Molecular Changes Underlying Hypertrophic Scarring Following Burns Involve Specific Deregulations at All Wound Healing Stages (Inflammation, Proliferation and Maturation).

Excessive connective tissue accumulation, a hallmark of hypertrophic scaring, results in progressive deterioration of the structure and function of organs. It can also be seen during tumor growth and other fibroproliferative disorders. These processes result from a wide spectrum of cross-talks between mesenchymal, epithelial and inflammatory/immune cells that have not yet been fully understood. In the present review, we aimed to describe the molecular features of fibroblasts and their interactions with immune and epithelial cells and extracellular matrix. We also compared different types of fibroblasts and their roles in skin repair and regeneration following burn injury. In summary, here we briefly review molecular changes underlying hypertrophic scarring following burns throughout all basic wound healing stages, i.e. during inflammation, proliferation and maturation.

Scar-reducing effects of gambogenic acid on skin wounds in rabbit ears.

Hypertrophic scar (HS) is a dermal fibroproliferative disease that often occurs following abnormal wound healing. To date, there is no satisfied treatment strategies for improvement of scar formation with few side effects. The effects of gambogenic acid (GNA) on scar hypertrophy has not been studied previously. The present study was undertaken to find out the scar-reducing effects of GNA (0.48, 0.96 or 1.92 mg/ml) on skin wounds in rabbit ears. Scar evaluation index (SEI), collagen I (Col1) and collagen III (Col3), microvascular density (MVD), CD4+T cells and macrophages, vascular endothelial growth factor receptor 2 (VEGFR2), fibroblast growth factor receptor 1 (FGFR1), phospho-VEGFR 2 (p-VEGFR2) and p-FGFR1, interleukin (IL)-1ß, IL-6, IL-10 and tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1 and connective tissue growth factor (CTGF) in scar tissue were detected using various methods, respectively. Our data showed that GNA significantly reduced SEI, and the expression of Col1 and Col3 in scar tissue in a concentration-dependent manner. Also, it decreased MVD, the infiltration of CD4+T cells and macrophages, and the levels of VEGFR2, p-VEGFR2, FGFR1, p-FGFR1, TGF-ß1, CTGF, IL-1ß, IL-6, TNF-α, in addition to upregulated IL-10 in scar tissue. As a result, this study revealed that GNA reduced HS formation, which was associated with the inhibition of neoangiogenesis, local inflammatory response and growth factor expression in scar tissue during wound healing. These findings suggested that GNA may be considered as a preventive and therapeutic candidate for HS.

Human CD206+ Macrophages Show Antifibrotic Effects on Human Fibroblasts through an IL-6-Dependent Mechanism In Vitro.

BACKGROUND: Pathologic scarring including keloid and hypertrophic scar causes aesthetic and physical problems, and there are clinical difficulties (e.g., posttreatment recurrence) in dealing with pathologic scarring. Understanding the mechanisms that underlie scar control in wound healing will help prevent and treat pathologic scarring. The authors focused on CD206+ macrophages in the wound-healing process, and hypothesized that CD206+ macrophages have antifibrotic effects on fibroblasts. METHODS: The authors established a co-culture system for CD206+ macrophages and fibroblasts (cell ratio, 1:1). The authors examined the CD206+ macrophages' antifibrotic effects on fibroblasts after a 72-hour culture, focusing on fibrosis-related genes. To identify key factor(s) in the interaction between CD206+ macrophages and fibroblasts, the authors analyzed cytokines in a conditioned medium of the co-culture system. RESULTS: Under co-culture with CD206+ macrophages, expression of the following in the fibroblasts was significantly down-regulated: type 1 (fold change, 0.38) and type 3 collagen (0.45), alpha smooth muscle actin (0.24), connective tissue growth factor (0.40), and transforming growth factor-beta (0.66); the expression of matrix metalloproteinase 1 was significantly up-regulated (1.92). Conditioned medium in the co-culture showed a high interleukin (IL)-6 concentration (419 ± 88 pg/ml). When IL-6 was added to fibroblasts, antifibrotic changes in gene expression (as observed under the co-culture) occurred in the fibroblasts. CONCLUSIONS: The authors' in vitro results revealed that CD206+ macrophages have antifibrotic effects on fibroblasts by means of a paracrine mechanism involving IL-6. Understanding these effects, especially in vivo, will help elucidate the mechanism of scar control in wound healing and contribute to the development of new scar treatments.

Prognostic tools for hypertrophic scar formation based on fundamental differences in systemic immunity.

Unpredictable hypertrophic scarring (HS) occurs after approximately 35% of all surgical procedures and causes significant physical and psychological complaints. Parallel to the need to understanding the mechanisms underlying HS formation, a prognostic tool is needed. The objective was to determine whether (systemic) immunological differences exist between patients who develop HS and those who develop normotrophic scars (NS) and to assess whether those differences can be used to identify patients prone to developing HS. A prospective cohort study with NS and HS groups in which (a) cytokine release by peripheral blood mononuclear cells (PBMC) and (b) the irritation threshold (IT) after an irritant (sodium lauryl sulphate) patch test was evaluated. Univariate regression analysis of PBMC cytokine secretion showed that low MCP-1, IL-8, IL-18 and IL-23 levels have a strong correlation with HS (P < .010-0.004; AUC = 0.790-0.883). Notably, combinations of two or three cytokines (TNF-a, MCP-1 and IL-23; AUC: 0.942, Nagelkerke R2 : 0.727) showed an improved AUC indicating a better correlation with HS than single cytokine analysis. These combination models produce good prognostic results over a broad probability range (sensitivity: 93.8%, specificity 86.7%, accuracy 90,25% between probability 0.3 and 0.7). Furthermore, the HS group had a lower IT than the NS group and an accuracy of 68%. In conclusion, very fundamental immunological differences exist between individuals who develop HS and those who do not, whereas the cytokine assay forms the basis of a predictive prognostic test for HS formation, the less invasive, easily performed irritant skin patch test is more accessible for daily practice.

Exacerbated and prolonged inflammation impairs wound healing and increases scarring.

Altered inflammation in the early stage has long been assumed to affect subsequent steps of the repair process that could influence proper wound healing and remodeling. However, the lack of explicit experimental data makes the connection between dysregulated wound inflammation and poor wound healing elusive. To bridge this gap, we used the established rabbit ear hypertrophic scar model for studying the causal effect of dysregulated inflammation. We induced an exacerbated and prolonged inflammatory state in these wounds with the combination of trauma-related stimulators of pathogen-associated molecular patterns from heat-killed Pseudomonas aeruginosa and damage-associated molecular patterns from a dermal homogenate. In stimulated wounds, a heightened and lengthened inflammation was observed based on quantitative measurements of IL-6 expression, tissue polymorphonuclear leukocytes infiltration, and tissue myeloperoxidase activity. Along with the high level of inflammation, wound healing parameters (epithelial gap and others) at postoperative day 7 and 16 were significantly altered in stimulated wounds compared to unstimulated controls. By postoperative day 35, scar elevation of stimulated wounds was higher than that of control wounds (scar elevation index: 1.90 vs. 1.39, p < 0.01). Moreover, treatment of these inflamed wounds with Indomethacin (at concentrations of 0.01, 0.1, and 0.4%) reduced scar elevation but with adverse effects of delayed wound closure and increased cartilage hypertrophy. In summary, successful establishment of this inflamed wound model provides a platform to understand these detrimental aspects of unchecked inflammation and to further test agents that can modulate local inflammation to improve wound outcomes.

The natural behavior of mononuclear phagocytes in HTS formation.

Hypertrophic scars (HTS) are caused by trauma or burn injuries to the deep dermis and are considered fibrosis in the skin. Monocytes, M1 and M2 macrophages are mononuclear phagocytes. Studies suggest that M2 macrophages are profibrotic and might contribute to HTS formation. Our lab has established a human HTS-like nude mouse model, in which the grafted human skin develops red, raised, and firm scarring, resembling HTS seen in humans. In this study, we observed the natural behavior of mononuclear phagocyte system in this nude mouse model of dermal fibrosis at multiple time points. Thirty athymic nude mice received human skin grafts and an equal number of mice received mouse skin grafts as controls. The grafted skin and blood were harvested at 1, 2, 3, 4, and 8 weeks. Wound area, thickness, collagen morphology and level, the cell number of myofibroblasts, M1- and M2-like macrophages in the grafted skin, as well as monocyte fraction in the blood were investigated at each time points. Xenografted mice developed contracted and thickened scars grossly. The xenografted skin resembled human HTS tissue based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level, and infiltration of myofibroblasts. In the blood, monocytes dramatically decreased at 1 week postgrafting and gradually returned to normal in the following 8 weeks. In the xenografted skin, M1-like macrophages were found predominantly at 1-2 weeks postgrafting; whereas, M2-like macrophages were abundant at later time points, 3-4 weeks postgrafting coincident with the development of fibrosis in the human skin tissues. This understanding of the natural behavior of mononuclear phagocytes in vivo in our mouse model provides evidence for the role of M2-like macrophages in fibrosis of human skin and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation.

Inhibition of mechanical stress-induced hypertrophic scar inflammation by emodin.

At least 50% of hypertrophic scarring (HS) is characterized by inflammation, for which there is currently no effective treatment available. Emodin is a major component of the widely used Chinese herb, rhubarb, which has been used to treat inflammation in several types of disease. However, few studies have investigated the efficacy of emodin in the treatment of HS. In the present study, a mouse model with mechanical stress­induced HS was used to investigate the effects of emodin (20, 40, 80, or 120 mg/ml) on HS, and to determine the potential underlying mechanisms. Treatment with emodin significantly attenuated HS inflammation, as determined by histopathological assessment of the scar elevation index, collagen structure and inflammation (P<0.05). Furthermore, treatment with emodin (40 mg/ml) markedly inhibited phosphoinositide 3­kinase (PI3K)/Akt activity (P<0.01) and this attenuation was associated with reduced expression levels of tumor necrosis factor­α, interleukin­6 and monocyte chemoattractant protein­1 (P<0.05) in the HS tissue. The results of the present study indicated that administration of emodin had therapeutic effects on the progression of HS and the underlying mechanism of this may be due to inhibition of the PI3K/Akt signaling pathway.

Toll-like receptors expressed by dermal fibroblasts contribute to hypertrophic scarring.

Hypertrophic scar (HTS), a fibroproliferative disorder (FPD), complicates burn wound healing. Although the pathogenesis is not understood, prolonged inflammation is a known contributing factor. Emerging evidence suggests that fibroblasts regulate immune/inflammatory responses through toll-like receptor 4 (TLR4) activated by lipopolysaccharide (LPS) through adaptor molecules, leading to nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinases activation, cytokine gene transcription and co-stimulatory molecule expression resulting in inflammation. This study explored the possible role of TLR4 in HTS formation. Paired normal and HTS tissue from burn patients was collected and dermal fibroblasts isolated and cultured. Immunohistochemical analysis of tissues demonstrated increased TLR4 staining in HTS tissue. Quantitative RT-PCR of three pairs of fibroblasts demonstrated mRNA levels for TLR4 and its legend myeloid differentiation factor 88 (MyD88) in HTS fibroblasts were increased significantly compared with normal fibroblasts. Flow cytometry showed increased TLR4 expression in HTS fibroblasts compared with normal. ELISA demonstrated protein levels for prostaglandin E2, interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 (MCP-1) were significantly increased in HTS fibroblasts compared to normal. When paired normal and HTS fibroblasts were stimulated with LPS, significant increases in mRNA and protein levels for MyD88, IL-6, IL-8, and MCP-1 were detected. However, when transfected with MyD88 small interfering RNA (siRNA), then stimulated with LPS, a significant decrease in mRNA and protein levels for these molecules compared to only LPS-stimulated fibroblasts was detected. In comparison, a scramble siRNA transfection did not affect mRNA or protein levels for these molecules. Results demonstrate LPS stimulates proinflammatory cytokine expression in dermal fibroblasts and MyD88 siRNA eliminates the expression. Therefore, controlling inflammation and manipulating TLR signaling in skin cells may result in novel treatment strategies for HTS and other FPD.

The temporal effects of anti-TGF-beta1, 2, and 3 monoclonal antibody on wound healing and hypertrophic scar formation.

BACKGROUND: A number of studies have implicated transforming growth factor (TGF)-beta1, 2, and 3 (TGF-beta) in wound healing and hypertrophic scarring. We propose that TGF-beta has a temporal effect on these processes. To test this hypothesis, we applied anti-TGF beta1, 2, and 3 monoclonal antibody topically to our dermal ulcer model in the rabbit ear. STUDY DESIGN: Rabbit ear wounds were treated intradermally with anti-TGF-beta1, 2, and 3 antibody at early, middle, and late time points. Treated and untreated control wounds were harvested at various time points and examined histologically to quantify wound healing and scar hypertrophy. Real-time polymerase chain reaction was performed to determine TGF-beta mRNA expression in the treated and control wounds. RESULTS: The early treatment group demonstrated decreased new epithelium and granulation tissue (p < 0.05 versus controls). Scars harvested on days 28 and 40 displayed no difference in scar hypertrophy. Both the middle and late treatment groups demonstrated a significant decrease in scar hypertrophy (p < 0.05). CONCLUSIONS: Treated wounds from the early treatment group displayed delayed wound healing, with no reduction in scar hypertrophy. Later treatment of wounds with the same antibody, beginning 7 days after wounding, resulted in a reduction in scar hypertrophy. These results support our hypothesis and clearly demonstrate that TGF-beta1, 2, and 3 have differential temporal effects during the wound-healing process, and are important for optimal wound healing in the first week after wounding; beyond 1 week, TGF-beta1, 2, and 3 play a critical role in hypertrophic scar formation.

[Immunological regulations of dendritic cell in abnormal scarring tissue].

OBJECTIVE: To investigate the relationship between dendritic cell(DC) and pathogenesis of abnormal scar. METHODS: The content of HLA-DR and CD1a molecules of DC, in 6 samples of hypertrophic scar (HS), keloid (K) and normal skin, were determined with the stain of avidin-biotin-peroxidase complex method (ABC). The effect of Triamcinolone Acetonide was evaluated with the measurement of the HLA-DR and CD1a molecules of DC in the epidermis of the HS. RESULTS: 1. The amounts of HLA-DR molecules of the positive DC were 806.67 +/- 101.72 and 870.00 +/- 134.24 in the HS and the K respectively, significantly higher than the controlled normal skin (510.01 +/- 45.17, P < 0.05). HLA-DR molecules showed an abnormal expression in the Kerationcytes and fibroblasts. 2. The amounts of CD1a molecules of the positive DC were 700.00 +/- 97.23 and 780.00 +/- 104.47 in the HS and the K respectively, significantly higher than the controlled normal skin (521.24 +/- 57.87)(P < 0.05). 3. The amounts of the HLA-DR molecules positive DC, in the positive kerationcytes and fibroblasts of hypertrophic scar, treated by Triamcinolone Acetonide, were 476.67 +/- 70.02 and 447.76 +/- 90.03 (P < 0.05) for 3 days and 7 days treatment respectively, significantly lower than the control. The amounts of CD1a molecules positive DC in the epidermis of hypertrophic scar with the injection of Triamcinolone Acetonide were significantly lower in 3 days and 7 days treatment (456.36 +/- 82.88 and 397.18 +/- 99.36, P < 0.05). CONCLUSION: 1. The results, with the high expression of HLA-DR and CD1a molecules, indicate that the HS and the K may have strong immune reactions. 2. Triamcinolone Acetonide may decrease the immune reactions of the HS, through the inhibition of the expressions of HLA-DR and CD1a molecules in the dendritric cell.

Response to tissue injury.

Cutaneous injury, whether by laser, chemical, or scalpel, results in scar formation. The normal response to such an insult occurs in the middle of a continuum of wound repair processes. On one end of the continuum are the overhealed responses (i.e., keloids). On the opposite end are tissue regeneration and scarless healing as seen in fetal wounds. This article reviews the molecular biology and mechanisms leading to these various clinical phenotypes and discusses future potential modalities that may replicate the scarless wound healing mechanism in adults.

[The study of expression and distribution of PCNA in hypertrophic scar tissue].

OBJECTIVE: To analyze the expression and the distribution of proliferation cell nuclear antigen (PCNA) in hypertrophic scar for identifying its certain characteristics during the fibroblast proliferation. METHODS: Bioptic samples were taken form 12 cases of hypertrophic scar, 8 cases of matured scars and 20 cases of controlled normal skin. Each sample was immunohistochemically stained and examined with monoclonal antibody[PCNA(pc10)] technique. The level of PCNA expression was decided for evaluation of the fibroblast proliferation. RESULTS: The level of PCNA in the hypertrophic scar(45.4 +/- 12.3) was significantly higher than mature scar(17.4 +/- 6.2), also normal skin(13.5 +/- 4.1, P < 0.01). But, there was no significant deference between the matured scar and the normal skin. The PCNA intracellular particles may sometimes be bound in the keratinocytes and the fibroblasts of some connective tissue. CONCLUSION: The fibroblast proliferation in the hypertrophic scar has kept a high level. During the process of hypertrophic scar formation it may play an important role.

[Morphological study of CD3+ dendritic epidermal T cells in human skin and scar].

OBJECTIVE: To investigate the CD3/TCR-bearing T cell in the epidermis and explore the influence of the dendritic epidermal T cells on regeneration and differentiation of epidermal cells. METHODS: The CD3/TCR-bearing T cells in epidermis of human skin and cicatrix were examined by means of histomorphology and immunohistochemistry. RESULTS: It was found that the density of CD3/TCR-bearing T cells in human skin and scar tissue had great dissimilarity. CD3+ dendritic epidermal T cells increased in hyperplastic scar. Such result was not observed in atrophic scar. CONCLUSION: Our findings indicate CD3+ dendritic epidermal T cells regulate the generation and differentiation of epidermal cells. CD3+ dendritic epidermal T cells may play an important role in the maintenance of the morphology of epidermal tissue.

Inverse correlation between CD34 expression and proline-4-hydroxylase immunoreactivity on spindle cells noted in hypertrophic scars and keloids.

The CD34 positive (CD34+) spindle cells constitute a special population of spindle cells which shows a unique distribution in the skin. So far, however, the functional role of CD34+ spindle cells and the regulation of CD34 expression on dermal spindle cells are totally unknown. We examined immunohistologically the pattern of the expression of CD34 and proline-4-hydroxylase, a marker for the fibroblasts that participate in active collagen synthesis, on dermal spindle cells at various stages of scar and keloidal tissues. Dermal spindle cells in the lesions of hypertrophic scar and those at inflammatory expanding borders of keloids totally lost CD34 expression, but they strongly expressed proline-4-hydroxylase. On the other hand, they expressed CD34, together with decreased immunoreactivity to anti-proline-4-hydroxylase antibody, in non-inflammatory scars or in a non-inflammatory central portion of keloid. In two cases of scars, in which inflammation began to subside, double immunofluorescence demonstrated that both CD 34 and proline-4-hydroxylase were expressed on the same spindle cells. CD34 expression, once disappeared from the lesions of hypertrophic scar or keloid, seems to return on CD34-proline-4-hydroxylase+ cells, when the initial inflammatory changes begin to regress. There is a reverse correlation between CD34 expression on spindle cells and the synthesis of type I collagen in the skin.

Characterization of T-cell subsets infiltrating post-burn hypertrophic scar tissues.

In this study, skin-infiltrating cells were characterized in both the active and remission phases of post-burn hypertrophic scar biopsies. Immunohistochemistry examination of active phase samples showed an abundant presence of Langerhans cells, T cells, macrophages, a low presence of natural killer cells and the lack of B lymphocytes. In active hypertrophic scars T lymphocytes infiltrate deep into the superficial dermis and are also observed in the epidermis: CD3+ cells were present at about 222 +/- 107 per 0.25 mm2. In particular the analysis of lymphocyte subpopulations showed that CD4+ T cells predominate in the dermis as well as in the epidermis of active hypertrophic scars whereas CD8+ cells were less well represented (CD4/CD8 ratio is 2.06). This distribution was also shown in remission phase samples and in normotrophic scar specimens, although the lymphocyte number was significantly lower. Approximately 70 per cent of T lymphocytes present in the tissue involved in active phase hypertrophic scar samples were activated (positive with anti-HLA-DR and IL-2 receptor antibodies) which is significantly higher than remission phase hypertrophic and normotrophic scars, in which positivity was 40 and 38 per cent, respectively. Upon activation, the lesional lymphocytes release several cytokines, locally and transiently, that interact with specific receptors in response to different stimulation. Central to the immune hypothesis of hypertrophic scars is that some of the T-cell lymphokines act on keratinocytes, fibroblasts and other cell types to induce changes characteristic of these scars. The presence and close proximity of activated T lymphocytes and antigen-presenting cells of various phenotypes in both the epidermis and dermis of hypertrophic tissues provides strong circumstantial evidence of a local immune response. However, the manner in which T cells achieve and maintain their activated state in hypertrophic tissues is not yet known, and both antigen-dependent and independent mechanisms may contribute.

Immunolocalization of TGF-beta 1 in human hypertrophic scar and normal dermal tissues.

Following severe thermal injury and other injuries to the deep dermis of the skin, patients frequently develop hypertrophic scarring (HSc) which is characterized by an over-abundance of extracellular matrix (ECM) proteins including collagen. Our previous work revealed a synchronous elevation in expression of mRNA for transforming growth factor (TGF)-beta 1, type I and type III procollagen in human HSc tissue, suggesting a possible role of locally synthesized TGF-beta 1 in matrix production. In this study the immunoreactive sites of TGF-beta 1 protein in hypertrophic and normal dermal (ND) tissue obtained from the same patients have been determined by an immunoperoxidase staining system. We used two TGF-beta 1-specific rabbit polyclonal antibodies known as anti-LC and anti-CC which were prepared to different synthetic preparations of a peptide corresponding to the first 1-30 amino acids of the amino-terminus of mature TGF-beta 1. These antibodies have affinity for two distinct epitopes of TGF-beta 1. The anti-LC antibody localized TGF-beta 1 to non-proliferating/differentiated epidermal cells, suprabasal keratinocytes in both normal and HSc tissues. The intensity of this staining was significantly higher (150 +/- 26 sq. micron vs 77 +/- 7 sq. micron, n = 5, P < 0.05) in normal tissues compared to HSc tissues. When the anti-CC antibody was used as the primary antibody, intense staining of focal regions was observed in the dermis of HSc tissue, but not in ND tissue obtained from the same patient. These foci contained collagen which was nodular, distributed in whorl-like arrangements and highly enriched with microvessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Similar ectopic expression of ICAM-1 and HLA class II molecules in hypertrophic scars following thermal injury.

In previous studies we have shown that HLA Class II antigens are expressed by keratinocytes and fibroblasts in hypertrophic scars. Because of the potential role of immunological events in the pathogenesis of hypertrophic scars, in the present study we have tested hypertrophic scars for the expression of intercellular adhesion molecule-1 (ICAM-1), a molecule which plays an important role in immunological phenomena. Immunoperoxidase staining with anti-ICAM-1 MoAb of 10 hypertrophic scar samples detected this molecule on epidermal keratinocytes and on about 30 per cent of fibroblasts at the site of lymphoid infiltration. The expression of ICAM-1 in hypertrophic scars was similar to that of HLA Class II antigens. Since the concomitant expression of ICAM-1 and HLA Class II by keratinocytes is known to enhance their antigen-presenting properties, the present results support the possibility that immunological events play a role in the disruption of the normal processes of wound healing and tissue remodelling which result in hypertrophic scars.

TNF production and hypertrophic scarring.

The percentage of TNF alpha- and beta-positive cells was analyzed in hypertrophic scar (N = 13), normotrophic scar (N = 7), and normal skin (N = 6) biopsies using monoclonal antibodies and immunoperoxidase staining of cryostat tissue sections. Samples were first characterized for infiltrating cells. In hypertrophic samples there was a significant increase in activated infiltrating cells, capable of producing TNF beta and IL-1 beta. In contrast, the percentage of TNF alpha-positive cells was significantly lower than that detected in normotrophic scars. In fact, in hypertrophic scar samples a positive staining with anti-TNF alpha mAb was restricted to 8% of tissue-infiltrating cells compared to 35.4% of the cells present in normotrophic scars; 12% of infiltrating cells were stained in normal skin sections. These results suggest that TNF alpha may be important for normal wound healing and that hypertrophic scarring might be partially a consequence of a low amount of TNF alpha.