Figure 4 ∣. Cell therapy benefits the mechanical properties of the infarct via remodeling of the extracellular matrix.
a, Schematic outline of experiments performed in panels (b-e). b, Representative picrosirius red-stained cardiac histological images from the infarct border zone of 3 weeks post-I/R mice subjected to MNC or saline injection. Fibrosis is shown in red. Scale bars = 100 μm. c, Quantitation of fibrotic area at the infarct border zone in MNC, freeze thaw-killed MNC, or saline-treated hearts, 3 weeks post-I/R. *p<0.05 by one-way ANOVA with Tukey’s post-hoc test with exact p-values shown in figure. Images in (b) and quantitation in (c) are from n=5 saline-treated, n=12 MNC-treated, or n=7 freeze-killed MNC-treated mice, with a minimum of 20 histological sections assessed from each individual mouse heart. d, Change in passive force generation over increasing stretch-lengthening (percent of L0) in isolated infarct strips from MNC or saline-treated hearts, 3 weeks post-I/R. *p<0.05 versus I/R/Sal. by Student’s 2-tailed t-test (exact p-values are shown in figure). Data from one uninjured control heart (no I/R or cell therapy) is also shown for comparison. e, Gene expression levels by RT-PCR for selected extracellular matrix (ECM) and matrix-associated genes in isolated infarct regions from MNC or saline-treated hearts, 3 weeks post-I/R. *p<0.05 versus I/R/Sal. by Student’s 2-tailed t-test (exact p-values shown in figure). f, Representative confocal micrographs of pre-fabricated collagen patches that were seeded and cultured for 5 days with either bone marrow-derived macrophages (BMDM) or peritoneal macrophages (PM) isolated from wild-type male and female mice, versus cell-free control patches cultured in media. Fluorescence signal is from native type I and II collagen using second harmonic generation microscopy. Scale bars = 100 μm. g, Schematic of experiments using activated cardiac macrophages isolated from post-I/R Ccr2-RFP × Cx3cr1-GFP knock-in mice that were then cultured with isolated cardiac fibroblasts (FB) for 72 hours. h-k, Fibroblast mRNA was used for RT-PCR to assess expression of smooth muscle α-actin (Acta2 / αSMA, h), lysyl oxidase (Lox, i), connective tissue growth factor (Ctgf, j) or collagen 1 alpha 2 (Col1a2, k). For panel 4i: *p<0.05 by Kruskal-Wallis with Dunn’s multiple comparisons test. For all other panels: *p<0.05 by one-way ANOVA with Tukey’s post-hoc test (exact p-values shown in figure). All numerical data are summarized as box and whisker plots indicating the median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively). Micrographs in f are representative of five different collagen patches seeded with cells pooled from n=4 mice (2 male and 2 female). Data in g-k are from 4-5 replicates generated over fibroblasts isolated from n=10 wild-type mice (6 male and 4 female) and macrophages isolated from n=6 Ccr2-RFP × Cx3cr1-GFP heterozygous knock-in mice (3 male and 3 female).