Figure 4.

Analysis of ligand-receptor pairing in single-cell transcriptomics data sets identifies the cardiac macrophage as primary paracrine inducer of fibroblast activation. A, Network plot depicting significantly upregulated paracrine and autocrine interactions among different cardiac cell fractions in WT and miR-21 cKO mice. Arrows indicate that the direction of communication, line thickness, and color saturation are proportional to the number of intercellular ligand-receptor pairs; autocrine signaling is depicted in black. Ligands and receptors are considered to be expressed when detected in at least 20% of a cell population. B, Detailed view of paracrine interactions from M1-like macrophages toward activated fibroblast (Postn+) cells depicting the individual ligand-receptor pairs. C, Dot plot showing expression of fibroblast-related genes. D, Pie graphs depicting the proportions of different fibroblast clusters. E, RNA velocity analysis of fibroblast clusters detected by single-cell sequencing. Underlying feature plots label cells that express Tcf21 (gray), Postn (green), Acta2 (red), or both Postn and Acta2 (yellow). F, Experimental strategy for coculture of adult mouse cardiac fibroblasts (AMCF) and bone marrow–derived macrophages. Macrophages were transfected with either LNA-anti–miR-21 or LNA-anti–miR-control, stimulated with LPS, washed and seeded around freshly isolated AMCFs for 48 hours. Monocultures of AMCF served as negative controls. Immunofluorescence staining of AMCFs was performed using antibodies against vimentin (marker for fibroblasts) and α-smooth muscle actin (ACTA2; marker for myofibroblasts). G, Percentage of α-smooth muscle actin–positive cells as a measure of myofibroblast formation. Scale bar, 25 µm. n=4 to 6 independent experiments performed in triplicates. Data are mean and individual values and were analyzed by using 2-way ANOVA with the Sidak posttest. ***P<0.001. LNA indicates locked nucleic acid; LPS, lipopolysaccharide; LR, ligand-receptor; miR-21 indicates microRNA-21; miR-21 cKO, macrophage-specific miR-21–deficient mice; TAC, transverse aortic constriction; UMAP, uniform manifold approximation and projection; and WT, wild type.