Quantification of cardiac Fibrosis.

<p>Two sections of heart stained with Sirius Red and examined to quantify the percentage of cardiac fibrosis. <b>A:</b> Fibrosis quantification in ApoE−/− and C57Bl6 mice 40 weeks post-irradiation <b>B:</b> Fibrosis quantification in ApoE−/− and C57Bl6 mice 60weeks after irradiation. Non-irradiated ApoE−/− vs irradiated ApoE−/− *p<0.05;*** p<0.001 and non-irradiated C57Bl6 vs irradiated C57Bl6 ^μp<0.05. (n = 4–6 animals in each group and 2 sections of heart per animal).</p

Macrophages typing and quantification.

<p><b>A</b> and <b>B</b>: Quantitative analysis of number of positive CD68 and CD11b macrophages per mm² heart tissue area in C57Bl6 and ApoE−/− mice respectively (n = 2 sections of heart per animal and 3–5 animals per each group and each time point); ApoE−/− 0 Gy vs irradiated groups at 40weeks,*P<0.05;**P<0.01.</p

Leucocytes characterization and IL-6 Expression.

<p><b>A:</b> Quantitative analysis of the number of leucocytes per mm² tissue section area in C57Bl6 and ApoE−/− mice 40weeks post-irradiation; C57Bl6 0 Gy vs irradiated groups,*P<0.05 (n = 2 sections per heart and 3–5 animal per group and each time point). <b>B</b>: CD45R immunofluorescence (green) and DAPI staining (blue) at 0 and 2 Gy, 40weeks post-irradiation (original magnification ×1000). <b>C.</b> IL-6 ELISA in cardiac tissues from ApoE−/− and C57Bl6 mice, 20, 40 and 60weeks post-irradiation at 0, 0.2 and 2 Gy. ApoE−/− 0 Gy vs irradiated groups at 20weeks,*P<0.05; ApoE−/− 0 Gy vs irradiated groups at 40weeks; ^μP<0.05. (n = 4–6 samples).</p

Survival curve, weight and heart weight of mice post-irradiation.

<p><b>A</b> and <b>B</b> Survival curves of C57Bl6 and ApoE−/−Statistical analysis by Mantel-Cox test: ApoE−/− irradiated mice at 0.2 Gy <i>versus</i> age-matched controls P = 0.0019. <b>C</b> and <b>D</b> Weight of C57Bl6 and ApoE−/−. <b>E</b> and <b>F</b> weight of heart (2 ventricles)/weight of mice post-irradiation. * P<0.05; 0 Gy vs irradiated groups at 60weeks. Each group comprised of 7 to 10 mice.</p

Histological analysis of control and irradiated hearts 20, 40 and 60weeks post-irradiation.

<p><b>A</b> and <b>B.</b> Cardiomyocyte surface area measurement was performed on 180–200 cardiomyocytes from 4 serial slides (HES heart sections) of C57Bl6 and ApoE−/− respectively (n = 3–5 animals in each group), 0 Gy 20weeks vs 0 Gy 40weeks; * P<0.05; 0 Gy 20weeks vs 0 Gy 60weeks; * P<0.05; 0 Gy vs irradiated groups at 20weeks; £ P<0.05, ^££ P<0.01; 0 Gy vs irradiated groups at 40weeks; ^μ P<0.05, ^μμ P<0.01, 0 Gy vs irradiated groups at 60weeks; ^$ P<0.05, ^$$$ P<0.001. <b>C</b> and <b>D.</b> Histological assessment of cardiac ventricular pathology by HES staining in C57Bl6 and ApoE−/− respectively (n = 4–6 animals in each group and 2 sections of heart per animal). Original magnification ×400, <b>C</b> in C57Bl6 and <b>D</b> in ApoE−/−.</p

The measurements of cardiac physiologic parameters: Heart rate, Ejection fraction (EF), Shortening fraction (FS), left ventricular end-diastolic dimension (LVEDD) as well as the interventricular septal thickness in diastole (IVSd), LV posterior wall thickness (LVPWd) and LVmass/Body weigh (LV mass/BW).

<p>Sham 20weeks compared by sham groups at each time point <b>^*</b>p≤0.05; <b>^{* *}</b>p≤0.01;<b>^***</b>p≤0.001.</p><p>0 Gy at 20weeks vs irradiated groups at this time point <b>^§</b> p≤0.05; <b>^§§</b>p≤0.01; <b>^{§ § §}</b>p≤0.001.</p><p>0 Gy at 40weeks vs irradiated groups at this time point ^† p≤0.05; ^††p≤0.01; ^†††p≤0.001.</p><p>0 Gy at 60weeks vs irradiated groups at this time point ^‡ p≤0.05; ^‡‡p≤0.01.</p><p>n = 7 animals in each group (20 and 40 weeks post-irradiation and n = 5 animals in each group (60 weeks post-irradiation).</p