Genetic Deletion of Galectin-3 Does Not Impair Full-Thickness Excisional Skin Healing  John T. Walker, Christopher G. Elliott, Thomas L. Forbes, Douglas W. Hamilton  Journal of Investigative Dermatology  Volume 136, Issue 5, Pages 1042-1050 (May 2016) DOI: 10.1016/j.jid.2016.01.014 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Galectin-3 expression peaks in the inflammatory phase and remains present into the proliferative phase, primarily expressed in arginase I-positive cells. 6-mm, full-thickness skin wounds were inflicted into the backs of wild-type and galectin-3 knockout mice. (a) Lgals3 expression was quantified in tissue from 1 to 7 days post wounding using quantitative PCR. Values are given as mean ± standard deviation. Four mice per time point. *P < 0.05; **P < 0.01. (b) Tissue isolated from unwounded skin and at 3 and 7 days post wounding was subjected to histological analysis of galectin-3 expression. Bar = 200 μm. WB, wound bed; WE, wound edge. Co-staining of tissue isolated at (c) day 3 and (d) day 7 after wounding shows overlap of galectin-3 primarily in arginase I-positive cells and in cells expressing vimentin. Bar = 50 μm. Journal of Investigative Dermatology 2016 136, 1042-1050DOI: (10.1016/j.jid.2016.01.014) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Genetic deletion of galectin-3 does not affect gross wound closure but does impair the rate of re-epithelialization by 7 days post wounding. (a) Wound size was measured up to 7 days post wounding in 6-mm wounds from 13 knockout (KO) and 12 sex-matched wild-type (WT) littermates. Representative wounds are shown in the side panel in a KO mouse and its sex-matched littermate pair at 0, 3, 5, and 7 days post wounding (top to bottom). Bar = 5 mm. Data are expressed as mean ± standard deviation. (b) Masson trichrome-stained sections were examined for the epithelial tongue length and thickness, and wound size. (c) Knockout mice displayed impaired length of the epithelial tongue and (d) an impaired percent of re-epithelialization at 7 days post wounding. Eight knockout and nine WT mice were quantified. Data are expressed as mean ± standard deviation. *P < 0.05; **P < 0.01. Journal of Investigative Dermatology 2016 136, 1042-1050DOI: (10.1016/j.jid.2016.01.014) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Genetic deletion of galetin-3 does not affect the immune cell infiltrate by 3 days post wounding or the overall composition of the wounds by 7 days post wounding. (a) Arginase I, NOS2, and neutrophil elastase were examined histologically from whole wounds, 3 days post wounding and quantified. Bar = 50 μm. Five wild-type (WT)/galectin-3 knockout (Gal3KO) sex-matched littermate pairs were analyzed. (b) Histological staining of galectin-3 confirmed deletion of galectin-3 in knockout mice, whereas there was no change in α-smooth muscle actin (SMA) staining. Bar = 500 μm. The density and average size of CD146-labeled blood vessels were quantified in seven knockout and five WT mice. Bar = 50 μm. (c) Masson trichrome- and Van Gieson-stained sections of day 7 wounds. Arrows show the edge of the wounds. Bar = 500 μm. (d) Masson trichrome and α-SMA staining of day 15 wounds and relative hydroxyproline content measured in day 15 wounds. Bar = 500 μm. Hydroxyproline content was measured in three sex-/age-matched WT/Gal3KO pairs. Journal of Investigative Dermatology 2016 136, 1042-1050DOI: (10.1016/j.jid.2016.01.014) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Transcript abundance of genes associated with fibrotic response and revascularization are unchanged by galectin-3 knockout, 7 days post wounding. RNA was isolated from noninvolved (NI), proximate (P), wound edge (WE), and wound bed (WB) tissues, 7 days post wounding, and quantified using quantitative PCR. Transcripts for fibrotic response genes Acta2 and Col1a2, and the vascular gene Kdr, were measured alongside Lgals3 and its related family members Lgals1 and Lgals7. Tissues were isolated from four sex-matched knockout/wild-type pairs. Data are expressed as mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001. Journal of Investigative Dermatology 2016 136, 1042-1050DOI: (10.1016/j.jid.2016.01.014) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Galectin-3 expression is decreased in human chronic wound tissue and is negatively correlated with neutrophil elastase expression. (a) Histological analysis of galectin-3, neutrophil elastase, mannose receptor, and indolamine 2,3-dioxygenase in the wound edge of human chronic wounds shown for 5 of 9 total patients examined. Bars = 1 mm. (b) High-magnification images of staining in the wound bed of patient M56. Bar = 100 μm. (c) Quantification of LGALS3 gene expression in 15 patients after lower extremity amputation in noninvolved, proximate, and wound edge tissues. *P < 0.05; **P < 0.01; ***P < 0.001. See Supplementary Tables 1 and 2 (online) for relevant patient information. Journal of Investigative Dermatology 2016 136, 1042-1050DOI: (10.1016/j.jid.2016.01.014) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Regulation of galectin-3 expression in human noninvolved and wound edge dermal fibroblasts by transforming growth factor (TGF)-β1 and tumor necrosis factor (TNF)-α. (a) Fibroblasts isolated from noninvolved and wound edge tissues from patients with chronic wounds were subjected to combinations of 5 ng/mL TGF-β1 and 1 ng/mL TNF-α for 24 hours. Cells isolated from four patients were used. (b) Dose response of wound edge dermal fibroblasts was performed from 0–5 ng/mL with or without addition of 10 μM of the ALK5 receptor inhibitor SB431542 for 24 hours. Cells isolated from five patients were used. (c) Effect of TGF-β1 and TNF-α on galectin-3 and α-smooth muscle actin (αSMA) protein content within wound edge fibroblasts was measured by western blot after 48 hours of stimulation. Cells isolated from four patients were used. Data are expressed as mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001. Journal of Investigative Dermatology 2016 136, 1042-1050DOI: (10.1016/j.jid.2016.01.014) Copyright © 2016 The Authors Terms and Conditions