Mice lacking the thrombin receptor, PAR1, have normal skin wound ...

American Journal ofPathology, Vol. 151, No. 5, November 1997 Copyright X American Society for Investigative Pathology

Short Communication Mice Lacking the Thrombin Receptor, PAR1, Have Normal Skin Wound Healing

Andrew J. Connolly,*t David Y. Suh,t Thomas K. Hunt,* and Shaun R. Coughlin*§ From the Cardiovascular Research Institute,* Departments of Pathology,t Surgery,* and Medicine,5 and Daiichi Research Center, University of California, San Francisco, San

Francisco, California

Thrombin's actions on platelets, macrophages, fibroblasts, and endothelial cells have prompted the hypothesis that thrombin may be important for inflammatory and fibroproliferative processes in wound healing. Protease-activated receptor 1 (PAR1) is a Gprotein-coupled receptor that mediates many of the celilular activities of thrombin. To test the role of this receptor in vivo, we generated PARl-deficient mice. Despite the observation that fibroblasts cultured from these mice lacked responsiveness to thrombin in vitro, we now report that there was no difference detected between wild-type and PARl-deficient mice in skin wound healing assays including time to closure of open wounds, tensile strength of healed incisional wounds, wound histology, and hydroxyproline/DNA content of wound implants. We conclude that PAR1 is not necessary for normal skin wound healing in mice. (Am JPathol 1997, 151:1199-1204)

Thrombin is a multifunctional serine protease generated at sites of vascular injury, including skin wounds. Active thrombin is sequestered in fibrin clots where it is protected from neutralization by plasma proteinase inhibitors, and thus thrombin may be released over time during clot organization and wound healing.1-3 In addition to its well described roles in coagulation, thrombin directly activates a number of cell types that play important roles in wound healing. Thrombin causes platelets to aggregate and to release the contents of their granules, including platelet-derived growth factor and serotonin.4 It acts on endothelial cells to increase vascular permeability,5 adhesion of platelets and leukocytes,67 and the release of mediators such as platelet-derived growth factor8 and monocyte chemotactic protein-1.9 Thrombin has been reported to be chemotactic for neutrophils10 and mono-

cytes.11 It stimulates proliferation of smooth muscle cells and fibroblasts.12-14 Thrombin's actions on these various cell types prompted the hypothesis that thrombin might participate not only in hemostasis but also in inflammatory and proliferative responses to injury.15 In support of this idea, application of exogenous thrombin has been reported to accelerate healing of rat skin incisions, as assessed by wound tensile strength, type collagen deposition, and growth of capillaries in wounds by 7 days after surgery.16 A small family of G-protein-coupled receptors capable of mediating cellular responses to proteases has been identified.17-20 The prototype for this family is the thrombin receptor, protease-activated receptor 1 (PAR1). It is activated when thrombin binds to and cleaves its aminoterminal exodomain, thereby unmasking a new amino terminus. This then acts as a tethered ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. 17921X22 PAR2 can mediate signaling to trypsin and tryptase but not thrombin.19 PAR3 is a recently described second thrombin receptor.20 PAR1 is expressed in a variety of human cell types, including platelets,17 fibroblasts,18,23 endothelial cells,24 and perhaps leukocytes.9 In mice, PAR1 is expressed in leukocytes, endothelial cells, smooth muscle cells, mesenchymal cells, and certain neurons.25 Synthetic peptides representing the first six or more amino acids of the new receptor amino terminus generated when thrombin cleaves PAR1 act as agonists for this receptor.172627 Such PARl-activating peptides reproduced many of thrombin's actions on human platelets,17 rodent and human fibroblasts,1623 and human endothelial cells.2428 The PARl-activating peptide is known to show some activity at PAR229,30 but not at the other known thrombin receptor PAR320; thus available data suggest that thrombin responses in human platelets, endothelial cells, and fibroblasts are mediated at least in part by PAR1. In vivo, PARl1-activating peptides recapitulated thrombin's ability Supported by NIH-DK50267 and the Daiichi Research Center (S. R. Coughlin), NIH-HL03234 (A. J. Connolly), and NIH-GM27345 (T. K. Hunt). Accepted for publication July 22, 1997. Address reprint requests to Dr. Shaun R. Coughlin, HSE-1300, University of California, San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143-0130.

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to stimulate leukocyte rolling in rat mesenteric blood vessels31 and acute inflammation of the rat paw.32 These observations suggested that PAR1 might promote inflammatory and proliferative responses to vascular injury, thereby potentially promoting wound repair. Toward defining the in vivo roles of PAR1, PARl-deficient mice were produced.3334 One-half or more par -/- mice died during embryonic development at E9.5 to E10.5, but those that survived to birth were grossly normal and showed no hemostatic defect. Indeed, platelets from parl -/- mice responded normally to thrombin, possibly via PAR3 signaling.20'33 By contrast, fibroblasts from parl -/- mice were unresponsive to thrombin,14'33 providing an opportunity to test the importance of PAR1 signaling in these cells in wound healing.

Materials and Methods Mice The mice used for these studies were all healthy adult male mice of a mixed strain, each 50% C57BL/6 and 50% 129Sv. They were raised and studied in microisolator cages in a mouse facility free from common pathogens. The parl +/+ and parl -/- mice grew at comparable rates and were matched for age (8 to 12 weeks) and weight (25 to 35 g). Mice had been genotyped by Southern blot33 at 4 weeks of age and were genotyped once again after being sacrificed. Before all wounding studies, mice were anesthetized by inhalation of methoxyfluorane and the wound sites were carefully shaved. All wounds were full thickness, extending through the epidermis and dermis to the panniculus carnosus. After wounding, mice were separated into individual cages containing sterilized pressed-cellulose bedding to limit disruption or soiling of wounds. The wounds and general health of the mice were monitored each day; no complications were encountered.

Open Wound Contraction Two full-thickness punch biopsies (0.5 cm diameter, 2.0 cm apart) were made overlying the scapulas using circular skin bioptomes. Five mice were used for each genotype. Compression with clean gauze was held on wounds for 2 minutes to achieve hemostasis. Wounds were neither dressed nor sutured. Each day for 11 days, the lengths and widths of the epidermal defects were measured and the wound areas calculated assuming an oval geometry.

Wound Tensile Strength Two full-thickness paramedian wound incisions (1.5 cm long, 2.5 cm apart) were made on the lower back using a scalpel. The wounds were made on the same 10 mice used in the punch biopsy study. Compression with clean gauze was held on wounds for 2 minutes to achieve hemostasis, and the wounds were closed with two interrupted 4-0 nylon sutures spaced each 0.5 cm. After

11 days, the mice were euthanized and a transverse, full-thickness, 1.0-cm-wide strip was taken through the center of each wound and mounted on a Sandblom tensiometer to assay breaking strength.35 Transverse, fullthickness, 1-cm-wide strips of skin were taken in a similar fashion from skin adjacent to the wounds in three mice of each genotype to act as control skin.

Wound Histology Two full-thickness paramedian wound incisions (1.0 cm long, 2.0 cm apart) were made overlying the scapulas using scissors. The wounds initially gaped 0.4 to 0.6 cm at the center and were neither dressed nor sutured. Two mice of each genotype were euthanized at the designated times (days 1, 3, 5, 7, and 10). The wounds and surrounding skin were excised, fixed in 10% neutral buffered formalin, and processed for paraffin embedding. Level sections were cut from the midtransverse plane of each wound and stained with hematoxylin and eosin (H&E) or Masson trichrome.

Wound Implant Biochemical Assays Implants were used as previously described.3637 Expanded polytetrafluoroethylene tubing with a pore size of 90 to 120 ,um, internal diameter of 1.2 mm, and wall thickness of 0.6 mm (Imprar, International Polymer Engineering, Tempe, AZ) was cut into 3.5-cm lengths and guided percutaneously by a cutting needle into the subcutaneous tissue of the left and right flanks of five mice of each genotype. The implants were left in place for 14 days before the mice were euthanized, and the implants were removed by exposing one end and pulling the implant out. A 1.0-cm length of each implant was assayed for DNA content by the method of Burton38; the remainder was assayed for total protein content by a ninhydrin assay39 and hydroxyproline content by the method of Grant.40 To demonstrate the sensitivity of these assays to inhibitors of wound healing, a positive control group was added that consists of parl +1+ mice injected with methylprednisolone (25 mg/kg body weight) at the time of surgery.

Results Physical Assessment of Wounds Wound healing was indistinguishable in wild-type versus parl -/- mice matched for sex, age, weight, and strain. The area of wounds was followed after punch excisional biopsy for 1 1 days (Figure la). The general trend for both +/+ and -/- mice was a linear reduction in wound area until complete closure at 10 to 11 days. Skin tensile strength was measured in wounds and nearby control skin 11 days after incisions were made and closed by suturing (Figure ib). The tensile strength of parl -/wounds was not different from that of parl +1+ wounds. The tensile strength of control skin from parl +1+ and -/- mice was also indistinguishable.

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+/+ and -/- mice for 14 days (Table 1). The DNA content gives a measure of cellularity within the implant, the hydroxyproline content gives an estimate of collagen production, and total protein content assays amounts of both cellular and extracellular protein. No differences were seen between parl +/+ and -Imice in these assays. To demonstrate the sensitivity of these assays to inhibitors of wound healing, a positive control group was added that consists of parl +/+ mice injected with methylprednisolone at the time of surgery. In this corticosteroid-treated group, reductions in DNA content and hydroxyproline content were more apparent than reductions in total protein, consistent with previous observations in corticosteroid-treated rats.36

+1+

E 0

0

B

Vol.

800 r

Discussion

"= 400

0 e 200

Co +1+

skcontrol skin

healing skin

Figure 1. Analysis of wound closure and tensile strength in healing skin wounds in parl +/+ and parl -/- mice. a: Wound areas measured daily after excisional biopsy of back skin. Data on 10 individual wounds (five mice, two per mouse) are shown. b: Tensile strength (mean + SEM; n = 10) of wound skin and nearby control skin in mice 11 days after incisions with primary closure by suturing.

Histological Assessment of Wounds The histology of healing open wounds for parl -/- and +/+ mice was indistinguishable at 1, 3, 5, and 7 days after wounds were made (Figure 2). The base of the wound was covered with blood clot and a fibrinopurulent exudate at 1 day. This was followed, in the sections at days 3 and 5, by macrophage infiltration and granulation tissue growth at the base and shoulders of the wound. The wound contracted and epidermis grew in from the edges, steadily displacing the eschar throughout days 1, 3, 5, and 7. The wounds had similar amounts of blood clot and neutrophil infiltration at 1 day, granulation tissue growth at 3 and 5 days, and epidermal growth throughout.

Biochemical Assessment of Wounds To assess the cellularity and collagen production of wounds in parl -/- and +/+ mice, biochemical assays were performed on expanded polytetrafluoroethylene implants that remained in the subcutaneous tissue of parl

Thrombin is generated in wounds and is a potent activator of cell types known to play key roles in wound healing. Many of these cellular responses to thrombin are mediated by PAR1, and we postulated that important aspects of wound healing might be mediated by thrombin stimulation of this receptor. In this study, wound healing was assessed physically, histologically, and biochemically, and no differences in healing were seen between parl +/+ and -/- mice. This study definitively indicates that PAR1 is not necessary for skin wound healing in mice. Thrombin responses were ablated in fibroblasts cultured from the lungs of parl -/- mice and from parl -/embryos.14 To the extent that such fibroblasts are representative of those in wounds, this study also suggests that thrombin signaling in fibroblasts is not necessary for wound healing in mice. The wound-healing assays used in this study are sensitive to alterations in healing caused by various perturbations. There was inhibition of wound healing in the open wound closure and ePTFE implant models in ob/ob mice37 and in open wound closure and incision tensile strength models in db/db mice.41 In rats, wound healing as measured by these assays was altered by systemic administration of corticosteroids,3642 cyclosporine A,43 and anti-Thyl.2 (T cell) antibody.44 In the present study, methylprednisolone had substantial effects (Table 1). The sensitivity of these assays suggests that important wound healing defects in parl -/- mice would have been detected had they been present. These studies do not exclude the possibility that thrombin and those cellular activities of thrombin that are mediated by receptors other than PAR1 might be important for wound healing. At least one thrombin-triggered cellular event, platelet activation in mice, is mediated by a second thrombin receptor,33 and at least one additional thrombin receptor, PAR3, has been identified.20 PAR3 expression appears to be restricted to megakaryocytes and platelets in normal adult mice, and in situ hybridization performed on tissue sections from healing open wounds revealed no expression of PAR3 (Ref. 20 and data not shown). It therefore seems likely that PAR3's role in wound healing, if any, would be via its actions on platelets. PAR3's role will become testable when mice

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taken from parl +/+ (A, C, E, and G) and parl -/ Figu re 2. Histology of healing skin incisions in parl +/+ and parl-/-mice. Midtransverse sections were wounds from two mice were studied in each group Four after and wounding. 7 H) and (G and days and 5 F), (E D), (B, D, F, and H) mice at 1 (A and B), 3 (C at each time point with similar results. Tissue sections were stained with H&E; each photographic field covers 1.7 x 1.2 mm of tissue section.

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Table 1.

Biochemical Assays DNA content

Mice

parl +/+ parl -/parl +/+ with methylprednisolone

Protein content

45.5 ± 14.0 1190 ± 129 43.9 ± 18.8 1090 ± 238 17.6 ± 2.4 911 ± 162

HP content

8.

21.0 ± 5.2 21.1 ± 6.0 14.8 ± 5.5

HP, hydroxyproline. Results (mean ± SEM; n = 10) are expressed as jig/cm expanded polytetrafluoroethylene implant.

with null mutations of the par3 gene alone or in the parl -/- background are available. Such mice will also point out which cellular activities of thrombin are not accounted for by known thrombin receptors, perhaps revealing new receptors with potential roles in wound healing. Despite these caveats, it should be noted that the hypothesis that thrombin might be important in wound healing was based largely on its ability to elicit relevant cellular events in vitro. As discussed above, activation of PAR1 is sufficient to elicit pro-inflammatory responses in endothelial cells and proliferative responses in mesenchymal cells, but a host of cytokines and growth factors can elicit the same responses. Moreover, thrombin is less effective as a mitogen for mouse fibroblasts than either serum or fibroblast growth factor in vitro, and parl -/fibroblasts respond mitogenically to serum as well as wild type.12'14 It would not be surprising if such redundancy rendered thrombin's cellular actions unnecessary for the inflammatory and fibroproliferative components of wound repair to take place normally. Humans with complete deficiencies in prothrombin, ie, aprothrombinemia, have not been identified.45 Prothrombin null mice, if viable, could provide a more direct test of the necessary roles of thrombin in wound healing.

Acknowledgments We thank Heinz Scheuenstuhl for technical assistance with biochemical assays of wound implants, Linda Prentice for assistance with histology, and Carmen Tam for assistance with in situ hybridization.

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