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Platelet GPVI and CLEC-2 in skin wound healing
wound contraction.1 In parallel, the number of M2 repar- ative macrophages increases, contributing to resolution of inflammation.1,6 Complete wound closure and re-organi- zation of collagen fibers restore skin integrity during the remodeling phase.1
Platelets play several roles in wound healing during the hemostatic, inflammatory, and vascular repair phases.7 Platelets secrete chemoattractants and growth factors that mediate cell recruitment and tissue repair, respectively.1,7,8 This is illustrated by the delay in healing of corneal epithelial abrasion in thrombocytopenic and P-selectin- deficient mice.9 Moreover, platelet-rich plasma, which contains growth factors, promotes skin wound healing in mice10 and is a possible therapeutic agent to facilitate wound repair.
Platelet immunoreceptor tyrosine-based activation motif (ITAM) receptors, GPVI and CLEC-2, share a com- mon Src/Syk/PLCγ2-dependent signaling pathway lead- ing to platelet activation.11 A primary role for GPVI and a secondary role for CLEC-2 in maintaining vascular integrity in the inflamed skin has been demonstrated.12,13 In addition, CLEC-2 and GPVI are key regulators of inflammation. GPVI promotes a pro-inflammatory phe- notype during glomerulonephritis,14 arthritis,15 and der- matitis.16 The CLEC-2-podoplanin axis is anti-inflamma- tory and protects against organ damage during lung and systemic inflammation.17,18
Due to the complex interplay between platelets and inflammation during wound healing, we hypothesize that GPVI and/or CLEC-2 regulate vascular integrity dur- ing wound repair and alter the healing process. In the present study, we show that deletion of both CLEC-2 and GPVI accelerates wound healing in a mouse model of full- thickness excisional skin wound repair. This is associated with a transient and self-limited bleeding (i.e. due to impaired vascular integrity), fibrin(ogen) matrix deposi- tion, a reduction in wound neutrophils and M1 macrophages, and increased angiogenesis during the inflammatory phase. Taken together, we show that impaired vascular integrity-induced bleeding is beneficial during a model of sterile wound healing.
Methods
Animals
Male and female wild-type (WT), platelet-specific CLEC-2- deficient (Clec1bfl/flPf4-Cre), GPVI knockout (Gp6-/-), and CLEC- 2/GPVI double-deficient (Clec1bfl/flPf4-Cre/Gp6-/-; DKO) mice aged 8-10 weeks were used. All experiments were performed in accordance with UK laws (Animal Scientific Procedures Act 1986) with the approval of the local ethics committee and UK Home Office under PPL P0E98D513 and P14D42F37, respective- ly.
Full-thickness excisional skin wound model
A single full-thickness excisional skin wound was made on the shaved flank skin of mice using a 4 mm-diameter biopsy punch (Kai Industries, Japan). Wounds were imaged using a Nikon COOLPIX B500 digital camera each day and wound size measured using calipers19 on a daily basis for up to nine days post injury. Wound area was calculated as described19 and pre- sented as the percentage of initial wound size.10 In a second set of experiments, anti-podoplanin antibody (clone 8.1.1, 100 μg)13,17,20 or IgG isotype control were intravenously injected 24
hours (h) before and again 24 h after wounding. Wound size was monitored for three days post injury.
Other associated methods
For details of other associated methods see the Online Supplementary Appendix.
Results
CLEC-2 and GPVI ligands are present within perivascular areas during skin wounding
In unchallenged WT mouse skin, collagen was abun- dantly expressed throughout the dermis and hypodermis, including around blood vessels, and podoplanin was pre- dominantly expressed on lymphatic endothelial cells and on perivascular cells (Online Supplementary Figure S1A and B). At day 3 post wounding, podoplanin expression was also observed surrounding blood vessels in WT and trans- genic mice (Figure 1A). These podoplanin-expressing cells included pericytes (neuron-glial antigen 2; NG2+) (Figure 1A), fibroblasts (vimentin+), infiltrating monocytes (Ly6C+), and macrophages (F4/80+) (Figure 1B). In addi- tion, podoplanin was up-regulated on migrating ker- atinocytes, and on stromal and infiltrating cells within the granulation tissue (Online Supplementary Figure S1C). The level of perivascular podoplanin was increased in Clec1bfl/flPf4-cre mice compared to WT mice (Figure 1A and B). In DKO mice, podoplanin expression was similar to that observed in WT and Gp6-/- mice (Figure 1A and B).
Three days after wound injury, platelets (CD41+) were observed in close proximity to the vessel wall (surround- ed by pericytes) in WT, Clec1bfl/flPf4-cre, and Gp6-/- mice (Figure 1A, arrow). In DKO mice, platelets were more widely distributed, including at the vessel wall and in the surrounding tissue (Figure 1A, star). The platelet count in Clec1bfl/flPf4-cre and DKO mice was approximately 25% and 40% lower than in WT, respectively (Online Supplementary Figure S1D) and was not altered by wound injury.
These results demonstrate that platelets are present in perivascular areas during the initial wound healing process in mouse skin where the ligands for GPVI and CLEC-2 are also observed. Deletion of CLEC-2 from platelets is associated with increased podoplanin-express- ing cells in the perivascular space during wound healing while concurrent ablation with GPVI reverses this pheno- type.
Cutaneous wound healing is accelerated in mice deficient in platelet CLEC-2 and GPVI
To determine the contribution of platelet ITAM recep- tors in wound repair, we monitored the time course of wound closure in WT and platelet ITAM receptor(s)-defi- cient mice. All mouse strains exhibited complete wound closure within nine days post injury (Figure 2A). However, mice deficient in both GPVI and CLEC-2 demonstrated accelerated repair (Figure 2B), character- ized by the presence of a dark scab and redness around the wound (Figure 2A), especially within the first three days after injury compared to WT and single ITAM-defi- cient mice. There was no discernible difference in wound appearance in any of the mouse strains after four days post injury (Figure 2A) and there was no difference in macroscopic wound size at day 9 post injury (Figure 2B).
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