Anomalous expression of HLA class II molecules on keratinocytes and fibroblasts in hypertrophic scars consequent to thermal injury.

Immunoperoxidase staining of skin sections obtained from 11 hypertrophic scars, six normotrophic scars and three samples of normal skin were performed using anti-HLA monoclonal antibodies (HLA-DR, -DQ, class I), anti-interleukin-2 receptor (IL-2R) and anti-CD1. Sections from all hypertrophic scars showed an anomalous expression of HLA-DR molecules on keratinocytes and fibroblasts. Moreover hypertrophic scars were characterized by dense infiltrates of IL-2R-positive cells and by the presence of abundant Langerhans (CD1+) cells in the epidermis and dermis. These results support the hypothesis that immunologic mechanisms play an important role in hypertrophic scarring and point to an involvement of cell-mediated immune phenomena.     

Activated keratinocytes in the epidermis of hypertrophic scars.

The etiology of hypertrophic scarring, a pathological end point of wound healing, is unknown. The scars most commonly occur when epithelialization has been delayed during, for example, the healing of deep dermal burn wounds. Hypertrophic scars are conventionally described as a dermal pathology in which the epidermis has only a passive role. In this study, the expression of keratin intermediate filament proteins and filaggrin has been investigated in the epidermis of hypertrophic scars and site-matched controls from the same patients. Hypertrophic scar epidermis was found to express the hyperproliferative keratins K6 and K16 in interfollicular epidermis in association with K17 and precocious expression of filaggrin. K16 mRNA was localized by in situ hybridization using a highly specific cRNA probe. In contrast to the immunohistochemical location of K16 protein, the K16 mRNA was found to be expressed in the basal cell layer of normal skin. In hypertrophic scars the mRNA distribution corroborated the abnormal K16 protein distribution. These results suggest the keratinocytes in hypertrophic scar epidermis have entered an alternative differentiation pathway and are expressing an activated phenotype. Activated keratinocytes are a feature of the early stages of wound healing producing growth factors that influence fibroblasts, endothelial cells, and the inflammatory response. We propose that cellular mechanisms in the pathogenesis of hypertrophic scarring are more complex than isolated dermal phenomena. The persistence of activated keratinocytes in hypertrophic scar epidermis implicates abnormal epidermal-mesenchymal interactions.     

Expression of keratin K2e in cutaneous and oral lesions: association with keratinocyte activation,proliferation, and keratinization

The cytoskeleton in keratinocytes is a complex of highly homologous structural proteins derived from two families of type I and type II polypeptides. Keratin K2e is a type II polypeptide that is expressed in epidermis late in differentiation. Here we report the influence of keratinocyte activation, proliferation, and keratinization on K2e expression in samples of cutaneous and oral lesions. The normal expression of K2e in the upper spinous and granular layers of interfollicular epidermis is increased in keloid scars but showed distinct down-regulation in psoriasis and hypertrophic scars where keratinocytes are known to undergo activation. Unlike normal and psoriatic skin, K2e expression in hypertrophic and keloid scars began in the deepest suprabasal layer. In cutaneous basal and squamous cell carcinomas, K2e was absent in most tumor islands but the overlying epidermis showed strong expression. No significant K2e expression in nonkeratinized or keratinized oral epithelia, including buccal mucosa, lateral border of tongue and gingiva was detected. In oral lichen planus K2e expression was undetectable, but in benign keratoses of lingual mucosa induction of K2e along with K1 and K10 was observed. In mild-to-moderate oral dysplasia with orthokeratinization, K2e was highly expressed compared with parakeratinized areas but in severe dysplasia as well as in oral squamous cell carcinoma, K2e expression was undetectable. Taken together, the data suggest that K2e expression in skin is sensitive to keratinocyte activation but its up-regulation in oral lesions is a reflection of the degree of orthokeratinization.     

Hypertrophic scarring is associated with epidermal abnormalities: an immunohistochemical study

The role of epidermal keratinocytes in the early phases of normal unimpaired wound healing has been studied extensively. However, little is known about the cell biological processes in the epidermis and the basal membrane zone during the later phases of dermal matrix formation and remodelling of the scar tissue. This study investigated epidermal growth and differentiation and maturation of the basal membrane zone. Biopsies were taken from (clinically) hypertrophic and non-hypertrophic scars at 3 and 12 months after a breast- reduction operation. Tissues were analysed using immunohistochemical techniques. The data showed that epidermal abnormalities with respect to differentiation persist up to 3 months, as witnessed by the expression of cytokeratin 16. Remarkably, hypertrophic scars that remained hypertrophic throughout the period of analysis (up to 12 months) showed significantly more cytokeratin 16 expression at 3 months, when compared either with normal scars or with hypertrophic scars that became normal after 12 months. Staining for Ki-67 antigen, a marker for cell proliferation, revealed an increase in basal keratinocyte proliferation rate in 3-month-old hypertrophic scars compared with non-hypertrophic scars. After 12 months, this difference had disappeared completely and the number of cycling basal cells had returned to normal values. Three-month-old hypertrophic scars showed more acanthosis than non-hypertrophic scars of the same age, irrespective of whether they remained hypertrophic or became normal scars. After 12 months, this difference was no longer present. Staining for various heparan sulphate proteoglycan epitopes revealed that restoration of the basal membrane was incomplete at 3 months, but was complete at 12 months with respect to this component. No differences in the expression of several components of the basal membrane zone (heparan sulphate proteoglycan, laminin, tenascin) were noted between hypertrophic and non-hypertrophic scars. These data show that in the early phase of hypertrophic scarring, epidermal abnormalities are found compared with normal wound healing. In addition, early (3 months) epidermal abnormalities are associated with the clinical outcome at 12 months. These findings raise the possibility that the epidermal compartment is involved in the pathogenic process. Copyright © 1998 John Wiley & Sons, Ltd.     

Mechanical evaluation of the resistance and elastance of post-burn scars after topical treatment with tretinoin

OBJECTIVE:

After burn injuries, scarred skin lacks elasticity, especially in hypertrophic scars. Topical treatment with tretinoin can improve the appearance and quality of the skin (i.e., texture, distensibility, color, and hydration). The objective of this prospective study was to examine the effects of treatment with 0.05% tretinoin for one year on the biomechanical behavior and histological changes undergone by facial skin with post-burn scarring. Setting: Tertiary, Institutional.

METHOD:

Fifteen female patients who had suffered partial thickness burns with more than two years of evolution were selected. Skin biopsies were obtained initially and after one year of treatment. The resistance and elastance of these skin biopsies were measured using a mechanical oscillation analysis system. The density of collagen fibers, elastic fibers, and versican were determined using immunohistochemical analysis.

RESULTS:

Tretinoin treatment significantly lowered skin resistance and elastance, which is a result that indicates higher distensibility of the skin. However, tretinoin treatment did not significantly affect the density of collagen fibers, elastic fibers, or versican.

CONCLUSION:

Topical tretinoin treatment alters the mechanical behavior of post-burn scarred skin by improving its distensibility and thus leads to improved quality of life for patients.     

瘢痕组织中α-平滑肌肌动蛋白的表达与细胞凋亡的关系

目的:观察α-平滑肌肌动蛋白在瘢痕组织中的表达,了解病理性瘢痕形成过程中凋亡所扮演的角色及其与肌成纤维细胞在真皮中变化的关系。方法:瘢痕标本来自烧伤后来我院进行整形手术的病人,同时取病人手术供皮区的正常皮肤作为对照。 8例瘢痕组织标本 (含 2例愈合较为平坦的瘢痕和 6例增生性瘢痕组织)被分成增殖期和成熟期两组。运用caspase-3mRNA及其蛋白的表达及TUNEL方法检测瘢痕及正常组织中的凋亡细胞,并以免疫组化法检测瘢痕及正常皮肤真皮内α-平滑肌肌动蛋白单克隆抗体的表达。结果:瘢痕组织中细胞凋亡的数目与正常组织明显不同。瘢痕内的TUNEL标记阳性细胞数多于正常组织;增殖期瘢痕内的细胞凋亡的数目多于成熟期。增殖期TUNEL标记阳性的细胞多于平坦瘢痕,而成熟期两者无显著差别,Caspase-3mRNA及其蛋白的表达与TUNEL标记结果具有一致性。随着瘢痕组织的成熟,α-平滑肌肌动蛋白单克隆抗体的表达逐渐降低,平坦的瘢痕组织中的表现尤为明显;增生性瘢痕中,增殖期与成熟期之间无显著差别。结论:正常伤口愈合过程中,肌成纤维细胞暂时性的表达,可引起伤口的收缩,随着真皮再塑形,含有α-平滑肌肌动蛋白的肌成纤维细胞因凋亡而消失,而病理性的愈合结局可能是它持续表达的结果。     

Enhanced expression of transforming growth factor-beta type I and type II receptors in wound granulation tissue and hypertrophic scar.

In the present study we have analyzed and compared, by immunohistochemistry and in situ hybridization, the expression pattern of the R4/ALK5 transforming growth factor (TGF)-beta type I receptor (RI) and the TGF-beta type II receptor (RII) in normal human skin, in wounded skin at various stages during the transition of wound granulation tissue to scar, and in long-persisting post-burn hypertrophic scars. In normal human skin, expression of RI and RII was clearly visible in the epidermis, in epidermal appendages, and in vascular cells, although only a small number of dermal fibroblasts revealed detectable levels of TGF-beta receptor expression. In contrast, granulation tissue fibroblasts showed strong expression of both TGF-beta receptor types, although in normal-healing excisional wounds their density decreased during granulation tissue remodeling. However, in post-burn hypertrophic scars, RI- and RII-overexpressing fibroblasts were found in high densities up to 20 months after injury. From these findings we suggest that the repair process of deep wounds involves the transformation of a subset of fibroblastic cells toward an increased TGF-beta responsiveness and a transient accumulation of these cells at the wound site. In addition, our study provides evidence that excessive scarring is associated with a failure to eliminate TGF-beta receptor-overexpressing fibroblasts during granulation tissue remodeling, which leads to a persistent autocrine, positive feedback loop that results in over-production of matrix proteins and subsequent fibrosis.     

Keratinocyte-derived growth factors play a role in the formation of hypertrophic scars

In predisposed individuals, wound healing can lead to hypertrophic scar or keloid formation, characterized by an overabundant extracellular matrix. It has recently been shown that hypertrophic scars are accompanied by abnormal keratinocyte differentiation and proliferation, and significantly increased acanthosis, compared with normal scars. This study addressed the question of whether the development of normal and hypertrophic scars is regulated by differences in the growth factor profiles of both the epidermis and the dermis. The presence of interleukin-1alpha (IL-1alpha), IL-1beta, tumour necrosis factor-alpha (TNF-alpha), platelet-derived growth factor (PDGF), transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) was investigated in biopsies taken from breast reduction scars at 3 and 12 months following surgery. The samples were analysed by immunohistological methods and categorized as scars that remained hypertrophic (HH), became normal (HN) or remained normal after 12 months (NN). The epidermal expression of IL-1alpha was significantly increased in NN scars compared with HN and HH scars 3 and 12 months following operation, whereas the dermal expression showed no difference. PDGF was significantly increased in the dermis of normal scars after 3 months and in both the epidermis and the dermis of hypertrophic scars after 12 months. IL-1beta, TNF-alpha, TGF-beta and bFGF showed no differences. It is hypothesized that impaired production of keratinocyte-derived growth factors, such as IL-1alpha, leads to a decrease in the catabolism of the dermal matrix, whereas augmented epidermal PDGF production leads to increased formation of the dermal matrix in hypertrophic scars. These observations support the possibility that the epidermis is involved in preventing the formation of hypertrophic scars.     

Effects of retinoic acid on keratinocyte proliferation and differentiation in a psoriatic skin model

Psoriasis is a skin disease characterized by the presence of red plaques on the skin. This pathology is well-known to be a retinoid-sensitive disease. Previous investigations have shown that retinoids can modulate epidermal proliferation with an antiproliferative potential in hyperproliferative skins. The aim of this study was to compare the development of psoriatic substitutes cultured in a retinoic acid supplemented medium with those cultured in medium receiving no supplement, to define the effects of this growth factor on keratinocyte proliferation and differentiation. The self-assembly method was used to create substitutes. Characterization of the psoriatic substitutes was performed by histological and immunolabeling analyses. Results showed that psoriatic keratinocyte substitutes cultured with retinoic acid have a thinner epidermis compared with psoriatic keratinocyte substitutes cultured without this supplement. Further, the expression of all tested cell differentiation markers was restored in psoriatic keratinocyte substitutes cultured in presence of retinoic acid. No significant change in epidermal thickness or in the expression of late differentiation markers was observed in healthy keratinocyte substitutes cultured with or without retinoic acid; however, some changes were reported for proliferation and early differentiation markers. Results suggest that retinoic acid can modulate epidermal differentiation and proliferation with an antiproliferative potential in psoriatic substitutes such as observed in psoriatic skin in vivo.     

Growth factors in porcine full and partial thickness burn repair. Differing targets and effects of keratinocyte growth factor, platelet-derived growth factor-BB, epidermal growth factor, and neu differentiation factor.

The topical application of recombinant growth factors such as epidermal growth factor, platelet-derived growth factor-BB homodimer (rPDGF-BB), keratinocyte growth factor (rKGF), and neu differentiation factor has resulted in significant acceleration of healing in several animal models of wound repair. In this study, we established highly reproducible and quantifiable full and deep partial thickness porcine burn models in which burns were escharectomized 4 or 5 days postburn and covered with an occlusive dressing to replicate the standard treatment in human burn patients. We then applied these growth factors to assess their efficacy on several parameters of wound repair: extracellular matrix and granulation tissue production, percent reepithelialization, and new epithelial area. In full thickness burns, only rPDGF-BB and the combination of rPDGF-BB and rKGF induced significant changes in burn repair. rPDGF-BB induced marked extracellular matrix and granulation tissue production (P = 0.013) such that the burn defect was filled within several days of escharectomy, but had no effect on new epithelial area or reepithelialization. The combination of rPDGF-BB and rKGF in full thickness burns resulted in a highly significant increase in extracellular matrix and granulation tissue area (P = 0.0009) and a significant increase in new epithelial area (P = 0.007), but had no effect on reepithelialization. In deep partial thickness burns, rKGF induced the most consistent changes. Daily application of rKGF induced a highly significant increase in new epithelial area (P < 0.0001) but induced only a modest increase in reepithelialization (83.7% rKGF-treated versus 70.2% control; P = 0.016) 12 days postburn. rKGF also doubled the number of fully reepithelialized burns (P = 0.02) at 13 days postburn, at least partially because of marked stimulation of both epidermal and follicular proliferation as assessed by proliferating cell nuclear antigen expression. In situ hybridization for KGFR in porcine burns revealed strong expression of KGFR on hair follicles and basal epidermis, confirming direct rKGF action on follicular as well as epidermal keratinocytes. Although the epithelial proliferation induced by rKGF resulted in marked neoepidermal psoriasiform hyperplasia with exaggerated rete ridges and neoepidermal and follicular maturation as assessed by expression of cytokeratin 10, a marker of keratinocyte terminal differentiation was not delayed and appeared to be accelerated in some rKGF-treated burns. Recombinant epidermal growth factor induced a trend toward increased new epithelial area in deep partial thickness burns, but had no effect on reepithelialization. The recombinant neu differentiation factor-alpha 2 isoform had no significant biological effects in either full or deep partial thickness burns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distributions of melanoma growth stimulatory activity of growth-regulated gene and the interleukin-8 receptor B in human wound repair.

The alpha-chemokines have been implicated as regulators of proliferation and differentiation of normal keratinocytes and as mediators of keratinocyte maturation and migration in inflammatory processes that involve the skin. Using the cutaneous wound repair model, we examined the sites and temporal sequence of the appearance of melanoma growth stimulatory activity or growth-regulated gene (MGSA/GRO;ligand) and the type B interleukin (IL)-8 receptor (IL-8RB) to which MGSA/GRO binds. Human burn tissues (n = 44) representing days 2 to 12 after injury were obtained during surgical debridement, fixed in 4% paraformaldehyde, and embedded in paraffin. Immunolocalizations were performed with polyclonal antisera for both ligand and receptor, as well as a monoclonal antibody for the IL-8 RB. Western blot analysis confirmed the presence of the IL-8 RB in immunoprecipitates of epidermal keratinocyte lysates. In normal skin, MGSA/GRO protein was restricted to sites populated by differentiated keratinocytes (suprabasal compartments, inner root sheath cells, and dermal sweat ducts). MGSA/GRO protein was barely detectable within epithelial margins and islands of burn wounds where the migrating/proliferating keratinocyte populations reside, but staining intensities increased as cells matured into the outer layers. Weak diffuse staining was detected in areas of neutrophilic infiltration (granulation tissue and overlying exudates). By contrast, in normal skin the IL-8 RB was detected in specific locations within epidermal and dermal compartments of healing wounds. In the dermis, polyvalent antibodies detected receptor immunoreactivity most prominently in dermal sweat ducts and endothelium of capillaries, whereas this immunoreactivity was inconspicuous in sections stained with the monoclonal antibody. Receptor immunostaining was noted in migrating/proliferating keratinocytes in epithelial margins and islands but was in the outer layers or in hypertrophic epidermis adjacent to wounds. This same pattern was observed in epidermal appendages such as hair follicles and eccrine sweat ducts. In granulation tissues, IL-8 RB was noted in numerous fibroblasts and in subpopulations of macrophages and smooth muscle. The presence of both MGSA/GRO and its receptor in human burn wounds implicate this cytokine as an autocrine or paracrine mediator of epidermal regeneration in both the inflammatory and proliferative phases of cutaneous wound repair.     

The antimicrobial protein REG3A regulates keratinocyte proliferation and differentiation after skin injury

Epithelial keratinocyte proliferation is an essential element of wound repair, and abnormal epithelial proliferation is an intrinsic element in the skin disorder psoriasis. The factors that trigger epithelial proliferation in these inflammatory processes are incompletely understood. Here we have shown that regenerating islet-derived protein 3-alpha (REG3A) is highly expressed in keratinocytes during psoriasis and wound repair and in imiquimod-induced psoriatic skin lesions. The expression of REG3A by keratinocytes is induced by interleukin-17 (IL-17) via activation of keratinocyte-encoded IL-17 receptor A (IL-17RA) and feeds back on keratinocytes to inhibit terminal differentiation and increase cell proliferation by binding to exostosin-like 3 (EXTL3) followed by activation of phosphatidylinositol 3 kinase (PI3K) and the kinase AKT. These findings reveal that REG3A, a secreted intestinal antimicrobial protein, can promote skin keratinocyte proliferation and can be induced by IL-17. This observation suggests that REG3A may mediate the epidermal hyperproliferation observed in normal wound repair and in psoriasis.     

FasL and TRAIL induce epidermal apoptosis and skin ulceration upon exposure to Leishmania major

Receptor-mediated apoptosis is proposed as an important regulator of keratinocyte homeostasis in human epidermis. We have previously reported that Fas/FasL interactions in epidermis are altered during cutaneous leishmaniasis (CL) and that keratinocyte death through apoptosis may play a pathogenic role for skin ulceration. To further investigate the alterations of apoptosis during CL, a keratinocyte cell line (HaCaT) and primary human epidermal keratinocytes were incubated with supernatants from Leishmania major-infected peripheral blood mononuclear cells. An apoptosis-specific microarray was used to assess mRNA expression in HaCaT cells exposed to supernatants derived from L. major-infected cultures. Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression were significantly up-regulated, and apoptosis was detected in both HaCaT and human epidermal keratinocyte cells. The keratinocyte apoptosis was partly inhibited through blocking of Fas or FasL and even more efficiently through TRAIL neutralization. Up-regulation of Fas on keratinocytes in epidermis and the presence of FasL-expressing macrophages and T cells in dermis were previously reported by us. In this study, keratinocytes expressing TRAIL, as well as the proapoptotic receptor TRAIL-R2, were detected in skin biopsies from CL cases. We propose that activation of Fas and TRAIL apoptosis pathways, in the presence of inflammatory mediators at the site of infection, leads to tissue destruction and ulceration during CL.     

Human Merkel cell regeneration in skin derived from cultured keratinocyte grafts.

Human Merkel cell regeneration in epidermis derived from cultured keratinocyte autografts was studied from 6 days to 6 years after transplantation. Cultured keratinocyte sheets derived from skin of the sole, axilla, groin, or scalp were transplanted to full-thickness wounds in 20 pediatric patients treated for massive burns or giant congenital nevi. Normal age- and site-matched skin as well as meshed split-thickness autografts from the same patients served as controls. Merkel cells were identified by immunohistochemistry using antibodies to cytokeratins #8 and #18. Cultured keratinocytes in vitro expressed no neuroendocrine markers, but nonspecific, simple-epithelial cytokeratin expression was observed in about 20% of cells. After transplantation, Merkel cells were identified only in cultured grafts derived from sole skin and appeared in the epidermis as early as 21 days postgrafting. Dermal Merkel cells were rarely observed, but their appearance invariably succeeded that of intraepidermal Merkel cells. Regenerated Merkel cells were never innervated, and their emergence was unrelated either spatially or temporally to epidermal reinnervation. In skin bridges of meshed split-thickness grafts, Merkel cells survived after degeneration of associated neurites, but no Merkel cells appeared within re-epithelialized interstices. Among the neuroendocrine markers tested, Merkel cells in cultured grafts, meshed skin grafts or normal pediatric skin expressed only neuron-specific enolase. They failed to stain for calcitonin, chromogranin A, Leu-7, synaptophysin, bombesin, or vasoactive intestinal polypeptide by immunohistochemistry. These findings suggest that: (a) Merkel cells derive from keratinocyte precursors which undergo neuroendocrine differentiation in the epidermis; (b) that keratinocyte stem cells are capable of undergoing Merkel cell differentiation postnatally; (c) that postnatal Merkel cell differentiation may be body-site dependent; and (d) that Merkel cell development and maintenance is independent of neural induction.     

The morphologic characteristics of scar tissues and the new clinicomorphologic classification of human skin scars

The morphological study of 200 intraoperative biopsy specimens of skin scars of various etiology identified criteria for the differential diagnosis and mechanisms responsible for formation of 4 major types of scar tissues and their structural and functional varieties. In addition to the earlier known normotrophic, hypertrophic, and keloid scar tissues; the special type of scar tissue - the fibrously altered derma) was defined. Most skin scars were found to comprise a combination of a few of scar tissues and a relationship was established between the clinical type of a scar and its histological structure. The principles and mechanisms responsible for changes occurring in the structural composition of scars as the latter mature are described. The new scar clinicomorphological classification provides the basis for the pathogenetically substantiated choice of methods for the prevention and treatment of scars and for the prediction of recurrent scarring.     

Development of fungal mycelia as a skin substitute: characterization of keratinocyte proliferation and matrix metalloproteinase expression during improvement in the wound-healing process

SACCHACHITIN membranes, prepared from the waste residue of the fruiting body of Ganoderma taugae, were used in our previous study to enhance skin wound healing in animal models. In the present study, the effects of the membrane on the growth of keratinocytes and the activity of matrix metalloproteinases (MMPs), as well as on the healing of skin wounds in humans, were estimated. Fresh human foreskin was employed as the source of the keratinocyte culture, and a modified keratinocyte-SFM medium supplemented with 0.2 ng/mL of recombinant epidermal growth factor and 30 microg/mL bovine pituitary extract was used to enhance the successful growth of keratinocytes under an atmosphere of 5% CO2, at 37 degrees C. The results indicated that 0.01% SACCHACHITIN enhanced the proliferation of keratinocytes in the culture on the fourth and fifth days, and cells showed neither morphological alteration nor disordered proliferation. This evidence clearly indicated that SACCHACHITIN was not cytotoxic to and was safe for the growth of keratinocytes. Thus, SACCHACHITIN might play a positive role in the proliferation and differentiation of keratinocytes around wounds and in accelerated wound healing of epidermal tissue. In addition, microscopic observations during the growth of keratinocytes showed that normal proliferation and differentiation took place along the margin of the SACCHACHITIN membrane. This indicates that SACCHACHITIN is possibly cytocompatible with keratinocytes. Electrophoretic analysis and inhibition tests for the binding effect of SACCHACHITIN on MMPs showed that SACCHACHITIN reduced MMPs in extracellular matrix degradation and facilitated establishment of an extracellular matrix around wounds; these effects resulted in rapid wound healing. SACCHACHITIN was used as a skin dressing for patients who had skin chronicle ulcer, which had not healed for over 7 months. Preliminary clinical observations showed that the wound improved and began to heal. An analysis of MMPs by ELISA in tissue of the wound indicated a significant decrease in MMP levels.