Dermal αSMA+ myofibroblasts orchestrate skin wound repair 
via β1 integrin and independent of type I collagen production (2022)
Figure 1

Figure 1. αSMA+ myofibroblasts are required for proper wound closure.

(A-E) Impaired wound closure in αSMA-TK mice. (A) IHC for αSMA+ cells in wound tissue sections at day 6 post-wounding. Representative images are shown. Scale bar, 50 µm. Black boxes denote zoomed regions displayed in bottom panels. (B) Quantification of αSMA+ cells at day 6 post-wounding. WT, N=6; TK, N=5 biological replicates. Two-tailed non-parametric Mann-Whitney test performed. (C) WT and αSMA-TK mice were treated with GCV for 32 days. Wounds were measured at indicated timepoints and wound area is presented as percentage of day 0 value. WT, N=5; TK, N=4 biological replicates per group, data are from one experiment. Two-way ANOVA performed comparing WT to TK mice. (D) Representative gross images of wounds. Scale bar, 5 mm. (E) αSMA-TK mice and WT littermates were placed on GCV regimen three days prior to wounding (day -3). On day 21, GCV treatment was stopped and the mice monitored until day 39. WT, N=7; TK, N=9 biological replicates. Data are from two independent experiments. Two-way ANOVA and Sidak's multiple comparison test were performed comparing WT to TK mice.

(F) Expression levels of ACTA2 in normal and diabetic human wound tissue. RNA-seq reads are derived from (Davis et al., 2020, Data ref: Davis et al, 2020). Normal, N=3; diabetic, N=4 biological replicates. Data are presented as a box and whisker plot of min to max values. Unpaired t-test performed.

(G-I) Impaired re-epithelialization of the wounds in αSMA-TK mice at day 17 post-wounding. (G) Representative images of keratin 5 (Krt5, red) and Hoechst (blue) stained wound sections at day 17 post-wounding. White boxes denote zoomed regions displayed in right panels, triangles (open: incomplete closure, filled: closed wounds) denote the epithelial tongues. Scale bar, 1000 µm. (H) Representative H&E images of wound sections. Wound bed is marked with black lines, triangles (open: incomplete closure, filled: closed wounds) denote the epithelial tongues. Epi, epidermis; derm, dermis; subq, subcutaneous regions denoted. Scale bar, 200 µm. (I) Measurements of the distances between epithelial tips in the wounded skins of WT and αSMA-TK mice at days 17 post wounding. WT, N=7; TK, N=8 biological replicates. Black line denotes median value. Mann-Whitney test performed.

(J-O) Impaired granulation tissue formation and angiogenesis in αSMA-TK mice. (J) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines, triangles (open: incomplete closure, filled: closed wounds) denote the epithelial tongues. Epi, epidermis; derm, dermis regions denoted. Scale bar: 200 μm. (K) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control (set to 100%). NA (not assessed) reflects the lack of granulation in αSMA-TK mice. WT, N=5; TK, N=4 biological replicates. (L) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (M) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=3; TK, N=5 biological replicates. (N) 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. (O) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=5; TK, N=6 biological replicates.

Data information: Data are reported as mean ± s.e.m. for (B, C, E, K, M and O). Data are presented as a box and whisker plot of min to max values for (F). Individual data points (circles) and the median (black line) are reported for (I). Exact p-values are reported. One sample t-test performed comparing values to WT average for (K, M, and O).