Pivotal role for alpha1-antichymotrypsin in skin repair

Abstract

α1-Antichymotrypsin (α1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair. By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of α1-ACT and the mouse homologue Spi-2, respectively. In both species, attenuated α1-ACT/Spi-2 activity and gene expression at the local wound site was associated with severe wound healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage.

FIGURE 1.

Spi-2/α1-ACT is expressed during the acute phase response post skin injury. A, quantitative RT-PCR analysis of RNA from wound tissue at indicated time points after injury revealed a dramatic increase in Spi-2 mRNA in wild-type (wt) mice (wounds per time point n = 4), and at all time points expression in wounds of db/db mice (wounds per time point n = 4) was attenuated. B, levels of α1-ACT mRNA were also strongly up-regulated in normal healing human wounds (wounds per time point n = 4); ns, not wounded skin (n = 4); h, hours; d, day. C, paraffin sections of normal healing wounds (day 5 post-injury) (left and middle) and intact skin (right) were hybridized to a digoxigenin-labeled antisense probe for Spi-2 mRNA (left and right) and sense probe; reaction product appears in purple; some sections were counterstained with nuclear fast red. D, double immunofluorescence staining of α1-ACT (red) and CD68 (green) in tissue of a healing wound (day 3 post-injury), a nonhealing human wound, and not wounded skin; DAPI counterstaining of nuclei (blue); dotted line indicates basement membrane; arrow indicates wound edge; arrowheads indicate double-positive cells. e, epidermis; d, dermis. Scale bars, 100 μm in C and D; 20 μm in D, right panel.

FIGURE 2.

Topical application of rα1-ACT accelerates granulation tissue formation and epithelialization in diabetic mice. Repetitive application of rα1-ACT (concentrations as indicated) significantly accelerated wound closure kinetics of db/db mice compared with vehicle-treated controls. A, presented are numbers of closed wounds versus total number of wounds for indicated conditions and time points post-injury. B, representative macroscopic appearance of wounds at indicated time points post-injury; day 5 post-injury, wounds treated with 2.0 mg/ml rα1-ACT revealed almost complete epithelialization; day 10 post-injury, almost all wounds treated with both concentrations are closed. C, representative H&E staining of wound tissues post-injury; in rα1-ACT treated wounds a highly vascularized and cellular granulation tissue developed that is covered by a hyper-thickened and closed epithelium; in contrast, in control mice scarce granulation tissue developed at wound edges and a thin, not closed, epithelium overlays the massive subcutaneous fat layer; right panel depicts magnifications of rectangles in left panel. D–G, morphometric analysis of wound tissue at different time points post-injury. D, area of granulation tissue (p = 0.01, control versus 2.0 mg/ml rα1-ACT day 5; p = 0.004, control versus 0.4 mg/ml rα1-ACT day 10; p = 0.003, control versus 2.0 mg/ml rα1-ACT day 10). E, length of epithelial tongue (p = 0.0001, control versus 2.0 mg/ml rα1-ACT day 5). F, distance between epithelial tips (p = 0.0004, control versus 2.0 mg/ml rα1-ACT day 5). G, distance between injured edges of panniculus carnosus (p = 0.04, control versus 0.4 mg/ml rα1-ACT day 5; p = 0.0006, control versus 2.0 mg/ml rα1-ACT day 5), at each time point and for each condition, 12 wounds from three different mice were analyzed; dashed line indicates granulation tissue; arrows indicate tip of epithelial tongue; arrowheads indicate ends of the injured panniculus carnosus at the wound edge; e, epidermis; d, dermis, gr, granulation tissue; sft, subcutaneous fat tissue. Scale bar, 400 μm. *, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p < 0.001.

FIGURE 3.

rα1-ACT treatment increases angiogenesis and myofibroblast differentiation in wounds of diabetic mice. A, immunofluorescence staining of cryosections from vehicle-treated (control) and rα1-ACT-treated (2 mg/ml) wounds, day 10 post-injury. B, morphometric quantification of the area within the granulation tissue that stained positive for α-SMA and CD31, day 10 post-injury (α-SMA, p = 0.008, control versus 2.0 mg/ml rα1-ACT; CD31, p = 0.003, control versus 2.0 mg/ml rα1-ACT); for each condition 12 wounds from three different mice were analyzed. Dashed line outlines granulation tissue, and dotted line indicates the epidermal-dermal junction; e, epidermis; gr, granulation tissue. Scale bar, 100 μm. **, p = 0.001 to 0.01.

FIGURE 4.

Endogenous, rα1-ACT activity is attenuated in exudates obtained from nonhealing versus healing human wounds. A, box plot diagram illustrates activity of α1-ACT protein in exudates obtained from healing (n = 5) and nonhealing (n = 15) human wounds. B, box plot diagram illustrating segregation of exudates obtained from patients with nonhealing wounds in two subgroups, based on endogenous α1-ACT activity levels <1 (n = 10) or >1 (n = 5) mg/ml; asterisk and circle illustrate divergent values.

FIGURE 5.

rα1-ACT is a target of wound proteases. A–C, Western blot analysis for α1-ACT; samples were subjected to reducing SDS-PAGE analysis, and integrity of α1-ACT protein was determined by detecting immunoreactive products with an α1-ACT-specific antibody. A, human plasma α1-ACT (600 ng/lane); exudates obtained from nonhealing (#1 and #2) or healing (#1, postoperative days (d) 2, 7, and 14) wounds. B and C, rα1-ACT (600 ng/lane) was incubated in wound exudates obtained from healing (B, #1) or nonhealing (C, #1–#5) wounds for time periods as indicated. Arrowheads indicate endogenous α1-ACT (α1-ACT), intact rα1-ACT (rα1-ACT intact), and fragments of rα1-ACT (rα1-ACT fragment). D, activity of endogenous α1-ACT in exudates obtained from nonhealing wounds (#1–5).

FIGURE 6.

LC-MS analysis of rα1-ACT fragments present in human nonhealing wounds. α1-ACT amino acid sequence of reactive center loop, identified cleavage sites (indicated by arrow), and α1-ACT fragments are shown.

FIGURE 7.

Neutrophil elastase inhibitor rescues the stability and activity of rα1-ACT in nonhealing human wounds. A, exudate (#1) obtained from a nonhealing wound was incubated with SSR 69071 to reach different final concentrations as indicated and then rα1-ACT was added. At the indicated time points, intact rα1-ACT protein was quantified by LC-MS analysis. B, SSR 69071 or PBS (control) was added to exudates obtained from nonhealing wounds (#1, #5, #11, and #12) and then rα1-ACT was added; after 12 h, rα1-ACT activity was quantified by ELISA.