Extended Data Fig. 7: P1 versus P7 macrophage and monocyte populations have distinct proliferation profiles and differentially interact with lymphatic endothelial cells.

Pooled UMAP plot showing the different major clusters (A). Stacked violin plots showing expression of marker genes for each cluster (B). UMAP of the macrophage and monocyte clusters separated based on timepoint and condition (P1 intact, P1MI1dpi, P1MI7dpi, P7 intact, P7MI1dpi and P7MI7dpi) (C). The percentage of cell cycle genes for each macrophage and monocyte cluster confirmed the existence of a proliferating population, with approximately half of the Mf2 cluster at phase S of cell division (D). The percentage of mitochondrial RNA (E) and the number of molecules (F) detected within cells confirmed the existence of an apoptotic population. Stacked violin plots showing expression of marker genes for each cluster suggests at least two tissue-resident macrophage populations, designated macrophage 1 (Mf1; Lyve1⁺;Ccr2⁻;Arg1⁻) and macrophage 2 (Mf2; Lyve1⁻;Ccr2⁺;Arg1⁺) (G). The relative percentage of each cell population at different timepoints and conditions as represented in a proportional bar chart (H). The unbiased differential expression analysis identified genes that were enriched in each cluster by comparing across all the other clusters (I). Comparison of total incoming path weights and total outgoing path weights across populations (J). Hierarchical network diagram of significant cell-cell interaction pathways, with arrows and edge colour indicating signalling direction ligand:receptor (K). Summed ligand weights across ligand and receptor target paths for top ligands in LECs (L) and macrophages (Mf) (M). Subclustering of endothelial cells to identify LECs (N). Unbiased GO term analysis showed pathways enriched at P7MI7dpi, vs P1MI7dpi but not between P1 and P7 (O). Genes implicated in enriched pathways (P). Relative abundance of macrophage subsets grouped by individual hearts following de-multiplexing of pooled samples (Q). Cumulative expression scores of cell cycle markers showed a decrease in Lyve1^−/− CCR2- macrophage proliferation, but no change in CCR2+ or monocytes (R). In vivo validation of increased apoptosis; CC3 co-expression with F4/80 within the infarct zone 7 days following MI at P2 was significantly increased in the Lyve1^−/− context (S). Violin plot indicating expression levels of CD44 were significantly reduced in the CCR2- macrophage cluster, but using vireo5³⁷ were unchanged following loss of Lyve1 (T). CM = cardiomyocytes, Art EC = arterial endothelial cells, VEC = venous endothelial cells, Endo = endocardium, Prol VEC = proliferating VEC, LEC = lymphatic endothelial cells, Fb = fibroblasts, Prol Fb = proliferating Fb, Epic = epicardium, SMC = smooth muscle cells, Peri = pericytes, Mf = macrophages, DC-like = dendritic cell-like, Gran = granulocytes. Data are presented as mean ± SD. Significant differences between Enriched Pathways in O and P were calculated using Fisher’s exact test. The box centre in violin plots in R and T indicates the median, the lower and upper hinges correspond to the first and third quartiles, and the whiskers extend to values with a distance from the hinges that is at most the inter-quartile range (IQR) multiplied by 1.5. Box plot parameters, including cell counts are available in Source Data. Bonferroni-corrected pairwise Wilcoxon Rank Sum test was used to determine significance in R and T. Unpaired Student’s t tests were used to determine significance in S. n = 4, 4 for S. Scale bar 200μm.

Source data