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.     

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.     

病理性瘢痕中原癌基因c-myc和c-fos的表达

目的　探讨原癌基因的表达与病理性瘢痕形成的相关性。方法　应用免疫组化SP法 ,检测c -myc和c -fos蛋白在增生性瘢痕、瘢痕疙瘩和正常皮肤组织中的表达和分布 ,并用图像定量分析比较其差异。结果　在增生性瘢痕和瘢痕疙瘩的成纤维细胞中c-myc、c -fos呈强阳性表达 ,两组间无明显差异 ,而与正常皮肤对照组均有显著性差异。结论　增生性瘢痕与瘢痕疙瘩中c -myc、c -fos蛋白表达升高 ,存在c -myc和c -fos原癌基因的激活 ,可能参与了成纤维细胞的分化增殖或表型转化、胶原合成与降解以及对细胞因子的调控 ,并导致瘢痕增生。     

Phosphorylation of extracellular signal-regulated protein kinase in cultured keloid fibroblasts when stimulated by platelet-derived growth factor BB.

Abnormal scars result in distressing symptoms and disfiguring blemishes; an understanding of the molecular events that cause such scars, particularly keloids, would make possible the optimisation of both wound healing and treatment. Extracellular signal-regulated protein kinase (ERK) has a crucial role in distinct signalling pathways in different cells, but to date we know of no study on its signalling events in keloid fibroblasts. The purpose of this study was to characterise the expression of tyrosine phosphorylation kinases, particularly that of ERK, in keloids at the protein level by immunoblotting analysis. Studies on phosphorylation were made on cell lysates of three cultures of five different keloid fibroblasts (n = 5), their relatively 'normal' fibroblasts in adjacent skin (rNHDF, n = 5), and normal human dermal fibroblasts (n = 1, standard control). The result showed that ERK signalling molecular protein was more highly phosphorylated in keloid fibroblast culture than in the other two cultures.     

E2F1蛋白在病理性瘢痕组织中的表达

目的　增生性瘢痕和瘢痕疙瘩是临床上常见的病理性瘢痕 ,是创伤后过度愈合反应的结果 ,以成纤维细胞的异常增殖及合成分泌大量细胞外基质为特征 ,其形成机理尚不清楚 ,研究表明基因失调是其中的关键。E2F基因是细胞周期G1向S期过渡的重要调控因子 ,在调节细胞周期进程和调节细胞增殖过程中起着关键作用。本实验的目的是检测E2F1基因在病理性瘢痕组织中的表达 ,以正常皮肤组织做对照 ,初步探讨E2F1在病理性瘢痕形成中的生物学作用。方法　利用免疫组化ABC法检测正常皮肤、成熟瘢痕、增生性瘢痕和瘢痕疙瘩组织中E2F1蛋白的表达 ,并进行统计学分析。结果　增生性瘢痕和瘢痕疙瘩组织中E2F1蛋白表达两组间无明显差异 ,与正常皮肤、成熟瘢痕对照组比较均有显著性差异 (P <0 .0 1)。结论　E2F1蛋白表达在病理性瘢痕组织中增高 ,促进瘢痕组织中细胞的增生 ,对病理性瘢痕的形成可能起着重要作用     

Non‐animal models of wound healing in cutaneous repair: In silico,in vitro,ex vivo,and in vivo models of wounds and scars in human skin

Tissue repair models are essential to explore the pathogenesis of wound healing and scar formation, identify new drug targets/biomarkers and to test new therapeutics. However, no animal model is an exact replicate of the clinical situation in man as in addition to differences in the healing of animal skin; the response to novel therapeutics can be variable when compared to human skin. The aim of this review is to evaluate currently available non‐animal wound repair models in human skin, including: in silico, in vitro, ex vivo, and in vivo. The appropriate use of these models is extremely relevant to wound‐healing research as it enables improved understanding of the basic mechanisms present in the wound healing cascade and aid in discovering better means to regulate them for enhanced healing or prevention of abnormal scarring. The advantage of in silico models is that they can be used as a first in virtue screening tool to predict the effect of a drug/stimulus on cells/tissues and help plan experimental research/clinical trial studies but remain theoretical until validated. In vitro models allow direct quantitative examination of an effect on specific cell types alone without incorporating other tissue‐matrix components, which limits their utility. Ex vivo models enable immediate and short‐term evaluation of a particular effect on cells and its surrounding tissue components compared with in vivo models that provide direct analysis of a stimulus in the living human subject before/during/after exposure to a stimulus. Despite clear advantages, there remains a lack of standardisation in design, evaluation and follow‐up, for acute/chronic wounds and scars in all models. In conclusion, ideal models of wound healing research are desirable and should mimic not only the structure but also the cellular and molecular interactions, of wound types in human skin. Future models may also include organ/skin‐on‐a‐chip with potential application in wound healing research.     

A wound contraction experimental model for studying keloids and wound-healing modulators

Preventing and treating hypertrophic and keloid scars is difficult because of the lack of knowledge about their genesis. Tissue repair can be studied with biocompatible matrices and ex vivo cultures of different cell types. We used an experimental model where collagen gels populated by human fibroblasts underwent progressive contraction, allowing the study of wound healing remodeling. The fibroblast-populated lattices showed the greater contraction of the gel populated by fibroblasts from keloids versus fibroblasts from normal skin. Moreover, fibroblast growth factor (FGF) and transforming growth factor beta (TGF-beta) involved in scar formation were added to the collagen gels populated by normal skin fibroblasts. TGF-beta caused an increase in gel contraction; FGF did not. The mean percentages of contraction of the gels populated by keloid fibroblasts were very similar to the percentages of gels populated by normal skin fibroblasts with added TGF-beta. These observations confirm the existing hypothesis that TGF-beta may be involved in keloid formation.     

The effect of the anti-allergic agent avil on abnormal scar fibroblasts.

Abnormal wound healing in humans leads to the formation of hypertrophic scar and keloids. These abnormal scars accumulate excessive extracellular matrix proteins through increased synthesis as well as decreased degradation. In order to find a therapeutic control for scar formation, we investigated the effect of avil (pheniramine maleate) on fibroblasts cultured from abnormal scars in comparison to normal skin. We observed a decrease in the proliferation rate in cells from normal skin (39%), hypertrophic scar (44%), keloid (63%) and in DNA synthesis in cells from normal skin (50%), hypertrophic scar (55%) and keloid (63%) treated with 8 mM avil (72 h). The rate of decrease in collagen synthesis in normal skin (44%), hypertrophic scar (74%) and keloid fibroblast (73%) correlated with changes in DNA synthesis.     

Transforming growth factor beta (TGFbeta) and keloid disease

Keloids are benign fibroproliferative diseases of unknown aetiology. They occur as a result of derangement of the normal wound healing process in susceptible individuals. Although several factors have been postulated in the aetiopathogenesis of this condition, there has been growing evidence to suggest a role for Transforming Growth Factor beta (TGFbeta) family members in its pathogenesis. TGFbeta has also been found to be associated with fibrotic diseases affecting different organs of the body including liver, kidney, lung as well as skin. In this review article, we will discuss the morphology and mechanism of action of TGFbeta and its isoforms and present the most up to date literature discussing the role of TGFbeta isoforms, their receptors, and intracellular signalling pathways (the SMAD pathway) in the pathogenesis of keloid disease. Understanding the role of TGFbeta in keloid disease could lead to the development of clinically useful therapeutic modalities for treatment of this condition.     

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.     

Up-regulation of α1-adrenoceptors in burn and keloid scars

Stimulation of α₁-adrenoceptors evokes inflammatory cytokine production, boosts neurogenic inflammation and pain, and influences cellular migration and proliferation. Hence, these receptors may play a role both in normal and abnormal wound healing. To investigate this, the distribution of α₁-adrenoceptors in skin biopsies of burn scars (N = 17), keloid scars (N = 12) and unscarred skin (N = 17) was assessed using immunohistochemistry. Staining intensity was greater on vascular smooth muscle in burn scars than in unscarred tissue, consistent with heightened expression of α₁-adrenoceptors. In addition, expression of α₁-adrenoceptors was greater on dermal nerve fibres, blood vessels and fibroblasts in keloid scars than in either burn scars or unscarred skin. These findings suggest that increased vascular expression of α₁-adrenoceptors could alter circulatory dynamics both in burn and keloid scars. In addition, the augmented expression of α₁-adrenoceptors in keloid tissue may contribute to processes that produce or maintain keloid scars, and might be a source of the uncomfortable sensations often associated with these scars.     

Multi‐dimensional models for functional testing of keloid scars: In silico,in vitro,organoid, organotypic,ex vivo organ culture,and in vivo models

Keloid scars are described as benign fibro‐proliferative dermal outgrowths that commonly occur in pigmented skin post cutaneous injury, and continue to grow beyond the boundary of the original wound margin. There is a lack of thorough understanding of keloid pathogenesis and thus keloid therapeutic options remain ill‐defined. In view of the poor response to current therapy and high recurrence rates, there is an unmet need in improving our knowledge and therefore in identifying targeted and effective treatment strategies in management of keloids. Keloid research however, is hampered by a lack of relevant animal models as keloids do not spontaneously occur in animals and are unique to human skin. Therefore, developing novel animal models and nonanimal models for functional evaluation of keloid cells and tissue for better understanding their pathobiology and response to putative candidate therapies are essential. Here, we present the key concepts and relevant emerging research on two‐dimensional and three‐dimensional cell and tissue models for functional testing of keloid scars. We will describe in detail current models including in vitro mono‐ and co‐cultures, multi‐cellular spheroids (organoids) and organotyopic cultures, ex vivo whole skin keloid tissue organ culture models as well as in vivo human patient models. Finally, we discuss the role played by time as the fourth dimension in a novel model that involves sequential temporal biopsies of human patients with keloids (a so called 4D in vivo human model). The use of these unique models will no doubt prove pivotal in identification of new drug targets as well as biomarkers, in functional testing of emerging novel therapeutics, and in enhancing our understanding of keloid disease biology.     

Detection of apoptosis in keloids and a comparative study on apoptosis between keloids, hypertrophic scars, normal healed flat scars, and dermatofibroma

Recent studies have suggested that the regulation of apoptosis during wound healing is important in scar establishment and the development of pathological scarring. In this study, we demonstrate that keloid fibroblasts can be identified as apoptotic cells because of their highly condensed chromatin and discrete nuclear fragments. To further reveal the phenomenon of apoptosis, we quantified the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in surgically resected tissues of keloids (N = 10), hypertrophic scars (N = 10), normal healed flat scars (N = 10), and dermatofibroma (N = 10). The number of TUNEL-positive cells was relatively low, but was significantly higher for the keloid group compared with the normally healed flat scar group (p = 0.004), suggesting reduced cell survival and increased apoptotic cell death in a subpopulation of keloid fibroblasts. Furthermore, the number of TUNEL-positive cells was significantly higher for the keloid group compared with the dermatofibroma group (p = 0.044), suggesting that a subpopulation of keloid fibroblasts may suppress tumorgenicity at a greater rate than dermatofibroma by undergoing cell death. Hypertrophic scars had significantly higher levels of apoptosis than normally healed flat scars (p = 0.033). Therefore, these results suggest that selected fibroblasts in keloids and hypertrophic scars undergo apoptosis, which may play a role in the process of pathological scarring.     

病理性瘢痕和正常皮肤成纤维细胞缝隙连接介导的细胞间通讯

目的 研究和比较不同来源的成纤维细胞缝隙连接介导的细胞间通讯的差异。方法 取手术切除的正常皮肤、增生性瘢痕、瘢痕疙瘩组织各6例,通过细胞培养6-8代后,应用粘附式细胞仪检测及比较各种来源的细胞缝隙连接介导的细胞间通讯。结果 正常皮肤成纤维细胞的细胞间通讯正常,增生性瘢痕成纤维细胞的细胞间通讯受到抑制,瘢痕疙瘩成纤维细胞的细胞间通讯被阻断。结论 细胞间通讯被证明与细胞生长的接触抑制及细胞的浸润性生长密切相关。细胞间通讯被阻断是瘢痕疙瘩呈“蟹足样”浸润性生长的细胞生物学机理之一。     

Normal Cutaneous Wound Healing: Clinical Correlation with Cellular and Molecular Events

BACKGROUND: Cutaneous wound healing is a normal physiologic function, observed and described for centuries by those afflicted with wounds and by those caring for them. Recently, tremendous progress has been made in discovering the cellular and molecular mechanisms responsible for wound healing. Counseling patients appropriately and planning future therapeutic interventions in delayed or abnormal wound healing may be improved by a thorough understanding of the relationship between clinical, cellular, and subcellular events occurring during the normal healing process. MATERIALS AND METHODS: A review of the wound healing literature from the past several decades, with a focus on the past 5 to 10 years in particular, along with illustrative case examples from our clinical practice over the past decade. RESULTS: Traditional clinical stages of wounding healing are still relevant, but more overlap between stages is likely a more accurate depiction of events. The role of cells such as platelets, macrophages, leukocytes, fibroblasts, endothelial cells, and keratinocytes is much better known, particularly during the inflammatory and proliferation stages of healing. Molecules such as interferon, integrins, proteoglycans and glycosaminoglycans, matrix metalloproteinases, and other regulatory cytokines play a critical role in the regulation of healing mechanisms. CONCLUSION: Cutaneous wound healing in normal hosts follows an orderly clinical process. The scientific underpinnings for healing are better understood than ever, although much remains to be discovered. Eventually, such improved understanding of cellular and subcellular physiology may lead to new or better forms of therapy for patients with acute, chronic, and surgical skin wounds.     

Observations of burn scars sustained by atomic bomb survivors; a preliminary study

A relatively high incidence of scar keloid and hypertrophic scar formation of a severe degree has occurred after healing of flash burns that probably were of deep second or third degree severity and in a people (Japanese), some of whom perhaps have a predisposition for the development of scar keloids. At this late date, the lack of complete, detailed, continuous studies and records on a large group of cases dating from the time of the occurrences of the injury and the large number of variables to be considered render impossible a complete evaluation and understanding of scar keloid formation. The inadequate treatment, poor nutrition, high incidence of severe infection and delayed healing should be considered as important contributing factors which affected the healing process to result in a high incidence of severe keloid or excessive scar formation. Scar keloids were found to occur in Japanese sustaining burns from other causes than the atomic bomb explosion. It would seem most probable that the scar keloids represent no peculiar effect of the atomic bomb explosion. Furthermore, it seems probable that a similar incidence of occurrence of scar keloids could have occurred in burns of the same severity from any other cause under similar conditions during the healing of the lesions in patients having the same general state of health.One gains the impression that the pathogenesis of keloids can be completely explained only by a better understanding of the detailed biophysical and biochemical processes which occur in the healing of skin lesions and how the initial conditions and possible later alterations in these steps influence the final result of the reparative process. Some individuals may be so constituted that they have a tendency to develop excessive amounts of scar tissue in the healing of wounds. The factors involved in the etiology of keloids are probably multiple.It is difficult to arrive at a differentiating working definition of scar keloids. It seems most probable that the differences between ordinary non-elevated scars, hypertrophic scars and scar keloids are only those of degree of amount of fibrous connective tissue produced during the healing process. From clinical data, histologic observations of various types of excised scars and reported experimental studies of the regeneration of skin in man, it would appear that the excess collagen production causing scar keloids and hypertrophic scars occurs when the lesion extends deep in the reticular layer of the dermis and, therefore, occurs usually in burns which extend to this depth initially, or later as a result of necrosis caused by infection or additional trauma. The necessity for early grafting of full thickness burns is again demonstrated. The necessity of preparation for the early care of burns in great numbers of casualties in the event of a catastrophe such as an atomic bombing of a populated area is obvious.     

CTGF在病理性瘢痕中的表达及意义

目的：了解细胞生长因子（connective tis sue growth factor，CTGF）在病理性瘢痕中的表达及意义，探讨它在病理性瘢痕发病机制中所起的作用.方法：对11例增生性瘢痕、10例瘢痕疙瘩及10例正常皮肤组织进行免疫组化（SP法）染色，观察CTGF在正常皮肤、增生性瘢痕、瘢痕疙瘩中的表达，以了解它们在不同组织中表达的差异性.结果：正常皮肤中CTGF的表达为阴性;增生性瘢痕、瘢痕疙瘩成纤维细胞中CTGF的表达与正常皮肤相比均有显著性差异（P＜0.01）;CTGF在瘢痕疙瘩成纤维细胞中的表达较增生性瘢痕为高，但两者之间没有统计学差异.结论：CTGF在增生性瘢痕的发病机制中发挥重要作用。