Ionizing Irradiation Induces Vascular Damage in the Aorta of Wild-Type Mice

Abstract

There has been a recent upsurge of interest in the effects of ionizing radiation exposure on the circulatory system, because a mounting body of epidemiological evidence suggests that irradiation induces cardio- and cerebrovascular disease at a much lower dose and lower dose rate than previously considered. The goal of our project is to determine whether dose protraction alters radiation effects on the circulatory system in a mouse model. To this end, the use of wild-type mice is pivotal albeit without manifestation of vascular diseases, because disease models (e.g., apolipoprotein E-deficient mice) are prone to hormetic responses following protracted exposures. As such, here, we first set out to analyze prelesional changes in the descending thoracic aorta of wild-type mice up to six months after a single acute exposure to 0 or 5 Gy of ¹³⁷Cs γ-rays. Scanning electron microscopy demonstrated that irradiation facilitated structural disorganizations and detachment of the aortic endothelium. The Miles assay with an albumin-binding dye Evans Blue revealed that irradiation enhanced vascular permeability. Immunofluorescence staining showed that irradiation led to partial loss of the aortic endothelium (evidenced by a lack of adhesion molecule CD31 and 4',6-diamidino-2-phenylindole (DAPI) signals), a decrease in endothelial nitric oxide synthase and adherens junction protein (vascular endothelial (VE)-cadherin) in the aortic endothelium, along with an increase in inflammation (tumor necrosis factor (TNF)-α) and macrophage (F4/80) markers in the aorta. These findings suggest that irradiation produces vascular damage manifested as endothelial cell loss and increased vascular permeability, and that the decreased adherens junction and the increased inflammation lead to macrophage recruitment implicated in the early stage of atherosclerosis.

Keywords: aorta; inflammation; ionizing radiation; vascular damage; vascular permeability.

Figure 1

Morphological changes in the aortic endothelium of B6J mice. (A) Representative FE-SEM images of (a) normal endothelium (6 months after 0 Gy), (b) detachment (1 month after 5 Gy), and (c) large detachment (1 month after 5 Gy). Boxed areas in the upper panels are shown at higher magnification in the lower panels. Scale bars as indicated. (B) Quantitative analysis for (a) the number of crests/field (10 mice/group analyzed), and (b,c) percentage of mice with detachment and large detachment, respectively (20 mice/group analyzed). (a) There was a difference among three post-irradiation timepoints in irradiated groups (ANOVA p = 0.003, pairwise p < 0.02 for 1 vs. 3 months and 1 vs. 6 months) but not in sham-irradiated controls (ANOVA p > 0.5). ** p < 0.001 for irradiated vs. sham-irradiated groups at each timepoint (by the two-sample t-test). ^† The degree of difference between irradiated and sham-irradiated groups at two timepoints (pairwise p = 0.015). (b,c) According to the Wald test, there was a difference among three post-irradiation timepoints in irradiated groups (p < 0.03 for 1 vs. 6 months and 3 vs. 6 months) but not in sham-irradiated controls (p > 0.5). ** p < 0.001, * 0.001 ≤ p < 0.05, or ns (nonsignificant, p > 0.1) for irradiated vs. sham-irradiated groups at each timepoint (by Fisher’s exact test).

Figure 2

Changes in vascular permeability in the descending thoracic aorta of B6J and ApoE^−/− mice evaluated with the Miles assay. (A) Representative images of extravasated dye. (B) Quantitative analysis for (a) stained area and (b) intensity (b) in B6J mice. 10 mice/group analyzed. According to the one-way ANOVA, a difference among three post-irradiation timepoints in irradiated groups was significant for stained area (ANOVA p = 0.03, pairwise p = 0.02 for 1 vs. 3 months) and nonsignificant for stained intensity (ANOVA p = 0.07), but a difference among four (1 non-irradiated and 3 sham-irradiated) control groups was nonsignificant for both stained area and intensity (ANOVA p > 0.5). ** p < 0.001 or * 0.001 ≤ p < 0.05 for irradiated vs. sham-irradiated groups at each timepoint (by the two-sample t-test). ^† 0.001 ≤ p < 0.05 for the degree of a difference between irradiated and sham-irradiated groups at two timepoints (pairwise p). (C) Quantitative analysis for (a) stained area and (b) intensity (b) in ApoE^−/− mice. 10–13 mice/group analyzed. ** p < 0.001, * 0.001 ≤ p < 0.05, or ns (nonsignificant, p > 0.1) between two groups (by the two-sample t-test). A difference between non-irradiated B6J (Figure 2(Ba,Bb)) and normal-fat diet-fed ApoE^−/− (Figure 2(Ca,Cb)) controls was not significant (p > 0.5). AU, arbitrary unit. C, non-irradiated or normal-fat diet-fed controls. HFD, high-fat diet.

Figure 3

Molecular changes in the aortic endothelium of B6J mice. (A) Representative merged images for double immunofluorescence of CD31, and (a) eNOS, (b) VE-cadherin, (c) TNF-α, or (d) F4/80, with cell nuclei counterstained with DAPI. (a,d) 1 month post-irradiation. (b,c) 3 months post-irradiation. Upper panels, 0 Gy. Lower panels, 5 Gy. Boxed areas in the left panels (tiled images) are shown at higher magnification in the right panels. Scale bars as indicated. (B) Quantitative analysis for (a) CD31 negativity, (b) DAPI negativity, (c) eNOS, (d) VE-cadherin, (e) TNF-α, and (f) F4/80. For CD31 negativity, DAPI negativity and VE-cadherin, there was no difference among three post-irradiation timepoints in irradiated groups (ANOVA p > 0.06) and in sham-irradiated controls (ANOVA p > 0.1). A difference among three post-irradiation timepoints was significant for eNOS (ANOVA p = 0.02, pairwise p = 0.01 for 1 vs. 6 months) and F4/80 (ANOVA p = 2 × 10⁻⁶, pairwise p < 2 × 10⁻⁵ for 1 vs. 3 months and 1 vs. 6 months) in irradiated groups but not in sham-irradiated controls (ANOVA p > 0.3). For TNF-α, a difference among three post-irradiation timepoints was significant in sham-irradiated controls (ANOVA p = 0.0005, pairwise p < 0.03 for 1 vs. 6 months and 3 vs. 6 months) but not in irradiated groups (ANOVA p > 0.9). ** p < 0.001, * 0.001 ≤ p < 0.05, or ns (nonsignificant, p > 0.1) between irradiated vs. sham-irradiated groups at each timepoint (by the two-sample t-test). ^†† p < 0.001, or ^† 0.001 ≤ p < 0.05 for the degree of a difference between irradiated and sham-irradiated groups at two timepoints (pairwise p). For CD31 negativity, DAPI negativity, eNOS, and VE-cadherin, there was no difference in the degree of intergroup differences between timepoints (ANOVA p > 0.1). 9–10 mice/group analyzed. AU, arbitrary unit.