Scarless wound healing: Current insights from the perspectives of TGF-β, KGF-1, and KGF-2

The process of wound healing involves a complex and vast interplay of growth factors and cytokines that coordinate the recruitment and interaction of various cell types. A series of events involving inflammation, proliferation, and remodeling eventually leads to the restoration of the damaged tissue. Abrogation in the regulation of these events has been shown to result in excessive scarring or non-healing wounds. While the process of wound healing is not fully elucidated, it has been documented that the early events of wound healing play a key role in the outcome of the wound. Furthermore, high levels of inflammation have been shown to lead to scarring. The regulation of these events may result in scarless wound healing, especially in adults. The inhibition of transforming growth factor-β (TGF-β) and the administration of keratinocyte growth factors (KGF), KGF-1 and KGF-2, has in recent years yielded positive results in the acceleration of wound closure and reduced scarring. Here, we encapsulate recent knowledge on the roles of TGF-β, KGF1, and KGF2 in wound healing and scar formation and highlight the areas that need further investigation. We also discuss potential future directions for the use of growth factors in wound management.     

The epithelial mitogen keratinocyte growth factor binds to collagens via the consensus sequence glycine-proline-hydroxyproline

The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that mesenchymal cell-derived keratinocyte growth factor (KGF), a key stimulator of epithelial cell proliferation during wound healing, preferentially binds to collagens I, III, and VI. Binding is inhibited in a dose-dependent manner by denatured single collagen chains and collagen cyanogen bromide peptides. This interaction is saturable with dissociation constants of approximately 10(-8) to 10(-9) m and estimated molar ratios of up to three molecules of KGF bound to one molecule of triple helical collagen. Furthermore, collagen-bound KGF stimulated the proliferation of transformed keratinocyte or HaCaT cells. Ligand blotting of collagen-derived peptides points to a limited set of collagenous consensus sequences that bind KGF. By using synthetic collagen peptides, we defined the consensus sequence (Gly-Pro-Hyp)(n) as the collagen binding motif. We conclude that the preferential binding of KGF to the abundant collagens leads to a spatial pattern of bioavailable KGF that is dictated by the local organization of the collagenous extracellular matrix. The defined collagenous consensus peptide or its analogue may be useful in wound healing by increasing KGF bioactivity and thus modulating local epithelial remodeling and regeneration.     

细胞因子对创伤愈合的影响

创伤愈合是一个复杂的生物学过程,涉及炎症细胞,修复细胞、细胞外基质以及细胞因子之间的相互作用。传统将这一过程分为炎症期、增值期、组织重构三个相互重叠的时期。细胞因子是一类对细胞生长、分化有明显调控作用的小分子生物活性多肽。是细胞与细胞外基质间重要的信号传导物。多种生长因子被释放到伤口部位被认为是创伤愈合所必需的。本文就细胞因子对创伤愈合的促进作用、细胞因子相互之间的协同作用,以及应用前景作以概述。     

Hepatocyte growth factor and keratinocyte growth factor enhance IL-1-induced IL-8 secretion through different mechanisms in Caco-2 epithelial cells

A variety of cytokines have been detected in inflamed intestinal mucosal tissues, including the pro-inflammatory cytokine, interleukin-1 (IL-1), along with growth factors involved in wound healing processes such as proliferation and cell migration. However, little is known about how IL-1 and growth factors interact with intestinal epithelial cells to regulate the production of inflammatory cytokines such as interleukin-8 (IL-8). Previously, we have shown that hepatocyte growth factor (HGF) could significantly enhance IL-1-stimulated IL-8 secretion by the Caco-2 colonic epithelial cell line, yet HGF, by itself, did not stimulate IL-8 secretion. In this report, a second growth factor, keratinocyte growth factor (KGF), was also found to significantly enhance IL-1-induced IL-8 secretion by Caco-2 cells, yet KGF, by itself, also had no effect. Simultaneous addition of both IL-1 and KGF was also required for the enhancing effect. Treatment of the Caco-2 cells with wortmannin or triciribine suppressed the enhancing effect of HGF, suggesting that the effect was mediated by signaling through phosphatidylinositol-3-kinase (PI3K) and the kinase AKT. The enhancing effect of KGF was not affected by wortmannin, but was suppressed by triciribine, suggesting that the effect of KGF was through a PI3K-independent activation of AKT. These results suggest that the growth factors HGF and KGF may play a role in enhancing IL-1-stimulated production of IL-8 by epithelial cells during mucosal inflammations. However, the mechanism by which the growth factors enhance the IL-1 response may be through different initial signaling pathways.     

p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades

One important action of growth factors is their participation in tissue repair; however, the signaling pathways involved are poorly understood. In a model of corneal wound healing, we found that two paracrine growth factors, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), induced rapid and marked activation and prompt nuclear accumulation of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2), but not of JNK (p-JNK1/2), in corneal epithelial cells. Interruption of p38 and ERK1/2 signaling pathways by pretreatment with inhibitors SB203580 and PD98059 and subsequent stimulation with HGF or KGF abolished the activation and nuclear localization. Inhibition of either one of these mitogen-activated protein kinases, p38 or ERK1/2, induced a robust cross-activation of the other. In immunofluorescence studies of wounded cornea, p-p38, unlike p-ERK1/2, was immediately detectable in epithelium after injury. Inhibition of p38 by SB203580 blocked migration of epithelial cells almost completely. In contrast, PD98059 seemed to slightly increase the migration, through concomitant activation of p38. Unlike ERK1/2, p38 did not significantly contribute to proliferation of epithelial cells. Inhibition of either the ERK1/2 or p38 pathway resulted in delayed corneal epithelial wound healing. Interruption of both signaling cascades additively inhibited the wound-healing process. These findings demonstrate that both p38 and ERK1/2 coordinate the dynamics of wound healing: while growth factor-stimulated p38 induces epithelial migration, ERK1/2 activation induces proliferation. The cross-talk between these two signal cascades and the selective action of p38 in migration appear to be important to corneal wound healing, and possibly wound healing in general, and may offer novel drug targets for tissue repair.     

Caveolin-1 and -2 expression is differentially regulated in cultured keratinocytes and within the regenerating epidermis of cutaneous wounds

Keratinocyte growth factor (KGF) and its receptor are involved in various types of epithelial repair processes. To gain insight into the molecular mechanisms of KGF action in the healing skin wound, we searched for genes which are regulated by this factor in cultured keratinocytes. Using the PCR-select technology we constructed a subtractive cDNA library. One of the KGF-regulated genes that we identified was shown to encode caveolin-1, a major component of caveolar membranes. Caveolin-1 is involved in a wide variety of cellular processes, particularly in the regulation of various signal transduction pathways. Caveolin-1 mRNA levels increased in cultured keratinocytes after KGF treatment. By in situ hybridization and immunohistochemistry we found a strong expression of caveolin-1 in the KGF-responsive basal keratinocytes of the epidermis and the hyperproliferative epithelium of the wound as well as in endothelial cells and in other cells of the granulation tissue. In 13-day wounds expression of caveolin-1 mRNA was restricted to the regenerated dermis. In addition to caveolin-1, the mRNA expression of caveolin-2, a second member of the caveolin family, was also induced in keratinocytes after stimulation with KGF but also with other growth factors and cytokines. In contrast to caveolin-1, caveolin-2 protein was expressed in all layers of the normal epidermis and in the suprabasal layers of the hyperproliferative wound epithelium. These results demonstrate a differential expression of caveolin-1 and -2 in proliferating versus differentiating keratinocytes.     

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions.     

角质细胞生长因子研究进展

角质细胞生长因子（KGF）属于成纤维细胞生长因子家族,它通过与受体（KGFR）的结合,特异性地刺激上皮细胞的增殖.KGF基因表达受到正负调控作用,正负调控作用的平衡对于KGF正常发挥功能具有重要意义.KGF具有多种生物学功能：参与组织、器官的发育,具有损伤防治功能,与癌症的发生有着密切的联系.     

Keratinocyte growth factor promotes cell motility during alveolar epithelial repair in vitro

Epithelia play a key role as protective barriers, and mechanisms of repair are crucial for restoring epithelial barrier integrity, especially in the lung. Cell spreading and migration are the first steps of reepithelialization. Keratinocyte growth factor (KGF) plays a key role in lung epithelial repair and protects against various injuries. We hypothesized that KGF may protect the lung not only by inducing proliferation but also by promoting epithelial repair via enhanced epithelial cell migration. In an in vitro wound-healing model, we found that KGF enhanced wound closure by 33%. KGF acted primarily by inducing lamellipodia emission (73.2 +/- 3.9% of KGF-treated cells had lamellipodia vs 61.3 +/- 3.4% of control cells) and increasing their relative surface area (59 +/- 2.7% with KGF vs 48 +/- 2.0% in controls). KGF reduced cytoskeleton stiffness as measured by magnetic twisting cytometry and increased cell motility (5.8 +/- 0.42 microm/h with KGF vs 3.7 +/- 0.41 microm/h in controls). KGF-increased cell motility was associated with increased fibronectin deposition during wound closure and with fibronectin reorganization into fibrils at the rear of the cells. Taken together, our findings strongly suggest that KGF may promote epithelial repair through several mechanisms involved in cell migration.     

第二军医大学长征医院口腔科

创伤愈合是一个复杂的生物学过程,涉及炎症细胞,修复细胞、细胞外基质以及细胞因子之间的相互作用。传统将这一过程分为炎症期、增值期、组织重构三个相互重叠的时期。细胞因子是一类对细胞生长、分化有明显调控作用的小分子生物活性多肽,是细胞与细胞外基质间重要的信号传导物。多种生长因子被释放到伤口部位被认为是创伤愈合所必需的。本文就细胞因子对创伤愈合的促进作用、细胞因子相互之间的协同作用,以及应用前景作以概述。     

Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc

When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.     

Tissue scaffolds functionalized with therapeutic elastin-like biopolymer particles

Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin-like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF–ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF–ELPs.     

Phospholipase C-delta extends intercellular signalling range and responses to injury-released growth factors in non-excitable cells

Abstract.   Objectives : Intercellular communication in non-excitable cells is restricted to a limited range close to the signal source. Here, we have examined whether modification of the intracellular microenvironment could prolong the spatial proposition of signal generation and could increase cell proliferation . Material and methods : Mathematical models and experimental studies of endothelial repair after controlled mechanical injury were used. The models predict the diffusion range of injury-released growth factors and identify important parameters involved in a signalling regenerative mode. Transfected human umbilical vein endothelial cells (HUVECs) were used to validate model results, by examining intercellular calcium signalling range, cell proliferation and wound healing rate. Results : The models predict that growth factors have a limited capacity of extracellular diffusion and that intercellular signals are specially sensitive to cell phospholipase C-delta (PLCδ) levels. As basal PLCδ levels are increased by transfection, a significantly increased intercellular calcium range, enhanced cell proliferation, and faster wound healing rate were observed. Conclusion : Our in silico and in vitro studies demonstrated that non-excitable endothelial cells respond to stimuli in a complex manner, in which intercellular communication is controlled by physicochemical properties of the stimulus and by the cell microenvironment. Such findings may have profound implications for our understanding of the tight nature of autocrine cell growth control, compensation to stress states and response to altered microenvironment, under pathological conditions.     

Keratinocyte growth factor accelerates wound closure in airway epithelium during cyclic mechanical strain.

The airway epithelium may be damaged by inhalation of noxious agents, in response to pathogens, or during endotracheal intubation and mechanical ventilation. Maintenance of an intact epithelium is important for lung fluid balance, and the loss of epithelium may stimulate inflammatory responses. Epithelial repair in the airways following injury must occur on a substrate that undergoes cyclic elongation and compression during respiration. We have previously shown that cyclic mechanical strain inhibits wound closure in the airway epithelium (Savla and Waters, 1998b). In this study, we investigated the stimulation of epithelial wound closure by keratinocyte growth factor (KGF) in vitro and the mechanisms by which KGF overcomes the inhibition due to mechanical strain. Primary cultures of normal human bronchial epithelial cells (NHBE) and a cell line of human airway epithelial cells, Calu 3, were grown on Silastic membranes, and a wound was scraped across the well. The wells were then exposed to cyclic strain using the Flexercell Strain Unit, and wound closure was measured. While cyclic elongation (20% maximum) and cyclic compression (approximately 2%) both inhibited wound closure in untreated wells, treatment with KGF (50 ng/ml) significantly accelerated wound closure and overcame the inhibition due to cyclic strain. Since wound closure involves cell spreading, migration, and proliferation, we investigated the effect of cyclic strain on cell area, cell-cell distance, and cell velocity at the wound edge. While the cell area increased in unstretched monolayers, the cell area of monolayers in compressed regions decreased significantly. Treatment with KGF increased the cell area in both cyclically elongated and compressed cells. Also, when cells were treated with KGF, cell velocity was significantly increased in both static and cyclically strained monolayers, and cyclic strain did not inhibit cell migration. These results suggest that KGF is an important factor in epithelial repair that is capable of overcoming the inhibition of repair due to physiological levels of cyclic strain.     

Suppression of keratin 15 expression by transforming growth factor beta in vitro and by cutaneous injury in vivo

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine which plays an important role in cutaneous wound repair. To gain insight into the mechanisms of action of this growth and differentiation factor in the skin, we searched for genes which are regulated by TGF-beta1 in cultured HaCaT keratinocytes. Using the differential display RT-PCR technology we identified a gene which was strongly downregulated by TGF-beta1. The identified cDNA includes sequences of the keratin 15 (K15) gene which encodes a component of the cytoskeleton of basal cells in stratified epithelia. Surprisingly, our cDNA also included an unknown sequence. Since this cDNA lacks an open reading frame, the corresponding mRNA is likely to be nonfunctional. However, we also demonstrate a strong negative regulation of the expression of the published, functional K15 variant. Expression of K15 was also suppressed by tumor necrosis factor alpha (TNF-alpha) and to a lesser extent by epidermal growth factor (EGF) and keratinocyte growth factor (KGF). By contrast, the major basal type I keratin, K14, was upregulated by TGF-beta1, whereas TNF-alpha, EGF, and KGF had no effect. Consistent with the in vitro data, we found a significant reduction of the K15 mRNA levels after skin injury, whereas K14 expression increased during the wound healing process. Immunostaining revealed the presence of K15 in all basal cells of the epidermis adjacent to the wound, but not in the hyperproliferative epithelium above the granulation tissue. These data demonstrate that K15 is excluded from the activated keratinocytes of the hyperthickened wound epidermis, possibly as a result of increased growth factor expression in injured skin.