Annexin A5 reduces infarct size and improves cardiac function after myocardial ischemia-reperfusion injury by suppression of the cardiac inflammatory response

Figure 1. Annexin A5 staining. Representative images of cross-sections from the whole heart (left) or the infarct area (right) two days after MI-R stained for human annexin A5 using a specific AnxA5 antibody, while counterstaining was performed using haematoxilin. AnxA5 treated mice showed a more intense staining compared to vehicle and sham mice. Left panel scale bar: 500 μm, right panel scale bar: 20 μm.

Figure 2. Contrast-enhanced MR imaging. Two days after MI-R no difference in infarct size was observed between the groups (n = 13–15). However, after three weeks the AnxA5 group displayed a reduced infarct size as compared to the vehicle group (A). Representative Gd-DPTA-enhanced MR images (B) two days and three weeks after MI-R of the vehicle and AnxA5 group, red line indicates epicardial border, green line indicates endocardial border and yellow line indicates infarct area. Data are mean ± SEM. #P < 0.05 vs. vehicle.

Figure 3. Cardiac MR imaging of LV volumes and function. Assessment of LV volumes and function two days and three weeks after MI-R (n = 12–15). AnxA5 therapy prevented the increase in end-diastolic volume (EDV) (A) and end-systolic volume (ESV) (B) accompanied by a preserved ejection fraction (EF) (C) as compared to vehicle three weeks post MI-R. Representative transversal short-axis MR images at end-diastole (ED) and end-systole (ES) two days (D) and three weeks (E) following MI-R in the sham, AnxA5 and vehicle groups, red line indicates epicardial border and green line indicates endocardial border. Data are mean ± SEM. #P < 0.05, ##P < 0.01, ###P < 0.001 all vs. vehicle, *P < 0.05 and ***P < 0.001 both vs. sham.

Figure 4. LV fibrous content and LV wall thickness. Histological analysis after three weeks (n = 9–10) showed a significant reduced LV fibrous content in the AnxA5 group compared to the vehicle group (A). Representative images (B) of Sirius red staining of cross-sections of the whole heart in the vehicle group and the AnxA5 group. Scale bar: 500 μm. LV wall thickness was significantly increased in the infarct area after AnxA5 treatment (C). Data are mean ± SEM. #P < 0.05 vs. vehicle, *P < 0.05, **P < 0.01, ***P < 0.001 all vs. sham.

Figure 5. Local inflammatory response and macrophage proliferation. Quantification of the number of infiltrated macrophages showed an decreased number of infiltrated macrophages in the border zones and infarct area two days (n = 3–5) following MI-R in the AnxA5 group (A). Mac3/Ki67 double staining revealed a significant reduction in the number of proliferating macrophages in the border zones and infarct area after two days in the AnxA5 treated mice as compared to vehicle (n = 5–6; B). Three weeks after MI-R the number of infiltrated macrophages is significantly reduced in the border zones and infarct area upon AnxA5 treatment (G). No differences were observed regarding the number of proliferating macrophages three weeks after MI-R between AnxA5 and vehicle groups (H). Representative images of Mac3 staining (C,D,I and J) and Mac3/Ki67 double staining (E,F,K and L) of the infarct area. Nuclei are shown in blue, Mac3 staining in green and Ki67 in red, arrowheads indicate positive cells. Scale bar: 50 μm. Data are mean ± SEM. #P,0.05, ##P < 0.01, ###P < 0.001 all vs. vehicle, *P < 0.05, **P < 0.01 and ***P < 0.001 all vs. sham.

Figure 6. In vitro effect of AnxA5 stimulation on bone-marrow derived macrophages. IL-6 production was significantly reduced following AnxA5 stimulation (n = 5) compared to vehicle. Data are mean ± SEM. ###P < 0.001 vs. vehicle.