Skin resident αSMA+ myofibroblasts are critical for wound repair.Figure 2(A) Analysis of multiplex immunostaining of full thickness skin wounds performed at indicated time points. Data are presented as the fractional area of each image positive for αSMA and negative for CD31. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates. One-way ANOVA with Dunnett's multiple comparison test performed. (B) FACS quantification of αSMA-RFP+ cells in wounds. Day 0, N=2; day 3, N=2; day 6, N=6; day 8, N=3; day 14, N=3 biological replicates. One-way ANOVA with Dunnett's multiple comparison test performed comparing each timepoint to day 0. (C) Schematic of bone marrow transplants between WT and αSMA-RFP mice. (D) Representative FACS plots of digested skin from bone marrow (BM) transplanted αSMA-RFP mice 17 days post-wounding. (E) FACS quantification of αSMA-RFP+ cells in wounds of BM transplanted mice. αSMA-RFP with αSMA-RFP BM, N=2; WT with αSMA -RFP BM, N=3; αSMA-RFP with WT BM, N=4 biological replicates. One-way ANOVA with Tukey's multiple comparison test performed comparing WT with αSMA-RFP BM and αSMA-RFP with WT BM to αSMA-RFP with αSMA-RFP BM. ( (F) Schematic of bone marrow transplants between WT and αSMA-TK mice. (G) Wound area measurements in WT and ⍺SMA-TK mice transplanted with WT or αSMA-TK BM. WT with WT BM and WT with αSMA-TK BM, N=5; αSMA-TK with WT BM and αSMA-TK with αSMA-TK BM, N=4 biological replicates. Two-way ANOVA performed with the indicated comparisons. (H-J) Multispectral imaging of changes in fibroblast subsets during wound repair. (H) Representative images (20x magnification) of orthogonal wound tissue sections at day 6 post-wounding displaying αSMA, FAP, and FSP1 overlaid with DAPI in the wound bed. Subcutaneous (subq), dermis (derm), epidermis (epi), and scar regions are denoted. Individual αSMA, FAP, and FSP1 channel images overlaid with DAPI. Scale bar, 50 µm. (I) Analysis of multiplex immunostaining of full thickness skin wounds performed at indicated time points. Data are presented as the fractional area of each image positive for the indicated markers and negative for CD31. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates. One-way ANOVA with Dunnett's multiple comparison test performed. (J) Percent overlap of αSMA with FAP and FSP1 throughout wound healing. αSMA+ denotes cells positive for αSMA but not FAP or FSP1. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates. (K) Wound closure in FAP-TK and wild-type littermate mice. N=4 biological replicates per group, data are from one experiment. Two-way ANOVA performed comparing WT to TK mice. (L) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines. Scale bar, 200 μm. (M) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control. WT, N=3; FAP-TK, N=3; WT, N=17, FSP1-TK, N=9 biological replicates. (N) Immunofluorescent staining for CD31 (red) and nuclei on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (O) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=4; FAP-TK, N=4; WT, N=3; FSP1-TK, N=3 biological replicates. (P) IHC for hypoxyprobe (brown) on wound tissue sections at day 17 post-wounding. Black boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (Q) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=3; FAP-TK, N=3; WT, N=3; FSP1-TK, N=4 biological replicates. (R) W (R) Wound closure in FSP1-TK and WT littermate mice. N=10 biological replicates per group, data are from two independent experiments. Two-way ANOVA performed comparing WT to TK mice.