Fig. 1: Time course of ITGA5 expression in infarct macrophages.

A–C Flow cytometry was used to assess ITGA5 expression in infarct macrophages. The gating strategy is shown in Supplementary Fig. 2. Representative images show the mean fluorescent intensity (MFI) for ITGA5 antibody (A) and isotype control (B) in CD45 + /CD11b + /Ly6G-/CD64 + /MerTK+ macrophages in sham animals and in infarcts. Quantitative analysis showed a 2-fold increase in ITGA5 mean fluorescent intensity (MFI) in macrophages 7 days after infarction (**p < 0.01; Sham: n = 5 biologically independent experiments, 7-day: n = 8 biologically independent experiments, 28-day: n = 11 biologically independent experiments). Statistical analysis was performed using one-way ANOVA followed by Bonferroni post-hoc test. To examine the time course of ITGA5 expression in infarct macrophages, we have also performed dual immunofluorescence in myocardial sections from control and infarcted CSF1R^EGFP macrophage reporter mice, combining ITGA5 staining (red) and CSF1R (GFP) labeling. Abundant ITGA5+ macrophages were noted in 7-day infarcts (D–I, arrows). CSF1R-negative cells with vascular, or fibroblast morphology (D–F, yellow arrow) also expressed ITGA5. J Quantitative analysis showed that the density of CSF1R+ macrophages was significantly higher in infarcted segments, when compared with remote non-infarcted myocardium from the same timepoint (^p < 0.05, ^^^p < 0.001, n = 6 biologically independent experiments in control (C), 24-hour, 7-day and 28-day groups, n = 5 biologically independent experiments in the 3-day group). Statistical analysis was performed using non-parametric ANOVA (Kruskal-Wallis) followed by Dunn’s post-hoc test. No significant increase in the density of myeloid cells was noted in non-infarcted remodeling segments. K An increase in the density of ITGA5+ macrophages was first noted 3 days after coronary occlusion, and peaked after 7 days of coronary occlusion, but was significantly reduced at the 28-day timepoint (*p < 0.05, **p < 0.01 ****p < 0.0001 vs. C ^p < 0.05, ^^^p < 0.001, vs. corresponding remote non-infarcted myocardium, n = 6 biologically independent experiments in control (C), 24-hour, 7-day and 28-day groups, n = 5 biologically independent experiments in 3-day group). Statistical analysis was performed using non-parametric ANOVA (Kruskal–Wallis) followed by Dunn’s post-hoc test. L–N Confocal microscopy showed that ITGA5 was localized not only on the macrophage surface (white arrows), but also in the cytoplasm (yellow arrow), likely reflecting de novo synthesis of ITGA5, followed by subsequent shuttling to the cell membrane. Representative images were selected from 50 different scanned fields from 6 biologically independent experiments. Scalebar = 50 μm (for panels D–F), =30 μm (panels G–I), = 10 μm (panels L–N). All data are shown as mean values +/- SEM. Source data are provided as a Source Data file.