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.     

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.     

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.     

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.     

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.     

Control of wound contraction. Basic and clinical features

Although a substantial amount of molecular and cellular data have been generated in an effort to understand the process of wound contraction and scar contracture formation, questions remain. What seems apparent is that the myofibroblast is not the only cell that generates contractile forces within wounds, but it does appear to be intrinsically linked to the development of hypertrophic scars. The supposition that the formation of scar contractures is solely the result of a continuation of wound contraction is an oversimplification. Figure 4 provides a model of the possible evolution of contractile forces during the wound healing process and their role in the development of scar contractures. Migration of fibroblasts into and through the extracellular matrix during the initial phase of wound healing, prior to the expression of alpha-SMA, appears to be a fundamental component of wound contraction. During this migration, the pulling of collagen fibrils into a streamlined pattern in their wake, and the associated production of collagenase, may facilitate a more normal arrangement of collagen. Once the wound has been repopulated and the chemotactic gradient that was established by inflammatory cells is decreased, fibroblast migration will cease. It is at this point that myofibroblasts appear and play a key role in the production of hypertrophic scars, given that their prolonged presence and over-representation are hallmarks of this pathology. One of the pivotal differences between wounds that proceed to normal scar compared with those that develop hypertrophic scars and scar contractures may be a lack (or late induction) of myofibroblast apoptotic cell death. The combined contribution of fibroblasts and myofibroblasts to abnormal extracellular matrix protein production results in an excessive and rigid scar. The isometric application of contractile forces by myofibroblasts probably contributes to the formation of the whorls, nodules, and scar contractures characteristic of hypertrophic scars. Because the prolonged presence of myofibroblasts, producing an imbalance in extracellular matrix proteins and proteases, probably exacerbates hypertrophic scars and wound contraction, accelerating the rate of apoptotic cell death to reduce the cell number to that seen in normal scar may be a useful strategy for providing effective and efficient treatment of scar contracture.     

定量PCR技术检测增生性瘢痕组织纤维连接蛋白的基因表达

目的探讨纤维连接蛋白的表达水平与创伤后增生性瘢痕形成的关系。方法采用定量ＰＣＲ技术，对纤维连接蛋白（Ｆｉｂｒｏｎｅｃｔｉｎ，ＦＮ）在正常皮肤和增生性瘢痕中ｍＲＮＡ表达水平的变化进行比较。结果在增生性瘢痕中，ＦＮ的ｍＲＮＡ表达水平较正常皮肤增高。结论实验结果表明细胞外基质成分的变化在创伤修复失控形成中起重要作用。     

定量PCR技术检测增生性瘢痕组织纤维连接蛋白的基因表达

目的 探讨纤维连接蛋白的表达水平与创伤后增生性瘢痕形成的关系。方法 采用定量ＰＣＲ技术，对纤维连接蛋白在正常皮肤和增生性瘢痕中ｍＲＮＡ表达水平的变化进行比较。结果 在增生性瘢痕中，ＦＮ的ｍＲＮＡ表达水平较正常皮肤增高。结论 实验结果表明细胞外基质成分的变化在创伤修复失控形成中起重要作用。     

定量PCR技术检测增生性瘢痕组织纤维连接蛋白的基因表达

目的探讨纤维连接蛋白的表达水平与创伤后增生性瘢痕形成的关系。方法采用定量PCR技术,对纤维连接蛋白(Fibronectin,FN)在正常皮肤和增生性瘢痕中mRNA表达水平的变化进行比较。结果在增生性瘢痕中,FN的mRNA表达水平较正常皮肤增高。结论实验结果表明细胞外基质成分的变化在创伤修复失控形成中起重要作用。     

血小板衍生生长因子对成纤维细胞合成胶原的影响

目的 探讨血小板衍生生长因子（ＰＤＧＦ－ＡＢ）促进伤口愈合的作用机理及其在瘢痕增生中的作用。方法 取体外培养的人正常皮肤及增生性瘢痕成纤维细胞,用原位杂交法观察ＰＤＧＦ对两种细胞表达Ｐｒｏα１（Ⅲ）ｍＲＮＡ的影响。结果;ＰＤＧＦ－ＡＢ对两种成纤维细胞表达Ｐｒｏ α１（Ⅲ）ｍＲＮＡ均有明显促进作用,且均呈剂量依赖性关系,但作用有差异。     

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.     

皮肤伤口减张器抑制切口瘢痕作用的临床观察

目的 探索皮肤伤口减张器在增生性瘢痕和瘢痕疙瘩切除术后,减少切口瘢痕形成和抑制瘢痕增生的作用。方法 选取10例增生性瘢痕和瘢痕疙瘩患者,行病理性瘢痕切除术,采用自身对照的方法,将瘢痕术后切口分为减张组和对照组。减张组瘢痕术后切口局部使用皮肤减张器,对照组瘢痕术后切口不使用减张器。定期随访3个月,比较两组瘢痕宽度、温哥华瘢痕量表评分,并进行统计分析。结果 与对照组相比,术后1、3个月减张组术后瘢痕宽度较对照组窄,温哥华瘢痕量表评分低,差异显著(P<0.05)。结论 皮肤伤口减张器对抑制手术后皮肤切口张力导致的瘢痕形成具有明确作用,能在一定程度上缩小瘢痕宽度、抑制瘢痕增生,从而提高增生性瘢痕和瘢痕疙瘩的手术治疗效果。     

mRNA Expression and Protein Distribution of Fibronectin Splice Variants and High-Molecular Weight Tenascin-C in Different Phases of Human Fracture Healing

Fracture healing is a reparative physiological process, which proceeds in stages, each characterized by the predominant tissue in the fracture gap. The tissue matrix is continuously reorganized by cell migration, proliferation, and differentiation. Adhesive proteins such as fibronectin and tenascin transmit information between matrix and cells. As a result of alternative splicing of pre-RNA, EDA + fibronectin, EDB + fibronectin, and high-molecular weight (hm) tenascin-C are generated. By definition, EDB + fibronectin is an oncofetal protein because it is extremely rare in normal adult tissue and plasma, whereas it is expressed in fetal and tumor tissues and during wound healing. In this study, we for the first time describe EDA + fibronectin, EDB + fibronectin, and hm tenascin-C expression in human fracture gap tissue during various stages of differentiation. We demonstrate mRNA expression of all three splice variants in the initial fibrin matrix with upregulation in the enchondral ossification/osteoid and woven bone stages. Of all variants, EDA + fibronectin mRNA has the highest concentration in all stages. For the analysis, we used LightCycler-based relative mRNA quantification and immunohistochemistry. Our data demonstrate that EDA + fibronectin and hm tenascin-C show a diffuse distribution pattern in fracture gap connective tissue, while EDB + fibronectin is focally concentrated in osteoblastic cells at the margins of woven bone. EDA + fibronectin and hm tenascin represent markers for active granulation processes, whereas EDB + fibronectin is specific for cells forming the enchondral and osteoid matrix. The possibility of stimulating fracture healing by EDB + fibronectin-cytokine complexes should be tested in further investigations.     

瘢痕疙瘩和增生性瘢痕表皮异常的实验研究

目的 探讨瘢痕疙瘩和增生性瘢痕的表皮异常。方法 采用免疫组织化学方法,检测腱糖蛋白C(Tn-C),角蛋白16(CK-16)和增殖相关核抗原Ki-67蛋白在瘢痕疙瘩、增生性瘢痕和正常成人表皮中的表达。取正常成人皮肤组织RNA,构建正义、反义Tn-C mRNA探针,运用原位杂交技术,观测瘢痕疙瘩、增生性瘢痕和正常皮肤表皮中Tn-C mRNA的表达。结果 瘢痕疙瘩、增生性瘢痕表皮角质形成细胞增生明显高于正常皮肤。Tn-C mRNA在瘢痕疙瘩表皮的表达明显高于其在增生性瘢痕及正常皮肤表皮中的表达。CK-16,Ki-67蛋白在瘢痕疙瘩和增生性瘢痕表皮中表达增加。结论 瘢痕疙瘩和增生性瘢痕的表皮存在增生分化异常,尤以瘢痕疙瘩更为明显。     

瘢痕成纤维细胞中cyclin D1、p16的表达及关系研究

目的:了解细胞周期蛋白D1、p16在病理性瘢痕中的表达及它们之间的相互关系,以探讨他们在瘢痕形成过程中的作用。方法:采用免疫组化(SP法)对8例成熟瘢痕、11例增生性瘢痕、11例瘢痕疙瘩及8例正常皮肤组织进行染色,观察CyclinD1和p16在不同组织中的表达。结果:正常皮肤及普通瘢痕成纤维细胞中CyclinD1、p16均为阴性;增生性瘢痕与瘢痕疙瘩成纤维细胞中CyclinD1、p16与正常皮肤相比均有极显著性差异(P<0.05);瘢痕疙瘩成纤维细胞CyclinD1表达高于增生性瘢痕,且有显著性差异(P<0.05);p16在瘢痕疙瘩成纤维细胞的表达比增生性瘢痕为高,但两者之间没有统计学差异.结论:CyclinD1、p16在病理性瘢痕的发生及发展中起重要的作用。在瘢痕疙瘩里p16的细胞抑制作用可能无法与CyclinD1的促细胞增殖作用相拮抗,所以细胞呈现持续增殖状态;而在增生性瘢痕里CyclinD1与p16可能处于相对的平衡状态,所以其生长具有一定的自限性。     

Basic fibroblast growth factor (bFGF) alleviates the scar of the rabbit ear model in wound healing

Studies suggest a possible antiscarring effect of basic fibroblast growth factor (bFGF) during wound healing. However, little is known about the precise pathological mechanisms of bFGF. In particular, there is only limited information available about the mechanism of exogenous administration of bFGF to scar formation. To investigate the effect of bFGF on the hypertrophic scar in the rabbit ear model and to clarify the mechanisms of bFGF on treatment for scar in wound healing, the rabbit ear model of wound healing was created and treated topically with bFGF once daily for 3 months; then we examined the changes of macroscopic and histopathological characteristics of scars and the expression of collagen and collagenase-1 (matrix metalloproteinase-1). The results of macroscopic and histologic characteristics revealed a significant difference between scars treated with bFGF and control scars. The expression of collagen in the scars treated with bFGF was decreased, as compared with the scars treated with saline. Further study revealed that bFGF could remarkably enhance expression of matrix metalloproteinase-1. bFGF could improve the quality of wound healing and remarkably alleviate the scar in the rabbit ear model in wound healing, which suggests that bFGF exerted a net negative effecton scar formation in wound healing. The evidence should contribute to a better understanding of the biological activities of bFGF during hypertrophic scar formation.     

性激素受体在瘢痕中的表达及与细胞周期调控蛋白相互关系的研究

目的　了解雄激素受体 (AR)、雌激素受体 (ER)在病理性瘢痕中的表达及其与细胞周期调节蛋白D1(cyclinD1)、p16之间的相互关系 ,以探讨他们在瘢痕形成过程中的作用及机制。方法　采用免疫组化方法 (SP法 )对 30例瘢痕标本进行研究 ,以正常皮肤组织为对照 ,观察上述指标的表达。结果　正常皮肤及普通瘢痕成纤维细胞中所有指标均为阴性 ;增生性瘢痕与瘢痕疙瘩成纤维细胞中cyclinD1、p16、AR与正常皮肤相比差异均有显著性意义 (P <0 0 5 ) ;瘢痕疙瘩成纤维细胞cyclinD1和AR的表达高于增生性瘢痕 ,且有显著性意义 (P <0 0 5 ) ;p16在瘢痕疙瘩成纤维细胞的表达比增生性瘢痕为高 ,但两者之间差异无显著性意义。在病理性瘢痕中cyclinD1和AR的表达具有明显的相关性。结论　AR在病理性瘢痕的发生及发展中起一定的作用 ,它可能是通过与其配体结合后促使与cyclinD1有关的基因表达而发挥作用的。在瘢痕疙瘩里可能存在cyclinD1的促细胞增生作用超过P16细胞抑制 ,所以细胞呈现持续增殖状态 ;而在增生性瘢痕里cyclinD1与p16可能处于相对的平衡状态 ,细胞生长具有一定的自限性。     

Extracellular matrix protein turnover during salamander limb regeneration

After amputation of a salamander limb, the extracellular matrix undergoes remodeling. The extracellular matrix that maintains the differentiated state of limb tissues is broken down and replaced by an extracellular matrix essential for dedifferentiation and blastema formation. We used monoclonal antibodies in immunohistochemistry methods and riboprobes in in situ hybridization to evaluate the upregulation of tenascin, type XII collagen, fibronectin, and the MT5 antigen. The Stump 1 antigen, an extracellular matrix protein that is abundant in the normal limb, is downregulated during regeneration and reappears late in regeneration as differentiation occurs. In the embryo, the Stump 1 antigen is also absent from the early limb bud and first appears during differentiation stages. Tenascin and fibronectin are also upregulated in the limb bud of the embryo, and these two extracellular matrix proteins appear to function during limb regeneration in adults and limb development in embryos. However, type XII collagen and the MT5 antigen are not found in the limb bud, indicating that type XII collagen and the MT5 antigen have roles in the regenerating limb but not in the embryo limb bud.     

Wound healing in oral mucosa results in reduced scar formation as compared with skin: Evidence from the red Duroc pig model and humans

Scar formation is a common, unwanted result of wound healing in skin, but the mechanisms that regulate it are still largely unknown. Interestingly, wound healing in the oral mucosa proceeds faster than in skin and clinical observations have suggested that mucosal wounds rarely scar. To test this concept, we created identical experimental wounds in the oral mucosa and skin in red Duroc pigs and compared wound healing and scar development over time. We also compared the pig oral mucosal wound healing to similar experimental wounds created in human subjects. The findings showed significantly reduced scar formation at both clinical and histological level in the pig oral mucosa as compared with skin 49 days after wounding. Additionally, the skin scars contained a significantly increased number of type I procollagen immunopositive cells and an increased fibronectin content, while the oral mucosal wounds demonstrated a prolonged accumulation of tenascin-C. Furthermore, the pig oral mucosal wounds showed similar molecular composition and clinical and histological scar scores to human oral mucosal wounds. Thus, the reduced scar formation in the pig oral mucosa provides a model to study the biological processes that regulate scarless wound healing to find novel approaches to prevent scar formation in skin.     

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.