Keratinocyte Re-epithelialization Research Articles

P11 A role for keratinocyte arginase in skin wound healing and barrier function

Abstract Introduction and aims Delayed cutaneous wound healing is a major problem that can result in social isolation and significant morbidity including amputation. There are many changes in cellular function in a chronic wound. Notably, the enzyme arginase (ARG)1 is markedly changed in wound healing. We have identified a role for ARG1 in keratinocyte re-epithelialization during wound closure, but the underlying mechanism driving re-epithelialization was not known. We aimed to determine the role of ARG1 in keratinocyte migration, differentiation and barrier formation. Methods Two-dimensional scratch and differentiation assays using immortalized keratinocytes (NTERT cells) treated with the ARG1 inhibitor, nor-NOHA, and short hairpin RNA-based knockdown cells were assessed. Gene and protein expression changes were highlighted. Expression of proteins was further validated using immunohistochemical profiling of human skin equivalents and human wound lesions by spatial transcriptomics. Results ARG1 was strongly expressed both 2–4h postscratching and at late stages of keratinocyte differentiation. Dysregulation of ARG1 expression was spatially highlighted in chronic lesions, further suggesting an involvement in keratinocyte migration and differentiation. ARG1 inhibition led to a significant delay in postscratch keratinocyte migration. Furthermore, upon ARG1 inhibition, a significant decrease was seen in the expression of late differentiation markers involucrin and filaggrin. ARG1 inhibition significantly decreased the expression of host defence genes β-defensin 3, lipocalin 2 and kallikrein 6. ARG1 triggers production of multiple downstream products and we confirmed a role for the pathway involving production of putrescine and urea, as the addition of these metabolites led to the rescue of these phenotypes. Conclusions ARG1 and its downstream metabolites play a major role in keratinocyte migration, differentiation, and barrier formation, which are vital functions for re-epithelialization and wound closure. Therefore, manipulation of the ARG1 pathway may be crucial in the management of chronic lesions.

AN OVERVIEW ON THERAPEUTIC SCREENING USING EXPERIMENTAL MODELS OF WOUND HEALING

The complex process of wound healing is how damaged tissue heals itself. It's a process that includes homeostasis, tissue integrity coordination, and the activation of intercellular pathways. The present review was based on the therapeutic screening using experimental models of wound healing. Hemostasis and clot formation are the two main complicated events that take place during this phase. The clot has aggregated platelets encased in a fibrin network. Oxygen is necessary for almost all wound-healing processes and cell metabolism, particularly to produce energy via ATP. It stops wounds from getting infected, stimulates the growth of new blood vessels, raises the differentiation, migration, and re-epithelialization of keratinocytes, boosts the production of collagen and fibroblasts, and encourages wound contraction. Wound healing models include incision, excision and burn model. In conclusion, our understanding of wound pathophysiology will be significantly advanced by combining these strategies with cutting-edge tissue, cell, and molecular "omics" technology. Developing novel therapeutic approaches for enhanced wound care is, in fact, one of the many promising areas of the future.

Wound healing effects of Asparagus lucidus Lindl extract through the phosphorylation of ERK1/2

BackgroundSkin is the outermost part of the human body and is essential in maintaining body homeostasis. In the event of skin injury, rapid wound repair is crucial to protect the body. In this study, we investigated the wound-healing properties of Asparagus lucidus Lindl extract by promoting keratinocyte proliferation.MethodsTo evaluate the effect of Asparagus lucidus Lindl extract on skin regeneration, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to measure keratinocyte proliferation, while an in vitro wound-healing assay was performed to evaluate wound closure through keratinocyte re-epithelialization. The intracellular mechanisms of the extract were studied using Western blot analysis to measure the phosphorylated forms of mitogen-activated protein kinases and protein kinase B. The mRNA expression of cell cycle-related genes was analyzed using quantitative real time-PCR analysis. A murine in vivo wound-healing assay was also conducted to observe the effect of the extract on wound closure.ResultsAsparagus lucidus Lindl extract induced 131.15% keratinocyte proliferation compared to the control in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The in vitro wound-healing assay showed that the extract improved wound closure by 216.94% through keratinocyte re-epithelialization. Western blot analysis revealed that the phosphorylated form of extracellular signal-regulated kinase 1/2 was increased by extract treatment. Quantitative real time-PCR analysis showed a dose-dependent increase in the mRNA expression of c-fos, c-jun, and VEGF. The in vivo wound-healing assay showed a significant increase (22.13%) of wound closure compared to the control on day 5.ConclusionAsparagus lucidus Lindl extract promotes keratinocyte proliferation by activating the extracellular signal-regulated kinase 1/2 pathway and up-regulating the mRNA expression of c-fos, c-jun, and vascular endothelial growth factor.

Transforming growth factor-β1 negatively regulates SOCS7 via EGR1 during wound healing

BackgroundTGF-β1 promotes keratinocyte migration and re-epithelialization of cutaneous wounds during the wound healing process. Decreased SOCS7 expression has been associated with increased healing potential. However, the relationship between TGF-β1 and SOCS7 in wound re-epithelialization remains unclear.ObjectivesTo investigate the relationship between TGF-β1 and SOCS7 in the re-epithelialization of keratinocytes during skin wound healing.MethodsThe expression of SOCS7 under different concentrations of TGF-β1 was detected by WB and qPCR. The migration ability of keratinocytes was detected by scratch and Transwell assay. Protein interactions were detected by ChIP and luciferase assay. The effect of SOCS7 on skin healing in mice was detected in animal model.ResultsIn this study, we found that SOCS7 was downregulated by TGF-β1 and that overexpression of SOCS7 led to suppression of TGF-β1-induced keratinocyte migration through inhibition of the PI3K/AKT and MEK/ERK pathways. Also, TGF-β1 negatively regulated SOCS7 expression at the transcriptional level through the binding of EGR1 to the EGR1/SP1 overlapping binding sites in the SOCS7 promoter.ConclusionTaken together, our findings show that TGF-β1-induced EGR1 expression is required for repression of SOCS7, which promotes keratinocyte migration and re-epithelialization during wound healing. Finally, our study identifies the TGF-β1/EGR1/SOCS7 pathway as a potential therapeutic target to promote wound healing.5iNumoHWC7LtyUXVHhpfUVVideo

Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing

Keratinocytes, the predominant cell type of the epidermis, migrate to reinstate the epithelial barrier during wound healing. Mechanical cues are known to regulate keratinocyte re-epithelialization and wound healing however, the underlying molecular transducers and biophysical mechanisms remain elusive. Here, we show through molecular, cellular and organismal studies that the mechanically-activated ion channel PIEZO1 regulates keratinocyte migration and wound healing. Epidermal-specific Piezo1 knockout mice exhibited faster wound closure while gain-of-function mice displayed slower wound closure compared to littermate controls.

Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing.

Keratinocytes, the predominant cell type of the epidermis, migrate to reinstate the epithelial barrier during wound healing. Mechanical cues are known to regulate keratinocyte re-epithelialization and wound healing; however, the underlying molecular transducers and biophysical mechanisms remain elusive. Here, we show through molecular, cellular, and organismal studies that the mechanically activated ion channel PIEZO1 regulates keratinocyte migration and wound healing. Epidermal-specific Piezo1 knockout mice exhibited faster wound closure while gain-of-function mice displayed slower wound closure compared to littermate controls. By imaging the spatiotemporal localization dynamics of endogenous PIEZO1 channels, we find that channel enrichment at some regions of the wound edge induces a localized cellular retraction that slows keratinocyte collective migration. In migrating single keratinocytes, PIEZO1 is enriched at the rear of the cell, where maximal retraction occurs, and we find that chemical activation of PIEZO1 enhances retraction during single as well as collective migration. Our findings uncover novel molecular mechanisms underlying single and collective keratinocyte migration that may suggest a potential pharmacological target for wound treatment. More broadly, we show that nanoscale spatiotemporal dynamics of Piezo1 channels can control tissue-scale events, a finding with implications beyond wound healing to processes as diverse as development, homeostasis, disease, and repair.

BET bromodomain inhibitors regulate keratinocyte plasticity.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

The Suppressive Effect of Leucine-Rich Glioma Inactivated 3 (LGI3) Peptide on Impaired Skin Barrier Function in a Murine Model Atopic Dermatitis.

This study aimed to restore the skin barrier function from atopic dermatitis (AD) via treatment with leucine-rich glioma inactivated 3 (LGI3) peptide. Male NC/Nga mice (7 weeks, 20 g) were randomly allocated into three groups (control, AD, and LGI3 group). After induction of AD skin lesions with Dermatophagoides farinae ointment, mice were treated with LGI3. The clinical score of AD was the highest and the dorsal skin thickness was the thickest in the AD group. In contrast, LGI3 treatment improved the clinical score and the dorsal skin thickness compared to the AD model. LGI3 treatment suppressed histopathological thickness of the epithelial cell layer of the dorsal skin. LGI3 treatment could indirectly reduce mast cell infiltration through restoring the barrier function of the skin. Additionally, the filaggrin expression was increased in immunohistochemical evaluation. In conclusion, the ameliorating effect and maintaining skin barrier homeostasis in the AD murine model treated with LGI3 could be attributed to complete re-epithelialization of keratinocytes. Hence, LGI3 might be considered as a new potential therapeutic target for restoring skin barrier function in AD.

Keratinocyte Growth Factor Modified Messenger RNA Accelerating Cell Proliferation and Migration of Keratinocytes.

Keratinocyte growth factor (KGF) plays a central role in wound healing as it induces cell proliferation and motility. The use of growth factors such as KGF is therefore viewed as a promising approach in wound therapy, although effective application remains a major problem because of inactivation and the resulting short half-life of applied growth factors in wound beds. Therefore, the rational of this study was to develop and investigate an innovative strategy to improve wound healing using an in vitro-transcribed modified KGF messenger RNA (mRNA). After transfection of cells, we evaluated the effects of the produced KGF protein on cell migration and reepithelialization of keratinocytes using a scratch assay. The results demonstrate that KGF-mRNA-transfected cells exhibited a high KGF protein release that is sufficient to significantly improve reepithelialization in the performed scratch assays. Transfection with growth factor mRNA therefore seems to be a promising therapeutic strategy, especially for difficult wounds, as it leads to a temporary increase of growth factor expression in the treated wound area without interfering with the DNA of the nucleus, as seen in gene therapeutic applications.

SR-B1 involvement in keratinocytes in vitro wound closure

Skin represents the most extended organ of human body, having as main function the protection of our body from outdoor stressors. Its protective ability is compromised when the skin is disrupted as a consequence of mechanical insults. For this purpose, cutaneous tissue is equipped with an efficient and fine mechanism involved in repairing the wounded area. Among the numerous players that take part in the wound healing process, SR-B1 has been recently shown to have a role in keratinocyte re-epithelialization. SR-B1 is a mediator of cholesterol uptake from HDLs, whereas it is implicated in other cellular processes such as vitamins absorption, vesicle trafficking or pathogen identification. The aim of this study was to investigate the mechanisms involved in SR-B1 role in skin wound closure. Our in vitro data demonstrated that SR-B1 influenced keratinocyte proliferation and migration through a downregulation of nuclear cyclin D1 levels and active MMP9 expression respectively possibly in an NF-kB-dependent mechanism. In addition, SR-B1 was also able to modulate keratinocyte morphology into a pro-migratory cytoskeleton rearrangement.The present in vitro study suggests a new role of SRB1 as a possible new key player in cutaneous wound healing mechanism.

Substrate deformations induce directed keratinocyte migration.

Cell migration is an essential part of many (patho)physiological processes, including keratinocyte re-epithelialization of healing wounds. Physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected human keratinocyte behaviour and that mechanical deformations in soft (approx. 1.2 kPa) PA gels produced by neighbouring cells appeared to influence the process of de novo epithelial sheet formation. To clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighbouring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632 or blebbistatin on cell response. Our results indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate deformations imposed by neighbouring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to substrate deformation-directed keratinocyte migration.

Newly Discovered Olfactory Receptors in Epidermal Keratinocytes Are Associated with Proliferation, Migration, and Re-Epithelialization of Keratinocytes

Skin contains receptors for various environmental factors. In this issue of the Journal, Busse et al. cloned a new olfactory receptor, OR2AT4, in keratinocytes. They show that the activation of OR2AT4 induces phosphorylation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinases, and that it accelerates wound healing. OR2AT4 may be a promising candidate as a target in clinical drug development.

A Synthetic Sandalwood Odorant Induces Wound-Healing Processes in Human Keratinocytes via the Olfactory Receptor OR2AT4

As the outermost barrier of the body, the skin is exposed to multiple environmental factors, including temperature, humidity, mechanical stress, and chemical stimuli such as odorants that are often used in cosmetic articles. Keratinocytes, the major cell type of the epidermal layer, express a variety of different sensory receptors that enable them to react to various environmental stimuli and process information in the skin. Here we report the identification of a novel type of chemoreceptors in human keratinocytes, the olfactory receptors (ORs). We cloned and functionally expressed the cutaneous OR, OR2AT4, and identified Sandalore, a synthetic sandalwood odorant, as an agonist of this receptor. Sandalore induces strong Ca(2+) signals in cultured human keratinocytes, which are mediated by OR2AT4, as demonstrated by receptor knockdown experiments using RNA interference. The activation of OR2AT4 induces a cAMP-dependent pathway and phosphorylation of extracellular signal-regulated kinases (Erk1/2) and p38 mitogen-activated protein kinases (p38 MAPK). Moreover, the long-term stimulation of keratinocytes with Sandalore positively affected cell proliferation and migration, and regeneration of keratinocyte monolayers in an in vitro wound scratch assay. These findings combined with our studies on human skin organ cultures strongly indicate that the OR 2AT4 is involved in human keratinocyte re-epithelialization during wound-healing processes.

Honey exposure stimulates wound repair of human dermal fibroblasts.

Honey is widely used for treating burns, ulcers and wounds, but the mechanisms of action are poorly known and the product is mainly used as an antimicrobial. We have examined here the wound healing properties of honey on human fibroblasts, using an in vitro scratch wound healing model. Three kinds of widely used monofloral honeys were used, viz. acacia (Robinia pseudacacia), buckwheat (Fagopyrum sp.), and manuka (Leptospermum scoparium). Data displayed an increased wound healing activity in fibroblasts, but with different efficiency and mechanisms of action among honeys. The effects of acacia and buckwheat emerged in both scratch wound and chemotaxis assays, while the effect of manuka was significant but lower. The use of inhibitors indicated on the whole an essential role of cytosolic calcium, an important role of ERK and p38, and a secondary role of PI3K. Acacia and buckwheat, but not manuka, induced significant increases in the release of interleukin-4 (IL-4), IL-6, and IL-8, indicating a correlation between interleukin upregulation and wound closure efficiency. This is consistent with our previous findings suggesting a higher ability of acacia and buckwheat to activate keratinocyte reepithelialization, with respect to manuka honey. In conclusion, our data indicate that acacia and buckwheat honeys are particularly efficient in facilitating fibroblast wound closure activities, suggesting new therapeutic possibilities for this natural product.

Epithelial mesenchymal transition traits in honey‐driven keratinocyte wound healing: Comparison among different honeys

Honey has been used since ancient times for wound repair, but the subjacent mechanisms are almost unknown. We have tried to elucidate the modulatory role of honey in an in vitro model of HaCaT keratinocyte re-epithelialization by using acacia, buckwheat, and manuka honeys. Scratch wound and migration assays showed similar increases of re-epithelialization rates and chemoattractant effects in the presence of different types of honey (0.1%, v/v). However, the use of kinase and calcium inhibitors suggested the occurrence of different mechanisms. All honeys activated cyclin-dependent kinase 2, focal adhesion kinase, and rasGAP SH3 binding protein 1. However, vasodilator-stimulated phosphoprotein, integrin-β3, cdc25C, and p42/44 mitogen activated protein kinase showed variable activation pattern. Re-epithelialization recapitulates traits of epithelial-mesenchymal transition (EMT) and the induction of this process was evaluated by a polymerase chain reaction array, revealing marked differences among honeys. Manuka induced few significant changes in the expression of EMT-regulatory genes, while the other two honeys acted on a wider number of genes and partially showed a common profile of up- and down-regulation. In conclusion, our findings have shown that honey-driven wound repair goes through the activation of keratinocyte re-epithelialization, but the ability of inducing EMT varies sensibly among honeys, according to their botanical origin.

Smad4 disruption accelerates keratinocyte reepithelialization in murine cutaneous wound repair

Keratinocyte reepithelialization is a rate-limiting event in cutaneous wound repair, which involves the migration and proliferation of keratinocytes to cover the denuded dermal surface. Transforming growth factor-β1 (TGF-β1) has the ability to induce epithelial cell migration while inhibiting proliferation, and controversial results have been generated regarding the effect of TGF-β signaling on reepithelialization. In this study, full-thickness skin wounds were made in keratinocyte-specific Smad4 knockout and the control mice. The wound closure, reepithelialization, keratinocyte proliferation, myofibroblast numbers and collagen deposition of were assessed. The results showed that the proliferation of keratinocytes increased, which accelerated the reepithelialization, and led to faster wound repair in the epidermis of Smad4 mutant mice. Upregulation of keratin 17, 14-3-3 sigma and phosphorylated AKT in the hyperproliferative epidermis may be correlated with the accelerated reepithelialization. We conclude that Smad4 plays an inhibitory role in the keratinocyte-mediated reepithelialization of wound healing.

Expression of WAVEs, the WASP (Wiskott-Aldrich syndrome protein) family of verprolin homologous proteins in human wound tissues and the biological influence on human keratinocytes

WAVEs (Wiskott-Aldrich syndrome protein family verprolin homologs) regulate actin polymerization and influence cellular motility. Here, we investigated the pattern of expression of WAVE-1, WAVE-2, and WAVE-3, in a cohort of human wound tissues together with normal skins and evaluated the role of these molecules in the reepithelialization and migration of keratinocytes. It was shown that there was a significant reduction of the WAVE-3 transcripts in chronic wound tissues, when compared with normal skin and acute wound tissue (p=0.002). Marginal reduction of the WAVE-1 and WAVE-2 transcripts in chronic wounds were also seen. Immunohistochemical analysis showed a significant reduction of all three WAVE proteins in chronic wound tissues in comparison with the acute. We created in vitro cell models using keratinocytes in which we overexpressed WAVE-2, and knocked down the expression of WAVE-1 and WAVE-3. Using ECIS assay, it was shown that knocking down WAVE-3 had a significant effect on the migration and reepithelialization of keratinocytes and that overexpression of WAVE-2 also increased the migration of keratinocytes. We further demonstrated that the impact of WAVE on cell migration was independent upon the PLCg and ERK pathways, but in the downstream requires PI3K pathway and ROCK pathways. In conclusion, the WAVE family proteins are essential for the reepithelialization of keratinocytes. Aberrant expression of WAVEs is linked to the healing process of clinical wounds and loss of WAVE-3 is a particular indicator of an abnormally healing wound. The WAVE proteins have a significant predicative and therapeutic value in wound healing.

MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing

Matrix metalloproteinases (MMPs) have been implicated in wound healing. To analyze the roles of MMP-9 and MMP-13 in wound healing, we generated full-thickness cutaneous wounds in MMP-9 knockout (KO), MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Macroscopic wound closure was delayed in all of the KO mice, as compared with wild-type mice. The rate of re-epithelialization was significantly delayed in MMP-9 KO and MMP-13 KO mice and remarkably delayed in MMP-9/13 double KO mice, as compared with wild-type mice. Both MMP-9 and MMP-13 were expressed by the leading edges of epidermal cells in wild-type mice, and the migration of keratinocytes was suppressed by treatment with an MMP inhibitor or transfection of small interfering RNAs for MMP-9 or MMP-13, as compared with controls. The vascular density in wound granulation was significantly lower in both MMP-13 KO and MMP-9/13 double KO mice than in wild-type mice. Degradation of connective tissue growth factor in wound tissue was transiently prevented in MMP-13 KO mice. Morphometric analyses demonstrated a reduction in both wound contraction and myofibroblast formation in both MMP-13 KO and MMP-9/13 double KO mice. Proliferation and transforming growth factor-beta1-induced myofibroblast differentiation of dermal fibroblasts from MMP-13 KO mice were decreased, as compared with wild-type dermal fibroblasts. These data suggest that MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in wound healing, while MMP-9 functions in keratinocyte migration.

Effects of bio-active ceramic resources in cutaneous wound healing and the role of TGF-β signaling

The wound healing process is a highly orchestrated process, which includes inflammation, re-epithelialization, granulation tissue formation, matrix formation and re-modeling. In this paper, we attempt to determine if bio-active ceramic resource powder particles had an effect on cutaneous wound healing. Furthermore, we investigated its related mechanism and the expression of Smads of cutaneous wound healing, which can be accelerated by bio-active ceramic ointment. Topically applied lesions of 5%, 10% and 15% bio-active ceramic ointment (AO) showed accelerated wound closure, re-epithelialization, and the related immediate down stream of TGF-beta (p-Smad2/3 and Smad3) was suppressed. In particular, 10% and 15% AO lesions became closed faster at Days 3 and 4 of post-wound and p-Smad2/3 was also suppressed. All AO lesions showed accelerated mild wound closure at Day 6, but there were no significant difference. Several papers reported that Smad3 may mediate the signaling pathways that is inhibitory to wound healing, as the deletion of Smad3 leads to enhanced re-epithelialization and contraction of the wound area. This study showed that topical, bio-active ceramic ointment applications accelerated wound closure, re-epithelialization and the suppression of Smad proteins (p-Smad2/3, Smad3). The data revealed that the suppression of Smad3, which was induced by bio-active ceramic resources powder particles affected re-epithelialization and cutaneous wound closure. At the end of this paper, we concluded that bio-active ceramic resources affect cutaneous wound healing by accelerating the re-epithelialization of keratinocytes and that is mediated by the suppression of related protein, Smad3.

Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissue-type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds to a degree indistinguishable from that observed in Plg-deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation.