The plasminogen receptor, Plg-RKT, plays a role in inflammation and fibrinolysis during cutaneous wound healing in mice

Wound healing is a complex physiologic process that proceeds in overlapping, sequential steps. Plasminogen promotes fibrinolysis and potentiates the inflammatory response during wound healing. We have tested the hypothesis that the novel plasminogen receptor, Plg-RKT, regulates key steps in wound healing. Standardized burn wounds were induced in mice and time dependence of wound closure was quantified. Healing in Plg-RKT−/− mice was significantly delayed during the proliferation phase. Expression of inflammatory cytokines was dysregulated in Plg-RKT−/− wound tissue. Consistent with dysregulated cytokine expression, a significant delay in wound healing during the proliferation phase was observed in mice in which Plg-RKT was specifically deleted in myeloid cells. Following wound closure, the epidermal thickness was less in Plg-RKT−/− wound tissue. Paradoxically, deletion of Plg-RKT, specifically in keratinocytes, significantly accelerated the rate of healing during the proliferation phase. Mechanistically, only two genes were upregulated in Plg-RKT−/− compared with Plg-RKT+/+ wound tissue, filaggrin, and caspase 14. Both filaggrin and caspase 14 promote epidermal differentiation and decrease proliferation, consistent with more rapid wound closure and decreased epidermal thickness during the remodeling phase. Fibrin clearance was significantly impaired in Plg-RKT−/− wound tissue. Genetic reduction of fibrinogen levels to 50% completely abrogated the effect of Plg-RKT deletion on the healing of burn wounds. Remarkably, the effects of Plg-RKT deletion on cytokine expression were modulated by reducing fibrinogen levels. In summary, Plg-RKT is a new regulator participating in different phases of cutaneous burn wound healing, which coordinately plays a role in the interrelated responses of inflammation, keratinocyte migration, and fibrinolysis.


Introduction
Wound healing is a fundamental and complex physiologic process that proceeds in overlapping, sequential steps 1 . Following burn wounding, vascular permeability increases resulting in the deposition of extravascular fibrin in the wounded area, and in the promotion of the inflammatory phase, which initially consists of infiltration by neutrophils followed by the recruitment of macrophages, the key regulators of the wound healing 2 . Macrophages release pro-inflammatory cytokines to promote additional leukocyte recruitment. Subsequently, the proliferation phase is characterized by keratinocyte proliferation and migration 3 as well as efferocytosis of apoptotic neutrophils by macrophages and subsequent polarization of macrophages to an anti-inflammatory phenotype, characterized by the secretion of antiinflammatory cytokines that stimulate the epithelial to mesenchymal transition, activate fibroblasts to proliferate and release collagen and stimulate angiogenesis 4 . In the remodeling phase, granulation tissue is formed 5 . The plasminogen activation system regulates activation of the circulating zymogen, plasminogen, to the broad spectrum protease, plasmin 6 . Previous studies in plasminogen deficient humans [7][8][9][10][11] and mice [12][13][14][15] have documented an essential requirement for plasminogen in normal wound healing. Plasmin directly promotes keratinocyte migration 16 and the role of plasmin was considered to be the promotion of keratinocyte migration by allowing keratinocytes to dissect their way through the fibrin-rich extracellular matrix (ECM) by cleaving components of the ECM 12,17 . However, recent studies from our group have demonstrated a novel key role for plasminogen in wound healing: plasminogen bound to macrophages and neutrophils is transported to the wound area, where the level of plasminogen is increased locally. This leads to the induction of intracellular signaling and cytokine release 18 . Notably, the recruitment of immune cells to cutaneous wounds is not affected by plasminogen deficiency 15,18 and thus, the role of plasminogen in the initial stages of inflammation is predominantly induction of intracellular signaling. Furthermore, although reepithelialization eventually occurs in plasminogen deficient mice, the wound area continues to exhibit excessive neutrophil accumulation and collagen deposition after wound closure, providing evidence for an additional requirement for plasminogen in the resolution of inflammation 15 .
Plg-R KT is a transmembrane plasminogen receptor that accounts for the majority of the plasminogen binding capacity of macrophages [19][20][21] and promotes plasminogen activation to plasmin by plasminogen activators on cell surfaces 19,22,23 . Plg-R KT is preferentially expressed on proinflammatory monocytes and macrophages 24 . In addition, plasmin-dependent cytokine release from macrophages is promoted by Plg-R KT 24,25 . In the present study, we investigated the role of Plg-R KT in wound healing using a standard burn wound model in mice with global deletion of Plg-R KT as well as in mice with Plg-R KT specifically deleted in either myeloid cells or in keratinocytes. The results of our study suggest that Plg-R KT plays a role in wound healing during the inflammatory, proliferative, and remodeling phases and that Plg-R KT regulates the function of both myeloid cells and keratinocytes during the wound healing process in addition to regulating fibrin clearance during wound healing.

Burn wound model
Mice were anesthetized with 3% isoflurane (inhaled). Burn wounds (one wound in each mouse) were made with a brass stave and wound healing was quantified as described 18 . Briefly, digital photographs of the wounded skin were taken and analyzed by tracing wound margins and calculating the pixel areas using ImageJ Version 1.41o software (National Institute of Health, Bethesda, USA). The remaining wound area was determined as the percent area of the original wound area. The sample size was chosen based on our previous experience with this model 15,18 . After separating mice by established genotype, mice from each group were assigned randomly to either untreated or burned groups. Blinding: information on genotype was not available to investigators performing burn wounding or to separate investigators who analyzed tissue samples. Burn wounding experiments were performed at least three times.

Immunohistochemistry and histology
Wounded skin or control unwounded skin was fixed in 4% paraformaldehyde, embedded in paraffin, and sections of 6 µm were cut perpendicular to the skin surface. Immunohistochemistry and histology were performed as in Supplemental Methods.

Western blotting
Wound tissue was lysed in RIPA buffer with antiprotease and anti-phosphatase cocktail (Thermo Fisher Scientific, Waltham, MA, USA), and western blotting was performed as in Supplemental Methods.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR)
Skin samples from the wounded area and control unwounded skin (100-200 mg) were cut into 1-2 mm 2 pieces and kept in RNAlater (Thermo Fischer Scientific) for 3 days at +4°C and quantitative RT-PCR was carried out as in Supplemental Methods.

mRNA sequencing
For mRNA sequencing, skin samples were incubated in RNAlater and homogenized in QIAzol (Qiagen, Hilden, Germany) using Precellys CK28R tubes on a Precellys 24 homogenizer. RNA was prepared and characterized as in Supplemental Methods and 5 µg of the pooled RNA was sent to Novogene (Hong Kong) for transcriptome sequencing and data analysis.

Statistics
A mixed-effects model (REML) was used to analyze repeated measures data due to differences in mouse numbers at different time points. Residual, homoscedasticity, and Q-Q plots were created and inspected to check for heteroscedasticity, normality of residuals, and Gaussian distribution of the dataset to make sure assumptions of repeated measures ANOVA was not violated. Robust ANOVA (rank-based estimation for linear models) which was performed using Rfit package 27 installed on RStudio (version 1.2.5042 28 ) with R 3.6.3 29 , was used to analyze several data sets that involved more than one independent variables for their interaction. Nonparametric comparisons were calculated using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, CA, USA. GAPDH normalized PCR data were tested if it was different from the ratio of 1 using the one-sample Wilcoxon test. Time to event data was analyzed in GraphPad Prism, using the Log Rank Mantel-Cox method. All tests were two-tailed.

Study approval
All animal experiments were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute and The Regional Ethics Committee of Umeå University.

Data sharing statement
For original data please contact Tor Ny, tor.ny@umu.se or Lindsey A. Miles, lmiles@scripps.edu. The data from RNA sequencing are available at Figshare: https://figshare. com/account/home#/projects/88871.

Plg-R KT deletion decreases healing of burn wounds
To determine whether deletion of Plg-R KT would affect wound healing rates, full-thickness standardized burn wounds (1 cm in diameter) were induced in Plg-R KT −/− male mice and Plg-R KT +/+ male littermates. Quantification of the wound area at different time points showed that from day 9 after injury, healing in Plg-R KT −/− mice was significantly delayed (Fig. 1a). Scab loss was significantly delayed in Plg-R KT −/− mice (12th median day of scab loss and 9.5th median day of scab loss for Plg-R KT −/− and Plg-R KT +/+ littermates, respectively (p = 0.0194, n = 5-6) ( Fig. 1b and compare day 11 images in 1C). On day 11, all wounds were covered with an early keratinocyte layer, although some retained a scab (see example in Fig. 1c). The morphologic analysis revealed a decrease in the area of the keratinocyte tongue protruding at wound edges at day 4 (23% less in Plg-R KT −/− mice compared with Plg-R KT +/+ littermates) (Fig. 1d). Interestingly, following wound closure (Day 20), the epidermal thickness was significantly increased in Plg-R KT +/+ tissue compared with untreated tissue, while epidermal thickness was not increased in Plg-R KT −/− tissue following wound closure (Fig. 1c, e). Epidermal thickness was also significantly greater in Plg-R KT +/+ tissue compared with Plg-R KT −/− tissue at day 20, but there was no difference in epidermal thickness between the two genotypes in unburned tissue.
These data indicate that Plg-R KT −/− mice and Plg-R KT +/+ mice respond differently to burn treatment with regard to epidermal thickness and may suggest that migration/ proliferation of keratinocytes is less in mice deficient in Plg-R KT and that Plg-R KT is required for optimal wound healing.

Plg-R KT regulates plasminogen accumulation during wound healing
We have previously demonstrated that plasminogen specifically accumulates in burn wounds and that the extent of accumulation directly correlates with circulating plasminogen concentrations 18 . For example, accumulation of plasminogen in burn wounds of heterozygous plasminogen deficient (Plg +/− ) mice (approximately 50% levels of circulating plasminogen 30 ) is reduced by 1.6-fold, compared with wild-type littermates 18 . The transport of plasminogen to wounds is predominantly accomplished by plasminogen binding to plasminogen binding sites on inflammatory cells 18 . It is noteworthy that intravenous injection of 2 mg plasminogen/mouse (to increase circulating plasminogen concentrations by 15.5 µM to a final circulating concentration of 17.5 µM, an~35-fold increase) results in only a 2.6-fold increase in wound plasminogen accumulation 18 . To detect Plg-R KT -dependent effects on plasminogen accumulation in wounds, we used a sensitive antibody inhibition approach. We used our blocking anti-Plg-R KT mAB that decreases recruitment of thioglycolate-elicited macrophages in vivo 23 . First, we verified that anti-Plg-R KT mAB exhibited the ability to block plasminogen binding to leukocytes in vivo. Plg −/− mice were injected with either anti-Plg-R KT mAB or isotype control 30 min before the burn wound was created, followed immediately by injection of Alexa 488labeled plasminogen. Twenty-four hours later, blood was collected and analyzed by FACS. A clear peak shift of plasminogen binding to leukocytes was observed after anti-Plg-R KT mAB treatment, relative to injection with isotype control, indicating that anti-Plg-R KT mAB blocked plasminogen binding to leukocytes in vivo (Fig. 2a). When plasminogen accumulation in the burn wounds was analyzed following injection of plasminogen into Plg +/+ and Plg −/− mice, a significant effect of treatment with anti-Plg-R KT mAB was shown by analysis with robust ANOVA (p = 0.003, f = 9.847) (resulting in an 18% and a 25% decrease for Plg +/+ and Plg −/− mice, respectively) ( Fig. 2b). Significant effects of treatment with anti-Plg-R KT mAB on plasminogen accumulation (Fig. 2b) in wound tissue of mice that received PBS were not detectable.
Thus, under these conditions, anti-Plg-R KT mAB blocked the ability of cells to transport human plasminogen to the wounds, but the ability of anti-Plg-R KT mAB to dissociate pre-bound mouse plasminogen was not detectable. Our data suggest that Plg-R KT allows cells to transport plasminogen to wound sites.

Plg-R KT expression increases during wound healing
We examined the expression and localization of Plg-R KT in wounded skin. Expression of Plg-R KT increased during wound healing and was maximal at day 11 and decreased by day 20 (Fig. 3a, left panels, and quantified in Fig. 3b). Plg-R KT was localized in hair follicles in unwounded skin (white arrows) and was prominently expressed in both hair follicles and keratinocytes (white arrowheads) on days 4, 11, and 20 following wounding. Plg-R KT expression was also confirmed in isolated primary keratinocytes ( Supplementary Fig. S1). Plg-R KT expression was not detected in wound tissue in Plg-R KT −/− littermates, as negative controls for the immunostaining (Fig. 3, right panels).
Specific deletion of Plg-R KT in myeloid cells decreases the rate of wound healing during the transition from the inflammatory phase to the proliferation phase We have shown previously that the bulk of plasminogen transport to wound sites is accomplished by inflammatory cells 18 . Therefore, we used immunohistochemistry and quantified the percentage of macrophages and neutrophils in the wounds at Days 4, 11, and 20 after burning. Macrophage recruitment to the wound sites was time-dependent and peaked at day 11 following wounding, but the genotypes did not respond differently to burn treatment with regard to time (Fig. 4a). There was also no genotype effect on the expression of the macrophage markers, F4/80 and arginase 1, as determined by qPCR (i.e., fold difference in expression was not greater than 1.6 at each time point tested (Fig. 4b, c, respectively), consistent with results of the immunohistochemical analyses. Neutrophils were most abundant in the wounds at days 4 and 11 following wounding, but there also was no significant genotype effect on the total number of neutrophils present at each time point (Fig. 4d).
In view of the effect of anti-Plg-R KT mAB in reducing plasminogen levels in wounds of both Plg +/+ and Plg −/− mice ( Fig. 2b), we directly assessed the potential role of macrophage/neutrophil Plg-R KT in wound healing. Burn wounds were induced in mPlg-R KT −/− mice, in which Plg-R KT was specifically deleted in myeloid cells 25 , and Plg-R KT flox/flox25 control mice. Quantification of the wound area at different time points showed that the genotypes responded differently to burn wounding with respect to time (p < 0.0001) with a prominent delay in wound healing at days 10 and 12 ( Fig. 4e) during the proliferation phase, when the maximal number of macrophages were present in the wounds of mice with global deletion of Plg-R KT (Fig. 4a), during the transition from the inflammatory to proliferation phases of wound healing. There was also a trend for delayed scab loss in mPlg-R KT −/− wounds (13th median day of scab loss and 11th median day of scab loss for mPlg-R KT −/− and Plg-R KT flox/flox mice, respectively, p = 0.097, n = 7) (Fig. 4f). Macrophage recruitment to mPlg-R KT −/− wound sites peaked at day 11 following wounding, but there was no significant effect of myeloidspecific deletion of Plg-R KT on macrophage accumulation at this time point, nor at earlier time points examined (Fig. 4g). Neutrophils were most abundant in the wounds on day 4 following wounding, but there also was no significant effect of cell-specific deletion of Plg-R KT on the total number of neutrophils present at each time point (Fig. 4h).

kPlg-R KT
−/− mice and Plg-R KT flox/flox control mice. Quantification of the wound area at different time points did not reveal a significant difference between the genotypes in response to burn to wound with respect to time. However, healing in kPlg-R KT −/− mice was significantly accelerated at day 13 during the proliferation phase (Fig.  5a). There was no effect of specific deletion of Plg-R KT in keratinocytes on scab loss, consistent with an effect of specific keratinocyte deletion of Plg-R KT only during the proliferation phase (Fig. 5b). There was a trend for decreased epidermal thickness in wounds of kPlg-R KT −/− mice compared with Plg-R KT flox/flox control mice (Fig. 5c). In contrast, there was no detectable effect of specific myeloid deletion of Plg-R KT on epidermal thickness compared to the Plg-R KT flox/flox control (Fig. 5d). Thus, keratinocyte Plg-R KT appears to regulate wound healing during the proliferation phase.

Effects of Plg-R KT deletion on gene expression during wound healing
We used RNA Sequencing to investigate differences in gene expression profiles of Plg-R KT −/− and Plg-R KT wound tissue harvested on day 11. Volcano plots are shown in Fig. 6a and genes whose expression changed by ≥ 1.5-fold with p < 0.05 are presented in Fig. 6b. Six of the genes that were downregulated are expressed in the epidermis (HBA½, Csfr3, MMP3, Tnc, and Timp1). Of downregulated genes, six were inflammation-related and eight were ECM related. Of downregulated inflammation-related genes, six are expressed in myeloid cells (Csf3r, Ilrl1, Cxcl5, and Ccr2) and/or regulate macrophage function (Il33, Cxcl5, Saa3) and one is expressed in the epidermis (Csfr3). Of note, IL33 (and its receptor Il1rl1) accelerates the development of M2 macrophages in wound sites in vivo.
Of downregulated ECM-related genes, six are expressed in myeloid cells (Mmp3, Serpine 1, Timp 1, Tnc, and Vcan) or regulate macrophage function (Postn) and three are expressed in epidermis (MMP3, Tnc, and Timp1). Thus, deletion of Plg-R KT has effects on the expression of genes involved in wound healing that are expressed by both myeloid cells and keratinocytes.
Effect of fibrinogen depletion on wound healing in Plg-R KT −/− mice Fibrin formation is an early hemostatic event following wounding that is then followed by subsequent plasmindependent fibrinolysis 31 . Plasminogen deficient mice exhibit impaired wound healing 12,14,18 and genetic deletion of fibrinogen corrects the defect in skin wound healing in these mice 17   Plg +/+ and Plg −/− mice (8-12 weeks of age) were intravenously injected with 100 µl of anti-Plg-R KT mAB7H1 (2.5 mg/ml) or with mouse IgG2A (2.5 mg/ml) as isotype control (n = 3 for each study group). Thirty minutes later, standard burn wounds were introduced and all mice were intravenously injected with 100 µl (2 mg) of Alexa Fluor 488-labeled human plasminogen or PBS (as control). At 24 h after wounding and injection, blood samples were collected. Erythrocytes were lysed immediately with a solution containing 0.15 M NH 4 Cl for 5 min, and the remaining leukocytes were washed and resuspended in 500 μl PBS. FACS analysis was performed using a Cytomics FC500 (Beckman Coulter, Indiana, USA) and leukocytes were defined by forward scatter and side scatter. A clear peak shift of plasminogen binding to leukocytes was observed after anti-Plg-R KT mAB treatment, relative to injection with isotype control. B Effect of anti-Plg-R KT mAB on the accumulation of plasminogen in wounds. Both Plg −/− and Plg +/+ littermates were injected intravenously with isotype control or anti-Plg-R KT mAB (2.5 mg/ml) 30 min before the burn injury, followed immediately by intravenous injection of human plasminogen (hplg) (2 mg). Wound tissue was collected 24 h after the injury. The plasminogen concentration in wound lysates was determined by specific ELISA. Data analysis by robust ANOVA showed a significant effect of the addition of plasminogen littermates, consistent with relatively greater vascular permeability in Plg-R KT +/+ wound tissue 32 . However fibrin clearance from day 4 to day 11 was markedly and significantly impaired in Plg-R KT −/− wound tissue compared with Plg-R KT +/+ wound tissue and, in addition, at day 11 there was a trend for an increased presence of fibrin in Plg-R KT −/− compared with Plg-R KT +/+ wound tissue. Fibrin clearance was essentially complete in wounds of both genotypes at day 20 (Fig. 7a, b). Thus, the time course of fibrin clearance was impaired in Plg-R KT −/− wound tissue. Western blot analysis also demonstrated increased fibrin content in Plg-R KT −/− the tissue on day 11 (Fig. 7c, d). Remarkably, genetic reduction of fibrinogen levels of Plg-R KT −/− mice to 50% completely abrogated the effect of Plg-R KT deletion on the healing of burn wounds (Fig. 7e).

Effects of concomitant deletion of Plg-R KT and fibrinogen on gene expression during wound healing
We used mRNA sequencing to examine the effect of Plg-R KT deletion on gene expression in the context of fibrinogen heterozygosity in wound tissue harvested at day 11. Volcano plots are shown in Fig. 8a and genes whose expression changed by ≥1.5-fold with p < 0.05 are presented in Fig. 8b. Strikingly, eleven inflammationrelated genes were up-regulated whose expression had not been affected in Plg-R KT −/− wound tissue with wild-type levels of fibrinogen. The fibrinogen +/− background did not influence the effect of Plg-R KT deletion on expression of Saa3 or of hemoglobin. Therefore, the presence of fibrin(ogen) appears to be necessary for Plg-R KT to exert its ability to promote wound healing.

Discussion
The present study provides mechanistic insight into the novel role of the plasminogen receptor, Plg-R KT , in cutaneous wound healing. Plg-R KT exerted pleiotropic, interrelated effects in the inflammatory, proliferative, and remodeling phases of wound healing. We found that (1) Plg-R KT was expressed in epidermis and expression increased during wound healing; (2) deletion of Plg-R KT decreased the rate of healing of burn wounds; (3) Plg-R KT regulated plasminogen accumulation in burn wounds; (4) Plg-R KT regulated expression of inflammatory cytokines during wound healing; (5) specific deletion of Plg-R KT in myeloid cells impaired wound healing during the transition from the inflammatory to proliferation phase; Plg-R KT was initially discovered in monocytes/macrophages 19 . Here, we found expression, also, in isolated primary keratinocytes as well as in keratinocytes and hair follicles within epidermal tissue. Notably, expression of Plg-R KT in keratinocytes increased as wound healing progressed, consistent with a role for Plg-R KT in the wound-healing program. This is the first example of changes in tissue Plg-R KT expression during the progression of a pathophysiological program.
Healing of cutaneous wounds is impaired in plasminogen deficient mice 12,15,17 . We have demonstrated previously that plasminogen bound to macrophages and neutrophils, is transported to the wound area, where the level of plasminogen is increased locally. This leads to the induction of intracellular signaling and cytokine release 18 . Thus, the role of plasminogen in the initial stages of inflammation is predominantly induction of intracellular signaling 18 . In the current study, deletion of the plasminogen receptor, Plg-R KT , significantly attenuated the rate of wound healing. And, when either plasminogen deficient or Plg-R KT −/− mice were treated with anti-Plg-R KT mAB, followed by exogenous plasminogen, the accumulation of plasminogen in wounds was decreased. Furthermore, in Plg-R KT −/− wound tissue, the expression of six inflammation-related genes that are expressed by myeloid cells (Csf3r, Ilrl1, Cxcl5, and Ccr2) and/or regulate macrophage function (Il33, Cxcl5, Saa3) was downregulated. These results are consistent with the concept that Plg-R KT is a key regulator of the effect of plasminogen on immune cell function in the healing wound. Of note, IL33 (and its receptor Il1rl1) accelerates the development of M2 macrophages in wound sites in vivo 33 . We have shown previously that plasminogen and Plg-R KT promote polarization of macrophages to an M2-like phenotype 25,34 . Thus, in addition to dysregulated cytokine expression, decreased development of M2-like macrophages may also contribute to the delayed healing curve in Plg-R KT −/− mice, resulting in a longer pro-inflammatory phase. Earlier studies in vitro documented plasmin-dependent stimulation of intracellular signaling pathways and cytokine release by monocytes and macrophages that depend on the interaction of plasmin with cell surfaces [35][36][37] and our recent studies suggest that Plg-R KT can mediate plasmin(ogen)-dependent intracellular signaling and cytokine release 24,25 . The current study shows that Plg-R KT regulates the expression of many cytokines that were not previously known to be regulated   by Plg-R KT . It is noteworthy that Plg-R KT contains only a four amino acid cytoplasmic domain. Thus, signaling mediated by Plg-R KT is likely to involve an interaction with additional transmembrane molecules, similar to the example of the GPI-linked urokinase receptor (uPAR) that is able to mediate intracellular signaling 38 . Despite functioning to transport plasminogen to the wound to promote cytokine release, recruitment of immune cells to cutaneous wounds is not affected by plasminogen deficiency 15,18 and we did not find an effect of Plg-R KT deficiency on recruitment of macrophages and neutrophils to wounds. Nonetheless, we did find a significant decrease in the rate of wound healing when Plg-R KT was specifically deleted in myeloid cells, consistent with a role for Plg-R KT -bound plasmin(ogen) in regulating cytokine release as well as macrophage polarization. The lack of effect of Plg-R KT and plasminogen on recruitment of macrophages to wound sites is notable considering the requirement for both Plg-R KT and plasminogen in experimental peritonitis 21,23,39 . Conversely, increased macrophage infiltration is observed in spontaneously thrombotic organs of plasminogen deficient mice 30,40 and in mammary glands of Plg-R KT −/− mice 26 . Thus, the roles of both plasminogen and Plg-R KT in macrophage recruitment depend on the pathophysiological setting.
Plasmin directly promotes keratinocyte migration in vitro 16 and keratinocyte migration is decreased in cutaneous wounds in plasminogen deficient mice 12,17 . In Plg-R KT −/− mice the area of the keratinocyte tongue protruding at wound edges was decreased at day 4, consistent with altered keratinocyte migration. Decreased keratinocyte migration may result from impaired ability to degrade the ECM in order to migrate due to decreased plasmin associated with the cell surface in the absence of Plg-R KT , as well as a dysregulated ECM due to impaired fibrin clearance and resulting from down-regulation of other ECM-related genes in Plg-R KT −/− wound tissue (Mmp3, Serpine 1, Timp 1, Tnc, and Vcan).
Following wound closure (day 20), the epidermal thickness was significantly less in Plg-R KT −/− tissue. There also was a trend for decreased epidermal thickness in kPlg-R KT −/− mice. Paradoxically, deletion of Plg-R KT specifically in keratinocytes significantly accelerated the rate of healing (determined by wound closure) during the proliferation phase. Mechanistically, only two genes were upregulated in Plg-R KT −/− compared with Plg-R KT +/+ wound tissue, filaggrin (Flg), and caspase 14 (Casp14). Filaggrin is essential for the regulation of epidermal homeostasis and is a marker for keratinocyte differentiation, thus higher filaggrin expression is correlated with lower proliferation 41 . Caspase 14 is a non-apoptotic caspase involved in epidermal differentiation and is the predominant caspase in epidermal stratum corneum 42 . Caspase 14 plays a role in keratinocyte differentiation and is required for cornification and regulates maturation of the epidermis by proteolytically processing filaggrin. Thus, in Plg-R KT −/− wounds, the thinner epidermis at day 20 and earlier wound closure may be attributed to, at least in part, to increased differentiation and decreased proliferation of keratinocytes, consistent with increased filaggrin and caspase 14 expressions.
Plasmin is the major enzyme responsible for fibrinolysis 30,40 . Initially (day 4) fibrin deposition was significantly greater in wound tissue of Plg-R KT +/+ littermates. The early increase in fibrin deposition in Plg-R KT +/+ mice may be due to relatively greater vascular permeability in Plg-R KT +/+ wound tissue 32 . Subsequently, clearance of the initial fibrin deposits in Plg-R KT +/+ wound tissue took place, while fibrin clearance was delayed in Plg-R KT −/− wounds. Genetic reduction of fibrinogen levels of Plg-R KT −/− mice to 50% completely reversed the effect of Plg-R KT deletion on the healing of burn wounds. This parallels the rescue of defective cutaneous wound healing in plasminogen deficient mice by concomitant deletion of fibrinogen 17 . Remarkably, the effect of Plg-R KT deletion on gene expression in wound tissue was markedly altered on the fibrinogen heterozygous background. Strikingly, 11 inflammation-related genes were upregulated. Extravascular fibrin(ogen) is a strong modifier of proinflammatory disease, acting through the integrin α M β 2 on macrophages [43][44][45][46] . The upregulation of Ccl3, Ccl4, Cxl2, Cxc13, and Tnf may result from decreasing an effect of persistent fibrin(ogen) on macrophages and is potentially a key factor in the ability of fibrin(ogen) deletion to reverse the defective wound healing phenotype of Plg-R KT −/− mice. Thus, defective fibrinolysis is likely to play a role in reduced keratinocyte migration as well as impaired stimulation of cytokine release by macrophages.
In summary, our results suggest that Plg-R KT has multiple functions in the wound-healing program (as outlined in Supplementary Table 1). Plg-R KT promotes plasminogen transport to the wound site and promotes plasmin (ogen)-dependent cytokine release as a key step in the proinflammatory phase of wound healing. Plg-R KT regulates the composition of the ECM, regulates gene expression by keratinocytes, and promotes proliferation of keratinocytes while decreasing keratinocyte differentiation, as key steps in the proliferation and resolution phases. Fibrinolysis, regulated by Plg-R KT , is a necessary (although not sufficient) step in the wound healing program that impacts both cytokine release by macrophages, keratinocyte migration, and ECM remodeling. The results of our study may also apply more broadly to healing in other tissue due to other types of injury including infection and inflammation.