Local arginase 1 activity is required for cutaneous wound healing

Abstract

Chronic nonhealing wounds in the elderly population are associated with a prolonged and excessive inflammatory response, which is widely hypothesized to impede healing. Previous studies have linked alterations in local L-arginine metabolism, principally mediated by the enzymes arginase (Arg) and inducible nitric oxide synthase (iNOS), to pathological wound healing. Over subsequent years, interest in Arg/iNOS has focused on the classical versus alternatively activated (M1/M2) macrophage paradigm. Although the role of iNOS during healing has been studied, Arg contribution to healing remains unclear. Here, we report that Arg is dynamically regulated during acute wound healing. Pharmacological inhibition of local Arg activity directly perturbed healing, as did Tie2-cre-mediated deletion of Arg1, revealing the importance of Arg1 during healing. Inhibition or depletion of Arg did not alter alternatively activated macrophage numbers but instead was associated with increased inflammation, including increased influx of iNOS(+) cells and defects in matrix deposition. Finally, we reveal that in preclinical murine models reduced Arg expression directly correlates with delayed healing, and as such may represent an important future therapeutic target.

Figure 1

Arginase1 (Arg1) is dynamically regulated during healing. (a) Images representing the experimental group mean for inducible nitric oxide synthase-positive (iNOS⁺) and Arg1⁺ cells in 1-, 5-, and 7-day excisional wound granulation tissue. (b) Quantification of iNOS⁺ and Arg1⁺ dermal inflammatory cells reveals differing temporal profiles. Immunohistochemical quantification data are derived from the mean of five randomly selected high-powered fields per wound and two wounds per mouse. (c) Arginase activity from isolated excisional wound tissue (measured through urea production) peaks at 5 days post wounding. (d) Western blot analysis of total Arg1 protein in excisional wounds reveals increased expression at 3, 5, and 10 days post wounding. (b) Data presented indicate mean+SEM of n=5–6 mice per group or (c, d) three replicates per group across two individual experiments. Bar=100 μm. (b) *P<0.05 comparing iNOS with Arg1, (c) *P<0.05 compared with days 1, 3, and 10.

Figure 2

Inhibition of arginase activity significantly delays cutaneous healing. (a) Representative hematoxylin and eosin–stained incisional wounds (day 3) from control and N(omega)-hydroxy-nor-l-arginine (nor-NOHA)-treated mice. Arrows indicate wound margins. Nor-NOHA treatment significantly delays wound closure quantified by (b) increased wound area at 3 and 7 days post wounding and (c) decreased re-epithelialization at 3 days post wounding. Images representing the experimental group mean for (d) wound neutrophils and (e) macrophages from control and nor-NOHA-treated mice. (f) Quantification reveals no effect of nor-NOHA on wound neutrophil numbers. (g) Quantification of wound macrophage numbers reveals a significant increase following nor-NOHA treatment at 3 and 7 days post wounding. Immunohistochemical quantification data are derived from the mean of five randomly selected high-powered fields per wound and two wounds per mouse. Data presented indicate mean+SEM, n=6 mice per group. Bar=400 μm (a), 50 μm (d, e). *P<0.05. PBS, phosphate-buffered saline.

Figure 3

Conditional ablation of Arg1 (T2C;Arg1^fl/fl) significantly delays cutaneous repair. (a) Representative macroscopic images of control (T2C) and T2C;Arg1^fl/fl wounds at 3, 7, and 14 days post wounding show delayed healing in T2C;Arg1^fl/fl wounds. (b) Representative hematoxylin and eosin–stained day-3 histology from T2C and T2C;Arg1^fl/fl wounds. Arrows indicate wound margins. Dermal cell–specific deletion of arginase 1 significantly delays wound closure quantified by (c) increased wound area and (d) delayed re-epithelialization over multiple time points. Bar=5 mm (a) and 400 μm (b). Mean+SEM, n=6–7 mice per group. **P<0.01, *P<0.05.

Figure 4

Conditional deletion of arginase 1 (Arg1) results in excessively prolonged and local inflammation. Images representing the experimental group mean for (a) neutrophil and (b) macrophage immunohistochemical analysis from day-3 wound granulation tissue. T2C;Arg1^fl/fl wounds display significantly increased numbers of (c) neutrophils and (d) macrophages at 3 and 7 days post wounding. Images representing the experimental group mean for (e) inducible nitric oxide synthase-positive (iNOS⁺) and (f) Ym1⁺ cells. T2C;Arg1^fl/fl wounds have increased numbers of (g) iNOS⁺ cells, with no difference in (h) Ym1⁺ cells. Immunohistochemical quantification data are derived from the mean of five randomly selected high-powered fields per wound and two wounds per mouse. Data presented indicate the mean+SEM of n=6–7 mice per group. Bar=50 μm. *P<0.05.

Figure 5

T2C;Arg1^fl/fl wounds display altered matrix deposition and protease activity. Wound protein collagen 1 content determined by (a) immunofluorescence with corresponding western blot analysis for (b) collagen (Col) 1 and 3 revealed a reduction in T2C;Arg1^fl/fl wounds. (c, d) T2C;Arg1^fl/fl wounds display increased expression of collagen species and MMP2 (quantitative PCR) at 7 days post wounding. (e) Images representing the experimental group mean for MMP2⁺ dermal cells at 7 days post wounding. (f) Quantification of MMP2⁺ dermal cells reveals significantly increased numbers in T2C;Arg1^fl/fl day 7 wounds. Immunohistochemical quantification data are derived from the mean of five randomly selected high-powered fields per wound and two wounds per mouse. Data presented indicate (f) mean+SEM of n=6–7 mice per group, (a) n=4 mice per group, or (c, d) three replicates per group and two individual experiments. Bar=200 μm (a) and 50 μm (e). **P<0.01, *P<0.05.

Figure 6

Reduced Arginase1 (Arg1) is a conserved feature of delayed healing in mouse and human wounds. Images representing the experimental group mean for (a) inducible nitric oxide synthase-positive (iNOS⁺) and (b) Arg1-positive (Arg1⁺) immunohistochemical analysis from control (young) and delayed healing aged and ovariectomized (Ovx) day-3 wounds. Aged and Ovx mice wounds are associated with increased numbers of (c) iNOS⁺ dermal cells and (d) reduced Arg1⁺ cells compared with control mice. Immunohistochemical quantification data are derived from the mean of five randomly selected high-powered fields per wound and two wounds per mouse. Data presented indicate the mean+SEM, n=6 mice per group. Bar=50 μm. *P<0.05.