Factor VII deficiency impairs cutaneous wound healing in mice

Abstract

Skin keratinocytes express tissue factor (TF) and are highly associated with skin wound healing. Although it has been demonstrated that perivascular TF expression in granulation tissue formed after dermal injury is downregulated during healing, studies of the mechanism of factor (F) VII, a TF ligand, in skin wound healing are lacking. We reported the use of a dermal punch model to demonstrate that low-expressing FVII mice (approximately 1% of wild type [WT]) exhibited impaired skin wound healing compared with WT controls. These low-FVII mice showed defective reepithelialization and reduced inflammatory cell infiltration at wound sites. This attenuated reepithelialization was associated with diminished expression of the transcription factor early growth response 1 (Egr-1). In vitro, Egr-1 was shown to be essential for the FVIIa-induced regulation of keratinocyte migration and inflammation. Both Egr-1 upregulation and downstream inflammatory cytokine appearance in keratinocytes depended on FVIIa/TF/protease-activated receptor 2 (PAR-2)-induced signaling and did not require subsequent generation of FXa and thrombin. The participation of Egr-1 in FVIIa-mediated regulation of keratinocyte function was confirmed by use of Egr-1-deficient mice, wherein a significant delay in skin wound healing after injury was observed, relative to WT mice. The results from these studies demonstrate an in vivo mechanistic relationship between FVIIa, Egr-1 and the inflammatory response in keratinocyte function during the wound healing process.

Figure 1

Time course of dermal wound healing in WT and FVII^tTA/tTA mice. Representative post wound photographs at d 1 (A), d 9 (B) and d 12 (C) in WT mice and at d 1 (D), d 9 (E) and d 12 (F) in FVII^tTA/tTA mice. The wound area is marked by red circle. The scale bar represents 1 cm in length. (G) The kinetics of skin wound closure in WT (solid line) and FVII^tTA/tTA (dashed line) mice. The values represent mean ± SEM, N = 7 for WT and N = 8 for FVII^tTA/tTA mice. *P < 0.05 at d 5, 7, 9 and 11 after injury.

Figure 2

Low FVII impairs reepithelialization and leukocyte migration during wound healing. (A–D) Immunohistochemical stains of d 5 wounds. Representative cytokeratin stains of wounded skin at d 5 postinjury in WT (A) and FVII^tTA/tTA (B) mice. Original magnification 40×. The arrows indicate the distance from the edge of the wound (e) to the tip (t) of the keratinocytes. Representative CD45 stains (brown) at d 5 after wound induction in WT (C) and FVII^tTA/tTA (D) mice. The orientation is marked with the location of the scar tissue (s) and hypodermis (h). Original magnification 200×.

Figure 3

FVIIa signaling is mediated through Egr-1 and depends on TF and PAR-2 in keratinocytes. (A) Egr-1 mRNA levels in keratinocyte in response to addition of murine FVIIa. (B) MIP-2 mRNA levels from keratinocytes obtained from WT (filled bars) and Egr-1^−/− (unfilled bars) mice after various combinations of mFVIIa (50 nmol/L) and LPS (10 μg/mL) were added to the culture. The Student t test was performed to compare WT and Egr-1^−/− keratinocytes within each treatment group. (C–D) Egr-1 and MIP-2 are downstream targets of PARs signaling in keratinocytes. Expression levels of Egr-1 and MIP-2 are measured in WT keratinocytes before and after stimulation with either PAR-1/AP, 100 μmol/L, or PAR-2/AP, 100 μmol/L. (E, F) FVIIa-induced signaling in keratinocyte is mediated by TF and PAR-2. Expression levels of Egr-1 (E) and MIP-2 (F) in keratinocyte derived from WT (filled bars), PAR-1^−/− (open bars), PAR-2^−/−(horizontal striped bars) and TF^−/−hTF (tg) (vertical striped bars) mice, before (− mFVIIa) and after (+ mFVIIa) addition of 50 nmol/L mFVIIa for 2 h. *P < 0.05, N ≥ 3 for each genotype.

Figure 4

FVIIa signaling is specific and does not require subsequent generation of FXa and thrombin in keratinocyte. Expression levels of Egr-1 (A) and MIP-2 (B) in WT keratinocytes either without pretreatment (filled bars), or preincubated with fondaparinux (5 μg/mL, open bars), or lepirudin (4 μg/mL, horizontal striped bars) for 1 h, before (− mFVIIa) and after (+ mFVIIa) addition of 50 nmol/L mFVIIa for 2 h. *P < 0.05, N ≥ 3 for each genotype.

Figure 5

Egr-1 is a critical mediator in wound healing and cell migration. (A) Kinetics of skin wound healing in WT (solid line) and Egr-1^−/− (dashed line) mice. The values represent the mean ± SEM (N = 4 for WT and N = 6 for Egr-1^−/− mice). *P < 0.05 at d 5, 7, 9, 11, 13 and 15. (B) Primary keratinocytes (top chamber) from Egr-1^−/− mice (unfilled bars) showed significantly reduced migration toward EGF (20 ng/mL in the bottom chamber) compared with WT keratinocytes (filled bars). (C, D) Inflammatory cell recruitment in WT (filled bars) and Egr-1^−/− mice (unfilled bars) after thioglycollate challenge. (C) Total peritoneal leukocytes after thioglycollate challenge from WT and Egr-1^−/− mice. Differential counts of peritoneal neutrophils (D) and macrophages (E). *P < 0.05, N = 3–8.