1 INTRODUCTION

There is a fine tuning between production and degradation of key extracellular matrix (ECM) components such as collagen and its hydrolysis in normal skin repair.1, 2 When the healing process ends earlier than required, there is insufficient deposition of connective tissue matrix resulting in weakness, often to the extent of tissue fragmentation. Chronic non-healing ulcers, such as those caused by diabetes or pressure injury, are examples of deficient healing propagated by several factors including insufficient matrix deposition. On the contrary, if healing continues postoptimal end point, excessive deposition of connective tissue occurs. This process results in over-healing wounds, described as fibroproliferative disorders. Both keloid and hypertrophic scars are considered as two devastating fibroproliferative disorders that happen most frequently after dermal injuries and surgical wounds. These pathological conditions develop due to excessive fibroblast proliferation and prolonged ECM deposition. It is reported that development of hypertrophic scarring is the result of greater ECM deposition than degradation.3 There is now growing evidence to suggest that keratinocytes also actively participate in the pathophysiology of scar formation.4 The role of fibroblasts is critical throughout the healing process, starting with the degradation of the fibrin clot, production of a vast array of cytokines and growth factors, granulation tissue formation and wound contraction. Another key function of fibroblasts is the production and remodelling of ECM components.5, 6 There should be a balance between inadequate fibroblast activity, which can lead to chronic wound formation, and excessive fibroblast activity, which can lead to fibrotic healing, given fibroblasts’ complex roles in wound healing. It remains unclear how activated cells at the wound site lessen their activities upon completion of wound epithelialization despite extensive discussion on the details of cellular interaction in wound healing initiation signals, which are mainly growth factors and cytokines.7-9 This review describes the role of cutaneous cell-cell communication in wound healing outcome and predominantly explores the roles of releasable factors from epithelial-mesenchymal interaction in modulating the late phase of wound healing and remodelling.

2 WOUND HEALING INITIATING FACTORS

The healing process of injuries passes through four distinct, yet interconnected stages: haemostasis, inflammation, proliferation and remodelling.10 Haemostasis initiates as soon as bleeding occurs regardless of injury type; platelets reach the site where they release a set of wound healing initiating cytokines and growth factors including transforming growth factor beta family and platelet-derived growth factors.11 The inflammatory phase is initiated by infiltrating immune cells. Later, fibroblasts migrate into the wound and trigger the proliferative phase. These cells later become the source of many cytokines and growth factors that orchestrate skin cell migration, proliferation, matrix production and degradation.7 A combination of these functional activities determines the final result of the healing process. Here, we focus on two important growth factors, TGF-β and PDGF family, as two key group of initiation signals for wound healing.

2.1 TGF-β and PDGF families as initiation factors for the wound healing process

During haemostasis of any injury, platelets degranulate and release platelet granules, also known as alpha granules, which contain PDGFs, members of TGF-β family, epidermal growth factors (EGF) and many other growth factors. Immune cells such as macrophages and neutrophils and stromal cells (eg smooth muscle cells and fibroblasts) are attracted to the wound once PDGF is released.12, 13 PDGFs are a family of hetero and homodimeric molecules, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD. As PDGFs participate in every stage of dermal wound healing, it is no surprise that they are synthesized by almost all cells that contribute in the healing process.8, 9 PDGFs are also involved in re-epithelialization by up-regulating the production of insulin-like growth factor-1 (IGF-1) that facilitates keratinocyte migration.14, 15

PDGFs stimulate fibroblast proliferation and promote ECM production, and also stimulate the contraction and reorganization of collagen scaffold, and switch fibroblasts to the myofibroblast phenotype.14, 16 This can be further supported by studies revealing increased tensile strength and collagen deposition due to PDGF delivery into rat incisional wounds.17 At the remodelling phase, PDGFs up-regulate matrix metalloproteinases, leading to the degradation of old collagen.9, 18 The dual role of PDGFs in connective tissue synthesis and degradation aligns with their expression levels in normal scars where PDGFs are increased and decreased at 3 and 12 months respectively. It is therefore suggested that PDGFs play a key role in pathogenesis of hypertrophic scars and keloids.19

Upon tissue injury, platelets release thier stored content of TGF-β1 at the site of injury.20 In the inflammatory phase of wound healing, TGF-β1 helps recruit pro-inflammatory monocytes and fibroblasts, assists conversion of monocytes to macrophages, and augments macrophage debridement.21-23 TGF-β protein family includes TGF-β1, 2 and 3, bone morphogenic proteins (BMPs) and activins. Of these molecules, TGF-β1 is the most important factor being involved in inflammation, angiogenesis, re-epithelialization and regeneration of connective tissue. A large variety of cell types can produce transforming growth factors, including platelets, macrophages, keratinocytes and fibroblasts.24, 25 These ligands bind a complex receptor composed of heterodimeric type I and type II subunits that function as serine-threonine kinases. Once the ligand-receptor complex formed, SMAD family activation transcription factors are expressed due to autophosphorylation. Members of TGF-β family are also involved in the processes of new collagen and fibronectin synthesis, the disintegration of old collagen and the conversion of fibroblasts into myofibroblasts, the primary cells involved in the contraction of collagen matrix thereby assisting wound closure during the fibroproliferative and remodelling phases.26-28 TGF-β1–specific role varies depending on its concentration; for instance, low concentrations of TGF-β1 stimulate fibroblast mitosis, and high concentrations stimulate differentiation of fibroblasts towards a pro-collagen and pro-matrix formation state.28 TGF-β1 over-expression is involved in keloid and hypertrophic scar formation and neutralization of TGF-β1 results in a better outcome for these dermal fibrotic conditions.29

3 EPIDERMAL-MESENCHYMAL INTERACTION

Interaction of mesenchymal and epidermal cells during and upon epithelialization influences the healing outcome. Disruption of this crosstalk leads to excessive or insufficient ECM depositions as seen in over-healing (hypertrophic scarring and keloid) and difficult-to-heal wounds, respectively.4, 30 Several studies have shown significant changes in the gene expression profile of fibroblasts when co-cultured with keratinocytes. These findings provide compelling evidence in support of the idea that epidermal cells orchestrate the healing process through modulation of the fibroblast phenotype and gene expression which result in ECM production, cell adhesion and tissue remodelling.31, 32 Crosstalk between keratinocytes and fibroblasts mainly occur through releasable factors. Thus, identifying the sources and functionalities of these factors in controlling synthesis and breakdown of ECM is essential in understanding the pathogenesis of abnormal wound healing.

3.1 Bilateral paracrine communications between fibroblasts and keratinocytes

A mechanism known as ‘double-paracrine’ communication regulates dermal cells function during the healing process.33 Indeed, communication or crosstalk between keratinocytes and dermal fibroblasts is an integral process during wound healing and normal skin maintenance.33 The importance of the interactions between dermal fibroblasts and keratinocytes is observed such that interrupted re-epithelialization is correlated to increased fibrosis in wounds.34

As a result of injury, fibroblasts become activated and produce and release ECM factors like collagen which act as an interim scaffold for tissue repair.33 As the healing process proceeds, fibroblasts switch from ECM synthesis towards increase MMPs production, which triggers ECM degradation and tissue remodelling.35, 36 This fine tuning between ECM synthesis and degradation is orchestrated by keratinocytes. Keratinocytes modulate fibroblasts via TGF-β and IL-1 paracrine signalling pathways and steer them to differentiate into myofibroblasts with remarkably enhanced capacity to produce and deposit collagen and ECM within the wound microenvironment.37 Once the wound is fully re-epithelialized, mature keratinocytes release a different set of factors that reduce ECM synthesis and increase its degradation. It has been shown by many research groups including ours that keratinocyte conditioned medium (KCM) can significantly inhibit expression of collagen type-1 and type-3 in dermal fibroblasts.38-42 It has also been demonstrated by several studies that keratinocytes can induce MMP-1 expression in fibroblasts.35, 43 As stated before, through protein purification and mass spectrometry peptide identification, we were able to isolate and identify a keratinocyte-releasable protein known as stratifin as a MMP-inducing factor for fibroblasts.2, 34, 44

Dermal fibroblast releasable factors can regulate expression of different genes and modulate functionality of keratinocytes. In previous sections, we have provided supporting evidence that fibroblasts can stimulate stratifin expression in keratinocytes, which in turn stimulates fibroblasts to overexpress MMPs and subside ECM production.45 Further, several factors are produced and secreted by fibroblasts during the process of wound healing that exert biological effect on keratinocytes. For instance, upon injury, fibroblast rapidly express a potent mitogen known as keratinocytes growth factor (KGF).46 Another factor that is also generated by fibroblasts is hepatocyte growth factor that induces epithelial cell migration and proliferation.47 Using DNA microarray methods, we further studied how keratinocyte-releasable factors affect fibroblast cytokines profile. We found that granulocyte colony-stimulating factor (G-CSF) expression is significantly increased in fibroblasts when co-cultured with keratinocytes compared to mono-cultured cells. This result was further verified by RT-PCR and Western blot.48 G-CSF is primarily known as a haematopoietic factor, but it also known to stimulate proliferation of keratinocyte, and hence promote wound healing. It is known that keratinocytes secret IL-1, which is a G-CSF expression stimulator in many immune cell types. As such, we investigated whether IL-1 can also stimulate fibroblasts to express G-CSF. We found that G-CSF expression was markedly overexpressed in fibroblasts upon treatment with recombinant IL-1. However, KCM-stimulated G-CSF expression in fibroblast was only partially suppressed by an IL-1 receptor antagonist, suggesting additional keratinocyte-releasable factors may also play a role in this process.48 A study by Shephard et al showed that G-CSF was up-regulated in human dermal fibroblasts when co-cultured with keratinocytes and also keratinocytes induced dermal fibroblasts to differentiate into myofibroblasts through an IL-1-dependent pathway.49 These studies together highlight the critical role of keratinocytes-fibroblasts crosstalk in orchestrating and modulating the wound healing process. As such, intercommunication between fibroblasts and keratinocytes should be considered as an important field of research to better understand the cutaneous wound healing physiology and pathology.

3.2 Expression of matrix metalloproteinases (MMPs) in fibroblasts stimulated by keratinocytes

Fibroblasts and keratinocytes interact through a series of mechanisms known as epidermal-mesenchymal communication. The outcome of wound healing is highly dependent on this communication. The importance of the interactions between dermal fibroblasts and keratinocytes is observed such that interrupted re-epithelialization is correlated to increased fibrosis in wounds.34 For instance, only 30% of wounds show abnormal collagen deposition and fibrosis when migration of keratinocytes results in complete re-epithelialization of the wound within 21 days, versus 78% fibrosis in site-matched wounds when epithelialization takes longer than 21 days.2

Optimal wound healing involves a fine tuning between production/deposition of collagen and its degradation. Our group previously found that when co-cultured with keratinocytes, fibroblasts expressed over 10-fold more MMP-1 compared to separately cultured fibroblasts.2 This interesting observation resulted in discovery of a keratinocyte-releasable factor with a potent collagenase stimulatory effect on fibroblasts. We identified this releasable factor as keratinocyte-derived anti-fibrogenic factor, which modulates degradation of type I and type III collagen accumulated within fibrotic tissue.2 In another study, we further characterized this factor by chromatography and trypsin digestion followed by subsequent peptide mapping and showed that it is a releasable form of stratifin, also known as 14-3-3 sigma (14-3-3σ) protein. Next, we generated recombinant stratifin via cloning its cDNA into a pGEX-6P-1 vector. When northern analysis was performed on fibroblasts that were treated with various concentrations of recombinant stratifin, the expression of collagenase mRNA in stratifin-treated fibroblasts was remarkably increased in a dose-dependent manner relative to that of control fibroblasts. Although different isoforms of 14-3-3 proteins are well known for their intercellular activities, biological function of the releasable forms of 14-3-3 protein family, particularly the σ isoform, was not reported before this set of experiments.2 14-3-3 proteins, including stratifin, are a large family of dimeric acidic highly conserved proteins. Stratifin, unlike other 14-3-3 proteins that are ubiquitously expressed, seems to be expressed specifically in stratified epithelial cells.50 Modulation of dermal fibroblasts function by keratinocyte-releasable stratifin was probably the first report of a biological activity of 14-3-3s through extracellular pathway.2 However, extracellular 14-3-3 proteins have now been found to be important in immune regulation and play a role in pathophysiology of variety of disease such as cancer, articular diseases and neurological disorders.51

In another set of studies, the pathway-specific microarrays showed that keratinocyte-released stratifin stimulates dermal fibroblasts through paracrine signalling pathways and significantly increased the expression of MMP-1, MMP-3, MMP-8, MMP-10 and MMP-24 in these cells. On the contrary, stratifin-treated fibroblasts showed decreased expression of type I collagen and fibronectin.32 These findings suggest that keratinocyte-releasable stratifin has an indispensable effect on dermal fibroblasts by regulating the ECM synthesis and deposition through suppressing the synthesis of major ECM components and stimulation of MMPs, specially MMP-1 and MMP-3.32, 52, 53 Further studies showed a bilateral paracrine relation between keratinocytes and fibroblasts in regulation of ECM and MMPs through stratifin. As keratinocyte-releasable stratifin modulates MMPs expression in fibroblasts, fibroblasts also influence the expression of 14-3-3 proteins in keratinocytes.45

To evaluate the mechanism of transmembrane signalling on fibroblasts in response to keratinocyte-releasable stratifin, our group previously performed a series of experiments using affinity purification and mass spectroscopy analysis, and the findings revealed that the stratifin associates with aminopeptidase N (APN), or CD13, at the cell surface. The transient knockdown of APN in fibroblasts eliminated the stratifin-mediated p38 MAP kinase activation and MMP-1 expression, implicating APN in a receptor-mediated transmembrane signalling event. Further experiments showed the importance of C-terminus as a potential APN-binding site. Furthermore, the dephosphorylation of APN ectodomains reduced its binding affinity to the stratifin.54 Further work has been done to identify potential binding epitopes and APN-mediated MMP activation mechanism.55 Moreover, CD13 was reported to be a cell-surface receptor for other isoforms of 14-3-3 proteins, including 14-3-3ε.56 It was shown that interaction between 14-3-3 proteins and APN can be suppressed by blocking the 14-3-3 binding groove either by small-molecule inhibitors (phosphonates) or peptidomimetics.57, 58

In another study, it was shown that a paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin may serve as a target in the regulation of MMP-1 expression in epidermal-mesenchymal communication.59

These results collectively suggest that at beginning of the healing process, high levels of ECM deposition factors are released from proliferating and migrating keratinocytes, and when epithelialization of the wound is complete, the multilayered differentiated keratinocytes release switch their phenotype to express ECM modulating factors like stratifin and probably other 14-3-3 proteins. This feedback mechanism, which is triggered via epithelialization by differentiated keratinocyte, would act as a stop signal for the active process of healing.52 Figure 1 shows keratinocyte-fibroblast crosstalk via a stratifin modulated pathway.

Keratinocyte-fibroblast crosstalk via stratifin pathway. Stratifin (14-3-3σ) is secreted from differentiated keratinocytes and affect fibroblast possibly via aminopeptidase N (APN), or CD13. This process activates P38-MAP kinase pathway with involvement of p38 MAPK-dependent activator protein (AP-1) which eventually up-regulates expression of members of matrix metalloproteinases including MMP-1, MMP-3, MMP-8, MMP-10 and MMP-24 in fibroblasts, resulting in degradation of ECM. Stratifin-treated fibroblasts also show decreased expression of ECM proteins such as type I and III collagen and fibronectin. Reciprocally, fibroblasts also can modulate the expression of 14-3-3 proteins in keratinocytes

It is important to note that although stratifin has significant MMP-1 stimulatory effects, when stratifin was eliminated from keratinocyte conditioned medium (KCM) through immunodepletion method, expression of MMP-1 was reduced to around 40–50% and did not reach zero. This suggests that stratifin is partially responsible for a MMP-1 stimulatory effects on fibroblasts.60 In fact, keratinocytes express other MMP-1 stimulatory factors, like IL-1.61, 62

3.3 Keratinocyte-releasable factors suppress modulatory effects of wound healing promoting factors in fibroblasts

Dermal fibrosis is induced by pro-healing factors (eg TGF-β1 and IGF-1) during the active healing process after severe injuries such as burns, surgical wounds and deep trauma.63 It is important to know how these two fibrogenic factors can be suppressed in the late stages of the healing process to stop fibrosis. Thus, a key question is whether keratinocytes can release factors that impede the fibrogenic effect of IGF-1 and TGF-β1. To address this question, in one of our previous studies, we tested the responses of dermal fibroblasts to either IGF-1, TGF-β1 or both in the presence or absence of KCM or stratifin. The results of that study showed that while MMP-1 expression was remarkably reduced in TGF-β1 and IGF-1-treated fibroblasts, expression of MMP-1 mRNA was significantly increased in fibroblasts treated with KCM, even in the presence of both TGF-β1 and IGF-1. This was at least partially caused by stratifin released from keratinocytes when fibroblasts were treated with stratifin-immuno-depleted KCM, MMP-1 mRNA expression was prominently reduced. In accordance with increased MMP-1 mRNA expression, we also found that MMP-1 was increased at protein level in stratifin-treated fibroblasts. These findings collectively suggest that MMP-1 expression is stimulated by both stratifin and KCM in fibroblasts even in the presence of either IGF-1, TGF-β1 or a combination of both.60

In a subsequent study, we evaluated the efficacy of stratifin in improving the fibrotic condition of wounds in a rabbit ear model. The wounds that were treated with stratifin showed reduced infiltrated CD31 endothelial and T cells, increased expression of MMP-1 and decreased collagen density relative to that of untreated controls. That study suggested that formation of hypertrophic scar can be improved or prevented by application of topical stratifin when applied at the appropriate time during the wound healing process.64

3.4 Role of secreted growth factors and cytokines in cutaneous cell-cell communication

In previous section, the role of releasable growth factors and cytokines as wound healing initiation factors was described. Here, we review other releasable factors that are the key mediators of cell-cell communication in the skin during the wound healing process. Among the keratinocyte-releasable inflammatory factors, interleukin 1 alpha (IL-1α) is reported to be an important mediator inducing fibroblast activation.65 A proper wound healing outcome depends on a balanced interaction between IL-1α and its receptor antagonist (IL-1RA). A study by Zheng et al. showed that fibroblast gene expression and differentiation are regulated by keratinocyte-derived paracrine signals of epidermal integrin α3β1 leading to secretion of IL-1α. Further study showed that α3β1/IL-1α/Cox-2 regulatory pathway is involved in controlling the TGF-β–mediated fibroblast phenotype during wound healing.66 In a comprehensive review article by Barbara Russo et al., the effects of keratinocytes on fibroblasts via releasable factors were well described.58 It is reported that IL-1 released by keratinocytes increases the production of keratinocyte growth factor (KGF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), which in turn they stimulate the expression of bcl-2, p53, KGF and GM-CSF, which are involved in keratinocyte proliferation and differentiation.67 Our research group has also previously shown that addition of recombinant IL-1 markedly increased G-CSF expression in fibroblasts; however, IL-1 receptor antagonist only partially abrogated KCM-stimulated G-CSF expression.68 This indicates the potential roles of additional keratinocyte-releasable factors. These findings underline the importance of crosstalk between keratinocytes and fibroblasts, suggesting that communication of these cells modulates production and secretion of key growth factors and cytokines during cutaneous wound healing and remodelling period.

Another group of growth factor in cutaneous wound healing is the family of fibroblast growth factors (FGFs) that control the migration, proliferation, differentiation and survival of different cell types (reviewed by Maddaluno et al.).69 FGFs are a family of cell signalling proteins produced by macrophages and are crucial for normal development in animal cells. Any irregularities in their function result in a range of developmental defects. Members of the FGF family bind to cell surface proteins containing heparin and heparan sulphate and other extracellular matrix (ECM) molecules, which are released locally upon injury or tissue remodelling.70 One of the members of FGF family, keratinocyte growth factor (KGF, also known as FGF7), is a potent mitogen for different types of epithelial cells. It is produced by mesenchymal cells and exerts its biological effects through the binding to its high-affinity FGF receptor 2 (FGFR2-IIIb). This receptor which is expressed by various types of epithelial cells including keratinocytes regulates epithelial cell migration and differentiation.71 Upon skin injury, the expression of KGF is strongly up-regulated which likely to be important in wound epithelialization. The interaction of KGF with its receptor seems to predominantly be through a paracrine fashion.

In a recent study, the role of platelet-derived TGF-β1 in cutaneous wound healing has been investigated using transgenic mouse model targeting the deletion of TGF-β1 in megakaryocytes and platelets.72 The histological analysis of healed wounds showed a significantly thinner epidermis and dermis in TGF-β1–deficient group. However, the loss of platelet-derived TGF-β1 did not impede the overall healing process. It is suggested that platelet-derived TGF-β1 contributes to epidermal and dermal thickening and cellular turnover in excisional wounds. In vitro part of this study demonstrated that treating dermal fibroblasts with TGF-β1 led to significant increase in extracellular collagen I deposition in a concentration-dependent manner. However, fibroblasts stimulated with platelet lysate containing high levels of platelet-derived TGF-β1 did not show enhanced collagen deposition or proliferation, suggesting that platelet-derived TGF-β1 is not a key promoter of these wound healing processes. On the contrary, result of an in vitro study showed that both TGF-β1 and platelet lysate increases the human keratinocytes proliferation, suggesting that platelet-derived TGF-β1 significantly contributes to primary human keratinocyte proliferation and confirming the in vivo findings.72

Stromal cell-derived factor-1 (SDF-1) or CXCL 12 is a secreted 89-amino acid protein that binds to chemokine receptor type 4 (CXCR4) which expresses by several cell types including fibroblasts. Results of a study by Quan et al. revealed that SDF-1 functions as a mitogen to stimulate keratinocyte proliferation.73 A high level of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both normal and diseased skin conditions.73 The role of the SDF-1/CXCR4 axis in cutaneous wound healing is, however, controversial. Some studies reported that topical application of SDF-1 on mouse excisional and diabetic wounds accelerated healing, while result of other study showed that SDF-1/CXCR4 signalling delayed healing in excisional and burn wounds when SDF-1 neutralizing antibodies or chemical inhibitors of CXCR4 were used. Bollag & Hill comprehensively discussed different possibilities in a review article and suggested that inhibition of SDF-1 signalling locally at the injury site or at the distance affected infiltrating cells in the wound site or inflammatory cells at the distance and resulted in different responses.74

3.5 Role of other mediators in cell-cell interaction in wound healing

In addition to soluble factors described above, there are some other ways that cells within wound environment communicate with each other. Pannexins (PANX) are glycoproteins that form functional single membrane channels and has three members in humans, PANX1, 2 and 3.75 PANX3 plays a key role in wound healing process by controlling keratinocytes and keratinocyte-mesenchyme crosstalk via hemichannel and endoplasmic reticulum Ca2+ channels. The role of this glycoprotein has been investigated in PANX 3 knockout mouse wound healing model, and the result showed that PANX3 deficiency delays re-epithelialization and wound closure by reducing keratinocyte proliferation, differentiation and collagen deposition.76 Moreover, the expression of key fibrotic markers, fibronectin, type 1 collagen and α-smooth muscle actin was lower in PANX 3 deficit wounds as compared to those of wild type mice. Further, it is shown that PANX3-associated hemichannel, gap junction and ER Ca2+ channels function as an intermediary factor to co-regulate inflammation reaction and mesenchymal tissue remodelling via interacting with the TNF-α and TGF-β signalling pathways.76

Hydration is one of the most important external factors affecting optimal healing. It is widely accepted that a well-hydrated wound microenvironment is essential to optimal healing and dry wound may lead to delay in the healing response due to impaired re-epithelialization.77 On the contrary, a less functional epidermis enhances trans-epidermal water loss (TEWL) and a reduction in moisture of skin. Many studies suggest that TEWL remains high in pathological scars, such as hypertrophic scars and keloids.78 Interestingly, reducing TEWL loss by restoring the epidermal integrity (high occlusion of the wounds) shows noticeable efficacy at reducing dermal inflammatory conditions, thereby inhibiting scar formation. Therefore, epidermal-dermal interactions seem to play an important role in regulating wound healing in a dehydrated condition, as well. Studies have shown that reduced hydration increases the expression of S100 protein family members, S100A8, S100A9 and S100A12, in epidermal keratinocyte and promote fibroblast activation and fibrosis in the dermis. This up-regulation was reported to be done through toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE), which are the main receptors foe S100 family protein.79, 80 Further, by establishing a keratinocyte-fibroblast co-culture and conditional medium treatment models, Zhao et al. found that a reduced hydration condition increased the expression and secretion of HMGB1 in keratinocytes, subsequently activating dermal fibroblasts.81 HMGB1, a 214-amino acid protein with 99% amino acid similarity between rodents and humans, was first reported as a non-histone nuclear protein that regulates gene expression and nucleosome stability by binding to the minor groove of DNA. However, recent studies have revealed that HMGB1 can be passively released into the extracellular space in response to a variety of stimuli, such as apoptosis, infection and inflammation.82 The extracellular HMGB1 acts as a pro-inflammatory cytokine by binding to its receptors and stimulating the release of inflammatory cytokines, such as TNF, IL1α, IL6 and IL8. In Zhao et al. study, HMGB1 secreted from keratinocytes in reduced hydration environment activated fibroblasts by promoting the nuclear import of MRTF-A, increased the nuclear accumulation of MRTF-A/SRF complexes and consequently enhanced α-smooth muscle actin promoter activation. Therefore, they suggested that HMGB1 is the epidermal-derived ‘alarming’ that activated fibroblasts in hypertrophic scar formation.81

Mechanical signalling networks or mechanotransduction is another important mechanism that affects wound healing process and scar formation. Skin cells in response to mechanical stress (eg intrinsic or extrinsic forces) can alter their morphology, function and signalling pathways. This phenomenon is known as mechanotransduction.83 Myofibroblasts, which are originated from fibroblast and characterized by expression of alpha smooth muscle actin (α-SMA), play an important role in this process. TGF-β1 is a strong stimulating factor for myofibroblasts and activated myofibroblasts also propagate this process in positive feedback loop by secreting more TGF-β1, creating a positive feedback loop that further exacerbates fibrosis. TGF-β1 triggers phenotypical and structural changes in myofibroblasts resulting in secretion of pro-fibrotic factors and contractility.84 Mechanical forces within the wound environment also regulates cell-matrix interactions via focal adhesion kinase (FAK) pathway.85 FAK pathway is indeed a link between mechanical stress and inflammatory pathways during the wound healing process. FAK activates extracellular-related kinase (ERK), which in turn triggers the secretion of pro-inflammatory chemokines such monocyte chemo-attractant protein-1 (MCP-1) resulting in recruitment of inflammatory cells, increased collagen deposition, myofibroblasts activation and fibrosis.86

4 DELAYED RE-EPITHELIALIZATION INFLUENCES THE HEALING OUTCOME

It is well known that failure to re-epithelialize is one of the most obvious clinical indicators of chronic non-healing wounds.87 Following injury, migration of keratinocytes starts from the wound edge into the wound gap to re-epithelialize injured tissue and thus reinstate the epidermal barrier. In non-healing wounds, keratinocytes stay on the wound edge and keep proliferating which results in epidermal thickening at the wound edge of an open wound.88 A study by Thomason et al. suggested that changes in desmosomal adhesiveness through protein kinase Cα (PKCα) are critical for keratinocytes migration and re-epithelialization of wound in mice.89 Keratinocyte migration is initiated when PKCα is activated and switches hyper-adhesive calcium-independent desmosomes to become more weakly adhesive calcium-dependent desmosomes. This conversion facilitates the stimulation of wound edge keratinocytes and promoting re-epithelialization by mobilizing kertinocytes.90 In PKCα knockout mice, desmosomes remained hyperactive and prevented keratinocyte migration resulting in delayed re-epithelialization and delayed healing. On the contrary, in double transgenic mice over-expressing PKCα, the opposite occurred and re-epithelialization was promoted. However, more work is required to investigate whether loss or overexpression of PKCα has the same outcome in re-epithelialization of human chronic non-healing wounds.89

In the view of the importance of rapid and effective re-epithelialization successful wound healing, targeting re-epithelialization could be a potentially helpful strategy for the treatment of chronic wounds. Xiao et al. took this new approach in their research for treatment of diabetic wounds. They developed a chitosan-collagen hydrogel loaded with integrin binding peptide (QHREDGS) that supports viability of keratinocyte and promotes their migration and therefore accelerates diabetic wound healing. Their results showed that even a low dose in a single application was sufficient to accelerate diabetic wound healing in mice by enhancing re-epithelialization and granulation tissue formation.91 The study of Reto et al. also revealed that the continuous treatment of chronic and difficult-to-heal wounds with a low flow rate pure oxygen improved diabetic wound healing in mice through accelerating wound closure and re-epithelization.92 Other wound healing studies demonstrated that gene therapy with MicroRNAs (miR) such as miR-21, miR-31 and miR-205 had a probable therapeutic effect on chronic wounds through promoting migration of keratinocytes and improving re-epithelialization of wounds.93, 94

As mentioned above, epidermal-dermal interactions have a great regulatory role on synthesis of ECM components. In large burn wounds, using autologous or allogeneic keratinocytes in the form of in vitro cultured sheets, cell transplantation before they form a sheet or suspension of non-cultured keratinocyte promotes wound healing process and attenuates scar formation.95 The effect of keratinocyte in the re-epithelialization of wound healing is not only providing physical protection on the wound surface but also secreting active ingredients that modulate the healing process. Therefore, keratinocytes are likely to be the main source of wound healing termination factors and any delay in reepithelialization results in disruption of normal epidermal-mesenchymal communication and that results in increasing fibroblast proliferation, ECM production and subsequent deposition, and lastly, less ECM degradation which is frequently seen in hypertrophic scarring and keloids.4, 30, 48, 96

5 CONCLUDING REMARKS

Complete and optimal wound healing involves a delicate balance between ECM deposition and degradation. Maintaining this balance requires highly accurate epidermal-mesenchymal communication and correct information exchange between keratinocytes and fibroblasts. Disruption in fibroblast-keratinocyte crosstalk, as seen in delayed epithelialization in open wounds, increases the risk of developing chronic wounds or hypertrophic scarring. Thus, the potential stop signal for wound healing should have a regulatory role on both ECM synthesis and degradation to reach the successful end for wound healing. Literature reviewed in this article collectively support a model in which differentiated keratinocytes secrete stratifin that stimulates collagenase production by dermal fibroblasts. This process then control degradation of the major dermal ECM components like collagen type I and III. As illustrated in Figure 1, stratifin and its receptor on fibroblasts may be potential targets for pharmacological intervention for controlling wound healing or development of hypertrophic scarring. Studies have shown that stratifin plays a critical role in wound healing process that is involved in both collagen deposition and its hydrolysis and plays an important role in keratinocyte-fibroblast communication. Here, we suggest that stratifin is a potent anti-fibrogenic factors secreted by keratinocytes and functions as a stop/slow down signal for the wound healing process. After successful re-epithelialization, stratifin is released from differentiated keratinocytes and stimulates MMPs expression in fibroblasts. It is also emphasized that this is a two-way communication and there is a double-paracrine relation between keratinocyte and fibroblasts in regulation of ECM turnover and MMPs expression through stratifin. Further, it has been reported that some of the keratinocyte-derived factors stimulate production and secretion of certain growth factors and cytokines by fibroblasts. These factors then reciprocally result in proliferation of keratinocyte in a double paracrine manner.97 Recent study reviewed 6271 abstracts from which 56 of these articles examined the potential role of keratinocytes on fibroblast functionality in terms of gene expression, protein production and phenotype modification, and the finding indicated that under physiological and fibrotic conditions, IL-1 secreted by keratinocytes stimulate fibroblasts which then produce keratinocyte growth factor (KGF) and metalloproteinases.65 It is suggested that defining the fibroblast phenotypes from different tissues and their contributions in wound healing may make it possible to control the healing process with a better outcome.97

Finally, these findings collectively suggest that interaction of keratinocyte and fibroblasts happens through a double paracrine manner and disruption of this communication could lead to undesired wound healing outcome such as dermal fibrotic condition.

CONFLICT OF INTEREST

A.G. is the leading inventor of serum stratifin as a biological marker for early detection of rheumatoid arthritis. Other authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

Nafise Amiri, Andrew P. Golin, Reza B. Jalili and Aziz Ghahary wrote the paper and have read and approved the final manuscript.