Chronic Internal Exposure to Low Dose 137Cs Induces Positive Impact on the Stability of Atherosclerotic Plaques by Reducing Inflammation in ApoE-/- Mice

Abstract

After Chernobyl and Fukushima Daï Chi, two major nuclear accidents, large amounts of radionuclides were released in the environment, mostly caesium 137 (137Cs). Populations living in contaminated territories are chronically exposed to radionuclides by ingestion of contaminated food. However, questions still remain regarding the effects of low dose ionizing radiation exposure on the development and progression of cardiovascular diseases. We therefore investigated the effects of a chronic internal exposure to 137Cs on atherosclerosis in predisposed ApoE-/- mice. Mice were exposed daily to 0, 4, 20 or 100 kBq/l 137Cs in drinking water, corresponding to range of concentrations found in contaminated territories, for 6 or 9 months. We evaluated plaque size and phenotype, inflammatory profile, and oxidative stress status in different experimental groups. Results did not show any differences in atherosclerosis progression between mice exposed to 137Cs and unexposed controls. However, 137Cs exposed mice developed more stable plaques with decreased macrophage content, associated with reduced aortic expression of pro-inflammatory factors (CRP, TNFα, MCP-1, IFNγ) and adhesion molecules (ICAM-1, VCAM-1 and E-selectin). Lesions of mice exposed to 137Cs were also characterized by enhanced collagen and smooth muscle cell content, concurrent with reduced matrix metalloproteinase MMP8 and MMP13 expression. These results suggest that low dose chronic exposure of 137Cs in ApoE-/- mice enhances atherosclerotic lesion stability by inhibiting pro-inflammatory cytokine and MMP production, resulting in collagen-rich plaques with greater smooth muscle cell and less macrophage content.

Fig 1. ¹³⁷Cs activity measurement correlates with ¹³⁷Cs absorption in the gastrocnemus muscle, validating our experimental model.

Activity is expressed as mean ± SEM of n = 8 per group per time.

Fig 2. ¹³⁷Cs exposure does not interfere with weight gain in ApoE^-/- mice.

Weight (in g) is expressed as mean ± SEM of n = 8 animals per group per time.

Fig 3. Atheromatous lesion area is not modified by a 6 or 9 month chronic internal exposure to ¹³⁷Cs.

A: Representative pictures of oil red O staining on aortic sinus cryosections after 6 months of exposure (magnification x 50). B: Quantification of the average lesion area performed using Histolab software. Lesion areas are expressed as mean ± SEM of n = 5 to 7 sections per animal.

Fig 4. A six months exposure to 20 and 100 kBq/l ¹³⁷Cs reduces macrophage content in atheromatous plaques.

A: Macrophages were detected by CD68 immunostaining (CD68⁺ cells: green; nuclei: blue). Representative pictures obtained at magnification x100. B: Quantification of CD68⁺ cell content. Results are expressed as mean ± SEM of CD68⁺ surface area per plaque surface area, proportional to Cs 0 control group (%). *p<0.05, **p<0.01 vs non-exposed group. n = 5 sections per animal.

Fig 5. Aortic mRNA expression of inflammatory cytokines and adhesion molecules following a 6 or 9 month exposure to ¹³⁷Cs.

Levels of CRP, TNFα, MCP-1, IFNγ, ICAM-1, VCAM-1 and E-Sel were determined by RT-qPCR. GAPDH or HPRT were amplified and used as endogenous control. At 6 months, IFNγ and MCP-1 expression was significantly decreased in animals exposed to 100 kBq/l ¹³⁷Cs compared to non-exposed animals; at 9 months, TNFα and ICAM-1 expression was significantly reduced. Results are expressed as mean of fold change ± SEM of n = 5–8 per group per time. *p < 0.05 and **p<0.01 versus Cs 0 control group.

Fig 6. Indices of atheromatous plaque stability are enhanced after 9 months exposure to 100 kBq/l ¹³⁷Cs.

A: αSMA immunostaining for smooth muscle cells (αSMA⁺ cells: green; nuclei: blue, n = 5 sections per animal) and B: αSMA quantification within the plaques. No difference is observed after 6 months exposure, however, after 9 months exposure, a significant increase in αSMA⁺ area is noted for the group exposed to 20 kBq/l. C: Picrosirius red staining for collagen was performed on aortic sinus cryosections. Representative images obtained at magnification x100. n = 5 sections per animal. D: Quantification of collagen in the plaque. After 6 and 9 months of exposure to 100 kBq/l ¹³⁷Cs, collagen content was increased in lesions. E: This result was paralleled by an increase in col3 mRNA expression, assessed by RT-qPCR, in the whole aorta. Results are expressed as mean ± SEM of n = 5–8. *p<0.05 vs Cs 0 control group.

Fig 7. Aortic expression of MMP-2, -3, -8 and -13 mRNA after 6 or 9 months exposure to ¹³⁷Cs.

MMP aorta expression was evaluated using milliplex kit (for MMP2, -3 and -8) and by RT-qPCR for MMP13. A and B: No differences were observed for MMP2 and MMP3 expression levels at every ¹³⁷Cs concentration and time exposure. C and D: concerning MMP8 and MMP13, we noticed a significant decrease for ApoE^-/- Cs100 after 6 months exposure. However, after 9 months, levels of MMP8 and -13 for this group are similar to control level. Results are expressed as mean ± SEM of n = 5–8 animals per group per time. **p<0.01 vs control. E: In-situ gelatinase activity, detected by enhanced fluorescence of fluorogenic gelatine substrate within the plaques and no difference were observed in MMP activity whatever the group.

Fig 8. Changes in oxidative stress and related enzyme expression after 6 or 9 months exposure to ¹³⁷Cs.

A: Representative images of superoxide production within the atheromatous plaques evaluated by DHE staining after 9 months exposition (magnification x 200). n = 5 sections per animal. B: Qualitative analyses did not revealed any differences in aortic mRNA expression of HO-1 or Nrf2 between exposed or non-exposed groups, measured by RT-qPCR. C: However, aortic mRNA expression of GPx was reduced at 6 months in animals exposed to 100 kBq/l ¹³⁷Cs. Moreover, GPx activity was significantly increased at 9 months, compared with non-exposed animals. Results are expressed as mean ± SEM of n = 5–8 animals per group per time. *p<0.05 vs Cs 0 control group.