Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.     

Fabrication, quality assurance, and assessment of cultured skin substitutes for treatment of skin wounds

Advances in treatment of skin wounds depend on demonstration of reduced morbidity or mortality either during or after hospitalization. Tissue engineering of skin grafts from cultured cells and biopolymers permits greater amounts of grafts from less donor tissue than conventional procedures. Autologous keratinocytes and fibroblasts isolated from epidermis and dermis of skin may be combined with collagen-based substrates to generate cultured skin substitutes (CSS) with epidermal and dermal components. By regulation of culture conditions, CSS form epidermal barrier and basement membrane, and release angiogenic factors that stimulate vascularization. Prototypes of CSS may be tested for safety and efficacy by grafting to athymic mice which do not reject human tissues. Clinical application of CSS requires establishment of quality assurance assessments, such as, epidermal barrier by measurement of surface hydration, and anatomy by standard histology. Medical benefits of tissue engineered skin for treatment of burns are evaluated quantitatively by the ratio of healed skin to donor skin, and qualitatively by the Vancouver Scar Scale. These benefits may also be extended to other medical conditions including chronic wounds and reconstructive surgery.     

Development of a 3D human in vitro skin co‐culture model for detecting irritants in real‐time

Tissue engineered materials for clinical purposes have led to the development of in vitro models as alternatives to animal testing. The aim of this study was to understand the paracrine interactions arising between keratinocytes and fibroblasts for detecting and discriminating between an irritant‐induced inflammatory reaction and cytotoxicy. We used two irritants [sodium dodecyl sulphate (SDS) and potassium diformate (Formi®)] at sub‐toxic concentrations and studied interleukin‐1 alpha (IL‐1α) release from human keratinocytes and activation of NF‐κB in human fibroblasts. NF‐κB activation in fibroblast 2D cultures required soluble factors released by prior incubation of keratinocytes with either SDS or Formi®. Neither cell type responded directly to either agent, confirming a paracrine mechanism. Fibroblasts were then cultured in 3D microfiber scaffolds and transfected with an NF‐κB reporter construct linked to GFP. Findings for 3D cultures were similar to those in 2D in that soluble factors released by prior incubation of keratinocytes with SDS or Formi® was required for NF‐κB activation in fibroblasts. Similarly, direct incubation with either agent did not directly activate NF‐κB. A technical advantage of using transfected cells in 3D was an ability to detect NF‐κB activation in live fibroblasts. To confirm paracrine signaling a twofold increase in IL‐1α was measured in keratinocyte‐conditioned medium after incubation with SDS or Formi®, which correlated with fibroblast NF‐κB activity. In summary, this work has value for developing 3D tissue engineered co‐culture models for the in vitro testing of irritant chemicals at sub‐toxic concentrations, as an alternative to in vivo models. Biotechnol. Bioeng. 2010;106: 794–803. © 2010 Wiley Periodicals, Inc.     

Investigation of the influence of keloid-derived keratinocytes on fibroblast growth and proliferation in vitro

Keloids are disfiguring, proliferative scars that represent a pathological response to cutaneous injury. The overabundant extracellular matrix formation, largely from collagen deposition, is characteristic of these lesions and has led to investigations into the role of the fibroblast in its pathogenesis. Curiously, the role of the epidermis in extracellular matrix collagen deposition of normal skin has been established, but a similar hypothesis in keloids has not been investigated. The aim of this study was to investigate the influence of keloid epithelial keratinocytes on the growth and proliferation of normal fibroblasts in an in vitro serum-free co-culture system. A permeable membrane separated two chambers; the upper chamber contained a fully differentiated stratified epithelium derived from the skin of excised earlobe keloid specimens, whereas the lower chamber contained a monolayer of normal or keloid fibroblasts. Both cell types were nourished by serum-free medium from the lower chamber.Epithelial keratinocytes from five separate earlobe keloid specimens were investigated. Four sets of quadruplicates were performed for each specimen co-cultured with normal fibroblasts or keloid-derived fibroblasts. Controls consisted of (1) normal keratinocytes co-cultured with normal fibroblasts, and (2) fibroblasts grown in serum-free media in the absence of keratinocytes in the upper chamber. Fibroblasts were indirectly quantified by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay, with results confirmed by DNA content measurement, at days 1 and 5 after the co- culture initiation.Significantly, increased proliferation was seen in fibroblasts co-cultured with keloid keratinocytes, as compared with the normal keratinocyte controls at day 5 (analysis of variance, p < 0.001). These results strongly suggest that the overlying epidermal keratinocytes of the keloid may have an important, previously unappreciated role in keloid pathogenesis using paracrine or epithelial-mesenchymal signaling.     

Basal Cell Carcinoma in Gorlin’s Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings suggest that defects in dermo/epidermal interactions could contribute to BCC susceptibility in NBCCS patients.     

In vivo and in vitro evidence of dermal fibroblasts influence on human epidermal pigmentation

Using chimeric human epidermal reconstructs, we previously demonstrated that epidermal pigmentation is dependent upon the phototype of melanocytes. We report here several lines of experimental evidence for dermal modulation of human epidermal pigmentation. First, phototype II-III epidermal reconstructs grafted on the back of immunotolerant Swiss nu/nu mice developed a patchy pigmentation dependent on the presence of colonizing human or mouse fibroblasts. Similarly, human white Caucasoid split-thickness skin xenografted on the same mouse strain became black within 3 months and histochemistry revealed a phototype VI pattern of melanin distribution. In vitro, human fibroblasts colonizing human dead de-epidermized dermis (DDD) induced a decrease in epidermal pigmentation whereas mouse (Swiss nu/nu) fibroblasts increased epidermal pigmentation. Conditioned medium from mice (Swiss nu/nu) fibroblasts also increased pigmentation whereas conditioned medium from human fibroblasts had no significant effect. Lastly, epidermal reconstructs made with normal or vitiligo keratinocytes and/or normal or vitiligo melanocytes from the same donor grown on DDD originating from several donors of the same clinical phototype did not pigment similarly and no specific dermal influence was noted for vitiligo. Thus, fibroblast secretion and acellular dermal connective tissue itself significantly influence melanocyte proliferation and melanin distribution/degradation. Our study suggests that murine fibroblasts are more potent than human fibroblasts in secreting soluble factors which can act directly on pigmentation, such as SCF, or activate keratinocytes to produce basement membrane proteins or melanogenic factors.     

Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal–epidermal engineered skin substitutes

Autologous engineered skin substitutes (ESS) containing melanocytes (hM) may restore pigmentation and photoprotection after grafting to full‐thickness skin wounds. In this study, normal hM were isolated from discard skin, propagated with or without tyrosinase inhibitors, cryopreserved, recovered into culture, and added to ESS (ESS‐P) before transplantation. ESS‐P were incubated in either UCMC160/161 or UCDM1 medium, scored for hM densities, and grafted to mice. The results showed that sufficient hM can be propagated to expand donor tissue by 100‐fold; incubation of hM in tyrosinase inhibitors reduced pigment levels but did not change hM recovery after cryopreservation; hM densities in ESS‐P were greater after incubation in UCDM1 than UCMC160 medium; hM were localized to the dermal–epidermal junction of ESS‐P; and UCDM1 medium promoted earlier pigment distribution and density. These results indicate that hM can be incorporated into ESS‐P efficiently to restore cutaneous pigmentation and UV photoprotection after full‐thickness skin loss conditions.     

Bidirectional cross talk between patient‐derived melanoma and cancer‐associated fibroblasts promotes invasion and proliferation

Tumor–stroma interactions are critical for epithelial‐derived tumors, and among the stromal cell types, cancer‐associated fibroblasts (CAFs) exhibit multiple functions that fuel growth, dissemination, and drug resistance. However, these interactions remain insufficiently characterized in non‐epithelial tumors such as malignant melanoma. We generated monocultures of melanoma cells and matching CAFs from patients’ metastatic lesions, distinguished by oncogenic drivers and immunoblotting of characteristic markers. RNA sequencing of CAFs revealed a homogenous epigenetic program that strongly resembled the signatures from epithelial cancers, including enrichment for an epithelial‐to‐mesenchymal transition (EMT). Melanoma CAFs in monoculture displayed robust invasive behavior while patient‐derived melanoma monocultures showed very little invasiveness. Instead, melanoma cells showed increased invasion when co‐cultured with CAFs. In turn, CAFs showed increased proliferation when exposed to melanoma conditioned media (CM), mediated in part by melanoma‐secreted transforming growth factor‐alpha that acted on CAFs via the epidermal growth factor receptor. This study provides evidence that bidirectional interactions between melanoma and CAFs regulate progression of metastatic melanoma.     

Impact of intercellular crosstalk between epidermal keratinocytes and dermal fibroblasts on skin homeostasis

Dermal fibroblasts seem critical for epidermal maturation and differentiation and recent work demonstrated that diseased fibroblasts may drive pathophysiological processes. Nevertheless, still very little is known about the actual crosstalk between epidermal keratinocytes and dermal fibroblasts and the impact of dermal fibroblasts on epidermal maturation and differentiation. Aiming for a more fundamental understanding of the impact of the cellular crosstalk between keratinocytes and fibroblasts on the skin homeostasis, we generated full-thickness skin equivalents with and without fibroblasts and subsequently analysed them for the expression of skin differentiation markers, their barrier function, skin lipid content and epidermal cell signalling. Skin equivalents without fibroblasts consistently showed an impaired differentiation and dysregulated expression of skin barrier and tight junction proteins, increased skin permeability, and a decreased skin lipid/protein ratio. Most interestingly, impaired Ras/Raf/ERK/MEK signalling was evident in skin equivalents without fibroblasts.Our data clearly indicate that the epidermal-dermal crosstalk between keratinocytes and fibroblasts is critical for adequate skin differentiation and that fibroblasts orchestrate epidermal differentiation processes.     

Stem cell recovering effect of copper‐free GHK in skin

The peptide Gly‐His‐Lys (GHK) is a naturally occurring copper(II)‐chelating motifs in human serum and cerebrospinal fluid. In industry, GHK (with or without copper) is used to make hair and skin care products. Copper‐GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. We also reported that copper‐GHK promotes the survival of basal stem cells in the skin. However, the effects of copper‐free GHK (GHK) have not been investigated well. In this study, the effects of GHK were studied using cultured normal human keratinocytes and skin equivalent (SE) models. In monolayer cultured keratinocytes, GHK increased the proliferation of keratinocytes. When GHK was added during the culture of SE models, the basal cells became more cuboidal than control model. In addition, there was linear and intense staining of α6 and β1 integrin along the basement membrane. The number of p63 and proliferating cell nuclear antigen positive cells was also significantly increased in GHK‐treated SEs than in control SEs. Western blot and slide culture experiment showed that GHK increased the expression of integrin by keratinocytes. All these results showed that GHK increased the stemness and proliferative potential of epidermal basal cells, which is associated with increased expression of integrin. In conclusion, copper‐free GHK showed similar effects with copper‐GHK. Thus, it can be said that copper‐free GHK can be used in industry to obtain the effects of copper‐GHK in vivo. Further study is necessary to explore the relationship between copper‐free GHK and copper‐GHK. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of keratinocytes and fibroblasts modulates the expression of matrix metalloproteinases-2 and -9 and their inhibitors

Disruption of epidermal-mesenchymal communication due to a delay in epithelialization, increases the frequency of developing fibrotic conditions in skin. As matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) are two key enzymes involved in wound healing and tissue remodeling, here we examined the efficacy of keratinocyte-fibroblast interaction on modulation of these enzymes and their inhibitors. The conditioned media derived from keratinocytes and fibroblasts grown in upper and lower chambers of a co-culture system, respectively, were analyzed for MMP-2 and -9. Keratinocyte or fibroblast conditioned medium (FCM) was used as a control. Gelatinolytic activity analyzed by zymography showed that keratinocytes mainly express MMP-9 and to a lesser extent MMP-2; while fibroblasts express only MMP-2. In a co-culture system, the activities of both MMP-2 and MMP-9 markedly increased in conditioned media collected from bottom chambers. These findings were consistent with the level of MMP-2 and MMP-9 measured by Western blot. Using the same experimental setting, the levels of tissue inhibitors of MMPs (TIMPs) secreted by keratinocytes and fibroblasts grown in the same co-culture system were also evaluated. Western blot showed that fibroblasts secrete only TIMP-1 and TIMP-2 whose levels were increased by co-culturing fibroblasts with keratinocytes. In contrary the level of TIMP-3, which was mainly expressed by keratinocytes, increased by co-culturing these cells with fibroblasts. In conclusion, interaction of fibroblast-keratinocyte modulates the levels of MMP-2 and -9 and their inhibitors produced by these cells and this interaction may be critical for a better healing quality at a late stage of the wound healing process.     

Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents

In vitro generated skin models find growing interest as promising tools in basic research and clinical application in regenerative medicine. Here, we present further details of an improved long-term skin equivalent (SE) enabling mechanistic studies on skin reconstruction and epidermal function. Growth conditions of fibroblasts in a 3D scaffold were analysed to optimise the dermal microenvironment by providing an authentic dermal matrix for regular tissue reconstruction and function of cocultured keratinocytes. These SEs demonstrate sustained epidermal viability - over 12 weeks - with regular differentiation as substantiated by in vivo-like patterns of all differentiation products, exemplified here by the cornified envelope components loricrin and repetin. The continuous expression of all major tight junction components in the granular layer, shown here for ZO-1 in coherence with the presence of epidermal barrier lipids, and ultrastructural accumulation of lamellar bodies, collectively indicate proper epidermal barrier structures. Remarkably, cocultured keratinocytes exerted an ongoing proliferation-stimulating effect on fibroblasts colonising the scaffold comparable to a cocktail of fibroblast growth factors. Consequently, precultivation of dermal equivalents (DEs) in basal or growth factor-enriched media had only minor effects on the quality of epidermal regeneration in cocultures. As to the role of fibroblast numbers, complete absence of dermal cells resulted in atrophic epithelia but the effect of cell numbers as low as 5 x 10(4)cells/cm(2) on epidermal tissue quality equalled that of the standard density (2 x 10(5)cells/cm(2)). Surprisingly, precultivation of fibroblasts in the DEs for 7 days (standard) showed no better effect on epidermal tissue reformation as compared to 2 days whereas a precultivation period of 14 days resulted in atrophic epidermal and dermal tissue development. These data demonstrate, (i) the strict dependence of epidermal tissue regeneration on the presence of fibroblasts, (ii) the mutual keratinocyte-fibroblast interactions for cell proliferation and organogenesis, and (iii) the importance of the proper microenvironment for epidermal tissue function and supposedly for establishment of a stem cell niche in vitro.     

Keratinocytes Control the Proliferation and Differentiation of Cultured Epidermal Melanocytes from Ultraviolet Radiation B‐Induced Pigmented Spots in the Dorsal Skin of Hairless Mice

Long‐term exposure of ultraviolet radiation B (UVB)‐induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR‐1 X HR//De) F₁. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts//melanocytes from UVB‐induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts//melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts//melanocytes and keratinocytes were prepared and co‐cultured in combination with control and irradiated mice in a serum‐free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non‐irradiated melanoblasts//melanocytes more greatly than those from non‐irradiated mice. In contrast, both non‐irradiated and irradiated adultmelanocytes proliferated and differentiated similarly when they were co‐cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB‐induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

Up‐Regulation and Redistribution of Bax in Ultraviolet B‐Irradiated Melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0–30 mJ/cm²). The levels of p53, Bax, and Bcl‐2, determined by immunoblotting, revealed a dose‐dependent increase in p53 and Bax, but the level of Bcl‐2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl‐2. These data suggest that the high constitutional level of Bcl‐2 may protect melanocytes from UVB‐induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.     

Endothelin‐1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet‐induced melanocyte homeostasis

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV‐induced melanocyte homeostasis. Selective ablation of endothelin‐1 (EDN1) in murine epidermis (EDN1^ep?/?) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non‐cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte‐derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV‐induced melanocyte activation and recapitulated the phenotype seen in EDN1^ep?/? mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV‐induced melanocyte homeostasis in vivo.     

Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.     

Interaction of keratinocytes and fibroblasts modulates the expression of matrix metalloproteinases-2 and -9 and their inhibitors

Disruption of epidermal-mesenchymal communication due to a delay in epithelialization, increases the frequency of developing fibrotic conditions in skin. As matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) are two key enzymes involved in wound healing and tissue remodeling, here we examined the efficacy of keratinocyte-fibroblast interaction on modulation of these enzymes and their inhibitors. The conditioned media derived from keratinocytes and fibroblasts grown in upper and lower chambers of a co-culture system, respectively, were analyzed for MMP-2 and -9. Keratinocyte or fibroblast conditioned medium (FCM) was used as a control. Gelatinolytic activity analyzed by zymography showed that keratinocytes mainly express MMP-9 and to a lesser extent MMP-2; while fibroblasts express only MMP-2. In a co-culture system, the activities of both MMP-2 and MMP-9 markedly increased in conditioned media collected from bottom chambers. These findings were consistent with the level of MMP-2 and MMP-9 measured by Western blot. Using the same experimental setting, the levels of tissue inhibitors of MMPs (TIMPs) secreted by keratinocytes and fibroblasts grown in the same co-culture system were also evaluated. Western blot showed that fibroblasts secrete only TIMP-1 and TIMP-2 whose levels were increased by co-culturing fibroblasts with keratinocytes. In contrary the level of TIMP-3, which was mainly expressed by keratinocytes, increased by co-culturing these cells with fibroblasts. In conclusion, interaction of fibroblast-keratinocyte modulates the levels of MMP-2 and -9 and their inhibitors produced by these cells and this interaction may be critical for a better healing quality at a late stage of the wound healing process. (Mol Cell Biochem 269: 209–216, 2005)     

Re‐epithelialization of large wound in paedomorphic and metamorphic axolotls

Axolotls (Ambystoma mexicanum ) may heal their skin wounds scar‐free in both paedomorphs and metamorphs. In previous studies on small punch skin wounds, rapid re‐epithelialisation was noted in these two axolotl morphs. However, large wound size in mammals may affect wound healing. In this study, large circumferential full thickness excision wounds on the hind limbs were created on juvenile paedomorphic and metamorphic axolotls. The results showed re‐epithelialisation was more quickly initiated in paedomorphs than in metamorphs after wounding. The migrating rate of epidermis on the wound bed was faster in paedomorphs than in metamorphs and thus completion of re‐epithelialisation was faster in paedomorphs than in metamorphs. Within these re‐epithelialisation periods, neither basement membrane nor dermis was reformed. Epidermal cell proliferation was detected by EdU‐labelling technique. In the normal unwounded skin, epidermal proliferation rate was higher in paedomorphs than in metamorphs. After wounding, the epidermal proliferation rate was significantly lower in the migrating front on the wound bed than in the normal skin in paedomorphs. The EdU‐labelling rate between normal skin and migration front was not different in metamorphs. Lacking of more proliferating epidermal cells on the wound bed indicated that the new epidermis here derived rather from migrating epidermal cells than from cell proliferation in situ. In conclusion, re‐epithelialisation in the large wound might be fully completed in both morphs despite it was initiated earlier and with faster rate in paedomorphs than in metamorphs. The new epidermis on the wound bed derived mainly from cell migration than by cell proliferation in the re‐epithelialisation period. J. Morphol. 278:228–235, 2017. © 2016 Wiley Periodicals,Inc.     

Effects of fibroblasts of different origin on long term maintenance of xenotransplanted human epidermal kerationocytes in immunodeficient mice

We examined effects of fibroblasts of different origin on long-term maintenance of xenotransplanted human epidermal keratinocytes. A suspension of cultured epidermal cells, originating from adult human trunk skin, was injected into double mutant immunodeficient (BALB/c nu/scid) mice subcutaneously, with or without cultured fibroblastic cells of different origin. At one week after transplantation, the epidermal cells generated epidermoid cysts consisting of human epidermis-like tissue. When the epidermal cells were injected alone or together with fibroblastic cells derived from human bone marrow, muscle fascia, or murine dermis, organized epidermoid cysts regressed within 6 weeks. In contrast, when the epidermal cells were injected together with human dermal fibroblasts, generated epidermoid cysts were maintained in vivo for more than 24 weeks. Histological examination showed that the reorganized epidermis, after injection of both epidermal keratinocytes and dermal fibroblasts, retained normal structures of the original epidermis during 6 to 24 weeks after transplantation. The results indicate that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human epidermal keratinocytes by supporting self renewal of the human epidermal tissue in vivo.