging from 0.2 Gy to 5 Gy does not induce HUVEC viability changes when measured at 6 h (a) and 24 h (b) postirradiation by the MTT method. However, it increased the gap distance when HUVECs were irradiated with radiation doses ranging from 0.5 to 5 Gy (c). The cell viability of the treatment groups is expressed as the percentage of the control; cell migration is measured by gap closure test. ∗A value of p < 0.05 is taken as statistical significance.

Figure 2

Figure 2

Cellular junction examination in HUVECs after exposure to gamma irradiation. Immunofluorescence examination indicates the damage of the cellular barrier 24 h after irradiation with 5 Gy (the impaired and disconnected junctions between two adjoined cells pointed by arrows in the lower panels in (a)) (×400). Scale bar: 50 _μ_m. While the gene expressions of junction-related molecules such as ZO-1, occludin, VE-cadherin, and connexin 40 do not change significantly (b), there are obvious reductions of protein expressions of ZO-1, occludin, VE-cadherin, and connexin 40 (c).

Figure 3

Figure 3

Gamma irradiation induces oxidative and nitrosative stresses in HUVECs. The intracellular ROS is increased significantly 6 h after irradiation with 5 Gy (a). NO2− concentration is increased significantly 6 h after irradiation with doses from 0.5 to 5 Gy (b). Western blot shows increased eNOS after irradiation with doses from 0.2 to 5 Gy (c). ∗A value of p < 0.05 is taken as statistical significance.

Figure 4

Figure 4

Gamma irradiation induces gene and protein expressions of cytokines. Irradiation with 2 or 5 Gy induces upregulation of the IL-6 gene 6 h after irradiation (a), but upregulation of the IL-6 protein is induced by irradiation with doses ranging from 0.2 to 5 Gy (b). Both gene (c) and protein (d) expressions of TNF-α are upregulated after irradiation with 2 or 5 Gy. ∗A value of p < 0.05 is taken as statistical significance.

Figure 5

Figure 5

Gamma irradiation activates MAPK/NF-_κ_B pathways. (a) The activation of MAPK pathway molecules p-p38, p38, p53, p21, and p27 in HUVECs after exposure to gamma irradiation was measured by western blot. (b) Protein levels of p-p65 and p65 were examined by western blot. (c) NF-_κ_B DNA-binding activity by TransAM™ NF-_κ_B Transcription Factor Assay kit. The absorbance at 450 nm was read on a microplate reader. Values were expressed as mean ± S.E.M. One-way analysis of variance (ANOVA) was used to determine statistical significance between groups followed by Tukey's post hoc test. ∗A value of p < 0.05 was taken as statistical significance.

Figure 6

Figure 6

BAY 11-7085 treatment on the barrier function examined by ECIS and FITC-dextran permeability assay. Irradiation with 5 Gy significantly reduces the resistance in HUVECs when compared to the nonirradiated (0 Gy) group (a). The treatment with BAY 11-7085 at 2.5 μ_M partially blocked the decreased resistance in HUVECs caused by 5 Gy gamma irradiation, while the treatment alone did not have any significant effects in HUVECs without gamma radiation exposure (a). The fluoresce intensity of FITC-labelled dextrans (4 kDa) was significantly higher after 5 Gy gamma irradiation, and the treatment of BAY 11-7085 partially inhibited this increase (b). ∗_p < 0.05 vs. 0 Gy; #p < 0.05 vs. 5 Gy.

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