Molecular dissection of abnormal wound healing processes resulting in keloid disease

Keloids are locally aggressive scars that typically invade into healthy surrounding skin and cause both physical and psychosocial distress to the patient. These pathological scars occur following minimal skin trauma after a variety of causes including burns and trauma. Although the pathogenesis of keloid disease is not well understood, it is considered to be the end product of an abnormal healing process. The aim of this review was to investigate the molecular and cellular pathobiology of keloid disease in relation to the normal wound healing process. The molecular aberrances in keloids that correlate with the molecular mechanisms in normal wound healing can be categorized into three groups: (1) extracellular matrix proteins and their degradation, (2) cytokines and growth factors, and (3) apoptotic pathways. With respect to cellular involvements, fibroblasts are the most well‐studied cell population. However, it is unclear whether the fibroblast is the causative cell; they are modulated by other cell populations in wound repair, such as keratinocytes and macrophages. This review presents a detailed account of individual phases of the healing process and how they may potentially be implicated in aberrant raised scar formation, which may help in clarifying the mechanisms involved in keloid disease pathogenesis.     

Resveratrol inhibits fibrogenesis and induces apoptosis in keloid fibroblasts

Keloids are benign dermal fibrotic tumors arising during the wound healing process. The mechanisms of keloid formation and development still remain unknown, and no effective treatment is available. Resveratrol, a dietary compound, has anticancer properties and, from recent studies, it has been suggested that resveratrol may have an antifibrogenic effect on organs such as the liver and kidney. Based on this idea, we investigated its effect on the regulation of extracellular matrix expression, proliferation, and apoptosis of keloid fibroblasts. Type I collagen, α‐smooth muscle actin, and heat shock protein 47 expression decreased in resveratrol‐treated keloid fibroblasts in a dose‐dependent manner. In addition, resveratrol diminished transforming growth factor‐β1 production by keloid fibroblasts. We also demonstrated that it suppressed their proliferation and induced apoptosis of the fibroblasts. Conversely, resveratrol did not decrease type I collagen, α‐smooth muscle actin, and heat shock protein 47 mRNA expression in normal skin fibroblasts and barely suppressed cell proliferation. Our data indicate that resveratrol may have an antifibrogenic effect on keloid fibroblasts without any adversely effects on normal skin fibroblasts, suggesting the potential application of resveratrol for the treatment of keloids.     

Gene expression patterns in isolated keloid fibroblasts

Keloid scars after skin trauma are a significant clinical problem, especially in black populations, in which the incidence of keloids has been estimated at 4-16%. Keloids are abnormal dermal proliferative scars secondary to dysregulated wound healing. Despite several biochemical studies on the role of extracellular matrix proteins and growth factors during keloid formation, we still do not know what molecules and signals induce this change. Fibroblasts are thought to be the major inductive cell for keloid scar formation. The aim of this study was to identify gene expression patterns that characterize keloid fibroblasts; identifying such genetic disequilibrium may shed light on the molecular signaling events responsible for keloid formation. In this study, we performed gene expression analysis of fibroblasts isolated from keloid lesions from three individuals in comparison with the fibroblasts isolated from normal skin using the Affymetrix U133a chip (22,284 genes and expression sequence tags). We found through J5 test score expression analysis that among 22,284 genes, there were 43 genes that were overexpressed and five genes were underexpressed in keloid fibroblasts when compared with dermal fibroblasts from persons without keloids. The overexpression of three genes not previously reported as being up-regulated in keloids (annexin A2, Transgelin, and RPS18) was confirmed by real-time polymerase chain reaction. Certain overexpressed genes were similar to previous biochemical observations on the protein levels of these overexpressed genes during keloid formation. We also report for the first time that a few tumor-related genes are overexpressed in keloid fibroblasts.     

Normal and keloid fibroblasts are differentially influenced by IFN‐γ and triamcinolone as well as by their combination

Impaired wound healing as well as imbalanced cell proliferation and extracellular matrix synthesis and degeneration can cause aberrant scarring. The most severe impacts of such scarring on patients' lives are stigmatization and physical restriction. Although, a broad variety of combinatorial approaches with, e.g., glucocorticoids, chemotherapeutics, and immunomodulators are used, there is still a high recurrence rate of keloids. The aim of this study was to investigate which influence interferon γ (IFN‐γ, 1.000–10.000 IU/mL) and/or triamcinolone acetonide (TA, 1 μg/mL) have on proliferation, cell viability, collagen type I synthesis, and cytokine secretion in healthy and keloid fibroblasts. It was shown that mono‐treatment with IFN‐γ or TA for 2 days induced a severe reduction of the proliferative potential in both cell species. The combinatory treatment (IFN‐γ plus TA) of keloid fibroblasts enhanced the anti‐proliferative effect of the mono‐treatments, whereas no additional anti‐proliferative effect was observed in normal fibroblasts. Furthermore, we observed that the combinatory treatment regimen reduced the expression of α‐smooth muscle actin (α‐SMA), an actin isotype contributing to cell‐generated mechanical tension, in keloid fibroblasts. In normal fibroblasts, α‐SMA was reduced by the mono‐treatment with IFN‐γ as well as by the combinatory treatment. The analysis of collagen‐type I synthesis revealed that TA did not reduce collagen type I synthesis in normal fibroblasts but in keloid fibroblasts. IFN‐γ reduced in both cell species the collagen type I synthesis. The combination of TA and IFN‐γ intensified the previously observed collagen type I synthesis reduction in keloid fibroblasts. The herein presented data suggest the combinatory application of IFN‐γ and TA as a promising therapy concept for keloids.     

Identification of unique gene expression patterns within different lesional sites of keloids

Keloid disease is a significant clinical problem, especially in black populations, with an estimated incidence of 4–16%. Keloids are fibroproliferative dermal tumors developing as a result of deregulated wound healing. The dynamic nature of keloids is illustrated by clinical regression in the center, while the margin remains active growing into the surrounding healthy skin. Therefore, the gene expression profiles of fibroblasts from different sites of the keloids were characterized using Affymetrix microarrays covering the whole human genome. This study revealed 105 genes that were differentially regulated (79 genes were up‐regulated and 26 down‐regulated) in a unique gene expression profile in different sites of keloids where progression or regression of the process was in progress. The apoptosis inhibitor AVEN was found to be up‐regulated at the active margin of keloids, while apoptosis‐inducing genes such as ADAM12 and genes inducing extracellular matrix (ECM) degradation such as matrix metalloproteinase‐19 were up‐regulated in the regressing keloid center. We identified genes previously not described in the development of keloids. Activating proapoptotic genes or inhibiting ECM‐inducing genes as INHBA or monocyte chemoattractant protein‐1 might be possible target genes for new treatment strategies for keloid disease.     

Keloid fibroblast responsiveness to epidermal growth factor and activation of downstream intracellular signaling pathways.

Keloids, which overgrow the boundaries of the original injury, represent aberrations in the fundamental process of wound healing that include over-abundant cell in-migration, cell proliferation, and inflammation, as well as increased extracellular matrix synthesis and defective remodeling. To understand the key events that result in the formation of these abnormal scars would open new avenues for better understanding of excessive repair, and might provide new therapeutic options. We examined epidermal growth factor receptor (EGFR)-induced cell motility in keloid fibroblasts, as this receptor initiates cell migration during normal wound repair. We show that keloid fibroblasts respond to EGF-induced cell migration but the response is somewhat diminished compared to normal adult fibroblasts (approximately 30% reduced); the mitogenic response was similarly blunted (approximately 5% reduced). Keloid fibroblasts express near normal levels of EGFR (82%), but show a much more attenuated activation of EGFR itself and the motility-associated phospholipase C-gamma. This was reflected in part by rapid loss of EGFR upon exposure to EGF. Interestingly, while extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) activation was relatively robust in keloid fibroblasts, the downstream triggering of the motility-associated calpain activity was blunted. This was reflected by high cell-substratum adhesiveness in the keloid fibroblasts. Thus, the blunted migratory response to EGF noted in keloid fibroblasts appears due to limited activation of two important biochemical switches for cell motility.     

Different properties of skin of different body sites: The root of keloid formation?

The purpose of this study was to examine extracellular matrix composition, vascularization, and immune cell population of skin sites prone to keloid formation. Keloids remain a complex problem, posing esthetical as well as functional difficulties for those affected. These scars tend to develop at anatomic sites of preference. Mechanical properties of skin vary with anatomic location and depend largely on extracellular matrix composition. These differences in extracellular matrix composition, but also vascularization and resident immune cell populations might play a role in the mechanism of keloid formation. To examine this hypothesis, skin samples of several anatomic locations were taken from 24 human donors within zero to 36 hours after they had deceased. Collagen content and cross‐links were determined through high‐performance liquid chromatography. The expression of several genes, involved in extracellular matrix production and degradation, was measured by means of real‐time PCR. (Immuno)histochemistry was performed to detect fibroblasts, collagen, elastin, blood vessels, Langerhans cells, and macrophages. Properties of skin of keloid predilections sites were compared to properties of skin from other locations (nonpredilection sites [NPS]). The results indicated that there are site specific variations in extracellular matrix properties (collagen and cross‐links) as well as macrophage numbers. Moreover, predilection sites (PS) for keloid formation contain larger amounts of collagen compared to NPS, but decreased numbers of macrophages, in particular classically activated CD40 positive macrophages. In conclusion, the altered (histological, protein, and genetic) properties of skin of keloid PS may cause a predisposition for and contribute to keloid formation.     

Sulforaphane suppresses cell growth and collagen expression of keloid fibroblasts

Keloids are fibroproliferative diseases characterized by the accumulation of an extracellular matrix including collagen. Various growth factors, or cytokines, and their receptors are overexpressed in keloids, and they are expected to be therapy targets. Sulforaphane, a dietary isothiocyanate, has recently shown anti‐tumor, anti‐inflammatory, and anti‐fibrotic properties. In this study, we found that sulforaphane inhibited cell growth and reduced collagen at the mRNA and protein levels in keloid fibroblasts. Moreover, sulforaphane markedly suppressed the expression of IL‐6 and α‐SMA and inhibited Stat3 and Smad3 signaling pathways in keloid fibroblast KF112 cells. Sulforaphane induced G2/M cell‐cycle arrest with the induction of p21 in KF112 cells. In addition, sulforaphane inhibited cell growth and suppressed the expression of collagen in keloid fibroblasts under a coculture with peripheral blood mononuclear cells. Furthermore, sulforaphane suppressed IL‐6, Stat3, and Smad3 signaling in the coculture system. This study suggests that sulforaphane may be a novel keloid treatment.     

Expression of apoptosis-associated genes by human dermal scar fibroblasts

The purpose of this study was to determine if aberrant apoptosis plays a role in pathologic wound healing as manifested by hypertrophic scarring and keloid formation. Apoptosis has recently been found to participate in the transition between granulation tissue and the development of definitive scar. The question that remains to be answered is what stimuli initiate apoptosis during wound healing. Hitherto, regulatory factors and pathways involved have been largely undefined. We investigated heterogeneity among fibroblasts derived from normal skin and keloid scar, by examining apoptotic profiles and pathways for these cells. Quantitative analysis of apoptotic cells using an Annexin-V-FITC binding assay showed that normal skin fibroblast cultures were found to have a two-fold higher percentage of apoptotic cells than did keloid fibroblast cultures. To study apoptotic pathways and related death-associated genes, a ribonuclease protection assay was performed for fibroblasts exposed to anti-Fas antibody and tumor necrosis factor-alpha to activate the Fas/TNF receptor apoptotic pathway. Compared with normal skin fibroblasts, keloid fibroblasts exhibited decreased expression of apoptosis-associated genes.     

瘢痕疙瘩成纤维细胞的基因组学研究

目的 寻找瘢痕疙瘩致病相关基因，探讨瘢痕疙瘩的发生机理。方法 利用含1100个人类肿瘤相关基因的cDNA芯片(cDNA—microarray)对耳垂和胸部瘢痕疙瘩及正常皮肤成纤维细胞进行检测，初步分析瘢痕疙瘩成纤维细胞与正常皮肤成纤维细胞基因总体表达的差异，并筛选出差异基因。结果 在耳垂及胸部瘢痕疙瘩成纤维细胞中，分别有8种和17种特异性表达基因被检出。在正常皮肤中特异性表达的细胞增殖抑制基因Mda-7，在耳垂及胸部瘢痕疙瘩成纤维细胞中均未被表达。结论 多种基因参与了瘢痕疙瘩的形成过程，瘢痕疙瘩成纤维细胞与正常皮肤成纤维细胞之间存在基因表达的差异，增殖因子受体PAR-1和增殖抑制基因Mda-7可能参与瘢痕疙瘩的形成。     

Suppression of transforming growth factor beta/smad signaling in keloid-derived fibroblasts by quercetin: implications for the treatment of excessive scars

BACKGROUND: Keloids are characterized by abnormal proliferation and overproduction of extracellular matrix. Quercetin, a dietary compound, has strong antioxidant and anticancer properties. Previous studies by the authors have shown that quercetin inhibits fibroblast proliferation, collagen production, and contraction of keloid and hypertrophic scar-derived fibroblasts. Quercetin also blocks the signal transduction of insulin-like growth factor-1 in keloid fibroblasts. This study assessed the effects of quercetin on the transforming growth factor (TGF)-beta/Smad-signaling pathway in keloid-derived fibroblasts, which may be an important biologic mechanism of this proliferative scarring. METHODS: Keloid fibroblasts were isolated from keloid tissue specimens. Cells were treated with quercetin at different concentrations, then harvested, and subjected to immunoblotting analysis. RESULTS: Quercetin significantly inhibited the expression of TGF-beta receptors 1 and 2 in keloid fibroblasts at three concentrations (low, medium, and high). Quercetin also strongly suppressed the basal expression of Smad2, Smad3, and Smad4 as well as the phosphorylation of Smad2 and Smad3 and the formation of the Smad2-Smad3-Smad4 complex. CONCLUSIONS: Taken together, these data suggest that quercetin effectively blocks the TGF-beta/Smad-signaling pathway in keloid fibroblasts. These data indicate that quercetin-based therapies for keloids should be investigated further.     

Decreased expression of inhibitory SMAD6 and SMAD7 in keloid scarring.

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterised by an abnormal deposition of extracellular matrix components, in particular collagen. There is evidence that transforming growth factor-beta (TGFbeta) is involved in keloid formation. SMAD proteins play a crucial role in TGFbeta signaling and in terminating the TGFbeta signal by a negative feedback loop through SMAD6 and 7. It is unclear how TGFbeta signaling is connected to the pathogenesis of keloids. Therefore, we investigated the expression of SMAD mRNA and proteins in keloids, in normal skin and in normal scars. Dermal fibroblasts were obtained from punch-biopsies of keloids, normal scars and normal skin. Cells were stimulated with TGFbeta1 and the expression of SMAD2, 3, 4, 6 and 7 mRNA was analysed by real time RT-PCR. Protein expression was determined by Western blot analysis. Our data demonstrate a decreased mRNA expression of the inhibitory SMAD6 and 7 in keloid fibroblasts as compared to normal scar (p<0.01) and normal skin fibroblasts (p<0.05). SMAD3 mRNA was found to be lower in keloids (p<0.01) and in normal scar fibroblasts (p<0.001) compared to normal skin fibroblasts. Our data showed for the first time a decreased expression of the inhibitory SMAD6 and SMAD7 in keloid fibroblasts. This could explain why TGFbeta signaling is not terminated in keloids leading to overexpression of extracellularmatrix in keloids. These data support a possible role of SMAD6 and 7 in the pathogenesis of keloids.     

Expression of transforming growth factor beta 1, 2, and 3 proteins in keloids.

Keloids represent a pathological response to cutaneous injury, creating disfiguring scars with no known satisfactory treatment. They are characterized by an excessive accumulation of extracellular matrix, especially collagen. Transforming growth factor beta (TGF-beta) has been implicated in the pathogenesis of keloids. The three TGF-beta isoforms identified in mammals (TGF-beta1, -beta2, and -beta3), are thought to have different biological activities in wound healing. TGF-beta1 and TGF-beta2 are believed to promote fibrosis and scar formation, whereas TGF-beta3 has been shown to be either scar inducing or reducing, depending on the study. The aim of this study was to characterize expression of TGF-beta isoforms in keloids at the protein level using Western blot analysis. The authors found that TGF-beta1 and -beta2 proteins were at higher levels in keloid fibroblast cultures compared with normal human dermal fibroblast cultures. In contrast, the expression of TGF-beta3 protein was comparable in both the normal (N = 3) and keloid (N = 3) cell lines. These findings, demonstrating increased TGF-beta1 and -beta2 protein expression in keloids relative to normal human dermal fibroblasts further support the roles of TGF-beta1 and -beta2 as fibrosis-inducing cytokines.     

Double‐edged effects of neuropeptide substance P on repair of cutaneous trauma

To explore further the role of substance P (SP) in wound healing and scar formation, SP concentrations in wounds of scalded rats were assayed. Expressions of apoptosis‐associated genes in fibroblasts cultured with SP were detected. SP concentrations in superficial wounds increased earlier than those in deep wounds. SP was associated with an increased proliferation and a decreased apoptosis of fibroblasts. It had a greater influence on keloid fibroblasts than on hypertrophic scar fibroblasts by elevating the expression of proliferating cell nuclear antigen and BCL‐2 in fibroblasts. Spantide completely suppressed the effects of SP on hypertrophic scar fibroblasts, and partly inhibited its effects on keloid scar fibroblasts. SP may play an important role in wound healing by promoting wound fibroblast proliferation and inhibiting apoptosis. It may also participate in pathological scar formation by modulating the expression of apoptosis‐associated genes. SP is postulated to play a dual role in wound repair.     

Perturbation of cell cycle expression in keloid fibroblast

The pathogenesis of keloid formation is poorly understood. The fibroblasts in keloid patients continue to multiply even after initial wound repair and are characterized by a persistent dermal fibroproliferative reaction and excessive extracellular matrix production. Most studies concentrate on the type of collagen produced within keloids and the cytokines that dominate the disease. There have been considerably fewer studies in the expression of messenger RNA level in key cell cycle genes of the keloid fibroblast. The aim of this study was to measure the messenger RNA expression of the key regulators of cell cycle, cell cycle cyclins, and cyclin-dependent kinases, and their inhibitors.     

Fibronectin gene expression differs in normal and abnormal human wound healing

The overproduction of fibronectin and type I collagen in keloids and hypertrophic scars implicates altered regulation of extracellular matrix components as an important aspect of these wound healing pathologies. However, little is known about the similarities and differences in extracellular matrix gene expression during normal and abnormal wound healing. This study compared the content of fibronectin messenger RNA and rates of fibronectin protein biosynthesis in fibroblasts derived from normal skin, normal scar, keloid, and hypertrophic scar. Fibronectin expression was enhanced in cells from both normal and abnormal wounds relative to cells from quiescent normal skin. Matched pairs of normal and keloid fibroblasts from the same individuals were also compared, and three of the four pairs showed higher fibronectin expression by the keloid cells at the levels of messenger RNA and protein synthesis. This was consistent with previous studies showing elevated steady state content of fibronectin in keloid cells relative to normal cells from the same individual. Fibronectin messenger RNA and protein content in the tissues from which these cells were derived was examined by in situ hybridization and immunohistochemistry. These studies revealed that in vivo, the steady state content of fibronectin messenger RNA and protein was highest in abnormal wounds, less in most normal scars, and lowest in normal skin. Thus, fibroblasts from keloids and hypertrophic scars overexpressed fibronectin in vivo relative to normal skin and normal scar and retain this characteristic in vitro relative to normal skin. Although normal scars contained little fibronectin protein and messenger RNA, cultured fibroblasts derived from these scars had contents of fibronectin messenger RNA and rates of biosynthesis in vitro similar to those of keloid fibroblasts. This indicates that the fibronectin regulatory pathway in scar fibroblasts is influenced by the tissue environment. These results are discussed with respect to the relationship of fibronectin expression in keloids, hypertrophic scars, and normal wounds in human beings.