CD226 deletion improves post-infarction healing via modulating macrophage polarization in mice

Figure 1. CD226 expression is increased in the heart after myocardial infarction (MI) and CD226 expressing macrophages accumulate in the infarcted heart. A. Representative immunoblots and summary data showing CD226 expression in infarcted heart tissue at different time points after MI. NS indicates no significance. ** P < 0.01 vs. sham and ## P < 0.01 versus MI day 4, n=6 per group. B. Representative images of immunofluorescence staining for CD226 in the infarcted heart of wild type (WT) mice 7 days after MI or sham surgery. Scale bars as indicated (left 20 μm, right 50 μm). C-E. Immunofluorescent costaining for CD226 (red) and Mac-3 (green) in the infarcted heart of WT and CD226 KO mice (C), and quantitative analysis of CD226+ cells (D) and CD226+ Mac3+ macrophages (E) in the infarcted heart at day 7 after MI. DAPI was used for nuclear staining (blue). Scale bar =20 μm. n=6 mice per group, ** P<0.01, *** P<0.001.

Figure 2. CD226 deletion ameliorates cardiac dysfunction after MI. A. Representative M-mode echocardiograms of infarcted hearts from WT and CD226 KO mice on day 7 after MI. B-C. Echocardiographic analysis of left ventricle end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD) and LV ejection fraction (LVEF) at pre-MI (baseline) and on 1 week and 5 weeks after MI. n=10 per group, * P<0.05 and ** P<0.01 vs. WT at corresponding time point. D. Representative LV pressure-volume loops of infarcted hearts from WT and CD226 KO mice 5 weeks after MI. E-G. LV end-systolic volume (LVESV), LV end-diastolic volume (LVEDV), LV end-systolic pressure (LVESP), LV end-diastolic pressure (LVEDP) and maximum of dP/dt were acquired from WT and CD226 KO mice at 5-week end point after MI or sham operation using Millar catheterization. n=8 per MI group, n=5 per sham group. * P<0.05, ** P<0.01 vs corresponding Sham, and # P < 0.01, ## P < 0.01 vs WT MI. Data are expressed as mean±SEM.

Figure 3. CD226 deletion prevents infarct expansion and ameliorates cardiac remodeling after MI. A. Gross observation of infarcted hearts (top) and the ratio of heart weight to tibia length (HW/TL) (bottom). B. Representative images of Masson trichrome staining of heart cross sections of WT and CD226 KO mice at 1 week and 5 weeks after MI. Scale bar =1 mm. C-D. Infarct scar circumference (C) was expressed as a percentage of LV including the septum, and wall thickness of infarct scar (D) at papillary muscle level was measured 1week and 5 weeks after MI. n=6 in each group at each time point, * P<0.05 vs. corresponding WT. E. CD226 KO mice exhibited more preserved myocardium (1 week post-MI) and increased collagen deposition (5 weeks post-MI) in the infarct zone, and decreased interstitial fibrosis in the border zone. Scale bar =50 μm. F-H. Ratio of myocyte to fibrosis 1 week after MI (F) and collagen density 5 weeks after MI (G) in the infarct zone, and collagen volume fraction in the border zone (H) were quantitatively estimated in 5 randomly chosen high-power fields (×200) in each section. Results obtained from the same heart were averaged and counted as n=1. n = 6 in each group at each…

Figure 4. CD226 deletion results in increased accumulation of F4/80+ CD206+ macrophages and diminished Mac-3+ iNOS+ macrophages in the ischemic heart after MI. A and B. Immunohistochemical staining for CD68 (A) and quantitative analysis of CD68+ macrophages (B) in the infarct zone and border zone of WT and CD226 KO mice at day 7 after MI. Scale bar=50 μm. n=5 mice per group, * P<0.05 vs. WT. C and D. Representative immunofluorescence photographs of Mac-3+ (red) and iNOS+ (green) macrophages and quantitative analysis of Mac-3+ iNOS+ M1 macrophages in the infarct, border zone and epicardium area of WT and CD226 KO mice at day 7 after MI. DAPI was used for nuclear staining (blue). Scale bar =50 μm. n=5 mice per group, ** P<0.01 vs. WT. E and F. Representative immunofluorescence photographs of F4/80+ (red) and CD206+ (green) macrophages and quantitative analysis of F4/80+ CD206+ M2 macrophages in the infarct, border zone and epicardium area of WT and CD226 KO mice at day 7 after MI. Scale bar =50 μm. n=5 mice per group, ** P<0.01 vs corresponding WT.

Figure 5. CD226 depletion restrains monocytes mobilization from the spleen and peripheral blood. A. Representative flow cytometric dot plots and histograms show Ly6Chigh and Ly6Clow monocytes in the peripheral blood of WT and CD226 KO mice 7 days after MI or sham surgery. Numbers indicate the percentage of Ly6Chigh or Ly6Clow cells within the cell population analyzed. B. Ly6Chigh and Ly6Clow monocytes proportion in the blood. * P < 0.05 vs. WT-MI. C. Mean fluorescent intensity (MFI) of Ly6C on peripheral blood Ly6Chigh monocytes at day 7 after MI. n = 6 mice per group. Data are represented as mean values ± SEM. ** P < 0.01 vs. WT-Sham, ## P < 0.01 vs. WT-MI. D. Representative flow cytometric dot plots and histograms show Ly6Chigh and Ly6Clow monocytes in the spleen of WT and CD226 KO mice. Numbers indicate the percentage of Ly6Chigh or Ly6Clow cells within the cell population analyzed. E. Ly6Chigh and Ly6Clow monocytes proportion in the spleen 7 days after MI or sham surgery. ** P < 0.01 vs. WT-MI. F. Number of Ly6Chigh monocytes in the spleen at day 7 after MI. n = 6 mice per group. Mean values ± SEM are represented. ## P < 0.01 vs WT-MI.

Figure 6. CD226 deletion promotes myofibroblasts accumulation and angiogenesis in the infarcted heart after MI. A-B. Representative staining for α-smooth muscle actin (α-SMA) and α-actinin, and quantitative analysis of α-SMA+ myofibroblasts in the infarcted heart 7 days after MI. CD226 KO mice exhibited an increase in the number of extravascular α-SMA+ spindle-shaped myofibroblasts in the infarct area. C. Masson trichrome staining of serial heart sections revealed higher density of collagen deposition with well-aligned collagen fibers in the infarct zone of CD226 KO mice. D. Representative immunofluorescence staining of CD31+ capillaries and Ki67+ CD31+ proliferating endothelial cells in the infarct border zone at day 7 after MI. E-F. Quantitative analysis of CD31+ capillaries and Ki67+ CD31+ proliferating endothelial cells in the infarct border zone 7 days after MI. n=4 mice per group, * P<0.05 and ** P<0.01 vs. WT. Data are expressed as mean±SEM.

Figure 7. CD226 deletion suppresses M1 polarization and potentiates M2 polarization in bone marrow derived macrophages (BMDM). BMDM were isolated from WT and CD226 KO mice. A. Time course changes of M1 polarization marker genes in BMDM, upon stimulation with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 6, 12 and 24 hours as indicated. CD226 deletion suppressed M1 polarization as evidenced by decreased mRNA levels of IL-1β, IL-6, IL-12p40 and TNF-α. n = 4 independent experiments. * P<0.05, ** P<0.01, *** P<0.01 vs. WT. B. Profile of M2 polarization marker genes in BMDM in the presence of IL-4 (10 ng/mL) for 6, 12 and 24 hours as indicated. CD226 deletion potentiated M2 macrophage polarization as evidenced by increased mRNA levels of Arg1, CD206, Fizz1, Ym1, IL-10. n = 4 independent experiments. * P<0.05, ** P<0.01, *** P<0.01 vs. WT.

Figure 8. Heat maps of differentially expressed proteins in the infarcted hearts of WT and CD226 KO mice. Inserted, scale of -fold change. Following mass spectrometry, we set an arbitrary threshold of 1.2-fold to declare differences, 17 proteins were up-regulated and 21 proteins were down-regulated in the infarcted hearts of CD226 KO mice compared with WT mice. n= 3 per group, and each sample was the mixture of two infarcted hearts of the same group.