Epithelial–mesenchymal interactions in tissue repair

After cutaneous injury a cascade of events is observed, which mediates tissue repair and eventually the re-establishment of the barrier function of the skin. Tissue repair has been divided into an inflammatory phase, a granulation phase with synthesis of new connective tissue and epithelial wound closure, and finally a scar-remodeling phase once the epidermal barrier has been re-established.

Throughout the whole repair process interaction between different cell types provides coordination of the individual events, allowing for a temporal and spatial control. In the mid- and late phase of wound healing, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts, which gradually shift the microenvironment away from an inflammatory to a synthesis-driven granulation tissue. Epithelial–mesenchymal interactions are also crucial for pre- and postnatal skin development, and they play an important role in tumor biology. During development, tightly controlled mutual interactions between the epidermis and the mesenchyme control the formation of limbs, skin architecture, and appendages through gradients of morphogens and differential sensitivity of target cells for these diffusible mediators. Thus, highly complex, cell-type-specific responses can be achieved. After a series of these interactions, limb or skin appendages eventually form (reviewed by Schmidt-Ullrich and Paus, 2005; Tickle, 2006). There is strong evidence that these patterns of epithelial–mesenchymal interactions persist in adult life and regulate skin homeostasis, part of the wound-healing response (Martin and Parkhurst, 2004), or become critically involved in tumor cell invasion and tumor progression (Bhowmick et al., 2004; Mueller and Fusenig, 2004).

The epidermal phenotype

Keratinocytes in skin represent a constantly renewing cellular compartment, and stem cells have raised continuous interest from a scientific as well as therapeutic aspect. Epidermal stem cells have been characterized largely on their functional properties and marker expression (Barrandon and Green, 1987; Cotsarelis et al., 1990; Oshima et al., 2001; Tumbar et al., 2004; Jensen and Watt, 2006), nevertheless their exact nature still remains somewhat elusive. The role of epithelial–mesenchymal interactions for the keratinocyte stem cell phenotype was demonstrated early (Rheinwald and Green, 1975). The feeder cell-culture system demonstrated clearly that the epidermal stem cell phenotype depends on the interaction with mesenchymal cells. Primary keratinocytes are seeded at clonal density onto growth-inhibited fibroblasts, which serve as feeder cells. Within 7–10 days, the cultures become confluent and keratinocytes can be passaged with high efficiency. There appears to be flexibility concerning the source of the fibroblasts, as murine fibroblasts can be substituted by human skin fibroblasts (Limat et al., 1990). Apparently, the mesenchymal feeder cells provide a microenvironment, which supports the stem cell phenotype and augments epidermal proliferation. Growth factors play a critical role and are added to the culture medium to support epidermal proliferation. However, even complex growth factor mixtures can hardly replace the net effect of mesenchymal feeder cells when keratinocytes are seeded at clonal densities. It became apparent that keratinocytes in these cultures instruct fibroblasts to synthesize and secrete growth factors and cytokines, such as keratinocyte growth factor (KGF)/fibroblast growth factor-7 (FGF7), IL-6, and GM-CSF (Waelti et al., 1992; Smola et al., 1993; Boxman et al., 1996). Expression of these factors can be induced by IL-1, and indeed, keratinocyte-derived IL-1 was identified as primary inductor (Waelti et al., 1992; Maas-Szabowski and Fusenig, 1996; Maas-Szabowski et al., 1999). Szabowski et al. (2000) had elegantly shown that in fibroblasts, the transcription factor activator protein-1 plays a central role in this paracrine growth factor loop. Activator protein-1-dependent KGF/FGF7, GM-CSF, pleiotrophin, and stromal cell-derived factor 1 expression regulate keratinocyte proliferation and differentiation in keratinocyte–fibroblast cocultures (Florin et al., 2005). The concept of double paracrine signaling was proposed where keratinocytes initiate growth factors in fibroblasts, which themselves stimulate keratinocyte proliferation.

Apart from paracrine growth factor regulation, the formation of a new basement membrane zone is another example where interaction between keratinocytes and fibroblasts are crucially involved. Basement membrane constituents and a fully organized basement membrane can influence the keratinocyte phenotype (El Ghalbzouri et al., 2004). Recombination experiments have detailed the expression pattern and cellular origin of basement membrane and extracellular matrix constituents in epidermal–dermal cultures (Regauer et al., 1990; Fleischmajer et al., 1997; Smola et al., 1998). Laminin-5 is of epithelial origin, collagen VII primarily produced by keratinocytes and to a lesser extent by fibroblasts (Marinkovich et al., 1993), whereas nidogen is exclusively derived from fibroblasts. The other basement membrane components are produced in both cell types with mutual induction patterns. Apart from matrix-derived signals, which help to maintain the epidermal stem cell phenotype, proteolytic processing can generate laminin-5 fragments, which can stimulate epidermal growth factor (EGF) receptor signaling and proliferation (Schenk et al., 2003). Extracellular matrix also modulates cellular functions directly and this has been extensively studied in fibroblasts (Eckes et al., 2006). This involves binding of matrix molecules to adhesion receptors, and mechanical tension plays a critical role in regulating the de novo synthesis of collagens. Interestingly, fibroblasts in collagen gels and also in the granulation tissue form gap junctions, which are also involved in the regulation of extracellular matrix production (Ehrlich and Diez, 2003; Ehrlich et al., 2006). In a polyvinyl alcohol sponge implant model, a gap junction uncoupler reduced the number of cells migrating into the sponges, the myofibroblast density and collagen deposition and organization (Ehrlich and Diez, 2003). Thus, epithelial–mesenchymal cocultures provide an environment where the epidermal stem cell phenotype and keratinocyte proliferation are supported by a synergistic mix of growth factors, extracellular matrix components, and direct cell–cell contacts. This concept has been excellently reviewed by Nelson and Bissell (2006).

Growth factor-mediated cross talk between keratinocytes and fibroblasts

Growth factor networks have been identified in the above-described tissue culture systems. In the meantime, the existence of at least some of these networks has been verified in vivo in normal and wounded skin. In particular, studies with genetically modified animals have advanced our understanding in this field, and the wound-healing phenotypes of different transgenic and knockout mice has been reviewed previously (Grose and Werner, 2004). Furthermore, a website has been set up, which summarizes these phenotypes (http://icbxw.ethz.ch/woundtran
sgenic/home.html).

One of the first growth factors, which was shown to be involved in mesenchymal–epithelial interactions, is KGF/FGF7, which is rapidly induced in fibroblasts after wounding and which exerts its effects through binding to its receptor FGFR2IIIb on keratinocytes. Some growth factors are predominantly expressed in epidermal cells and exert their actions on mesenchymal cells, such as platelet-derived growth factor (PDGF). For other growth factors, expression was detected in both keratinocytes and the mesenchymal cell compartment and the effects were observed in both cell compartments in an autocrine and paracrine fashion. Activins are typical examples (for review, see Werner and Grose, 2003).

Paracrine acting mesenchymally derived growth factors

As mentioned above, KGF/FGF7 is the prototype of a mesenchymally derived growth factor, which stimulates epidermal proliferation (Finch et al., 1989; Rubin et al., 1989). On injury, KGF/FGF7 expression is strongly upregulated in mesencymal cells (Werner et al., 1992; Marchese et al., 1995). IL-1, tumor necrosis factor- (TNF-), PDGF, and serum were shown to stimulate KGF expression, and all these inducers are present at the early stages of wound healing (Brauchle et al., 1994; Chedid et al., 1994). Expression of a dominant-negative FGFR2IIIb mutant in basal keratinocytes of transgenic mice caused epidermal thinning, hair follicle abnormalities, dermal fibrosis, and a severe delay in wound re-epithelialization (Werner et al., 1994). However, the dominant-negative mutant blocks signaling by all FGF receptors in response to common ligands, and the type of FGF and FGFR, which is involved in wound healing, remained therefore unclear. Subsequent analysis of mice deficient for FGF7/KGF showed that healing of incisional wounds is not impaired (Guo et al., 1996), suggesting that other factors like fibroblast-derived FGF10, which also signals through the FGFR2IIIb receptor, may compensate in this experimental model, which requires little epidermal healing. FGF10 is also expressed in fibroblasts, but unlike FGF7/KGF, FGF10 expression is not induced during wound healing. Transforming growth factor- (TGF-) and TNF- repress FGF10 expression, suggesting a differential regulation of FGF10 and FGF7 (Beer et al., 1997a). Recently, another FGF family member, FGF22, has been identified, which also can activate FGFR2IIIb (Zhang et al., 2006). Interestingly, the predominant cell type expressing FGF22 are keratinocytes (Nakatake et al., 2001; Beyer et al., 2003). Therefore, FGF22 is considered to act in an autocrine fashion, and high expression of this FGF was seen in the late stages of the wound-healing response. The activity of these FGFRIIIb ligands may be enhanced by the FGF-binding protein. FGF-binding protein is synthesized in the epidermis, and it is upregulated during wound healing. It binds FGF7, -10, -22 and enhances activity of ligands at low concentrations (Beer et al., 2005).

IL-6 has been shown to stimulate epidermal cell proliferation (Grossman et al., 1989), and its expression is strongly upregulated in keratinocyte–fibroblast cocultures in comparison to monocultures. In mice deficient for IL-6, excisional wound healing was severely retarded. This is very well in line with its presumed role in mesenchymal–epidermal cell–cell communication (Gallucci et al., 2000; Lin et al., 2003). In addition, fewer leukocytes were observed in the wounds of IL-6-deficient mice as well as decreased angiogenesis and collagen accumulation. The phenotype could be mimicked by injecting a neutralizing mAb into wild-type mice (Lin et al., 2003) and rescued by injection of a single dose of IL-6 into IL-6-deficient mice (Gallucci et al., 2000).

In addition to KGF/FGF7 and IL-6, other factors, such as hepatocyte growth factor and GM-CSF are primarily produced by mesenchymal cells and act on keratinocytes following the same paracrine pattern (Smola et al., 1993; Maas-Szabowski and Fusenig, 1996; Szabowski et al., 2000; Gron et al., 2002).

Keratinocyte-derived growth factors

KGF/FGF7 and IL-6 are predominantly expressed in the mesenchyme and target keratinocytes. There are also examples where keratinocytes are the primary source of growth factors, which may stimulate not only keratinocytes themselves but also fibroblasts or other mesenchymal cell types.

TGF- is predominantly expressed in keratinocytes and has a profound autocrine effect on keratinocytes in wound healing. Expression of this growth factor is upregulated in keratinocytes after skin injury (Antoniades et al., 1993). EGF as well as TGF- activity were detected in wound fluid from rats (Grotendorst et al., 1989) and humans (Ono et al., 1995). Furthermore, keratinocytes bordering the epithelial defect as well as epithelial cells in hair follicles were found to express high levels of TGF-, particularly in the strongly proliferating cells. High TGF- expression is also a hallmark of psoriatic epidermis and appears to be associated with keratinocyte hyperproliferation (Gottlieb et al., 1988; Elder et al., 1989). Surprisingly, wound-healing experiments in TGF--deficient mice showed no abnormal wound-healing phenotype (Luetteke et al., 1993; Mann et al., 1993) and only on closer examination and in wound-healing models where epithelial migration is critical for healing small differences were observed (Kim et al., 2001). Less clear is the role of TGF- on mesenchymal cells in wound healing. It is a strong mitogen for fibroblasts (Rosenthal et al., 1986) and augments angiogenesis (Schreiber et al., 1986). However, the lack of an obvious mesenchymal phenotype in TGF--deficient mice suggests that this growth factor is dispensable for fibroblast proliferation and/or angiogenesis and that its loss can be compensated by other EGFR ligands. Heparin-binding EGF might be a possible candidate, since it was also detected in wound fluid of partial thickness wounds and of pediatric burn patients (Marikovsky et al., 1993) as well as in migrating keratinocytes in a mouse burn wound model (Cribbs et al., 2002). Heparin-binding EGF is mitogenic for both keratinocytes (Cribbs et al., 2002) and fibroblasts and can synergize with IGF I (Marikovsky et al., 1993). Furthermore, keratinocyte-specific inactivation of the hb-egf gene resulted in delayed epithelial wound closure in mice, suggesting that heparin-binding EGF is involved in epithelialization by accelerating keratinocyte migration, rather than proliferation (Shirakata et al., 2005).

PDGF is another predominantly keratinocyte-derived growth factor acting in a paracrine manner. This growth factor is immediately released after wounding by degranulating platelets. Without the time-consuming de novo synthesis at the wound site, PDGF activity is readily available within minutes after injury. Later during tissue repair, PDGF is synthesized in the wound tissue. PDGF transcripts and protein were detected in the epithelium (Antoniades et al., 1991; Ansel et al., 1993; Beer et al., 1997a, 1997b), whereas transcripts in mesenchymal cells were described by Antoniades et al. (1991) also. The corresponding receptors were detected in mesenchymal cells (Ansel et al., 1993) and in one report on keratinocytes as well (Antoniades et al., 1991). Fibroblast- and keratinocyte-derived PDGF-AA has little effect on these cells, as the predominant PDGF receptor- is little responsive to the PDGF-AA isoform (Bonner et al., 1991) also. On the other hand, different cell types in healing skin wounds express the PDGF-BB isoform for which the fibroblasts are responsive and which is able to induce KGF in mesenchymal cells (Brauchle et al., 1994; Beer et al., 1997a, 1997b). The expression pattern suggests a paracrine signaling from the epidermis to mesenchymal cells as well as autocrine signaling in the granulation tissue. Moreover, neutralizing PDGF antibodies reduced the mitogenic activity of acute wound fluid by 45% when tested on fibroblasts (Katz et al., 1991), indirectly supporting this assumption.

Growth factors expressed in keratinocytes and mesencymal cells

Activins are members of the TGF- family of growth and differentiation factors. They are homo- or heterodimeric polypeptides consisting of _A, _B, _C, _D, and _E chains. The _A and _B chains are expressed in keratinocytes as well as in the mesenchyme on wounding (Hubner et al., 1996b). The _C and _E chains are predominantly found in other tissues (Vejda et al., 2002) and the _D subunit is present only in Xenopus laevis but not in mammals. Epidermal overexpression of the activin _A under the control of a keratin 14 promoter caused a dermal phenotype mostly characterized by a loss of adipose tissue, which was replaced by a fibrosing dermis, and a hyperproliferating epidermis (Munz et al., 1999). The epidermal phenotype was unexpected, as activin was shown to be growth inhibitory for keratinocytes in vitro (Seishima et al., 1999). Therefore, it seems likely that the phenotype is at least in part mediated via mesenchymal cells. Of particular interest was the enhanced wound healing in activin-overexpressing mice. Granulation tissue formation was particularly affected, but faster re-epithelialization was also observed. As a consequence, however, the mice also showed an extended scar area (Munz et al., 1999; S.W. unpublished data). To study the role of endogenous activin in wound repair, mice overexpressing the soluble activin antagonist follistatin in the epidermis were generated (Wankell et al., 2001). These mice showed a severe delay in wound healing, and granulation tissue formation was particularly affected. However, the wounds finally healed, and the resulting scar was smaller than in control animals. These findings demonstrated that the levels of bioactive activin in a wound determine the speed of wound repair but also the scarring response. To determine if activin affects wound healing via keratinocytes or mesenchymal cells or both, transgenic mice expressing a dominant-negative activin receptor mutant in keratinocytes were generated. Their skin architecture was almost unchanged compared with control mice. Wound re-epithelialization was slightly delayed owing to reduced keratinocyte migration. However, the strong inhibition of granulation tissue formation seen in the follistatin-overexpressing mice was not observed in the transgenic mice expressing the dominant-negative activin receptor mutant in keratinocytes, demonstrating that activin exerts its effect on wound healing predominantly via stromal cells (Bamberger et al., 2005).

The fibroblast phenotype in epidermal–mesenchymal interactions

Most of the studies described above analyzed the phenotype of the epidermis, as delayed wound closure is often due to reduced keratinocyte migration or proliferation. By contrast, much less is known about the changes that mesenchymal cells undergo in healing skin wounds. From wound-healing studies, it has long been known that myofibroblasts start to appear in the granulation tissue around the mid-phase of wound healing. This coincides with a strong induction of contractile properties so that cells align parallel to mechanical tension that is building up in the granulation tissue. Myofibroblasts appear to differentiate from fibroblasts by acquiring the smooth muscle cell actin isoform -smooth muscle action (-SMA). Several studies investigated the mechanisms on how fibroblasts become myofibroblasts, and it seems that the two most important stimuli are mechanical tension (Hinz et al., 2001) and TGF- activity (Desmouliere et al., 1993).

To analyze how fibroblasts respond to keratinocyte-derived stimuli, the messenger RNA (mRNA) expression pattern of fibroblasts in monoculture was compared with the fibroblasts cocultured with keratinocytes (Shephard et al., 2004b). To reduce the complexity of the experimental system HaCaT keratinocytes were cultured on a feeder layer of fibroblasts. HaCaT cells in these experiments had the advantage that the epidermal phenotype was relatively stable although normal epidermal architecture is observed only after transplantation onto nude mice (Boukamp et al., 1997) or after addition of TGF- in organotypic cultures (Maas-Szabowski et al., 2003). When HaCaT keratinocytes reached 50% confluence, cocultured fibroblasts were isolated by differential EDTA treatment. Comparing the RNA expression pattern of cocultured and control fibroblasts revealed differential expression of growth factor, extracellular matrix, protease, and intracellular structural protein transcripts (Table 1). The intracellular structural proteins suggested that fibroblasts differentiated into myofibroblasts as shown by a time-dependent increase of -SMA-positive mesenchymal cells. The expression data showed that several genes characteristic of TGF- signaling were upregulated. They encode, among others, plasminogen-activator inhibitor-1, -SMA and collagen type 1. HaCaT keratinocytes as well as fibroblast monocultures produced low amounts of TGF-, which was mostly in the latent form. In cocultures, however, there was a strong increase in latent and active TGF-. Surprisingly, high levels of active TGF- protein were already detected in cocultures after 24 and 48 hours, whereas the differentiation into myofibroblasts started to be seen only after 4 days and later. This raised the question whether there were inhibitors in the keratinocyte–fibroblast cocultures, which may delay the TGF- effects. And indeed, NFB was strongly induced in cocultured fibroblasts within the first 2 hours, persisting for the first 2 days, and slowly waning off until day 7. NFB activation was shown to interfere with TGF- signaling (Bitzer et al., 2000; Nagarajan et al., 2000; Verrecchia et al., 2000). Further experiments supported the assumption of a role of NFB as an inhibitor of TGF--mediated effects in fibroblasts, as exogenously added IL-1 was able to completely suppress -SMA induction in cocultures. Further, inhibition of keratinocyte-derived IL-1 increased -SMA expression (Shephard et al., 2004b). When keloid keratinocytes and fibroblasts were used in cocultures, increased expression of TGF- was observed, which is responsible for the enhanced proliferation rate of the mesenchymal cells (Xia et al., 2004). In these cocultures keloid keratinocytes expressed more TGF-1 and -3 than normal keratinocytes. Keloid fibroblasts had increased the levels for TGF-1- and 2 mRNA and produced more collagen I, connective tissue growth factor, and IGF-II/mannose-6-phosphate receptor. Interestingly, keloid fibroblasts also showed a constitutive increase in NFB-binding activity with and without TNF- treatment when compared with normal skin fibroblasts (Messadi et al., 2004). Whether targeting of the NFB pathway represents a novel therapeutic intervention for the treatment of keloid scarring remains to be elucidated. At present, the in vitro data suggest that keratinocytes and fibroblasts in cocultures produce significant amounts of active TGF-, but proinflammatory cytokines determine the degree of cellular responsiveness in fibroblasts.

Table 1 - Differential mRNA expression pattern of fibroblasts cocultured with HaCaT keratinocytes for 4 days.

Full table

Keratinocyte–fibroblast interactions increase contractile activity in mesenchymal cells

Apart from TGF- signals, myofibroblast differentiation is also regulated by mechanical tension (reviewed by Tomasek et al., 2002). In cocultures, fibroblasts readily acquired contractile properties already after 1 and 2 days when compared with control cultures (Shephard et al., 2004a). In these experiments, the cells were cultured on deformable silicone substrates. When cells start to exert contractile forces, the underlying substrate forms wrinkles and this can be observed in phase contrast microscopy. Contractile activity persisted in cocultures for the observational period of 7 days. Wrinkles were predominantly formed where keratinocytes and fibroblasts were in close proximity, reminiscent of -SMA induction, which also required close proximity of both cell types. At early time points, the underlying mechanism was unclear, as fibroblasts had not yet acquired -SMA expression. The initial complementary DNA array data suggested that endothelin-1 might have induced contraction of fibroblasts in the cocultures. Endothelin-1 peptide was upregulated in cocultures from early time points on, and inhibition with PD156252, a specific inhibitor at the receptor level, was able to prevent contraction in cocultured fibroblasts. Interestingly, inhibition occurred only at early time points. At later time points when -SMA was induced, contraction was unaffected by endothelin inhibition. PD156252 inhibited induction of -SMA expression in cocultured fibroblasts to some extent though not completely. Only the combination of endothelin and TGF- inhibition suppressed -SMA induction. Thus there appears to be a cooperative effect of mechanical tension and TGF- sensitivity in keratinocyte–fibroblast cocultures.

Apart from whole cells, a number of growth factors were shown to modulate contractile activity of fibroblasts in collagen gels. The PDGF-AA and -BB isoforms and TGF-1 led to efficient collagen–gel contraction, although TGF- treatment resulted in a delayed onset and a slower rate of contraction. IL-1 on the other hand inhibited collagen–gel contraction and caused degradation of the collagen gels at later time points, most likely due to enhanced matrix metalloproteinase activity (Tingstrom et al., 1992). Furthermore, mechanical tension was shown to inhibit the normally observed apoptosis in collagen gel-embedded fibroblasts (Fluck et al., 1998; Grinnell et al., 1999; Niland et al., 2001), and the gene expression pattern was differentially regulated by mechanical tension in normal fibroblasts (Kessler et al., 2001).

IL-1, TNF-, and TGF- expression during wound healing

The findings described above raise the question how the key players for the interaction between keratinocytes and fibroblasts are regulated in wounded skin. IL-1 and TNF- expression were analyzed in excisional wound-healing experiments in mice. Within 8 hours there was a strong induction of IL-1 and slightly later TNF- mRNA, which persisted until the wounds were in the mid-phase of healing (Hubner et al., 1996a). Granulocytes are the first cells to appear at the wound site and produce high levels of these proinflammatory cytokines (Feiken et al., 1995). Later, macrophages in the granulation tissue and keratinocytes become the predominant source (Hubner et al., 1996a). Blocking TNF signaling resulted in accelerated healing of excisional wounds. In TNF-receptor p55 knockout mice, enhancement of angiogenesis, collagen production, and epithelial wound closure was observed. Moreover, the expression of TGF-1 and connective tissue growth factor were enhanced and higher levels of vascular endothelial growth factor, vascular endothelial growth factor receptor-1, and vascular endothelial growth factor-2 were also observed. Overall, the inflammatory activity of these wounds was markedly reduced compared with controls (Mori et al., 2002). These findings suggest that inflammatory cytokine signals can balance TGF- expression and TGF--mediated effects on healing wounds.

TGF- expression was analyzed in full-thickness excisional mouse wounds at different stages after injury, and all three isoforms were detected. Nevertheless, each isoform had a distinct and characteristic expression pattern and time course in wound healing. TGF-1 and -2 expressions were rapidly induced in suprabasal wound keratinocytes and in the stroma, followed later by the expression of TGF-3 throughout the hyperproliferative epithelium, and also in the granulation tissue (Kane et al., 1991; Levine et al., 1993; Schmid et al., 1993; Frank et al., 1996). There is also a rapid release of TGF- at the site of injury from degranulating thrombocytes (Assoian et al., 1983), suggesting that at early time points, TGF- is supplied by thrombocytes, whereas later, keratinocytes, macrophages, and fibroblasts become the predominant source of TGF- in wounds. In situ measurement of TGF- bioactivity identified a second peak of active TGF- (Yang et al., 1999), which coincides with the appearance of myofibroblasts.

The phenotype of TGF- inhibition in wound healing is complex. Neutralization of TGF-1 and -2 with neutralizing antibodies and antisense-oligonucleotides resulted in reduced scarring (Shah et al., 1994; Choi et al., 1996). The analysis of wound healing in TGF-1-deficient mice was very difficult, as these mice show a severe multifocal inflammatory phenotype after 3 weeks of age, leading to multiple organ failure and death at <30 days of life (Kulkarni et al., 1993). When mice were wounded at day 10, initial wound healing was normal before high numbers of inflammatory cells accumulated, and vascular density and collagen deposition were reduced, and epithelial closure was delayed (Brown et al., 1995). The conclusion that other TGF-s were compensating at early time points is difficult to draw. Maternal transmission of TGF- through the milk – before complete weaning (Letterio et al., 1994) – may have ameliorated inflammation at early time points. Later, inflammation and the subsequent wasting may have prevailed and perturbed wound healing. Blocking TGF- signaling in keratinocytes or fibroblasts was achieved by tissue-specific expression of a dominant-negative receptor approach or by using a conditional knockout strategy, respectively. Wound closure occurred faster through increased keratinocyte proliferation and decreased apoptosis in transgenic animals, which overexpressed a dominant-negative type II TGF- receptor mutant in basal keratinocytes (Amendt et al., 2002). In these animals, signaling of all TGF- isoforms is abolished in basal keratinocytes. When the TGF- type II receptor was inactivated in fibroblasts, the granulation tissue in excisional wounds had less tensile strength at day 7. The epidermal wound closure rate was unaffected, but, interestingly, the number of suprabasal keratinocytes was increased, suggesting indirect paracrine effects. In these mice, the cell type-specific inactivation was achieved by crossing mice with two floxed TGF- type II receptor alleles with mice expressing the Cre recombinase under the control of the fibroblast specific protein-1 promoter, which becomes active in cells of the fibroblastic lineage after day 9 of embryonic development (Bhowmick et al., 2002). These studies demonstrated that TGF- activity is peaking early after wounding and later in the wound-healing process. Major TGF- effects are seen in the granulation tissue with increased extracellular matrix production; however, it appears that they occur at the expense of epidermal wound closure rates. Thus the balance of proinflammatory cytokines and TGF- activity appears to control major aspects of keratinocyte–fibroblast interactions in vivo.

Diversity of fibroblasts

Fibroblasts are poorly characterized, and there is a large variability of fibroblast phenotypes. Fibroblasts can be derived from many organs; several mesenchymal cells such as adipocytes can de-differentiate into fibroblastic cells, and dermal fibroblasts can acquire a new differentiation state, for example, they can differentiate into myofibroblasts. In keloids, benign tumors characterized by excessive production of extracellular matrix, and in hypertrophic scars, lesional fibroblasts and keratinocytes differ from those of nonlesional skin (Ehrlich et al., 1994; Machesney et al., 1998). mRNA for activin is increased in keloids, and activin A and follistatin were found in basal keratinocytes (Mukhopadhyay et al., 2006). On the mesenchymal side, keloid and hypertrohic scar fibroblasts show a strong expression of -SMA (Ehrlich et al., 1994). The phenotype of keloid and control fibroblasts is also evident in tissue culture. High baseline expression of activin A, follistatin, -SMA, collagen I, thrombospondin-1, and the ectodermal dysplasia-A fibronectin variant distinguishes keloid fibroblasts from normal fibroblasts in culture (Chipev et al., 2000; Chipev and Simon, 2002; Mukhopadhyay et al., 2006). Moreover, hypertrophic scar fibroblasts showed increased fibrin gel (Younai et al., 1996) and collagen–gel contraction (Phan et al., 2003). There is also evidence that neutralizing anti-TGF- antibodies can partially revert the keloid fibroblast phenotype (Younai et al., 1994). To complicate the situation, several groups have shown that lesional keratinocytes also differ from normal keratinocytes. Keloid keratinocytes are more effective in inducing fibroblast proliferation and matrix production in both keloid and control fibroblasts (Xia et al., 2004). Moreover, keratinocytes from keloids are characterized by a higher expression of TGF-1, -2, TGF- receptor 1, and Smad2 in cocultures, and they express the hyperproliferative keratins K6, K16, and K17 with premature expression of filaggrin (Machesney et al., 1998). At present, it is unclear how keratinocyte–fibroblast interactions in keloids or hypertrophic scars differ from normal cells or whether keratinocytes or fibroblasts are the primary cause for the keloid or hypertrophic scar phenotype. Still, it appears that the phenotype or differentiation state is stable for a long time in patients and in tissue culture.

Differences in fibroblast differentiation may be more subtle. Comparing dermal fibroblasts from different layers of human skin showed differences in collagen type I and III mRNA expression (Ali-Bahar et al., 2004). Fibroblasts derived from deeper layers showed reduced collagen expression compared with fibroblasts derived from more superficial layers. Fibroblast diversity was further demonstrated by expression data for fibroblasts derived from different body sites (Chang et al., 2002; Rinn et al., 2006). The analysis showed that fibroblasts retain positional information and their topographic differentiation in tissue culture. The HOX gene expression pattern in fibroblasts appears to be a signature from which body site the cells were derived (Chang et al., 2002). It is unclear at present how these differences may influence keratinocyte–fibroblast interactions. Recombination experiments have shown that keratinocytes maintain their gross anatomical site-specific differentiation pattern (Limat et al., 1996; Okazaki et al., 2003). On closer examination, there is evidence that keratinocyte differentiation can be modulated by fibroblast-derived information cues (Okazaki et al., 2003). When comparing the effects of different fibroblast lineages, the effects on the epithelial phenotype become more pronounced (Reynolds and Jahoda, 1992; Aoki et al., 2004). For rodent dermal papilla cells isolated from whiskers, the dermal papilla cell differentiation is sufficiently stable to survive several passages in tissue culture, and the cells are still able to induce hair follicle development with the necessary reprogramming of interfollicular keratinocytes into hair follicles (Reynolds and Jahoda, 1992). In the other example, Aoki et al. (2004) compared the effect of bone marrow stromal cells, subcutaneous preadipocytes, and dermal fibroblasts on the epidermal architecture of keratinocytes in three-dimensional cultures. All mesenchymal cell types supported epidermal proliferation and differentiation. Bone marrow stromal cells and preadipocytes induced in growth of keratinocytes into the fibroblast-populated dermal equivalents, a process the authors describe as "rete ridge-like" and "epidermal ridge-like" structure formation.

The last years have seen significant advances in the definition of the cells currently summarized as fibroblasts. It will be interesting with this knowledge in mind to review observations on anatomical site variances on wound healing rates and skin transplantation outcomes in patients. Fibroblast differentiation and resulting differences in keratinocyte–fibroblast interactions may have a central role in the clinics of wound-healing and could represent a novel target for therapeutic interventions (Table 1).

Conflict of Interest

The authors state no conflict of interest.

Notes

Editor's Note

The following 3 articles by Werner et al., Stramer et al., and Clark et al. continue the Wound Healing Perspectives Series which began in the March 2007 issue of the journal.

References

1. Ali-Bahar M, Bauer B, Tredget EE, Ghahary A (2004) Dermal fibroblasts from different layers of human skin are heterogeneous in expression of collagenase and types I and III procollagen mRNA. Wound Repair Regen 12:175–182 | Article | PubMed |
2. Amendt C, Mann A, Schirmacher P, Blessing M (2002) Resistance of keratinocytes to TGFbeta-mediated growth restriction and apoptosis induction accelerates re-epithelialization in skin wounds. J Cell Sci 115:2189–2198 | PubMed | ISI | ChemPort |
3. Ansel JC, Tiesman JP, Olerud JE, Krueger JG, Krane JF, Tara DC et al. (1993) Human keratinocytes are a major source of cutaneous platelet-derived growth factor. J Clin Invest 92:671–678 | PubMed | ISI | ChemPort |
4. Antoniades HN, Galanopoulos T, Neville-Golden J, Kiritsy CP, Lynch SE (1991) Injury induces in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor mRNAs in skin epithelial cells and PDGF mRNA in connective tissue fibroblasts. Proc Natl Acad Sci USA 88:565–569 | Article | PubMed | ChemPort |
5. Antoniades HN, Galanopoulos T, Neville-Golden J, Kiritsy CP, Lynch SE (1993) Expression of growth factor and receptor mRNAs in skin epithelial cells following acute cutaneous injury. Am J Pathol 142:1099–1110 | PubMed | ISI | ChemPort |
6. Aoki S, Toda S, Ando T, Sugihara H (2004) Bone marrow stromal cells, preadipocytes, and dermal fibroblasts promote epidermal regeneration in their distinctive fashions. Mol Biol Cell 15:4647–4657 | Article | PubMed | ChemPort |
7. Assoian R, Komoriya A, Meyers C, Miller D, Sporn M (1983) Transforming growth factor-beta in human platelets. Identification of a major storage site, purification, and characterization. J Biol Chem 258:7155–7160 | PubMed | ISI | ChemPort |
8. Bamberger C, Scharer A, Antsiferova M, Tychsen B, Pankow S, Muller M et al. (2005) Activin controls skin morphogenesis and wound repair predominantly via stromal cells and in a concentration-dependent manner via keratinocytes. Am J Pathol 167:733–747 | PubMed | ISI | ChemPort |
9. Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84:2302–2306 | Article | PubMed | ChemPort |
10. Beer HD, Bittner M, Niklaus G, Munding C, Max N, Goppelt A et al. (2005) The fibroblast growth factor binding protein is a novel interaction partner of FGF-7, FGF-10 and FGF-22 and regulates FGF activity: implications for epithelial repair. Oncogene 24:5269–5277 | Article | PubMed | ChemPort |
11. Beer HD, Florence C, Dammeier J, McGuire L, Werner S, Duan DR et al. (1997a) Mouse fibroblast growth factor 10: cDNA cloning, protein characterization, and regulation of mRNA expression. Oncogene 15:2211–2218 | Article | PubMed | ISI | ChemPort |
12. Beer HD, Longaker MT, Werner S (1997b) Reduced expression of PDGF and PDGF receptors during impaired wound healing. J Invest Dermatol 109:132–138 | Article | PubMed | ISI | ChemPort |
13. Beyer TA, Werner S, Dickson C, Grose R (2003) Fibroblast growth factor 22 and its potential role during skin development and repair. Exp Cell Res 287:228–236 | Article | PubMed | ISI | ChemPort |
14. Bhowmick NA, Chytil A, Carlisle C, Neilson EG, Davidson JM, Moses HL et al. (2002) The conditional knock-out of the transforming growth factor type II receptor in fibroblasts results in impaired wound healing. Wound Repair Regen 10:A4 (abstract)
15. Bhowmick NA, Neilson EG, Moses HL (2004) Stromal fibroblasts in cancer initiation and progression. Nature 432:332–337 | Article | PubMed | ISI | ChemPort |
16. Bitzer M, von Gersdorff G, Liang D, Dominguez-Rosales A, Beg AA, Rojkind M et al. (2000) A mechanism of suppression of TGF-beta/SMAD signaling by NF-kappa B/RelA. Genes Dev 14:187–197 | PubMed | ISI | ChemPort |
17. Bonner JC, Osornio-Vargas AR, Badgett A, Brody AR (1991) Differential proliferation of rat lung fibroblasts induced by the platelet-derived growth factor-AA, -AB, and -BB isoforms secreted by rat alveolar macrophages. Am J Respir Cell Mol Biol 5:539–547 | PubMed | ISI | ChemPort |
18. Boukamp P, Popp S, Altmeyer S, Hulsen A, Fasching C, Cremer T et al. (1997) Sustained nontumorigenic phenotype correlates with a largely stable chromosome content during long-term culture of the human keratinocyte line HaCaT. Genes Chromosomes Cancer 19:201–214 | Article | PubMed | ISI | ChemPort |
19. Boxman IL, Ruwhof C, Boerman OC, Lowik CW, Ponec M (1996) Role of fibroblasts in the regulation of pro-inflammatory interleukin IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1. Arch Dermatol Res 288:391–398 | Article | PubMed | ISI | ChemPort |
20. Brauchle M, Angermeyer K, Hubner G, Werner S (1994) Large induction of keratinocyte growth factor expression by serum growth factors and pro-inflammatory cytokines in cultured fibroblasts. Oncogene 9:3199–3204 | PubMed | ISI | ChemPort |
21. Brown RL, Ormsby I, Doetschman TC, Greenhalgh DG (1995) Wound healing in the transforming growth factor-beta 1-deficient mouse. Wound Repair Regen 3:25–36 | Article | PubMed | ChemPort |
22. Chang HY, Chi JT, Dudoit S, Bondre C, van de Rinn M, Botstein D et al. (2002) Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci USA 99:12877–12882 | Article | PubMed | ChemPort |
23. Chedid M, Rubin JS, Csaky KG, Aaronson SA (1994) Regulation of keratinocyte growth factor gene expression by interleukin 1. J Biol Chem 269:10753–10757 | PubMed | ISI | ChemPort |
24. Chipev CC, Simman R, Hatch G, Katz AE, Siegel DM, Simon M et al. (2000) Myofibroblast phenotype and apoptosis in keloid and palmar fibroblasts in vitro. Cell Death Differ 7:166–176 | Article | PubMed | ChemPort |
25. Chipev CC, Simon M (2002) Phenotypic differences between dermal fibroblasts from different body sites determine their responses to tension and TGFbeta1. BMC Dermatol 2:13 | Article | PubMed |
26. Choi BM, Kwak HJ, Jun CD, Park SD, Kim KY, Kim HR et al. (1996) Control of scarring in adult wounds using antisense transforming growth factor-beta 1 oligodeoxynucleotides. Immunol Cell Biol 74:144–150 | PubMed | ChemPort |
27. Cotsarelis G, Sun TT, Lavker RM (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61:1329–1337 | Article | PubMed | ISI | ChemPort |
28. Cribbs RK, Harding PA, Luquette MH, Besner GE (2002) Endogenous production of heparin-binding EGF-like growth factor during murine partial-thickness burn wound healing. J Burn Care Rehabil 23:116–125 | Article | PubMed |
29. Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G (1993) Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 122:103–111 | Article | PubMed | ISI | ChemPort |
30. Eckes B, Zweers MC, Zhang ZG, Hallinger R, Mauch C, Aumailley M et al. (2006) Mechanical tension and integrin alpha 2 beta 1 regulate fibroblast functions. J Investig Dermatol Symp Proc 11:66–72 | Article | PubMed | ChemPort |
31. Ehrlich HP, Allison GM, Leggett M (2006) The myofibroblast, cadherin, alpha smooth muscle actin and the collagen effect. Cell Biochem Funct 24:63–70 | Article | PubMed | ChemPort |
32. Ehrlich HP, Desmouliere A, Diegelmann RF, Cohen IK, Compton CC, Garner WL et al. (1994) Morphological and immunochemical differences between keloid and hypertrophic scar. Am J Pathol 145:105–113 | PubMed | ISI | ChemPort |
33. Ehrlich HP, Diez T (2003) Role for gap junctional intercellular communications in wound repair. Wound Repair Regen 11:481–489 | Article | PubMed |
34. El Ghalbzouri A, Hensbergen P, Gibbs S, Kempenaar J, van de Rinn M, Ponec M et al. (2004) Fibroblasts facilitate re-epithelialization in wounded human skin equivalents. Lab Invest 84:102–112 | Article | PubMed | ChemPort |
35. Elder JT, Fisher GJ, Lindquist PB, Bennett GL, Pittelkow MR et al. (1989) Overexpression of transforming growth factor alpha in psoriatic epidermis. Science 243:811–814 | Article | PubMed | ISI | ChemPort |
36. Feiken E, Romer J, Eriksen J, Lund LR (1995) Neutrophils express tumor necrosis factor-alpha during mouse skin wound healing. J Invest Dermatol 105:120–123 | Article | PubMed | ISI | ChemPort |
37. Finch PW, Rubin JS, Miki T, Ron D, Aaronson SA (1989) Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 245:752–755 | Article | PubMed | ISI | ChemPort |
38. Fleischmajer R, Kuhn K, Sato Y, MacDonald ED, Perlish JS, Pan TC et al. (1997) There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model. J Invest Dermatol 109:527–533 | Article | PubMed | ISI | ChemPort |
39. Florin L, Maas-Szabowski N, Werner S, Szabowski A, Angel P (2005) Increased keratinocyte proliferation by JUN-dependent expression of PTN and SDF-1 in fibroblasts. J Cell Sci 118:1981–1989 | Article | PubMed | ISI | ChemPort |
40. Fluck J, Querfeld C, Cremer A, Niland S, Krieg T, Sollberg S (1998) Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels. J Invest Dermatol 110:153–157 | Article | PubMed | ISI | ChemPort |
41. Frank S, Madlener M, Werner S (1996) Transforming growth factors 1, 2, and 3 and their receptors are differentially regulated during normal and impaired wound healing. J Biol Chem 271:10188–10193 | Article | PubMed | ISI | ChemPort |
42. Gallucci RM, Simeonova PP, Matheson JM, Kommineni C, Guriel JL, Sugawara T et al. (2000) Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice. FASEB J 14:2525–2531 | Article | PubMed | ISI | ChemPort |
43. Gottlieb AB, Chang CK, Posnett DN, Fanelli B, Tam JP (1988) Detection of transforming growth factor alpha in normal, malignant, and hyperproliferative human keratinocytes. J Exp Med 167:670–675 | Article | PubMed | ISI | ChemPort |
44. Grinnell F, Zhu M, Carlson MA, Abrams JM (1999) Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue. Exp Cell Res 248:608–619 | Article | PubMed | ISI | ChemPort |
45. Gron B, Stoltze K, Andersson A, Dabelsteen E (2002) Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes. APMIS 110:892–898 | Article | PubMed | ChemPort |
46. Grose R, Werner S (2004) Wound-healing studies in transgenic and knockout mice. Mol Biotechnol 28:147–166 | Article | PubMed | ISI | ChemPort |
47. Grossman RM, Krueger J, Yourish D, Granelli-Piperno A, Murphy DP, May LT et al. (1989) Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes. Proc Natl Acad Sci USA 86:6367–6371 | Article | PubMed | ChemPort |
48. Grotendorst GR, Soma Y, Takehara K, Charette M (1989) EGF and TGF-alpha are potent chemoattractants for endothelial cells and EGF-like peptides are present at sites of tissue regeneration. J Cell Physiol 139:617–623 | Article | PubMed | ChemPort |
49. Guo L, Degenstein L, Fuchs E (1996) Keratinocyte growth factor is required for hair development but not for wound healing. Genes Dev 10:165–175 | PubMed | ISI | ChemPort |
50. Hinz B, Mastrangelo D, Iselin CE, Chaponnier C, Gabbiani G (2001) Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am J Pathol 159:1009–1020 | PubMed | ISI | ChemPort |
51. Hubner G, Brauchle M, Rauchle M, Smola H, Madlener M, Fassler R et al. (1996a) Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice. Cytokine 8:548–556 | Article | PubMed | ISI | ChemPort |
52. Hubner G, Hu Q, Smola H, Werner S (1996b) Strong induction of activin expression after injury suggests an important role of activin in wound repair. Dev Biol 173:490–498 | Article | PubMed | ISI | ChemPort |
53. Jensen KB, Watt FM (2006) Single-cell expression profiling of human epidermal stem and transit-amplifying cells: Lrig1 is a regulator of stem cell quiescence. Proc Natl Acad Sci USA 103:11958–11963 | Article | PubMed | ChemPort |
54. Kane CJ, Hebda PA, Mansbridge JN, Hanawalt PC (1991) Direct evidence for spatial and temporal regulation of transforming growth factor beta 1 expression during cutaneous wound healing. J Cell Physiol 148:157–173 | Article | PubMed | ISI | ChemPort |
55. Katz MH, Alvarez AF, Kirsner RS, Eaglstein WH, Falanga V (1991) Human wound fluid from acute wounds stimulates fibroblast and endothelial cell growth. J Am Acad Dermatol 25:1054–1058 | PubMed | ISI | ChemPort |
56. Kessler D, Dethlefsen S, Haase I, Plomann M, Hirche F, Krieg T et al. (2001) Fibroblasts in mechanically stressed collagen lattices assume a "synthetic" phenotype. J Biol Chem 276:36575–36585 | Article | PubMed | ISI | ChemPort |
57. Kim I, Mogford JE, Chao JD, Mustoe TA (2001) Wound epithelialization deficits in the transforming growth factor-alpha knockout mouse. Wound Repair Regen 9:386–390 | Article | PubMed | ChemPort |
58. Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC et al. (1993) Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci USA 90:770–774 | Article | PubMed | ChemPort |
59. Letterio JJ, Geiser AG, Kulkarni AB, Roche NS, Sporn MB, Roberts AB et al. (1994) Maternal rescue of transforming growth factor-beta1 null mice. Science 264:1936–1938 | Article | PubMed | ISI | ChemPort |
60. Levine JH, Moses HL, Gold LI, Nanney LB (1993) Spatial and temporal patterns of immunoreactive transforming growth factor 1, 2, and 3 during excisional wound repair. Am J Pathol 143:368–380 | PubMed | ISI | ChemPort |
61. Limat A, Hunziker T, Boillat C, Noser F, Wiesmann U (1990) Postmitotic human dermal fibroblasts preserve intact feeder properties for epithelial cell growth after long-term cryopreservation. In vitro Cell Dev Biol 26:709–712 | PubMed | ChemPort |
62. Limat A, Stockhammer E, Breitkreutz D, Schaffner T, Egelrud T, Salomon D et al. (1996) Endogeneously regulated site-specific differentiation of human palmar skin keratinocytes in organotypic cocultures and in nude mouse transplants. Eur J Cell Biol 69:245–258 | PubMed | ISI | ChemPort |
63. Lin ZQ, Kondo T, Ishida Y, Takayasu T, Mukaida N (2003) Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice. J Leukoc Biol 73:713–721 | Article | PubMed | ISI | ChemPort |
64. Luetteke NC, Qiu TH, Peiffer RL, Oliver P, Smithies O, Lee DC et al. (1993) TGF alpha deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice. Cell 73:263–278 | Article | PubMed | ISI | ChemPort |
65. Maas-Szabowski N, Fusenig NE (1996) Interleukin-1-induced growth factor expression in postmitotic and resting fibroblasts. J Invest Dermatol 107:849–855 | Article | PubMed | ChemPort |
66. Maas-Szabowski N, Shimotoyodome A, Fusenig NE (1999) Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism. J Cell Sci 112:1843–1853 | PubMed | ISI |
67. Maas-Szabowski N, Starker A, Fusenig NE (2003) Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-alpha. J Cell Sci 116:2937–2948 | Article | PubMed | ISI | ChemPort |
68. Machesney M, Tidman N, Waseem A, Kirby L, Leigh I (1998) Activated keratinocytes in the epidermis of hypertrophic scars. Am J Pathol 152:1133–1141 | PubMed | ISI | ChemPort |
69. Mann GB, Fowler KJ, Gabriel A, Nice EC, Williams RL, Dunn AR et al. (1993) Mice with a null mutation of the TGF alpha gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation. Cell 73:249–261 | Article | PubMed | ISI | ChemPort |
70. Marchese C, Chedid M, Dirsch OR, Csaky KG, Santanelli F, Latini C et al. (1995) Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin. J Exp Med 182:1369–1376 | Article | PubMed | ISI | ChemPort |
71. Marikovsky M, Breuing K, Liu PY, Eriksson E, Higashiyama S, Farber P et al. (1993) Appearance of heparin-binding EGF-like growth factor in wound fluid as a response to injury. Proc Natl Acad Sci USA 90:3889–3893 | Article | PubMed | ChemPort |
72. Marinkovich MP, Keene DR, Rimberg CS, Burgeson RE (1993) Cellular origin of the dermal–epidermal basement membrane. Dev Dyn 197:255–267 | PubMed | ISI | ChemPort |
73. Martin P, Parkhurst SM (2004) Parallels between tissue repair and embryo morphogenesis. Development 131:3021–3034 | Article | PubMed | ISI | ChemPort |
74. Messadi DV, Doung HS, Zhang Q, Kelly AP, Tuan TL, Reichenberger E, Le AD (2004) Activation of NFkappaB signal pathways in keloid fibroblasts. Arch Dermatol Res 296:125–133 | PubMed | ChemPort |
75. Mori R, Kondo T, Ohshima T, Ishida Y, Mukaida N (2002) Accelerated wound healing in tumor necrosis-factor receptor p55-deficient mice with reduced leukocyte infiltration. FASEB J 16:963–974 | Article | PubMed | ChemPort |
76. Mueller MM, Fusenig NE (2004) Friends or foes – bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 4:839–849 | Article | PubMed | ISI | ChemPort |
77. Mukhopadhyay A, Chan SY, Lim IJ, Phillips D, Phan TT (2006) The role of the activin system in keloid pathogenesis. Am J Physiol Cell Physiol, doi:10.1152ajpcell.00373.2006, in press
78. Munz B, Smola H, Engelhardt F, Bleuel K, Brauchle M, Lein I et al. (1999) Overexpression of activin A in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair. EMBO J 18:5205–5215 | Article | PubMed | ISI | ChemPort |
79. Nagarajan RP, Chen F, Li W, Vig E, Harrington MA, Nakshatri H et al. (2000) Repression of transforming-growth-factor-beta-mediated transcription by nuclear factor kappaB. Biochem J 348:591–596 | Article | PubMed | ChemPort |
80. Nakatake Y, Hoshikawa M, Asaki T, Kassai Y, Itoh N (2001) Identification of a novel fibroblast growth factor, FGF-22, preferentially expressed in the inner root sheath of the hair follicle. Biochim Biophys Acta 1517:460–463 | PubMed | ChemPort |
81. Nelson CM, Bissell MJ (2006) Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol 22:287–309 | Article | PubMed | ChemPort |
82. Niland S, Cremer A, Fluck J, Eble JA, Krieg T, Sollberg S (2001) Contraction-dependent apoptosis of normal dermal fibroblasts. J Invest Dermatol 116:686–692 | Article | PubMed | ChemPort |
83. Okazaki M, Yoshimura K, Suzuki Y, Harii K (2003) Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model. Plast Reconstr Surg 112:784–792 | Article | PubMed |
84. Ono I, Gunji H, Zhang JZ, Maruyama K, Kaneko F (1995) Studies on cytokines related to wound healing in donor site wound fluid. J Dermatol Sci 10:241–245 | Article | PubMed | ChemPort |
85. Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y (2001) Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104:233–245 | Article | PubMed | ISI | ChemPort |
86. Phan TT, Sun L, Bay BH, Chan SY, Lee ST (2003) Dietary compounds inhibit proliferation and contraction of keloid and hypertrophic scar-derived fibroblasts in vitro: therapeutic implication for excessive scarring. J Trauma 54:1212–1224 | PubMed | ChemPort |
87. Regauer S, Seiler GR, Barrandon Y, Easley KW, Compton CC (1990) Epithelial origin of cutaneous anchoring fibrils. J Cell Biol 111:2109–2115 | Article | PubMed | ISI | ChemPort |
88. Reynolds AJ, Jahoda CA (1992) Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis. Development 115:587–593 | PubMed | ISI | ChemPort |
89. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343 | Article | PubMed | ISI | ChemPort |
90. Rinn JL, Bondre C, Gladstone HB, Brown PO, Chang HY (2006) Anatomic demarcation by positional variation in fibroblast gene expression programs. PLoS Genet 2:e119. (DOI: 10.1371/journal.pgen.0020119) | Article | PubMed | ChemPort |
91. Rosenthal A, Lindquist PB, Bringman TS, Goeddel DV, Derynck R (1986) Expression in rat fibroblasts of a human transforming growth factor-alpha cDNA results in transformation. Cell 46:301–309 | Article | PubMed | ChemPort |
92. Rubin JS, Osada H, Finch PW, Taylor WG, Rudikoff S, Aaronson SA et al. (1989) Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc Natl Acad Sci USA 86:802–806 | Article | PubMed | ChemPort |
93. Schenk S, Hintermann E, Bilban M, Koshikawa N, Hojilla C, Khokha R et al. (2003) Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution. J Cell Biol 161:197–209 | Article | PubMed | ISI | ChemPort |
94. Schmid P, Cox D, Bilbe G, McMaster G, Morrison C, Stahelin H et al. (1993) TGF-betas and TGF-beta type II receptor in human epidermis: differential expression in acute and chronic skin wounds. Pathol 171:191–197 | ChemPort |
95. Schmidt-Ullrich R, Paus R (2005) Molecular principles of hair follicle induction and morphogenesis. Bioessays 27:247–261 | Article | PubMed | ISI | ChemPort |
96. Schreiber AB, Winkler ME, Derynck R (1986) Transforming growth factor-alpha: a more potent angiogenic mediator than epidermal growth factor. Science 232:1250–1253 | Article | PubMed | ISI | ChemPort |
97. Seishima M, Nojiri M, Esaki C, Yoneda K, Eto Y, Kitajima Y et al. (1999) Activin A induces terminal differentiation of cultured human keratinocytes. J Invest Dermatol 112:432–436 | Article | PubMed | ISI | ChemPort |
98. Shah M, Foreman DM, Ferguson MWJ (1994) Neutralising antibody to TGF-beta 1,2 reduces cutaneous scarring in adult rodents. J Cell Sci 107:1137–1157 | PubMed | ISI | ChemPort |
99. Shephard P, Hinz B, Smola-Hess S, Meister JJ, Krieg T, Smola H (2004a) Dissecting the roles of endothelin, TGF-beta and GM-CSF on myofibroblast differentiation by keratinocytes. Thromb Haemost 92:262–274 | PubMed | ChemPort |
100. Shephard P, Martin G, Smola-Hess S, Brunner G, Krieg T, Smola H (2004b) Myofibroblast differentiation is induced in keratinocyte–fibroblast co-cultures and is antagonistically regulated by endogenous transforming growth factor-beta and interleukin-1. Am J Pathol 164:2055–2066 | ChemPort |
101. Shirakata Y, Kimura R, Nanba D, Iwamoto R, Tokumaru S, Morimoto C et al. (2005) Heparin-binding EGF-like growth factor accelerates keratinocyte migration and skin wound healing. J Cell Sci 118:2363–2370 | Article | PubMed | ISI | ChemPort |
102. Smola H, Stark HJ, Thiekotter G, Mirancea N, Krieg T, Fusenig NE (1998) Dynamics of basement membrane formation by keratinocyte–fibroblast interactions in organotypic skin culture. Exp Cell Res 239:399–410 | Article | PubMed | ISI | ChemPort |
103. Smola H, Thiekotter G, Fusenig NE (1993) Mutual induction of growth factor gene expression by epidermal–dermal cell interaction. J Cell Biol 122:417–429 | Article | PubMed | ISI | ChemPort |
104. Szabowski A, Maas-Szabowski N, Andrecht S, Kolbus A, Schorpp-Kistner M, Fusenig NE et al. (2000) c-Jun and JunB antagonistically control cytokine-regulated mesenchymal–epidermal interaction in skin. Cell 103:745–755 | Article | PubMed | ISI | ChemPort |
105. Tickle C (2006) Making digit patterns in the vertebrate limb. Nat Rev Mol Cell Biol 7:45–53 | Article | PubMed | ChemPort |
106. Tingstrom A, Heldin CH, Rubin K (1992) Regulation of fibroblast-mediated collagen gel contraction by platelet-derived growth factor, interleukin-1 alpha and transforming growth factor-beta 1. J Cell Sci 102:315–322 | PubMed |
107. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3:349–363 | Article | PubMed | ISI | ChemPort |
108. Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M et al. (2004) Defining the epithelial stem cell niche in skin. Science 303:359–363 | Article | PubMed | ISI | ChemPort |
109. Vejda S, Cranfield M, Peter B, Mellor SL, Groome N, Schulte-Hermann R et al. (2002) Expression and dimerization of the rat activin subunits betaC and betaE: evidence for the formation of novel activin dimers. J Mol Endocrinol 28:137–148 | Article | PubMed | ISI | ChemPort |
110. Verrecchia F, Pessah M, Atfi A, Mauviel A (2000) Tumor necrosis factor-alpha inhibits transforming growth factor-beta/Smad signaling in human dermal fibroblasts via AP-1 activation. J Biol Chem 275:30226–30231 | Article | PubMed | ISI | ChemPort |
111. Waelti ER, Inaebnit SP, Rast HP, Hunziker T, Limat A, Braathen LR et al. (1992) Co-culture of human keratinocytes on post-mitotic human dermal fibroblast feeder cells: production of large amounts of interleukin 6. J Invest Dermatol 98:805–808 | Article | PubMed | ISI | ChemPort |
112. Wankell M, Munz B, Hubner G, Hans W, Wolf E, Goppelt A et al. (2001) Impaired wound healing in transgenic mice overexpressing the activin antagonist follistatin in the epidermis. EMBO J 20:5361–5372 | Article | PubMed | ISI | ChemPort |
113. Werner S, Grose R (2003) Regulation of wound healing by growth factors and cytokines. Physiol Rev 83:835–870 | PubMed | ISI | ChemPort |
114. Werner S, Peters KG, Longaker MT, Fuller-Pace F, Banda MJ, Williams LT et al. (1992) Large induction of keratinocyte growth factor expression in the dermis during wound healing. Proc Natl Acad Sci USA 89:6896–6900 | Article | PubMed | ChemPort |
115. Werner S, Smola H, Liao X, Longaker MT, Krieg T, Hofschneider PH et al. (1994) The function of KGF in morphogenesis of epithelium and reepithelialization of wounds. Science 266:819–822 | Article | PubMed | ISI | ChemPort |
116. Xia W, Phan TT, Lim IJ, Longaker MT, Yang GP (2004) Complex epithelial–mesenchymal interactions modulate transforming growth factor-beta expression in keloid-derived cells. Wound Repair Regen 12:546–556 | Article | PubMed |
117. Yang L, Qiu CX, Ludlow A, Ferguson MW, Brunner G (1999) Active transforming growth factor-beta in wound repair: determination using a new assay. Am J Pathol 154:105–111 | PubMed | ISI | ChemPort |
118. Younai S, Nichter LS, Wellisz T, Reinisch J, Nimni ME, Tuan TL (1994) Modulation of collagen synthesis by transforming growth factor beta in keloid and hypertrophic scar fibroblasts. Ann Plast Surg 33:148–151 | PubMed | ISI | ChemPort |
119. Younai S, Venters G, Vu S, Nichter L, Nimni ME, Tuan TL (1996) Role of growth factors in scar contraction: an in vitro analysis. Ann Plast Surg 36:495–501 | PubMed | ChemPort |
120. Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM (2006) Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem 281:15694–15700 | Article | PubMed | ChemPort |