transforming-growth-factor-beta and Keloid

Keloid disorder, a group of fibroproliferative skin diseases, is characterized by unremitting accumulation of the extracellular matrix (ECM) of connective tissue, primarily collagen, to develop cutaneous tumors on the predilection sites of skin. There is a strong genetic predisposition for keloid formation, and individuals of African and Asian ancestry are particularly prone. The principal cell type responsible for ECM accumulation is the myofibroblast derived from quiescent resident skin fibroblasts either through trans-differentiation or from keloid progenitor stem cells with capacity for multi-lineage differentiation and self-renewal. The biosynthetic pathways leading to ECM accumulation are activated by several cytokines, but particularly by TGF-β signalling. The mechanical properties of the cellular microenvironment also play a critical role in the cell's response to TGF-β, as demonstrated by culturing of fibroblasts derived from keloids and control skin on substrata with different degrees of stiffness. These studies also demonstrated that culturing of fibroblasts on tissue culture plastic in vitro does not reflect their biosynthetic capacity in vivo. Collectively, our current understanding of the pathogenesis of keloids suggests a complex network of interacting cellular, molecular and mechanical factors, with distinct pathways leading to myofibroblast differentiation and activation. Keloids can serve as a model system of fibrotic diseases, a group of currently intractable disorders, and deciphering of the critical pathogenetic steps leading to ECM accumulation is expected to identify targets for pharmacologic intervention, not only for keloids but also for a number of other, both genetic and acquired, fibrotic diseases.

Topics: Animals; Cell Differentiation; Cell Transdifferentiation; Extracellular Matrix; Fibroblasts; Fibronectins; Gene Expression; Humans; Keloid; Myofibroblasts; Signal Transduction; Stem Cells; Transcriptome; Transforming Growth Factor beta

Aberrant scar formation, which includes keloid and hypertrophic scars, is associated with a pathological disorganized wound healing process with chronic inflammation. The TGF-β/Smad signaling pathway is the most canonical pathway through which the formation of collagen in the fibroblasts and myofibroblasts is regulated. Sustained activation of the TGF-β/Smad signaling pathway results in the long-term overactivation of fibroblasts and myofibroblasts, which is necessary for the excessive collagen formation in aberrant scars. There are two categories of therapeutic strategies that aim to target the TGF-β/Smad signaling pathway in fibroblasts and myofibroblasts to interfere with their cellular functions and reduce cell proliferation. The first therapeutic strategy includes medications, and the second strategy is composed of genetic and cellular therapeutics. Therefore, the focus of this review is to critically evaluate these two main therapeutic strategies that target the TGF-β/Smad pathway to attenuate abnormal skin scar formation.

Topics: Animals; Cell- and Tissue-Based Therapy; Cicatrix, Hypertrophic; Dermatologic Agents; Genetic Therapy; Humans; Keloid; Molecular Targeted Therapy; Signal Transduction; Skin; Smad Proteins; Transforming Growth Factor beta; Wound Healing

Keloid scars are a dysregulated response to cutaneous wound healing and are characterized by excessive deposition of collagen. Clinical and histological aspects are typical but they are often confused with hypertrophic scars. Principal pathogenesis is abnormal regulation of the collagen equilibrium because of TGFβ. In this first part, clinical characteristics, physiopathology and histology of keloid scars are explained.

Topics: Apoptosis; Collagen; Fibroblasts; Humans; Keloid; Risk Factors; Transforming Growth Factor beta

Keloids are common cutaneous pathological scars that are characterised by the histological accumulation of fibroblasts, collagen fibres, and clinically significant invasive growth. Although increasing lines of research on keloids have revealed genetic and environmental factors that contribute to their formation, the etiology of these scars remains unclear. Several studies have suggested the involvement of lipid metabolism, from a nutritional point of view. However, the role that lipid metabolism plays in the pathogenesis and progression of keloids has not previously been reviewed. The progress that has been made in understanding the roles of the pro- and anti-inflammatory lipid mediators in inflammation, and how they relate to the formation and progression of keloids, is also outlined. In particular, the possible relationships between mechanotransduction and lipid metabolites in keloids are explored. Mechanotransduction is the process by which physical forces are converted into biochemical signals that are then integrated into cellular responses. It is possible that lipid rafts and caveolae provide the location of lipid signaling and interactions between these signaling pathways and mechanotransduction. Moreover, interactions between lipid signaling pathway molecules and mechanotransduction molecules have been observed. A better understanding of the lipid profile changes and the functional roles lipid metabolism plays in keloids will help to identify target molecules for the development of novel interventions that can prevent, reduce, or even reverse pathological scar formation and/or progression.

Topics: Cicatrix; Fibroblasts; Humans; Inflammation; Keloid; Lipid Metabolism; Mechanotransduction, Cellular; Signal Transduction; Transforming Growth Factor beta

Fibrotic skin disorders may be debilitating and impair quality of life. There are few effective treatment options for cutaneous fibrotic diseases. In this review, we discuss our current understanding of the role of microRNAs (miRNAs) in skin fibrosis. miRNAs are a class of small, non-coding RNAs involved in skin fibrosis. These small RNAs range from 18 to 25 nucleotides in length and modify gene expression by binding to target messenger RNA (mRNA), causing degradation of the target mRNA or inhibiting the translation into proteins. We present an overview of the biogenesis, maturation and function of miRNAs. We highlight miRNA’s role in key skin fibrotic processes including: transforming growth factor-beta signaling, extracellular matrix deposition, and fibroblast proliferation and differentiation. Some miRNAs are profibrotic and their upregulation favors these processes contributing to fibrosis, while anti-fibrotic miRNAs inhibit these processes and may be reduced in fibrosis. Finally, we describe the diagnostic and therapeutic significance of miRNAs in the management of skin fibrosis. The discovery that miRNAs are detectable in serum, plasma, and other bodily fluids, and are relatively stable, suggests that miRNAs may serve as valuable biomarkers to monitor disease progression and response to treatment. In the treatment of skin fibrosis, antifibrotic miRNAs may be upregulated using mimics and viral vectors. Conversely, profibrotic miRNAs may be downregulated by employing anti-miRNAs, sponges, erasers and masks. We believe that miRNA-based therapies hold promise as important treatments and may transform the management of fibrotic skin diseases by physicians.

Topics: Cell Differentiation; Cell Proliferation; Humans; Keloid; MicroRNAs; Oligonucleotides; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

To review the current mechanisms and biologic processes leading to the formation of pathologic scars.. A computerised literature search was carried out using MEDLINE for all published articles on ''pathologic scarring''. The medical subject headings ''scarring'' were combined with ''mechanisms''. A review of selected relevant literature was then undertaken.. Scarless embryonal healing tends to be characterised by minimal inflammatory reaction mediated by reduced IL6,IL8 and hyaluronidase while there are elevated levels of hyaluronic acid MMP1to3, as well as IL10.The multifunctional cytokine TGF-B, its several isoforms as well as its postreceptor signalling mechanisms appears to play the key role in the scarring process . There is also evidence to show that PDGF, IGF and other cytokines regulate scarring . While conventional antiscarring agents target the fibroplasia phase, others such as tamoxifen ,calcium channel blockers, and imidazolaquinolines targets various phases of the scarring process .. It appears that multiple mechanisms are involved in the phenotypical appearance of abnormal scarring. A deeper understanding of these mechanisms is pivotal to the development of better antiscarring therapies in the very near future .

Topics: Cicatrix; Cytokines; Fibrosis; Humans; Inflammation; Keloid; Skin; Skin Physiological Phenomena; Transforming Growth Factor beta; Wound Healing

Organ fibrosis has been viewed as one of the major medical problems, which can lead to progressive dysfunction of the liver, lung, kidney, skin, heart, and eventually death of patients. Fibrosis is initiated by a variety of pathological, physiological, biochemical, and physical factors. Regardless of their different etiologies, they all share a common pathogenetic process: excessive activation of the key profibrotic cytokine, transforming growth factor-beta (TGF-beta). Peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor of the nuclear receptor superfamily, has received particular attention in recent years, because the activation of PPARgamma by both natural and synthetic agonists could effectively inhibit TGF-beta-induced profibrotic effects in many organs.. The English-language medical databases, PubMed, Elsevier and SpringerLink were searched for articles on PPARgamma, TGF-beta, and fibrosis, and related topics.. TGF-beta is recognized as a key profibrotic cytokine. Excessive activation of TGF-beta increases synthesis of extracellular matrix proteins and decreases their degradation, associated with a gradual destruction of normal tissue architecture and function, whereas PPARgamma agonists inhibit TGF-beta signal transduction and are effective antifibrogenic agents in many organs including the liver, lung, kidney, skin and heart.. The main antifibrotic activity of PPARgamma agonists is to suppress the TGF-beta signaling pathway by so-called PPARgamma-dependent effect. In addition, PPARgamma agonists, especially 15d-PGJ2, also exert potentially antifibrotic activity independent of PPARgamma activation. TGF-beta1/Smads signaling not only plays many essential roles in multiple developmental processes, but also forms cross-talk networks with other signal pathways, and their inhibition by PPARgamma agonists certainly affects the cytokine networks and causes non-suspected side-effects. Anti-TGF-beta therapies with PPARgamma agonists may have to be carefully tailored to be tissue- and target gene-specific to minimize side-effects, indicating a great challenge to the medical research at present.

Topics: Epithelial-Mesenchymal Transition; Fibrosis; Humans; Keloid; Kidney; Liver Cirrhosis; Myocardium; PPAR gamma; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

The complex biological and physiological mechanisms that result in poor quality scarring are still not fully understood. This review looks at current evidence of the role of transforming growth factor-beta (TGFβ) in this pathological process.

Topics: Biomarkers; Cicatrix; Cicatrix, Hypertrophic; Female; Humans; Keloid; Male; Risk Factors; Transforming Growth Factor beta; Wound Healing

Keloid scarring, also known as keloid disease (KD), is a common, abnormally raised fibroproliferative cutaneous lesion that can occur following even minor skin trauma. The aetiopathogenesis of KD has remained an enigma todate compounded by an ill-defined clinical management. There is strong evidence suggesting a genetic susceptibility in individuals affected by KD, including familial heritability, common occurrence in twins and high prevalence in certain ethnic populations. This review aims to address the genetic aspects of KD that have been described in present literature that include inheritance patterns, linkage studies, case-control association studies, whole genome gene expression microarray studies and gene pathways that were significant in KD. In addition to our clinical and scientific background in KD, we used search engines, Scopus, Scirus and PubMed, which searched for key terms covering various genetic aspects of KD. Additionally, genes reported in seven whole genome gene expression microarray studies were separately compared in detail. Our findings indicate a varied inheritance pattern in KD (predominantly autosomal dominant), linkage loci (chromosomes 2q23 and 7p11), several human leukocyte antigen (HLA) alleles (HLA-DRB1*15, HLA-DQA1*0104, DQ-B1*0501 and DQB1*0503), negative candidate gene case-control association studies and at least 25 dysregulated genes reported in multiple microarray studies. The major pathways reportedly proposed to be involved in KD include apoptosis, mitogen-activated protein kinase, transforming growth factor-beta, interleukin-6 and plasminogen activator inhibitor-1. In summary, involvement of more than one gene is likely to be responsible for susceptibility to KD. A better understanding of the genes involved in KD may potentially lead to the development of more effective diagnostic, therapeutic and prognostic measures.

Topics: Apoptosis; Cicatrix; Clinical Trials as Topic; Genetic Predisposition to Disease; Genome-Wide Association Study; HLA Antigens; Humans; Inheritance Patterns; Keloid; Mitogen-Activated Protein Kinases; Oligonucleotide Array Sequence Analysis; Plasminogen Activator Inhibitor 1; Polymorphism, Genetic; Transforming Growth Factor beta

Wound healing is a fundamental complex-tissue reaction leading to skin reconstitution and thereby ensuring survival. While, fetal wounds heal without scarring, a normal "fine line" scar is the clinical outcome of an undisturbed wound healing in adults. Alterations in the orchestrated wound healing process result in hypertrophic or keloid scarring. Research in the past decades attempted to identify genetic, cellular, and molecular factors responsible for these alterations. These attempts lead to several new developments in treatments for keloids, such as, imiquimod, inhibition of transforming growth factor beta, and recombinant interleukin-10. The urgent need for better therapeutics is underlined by recent data substantiating an impaired quality of life in keloid and hypertrophic scar patients. Despite the increasing knowledge about the molecular regulation of scar formation no unifying theory explaining keloid development has been put forward until today. This review aims to give an overview about the genetic and molecular background of keloids and focus of the current research on keloid scarring with special emphasis on new forthcoming treatments. Clinical aspects and the spectrum of scarring are summarized.

Topics: Adult; Aminoquinolines; Cicatrix; Female; Genetic Predisposition to Disease; Humans; Imiquimod; Immunity, Cellular; Interleukin-10; Keloid; Polymorphism, Genetic; Pregnancy; Quality of Life; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

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.

Topics: Collagen; Humans; Keloid; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Keloid management can be difficult and frustrating, and the mechanisms underlying keloid formation are only partially understood.. Using original and current literature in this field, this comprehensive review presents the major concepts of keloid pathogenesis and the treatment options stemming from them.. Mechanisms for keloid formation include alterations in growth factors, collagen turnover, tension alignment, and genetic and immunologic contributions. Treatment strategies for keloids include established (e.g., surgery, steroid, radiation) and experimental (e.g., interferon, 5-fluorouracil, retinoid) regimens.. The scientific basis and empiric evidence supporting the use of various agents is presented. Combination therapy, using surgical excision followed by intradermal steroid or other adjuvant therapy, currently appears to be the most efficacious and safe current regimen for keloid management.

Topics: Antimetabolites; Collagen; Combined Modality Therapy; Extracellular Matrix; Fibroblasts; Fluorouracil; Humans; Injections, Intralesional; Interferons; Keloid; Laser Therapy; Pressure; Retinoids; Sebum; Silicone Gels; Stress, Mechanical; Transforming Growth Factor beta; Triamcinolone Acetonide

Hypertropic and keloid scars cause both functional and cosmetic problems for those afflicted. Although people of all ages suffer from these conditions, the patients are often young and otherwise healthy, and become burdened with an activity limiting lesion or psychosocial stresses associated with a perceived aesthetic defect. Currently available treatment modalities are often inconvenient, occasionally painful, and have unwanted side effects. Despite the highest standard of care, treatment protocols are prone to failure with high rates of scar recurrence. Hypertropic and keloid scars are the result of an abnormal healing response and may result from an extended inflammatory phase in the wound healing process. Regardless of the causes, which remain elusive, excessive collagen deposition occurs relative to normal wounds. This extracellular matrix collagen accumulation makes a logical target for pharmacological interventions, and researchers are attempting to modify collagen-synthetic and -degradative pathways. In addition, growth factors and cytokines have been implicated in scar formation, and these factors are targeted for potential therapeutic use in scar management. Cytotoxic agents are also being evaluated for their potential utility in the reduction of tissue bulk associated with these excessive scar states. Given the wide range of potential therapeutic agents, the future market for scar therapy remains highly promising.

Topics: Antibiotics, Antineoplastic; Bleomycin; Cell Proliferation; Cicatrix, Hypertrophic; Collagen; Cytokines; Enzyme Inhibitors; Growth Substances; GTP-Binding Proteins; Humans; Interferon-gamma; Keloid; Procollagen-Proline Dioxygenase; Protein Glutamine gamma Glutamyltransferase 2; Randomized Controlled Trials as Topic; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta3; Transglutaminases

The precise mechanisms of normal and abnormal scar formation have long remained a mystery despite the extensive literature regarding wound healing. Only recently have researchers begun to delineate the complex biochemical signaling pathways that regulate these processes. This article reviews basic wound healing, while focusing on medicine's latest understanding of the development and treatment of keloids and hypertrophic scars.. The importance of the transforming growth factor-beta signaling pathways and the related downstream effector molecules has proven to offer a new detailed view of scar biology. Regulation of scar metabolism with regards to collagen and wound matrix degradation is likewise showing promise in generating alternate therapies to treat abnormal scars.. Understanding the exact process of normal and abnormal scar formation will help define better ways to successfully manage and potentially prevent abnormal healing like hypertrophic scars and keloids.

Topics: Cicatrix, Hypertrophic; Humans; Keloid; Risk Factors; Signal Transduction; Transforming Growth Factor beta; Wound Healing

Aberrant wound healing results in unsightly scar, hypertrophic scar, and keloid formation, causing functional and cosmetic deformities, discomfort, psychological stress, and patient dissatisfaction. Scar prevention and management, both surgical and nonsurgical, continue to be important issues for the otolaryngologist.. Both animal and human models continue to point to the integral role of transforming growth factor-beta in aberrant healing. Multiple extracts have promising results as therapies for scarring and are widely marketed but need to be further investigated. Scar prevention advancements include refinements in surgical technique, nutritional supplementation, and optimal wound care. Steroid injections continue to play a major role in the regression of scars and keloids. Dermatography assists in the minimization of scar appearance. Dermatography, laser therapies, intralesional 5-fluorouracil, and adjuvant radiotherapy are emerging therapies. Topical vitamin E utility is revisited. New surgical scar revision techniques include modified excision techniques and skin grafting.. Despite optimal efforts to avoid scar formation, aberrant wound healing may occur. The use of topical agents and intralesional steroid injections can minimize early scar formation. Strategies for prevention and management of keloids and hypertrophic scars continue to develop, as the basic science mechanisms underlying aberrant wound healing are elucidated.

Topics: Cicatrix, Hypertrophic; Cryotherapy; Humans; Keloid; Laser Therapy; Occlusive Dressings; Radiotherapy; Skin Transplantation; Transforming Growth Factor beta; Wound Healing

Keloidal scars are abnormal scars of uncertain etiology with a predilection for certain racial groups. Although many articles have been published on the management of these scars, there are no definitive treatment protocols. Our objective was to examine the scientific quality of the literature on therapy for keloidal scars. There are many problems with the study designs of existing keloidal scar research. These include lack of consistent disease definitions and outcome measures, inadequate follow-up, and inconsistent therapeutic interventions. Suggestions are given for future studies.

Topics: Cicatrix, Hypertrophic; Cytokines; Diagnosis, Differential; Humans; Keloid; Skin; Transforming Growth Factor beta; Wound Healing

Topics: Animals; Collagen; Fibroblasts; Humans; Keloid; Receptor, Platelet-Derived Growth Factor alpha; Transforming Growth Factor beta

Excess scar formation secondary to traumatic or surgical injuries can have devastating consequences, ranging from body disfigurement to organ dysfunction. Hypertrophic scars and keloids are skin fibrotic conditions that can be caused by minor insults to skin, such as acne or ear piercing, or by severe injuries such as burns. Differences between keloids, hypertrophic scars and normal scars include distinct scar appearance, histologic morphology and cellular function in response to growth factors. Recent advances in our understanding of the wound healing process reveal possible causes for hypertrophic scars and keloids. This information might assist in the development of efficacious treatment for hypertrophic scar and keloid formation.

Topics: Animals; Cicatrix, Hypertrophic; Extracellular Matrix; Fibroblasts; Forecasting; Growth Substances; Humans; Keloid; Signal Transduction; Transforming Growth Factor beta

Growth factors are becoming extremely valuable tools in our attempts to understand the mechanisms that modulate cellular activities. Their targeting to appropriate cells and maintaining adequate pharmacological levels becomes essential, particularly in view of the different effects that these compounds have on various cells and the dose dependence of their response. Within this context, this review focuses primarily on the delivery of growth factors involved in the processes of wound healing and tissue repair.

Topics: Animals; Biocompatible Materials; Biomedical Engineering; Bone Morphogenetic Proteins; Cicatrix, Hypertrophic; Drug Delivery Systems; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fracture Healing; Growth Substances; Humans; Keloid; Nerve Growth Factors; Peptides; Platelet-Derived Growth Factor; Transforming Growth Factor beta; Wound Healing

The formation of hypertrophic and keloid scars after cutaneous wounding is of particular relevance to the practice of maxillofacial surgery. This paper reviews current knowledge of the local and systemic factors underlying the formation of these scars and outlines the current and potential treatment modalities for these lesions.

Topics: Cicatrix, Hypertrophic; Collagen; Cytokines; Extracellular Matrix; Facial Injuries; Humans; Interleukin-1; Keloid; Transforming Growth Factor beta; Wound Healing

Keloid scars are characterized by the excessive proliferation of fibroblasts and an imbalance between the production and degradation of collagen, leading to its buildup in the dermis. There is no "gold standard" treatment for this condition, and the recurrence is frequent after surgical procedures removal. In vitro studies have demonstrated that photobiomodulation (PBM) using the blue wavelength reduces the proliferation speed and the number of fibroblasts as well as the expression of TGF-β. There are no protocols studied and established for the treatment of keloids with blue LED. Therefore, the purpose of this study is to determine the effects of the combination of PBM with blue light and the intralesional administration of the corticoid triamcinolone hexacetonide on the quality of the remaining scar by Vancouver Scar Scale in the postoperative period of keloid surgery. A randomized, controlled, double-blind, clinical trial will be conducted involving two groups: 1) Sham (n = 29): intralesional administration of corticoid (IAC) and sham PBM in the preoperative and postoperative periods of keloid removal surgery; and 2) active PBM combined with IAC (n = 29) in the preoperative and postoperative periods of keloid removal surgery. Transcutaneous PBM will be performed on the keloid region in the preoperative period and on the remaining scar in the postoperative period using blue LED (470 nm, 400 mW, 4J per point on 10 linear points). The patients will answer two questionnaires: one for the assessment of quality of life (Qualifibro-UNIFESP) and one for the assessment of satisfaction with the scar (PSAQ). The team of five plastic surgeons will answer the Vancouver Scar Scale (VSS). All questionnaires will be administered one, three, six, and twelve months postoperatively. The keloids will be molded in silicone prior to the onset of treatment and prior to excision to assess pre-treatment and post-treatment size. The same will be performed for the remaining scar at one, three, six, and twelve months postoperatively. The removed keloid will be submitted to histopathological analysis for the determination of the quantity of fibroblasts, the organization and distribution of collagen (picrosirius staining), and the genic expression of TGF-β (qPCR). All data will be submitted to statistical analysis. Trial registration: This study is registered in ClinicalTrials.gov (ID: NCT04824612).

Topics: Adrenal Cortex Hormones; Adult; Combined Modality Therapy; Double-Blind Method; Female; Humans; Injections, Intralesional; Keloid; Low-Level Light Therapy; Male; Middle Aged; Patient Satisfaction; Postoperative Care; Preoperative Care; Prospective Studies; Quality of Life; Transforming Growth Factor beta; Treatment Outcome; Triamcinolone Acetonide; Young Adult

The biochemical characteristics of keloid-derived fibroblasts differ from those of adjacent normal fibroblasts, and these differences are thought to be the cause of abnormal fibrosis. Therefore, we investigated the characteristic proteins that are differentially expressed in keloid-derived fibroblasts using proteomics tools.. We attempted to investigate the novel proteins that play important roles in the pathophysiology of keloids.. Proteomics analysis was performed to identify differentially expressed proteins in keloid-derived fibroblasts. Keloid-derived fibroblasts and adjacent normal fibroblasts were analyzed with 2-DAGE. We validated these proteins with immunoblot analysis, real-time RT-PCR, and immunohistochemistry.. Sixteen differentially expressed protein spots were identified in keloid-derived fibroblasts. Among them, heat shock protein 70 (Hsp70) was specifically upregulated in keloid-derived fibroblasts. Also, immunohistochemistry and western blot analysis revealed increased Hsp70, TGF- β , and PCNA expressions in keloids compared to normal tissue.. Hsp70 is overexpressed in keloid fibroblasts and tissue. The overexpression of Hsp70 may be involved in the pathogenesis of keloids, and the inhibition of Hsp70 could be a new therapeutic tool for the treatment of keloids.

Topics: Adult; Aged; Cells, Cultured; Female; Fibroblasts; Gene Expression Profiling; HSP70 Heat-Shock Proteins; Humans; Keloid; Male; Middle Aged; Proliferating Cell Nuclear Antigen; Proteomics; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor-beta (TGF-β) plays an instrumental role in forming scars and keloids. TGF-β isoforms exhibit differential expression, indicating distinct wound healing and scar formation functions. However, the role of TGF-β1 and TGF-β3 in wound healing and scar formation remains unclear. This study aimed to compare the specific roles of TGF-β1 and TGF-β3 in wound healing and scar formation by biomolecular analysis.. The study was conducted by cell isolation and culture cells from a total of 20 human samples. Normal human fibroblasts (NHF) were isolated from normal human samples and myofibroblasts from the different scar types, namely hypertrophic (HT) and keloid (K) scars. NHF and cells from the HT, and K scar, each of which were divided into 3 sample groups: the untreated control, TGF-β1 (10 µg/mL)-treated group, and TGF-β3 (10 µg/mL)-treated group. The results of confocal microscopy and fluorescence-activated cell sorting experiments were compared.. Both the HT and K groups had higher α-smooth muscle actin (α-SMA) expression than the NHF group in the untreated control group. In comparison with the untreated group, NHFs showed a significant increase in α-SMA expression in the TGF-β1-treated group. HT showed a high α-SMA level, which was statistically significant compared with the normal fibroblasts. In the TGF-β3-treated group, α-SMA expression was slightly increased in NHF as compared with the untreated group. TGF-β3 treated HT exhibited a greater reduction in α-SMA expression than in the TGF-β1 treated HT. K, on the other hand, had only a minimal effect on the treatment of TGF-β1 and TGF-β3.. The findings suggest that TGF-β3 may play a regulatory role in the wound repair process, which could be useful in the development of scar-reducing therapies for patients with scar-related cosmetic concerns.

Topics: Cicatrix, Hypertrophic; Fibroblasts; Humans; Hypertrophy; Keloid; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta3; Transforming Growth Factors

Keloid is a type of benign fibrous proliferative tumor characterized by excessive scarring. C1q/TNF-related protein 3 (CTRP3) has been proven to possess antifibrotic effect. Here, we explored the role of CTRP3 in keloid. In the current research, we examined the influence of CTRP3 on keloid fibroblasts (KFs) and investigated the potential molecular mechanism.. KF tissue specimens and adjacent normal fibroblast (NF) tissues were collected cultured from 10 keloid participants. For the TGF-. CTRP3 was downregulated in keloid tissues and KFs. CTRP3 overexpression significantly controlled TGF-

Topics: Cell Proliferation; Cells, Cultured; Fibroblasts; Humans; Keloid; RNA, Small Interfering; Transforming Growth Factor beta; Transforming Growth Factor beta1

Skin fibrosis is a common pathological manifestation in systemic sclerosis (SSc), keloid, and localized scleroderma (LS) characterized by fibroblast activation and excessive extracellular matrix (ECM) deposition. However, few effective drugs are available to treat skin fibrosis due to its unclear mechanisms. In our study, we reanalyzed skin RNA-sequencing data of Caucasian, African, and Hispanic SSc patients from the Gene Expression Omnibus (GEO) database. We found that the focal adhesion pathway was up-regulated and Zyxin appeared to be the primary focal adhesion protein involved in skin fibrosis, and we further verified its expression in Chinese skin tissues of several fibrotic diseases, including SSc, keloid, and LS. Moreover, we found Zyxin inhibition could significantly alleviate skin fibrosis using Zyxin knock-down and knock-out mice, nude mouse model and skin explants of human keloid. Double immunofluorescence staining showed that Zyxin was highly expressed in fibroblasts. Further analysis revealed pro-fibrotic gene expression and collagen production increased in Zyxin over-expressed fibroblasts, and decreased in Zyxin interfered SSc fibroblasts. In addition, transcriptome and cell culture analyses revealed Zyxin inhibition could effectively attenuate skin fibrosis by regulating the FAK/PI3K/AKT and TGF-β signaling pathways via integrins. These results suggest Zyxin appears a potential new therapeutic target for skin fibrosis.

Topics: Animals; Fibroblasts; Fibrosis; Humans; Integrins; Keloid; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta; Zyxin

Topics: Cells, Cultured; Fibroblasts; Fibrosis; Humans; Keloid; Metformin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Cells, Cultured; Fibroblasts; Fibrosis; Humans; Keloid; Metformin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Wound healing is a complicated cascading process; disequilibrium among reparative processes leads to the formation of pathologic scars. Herein, we explored the role of mortalin in scar formation and its association with the interleukin-1α receptor using in vitro and in vivo models. To investigate the effects of mortalin, we performed an MTT cell viability assay, qRT-PCR, and Western blot analyses, in addition to immunofluorescence and immunoprecipitation studies using cultured fibroblasts. A rat incisional wound model was used to evaluate the effect of a mortalin-specific shRNA (dE1-RGD/GFP/shMot) Ad vector in scar tissue. In vitro, the mortalin-treated human dermal fibroblast displayed a significant increase in proliferation of type I collagen, α-smooth muscle actin, transforming growth factor-β, phospho-Smad2/3-complex, and NF-κB levels. Immunofluorescence staining revealed markedly increased mortalin and interleukin-1α receptor protein in keloid tissue compared to those in normal tissue, suggesting that the association between mortalin and IL-1α receptor was responsible for the fibrogenic effect. In vivo, mortalin-specific shRNA-expressing Ad vectors significantly decreased the scar size and type-I-collagen, α-SMA, and phospho-Smad2/3-complex expression in rat incisional scar tissue. Thus, dE1-RGD/GEP/shMot can inhibit the TGF-β/α-SMA axis and NF-κB signal pathways in scar formation, and blocking endogenous mortalin could be a potential therapeutic target for keloids.

Topics: Animals; Cells, Cultured; Collagen Type I; Fibroblasts; HSP70 Heat-Shock Proteins; Humans; Interleukin-1alpha; Keloid; NF-kappa B; Oligopeptides; Rats; RNA, Small Interfering; Transforming Growth Factor beta

Keloids are a common type of pathological scar as a result of skin healing, which are extremely difficult to prevent and treat without recurrence. The pathological mechanism of keloids is the excessive proliferation of fibroblasts, which synthesize more extracellular matrices (ECMs), including type I/III collagen (COL-1/3), mucopolysaccharides, connective tissue growth factor (CTGF, also known as cellular communication network factor 2 (CCN2)), and fibronectin (FN) in scar tissue, mostly through the abnormal activation of transforming growth factor-‍β (TGF-‍β)/Smads pathway (Finnson et al., 2013; Song et al., 2018). Genetic factors, including race and skin tone, are considered to contribute to keloid formation. The reported incidence of keloids in black people is as high as 16%, whereas white people are less affected. The prevalence ratio of colored people to white people is 5:1‍‍-‍‍15:1 (Rockwell et al., 1989; LaRanger et al., 2019). In addition, keloids have not been reported in albinism patients of any race, and those with darker skin in the same race are more likely to develop this disease (LaRanger et al., 2019). Skin melanocyte activity is significantly different among people with different skin tones. The more active the melanocyte function, the more melanin is produced and the darker the skin. Similarly, in the same individual, the incidence of keloids increases during periods when melanocytes are active, such as adolescence and pregnancy. Keloids rarely appear in areas where melanocytes synthesize less melanin, such as in the palms and soles. Thus, the formation of keloids seems to be closely related to melanocyte activity.

Topics: Adolescent; Cells, Cultured; Exosomes; Fibroblasts; Humans; Keloid; Melanins; Melanocytes; Pilot Projects; Skin; Transforming Growth Factor beta

Fibrosis is a common pathophysiological response of many tissues and organs subjected to chronic injury. Despite the diverse aetiology of keloid, lacaziosis and localized scleroderma, the process of fibrosis is present in the pathogenesis of all of these three entities beyond other individual clinical and histological distinct characteristics. Fibrosis was studied in 20 samples each of these three chronic cutaneous inflammatory diseases. An immunohistochemical study was carried out to explore the presence of α-smooth muscle actin (α-SMA) and vimentin cytoskeleton antigens, CD31, CD34, Ki67, p16; CD105, CD163, CD206 and FOXP3 antigens; and the central fibrotic cytokine TGF-β. Higher expression of vimentin in comparison to α-SMA in all three lesion types was found. CD31- and CD34-positive blood vessel endothelial cells were observed throughout the reticular dermis. Ki67 expression was low and almost absent in scleroderma. p16-positive levels were higher than ki67 and observed in reticular dermis of keloidal collagen in keloids, in collagen bundles in scleroderma and in the external layers of the granulomas in lacaziosis. The presence of α-actin positive cells and rarely CD34 positive cells, observed primarily in keloids, may be related to higher p16 antigen expression, a measure of cell senescence. Low FOXP3 expression was observed in all lesion types. CD105-positive cells were mainly found in perivascular tissue in close contact with the adventitia in keloids and scleroderma, while, in lacaziosis, these cells were chiefly observed in conjunction with collagen deposition in the external granuloma layer. We did not find high involvement of CD163 or CD206-positive cells in the fibrotic process. TGF-β was notable only in keloid and lacaziosis lesions. In conclusion, we have suggested vimentin to be the main myofibroblast general marker of the fibrotic process in all three studied diseases, while endothelial-to-mesenchymal transition (EndoMT) and mesenchymal stem cells (MSCs) and M2 macrophages may not play an important role.

Topics: Endothelial Cells; Fibroblasts; Fibrosis; Forkhead Transcription Factors; Humans; Keloid; Ki-67 Antigen; Lobomycosis; Scleroderma, Localized; Skin; Transforming Growth Factor beta; Vimentin

Keloids are a benign dermal fibrotic disorder with features similar to malignant tumors. keloids remain a therapeutic challenge and lack medical therapies, which is partially due to the incomplete understanding of the pathogenesis mechanism. We performed single-cell RNA-sequencing of 28,064 cells from keloid skin tissue and adjacent relatively normal tissue. Unbiased clustering revealed substantial cellular heterogeneity of keloid tissue, which included 21 clusters assigned to 11 cell lineages. We observed significant expansion of fibroblast and vascular endothelial cell subpopulations in keloids, reflecting their strong association with keloid pathogenesis. Comparative analyses were performed to identify the dysregulated pathways, regulators and ligand-receptor interactions in keloid fibroblasts and vascular endothelial cells. Our results highlight the roles of TGFβ and Eph-ephrin signaling pathways in both the aberrant fibrogenesis and angiogenesis of keloids. Critical regulators probably involved in the fibrogenesis of keloid fibroblasts, such as TWIST1, FOXO3 and SMAD3, were identified. TWIST1 inhibitor harmine could significantly suppress the fibrogenesis of keloid fibroblasts. In addition, tumor-related pathways were activated in keloid fibroblasts and vascular endothelial cells, which may be responsible for the malignant features of keloids. Our study put insights into the pathogenesis of keloids and provides potential targets for medical therapies.

Topics: Adult; Carcinogenesis; Cell Differentiation; Cell Lineage; Cells, Cultured; Endothelial Cells; Ephrins; Female; Fibroblasts; Fibrosis; Forkhead Box Protein O3; Humans; Keloid; Sequence Analysis, RNA; Signal Transduction; Single-Cell Analysis; Skin; Smad3 Protein; Transforming Growth Factor beta; Twist-Related Protein 1

Keloid is a benign fibroproliferative skin tumor. The respective functions of fibroblasts and vascular endothelial cells in keloid have not been fully studied. The purpose of this study is to identify the respective roles and key genes of fibroblasts and vascular endothelial cells in keloids, which can be used as new targets for diagnosis or treatment.The microarray datasets of keloid fibroblasts and vascular endothelial cells were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened out. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for functional enrichment analysis. The search tool for retrieval of interacting genes and Cytoscape were used to construct protein-protein interaction (PPI) networks and analyze gene modules. The hub genes were screened out, and the relevant interaction networks and biological process analysis were carried out.In fibroblasts, the DEGs were significantly enriched in collagen fibril organization, extracellular matrix organization and ECM-receptor interaction. The PPI network was constructed, and the most significant module was selected, which is mainly enriched in ECM-receptor interaction. In vascular endothelial cells, the DEGs were significantly enriched in cytokine activity, growth factor activity and transforming growth factor-β (TGF-β) signaling pathway. Module analysis was mainly enriched in TGF-β signaling pathway. Hub genes were screened out separately.In summary, the DEGs and hub genes discovered in this study may help us understand the molecular mechanisms of keloid, and provide potential targets for diagnosis and treatment.

Topics: Computational Biology; Endothelial Cells; Fibroblasts; Gene Expression Profiling; Gene Ontology; Gene Regulatory Networks; Humans; Keloid; Protein Interaction Mapping; Protein Interaction Maps; Transcriptome; Transforming Growth Factor beta; Wound Healing

Accumulation of excessive extracellular matrix (ECM) and aberrant transforming growth factor β (TGF-β) signaling pathway function can be potential therapeutic targets for keloid treatment. In this study, we examined the antifibrotic effect of metformin as a suppressor of TGF-β signaling pathways in human dermal fibroblasts (HDFs) and keloid spheroids. Human dermal fibroblasts were stimulated with TGF-β (10 ng/mL) and treated with metformin (10 mM). The mRNA and protein expression of ECM components were evaluated by quantitative polymerase chain reaction, western blot, and immunofluorescence assay. In addition, we immunohistochemically examined the expression levels of ECM proteins in keloid spheroids. After addition of metformin (10 mM), collagen types I and III and elastin mRNA levels were significantly decreased in HDFs, and collagen type I protein level was significantly decreased. In addition, the expression levels of collagen types I and III, fibronectin, and elastin were significantly reduced in keloid spheroids after treatment with metformin (100 mM). Collagen types I and III and p-Smad2/3 complex proteins were decreased in metformin-treated keloid spheroids. These findings indicated that metformin inhibits the expression of ECM components in TGF-β-stimulated HDFs and keloid spheroids. Therefore, we suggest the potential of metformin as an effective agent for the treatment of keloids.

Topics: Cells, Cultured; Fibroblasts; Fibrosis; Humans; Keloid; Metformin; Transforming Growth Factor beta; Transforming Growth Factor beta1

The successful treatment of keloids is a great challenge in the plastic surgery field. Activating transcription factor 3 (ATF3) is discovered as an adaptive responsive gene, which plays a critical role in fibroblast activation. This study aimed to investigate the expression and biological role of ATF3 in the pathogenesis of keloids. ATF3 expression in normal skins and keloids was evaluated by real-time PCR, western blot and immunohistochemistry. Effects of ATF3 on cell growth, apoptosis, invasion and collagen production were evaluated in keloid fibroblast cells overexpressing or downregulating ATF3. ATF3 expression was significantly elevated in keloid tissues when compared with that of normal skins and parakeloidal skin tissues. Moreover, ATF3 promoted cell proliferation and collagen production in keloid fibroblast cells. Conversely, transfection with siRNA targeting ATF3 led to decreased cell viability and collagen synthesis via inhibiting transforming growth factor-β1 (TGF-β1) and fibroblast growth factor 2/8 (FGF2/8) production in keloid fibroblasts. ATF3 could reduce the apoptosis rate of keloid fibroblast cells. Molecularly, we found that ATF3 promoted BCL2 level and inhibit the expression of BCL2 associated agonist of cell death (Bad), Caspase3 and Caspase9 in keloid fibroblast cells. ATF3 also enhanced the invasive potential via upregulating the expression of Matrix Metalloproteinases (MMP) family members (MMP1, MMP2, MMP9 and MMP13). ATF3 could induce activation of TGF-β/Smad signaling pathway in fibroblasts. Collectively, ATF3 could promote growth and invasion, and inhibit apoptosis via TGF-β/Smad pathway in keloid fibroblast cells, suggesting that ATF3 might be considered as a novel therapeutic target for the management of keloid.

Topics: Activating Transcription Factor 3; Apoptosis; Cell Proliferation; Cells, Cultured; Collagen; Fibroblasts; Humans; Keloid; Signal Transduction; Skin; Smad Proteins; Transcriptome; Transforming Growth Factor beta

Keloids are dermal fibroproliferative disorders that characterized by over deposition of components of the extracellular matrix. Interleukin 37 (IL-37) is known by its ability to inhibit the proliferation of keloid fibroblasts by inhibiting extracellular matrix production induced by transforming growth factor β (TGF-β). Thus, Il-37 is suggested to be used as an early preventive treatment for keloids.. This study aimed to evaluate the correlation between serum levels of IL37 level and the keloid severity.. This is a cross-sectional analytic study involving thirty-two patients diagnosed clinically as having Keloid. An assessment of keloid severity was conducted by using Vancouver Scar Scale (VSS). Blood samples were collected from every patient to measure and assess the serum levels of IL37.. A negative correlation was found between IL37 level and the keloid severity (P = .0001; r = -.737). Also, there was a nonsignificant correlation between IL37 levels in patient with keloid and age, gender, duration of lesions, and family history.. Lower level of plasma IL 37 could be an indicator of the severity of Keloids.

Topics: Cell Proliferation; Cells, Cultured; Cross-Sectional Studies; Extracellular Matrix; Fibroblasts; Humans; Interleukin-1; Keloid; Transforming Growth Factor beta

Keloid is a benign dermal tumor with excessive hyperplasia and deposition of collagen. As a common tumor suppressor gene, miR-133a-3p has not been studied in keloid. This study will delve into the specific mechanism of miR-133a-3p in keloid.. Normal skin fibroblasts and keloid fibroblasts (KFs) were first isolated from patients' normal skin and keloid, and cells were identified by morphological observation and immunofluorescence. The expressions of miR-133a-3p and extracellular matrix (ECM)-associated markers (Collagen I, III and α smooth muscle activin) were detected by Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Cell viability and apoptosis of KFs were examined by Cell Counting Kit-8 assay, flow cytometry, and Caspase-3 colorimetry. TargetScan predicted target gene for miR-133a-3p was verified by luciferase assay, qRT-PCR and Western Blot (WB). WB was used to study protein expression of TGFBR1, phosphorylated -Smad2 (p-Smad2) and Smad2. Finally, a series of rescue experiments were performed to verify the intervention of target genes on miR-133a-3p.. MiR-133a-3p was lowly expressed in keloid tissue and KFs. Overexpression of miR-133a-3p inhibited the expression of ECM-associated markers, reduced KFs viability, and promoted apoptosis. It was verified that interference regulator 5 (IRF5) is miR-133a-3p target gene. The rescue experiments showed that IRF5 reversed the effect of miR-133a-3p mimic on inhibiting fibrosis, and reversed the effects on promoting apoptosis and reducing cell proliferation.. Overexpressed miR-133a-3p inhibits fibrosis by down-regulating IRF5 and thus inhibiting the TGF-β/Smad2 pathway. And it also promotes KFs apoptosis and reduces proliferation.

Topics: Adolescent; Adult; Apoptosis; Base Sequence; Biomarkers; Cell Proliferation; Cell Survival; Extracellular Matrix; Female; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Interferon Regulatory Factors; Keloid; Male; MicroRNAs; Middle Aged; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Young Adult

Excessive fibrosis and extracellular matrix deposition resulting from upregulation of target genes expression mediated by transforming growth factor-beta (TGF-β)/SMAD and hypoxia inducible factor-1 (HIF-1) signaling pathways are the main mechanisms that drive keloid formation. Sumoylation is a protein posttranslational modification that regulates the function of proteins in many biological processes. In the present study, we aimed to investigate the mechanism underlying the effects of sumoylation on the TGF-β/SMAD and HIF-1 signaling pathways in keloids. We used 2-D08 to block sumoylation and silenced the expression of sentrin sumo-specific protease 1 (SENP1) to enhance sumoylation in human foreskin fibroblasts (HFFs) and human keloid fibroblasts (HKFs). We also reduced and increased intracellular SUMO1 levels by silencing SUMO1 and transfecting cells with a SUMO1 overexpression lentivirus, respectively. Sumoylation has the ability to amplify TGF-β/SMAD and HIF-1 signals in keloids, while SUMO1, especially the SUMO1-RanGAP1 complex, is the key molecule affecting the TGF-β/SMAD and HIF-1 signaling pathways. In addition, we also found that hypoxia promotes sumoylation in keloids and that HIF-1α is covalently modified by SUMO1 at Lys 391 and Lys 477 in HKFs. In summary, we elucidated the role and molecular mechanism of sumoylation in the formation of keloids, providing a new perspective for a potential therapeutic target of keloids.

Topics: Extracellular Matrix; Fibroblasts; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Keloid; Signal Transduction; Smad Proteins; SUMO-1 Protein; Sumoylation; Transforming Growth Factor beta; Up-Regulation

Topics: Adult; Aged; Case-Control Studies; Cell Differentiation; Cell Proliferation; Cells, Cultured; Collagen; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Keloid; Male; Middle Aged; Myofibroblasts; Nonmuscle Myosin Type IIA; RNA Interference; Signal Transduction; Skin; Transforming Growth Factor beta

A keloid is defined as an overgrowth of the dense fibrous tissues that form around a wound. Since they destroy the vascular network, keloid tissues often exhibit anoxic conditions. Hypoxia-inducible factor-1α (HIF-1α) is a core factor that mediates hypoxia stress responses and regulates the hypoxic cellular and biological behaviors. In this study, we found that the expression level of HIF-1α in keloid tissue was significantly higher than that in the normal skin tissue. Hypoxia-induced HIF-1α expression significantly inhibited cellular apoptosis and promoted cellular proliferation in keloid fibroblasts but not in normal fibroblasts. Specifically, HIF-1α activated the TGF-β/Smad and TLR4/MyD88/NF-κB pathways, and the interaction of these two pathways may promote the development of keloids. Moreover,

Topics: Animals; Apoptosis; Cell Hypoxia; Cell Proliferation; Fibroblasts; Gene Expression Regulation; Heterografts; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Keloid; Mice; Myeloid Differentiation Factor 88; Primary Cell Culture; Signal Transduction; Smad Proteins; Toll-Like Receptor 4; Transcription Factor RelA; Transforming Growth Factor beta

Keloids are a skin disorder where the skin goes beyond the original border of the wound or trauma, resulting in functional and cosmetic deformities, displeasure, itching, pain, psychological stress, and patient dissatisfaction. This study aimed to explore the therapeutic effect of interleukin-10 (IL-10) on the proliferation of keloid fibroblasts.. Keloid fibroblasts were isolated, primarily cultured, and treated with IL-10 at different concentrations. Normal skin fibroblasts were used as normal control. Immunofluorescent staining was performed to identify the establishment of keloid, as well as normal skin fibroblast. Cell Counting Kit-8 (CCK-8) was carried out to monitor the proliferative variation, while Western blot was conducted to detect the expression variation of key members involved in the TGF-β/Smad signaling pathway.. Identified by the IF staining of Vimentin, a classical biomarker of fibroblast, both primary culture of keloid and normal skin fibroblasts have been established. Compared with control, the proliferation of Keloid fibroblasts was shown to be significantly suppressed on treatment with IL-10 in a time and dose-dependent manner. Expression of P-Smad2/3 and Smad4 were increasingly down-regulated, whereas Smad-7 was up-regulated with the increasing concentration of IL-10. By contrast, the variation of Smad 2/3 expressions was hardly influenced. Furthermore, the Collagen Type I and Collagen Type II were found to be markedly decreased after treatment with IL-10.. IL-10 was shown to be able to significantly inhibit the proliferation of keloid fibroblasts, which was explicitly and strongly suggestive of its potential therapeutic effect in the management of keloid.

Topics: Adult; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Female; Fibroblasts; Humans; Interleukin-10; Keloid; Male; Primary Cell Culture; Signal Transduction; Smad Proteins; Time Factors; Transforming Growth Factor beta; Young Adult

Pathological scarring is a result of the hypertrophy of scar tissue during tissue repair following trauma. The aim of the present study was to assess the effect of ubiquitin‑specific protease 4 (USP4) silencing on pathological scarring, and to evaluate the mechanistic basis for the effect. An MTT assay was used to assess cell viability. Immunoprecipitation (IP) was used to determine ubiquitination levels of the TGF‑β receptor (TβR)I and Smad7. Tumor formation was assessed by injecting keloid fibroblasts. Hematoxylin and eosin staining was used to detect pathological changes in tumor tissue. Reverse transcription quantitative polymerase chain reaction and western blot analysis assays were used to evaluate the expression levels of TβRI and Smad7. Compared with the untreated control animals, cell viability and the expression of TβRI and Smad7 increased significantly in animals treated with TGF‑β. Short hairpin RNA for USP4 (shUSP4) decreased the cell viability of negative control cells, TGF‑β‑induced cellular proliferation, and the expression of TβRI and Smad7. IP experiments indicated that the ubiquitination level of TβRI was decreased following USP4 silencing. There was no remarkable difference in the structure of scar tissue among the various animal groups at 14 days following treatment, while the necrotic area of the scar tissue in the shUSP4 and vialinin A (USP inhibitor)‑treated animals increased significantly at the 28th and 42nd day compared with the control animals. At days 14, 28 and 42, the expression levels of TβRI and Smad7 in the shUSP4 and vialinin A‑treated animals were significantly decreased compared with the control animals (P<0.05). In summary, interference with or inhibition of USP4 prevented the activity of the TGF‑β/Smad pathway signaling and inhibited the formation of pathological scars.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cicatrix; Fibroblasts; Gene Expression Regulation; Humans; Keloid; Mice; Mice, Inbred BALB C; Mice, Nude; Receptor, Transforming Growth Factor-beta Type I; RNA, Small Interfering; Signal Transduction; Smad7 Protein; Terphenyl Compounds; Transforming Growth Factor beta; Transplantation, Heterologous; Ubiquitin-Specific Proteases

A keloid is a benign fibroproliferative skin tumor that results from abnormal wound healing after injury and tends to grow beyond the boundary of the original wound; the mechanism of keloid formation is still unclear. MicroRNA-21 (MiR-21) is a representative microRNA that plays a key role in a variety of fibrotic diseases via the transforming growth factor-β/Smad signaling pathway. The aim of our study was to explore the mechanism of keloid formation. First, we found that the expression of miR-21 in keloids and keloid fibroblasts was significantly upregulated by microRNA microarray and real-time polymerase chain reaction. Additionally, at the protein level, our study confirmed that the overexpression of miR-21 could promote the process of keloid fibrosis to some extent and also indicated that a low expression of miR-21 could inhibit the process of keloid fibrosis. Finally, the results proved that miR-21 could participate in the keloid fibrosis process through negative regulation of its downstream target gene Smad7 via the transforming growth factor-β/Smad signaling pathway, which provides a guiding framework for further studies and new theoretical support for keloid clinical treatment.

Topics: Adult; Apoptosis; Cell Proliferation; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type III; Down-Regulation; Female; Fibroblasts; Humans; Keloid; Male; Microarray Analysis; MicroRNAs; Middle Aged; Real-Time Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta; Up-Regulation; Young Adult

The mechanistic details of keloid formation are still not understood. Given that the immune system is engaged in skin lesion repair, we examined the CD14

Topics: Adult; Cell Differentiation; Female; Forkhead Transcription Factors; Humans; Interleukin-10; Keloid; Lipopolysaccharide Receptors; Macrophages; Male; Middle Aged; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

A wide range of therapeutic options exists for the treatment of keloids, all of which have their own strengths; however, a high risk of side‑effects and frequent recurrence remains. Therefore, the present study aimed to identify improved therapeutic approaches or drugs for the treatment of keloids. Ginsenoside Rg3 (Rg3) has been reported to exert numerous antitumor effects, thus indicating that Rg3 may be a potential therapeutic agent that targets keloids. The present study determined the effects of Rg3 on human keloid fibroblasts (KFs) in vitro, and further explored the associated molecular and cellular mechanisms. Keloid scar specimens were obtained from patients, aged between 22 and 35 years, without systemic diseases and primary cells were isolated from keloid tissues. In each assay, KFs were divided into three groups and were cultured in medium with or without various concentrations of Rg3 (50 or 100 µg/ml). Cell viability assay, flow cytometry, quantitative polymerase chain reaction, cell migration assay, immunofluorescence staining, western blot analysis, Transwell cell invasion assay and immunohistochemical analysis were used to analyze the KFs and keloid explant cultures. The results of the present study demonstrated that Rg3 was able to exert an inhibitory effect on the transforming growth factor‑β/Smad and extracellular signal‑regulated kinase signaling pathways in KFs. The proliferation, migration, invasion, angiogenesis and collagen synthesis of KFs were markedly suppressed following treatment with Rg3. Furthermore, the results of an ex vivo assay indicated that Rg3 inhibited angiogenesis and reduced collagen accumulation in keloids. Significant statistical differences existed between the control and Rg3‑treated groups (P<0.05). All of these experimental results suggested that Rg3 may serve as a reliable drug for the treatment of patients with keloids.

Topics: Adult; Apoptosis; Cell Movement; Cell Proliferation; Collagen; Extracellular Matrix; Female; Fibroblasts; Fibrosis; Gene Expression Regulation; Ginsenosides; Humans; Keloid; Male; MAP Kinase Signaling System; Neovascularization, Physiologic; Smad Proteins; Transforming Growth Factor beta; Young Adult

Mesenchymal stem cells are a valuable cell source in regenerative medicine, and conditioned medium obtained from mesenchymal stem cells reportedly inhibits inflammation. Keloids are characterized by abnormal fibrosis, caused by fibroblasts in response to inflammation. In this study, the authors evaluated whether conditioned medium obtained from amnion-derived mesenchymal stem cells suppressed activation of keloid fibroblasts.. Keloid (n = 7), mature (n = 5), and normal (n = 5) fibroblasts were harvested from patients. Fibroblasts were stimulated with transforming growth factor (TGF)-β, and the effects of conditioned medium obtained from amnion-derived mesenchymal stem cells on cell proliferation, activation, and expression of extracellular matrix-related genes were analyzed. The effect of concentrating the conditioned medium by ultrafiltration on fibroblast activation was also analyzed.. Conditioned medium obtained from amnion-derived mesenchymal stem cells significantly up-regulated proliferation of mature fibroblasts but tended to suppress that of keloid fibroblasts. Conditioned medium obtained from amnion-derived mesenchymal stem cells significantly suppressed the TGF-β-induced up-regulation of α-smooth muscle actin in keloid and normal fibroblasts and collagen I in keloid fibroblasts, but not in mature fibroblasts. The conditioned medium obtained from amnion-derived mesenchymal stem cells concentrated by ultrafiltration and the filtrate significantly suppressed TGF-β-induced α-smooth muscle actin expression.. Conditioned medium obtained from amnion-derived mesenchymal stem cells prevents proliferation and activation of keloid fibroblasts and is a promising keloid treatment for administration as a topical agent.. Therapeutic, V.

Topics: Actins; Administration, Cutaneous; Adolescent; Adult; Amnion; Cell Proliferation; Cell- and Tissue-Based Therapy; Cells, Cultured; Culture Media, Conditioned; Female; Fibroblasts; Humans; Keloid; Male; Mesenchymal Stem Cells; Middle Aged; Transforming Growth Factor beta; Ultrafiltration; Up-Regulation; Young Adult

Keloid scars are currently considered a chronic inflammatory process and no longer a benign skin tumor. Keloids are defined as highly inflamed, hyperproliferative pathological scars. Growth factors and cytokines have important functions in the keloid inflammatory etiopathogenesis. The aim of this study was to analyze the in situ expression of cytokines and growth factors in keloid scars in comparison with that in normal scars. Among them, we specifically assessed TGF-β, FGF, IL-33, IL-22, ARG-1, ARG-2, iNOS, VIP, VIP-R1, TAC, and TAC-R1. A total of 98 biopsies were evaluated, including of 53 keloid and 45 normal scars. The age of patients with keloids ranged from 11 to 73 years, with a mean age of 28 years and predominance of the female gender (58.5% of the total patients). Around 64.15% of the patients belonged to the black ethnic group. Evaluated keloids were most commonly located in the earlobe because of ear piercing, representing 73.6% of the cases. We found significantly greater expression of TGF-β, IL-22, and ARG-1 in keloids when compared with that in normal scars. As for IL-33, ARG-2, and VIP-R1, despite the higher number of mRNA copies found in keloids, this difference was not significant. Furthermore, FGF, iNOS, VIP, TAC, and TAC-R1 mRNA levels were not detectable, and therefore these results were inconclusive in this study. Considering these results, understanding the cellular and molecular mechanisms that control the inflammatory response during cutaneous healing may promote the development of strategies to improve the treatment of patients with keloids.

Topics: Adult; Arginase; Biomarkers; Biopsy; Case-Control Studies; Female; Gene Expression; Humans; In Situ Hybridization; Interleukin-22; Interleukins; Keloid; Male; RNA, Messenger; Transforming Growth Factor beta

Keloid may induce severe impairment of life quality for the patients, although keloid is a cutaneous benign tumor. Collagen triple helix repeat containing protein 1 (Cthrc1) was identified as a novel gene that was originally found in adventitial fibroblasts after arterial injury. To address the role of Cthrc1 in keloid, the expression level of Cthrc1 was assessed in normal skin and keloid tissue, as well as in normal fibroblasts (NFs) and keloid fibroblasts (KFs) by using quantitative PCR, Western blotting and immunohistochemical analysis. The results showed that Cthrc1 was increased in keloid tissue and KFs as compared with normal skin and NFs. Meanwhile, CCK8 and Transwell assays found the cellular proliferation and migration of KFs were increased as compared with NFs. Further, to verify the function of Cthrc1 in NFs and KFs, we increased Cthrc1 expression by transfecting lentivirus (LV) vectors LV-Cthrc1. The cellular proliferation and migration, collagen synthesis and the influence on TGF-β and YAP signaling were tested. The cellular proliferation and migration were increased in NFs-Cthrc1 as compared with NFs-control. Meanwhile, YAP expression and nuclear-location was increased in NFs-Cthrc1. On the contrary, when Cthrc1 was overexpressed in KFs, the cellular migration was suppressed and YAP expression was reduced and transferred to cytoplasm in KFs-Cthrc1 as compared with KFs-control. But the expression level of collagen I was decreased and pSmad2/3 nucleus transfer was suppressed in both NFs-Cthrc1 and KFs-Cthrc1 by using Western blotting and immunofluorescence. Increased Cthrc1 activated NFs by promoting YAP nucleus translocation, whereas suppressed KFs by inhibiting YAP nucleus translocation. Enhanced Cthrc1 decreased collagen I in both NFs and KFs by inhibiting TGF-β/Smad pathway. In conclusion, Cthrc1 may play a role in the pathogenesis of keloid by inhibiting collagen synthesis and fibroblasts migration via suppressing TGF-β/Smad pathway and YAP nucleus translocation.

Topics: Adaptor Proteins, Signal Transducing; Adventitia; Extracellular Matrix Proteins; Fibroblasts; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Keloid; Lentivirus; Neoplasms; Phosphoproteins; Signal Transduction; Skin Neoplasms; Smad Proteins; Transcription Factors; Transfection; Transforming Growth Factor beta; YAP-Signaling Proteins

Keloids are characterized by excessive collagen deposition and growth beyond the edges of the initial injury, and cytokines may be related to their formation. The objective of this study was to evaluate the collagen fibers, analyze in situ expression of cytokines in keloid lesions, and compare to the control group. Results showed that there was a predominance of women and nonwhite and direct black ancestry. Keloid showed a significant increase in total and type III collagen. Significantly, the expression of mRNA for TGF-

Topics: Adolescent; Adult; Collagen; Collagen Type I; Collagen Type III; Cytokines; Female; Humans; Interferon-gamma; Interleukin-10; Keloid; Male; Middle Aged; RNA, Messenger; Transforming Growth Factor beta; Young Adult

Mortalin (Mot) is a mitochondrial chaperone of the heat shock protein 70 family and it's pro-proliferative and anti-apoptosis functions could be associated with keloid pathogenesis, and blocking of mortalin and its interaction with p53 might be a potential novel target for the treatment of keloid. Therefore, we generated mortalin-specific small hairpin (sh) RNAs (dE1-RGD/GFP/shMot) and introduced into keloid spheroids for examination of its apoptotic and anti-fibrotic effect. On keloid tissues, mortalin expression was higher than adjacent normal tissues and it's protein expressions were activated keloid fibroblasts (KFs). After primary keloid spheroid were transduced with dE1-RGD/GFP/shMot for knockdown of mortalin, expression of type I, III collagen, fibronectin, and elastin was significantly reduced and transforming growth factor-β1, epidermal growth factor receptor (EGFR), Extracellular Signal-Regulated Kinases 1 and 2 (Erk 1/2), and Smad 2/3 complex protein expression were decreased. In addition, increased TUNEL activities and cytochrome C were observed. Further, for examine of mortalin and p53 interaction, we performed immunofluorescence analysis. Knockdown of mortalin relocated p53 to the cell nucleus in primary keloid spheroids by dE1-RGD/GFP/shMot transduction. These results support the utility of knockdown of mortalin to induce apoptosis and reduce ECMs expression in keloid spheroid, which may be highly beneficial in treating keloids.

Topics: Adenoviridae; Apoptosis; Cell Nucleus; Collagen Type I; Collagen Type III; Elastin; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Fibronectins; Fibrosis; HSP70 Heat-Shock Proteins; Humans; Keloid; Proliferating Cell Nuclear Antigen; Protein Binding; RNA, Small Interfering; Spheroids, Cellular; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The keloid lesion is recognised as a spatially heterogeneous mass both in cellular and acellular composition and biological activity. Here, we have utilised a bioinformatic approach to determine whether this spatial heterogeneity is also evident at the molecular level and to identify key upstream regulators of signalling pathways enriched in the lesion in a spatially-restricted manner. Differentially expressed genes (20% change, p < 0.05) obtained from microarray datasets derived from whole keloid biopsies and ex vivo-cultured keloid fibroblasts, both from distinct regions of the keloid lesion (leading edge, centre, and top) have been analysed to show that the TGFβ family plays a significant but spatially dependent role in regulation of keloid gene expression. Furthermore, we have identified additional upstream signalling molecules involved in driving keloid biology and provide information on therapeutic targets whose modulation might be expected to lead to significant therapeutic efficacy.

Topics: Biopsy; Cells, Cultured; Computational Biology; Fibroblasts; Gene Expression; Humans; Keloid; Principal Component Analysis; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β)/Smad signaling plays a key role in excessive fibrosis and keloid formations. Smad7 is a negative feedback regulator that prevents activation of TGF-β/Smad signaling. However, the regulatory mechanism for Smad7 in the keloid pathogenic process remains elusive. Here, we show that expression of TIEG1 is markedly higher in keloid fibroblasts, whereas protein, mRNA, and promoter activity levels of Smad7 are decreased. When TIEG1 was knocked down with small interfering RNA, both the promoter activity and protein expression of Smad7 were increased, whereas collagen production and the proliferation, migration, and invasion of keloid fibroblasts were decreased. In contrast, TIEG1 overexpression led to a decrease in Smad7 expression and Smad7 promoter activity. Upon TGF-β1 stimulation, TIEG1 promoted Smad2 phosphorylation by down-regulating Smad7. Luciferase reporter assays and chromatin immunoprecipitation assays further showed that TIEG1 can directly bind a GC-box/Sp1 site located between nucleotides -1392 and -1382 in the Smad7 promoter to repress Smad7 promoter activity. Taken together, these findings show that TIEG1 is highly expressed in human keloids and that it directly binds and represses Smad7 promoter-mediated activation of TGF-β/Smad2 signaling, thus providing clues for development of TIEG1 blocking strategies for therapy or prophylaxis of keloids.

Topics: Adolescent; Adult; Case-Control Studies; Collagen; Down-Regulation; Early Growth Response Transcription Factors; Female; Fibroblasts; Gene Knockdown Techniques; Humans; Keloid; Kruppel-Like Transcription Factors; Male; Phosphorylation; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad7 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1; Young Adult

Keloid disease (KD) is a fibroproliferative cutaneous tumour characterised by heterogeneity, excess collagen deposition and aggressive local invasion. Lack of a validated animal model and resistance to a multitude of current therapies has resulted in unsatisfactory clinical outcomes of KD management. In order to address KD from a new perspective, we applied for the first time a site-specific in situ microdissection and gene expression profiling approach, through combined laser capture microdissection and transcriptomic array. The aim here was to analyse the utility of this approach compared with established methods of investigation, including whole tissue biopsy and monolayer cell culture techniques. This study was designed to approach KD from a hypothesis-free and compartment-specific angle, using state-of-the-art microdissection and gene expression profiling technology. We sought to characterise expression differences between specific keloid lesional sites and elucidate potential contributions of significantly dysregulated genes to mechanisms underlying keloid pathobiology, thus informing future explorative research into KD. Here, we highlight the advantages of our in situ microdissection strategy in generating expression data with improved sensitivity and accuracy over traditional methods. This methodological approach supports an active role for the epidermis in the pathogenesis of KD through identification of genes and upstream regulators implicated in epithelial-mesenchymal transition, inflammation and immune modulation. We describe dermal expression patterns crucial to collagen deposition that are associated with TGFβ-mediated signalling, which have not previously been examined in KD. Additionally, this study supports the previously proposed presence of a cancer-like stem cell population in KD and explores the possible contribution of gene dysregulation to the resistance of KD to conventional therapy. Through this innovative in situ microdissection gene profiling approach, we provide better-defined gene signatures of distinct KD regions, thereby addressing KD heterogeneity, facilitating differential diagnosis with other cutaneous fibroses via transcriptional fingerprinting, and highlighting key areas for future KD research.

Topics: Biopsy; Collagen; Epidermis; Epithelial-Mesenchymal Transition; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Humans; Inflammation; Keloid; Keratinocytes; Laser Capture Microdissection; Microarray Analysis; Neoplastic Stem Cells; Organ Specificity; Primary Cell Culture; Signal Transduction; Transcriptome; Transforming Growth Factor beta

Chemokine-like factor 1 (CKLF-1) is a novel cytokine which have a crucial role in immune and inflammatory responses. In this study, the expression level of CKLF-1 was measured to assess the difference between keloid patients and people without keloid. Fifty samples were taken from 30 patients: 10 keloid patients; 10 scar patients; and 10 patients without obvious scarring. Patients were randomly selected from the hospitalized patients of Peking Union Medical College Hospital from September 2013 to July 2015. Five groups of samples were established: keloid samples from keloid patients (K); normal skin samples from keloid patients (KS); scar samples from scar patients (C); normal skin samples from scar patients (CS); and normal skin samples from patients without obvious scarring (S). Hematoxylin and eosin (H&E) staining was used to observe morphological changes. CKLF-1, IL-6, IL-8, IL-18, and TGF-β were detected by immunohistochemical and western blot technology. The expression of CKLF-1's mRNA was also measured by the real-time quantitative polymerase chain reaction (RT-qPCR). Compared to the K group, the other 4 groups presented significantly less inflammatory infiltration and lower expression levels of CKLF-1, IL-6, IL-8, IL-18, and TGF-β. Among the 3 normal skin groups, the expression level of CKLF-1 was significantly higher in the KS group than in the CS or S group. The mRNA expression was also obvious in the K and KS groups. CKLF-1 and other inflammatory factors were overexpressed in the samples from keloid patients, indicating that the formation of keloid may be related to inflammation and that CKLF-1 may play an important role in this process.

Topics: Adolescent; Adult; Case-Control Studies; Chemokines; Cicatrix; Female; Genetic Predisposition to Disease; Humans; Interleukins; Keloid; Male; MARVEL Domain-Containing Proteins; Middle Aged; Skin; Transforming Growth Factor beta; Young Adult

Keloid disease is characterized by hyperproliferation of responsive fibroblasts with vigorously continuous synthesis of extracellular matrix (ECM) components. Although the process by which keloids develop is poorly understood, most theories of the etiology are referred to fibroblast dysfunction. A central event in dermal repair is the release of growth factors in response to skin injury, which leads to the dysregulation of several crucial pathways that initiate the activation of keloid fibroblasts (KFs) and promote ECM accumulation. Hence, strategies aimed at reducing the production of these cytokines and/or disrupting their intracellular signal transduction have potential clinical significance for curing keloid. As the first oral multikinase inhibitor, sorafenib blocks a number of intracellular signaling pathways which are also pivotal for keloid pathogenesis. Therefore, evaluation of the effects of sorafenib on keloid disease seems timely and pertinent. In this study, we reported the identification of sorafenib that antagonized TGF-β/Smad and MAPK/ERK signaling pathways in primary KFs. Impressively, treatment with sorafenib inhibited KF cell proliferation, migration, and invasion, and simultaneously reduced collagen production in KFs. Furthermore, we present ex vivo evidence that sorafenib induced the arrest of KF migration, the inhibition of angiogenesis, and the reduction of collagen accumulation. These preclinical observations suggest that sorafenib deserves systematic exploration as a candidate agent for the future treatment of keloids.. The intracellular TGF-β/Smad and MAPK/ERK signaling pathways is blocked by sorafenib. Sorafenib inhibits the proliferation, migration, invasion, and ECM deposition in keloid fibroblasts. Sorafenib reduces KF migration and concomitantly angiogenesis in keloid explants. Sorafenib is a promising agent for the treatment of keloids and hypertrophic scars.

Topics: Adult; Cell Cycle; Cell Movement; Cell Proliferation; Cells, Cultured; Female; Fibroblasts; Humans; Keloid; Male; Middle Aged; Mitogen-Activated Protein Kinases; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Signal Transduction; Sincalide; Smad Proteins; Sorafenib; Transforming Growth Factor beta; Young Adult

Keloids result from aberrations in the normal wound healing cascade and can lead to pruritus, contractures and pain. The underlying mechanisms of excessive scarring are not yet understood, and most therapeutic strategies remain unsatisfactory. Psoriasin (S100A7) and koebnerisin (S100A15) are released by keratinocytes during physiological wound healing. We found S100 production is markedly decreased in keloid scar tissue. The disturbed epidermal S100 expression might contribute to keloid formation; thus, we studied their effect on dermal fibroblasts and extracellular matrix (ECM) production.. S100 peptides, ECM regulation and distribution were analysed in normal and keloid tissue by quantitative PCR (qPCR), immunoblotting and immunofluorescent staining. Isolated dermal fibroblasts were incubated with S100 proteins, and the regulation of ECM and transforming growth factor (TGF)-β was determined using qPCR. Fibroblast proliferation and viability were determined by the 5-bromo-2'-deoxyuridine assay and crystal violet assay.. Keloid tissue featured a pronounced expression of ECMs, such as collagen types 1 and 3, whereas the production of psoriasin and koebnerisin was markedly decreased in keloid-derived cells and keloid tissue. Both S100 proteins inhibited the expression of collagens, fibronectin-1, α-smooth-muscle actin and TGF-β by fibroblasts. Further, they also suppressed fibroblast proliferation.. Psoriasin and koebnerisin show antifibrotic effects and may lead to novel preventive and therapeutic strategies for fibroproliferative diseases.

Topics: Cell Proliferation; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type III; Extracellular Matrix; Fibroblasts; Humans; Keloid; Peptides; Recombinant Proteins; S100 Calcium Binding Protein A7; S100 Proteins; Skin; Transforming Growth Factor beta

The 90-kDa heat-shock protein (heat-shock protein 90) is an abundant cytosolic chaperone, and inhibition of heat-shock protein 90 by 17-allylamino-17-demethoxygeldanamycin (17-AAG) compromises transforming growth factor (TGF)-β-mediated transcriptional responses by enhancing TGF-β receptor I and II degradation, thus preventing Smad2/3 activation. In this study, the authors evaluated whether heat-shock protein 90 regulates TGF-β signaling in the pathogenesis and treatment of keloids.. Keloid fibroblasts were treated with 17-AAG (10 μM), and mRNA levels of collagen types I and III were determined by real-time reverse- transcriptase polymerase chain reaction. Also, secreted TGF-β1 was assessed by enzyme-linked immunosorbent assay. The effect of 17-AAG on protein levels of Smad2/3 complex was determined by Western blot analysis. In addition, in 17-AAG-treated keloid spheroids, the collagen deposition and expression of major extracellular matrix proteins were investigated by means of Masson trichrome staining and immunohistochemistry.. The authors found that heat-shock protein 90 is overexpressed in human keloid tissue compared with adjacent normal tissue, and 17-AAG decreased mRNA levels of type I collagen, secreted TGF-ß1, and Smad2/3 complex protein expression in keloid fibroblasts. Masson trichrome staining revealed that collagen deposition was decreased in 17-AAG-treated keloid spheroids, and immunohistochemical analysis showed that expression of collagen types I and III, elastin, and fibronectin was markedly decreased in 17-AAG-treated keloid spheroids.. These results suggest that the antifibrotic action of heat-shock protein 90 inhibitors such as 17-AAG may have therapeutic effects on keloids.

Topics: Benzoquinones; Biomarkers; Blotting, Western; Cells, Cultured; Collagen Type I; Collagen Type III; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Fibroblasts; HSP90 Heat-Shock Proteins; Humans; Immunohistochemistry; Keloid; Lactams, Macrocyclic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Spheroids, Cellular; Transforming Growth Factor beta

Hypertrophic scars and keloids remain a challenge in surgery. We appreciate that our understanding of the process at cellular and molecular level, profound as it is, when it comes to the clinical evidence much is left to be desired. Although the bench to bedside conundrum remains, the science of translational research calls for an even higher level of cooperation between the scientist and the clinician for the impetus to succeed.The clinicians alerted us to the possible theories in the pathogenesis of keloid formation, inter alia, the ischemia theory, mast cell theory, immune theory, transforming growth factor β interaction, mechanical theory, and the melanocyte stimulating hormone theory. All of the above presupposed a stimulus that would result in an uncontrolled upregulation of collagen and extracellular matrix expression in the pathogenesis of the keloid. This bedside to bench initiative, as in true science, realized more ponderables than possibilities.By the same token, research into the epidermal-mesenchymal signaling, molecular biology, genomics, and stem cell research holds much promise in the bench top arena. To assess efficacy, many scar assessment scores exist in the literature. The clinical measurement of scar maturity can aid in determining end points for therapeutics. Tissue oxygen tension and color assessment of scars by standardized photography proved to be useful.In surgery, the use of dermal substitutes holds some promise as we surmise that quality scars that arise from dermal elements, molecular and enzyme behavior, and balance. Although a systematic review shows some benefit for earlier closure and healing of wounds, no such review exists at this point in time for the use of dermal substitutes in scars.Adipose-derived stem cell, as it pertains to scars, will hopefully realize the potential of skin regeneration rather than by repair in which we are familiar with as well as the undesirable scarring as a result of healing through the inflammatory response.Translational research will bear the fruit of coordinating bench to bedside and vice versa in the interest of progress into the field of regenerative healing that will benefit the patient who otherwise suffers the myriad of scar complications.

Topics: Cicatrix, Hypertrophic; Dermatologic Surgical Procedures; Epidermis; Humans; Keloid; Skin Physiological Phenomena; Transforming Growth Factor beta

Systemic sclerosis (SSc) is still an enigmatic disease of unknown etiology, although the pathophysiology is thought to be based on vascular alterations as well as immunological and fibrotic processes. Here we present the case of a female patient with diffuse SSc (dSSc), who developed multiple subcutaneous nodules. Histologic evaluation confirmed the diagnosis of nodular scleroderma, a very rare condition. Histological analysis of biopsies was combined with ultrastructural analysis by transmission electron microscopy and immunohistochemistry/immunofluorescence, using antibodies against different collagens and non-collagenous ECM proteins. Collagen fibrils were deposited at very high density in nodules as well as in adjacent extra nodular skin. Within nodules, a large fraction of immature collagen fibrils was detected with smaller and highly variable diameter. Activated fibroblasts were present, however no myofibroblasts were identified. Cartilage Oligomeric Matrix Protein (COMP), collagen XII and fibrillin-1 were all deposited at increased amounts within nodules and their distribution differed markedly from that in healthy skin. The excessive deposition of COMP within nodules closely resembled the distribution of COMP in keloids. Nodules-like keloids-were characterized by lack of myofibroblasts. By virtue of its structural properties and the capacity to avidly bind collagen I and XII, COMP is thought to reorganize and compact the collagen network, leading to a tissue with locally increased biomechanical tension acting on fibroblasts. In addition, COMP may present active TGFβ to fibroblasts. Both mechanisms in concert can activate fibroblast proliferation and production of extracellular matrix, resulting in a sustained activation loop.

Topics: Cartilage Oligomeric Matrix Protein; Cells, Cultured; Collagen Type XII; Extracellular Matrix; Female; Fibrillin-1; Fibrillins; Fibroblasts; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Keloid; Microfilament Proteins; Middle Aged; Scleroderma, Diffuse; Skin; Transforming Growth Factor beta

To investigate the effect of 5-aza-2-deoxycytidine on the TGF-beta/smad signal transduction pathway in human keloid fibroblasts (KFSs).. Firstly, immunohistochemical method was used to detect the positive expression rate of phospho-smad2 and phospho-smad3 in the specimens of 15 cases of keloid and 15 cases of normal skin. The keloid fibroblasts were cultured in vitro with 5-aza-2-deoxycytidine(experimental group) or with DMEM (control group). The effect of 5-aza-2-deoxycytidine on the cell cycle and apoptosis of fibroblasts was analysed with flow cytometry ( FCM). Transforming growth factor (TGF)-beta1, Smad7, phospho-smad2 and phospho-smad3 were analyzed by Western Blot, and Immunofluorescence.. It was found that the positive expression of phospho-smad2 and phospho-smad3 in keloid were higher than those in normal skin. The FCM showed that the proportion of cells in G0/G1 stage was increased, and so does the proportion of apoptosis cells in keloid fibroblasts intervened by 5-aza-2-deoxycytidine. The expression of TGF-beta1, phospho-smad2 and phospho-smad3 protein were significantly suppressed while the expression of smad7 protein increased in keloid fibroblasts with 5-aza-2-deoxycytidine. In addition, 5-aza-2-deoxycytidine reversed phosphorylation and nuclear translocation of smad2 and smad3.. 5-aza-2-deoxycytidine, methylase inhibitors, inhibits cell proliferation and promotes apoptosis of KFSs, which may be associated with the suppression of TGF-beta/smad signal pathway.

Topics: Apoptosis; Azacitidine; Cell Proliferation; Cells, Cultured; Decitabine; Enzyme Inhibitors; Female; Fibroblasts; Humans; Keloid; Male; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta

Keloid formation is closely related with transforming growth factor (TGF)-β-induced Smad signal transduction. Recent studies have shown that toll-like receptor4 (TLR4) may mediate liver and kidney fibrosis, and activation of TLR7 has anti-scarring effect. The role of TLR4/7 signalling in keloid formation, however, remains unknown. Our previous tests have found that mute Smad4 inhibited scar. We then speculated that keloid may be affected by TLR4/7 through TGF-β-induced Smad signal transduction.. The aim of this study was to evaluate effects of TLR4/7 on the TGF-β-induced Smad signal transduction pathway in human keloid, and provide information for the mechanism and therapy of keloid.. Normal scar samples with normal fibroblasts (NFs) served as control samples and keloid samples with keloid fibroblasts (KFs) served as experiment samples. Expression of collagen, connective tissue growth factor (CTGF), Smad4 and Smad7 and TLR4/7 were tested by immunohistochemistry, reverse transcription polymerase chain reaction (PCR) (RT-PCR) and Western blotting, respectively.. Expression of collagen, CTGF, Smad4 and TLR4 increased significantly while expression of Smad7 and TLR7 decreased in KFs while compared to NFs in keloid scar group (KFs), which were decreased in the normal scar group (NFs). However, expression of Smad7 and TLR7 decreased in the keloid scar group (KFs) while compared to the NFs.. TLRs participate in fibrosis of scar tissue through the TLRs-TGF-β-Smads signal pathway. Higher expression of TLR4 in keloid increased expression of TGF-β, CTGF and collagen through the Smad4 signal pathway. Activation of TLR7 or Smad7 may inhibit scar formation.

Topics: Adolescent; Adult; Blotting, Western; Collagen; Connective Tissue Growth Factor; Female; Humans; Immunohistochemistry; Keloid; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Toll-Like Receptor 4; Toll-Like Receptor 7; Transforming Growth Factor beta; Young Adult

Keloids are characterised by the excessive accumulation of extracellular matrix (ECM), especially overabundant collagen formation. In keloid fibroblasts (KFs), transforming growth factor (TGF)-β-dependent signalling is closely associated with a variety of keloid pathologic responses such as ECM production and fibroblast overgrowth. Thus, inhibition of TGF-β signalling would be a potential therapeutic approach to prevent keloid scar formation. Thereby, we aimed to identify a novel TGF-β signalling blocker among natural products using a simplified screening approach. We discovered that the extract of Aneilema keisak (A.K-Ex) lowered TGF-β-dependent signalling by reducing Smad2 protein levels. Given that KFs showed altered dependency on TGF-β for survival and proliferation, A.K-Ex-mediated reduction in Smad2 protein levels significantly inhibited the major characteristics of KFs such as cell growth, migration and collagen synthesis, suggesting that A.K-Ex exhibits possible therapeutic activity on keloids.

Topics: Cell Movement; Cell Proliferation; Cells, Cultured; Collagen; Commelinaceae; Down-Regulation; Fibroblasts; Humans; Keloid; Plant Extracts; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

The transforming growth factor (TGF)-β/Smad pathway plays a key role in keloid development. We have previously demonstrated that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits liver fibrosis and reduces invasion capacity of HepG2 cells by mediating the TGF-β/Smad pathway. We therefore hypothesize that CASE may also exert antifibrotic effects in keloids by mediating the TGF-β/Smad pathway.. To investigate the effects of CASE on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and to explore the effects of CASE on the TGF-β/Smad signal pathway in order to elucidate its mechanisms of action.. The inhibitory effects of CASE on keloid fibroblasts were evaluated. Cell proliferation was studied by MTT assay; cell invasion was observed utilizing Transwell invasion chambers; and collagen synthesis in keloid fibroblasts was measured by (3) H-proline incorporation assay. Expression of proteins induced by TGF-β1 and their intracellular localization in keloid fibroblasts were investigated by Western blot and immunofluorescence, respectively. Plasminogen activator inhibitor-1 (PAI-1) transcriptional activity was measured by real-time reverse transcription-polymerase chain reaction.. CASE significantly inhibited cell proliferation induced by newborn bovine serum as well as collagen synthesis and cell invasion induced by TGF-β1 in keloid fibroblasts, while it showed weak effects on normal fibroblasts. The phosphorylation of Smad2/3 was markedly reduced by CASE treatment, while CASE exhibited stronger inhibitory effects on linker region phosphorylation (pSmad2L and pSmad3L) compared with effects on C-terminal region phosphorylation (pSmad2C and pSmad3C). In addition, CASE blocked formation of Smad2/3/4 complexes and their nuclear translocation, but upregulated Smad7 expression in a dose-dependent manner. PAI-1 mRNA and protein levels were also suppressed by CASE treatment.. These results suggest that CASE exhibits inhibitory effects on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and its mechanisms of action may involve the TGF-β/Smad pathway.

Topics: Adult; Animals; Cattle; Cell Proliferation; Collagen; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Female; Fibroblasts; Humans; Keloid; Male; Middle Aged; Phosphorylation; Salvia miltiorrhiza; Serum; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1; Young Adult

Keloid scars are large protruding claw-shaped lesions that develop beyond the confines of the wound and uniquely appears only in humans. For thousands of years ginseng has been used in the traditional medicine in oriental countries. It occupies a prominent position in the list of the best-selling medicinal herbs in the world. Panax ginseng often called Asian or Korean ginseng, is the most extensively used and the best grade of ginseng and the term of 'ginseng' generally refers to Panax ginseng. Previous studies have revealed that ginseng inhibits NF-kappa B, TGF-β, IL-6, ACE and MMP-2 and these factors play a pivotal role in keloid formation pathogenesis. Therefore it could be reasoned that ginseng could be effective for the treatment of the keloid scars. Clinical studies by topical applications of iPanax notoginseng (800 µg/ml) are warranted.

Topics: Acetylcholinesterase; Animals; Ginsenosides; Humans; Interleukin-6; Keloid; Matrix Metalloproteinase 2; NF-kappa B; Panax; Transforming Growth Factor beta

  Keloid is a unique proliferative disorder of fibroblasts resulting from derailment of the typical wound healing process. Due to lack of animal models for therapeutic testing, treatment of keloids remains a clinical challenge. Transforming growth factor (TGF)-β1-related signalling plays a key role in keloid formation. As tacrolimus (FK506) has been reported to inhibit the effects of TGF-β1 on cultured fibroblasts, we hypothesized that FK506 may be useful in treating keloids.. To explore the effects of FK506 on TGF-β1-stimulated keloid fibroblasts (KFs) in terms of proliferation, migration and collagen production and to investigate the regulatory pathways involved.. Fibroblasts derived from keloids were treated with TGF-β1 with or without FK506. Relevant assays including 5-bromo-2'-deoxyuridine incorporation assay, in vitro scratch assay, reverse transcription-polymerase chain reaction (PCR), quantitative PCR and Western blotting were performed.. The proliferation and migration of KFs were significantly higher than those of normal fibroblasts. FK506 markedly inhibited KF proliferation, migration and collagen production enhanced by TGF-β1. The increase in TGF-β receptor I and II expression in TGF-β1-treated KFs was suppressed by FK506 treatment. TGF-β1 increased the phosphorylation of Smad2/3 and Smad4 in KFs, and this enhancing effect was abrogated by FK506. In addition, FK506 significantly increased the expression of Smad7 which was suppressed by TGF-β1 treatment.. Our results demonstrate that FK506 effectively blocks the TGF-β/Smad signalling pathway in KFs by downregulation of TGF-β receptors and suggest that FK506 may be included in the armamentarium for treating keloids.

Topics: Adult; Cell Movement; Cell Proliferation; Cells, Cultured; Collagen; Down-Regulation; Female; Fibroblasts; Humans; Immunosuppressive Agents; Keloid; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Tacrolimus; Transforming Growth Factor beta

SMAD3 is a key player in the TGFβ signaling pathway as a primary inducer of fibrosis. The inhibition of SMAD3 production is one strategy to alleviate fibrosis in keloid fibroblasts. In the present study, antisense oligonucleotides (ASOs) against SMAD3 were designed to specifically block the expression of SMAD3. The cationic lipid nanoparticles (cLNs) were formulated to enhance an intracellular activity of SMAD3 ASOs in keloid fibroblasts. This formulation was prepared using melt-homogenization method, composed of 3-[N-(N',N'-dimethylaminoethane)-carbamol] cholesterol (DC-Chol), dioleoylphosphatidylethanolamine (DOPE), Tween20, and trimyristin as a lipid core (1:1:1:1.3, w/w). The size and zeta potential of cLNs and cLN/ASO complexes were measured using light scattering. AFM was used to confirm the morphology and the size distribution of cLNs and cLN/ASO complexes. The prepared cLNs had a nano-scale sized spherical shape with highly positive charge, which were physically stable without aggregation during the storage. The cLN/SMAD3 ASO complexes were successfully generated and internalized onto keloid fibroblasts without toxicity. After the treatment with cLN/ASO complexes, SMAD3 was inhibited and collagen type I was also significantly suppressed in keloid fibroblasts. These results suggest that SMAD3 ASOs complexed with cLNs have a therapeutic potential to suppress collagen deposition in fibrotic diseases. Therefore, this strategy might be developed to lead to anti-fibrotic therapies.

Topics: Cells, Cultured; Collagen Type I; Drug Stability; Drug Storage; Fibroblasts; Fibrosis; Humans; Keloid; Light; Lipids; Nanoparticles; Oligonucleotides, Antisense; Particle Size; Scattering, Radiation; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Keloid is a complex condition with environmental and genetic risk-contributing factors. Two candidate genes, TGFβ1 and SMAD4, located in the same signaling pathway are highly expressed in the keloid fibroblast cells. In a case-control design, TGFβ1 haplotypes showed association with the risk of keloid in the present study. The CC haplotype, composed of both c.29C>T and -509T>C variants, was observed more frequently among cases (Corrected p = 0.037, OR = 2.07, 95 % CI = 0.87-4.93), showing a 4.5-fold increased risk for keloid. The AG genotype of the SMAD4 c.5131A>G variant showed a trend of significance (p = 0.0573, OR = 1.75, 95 % CI = 0.99-3.13). Taken together, either of these variants is most probably causative at the expression level or is in linkage disequilibrium with other causative variants in a complex pattern together with the environmental factors that contribute to the condition. To the best of our knowledge, there is only one documented report on a relationship between TGFβ1 and keloid with no association within the Caucasian population, while there have not been any reports for SMAD4. Therefore, the present study is likely the first research showing a significant association between TGFβ1 variants and keloids in the Malay population.

Topics: Adolescent; Adult; Case-Control Studies; Child; Female; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Keloid; Malaysia; Male; Middle Aged; Polymorphism, Genetic; Smad4 Protein; Transforming Growth Factor beta; Young Adult

Keloids are the result of excessive scar tissue formation. Besides their poor aesthetic appearance, keloids can be associated with severe clinical symptoms such as pain, itching, and rigidity. Unfortunately, most therapeutic approaches remain clinically unsatisfactory. Recently, injections with botulinum toxin A (BTA) were proposed for the treatment of established keloids in a clinical trial. In this study, we aimed to verify the effects of intralesional BTA for the treatment of therapy-resistant keloids using objective measurements. In addition, the underlying molecular mechanisms were investigated using cultured keloid-derived fibroblasts.. Four patients received BTA (doses varying from 70 to 140 Speywood units per session) injected directly into their keloids every 2 months for up to 6 months. Differences in height and volume were evaluated clinically and measured with a 3-D optical profiling system. Keloid-derived fibroblasts were treated with different concentrations of BTA, and expression of collagen (COL)1A1, COL1A2, COL3A1, TGF-β1, TGF-β2, TGF-β3, fibronectin-1, laminin-β2, and α-SMA was determined by real-time quantitative PCR. MTT and BrdU assays were used to analyze the effects of BTA on fibroblast proliferation and metabolism.. Intralesional administration of BTA did not result in regression of keloid tissue. No differences in expression of ECM markers, collagen synthesis, or TGF-β could be observed after BTA treatment of keloid fibroblasts. In addition, cell proliferation and metabolism of keloid fibroblasts was not affected by BTA treatment.. The suggested clinical efficiency of intralesional BTA for the therapy of existent keloids could not be confirmed in this study. Based on our data, the potential mechanisms of action of BTA on keloid-derived fibroblasts remain unclear.

Topics: Actins; Botulinum Toxins, Type A; Cell Proliferation; Collagen; Female; Fibroblasts; Fibronectins; Humans; Injections, Intralesional; Keloid; Laminin; Male; Transforming Growth Factor beta

Keloids and hypertrophic scars are significant symptomatic clinical problems characterized by the excessive and abnormal deposition of collagen-based extracellular matrix (ECM) components. However, the molecular basis of keloid and hypertrophic scar formation has not been fully elucidated. Here, we demonstrated that down-regulation of the transcription factor Smad interacting protein 1 (SIP1) could be relevant to keloid and hypertrophic scar formation. The results of the present study show that the level of SIP1 mRNA is significantly decreased in pathological scar tissues and in normal skin and pathological scar fibroblasts treated with transforming growth factor β1 (TGF-β1). In contrast, the expression of SIP1 mRNA is not decreased in normotrophic scar samples. The SIP1 mRNA level inversely correlates with the mRNA level of type I collagen (COL1A2) and directly correlates with the mRNA level of matrix metalloproteinase-1 (MMP1). Overexpression of SIP1 in keloid and hypertrophic scar fibroblasts represses TGF-β1-stimulated COL1A2 expression and induces MMP1 expression. Alternatively, knockdown of SIP1 in normal skin fibroblasts enhance TGF-β1-induced COL1A2 levels. These findings suggest that SIP1 could be a regulator of skin fibrosis, and depletion of SIP1 in pathological scar tissues could result in an up-regulation of collagen and down-regulation of matrix metalloproteinase, leading to an abnormal accumulation of ECM along with fibrosis and pathological scar formation.

Topics: Adolescent; Adult; Aged; Child; Cicatrix; Collagen; Collagen Type I; Down-Regulation; Female; Fibroblasts; Fibrosis; Humans; Intracellular Signaling Peptides and Proteins; Keloid; Male; Matrix Metalloproteinase 1; Middle Aged; RNA-Binding Proteins; RNA, Messenger; Skin; Transforming Growth Factor beta; Young Adult

Wound healing can result in the development of keloid scars that contain atypical fibroblasts and an overabundance of extracellular matrix components. Hyperbaric oxygenation (HBO) refers to exposure to pure oxygen under increased atmospheric pressure and is recognized as a valuable supplementary method of treatment for problematic wounds. The effect of HBO in the expression of insulin-like growth factor type 1 (ILGF-1) and transforming growth factor β (TGF-β) messenger RNAs was determined by semiquantitative RT-PCR in fibroblasts obtained from keloid scars and nonwound involved skin fibroblast from the same patient. ILGF-1 and TGF-β are the principal mitogens during wound regeneration. We found a decrease in the growth of fibroblasts and in the expression of ILGF-1 and TGF-β messengers in keloid and nonkeloid fibroblast after chronic exposition to hyperbaric oxygenation compared with normal oxygen partial pressure.

Topics: Fibroblasts; Humans; Hyperbaric Oxygenation; Insulin-Like Growth Factor I; Keloid; RNA, Messenger; Transforming Growth Factor beta; Wound Healing

Dermal wound healing depends on highly complex interplay among various cytokines and cell types. Disruption of this process can result in impaired healing in the form of excessive scarring, as is the case in fibrotic diseases such as keloid and scleroderma. In the present study, we found Fussel-15, a new member of the Ski/Sno family of TGF-β/BMP signaling repressors, to be expressed in early wound healing and constantly overexpressed in keloid-derived and scleroderma-derived fibroblasts. Comparing the results of three-dimensional free-floating and attached-released in vitro wound healing assays, we observed that Fussel-15 is expressed during the migratory phase in the free-floating assay, indicating that Fussel-15 might play a role during fibroblast migration. Fussel-15-transfected fibroblasts showed greater migration ability in a scratch wound healing assay, compared with control-transfected cells. This migratory phenotype due to Fussel-15 was confirmed by increased peripheral F-actin localization and modifications in size, amount, and distribution of focal adhesion complexes, which were observed using F-actin and focal adhesion kinase (FAK) immunofluorescence staining, respectively. The present results suggest that expression of Fussel-15 during wound healing might promote fibroblast migration. Permanent expression of Fussel-15 in keloid and skin sclerosis fibroblasts could be involved in the pathogenesis of these conditions, but the molecular mechanism underlying this up-regulation remains to be determined.

Topics: Actins; Biological Assay; Cell Adhesion; Cell Line; Cell Movement; Cell Proliferation; Co-Repressor Proteins; Collagen; Dermis; Extracellular Matrix; Fibroblasts; Focal Adhesions; Gene Expression Regulation; Humans; Keloid; Protein Transport; Repressor Proteins; Scleroderma, Localized; Transforming Growth Factor beta; Wound Healing

Keloids are recognized as benign tumours characterized by fibroblastic proliferation and accumulation of extracellular matrix, especially collagen deposition. The transforming growth factor (TGF)-beta(1)/Smad pathway plays an important role in keloid pathogenesis; however the underlying mechanisms are not fully understood.. To define further the mechanisms of TGF-beta(1)/Smad signal transduction mediated by mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 pathways, in keloid fibroblasts.. In the absence or presence of three MAPK (ERK, JNK and p38)-specific inhibitors, keloid fibroblasts were stimulated with exogenous TGF-beta(1) to activate Smad signalling. Smad protein expression was measured by immunoprecipitation/immunoblotting and immunofluorescence; plasminogen activator inhibitor (PAI)-1 transcriptional activity was measured by real-time reverse transcriptase-polymerase chain reaction analysis.. TGF-beta(1) induced Smad2/3 phosphorylation at both the C-terminal and the linker region, thus promoting formation of the Smad2/3/4 complex and nuclear translocation, and PAI-1 mRNA expression in keloid fibroblasts; in addition, TGF-beta(1) decreased inhibitory Smad7 expression. Meanwhile, the p38 inhibitor significantly inhibited Smad2/3 phosphorylation, especially at the linker region, and furthermore blocked Smad2/3/4 complex formation, and thus decreased PAI-1 mRNA expression; decreased Smad7 expression induced by TGF-beta(1) was also reversed by the p38 inhibitor. The ERK and JNK inhibitors interrupted Smad2/3/4 complex translocation into the nucleus and consequently decreased PAI-1 mRNA expression.. These results suggested that the ERK, JNK and p38 pathways mediate TGF-beta(1)/Smad signal transduction and might be considered as specific targets of drug therapy for keloids.

Topics: Analysis of Variance; Fibroblasts; Fluoroimmunoassay; Humans; Immunoblotting; Keloid; Mitogen-Activated Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Topics: Adult; Aged; Aged, 80 and over; DNA Primers; Female; Fibroblasts; Gene Expression Regulation; Humans; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Keloid; LIM Domain Proteins; Male; Middle Aged; Models, Biological; Protein Structure, Tertiary; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin; Transcription, Genetic; Transforming Growth Factor beta

African-Americans experience an excessive prevalence of a number of apparently disparate disorders that all appear to be, at least in part, mediated by the over-expression or activation of transforming growth factor-beta (TGF-beta) signaling pathways, and that certain genotypes including the codon 10 polymorphism occur more commonly among African-Americans and appears to predispose to these disorders. These disorders, fibrosing in nature, include hypertension, focal glomerulosclerosis, diabetic nephropathy, end stage renal disease, sarcoidosis, uterine leiomyoma, keloids, myocardial fibrosis, and glaucoma. The specific polymorphism for TGF-beta, codon 10, has been implicated in glomerulosclerosis and diabetic nephropathy as well as cardiac transplant rejection. Although TGF-beta over-expression is not the sole factor in these disorders, it is suggested that by designing future clinical studies that consider genomic differences in TGF-beta expression, a more complete understanding of these clinical disorders will be possible. A more thorough understanding of the genetic basis of disease will like promote improved therapeutic regimens and may help reduce the disparate health outcomes for African-Americans as well as improve treatment of individuals of various and diverse ethnic backgrounds.

Topics: Black People; Female; Glaucoma; Heart Transplantation; Humans; Keloid; Leiomyoma; Male; Sarcoidosis; Transforming Growth Factor beta

Keloid is a fibrotic disease characterized by abnormal accumulation of extracellular matrix (ECM) in the dermis. It is a late spreading skin overgrowth and may be considered a plastic surgeon's nightmare. In nature, curcuminoid is composed of curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (bDMC). Curcuminoids have been found to inhibit fibrosis. However, their role in the synthesis of ECM in the keloid fibroblasts (KFs) has remained unclear. In this series of studies, a total of seven primary KFs cultures were used as the KFs model for investigating the inhibitory effect of curcuminoids on the expression of ECM and TGF-β1. A sensitive and reproducible HPLC method was developed to provide a quantitative analysis on the cellular uptake of curcuminoids onto the KF cells. The level of ECM in the primary KFs was elevated. The elevation of ECM and TGF-β1/p-SMAD-2 level was substantially blocked by the cellular uptake of curcumin in a dose-dependent manner in all the seven primary KFs. The results have led to the conclusion that the excessive production of ECM in the KF cells could be blocked and/or rapidly decreased by curcumin.

Topics: Adult; Aged; Cell Culture Techniques; Cells, Cultured; Chromatography, High Pressure Liquid; Curcumin; Extracellular Matrix Proteins; Female; Fibroblasts; Humans; Keloid; Male; Middle Aged; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Up-Regulation

LL-37 is a human cathelicidin antimicrobial peptide that is released in the skin after injury and acts to defend against infection and modulate the local cellular immune response. We observed in human dermal keloids that fibrosis was inversely related to the expression of cathelicidin and sought to determine how LL-37 influenced expression of types I and III collagen genes in dermal fibroblasts. At nano-molar concentrations, LL-37 inhibited baseline and transforming growth factor-beta-induced collagen expression. At these concentrations, LL-37 also induced phosphorylation of extracellular signal-regulated kinase (ERK) within 30 minutes. Activation of ERK, and the activation of a G-protein-dependent pathway, was essential for inhibition of collagen expression as pertussis toxin or an inhibitor of ERK blocked the inhibitory effects of LL-37. c-Jun N-terminal kinase and p38 mitogen-activated protein kinase inhibitors did not alter the effects of cathelicidin. Silencing of the Ets-1 reversed inhibitory effects of LL-37. Taken together, these findings show that LL-37 can directly act on dermal fibroblasts and may have antifibrotic action during the wound repair process.

Topics: Antimicrobial Cationic Peptides; Cathelicidins; Cells, Cultured; Collagen; Fibroblasts; Humans; Keloid; MAP Kinase Signaling System; Pertussis Toxin; Proto-Oncogene Protein c-ets-1; Skin; Transforming Growth Factor beta

Numerous tissue niches in the human body, such as skin, are now recognized to harbour adult stem cells. In this study, we analyze multipotent human dermis-derived progenitor cell populations, isolated and propagated from mechanically and enzymatically processed adult scalp skin. The populations encompass Nestin-positive and -negative cells, which may serve as a convenient and abundant source for various therapeutic applications in regenerative medicine. Here, we show that these cultures exhibit a strong tendency to differentiate into mesodermal derivatives, particularly myofibroblasts, when maintained in media containing serum. Since undesired and excessive myofibroblast formation is a frequent postsurgical complication, we sought culture conditions that would prevent myofibroblast formation. In particular, we analyzed the effect of growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor AB (PDGF AB). Our results demonstrate that bFGF is a potent inhibitor of mesodermal differentiation, whereas PDFG AB favours myofibroblast formation and up-regulates expression of TGFbeta receptors I and II. This interesting discovery may help in the prevention and treatment of tissue fibrosis and in particular in the eradication of hypertrophic and keloid scars.

Topics: Cell Culture Techniques; Cell Differentiation; Cicatrix; Dermis; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Hypertrophy; Immunohistochemistry; Keloid; Platelet-Derived Growth Factor; Regeneration; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Transforming Growth Factor beta

Hypertrophic scars and keloid are dermal proliferative disorders in wound healing. Transforming growth factor beta (TGF-beta) has been implicated in scar formation through the activation of fibroblasts and the acceleration of collagen deposition. Our study aimed to design a novel truncated (27-123 residues) type II TGF-beta receptor (tTGFbetaRII) and to determine its effects on the proliferation of keloid fibroblasts and the collagen synthesis as well as TGF-beta I expression of the cells. The coding sequences of TGF-beta I and tTGFbetaRII were amplified using RT-PCR and then cloned into pGBKT7 and pGADT7 vectors. A yeast two-hybrid experiment and a glutathione S-transferase (GST)-pull down assay were performed to verify the affinity of tTGFbetaRII to TGF-beta I. Our results indicated that treatment with tTGFbetaRII inhibited the growth of keloid fibroblasts and suppressed the synthesis of type I collagen in keloid fibroblasts in a concentration-dependent manner. Moreover, northern and western blot analysis revealed a decline of the TGF-beta I expression at both mRNA and protein levels after exposure to 5, 10 or 20 mug/ml of tTGFbetaRII. Together, our data suggested that the exogenous tTGFbetaRII can efficiently trap TGF-beta I from access to wild-type receptors and can suppress TGF-beta I triggered signals. Thus it may potentially be clinically applied to scar therapy.

Topics: Cell Proliferation; Cells, Cultured; Collagen; Dose-Response Relationship, Drug; Down-Regulation; Fibroblasts; Gene Expression Regulation; Genetic Vectors; Humans; Keloid; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Saccharomyces cerevisiae; Skin; Transfection; Transforming Growth Factor beta; Wound Healing

Keloids arise from the aberrant wound healing process and nitric oxide (NO) plays an important role in the inflammation stage of wound healing. In order to better define the potential effect of NO/cGMP signal pathway in the keloid pathogenesis, the enhancing effect of exogenous NO (released from NO donor) on collagen expression in the keloid fibroblast (KF) as well as on the induction of collagen type I protein and TGF-beta1 expression in the KF were studied in this investigation. The DETA NONOate, an NO donor, was added to the KF, as the exogenous NO, to release NO in the culture medium. The expression of collagens was then determined by assaying the total soluble collagens and collagen type I in the KF. The cellular concentration of cGMP was measured by EIA in the KF. Exogenous NO was found to enhance the expression of collagens and elevate the cellular levels of cGMP. Moreover, to evaluate the effect of the elevated cellular cGMP levels on the expression of collagen and TGF-beta1, both cGMP and TGF-beta1 were measured by ELISA. The inhibitors for phosphodiesterase (PDE), such as IBMX (3-isobutyl-1-methylxanthine), Vinpocetine, EHNA, Milrinone and Zapriast, which have been reported to reduce the ability of PDE and subsequently produce an increase of cellular cGMP, induce the production of autocrine TGF-beta1 as well as the synthesis of collagen in the KF. In this investigation, the inhibition of the PDE enzyme activity was observed to enhance the effect on the collagen synthesis, and was induced by exogenous NO. Taken together, these results have suggested that the NO/cGMP pathway could positively influence the progression of keloid formation, via the TGF-beta1 expression in the KF.

Topics: 1-Methyl-3-isobutylxanthine; Adult; Aged; Blotting, Western; Cells, Cultured; Collagen Type I; Cyclic GMP; Female; Fibroblasts; Humans; Keloid; Male; Middle Aged; Nitric Oxide; Transforming Growth Factor beta

Topics: Cells, Cultured; Collagen; Cytokine TWEAK; Drug Interactions; Fibroblasts; Gels; Humans; Keloid; Transforming Growth Factor beta; Tumor Necrosis Factors

Keloids represent a dysregulated response to cutaneous wounding that result in an excessive deposition of extracellular matrix (ECM), especially types I and III collagen. In keloid scars, the ratio of 'type I to type III collagen' varies compared to normal skin. Transforming growth factor beta (TGF-beta) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of keloid fibroblasts. However, the underlying mechanisms are poorly understood. RNA interference (RNAi) is an evolutionally conserved mechanism for repressing targeted gene expression. In mammalian cells, RNAi is mediated by small interfering RNA (siRNA). In this paper, we examined the function of Sma and Drosophila mothers against decapentaplegic homolog 3 (Smad3), recently characterized as intracellular effector of TGF-beta signalling, in keloid fibroblasts using siRNA.. Dermal fibroblasts obtained from one female patient aged 21 years were maintained in Dulbecco's modified Eagle's medium. Cells (<6 passages) were treated with or without Smad3 siRNA and the expression levels of related genes were examined by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot. Statistical analysis was performed using one-way ANOVA (Dunnett correction) and the Excel 7.0 software, with significance set at p<0.05.. The knockdown of Smad3 expression in mRNA and protein levels was confirmed using RT-PCR and Western Blot. Compared to blank, the mRNA levels of types I and III procollagen were also significantly and uniquely decreased following the reduction of Smad3 by siRNA (p<0.05). The results indicate that Smad3 plays an important role in the TGF-beta induced fibrosis in keloid. Downregulation of Smad3 expression in keloid fibroblasts can significantly decrease procollagen gene expression. SiRNA targeting Smad3 was an efficient reagent to reduce ECM deposition and attenuate process of fibrosis. It could be a new promising therapeutic approach to improve skin wound healing and inhibit progression of fibrotic conditions by interrupting the TGF-beta signal pathway.

Topics: Adult; Cells, Cultured; Collagen Type I; Collagen Type III; Female; Fibroblasts; Humans; Keloid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Smad3 Protein; Transforming Growth Factor beta

We examined the transcriptional response to serum stimulation as an in vitro model of wound healing in keloid fibroblasts to identify molecular mechanisms leading to their aberrant growth.. Keloids are proliferative dermal growths representing a pathologic wound healing response. Although several groups have shown increased expression of profibrotic factors in keloids, there is little known about why they are expressed at higher levels than normal.. Fibroblasts derived from keloids and normal scar were subjected to serum stimulation as an in vitro model to mimic a component of the wound microenvironment to examine differential gene expression in keloid derived fibroblasts versus normal human fibroblasts. A promoter analysis was performed to identify specific enhancers involved in mediating the differential response of connective tissue growth factor (CTGF, CCN2). Point mutations in the enhancers were performed to confirm their role. Finally, we examined activation of transcription factors known to bind the targeted enhancers.. Transcription of CCN2 after serum stimulation was significantly higher in keloid versus normal fibroblasts. Promoter analysis demonstrates the fragment from -625/-140 conferred increased serum responsiveness. Mutational analysis showed an AP-1 and SMAD binding site were both necessary for serum responsiveness. Preventing activation of either transcriptional complex will block CCN2 transcription. Additional experiments suggest that a single complex that includes components of the AP-1 and SMAD binding complexes is responsible for transactivation in response to serum. The key difference between keloid and normal fibroblasts appears to be the degree of activation of c-Jun.. We suggest that altered responsiveness to cellular stress, based upon current data using serum stimulation and past data on response to mechanical strain, is a key defect leading to keloid formation.

Topics: Adolescent; Adult; Binding Sites; Cell Culture Techniques; Connective Tissue Growth Factor; Cysteine-Rich Protein 61; Fibroblasts; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Keloid; RNA, Messenger; Serum; Smad Proteins, Receptor-Regulated; Transcription Factor AP-1; Transforming Growth Factor beta

Clinicians have observed that keloids preferentially form in body areas subject to increased skin tension. We hypothesized a difference exists in the transcriptional response of keloid fibroblasts to mechanical strain compared with normal fibroblasts. Normal and keloid fibroblasts were seeded in a device calibrated to deliver a known level of equibiaxial strain. We examined the transcriptional response of TGF-beta isoforms and collagen Ialpha, genes differentially expressed in keloids. Keloid fibroblasts produced more mRNA for TGF-beta1, TGF-beta2, and collagen Ialpha after mechanical strain compared to normals, and this was correlated with protein production. Inhibiting the major mechanical signal transduction pathway with the ERK inhibitor, U0126, blocked upregulation of gene expression. In addition, keloid fibroblasts formed more focal adhesion complexes as measured by immunofluorescence for focal adhesion kinase, integrin beta1, and vinculin. Finally, there is increased activation of focal adhesion kinase when we detected the phosphorylated form of focal adhesion kinase with immunofluorescence and immunoblotting. In summary, keloid fibroblasts have an exaggerated response to mechanical strain compared to normal fibroblasts leading to increased production of pro-fibrotic growth factors. This may be one molecular mechanism for the development of keloids.

Topics: Adolescent; Adult; Aged; Child; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesions; Humans; Integrin beta Chains; Keloid; Male; Protein Isoforms; Signal Transduction; Skin; Stress, Mechanical; Transforming Growth Factor beta; Vinculin

Keloids are abnormal fibrous growths of the dermis that develop only in response to wounding and represent a form of benign skin tumor. Previous studies have shown increased protein levels of TGF-beta in keloid tissue, suggesting a strong association with keloid formation leading us to examine mechanisms for why it is more highly expressed in keloids. Here, we use serum stimulation as an in vitro model to mimic a component of the wound microenvironment and examine differential gene expression in keloid human fibroblasts (KFs) vs. normal human fibroblasts (NFs). Transcription of TGF-beta2 was rapid and peaked between 1 and 6 h after serum stimulation in KFs vs. NFs. We confirmed increased TGF-beta activity in the conditioned medium from KFs, but not NFs. Inhibition of second messenger signaling pathways demonstrated that only the p38 MAPK inhibitor SB-203580 could block upregulation of TGF-beta2 following serum stimulation in KFs. Immunoblotting demonstrated that p38 MAPK was phosphorylated within 15 min and was maintained at a high level in KFs but not in NFs. The transcription factors activating transcription factor-2 and Elk-1 are activated by p38 MAPK, and also showed rapid and prolonged phosphorylation kinetics in KFs but not in NFs. In conclusion, increased TGF-beta2 transcription in response to serum stimulation in KFs appears to be mediated by the p38 MAPK pathway. This suggests the mechanism of keloid pathogenesis may be due in part to an inherent difference in how the fibroblasts respond to wounding.

Topics: Cells, Cultured; Fibroblasts; Humans; Keloid; p38 Mitogen-Activated Protein Kinases; Serum; Signal Transduction; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta2

Transforming growth factor beta 1 (TGF-beta1) upregulation has been implicated in hypertrophic scars and keloids, but it is unclear if it is the cause or an effect of excessive scar formation. In this study, we overexpressed TGF-beta1 in fibroblasts and characterized its role. Normal human dermal fibroblasts were genetically modified to overexpress TGF-beta1 as the wild-type latent molecule or as a mutant constitutively active molecule. TGF-beta1 secretion was measured, as were the effects of TGF-beta1 upregulation on cell proliferation, expression of smooth muscle cell alpha actin (SMC alpha-actin) and ability to contract collagen lattices. Fibroblasts were implanted intradermally into athymic mice and tissue formation was analyzed over time by histology and immunostaining. Gene-modified fibroblasts secreted approximately 20 times the TGF-beta1 released by control cells, but only cells expressing mutant TGF-beta1 secreted it in the active form. Fibroblasts expressing the active TGF-beta1 gene had increased levels of SMC alpha-actin and enhanced ability to contract a collagen lattice. After intradermal injection into athymic mice, only fibroblasts expressing active TGF-beta1 formed "keloid-like" nodules containing collagen, which persisted longer than implants of the other cell types. We conclude that upregulation of TGF-beta1 by fibroblasts may be necessary, but is not sufficient for excessive scarring. Needed are other signals to activate TGF-beta1 and prolong cell persistence.

Topics: Actins; Animals; Cell Proliferation; Cells, Cultured; Cicatrix, Hypertrophic; Fibroblasts; Humans; Keloid; Mice; Mice, Nude; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Keloids are benign tumors that occur only in response to injury, for which there is no effective treatment. We demonstrated previously that keloid keratinocytes (KKs) promote fibroblast proliferation more than normal keratinocytes (NKs) and that transforming growth factor (TGF)-beta is a component of that signal. We used the transcriptional response to serum stimulation to examine how TGF-beta expression is stimulated in KKs.. Quiescent KKs and NKs were stimulated using serum; harvested using RNA at 0, 1, 6, 12, and 24 h; and analyzed using quantitative real-time polymerase chain reaction. TGF-beta activity in the conditioned medium was measured with an MLEC/PAI-luciferase assay. Inhibition of ERK1/2, p38 kinase, and JNK pathways was performed with PD98059, SB203580, and SP600125, respectively.. Increased transcription of TGF-beta2 occurs within 1 h of serum stimulation in KKs but not in NKs. In contrast, TGF-beta3 transcription was suppressed in KKs compared with NKs. No significant differences were observed in the transcriptional response of TGF-beta1. Increased TGF-beta2 mRNA correlated with increased TGF-beta biological activity in the conditioned medium. Inhibition of the ERK, p38 kinase or JNK signal transduction pathways blocked the transcriptional up-regulation of TGF-beta2, TbetaR1, and TbetaR2 in KKs.. KKs produce more TGF-beta2 mRNA than NKs in response to serum stimulation, resulting in increased TGF-beta activity in conditioned medium. Combining these results with our previous data lead us to propose a model of keloid formation characterized by an exaggerated response to cellular stress and abnormal epithelial-mesenchymal signaling promoting keloid formation.

Topics: Adolescent; Adult; Anthracenes; Blood Proteins; Culture Media, Conditioned; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Keloid; Keratinocytes; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Pyridines; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta2; Transforming Growth Factor beta3

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.

Topics: Blotting, Western; Cells, Cultured; Fibroblasts; Humans; Keloid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad6 Protein; Smad7 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Hypertrophic scars and keloids represent a dysregulated response to cutaneous wounds, which results in an excessive deposition of collagen. Transforming growth factor-beta (TGF-beta) is the key regulator in the pathogenesis of fibrosis. Accumulating evidence suggests that Wnt signalling and its effector beta-catenin also play an important role in wound healing. The role of Wnt/beta-catenin signalling in TGF-beta induced collagen deposition in hypertrophic scars and keloids was studied. Transcriptional assays and Western blotting was performed using fibroblast cell lines established from normal skin and hypertrophic scar tissue. Immunohistochemical studies were performed using scar tissues. We provide evidence that TGF-beta induces activation of beta-catenin mediated transcription in human dermal fibroblasts via the Smad3 and p38 MAPK pathways. Immunohistochemical studies demonstrated that beta-catenin protein levels are elevated in hypertrophic scar and keloid tissues. This finding may be relevant to the pathogenesis of hypertrophic scars and keloids.

Topics: Adolescent; beta Catenin; Blotting, Western; Cell Line; Child; Cicatrix, Hypertrophic; Collagen; Female; Fibroblasts; Humans; Immunohistochemistry; Keloid; p38 Mitogen-Activated Protein Kinases; Signal Transduction; Skin; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation; Wnt Proteins; Wound Healing

Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially types I and III collagen. Transforming growth factor (TGF)-beta plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of keloid fibroblast. However, the underlying mechanisms are poorly understood. In this study, the authors examined the function of Smad2, a recently characterized intracellular effector of TGF-beta signaling, in keloid fibroblasts using small interfering RNA (siRNA).. Three pairs of siRNA duplexes targeting human Smad2 were designed; the most efficient one was selected and used for further research. Keloid fibroblasts were treated with or without Smad2 siRNA, and the expression levels of related genes were examined by reverse-transcriptase polymerase chain reaction and immunofluorescence.. The down-regulation of Smad2 by siRNA led to a significant decrease in mRNA levels of Smad2 in both a dose-dependent and time-dependent manner. The knockdown of Smad2 expression in protein level was confirmed using immunofluorescence. The mRNA levels of types I and III procollagen were also significantly and uniquely decreased following the reduction of Smad2 by siRNA.. The results indicate that Smad2 plays an important role in TGF-beta-induced fibrosis in keloids. Down-regulation of Smad2 expression in keloid fibroblasts can significantly decrease procollagen gene expression. Also, siRNA targeting Smad2 was an efficient reagent with which to reduce extracellular matrix deposition and attenuate process of fibrosis. It could be a new, promising therapeutic approach for improving skin wound healing and inhibiting progression of fibrotic conditions by interrupting the TGF-beta signaling pathway.

Topics: Adult; Cells, Cultured; Collagen; Down-Regulation; Female; Fibroblasts; Fluorescent Antibody Technique; Gene Silencing; Humans; Keloid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Topics: Adult; Cells, Cultured; Collagen Type I; Fibroblasts; Genetic Therapy; Humans; Keloid; Oligodeoxyribonucleotides; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Flashlamp pulsed-dye lasers (PDLs) has shown effectiveness in the treatment of keloids. In this study, we investigated whether PDL treatments decreased transforming growth factor-beta1 (TGF-beta1)-induction and up-regulation of matrix metalloproteinase (MMP) expression in keloid regression.. Keloid tissues obtained from 10 patients with intra-lesional or punch biopsies before and 7 days after PDL treatments [fluence per pulse was 10-18 J/cm2 (mean 14.0 J/cm2)]. Immunohistochemical (IHC) staining of TGF-beta1 and MMP-1 and MMP-13 expressions in keloid tissue was performed. Western blot analysis of MMP-1 and MMP-13 expressions in extracellular matrix was evaluated.. IHC staining indicated that expression of TGF-beta1 was significantly reduced in keloid tissues after PDL irradiation. MMP-13 but not MMP-1 expression on IHC staining significantly increased in extracellular matrix of keloid tissues after PDL treatment. Western blot analysis also showed MMP-13 but not MMP-1 significant increased in keloid tissues after PDL treatment.. Regression of keloids regressed after PDL treatments are associated with down-regulation of TGF-beta1 expression and up-regulation of MMP-13 activity.

Topics: Adolescent; Adult; Child; Collagenases; Down-Regulation; Female; Humans; Keloid; Laser Therapy; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Middle Aged; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Keloid scars represent a pathological response to cutaneous injury. Overproliferation of fibroblasts and overproduction of collagen characterize these abnormal scars. The pathology of these scars remains poorly understood. The role of epithelial-mesenchymal interactions in keloid pathogenesis and scar contracture has recently been explored.. To test our hypothesis that epithelial-mesenchymal interactions play a major role in modulating keloid scar contracture.. A coculture model was employed wherein keloid and normal keratinocytes were cocultured with keloid or normal fibroblasts, and the conditioned media from day 5 cocultures were collected to study the effect of the paracrine secretions on contraction of an in vitro fibroblast-populated collagen lattice (FPCL) model.. Keloid keratinocyte/keloid fibroblast coculture conditioned media brought about increased contraction of the collagen lattice compared with non-cocultured conditioned media. When keloid fibroblasts populated the collagen lattice, significantly increased lattice contraction was induced compared with lattices populated by normal fibroblasts. The addition of antitransforming growth factor (TGF)-beta neutralizing antibody to the conditioned media produced an attenuation of the contraction of the FPCLs. When keloid and normal fibroblasts were cultured on chamber slides and treated with conditioned media from coculture and non-coculture series, immunohistochemical analysis demonstrated an increased expression of alpha-smooth muscle actin (a marker for fibroblast differentiation into myofibroblasts) in fibroblasts exposed to conditioned media from coculture.. These data indicate that epithelial-mesenchymal interactions are likely to play a major role in scar contracture and scar pathogenesis, and underscore the role of TGF-beta1 as a key player in keloid pathogenesis.

Topics: Actins; Antibodies; Cells, Cultured; Coculture Techniques; Collagen; Culture Media, Conditioned; Epithelium; Fibroblasts; Humans; Immunohistochemistry; Keloid; Keratinocytes; Models, Biological; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta induces fibroblast contraction, which is implicated in wound healing and keloid formation. SB-431542 is a novel specific inhibitor of TGF-beta type I receptor kinase activity.. We sought to determine whether SB-431542 inhibited TGF-beta-induced fibroblast contraction.. We used an in vitro type I collagen gel contraction assay with normal or keloid dermal fibroblasts incorporated.. TGF-beta induced contraction of collagen gels with normal dermal fibroblasts incorporated, which was efficiently suppressed by SB-431542. Keloid fibroblasts showed higher basal contraction of collagen gels in the absence of TGF-beta than normal fibroblasts, which was enhanced by addition of TGF-beta. SB-431542 suppressed both the basal and TGF-beta-enhanced contraction of collagen gels by keloid fibroblasts. These inhibitory effects of SB-431542 were associated with suppression of TGF-beta-induced alpha-smooth muscle actin (alpha-SMA) expression and phosphorylation of Smad2 in normal and keloid fibroblasts.. SB-431542 can suppress TGF-beta-induced contraction of collagen gel by normal and keloid dermal fibroblasts. Importantly, SB-431542 can inhibit basal contraction of collagen gel by keloid fibroblasts. These results suggest that an inhibitor of TGF-beta type I receptor kinase activity may have therapeutic potential for excessive skin contraction as observed in keloid.

Topics: Actins; Benzamides; Collagen; Dioxoles; Fibroblasts; Gels; Humans; Keloid; Transforming Growth Factor beta

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterized by an abnormal deposition of extracellular matrix components, particularly collagen. There is uncertain evidence that transforming growth factor-beta (TGFss) is involved in keloid formation. Therefore we investigated the expression of TGFss1, 2 and 3 and their receptors in keloids, hypertrophic scars and normal skin. Dermal fibroblasts were obtained from punch biopsies of patients with keloids and hypertrophic scars and from normal skin of healthy individuals. Total RNA was isolated and the expression of TGFss1, 2 and 3 and of TGFss receptors I and II (TGFssRI and II) was analysed by real-time PCR using the Lightcycler technique. Our data demonstrate significantly lower TGFss2 mRNA expression in hypertrophic scar fibroblasts as compared with fibroblasts derived from keloids and normal skin (p<0.05). In contrast, TGFss3 mRNA expression was significantly lower in keloid fibroblasts in comparison with fibroblasts derived from hypertrophic scar and normal skin (p<0.01). TGFssRI mRNA expression was significantly decreased in hypertrophic scar fibroblasts (p<0.01) and TGFssRII mRNA expression was decreased in keloids compared with hypertrophic scar fibroblasts (p<0.001). The ratio of TGFssRI/TGFssRII expression was increased in keloids compared with hypertrophic scar and normal skin fibroblasts. As recently supposed, an increased TGFssRI/TGFssRII ratio could promote fibrosis. Therefore our data support a possible role of TGFssRI and TGFssRII in combination with a certain TGFss expression pattern as fibrosis-inducing factors in keloids.

Topics: Case-Control Studies; Cicatrix, Hypertrophic; DNA Primers; Fibroblasts; Humans; Immunohistochemistry; Keloid; Polymerase Chain Reaction; Receptors, Transforming Growth Factor beta; RNA, Messenger; Skin; Transforming Growth Factor beta; Wound Healing

Keloid disease (KD) is a fibroproliferative dermal tumour of unknown aetiology. The increased familial clustering in KD, its increased prevalence in certain races and its presence in identical twins suggest a strong genetic predisposition to keloid formation. Transforming growth factor beta isoforms (TGFbeta) play a central role in wound healing and fibrosis and have been implicated in KD pathogenesis. Recent data has suggested that TGFbeta(3) has an important role in scar formation. There is little known about the genetic variation present within the TGFbeta(3) gene, which contains seven exons and six introns spanning 43,000 base pairs of the human genome. Exons one to seven and the promoter region (1000 bp upstream from exon 1 in the 5'-flanking regions) were screened in 95 Caucasian KD cases and 95 Caucasian controls for the presence of novel mutations using a high throughput DHPLC mutation detection technology. There were no mutations identified in any of the exonic regions, however, multiple nondisease associated mutations were found in the promoter region of the TGFbeta(3) gene. These data demonstrate that there is no association between the exonic and promoter regions of TGFbeta(3) gene and keloid scarring in our cohort of Caucasian patients.

Topics: Adolescent; Adult; Chromatography, High Pressure Liquid; DNA Mutational Analysis; Exons; Female; Genetic Predisposition to Disease; Humans; Keloid; Male; Middle Aged; Mutation; Polymerase Chain Reaction; Promoter Regions, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta3

A keloid is a specific skin lesion that expands beyond the boundaries of the original injury as it heals. Histologically, it is characterized by the excessive accumulation of collagen. However, the reasons for the expansion and the invasive nature of keloids remain unknown.. We evaluated collagen degradation and migration by cultured keloid fibroblasts based on the assumption that these variables were of functional relevance to the expanding and invasive nature of keloid lesions.. Collagen production was investigated by the detection of type 1 collagen (procollagen type 1C peptide: P1P). Matrix metalloproteinase (MMP)-1 (interstitial collagenase) and MMP-2 (gelatinase-A), were investigated as elements of the collagen degradation system. Enzyme immunoassays were performed to measure the production of P1P, MMP-1, MMP-2, and tissue inhibitor of metalloproteinase (TIMP)-1. To assess the production of MMP-2 its gelatinolytic activity was measured by zymography using gelatin-containing gels. The participation of transforming growth factor-beta1 (TGF-beta1) in the production and degradation of collagen was also investigated. Finally, the migratory activity of keloid fibroblasts was evaluated using a colony dispersion assay.. The production of type 1 collagen, MMP-1, MMP-2, and TIMP-1 by keloid fibroblasts was 3-fold, 6-fold, 2.4-fold, and 2-fold greater than that of normal dermal fibroblasts, respectively. Production of P1P was increased when TGF-beta1 was added to cultures of keloid fibroblasts, while it was decreased when anti-TGF-beta1 antibody was added to the cultures. In contrast, the production of MMP-1 was decreased by the addition of TGF-beta1 to cultured keloid fibroblasts, while it was increased when anti-TGF-beta1 antibody was added to the cultures. The production of MMP-2 increased after treatment with TGF-beta1, but did not change significantly when anti-TGF-beta1 antibody was added to the cultures. Production of TIMP-1 did not change significantly when either TGF-beta1 or anti-TGF-beta1 antibody was added to the cultures. Keloid fibroblasts showed a 2.5-fold increase of migratory activity compared with normal dermal fibroblasts, while the migratory activity of these fibroblasts was reduced to the control level by treatment with a broad-spectrum MMP inhibitor (GM 6001).. Cultured keloid fibroblasts showed increased production of collagen and MMPs, and TGF-beta1 played a role in this regulation of production. In addition, increased production of MMPs had a role in the high migratory activity of cultured keloid fibroblasts.

Topics: Cell Movement; Cells, Cultured; Collagen Type I; Culture Media, Conditioned; Female; Fibroblasts; Growth Substances; Humans; Keloid; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinases; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Keloids are tumor-like lesions that result from excessive scar formation during healing of wounds. Histologically, keloids show an increased blood vessel density compared with normal dermis or normal scars. However, the angiogenic activity of keloid fibroblasts remains unknown. In this study, we investigated angiogenic activity of keloid fibroblasts. Transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor (VEGF) were investigated as elements of the angiogenic factors. Expressions of TGF-beta1 and VEGF in conditioned medium were measured with enzyme-linked immunosorbent assay (EIA) and Northern blot analysis. Participation of TGF-beta1 in the production of VEGF was also investigated with addition of TGF-beta1 and a neutralizing anti-TGF-beta1 antibody. A modified Boyden chamber assay was performed to assess the chemotactic activity of vascular endothelial cells. Angiogenic activity in vivo was evaluated by neovascularization of nodules formed by implantation of fibroblasts into severe combined immunodeficiency (SCID) mice. EIA showed that the concentrations of TGF-beta1 and VEGF in conditioned medium were increased 2.5- and 6-fold, respectively, after the culture of keloid fibroblasts compared with normal fibroblasts. Northern blot analysis revealed that the expression of TGF-beta1 and VEGF mRNA was upregulated 3.6- and 6-fold, respectively, in keloid fibroblasts compared with normal fibroblasts. Addition of TGF-beta1 to keloid fibroblast cultures increased VEGF production by 3.5-fold, while there was a 6-fold in culture of normal fibroblasts. A neutralizing anti-TGF-beta1 antibody reduced VEGF secretion to control levels, suggesting that TGF-beta1 mediated the upregulation of VEGF expression. A modified Boyden chamber assay demonstrated that the chemotactic activity of vascular endothelial cells was more strongly (sevenfold) induced by keloid fibroblast-conditioned medium than by normal fibroblast-conditioned medium. Anti-VEGF antibody inhibited chemotaxis to basal levels. When SCID mice underwent implantation of fibroblasts into the back, the nodules formed by keloid fibroblasts were three times larger than those formed by normal fibroblasts. Although abundant neovascularization was observed in keloid fibroblast nodules, neovascularization was scarce in normal fibroblast nodules. Both in vitro and in vivo studies confirmed the significantly higher angiogenic activity of keloid fibroblasts compared with normal fibroblasts, and

Topics: Adolescent; Adult; Animals; Antibodies; Blotting, Northern; Cells, Cultured; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Humans; Keloid; Male; Mice; Mice, SCID; Neovascularization, Pathologic; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

Hypertrophic scars and keloids respond to dermal disruption with excessive collagen deposition and increased transforming growth factor (TFG)-beta expression. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta activity that is associated with scar and fibrosis. The authors hypothesize that there is increased expression of CTGF by hypertrophic scar and keloid fibroblasts in response to TGF-beta stimulation.. Primary fibroblasts were isolated in culture from human hypertrophic scar (n = 2), keloid (n = 2), and normal skin (n = 2). After 18 hours of serum starvation, the cells were stimulated with 10 ng/ml of TGF-beta1, TGF-beta2, and TGF-beta3 for 24 hours. Quantitative real-time polymerase chain reaction was performed on extracted RNA samples to assay for CTGF mRNA expression.. Baseline CTGF expression was increased 20-fold in unstimulated hypertrophic scar fibroblasts and 15-fold in keloid fibroblasts compared with normal fibroblasts. CTGF expression increased greater than 150-fold when stimulated with TGF-beta1 (p < 0.002) and greater than 100-fold when stimulated by TGF-beta2 or TGF-beta3 compared with normal fibroblasts (p < 0.02 and p < 0.002, respectively). CTGF expression was greatest after TGF-beta1 stimulation in hypertrophic scar fibroblasts compared with TGF-beta2 (p < 0.04) and TGF-beta3 (p < 0.02). Keloid fibroblast CTGF expression also increased greater than 100-fold after stimulation with TGF-beta1 (p = 0.16) and greater than 75-fold after addition of TGF-beta2 and TGF-beta3 (p = 0.06 and p = 0.22, respectively).. Hypertrophic scar fibroblasts have both intrinsic up-regulation of CTGF transcription and an exaggerated capacity for CTGF transcription in response to TGF-beta stimulation. These data suggest that blockage of CTGF activity may reduce pathologic scar formation.

Topics: Adult; Cells, Cultured; Child; Cicatrix, Hypertrophic; Connective Tissue Growth Factor; Female; Fibroblasts; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Keloid; Male; Transforming Growth Factor beta

After studying this article, the participant should be able to: 1. Have a greater appreciation of the extent of differences and similarities between keloid and hypertrophic scarring. 2. Have a greater appreciation of the significance of the stage of maturation of a keloid or hypertrophic scar with regard to its morphologic, biochemical, and molecular profile. 3. More critically review basic science research that is based on poorly characterized scar tissue. 4. More critically review clinical studies that are based on poorly characterized scar tissue.. Hypertrophic and keloid scars remain extremely challenging, particularly in their variable response to treatment. The understanding of hypertrophic and keloid scarring is evolving from a position where they were regarded as different stages of the same process to the contemporary perspective of two separate entities. This article reviews the differences in the two forms of scarring and discusses the implications for future research.. The authors conducted a MEDLINE search of all English language reviews linking key words "hypertrophic," "keloid," and "scarring.". Over the past four decades, there has been considerable clinical and experimental research looking at the biological nature and therapeutic response of keloid and hypertrophic scarring. As more differences are emerging regarding the fundamental biology of the scars, investigators are giving more detailed characterization of their source material. It is evident that even within the broad categories of hypertrophic and keloid scarring there is a heterogenous distribution of pathologic connective tissue matrix biology.. Considerable advances have been made in our understanding of the fundamental biology of scarring. As research methodology becomes even more sophisticated, it will be even more crucial to extensively characterize source material, recognizing major differences not only between keloid and hypertrophic scar but also between scars of varying stages of maturation and histomorphological, biochemical, and molecular variations within individual scars.

Topics: Apoptosis; Cicatrix, Hypertrophic; Extracellular Matrix; Fibroblasts; Humans; Immunohistochemistry; Keloid; Keratinocytes; Microcirculation; Polymorphism, Genetic; Transforming Growth Factor beta

Keloids have been treated with flashlamp pulsed dye lasers (PDLs) with good results. We investigated whether PDL treatments induced keloid regression by decreasing growth factor-beta(1) (TGF-beta(1)) induction, thereby reducing fibroblast proliferation and collagen deposition.. Clinical evaluation and photography documented keloid height/texture, erythema, and pliability before and after PDL treatments scheduled at 2-month intervals in 30 patients. Fluence per pulse was 10-18 J/cm(2) (mean 14.0 J/cm(2)). Immunohistochemical (IHC) staining of TGF-beta(1), proliferating cell nuclear antigen (PCNA), and collagen (types I and III) in extra-cellular matrix was performed on 10 intra-lesional or punch biopsies obtained before and 7 days after PDL treatments.. Twelve months after final PDL treatments, keloid regression ( >/= 50%) had occurred in 26/30 patients in whom erythema and surface irregularities had been reduced and pliability had been increased. In 4/30 patients, no changes in keloids had occurred after 12 months. Multiple treatments ( > 6) yielded better results than fewer treatments: 79% versus 50%, respectively. Marked keloid regression ( >/= 90%) occurred in two patients who had received more than 10 treatments. IHC staining indicated that expression of TGF-beta(1), PCNA and collagen type III, but not type I, was significantly reduced in keloid fibroblasts after PDL irradiation.. Keloids regressed following PDL-induced reduction in TGF-beta(1) expression, fibroblast proliferation, and collagen type III deposition. More than six PDL treatments at 2-month intervals provided the best results.

Topics: Adolescent; Adult; Child; Collagen Type III; Down-Regulation; Extracellular Matrix; Female; Fibroblasts; Humans; Keloid; Laser Therapy; Male; Middle Aged; Proliferating Cell Nuclear Antigen; Transforming Growth Factor beta; Treatment Outcome

Keloid disease and hypertrophic scars are dermal tumors that are often familial and typically occur in certain races. Their exact etiology is still unknown. Transforming growth factor beta1 (TGF-beta1) plays a central role in wound healing and fibrosis and has been implicated in the pathogenesis of keloid disease and hypertrophic scar. The aims of this study were to measure the plasma level of TGF-beta1 in patients compared with controls, and to investigate the association of five common single nucleotide polymorphisms in TGF-beta1 with the risk of keloid disease and hypertrophic scar formation. Platelet-poor plasma levels of TGF-beta1 in 60 patients (15 with hypertrophic scar and 45 with keloid disease) and 18 controls were measured using an enzyme-linked immunoabsorbent assay technique. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping TGF-beta1 polymorphisms. DNA samples from 133 patients (101 with keloid disease and 32 with hypertrophic scar) and 200 controls were examined. All patients and controls were Caucasians of Northern European extraction. There was no statistically significant difference in TGF-beta1 plasma levels between patients with keloid disease and hypertrophic scar and controls. There was also no statistically significant difference in genotype or allele frequency distributions between patients and controls for codons 10, 25, and 263 and for -509 and -800 single nucleotide polymorphisms of the TGF-beta1 gene. These results suggest that TGF-beta1 plasma levels and common polymorphisms are not associated with a risk of keloid disease and hypertrophic scar formation. This lack of association may be significant in view of the importance attached to the role of TGF-beta1 in dermal scarring. To the authors' knowledge, this is the first report of a case-control association study in keloid disease and hypertrophic scars using any single nucleotide polymorphisms.

Topics: Adolescent; Adult; Cicatrix, Hypertrophic; Codon; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Keloid; Male; Middle Aged; Polymorphism, Single Nucleotide; Risk; Transforming Growth Factor beta; Transforming Growth Factor beta1; White People; Wound Healing

Heparin decreases dermal fibroblast proliferation and collagen production according to several studies. Heparin may mediate these effects by altering the levels of growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor-beta 1 (TGF-b1). This study sought to delineate the effect of heparin on proliferation and bFGF and TGF-b1 production by human normal, keloid, and fetal dermal fibroblasts.. Human normal, keloid, and fetal dermal fibroblasts were propagated in a serum-free in vitro model, with exposure to 0 microg/ml, 50 microg/ml, 300 microg/ml, or 600 microg/ml heparin for 0, 24, 72, or 96 hours. Cell counts were determined by phase contrast microscopy. Levels of bFGF and TGF-b1 in the supernatants were determined by enzyme-linked immunosorbant assay (ELISA).. Heparin inhibited keloid and fetal fibroblast proliferation. All doses of heparin significantly stimulated production of bFGF by normal (341% to 1137% increase), keloid (237% to 1955% increase), and fetal fibroblasts (292% to 1866% increase) at all time points (p<0.05). Heparin (300 microg/ml and 600 microg/ml) also stimulated production of TGF-b1 by normal (56% to 75%), keloid (105% to 269%), and fetal fibroblasts (25% to 57%), with statistical significance (p<0.05) at various time points. 600 microg/ml heparin generally caused the greatest increase in growth factor levels.. Heparin inhibits proliferation by keloid and fetal fibroblasts and significantly stimulates production of bFGF and TGF-b1 by normal, keloid, and fetal dermal fibroblasts. These effects of heparin on dermal fibroblasts may have implications for wound healing in vivo.

Topics: Cell Division; Cells, Cultured; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Fetus; Fibroblast Growth Factor 2; Fibroblasts; Heparin; Humans; Keloid; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

Keloids are benign dermal tumors, characterized by invasive growth of fibroblasts and concomitant increased biosynthesis of extracellular matrix components, with unclear etiology. We previously demonstrated that keloid fibroblasts overexpress insulin-like growth factor-I receptor. In investigating the role of insulin-like growth factor-I receptor overexpression, insulin-like growth factor-I and transforming growth factor-beta interaction was examined in relation to extracellular matrix protein production in cultured human and mouse fibroblasts. Western blotting revealed that collagen type I was expressed in keloid and normal fibroblasts, and its expression was increased by transforming growth factor-beta stimulation more significantly in keloid rather than in normal fibroblasts. Insulin-like growth factor-I and transforming growth factor-beta1 costimulation markedly increased extracellular matrix proteins (collagen type I, fibronectin, and plasminogen activator inhibitor-1) compared with cultures with transforming growth factor-beta1 alone. Insulin-like growth factor-I treatment alone had no stimulatory effect. Real-time reverse transcription-polymerase chain reaction confirmed parallel collagen type I messenger RNA level changes. Luciferase assays were conducted to investigate intracellular signaling pathways in this synergistic stimulation using a mouse fibroblast cell line. Transforming growth factor-beta1 (1 or 10 ng per ml) increased the specific signaling activity approximately 10-fold, whereas the increase with insulin-like growth factor-I (100 ng per ml) was less than 2-fold compared with basal activity; however, the combination of transforming growth factor-beta1 and insulin-like growth factor-I resulted in an approximately 25-fold increase. Insulin-like growth factor-I markedly enhanced transforming growth factor-beta-induced phosphorylation of p38 mitogen-activated protein kinase and activating transcription factor-2. Luciferase assay showed that this synergistic effect was attenuated by the p38 mitogen-activated protein kinase specific inhibitor SB203580 or phosphatidylinositol 3-kinase inhibitor wortmannin, but not by the mitogen-activated protein kinase/extracellular-signal-regulated protein kinase kinase inhibitor PD98059. These results indicate that insulin-like growth factor-I enhances transforming growth factor-beta-induced keloid formation through transforming growth factor-beta postreceptor signal cross-talk, mainly via the p38 mitogen

Topics: 3T3 Cells; Activating Transcription Factor 2; Adolescent; Adult; Aged; Animals; Cells, Cultured; Child; Collagen Type I; Cyclic AMP Response Element-Binding Protein; Dermis; Extracellular Matrix Proteins; Female; Fibroblasts; Humans; Insulin-Like Growth Factor I; Keloid; Male; Mice; Mice, Inbred BALB C; Middle Aged; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; RNA, Messenger; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Interferon-gamma (IFN-gamma) has been noted as a potential therapeutic agent for various fibrotic disorders, in part, through its antagonistic effect on a fibrogenic cytokine, transforming growth factor-beta (TGF-beta). Keloid is a fibrotic skin disorder that results in an excessive deposition of extracellular matrix, which is associated with altered-expression of or -responses to TGF-beta in dermal fibroblasts.. We sought to determine whether IFN-gamma antagonized TGF-beta-mediated fibrotic response in keloid-derived dermal fibroblasts.. Type I collagen production, fibroblast contractile activity, and alpha-smooth muscle actin (alpha-SMA) expression were assessed by using Western blotting, an in vitro type I collagen gel contraction assay, and immunofluorescence study in normal and keloid-derived human dermal fibroblasts in the presence or absence of IFN-gamma and/or TGF-beta.. In contrast to normal dermal fibroblasts, IFN-gamma did not inhibit TGF-beta-induced type I collagen production, contractile activity, and alpha-SMA expression in keloid-derived dermal fibroblasts. In addition, keloid-derived dermal fibroblasts constitutively expressed type I collagen and alpha-SMA with increased capacity to contract a collagen matrix.. IFN-gamma failed to antagonize TGF-beta-mediated fibrotic response in keloid-derived dermal fibroblasts. Thus, IFN-gamma may not be therapeutically useful for keloid and clarification of the molecular mechanisms underlying the IFN-gamma resistance should be investigated for therapeutic application of IFN-gamma for keloid.

Topics: Actins; Blotting, Western; Case-Control Studies; Cells, Cultured; Collagen; Collagen Type I; Fibroblasts; Fibrosis; Humans; Interferon-gamma; Keloid; Muscle, Smooth; Protein-Lysine 6-Oxidase; Skin; Transforming Growth Factor beta

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.

Topics: Cells, Cultured; Collagen; Fibroblast Growth Factors; Fibroblasts; Humans; Keloid; Microscopy, Electron, Scanning; Models, Biological; Skin; Transforming Growth Factor beta; Wound Healing

Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of diseases. Keloid is an intricate lesion that is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skin with the aid of a cDNA microarray to explore the molecular mechanism underlying keloid formation.. The polymerase chain reaction products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs were isolated from freshly excised human keloids and normal skins and then were purified to mRNAs by Oligotex. Both the mRNAs from keloids and normal skins were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues.. Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250 genes, including TGF-beta1 and NGF, were upregulated (2.98%) and 152 downregulated (1.81%). Analyses of collagen, fibronectin, proteoglycan, growth factors, and apoptosis-related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with the published biochemical and clinical observations of keloids. Higher expression of TGF-beta(1) and NGF in keloids versus normal skins was also testified with reverse transcription polymerase chain reaction method.. DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help to understand the molecular mechanism of keloid formation.

Topics: Adult; Child, Preschool; Female; Gene Expression; Gene Expression Profiling; Humans; Keloid; Male; Nerve Growth Factor; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1

To explore the effect of connective tissue growth factor on the pathogenesis of hypertrophic scar and keloid tissue.. The content of hydroxyproline was determined and the expression of connective tissue growth factor gene was detected by the reverse transcription-polymerase chain reaction and image analysis technique in 5 normal skins, 15 hypertrophic scars and 7 keloid tissues.. The contents of hydroxyproline in the hypertrophic scar (84.10 +/- 1.76) and keloid tissue (92.38 +/- 2.04) were significantly higher than that of normal skin tissue (26.52 +/- 4.10) (P < 0.01). The index of connective tissue growth factor mRNA in the hypertrophic scar (0.78 +/- 0.63) and keloid tissue (0.84 +/- 0.04) were higher than that of normal skin tissue (0.09 +/- 0.25) (P < 0.01).. Connective tissue growth factor may play an important role in promoting the fibrotic process of hypertrophic scar and keloid tissue.

Topics: Adolescent; Adult; Cicatrix, Hypertrophic; Connective Tissue Growth Factor; Female; Gene Expression; Humans; Hydroxyproline; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Keloid; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin; Transforming Growth Factor beta

Interactions between epidermal keratinocytes and dermal fibroblasts play an important role in regulating tissue homeostasis and repair. Nevertheless, little is known about the role of keratinocytes in the pathogenesis of keloid. In this study, we investigated the influence of normal skin- and keloid-derived keratinocytes on normal skin- and keloid-derived fibroblasts utilizing a serum-free indirect coculture system. The keloid-derived fibroblasts showed a greater proliferation and minimal apoptosis when cocultured with normal skin- or keloid-derived keratinocytes, and the results were most significant in the latter. This difference was not observed when the fibroblasts were treated with conditioned medium obtained from normal skin- and keloid-derived keratinocytes. Nevertheless, conditioned medium-treated groups showed more proliferation and less apoptosis compared to the nonconditioned medium-treated control groups. We also analyzed the profile of factors involved in cell growth and apoptosis in fibroblasts cocultured with keratinocytes. Extracellular signal-regulated kinase and c-Jun N-terminal kinase phosphorylations and expression of Bcl-2 and transforming growth factor-beta1 were all significantly upregulated in the fibroblasts cocultured with keloid-derived keratinocytes. Together, these results strongly suggest that the overlying keratinocytes of the keloid lesion play an important role in keloidogenesis by promoting more proliferation and less apoptosis in the underlying fibroblasts through paracrine and double paracrine effects.

Topics: Adolescent; Adult; Apoptosis; Cell Communication; Cell Division; Cells, Cultured; Child; Coculture Techniques; Culture Media, Conditioned; Female; Fibroblasts; Humans; JNK Mitogen-Activated Protein Kinases; Keloid; Keratinocytes; Male; Middle Aged; Mitogen-Activated Protein Kinases; Paracrine Communication; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Keloid disease (KD) is a benign fibroproliferative scarring condition of unknown aetiopathogenesis. There is a familial predisposition to keloid scarring. The genes involved in the pathogenesis of abnormal dermal scarring have yet to be identified. Transforming growth factor beta (TGF beta) is a family of multifunctional cytokines, which play a central role in wound healing and fibrosis. The TGF beta 2 isoform is a member of this cytokine family and has previously been implicated in KD pathogenesis. We tested for an association between KD and two novel polymorphisms within the TGF beta 2 gene: an insertion polymorphism within the 59-untranslated region, 109 base pairs away from the initiation codon, and a single nucleotide polymorphism in exon one. We examined DNA samples from 101 patients with KD and 187 ethnically matched controls. No statistically significant differences in TGF beta 2 genotype or allele frequency distribution were observed between the patients and the controls. We believe this to be the first report of a case-control association study in KD and TGF beta 2 polymorphisms.

Topics: Cicatrix, Hypertrophic; Genetic Predisposition to Disease; Genotype; Humans; Keloid; Polymorphism, Genetic; Transforming Growth Factor beta

Triamcinolone acetonide has been shown to decrease both cellular proliferation and collagen production by dermal fibroblasts. An alteration of cytokine levels may mediate these effects.. To delineate the effect of triamcinolone acetonide on both cellular proliferation and the production of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1) by human fibroblasts grown in a serum-free in vitro model.. Human normal and keloid dermal fibroblasts were propagated in a serum-free in vitro model with exposure to 0, 5, 10, or 20 microm triamcinolone acetonide for 0, 24, 72, or 96 hours. Cell counts were determined by phase contrast microscopy. Levels of bFGF and TGF-beta1 in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA).. In our study, 20 microm triamcinolone acetonide caused statistically significant increases in the peak levels of bFGF for normal and keloid fibroblast cell lines (P < 0.05). It also caused statistically significant decreases in the level of TGF-beta1 for normal and keloid fibroblast cell lines. For the keloid fibroblasts, 10 microm triamcinolone acetonide also caused a statistically significant decrease in the level of TGF-beta1.. We conclude from these results that triamcinolone acetonide increases the production of bFGF and decreases production of TGF-beta1 by human dermal fibroblasts.

Topics: Analysis of Variance; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Fibroblasts; Glucocorticoids; Humans; Keloid; Skin; Transforming Growth Factor beta; Triamcinolone

Wounds in the nonglabrous skin of keloid-prone individuals tend to cause large disordered accumulations of collagen which extend beyond the original margins of the wound. In addition to abnormalities in keloid fibroblasts, comparison of dermal fibroblasts derived from nonwounded glabrous or nonglabrous skin revealed differences that may account for the observed location of keloids.. Fibroblast apoptosis and the cellular content of alpha-smooth-muscle actin, TGFbeta1 receptorII and ED-A fibronectin were estimated by FACS analysis. The effects of TGFbeta1 and serum were examined.. In monolayer cultures non-glabrous fibroblasts were slower growing, had higher granularity and accumulated more alpha-smooth-muscle actin than fibroblasts from glabrous tissues. Keloid fibroblasts had the highest level of alpha-smooth-muscle actin in parallel with their expression level of ED-A fibronectin. TGFbeta1 positively regulated alpha-smooth-muscle actin expression in all fibroblast cultures, although its effects on apoptosis in fibroblasts from glabrous and non-glabrous tissues were found to differ. The presence of collagen I in the ECM resulted in reduction of alpha-smooth-muscle actin. A considerable percentage of the apoptotic fibroblasts in attached gels were alpha-smooth-muscle actin positive. The extent of apoptosis correlated positively with increased cell and matrix relaxation. TGFbeta1 was unable to overcome this apoptotic effect of matrix relaxation.. The presence of myofibroblasts and the apoptosis level can be regulated by both TGFbeta1 and by the extracellular matrix. However, reduction of tension in the matrix is the critical determinant. This predicts that the tension in the wound bed determines the type of scar at different body sites.

Topics: Actins; Apoptosis; Biopsy; Cells, Cultured; Collagen Type I; Dermis; Fibroblasts; Fibronectins; Humans; Keloid; Muscle, Smooth; Phenotype; Pressure; Stress, Mechanical; Transforming Growth Factor beta; Wound Healing

Topics: Adolescent; Adult; Apoptosis; Blotting, Western; Cell Communication; Cell Division; Cells, Cultured; Culture Media; Fas Ligand Protein; fas Receptor; Fibroblasts; Genes, bcl-2; Humans; Keloid; Keratinocytes; Membrane Glycoproteins; Middle Aged; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

To study the effect of overexpression of truncated type II TGF-beta receptor on transforming growth factor-beta 1(TGF-beta 1) autoproduction in normal dermal fibroblasts.. In vitro cultured dermal fibroblasts were treated with recombinant human TGF-beta 1(rhTGF-beta 1) (5 ng/ml) or recombinant adenovirus containing truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects on regulating gene expression of TGF-beta 1 were observed with Northern blotting.. rhTGF-beta 1 up-regulated the gene expression of TGF-beta 1 and type I procollagen. Overexpression of truncated receptor II down-regulated the gene expression of TGF-beta 1.. Overexpression of the truncated TGF-beta receptor II decreases TGF-beta 1 autoproduction via blocking TGF-beta receptor signal. The results may provided a new strategy for scar gene therapy.

Topics: Cell Division; Cells, Cultured; Fibroblasts; Gene Expression; Genetic Therapy; Humans; Keloid; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially collagen. However, the molecular mechanisms regulating this pathologic collagen deposition still remain to be elucidated. A previous study by this group demonstrated that transforming growth factor (TGF)-beta1 and -beta2 ligands were expressed at greater levels in keloid fibroblasts when compared with normal human dermal fibroblasts (NHDFs), suggesting that TGF-beta may play a fibrosis-promoting role in keloid pathogenesis.To explore the biomolecular mechanisms of TGF-beta in keloid formation, the authors first compared the expression levels of the type I and type II TGF-beta receptors in keloid fibroblasts and NHDFs. Next, they investigated the phosphorylation of Smad 3, an intracellular TGF-beta signaling molecule, in keloid fibroblasts and NHDFs. Finally, they examined the regulation of TGF-beta receptor II by TGF-beta1, TGF-beta2, and TGF-beta3 ligands. Our findings demonstrated an increased expression of TGF-beta receptors (types I and II) and increased phosphorylation of Smad 3 in keloid fibroblasts relative to NHDFs. These data support a possible role of TGF-beta and its receptors as fibrosis-inducing growth factors in keloids. In addition, all three isoforms of recombinant human TGF-beta proteins could further stimulate the expression of TGF-beta receptor II in both keloids and NHDFs. Taken together, these results substantiate the hypothesis that the elevated levels of TGF-beta ligands and receptors present in keloids may support increased signaling and a potential role for TGF-beta in keloid pathogenesis.

Topics: Activin Receptors, Type I; Adolescent; DNA-Binding Proteins; Fibroblasts; Humans; Keloid; Middle Aged; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Signal Transduction; Skin; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta; Up-Regulation

To study the effect of antisense TGF-beta 1 on keloid fibroblast proliferation in vitro.. The antisense TGF-beta 1 was transfected into the keloid fibroblast by liposome. The cell count technique was used to test the effect of antisense TGF-beta 1 on keloid fibroblast proliferation in vitro. Cell apoptosis was tested by FCM.. Antisense TGF-beta 1 can inhibit the proliferation of keloid fibroblast in vitro; Antisense TGF-beta 1 can increase the apoptosis ratio of keloid fibroblast in vitro.. Antisense TGF-beta 1 can induce keloid fibroblast apoptosis and inhibit keloid fibroblast proliferation in vitro, which may contribute to the inhibition of keloid formation in vivo.

Topics: Antisense Elements (Genetics); Apoptosis; Cell Division; Cells, Cultured; Fibroblasts; Humans; Keloid; Transforming Growth Factor beta; Transforming Growth Factor beta1

Keloid formation is a wound healing response, which fails to resolve and leads to formation of a raised collagen mass extending beyond the original wound margins. Keloids are typically excluded from palms and soles. Therefore we compared keloid and palmar fibroblasts in vitro using fibroblasts from nonaffected individuals as controls. Collagen I, alpha-smooth muscle actin and thrombospondin-1 were found at higher levels in keloid than in palmar fibroblasts. These differences were ameliorated by addition of TGFbeta1. The potential for resolution of the wound healing response was estimated analyzing apoptosis during serum starvation. Annexin V and TUNEL assays showed that palmar fibroblasts underwent faster apoptosis, than did the keloid fibroblasts, and started detaching. Addition of TGFbeta1 counteracted this effect. The weak expression of the myofibroblast phenotype and the advanced apoptosis of palmar fibroblasts suggest mechanisms for the exclusion of keloids from palmar sites.

Topics: Apoptosis; Cell Differentiation; Cells, Cultured; Collagen; Fibroblasts; Humans; Keloid; Muscles; Transforming Growth Factor beta; Wound Healing

An in vitro model was used to determine the effect of superpulsed CO2 laser energy on normal dermal and keloid-producing fibroblast proliferation and release of growth factors. Growth factors assayed included basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1). bFGF is mitogenic, inhibits collagen production, and stabilizes cellular phenotype. TGF-beta1 stimulates growth and collagen secretion and is thought to be integral to keloid formation. Growth in a serum-free medium allowed measurement of these growth factors without confounding variables. Keloid and normal dermal fibroblasts cell lines were established from facial skin samples using standard explant techniques. Samples consisted of three separate keloid and three separate normal dermal fibroblast cell lines. Cells were used at passage 4 to seed 24-well trays at a concentration of 6 x 10(4) cells per milliliter in serum-free medium. At 48 hours, 18.8 percent of each cell well was exposed to a fluence of 2.4, 4.7, and 7.3 J/cm2 using the superpulsed CO2 laser. Cell viability and counts were established at four time points: 0 (time of superpulsed CO2 laser treatment), 24, 72, and 120 hours. Supernatants were collected and assessed for bFGF and TGF-beta1 using a sandwich enzyme immunoassay. All cell lines demonstrated logarithmic growth through 120 hours (conclusion of experiment), with a statistically significant shorter population doubling time for keloid fibroblasts (p < 0.05). Use of the superpulsed CO2 laser shortened population doubling times relative to that of controls; the differences were statistically significant in keloid dermal fibroblasts when fluences of 2.4 and 4.7 J/cm2 were used (p < 0.05 and 0.01, respectively). bFGF was present in greater levels in normal dermal fibroblasts than in keloid dermal fibroblasts. Application of superpulsed CO2 demonstrated a trend toward increased bFGF secretion in both fibroblast types; the increase was significant in the keloid group at 4.7J/cm2. A consistent trend in suppression of TGF-beta1 was seen in both groups exposed to superpulsed CO2, with the maximal effect occurring at 4.7 J/cm2. Serum-free culture sustains logarithmic cell growth and allows growth factor measurement without confounding variables from serum-containing media. Superpulsed CO2 enhances fibroblast replication and seems to stimulate bFGF secretion and to inhibit TGF-beta1 secretion. Given the function of these growth factors, the application of

Topics: Cell Division; Cells, Cultured; Culture Media, Serum-Free; Dermis; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Fibroblasts; Humans; Keloid; Lasers; Transforming Growth Factor beta

The pathogenesis of keloid remains poorly understood. As no effective therapy for keloid is as yet available, an insight into its pathogenesis may lead to novel approaches. Apoptosis has been found to mediate the decrease in cellularity during the transition between granulation tissue and scar. Here, we report that in contrast to hypertrophic scar-derived and normal skin-derived fibroblasts, keloid-derived fibroblasts are significantly resistant to both Fas-mediated and staurosporine-induced apoptosis. The caspases-3, -8, and -9 were not activated indicating that the block in the apoptotic pathway in keloid is upstream of the caspases. There were no significant differences in the level of expression of Fas, Bcl-2, and Bax between the three groups but addition of transforming growth factor (TGF)-beta1 significantly inhibited Fas-mediated apoptosis in hypertrophic scar-derived and normal skin-derived fibroblasts and neutralization of autocrine TGF-beta1 with anti-TGF-beta1 antibody abrogated the resistance of keloid-derived fibroblasts. Anti-apoptotic activity was not observed with TGF-beta2. This is the first study linking refractory Fas-mediated apoptosis to cellular phenotype in keloids and indicating a pivotal role for the anti-apoptotic effect of TGF-beta1 in this resistance. Hence, it becomes important to treat keloids as a separate entity different from hypertrophic scars and enhancement of Fas-sensitivity could be a promising therapeutic target.

Topics: Antibodies; Apoptosis; Autocrine Communication; bcl-2-Associated X Protein; bcl-X Protein; Caspases; Cells, Cultured; Drug Resistance; Enzyme Activation; fas Receptor; Fibroblasts; Humans; Keloid; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Staurosporine; Transforming Growth Factor beta; Transforming Growth Factor beta1

In the third part of this study a basic lipid model (regarding phospholipids, triglycerides, cholesterol esters and free fatty acids) for keloids (n=20), compared with normal skin of keloid prone and non-keloid prone patients (n=20 of each), was constructed according to standard methods, to serve as a sound foundation for essential fatty acid supplementation strategies in the prevention and treatment of keloid formations. Essential fatty acid deficiency (EFAD) of the omega-6 series (linoleic acid (LA), g-linolenic acid (GLA), and dihomo-g-linolenic acid (DGLA)) and the omega-3 series (a-linolenic acid (ALA) and eicosapentaenoic acid (EPA)), but enhanced arachidonic acid (AA) levels, were prevalent in keloid formations. Enhanced AA, but a deficiency of AA precursors (LA, GLA and DGLA) and inflammatory competitors (DGLA and EPA), are inevitably responsible for the overproduction of pro-inflammatory metabolites (prostaglandin E(2)(PGE(2))) participating in the pathogenesis of inflammation. Of particular interest was the extremely high free oleic acid (OA) levels present, apart from the high free AA levels, in the keloid formations. OA stimulates PKC activity which, in turn, activates PLA(2)activity for the release or further release of AA from membrane pools. Interactions between EFAs, eicosanoids, cytokines, growth factors and free radicals can modulate the immune response and the immune system in undoubtedly involved in keloid formation. The histopathology of keloids can be adequately explained by: persistence of inflammatory- and cytokine-mediated reactions in the keloid/dermal interface and peripheral areas, where fibroblast proliferation and continuous depletion of membrane linoleic acid occur; microvascular regeneration and circulation of sufficient EFAs in the interface and peripheral areas, where maintenance of metabolic active fibroblasts for collagen production occur; microvessel occlusion and hypoxia in the central areas, where deprivation of EFAs and oxygen with consequent fibroblast apoptosis occur, while excessive collagen remain. All these factors contribute to different fibroblast populations present in: the keloid / dermal interface and peripheral areas where increases in fibroblast proliferation and endogenous TGF-b occur, and these metabolic active fibroblast populations are responsible for enhanced collagen production: the central areas where fibroblast populations under hypoxic conditions occur, and these fibroblasts are responsible for

Topics: Apoptosis; Arachidonic Acid; Biopsy; Black People; Case-Control Studies; Cell Division; Cholesterol Esters; Chromatography, Gas; Chromatography, Thin Layer; Eicosapentaenoic Acid; Fatty Acids, Nonesterified; Fibroblasts; Groin; Humans; Hypoxia; Keloid; Linoleic Acid; Lipid Metabolism; Methylation; Models, Biological; Oleic Acid; Oxygen; Phospholipases A; Phospholipids; Protein Kinase C; Rural Population; South Africa; Transforming Growth Factor beta; Triglycerides

Cytokines, such as the transforming growth factor beta (TGF-beta) isoforms, have been linked to the formation of proliferative scars. This study examines the stimulating effects of exogenous TGF-beta2 on cultured keloid, burn hypertrophic scar, and normal skin fibroblasts and whether such effects can be suppressed with TGF-beta2 antibody.. In vitro, the fibroblast-populated collagen lattice (FPCL) is used in the evaluation of fibroblast activation by measuring contraction of the lattice over time. Primary cultures of fibroblasts were grown from keloids, burn hypertrophic scars, and normal skin using standard cell culture techniques. TGF-beta2 (10 ng/ml) was added to each of the three types of cell cultures and placed on prefabricated FPCLs. Each was tested against their normal control counterparts. TGF-beta2 antibody (100 ng/ml) was then placed on the TGF-beta2-treated FPCLs. All lattices were allowed to contract and areas were measured for 5 days.. Compared to controls, keloid fibroblasts were most affected by the addition of exogenous TGF-beta2. Normal skin fibroblasts did not show a significant increase in contraction early on, yet a significant difference was seen as time progressed. The addition of TGF-beta2 antibody inhibited the function of keloid and burn hypertrophic scar fibroblasts. It also reversed the increased contraction of the TFG-beta2-treated proliferative scar fibroblasts.. By utilizing an in vitro model, we have demonstrated that TGF-beta2 antibody reverses the increased contraction of FPCLs by proliferative scar fibroblasts treated with TGF-beta2. This points to a possible treatment modality in patients afflicted with this disfiguring problem.

Topics: Antibodies; Burns; Cell Division; Cells, Cultured; Cicatrix; Fibroblasts; Humans; Keloid; Reference Values; Transforming Growth Factor beta

Keloids, hypertrophic scars, and burn hypertrophic scars are all forms of proliferative scarring characterized by overabundant matrix formation. Recently these dermal proliferative disorders have been linked clinically to the cytokine transforming growth factor beta (TGF-beta), and in vitro tests have shown it to be responsible for the activation of fibroblasts and their production and deposition of collagen. Using an established in vivo animal model of proliferative scarring, the effects of this cytokine, specifically the isoform TGF-beta2, on these scars were examined. Proliferative scar specimens were implanted into athymic, asplenic nude rats and isolated in sandwich island flaps based on the superficial inferior epigastric pedicle. After establishment of the transferred flap, the scars were injected with varying doses of TGF-beta2 or vehicle for 5 consecutive days and then again on days 10, 15, and 20. The specimens were measured weekly during the period of dosing, and a biopsy was acquired on days 30 and 60. Fibroblasts from the explanted biopsies and the original scars were grown in cell culture, and cell proliferation studies were performed and the results compared. There was a dose response to TGF-beta2, with 200 ng showing the greatest effect. From the original scar specimens, keloid scars demonstrated the greatest cell proliferation kinetics--significantly faster than nonburn and burn hypertrophic scars. After treatment with TGF-beta2, both keloids and burn hypertrophic scars showed an increase in their cell proliferation kinetics compared with vehicle alone. This was not demonstrated with the nonburn hypertrophic scars. Elevated levels of TGF-beta2 are a major contributing factor to the process of proliferative scars, but because nonburn hypertrophic scars do not result in an equally increased response to this cytokine, a truly causative role for this cytokine cannot be promulgated. Rather, it is the combination of the proliferative scar fibroblasts' abnormal response to TGF-beta2 stimulation and elevated levels of this cytokine that controls more accurately the process of keloid and burn hypertrophic scar formation.

Topics: Animals; Cell Division; Cells, Cultured; Cicatrix, Hypertrophic; Fibroblasts; Humans; Keloid; Protein Isoforms; Rats; Rats, Nude; Surgical Flaps; Transforming Growth Factor beta

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.

Topics: Adolescent; Adult; Blotting, Western; Fibroblasts; Humans; Keloid; Middle Aged; Protein Isoforms; Skin; Transforming Growth Factor beta

To describe two new cases of papillary carcinoma of the thyroid with exuberant nodular fasciitis-like stroma, one of which was characterized by previously unreported transformation into a poorly differentiated lesion. Moreover, we explore the presence of TGF-beta to help to clarify the pathogenesis of the collagen formation.. The case characterized by an aggressive behaviour exhibited areas of transformation into a poorly differentiated (insular) carcinoma of the thyroid. In both cases, as revealed by immunohistochemistry, neoplastic cells produced and secreted high amounts of TGF-beta. On the contrary, TGF-beta immunoreaction was never present in the normal thyroid or in papillary carcinomas without collagen bundles, while a weak, exclusively intracellular reaction was present in a patchy manner in cases showing intratumoral fibrous bundles.. The rare variant of papillary thyroid carcinoma characterized by exuberant stroma may give rise to more aggressive lesions, as do other histotypes of differentiated thyroid carcinomas. TGF-beta, the fundamental cytokine which mediates scarring and activation of myofibroblasts, most probably induces the exuberant stroma.

Topics: Adult; Aged; Carcinoma, Papillary; Cell Transformation, Neoplastic; Female; Humans; Immunoenzyme Techniques; Keloid; Male; Stromal Cells; Thyroid Gland; Thyroid Neoplasms; Transforming Growth Factor beta

We evidenced in vitro proopiomelanocortin (POMC) mRNA-transcription in human dermal fibroblasts using Northern blot hybridization. Modulation of POMC gene expression by cytokines (transforming growth factor-beta, TGF-beta, and tumor necrosis factor-alpha, TNF-alpha) was investigated by incubating human normal fibroblasts with 1 and 10 ng/ml cytokines, either alone or in combination, for 24 hours. Our results show that dermal fibroblasts express POMC at significant levels under unstimulated conditions. POMC steady-state levels were significantly reduced by addition of TGF-beta. On the other hand, TNF-alpha exerted a stimulatory effect on POMC mRNA transcription, partially counteracting the effect of TGF-beta. These data provide the first demonstration of POMC gene expression in cultured skin fibroblasts. The opposite regulatory effect of TGF-beta and TNF-alpha, two cytokines primarily involved in extracellular matrix regulation, suggests a possible role for POMC-derived peptides in fibroblast activity. We also investigated POMC mRNA expression in keloid-derived fibroblasts in culture, and its regulation by TGF-beta added at the highest concentration documented for inhibition. Keloid-derived fibroblasts showed clearly detectable levels of POMC mRNA in basal conditions, and no alteration of POMC gene expression was observed when TGF-beta was added in culture. The altered TGF-beta regulation of POMC mRNA levels suggest that POMC-derived peptides may play a role in the pathogenesis of keloid formation through an autocrine/paracrine network, resulting in modulation of extracellular matrix synthesis.

Topics: Cells, Cultured; Fibroblasts; Gene Expression Regulation; Humans; Keloid; Keratinocytes; Kinetics; Pro-Opiomelanocortin; Recombinant Proteins; Skin Physiological Phenomena; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The expression of mRNA for TGF-beta 1, type I, III procollagen in keloid and hypertrophic scar was investigated in order to elucidate the pathogenesis of and the difference between keloid and hypertrophic scar.. Dot-blot hybridization analysis was used to examine the type I, III procollagen and the expression levels of steady-state mRNA. In situ hybridization allowed direct assessment of the distribution of TGF-beta 1 mRNA in the tissue.. 1. Our study indicated that the expression level of mRNA for TGF-beta 1 significantly increased in keloid and hypertrophic scar compared with normal scar and normal skin. 2. In keloid tissues, type I precollagen mRNA expression was selectively increased. However, in hypertrophic scar, type I and III collagen mRNAs expressions were simultaneously increased.. TGF-beta 1 plays an important role in the pathogenesis of keloid and hypertrophic scarring. It implicates that distinct molecular mechanisms are operative in the development of keloid and hypertrophic scarring.

Topics: Cicatrix, Hypertrophic; Humans; Keloid; Procollagen; RNA, Messenger; Transforming Growth Factor beta

Keloids occur only in humans and are characterized by fibroblast overproduction of collagen types I and III. Keloid fibroblasts have been shown to make elevated levels of transforming growth factor beta (TGF-beta), a growth factor known to promote extracellular matrix production and fibrosis. Thus, the pathophysiology underlying keloid formation may be driven by the biological activity of TGF-beta. Tamoxifen, a synthetic, nonsteroidal antiestrogen has been shown to inhibit keloid fibroblast proliferation and decrease collagen production. The purpose of this study was to determine if a mechanism by which tamoxifen decreases keloid collagen production is through a downregulation of TGF-beta. Through a luciferase TGF-beta bioassay we found that 4 microM of tamoxifen generated a 49% reduction in total TGF-beta activity and 8 microM generated an 85% reduction compared with controls. Thus we propose that one of the mechanisms by which tamoxifen decreases keloid fibroblast collagen synthesis is by decreasing TGF-beta production.

Topics: Cell Culture Techniques; Down-Regulation; Estrogen Antagonists; Fibroblasts; Humans; Keloid; Luciferases; Tamoxifen; Transforming Growth Factor beta

Originally described as a product of the pituitary gland, propiomelanocortin (POMC) has recently been identified in other tissues, such as in human skin, where it may accumulate in response to various stimuli. Thus far, epidermal keratinocytes, as well as melanocytes and macrophages, have been shown to express POMC. This study investigated the expression of POMC mRNA in cultured dermal fibroblasts derived from either normal skin or keloids. Using Northern blot hybridization with a POMC cDNA generated by RT-PCR of mRNA isolated from cardiac muscle, we demonstrated that dermal fibroblasts express POMC, as significant levels of mRNA were detected in unstimulated cells in culture. POMC transcript steady-state levels were strongly reduced by transforming growth factor-beta (TGF-beta), whereas tumor necrosis factor-alpha (TNF-alpha) counteracted the effect of TGF-beta and exerted a stimulatory activity on POMC mRNA levels. Reduction of POMC transcript levels by TGF-beta was also observed in cultured keratinocytes. Clearly detectable levels of POMC mRNA were detected in cultured keloid-derived fibroblasts; however, little, if any, regulation by TGF-beta was observed. These data represent the first demonstration of POMC expression by fibroblasts and down-regulation by TGF-beta. Furthermore, our results indicate altered TGF-beta regulation of POMC gene expression in keloid-derived fibroblasts, suggesting that POMC may play a role in the pathogenesis of keloid formation.

Topics: Cells, Cultured; DNA Probes; DNA, Complementary; Epidermal Cells; Fibroblasts; Gene Expression Regulation; Humans; Keloid; Keratinocytes; Male; Pro-Opiomelanocortin; Recombinant Proteins; Skin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We studied the effects of electromagnetic fields (EMFs) on cell proliferation and collagen synthesis in both normal and keloid fibroblasts in vitro. Treatment of keloid fibroblasts with 60-Hz EMFs for 10 days resulted in the inhibition of cell proliferation, whereas no significant change in cell proliferation of normal fibroblasts was observed. EMFs inhibited collagen synthesis in keloid fibroblasts but not in normal fibroblasts without altering the ratio of type III to type I collagen, indicating that EMFs inhibit type I and type III collagen synthesis to the same extent. EMFs also decreased the expression of transforming growth factor beta (TGF-beta) in keloid fibroblasts. These results suggest that EMFs may have a useful therapeutic potential for the treatment of keloid.

Topics: Adolescent; Adult; Blotting, Northern; Cell Division; Collagen; Electromagnetic Fields; Female; Fibroblasts; Humans; Keloid; Male; Middle Aged; RNA; Transforming Growth Factor beta

Excessive scar contracture by wound fibroblasts can have devastating consequences, ranging from body disfigurement to joint immobility. The ability of fibroblasts isolated from lesions of hypertrophic scars, keloids, normal skin, or normal scars in contracting the provisional wound matrix (i.e., fibrin clot) was compared and analyzed. Hypertrophic scar fibroblasts showed a consistently higher basal level of fibrin matrix gel (FMG) contraction than other fibroblasts. This heightened basal level of contractility may be attributed partially to the autocrine effect of transforming growth factor-beta 1 (TGF-beta 1). Normal and keloid fibroblasts exhibited similar basal rates of FMG contraction, and both responded to platelet-derived growth factor (PDGF) and TGF-beta by increasing FMG contraction two- to threefold. However, 45% of the TGF-beta-induced increase in FMG contraction by keloid fibroblasts, but not normal fibroblasts, was mediated by the autocrine production of PDGF. Therefore, fibroblasts isolated from different scars exhibit varied degrees of FMG contraction. In addition, the mechanism underlying growth factor-mediated contraction differed vastly among fibroblasts of different scar origin. The significance of these differences in growth factor-mediated FMG contraction is discussed.

Topics: Cicatrix; Culture Techniques; Fibroblasts; Humans; Keloid; Transforming Growth Factor beta

Keloids are characterized by an overabundant deposition of collagen, and they recur frequently following excision. Fibroblasts isolated from keloid tissue and maintained in cell culture continue to express an increased capacity to produce collagen. In an effort to define the mechanisms responsible for keloid formation, the potential of exogenous transforming growth factor beta 1 (TGF-beta 1) to differentially affect DNA synthesis and collagen expression in cultured human fibroblasts derived from keloid or normal dermis was investigated. In this study, TGF-beta 1 at a concentration of 5.0 ng/ml was found to stimulate DNA synthesis of keloid-derived fibroblasts to a greater extent than fibroblasts derived from normal dermis. With a microassay to measure levels of collagenase-digestible radiolabeled proteins, TGF-beta 1 was found to elicit a greater increase in absolute collagen synthesis in keloid-derived fibroblasts compared with fibroblasts derived from normal dermis. Examination of tRNA(pro) pool-specific activities indicated that these observed differences in rates of collagen synthesis were not the result of unequal rates of proline transport or pool size. Likewise, TGF-beta 1 did not alter the uptake of vitamin C, an essential cofactor and mediator needed for maximal collagen expression. The increase in collagen synthesis by keloid-derived fibroblasts treated with TGF-beta 1 was accompanied by a corresponding increase in procollagen type I mRNA levels, indicating that the differential response of keloid and normal dermal fibroblasts to this growth factor is occurring primarily at a pretranslational level. These results suggest a unique sensitivity of keloid fibroblasts to TGF-beta 1 and thus a possible role for this mediator in keloid pathogenesis.

Topics: Cell Division; Cells, Cultured; Collagen; Fibroblasts; Humans; Keloid; Transforming Growth Factor beta

Keloids are pathological lesions characterized by enhanced expression of extracellular matrix molecules including fibronectin. A molecular dissection of the human fibronectin promoter was performed to identify DNA-protein interactions that correlate with altered fibronectin gene expression by keloid fibroblasts. DNA mobility shift patterns generated by nuclear extracts from skin, scar, and keloid fibroblasts were identical at a consensus CRE at -170 of the human fibronectin promoter whereas extracts from keloid fibroblasts formed complexes at a CRE/AP-1-like sequence at -415 that differed from those generated by skin and scar fibroblast extracts. The DNA-protein interactions identified at -415 were sensitive to altered serum concentrations in skin and scar but not keloid fibroblast cultures. The effects of forskolin and TGF-beta on fibronectin expression correlated with changes in the DNA-protein complexes assembled on the -170 and -415 cis elements, respectively. Oligonucleotides containing consensus CRE and AP-1 sequences did not compete for binding of nuclear proteins to the CRE/AP-1-like domain at -415, suggesting that this is a unique cis element. These studies indicate that the human fibronectin promoter contains two cis elements on which related but nonidentical complexes form. Alterations in the complexes interacting with the sequence at -415 may be responsible for the differences in fibronectin gene expression among quiescent skin, mature scar, and keloid fibroblasts.

Topics: Adolescent; Adult; Base Sequence; Binding Sites; Blood; Cells, Cultured; Child; Child, Preschool; Cicatrix; Colforsin; DNA; DNA-Binding Proteins; Female; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Keloid; Male; Middle Aged; Molecular Sequence Data; Promoter Regions, Genetic; RNA, Messenger; Skin; Transforming Growth Factor beta

Hyaluronic acid (HA), an important component of the tissue extracellular matrix, is a ubiquitous glycosaminoglycan (GAG) that forms a pericellular coat on the surface of cells. It has been speculated that this pericellular HA boundary may localize cytokines, such as transforming growth factor-beta 1, which is known to stimulate collagen production. The purpose of this study was to examine the role of HA and its cell surface receptor (CD44), an active participant in HA degradation, as they relate to keloid formation. Dermal excisions from both normal patients (n = 13) and keloid patients (n = 13) were analyzed for HA content using an alcian blue staining technique. Fibroblast cell cultures were used to quantitate HA synthesis and CD44 receptor density. Histological analyses showed a greater HA content in keloid tissue compared with normal dermal tissue. In agreement with this observation, keloid fibroblasts were found to synthesize significantly more HA than normal dermal fibroblasts (2469 +/- 483 cpm versus 1122 +/- 256 cpm, P = .02). Treatment of keloid fibroblasts with triamcinolone acetonide reduced the level of HA synthesis to that of normal fibroblasts (1560 +/- 477 cpm versus 1293 +/- 264 cpm, P = .6). However, there was no significant difference in HA receptor density on keloid cells compared with normal skin fibroblasts. Therefore, the increased HA deposits found in keloids are attributable to increased synthesis rather than to decreased degradation mediated by the CD44 receptor.

Topics: Adolescent; Adult; Alcian Blue; Cells, Cultured; Collagen; Coloring Agents; Extracellular Matrix; Fibroblasts; Flow Cytometry; Gene Expression Regulation; Humans; Hyaluronan Receptors; Hyaluronic Acid; Keloid; Skin; Transforming Growth Factor beta; Triamcinolone Acetonide

We investigated the effects of several growth factors on [3H]thymidine incorporation and procollagen type I carboxyterminal propeptide (P1CP) production, which reflects type I collagen metabolism, in keloid and normal fibroblasts.. Six fibroblast cell strains, derived from keloid or normal skin, exhibited similar growth responses to platelet-derived growth factor, transforming growth factor beta 1 (TGF-beta 1), gamma-interferon (gamma-IFN) and histamine. In contrast, keloid fibroblasts showed significantly greater growth response to epidermal growth factor (EGF) than normal fibroblasts. P1CP production was 4.4 times higher in 6 strains of keloid fibroblasts than in 6 controls. Treatment with gamma-IFN (100 U/ml) decreased P1CP production in both groups; the effect was significantly greater in keloid fibroblasts. TGF-beta 1 treatment upregulated P1CP production in both groups. Treatment with histamine increased P1CP production in keloid fibroblasts, although it did not change that in the controls.. EGF and histamine may play some role in the development of keloids.

Topics: Cell Division; Cells, Cultured; Collagen; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Fibroblasts; Growth Substances; Histamine; Humans; Interferon-gamma; Keloid; Male; Peptide Fragments; Platelet-Derived Growth Factor; Procollagen; Recombinant Proteins; Transforming Growth Factor beta

Keloid and hypertrophic scars are fibrous growths characterized by overabundant collagen deposition. We examined the effect of transforming growth factor-beta (TGF-beta), a known stimulant for the production of connective tissue matrices, on the rate of collagen synthesis in keloid fibroblasts (KFs), hypertrophic scar fibroblasts (HSFs), and normal skin fibroblasts (NSFs). Fibroblasts were cultured in three-dimensional fibrin-gel matrices in the presence or absence of TGF-beta (5 ng/ml) or anti-TGF-beta neutralizing antibody (50 micrograms/ml). Secreted collagen levels, labeled with 3H-proline, were measured after 48 hours. KFs produced up to 12 times more collagen than NSFs, and up to 4 times more than HSFs. Although KFs increased their rate of collagen production by up to 2.7 times in response to TGF-beta, HSFs and NSFs did not (p = 0.065). Anti-TGF-beta antibody reduced the rate of collagen synthesis of KFs by 40% (p = 0.003), although it did not suppress collagen production in HSFs (p = 0.06) and NSFs (p = 0.75). We conclude that although KFs and HSFs are similar in that they both overproduce collagen, they are different in that only KFs display a marked sensitivity to TGF-beta, which is abundant during the proliferative phase of wound healing.

Topics: Antibodies; Cells, Cultured; Cicatrix, Hypertrophic; Collagen; Fibroblasts; Humans; In Vitro Techniques; Keloid; Skin; Time Factors; Transforming Growth Factor beta; Wound Healing

Topics: Animals; Antibodies; Cells, Cultured; Cicatrix, Hypertrophic; Collagen; Extracellular Matrix; Fibroblasts; Humans; In Vitro Techniques; Keloid; Skin; Transforming Growth Factor beta; Wound Healing

Treatment with transforming growth factor beta 1 (TGF-beta 1) results in stimulation of total protein synthesis in normal dermal fibroblasts but not in keloid fibroblasts, suggesting that the TGF-beta regulatory program is altered in keloid fibroblasts. However, both keloid and normal fibroblasts treated with TGF-beta 1 exhibit accelerated fibronectin biosynthesis, indicating that keloid cells can respond to TGF-beta 1. In the absence of serum, the TGF-beta 1-induced increase in fibronectin biosynthesis occurs more rapidly in keloid fibroblasts, also suggesting modification of this regulatory pathway. The TGF-beta 1-mediated increase in keloid fibronectin production is independent of the steroid regulatory pathway for fibronectin, which accelerates synthesis by means of a post-transcriptional mechanism. Thus, TGF-beta 1 stimulation of fibronectin production in keloid cells is likely to involve a transcriptional mechanism and keloid overproduction of extracellular matrix components may be due to an inherent modification of the TGF-beta regulatory program.

Topics: Adolescent; Adult; Cell Line, Transformed; Female; Fibroblasts; Gene Expression Regulation; Humans; Keloid; Male; Middle Aged; Transforming Growth Factor beta

Untreated, clinically active keloids were examined as model system to study the spatial expression of extracellular matrix and transforming growth factor-beta 1 (TGF-beta 1) genes in fibrotic skin diseases. In situ hybridizations localized active expression of type I and VI collagen genes to the areas containing an abundance of fibroblasts and apparently representing the expanding border of the lesions. Within this zone, microvascular endothelial cells also expressed the type I collagen genes, as evaluated by simultaneous use of in situ hybridization for collagen gene expression and immunolocalization for factor VIII-related antigen, a marker for endothelial cell differentiation. Slot-blot hybridizations of RNA isolated from this zone suggested that the expression of type I and IV collagen genes was selectively enhanced, as compared to type III collagen gene expression. TGF-beta 1 protein and mRNA were also detected in areas active in type I and type VI collagen gene expression, indicating that TGF-beta 1 gene is transcribed and the corresponding protein is deposited in areas of elevated collagen gene expression, including microvascular endothelial cells. We conclude that the initial step in the development of fibrotic reaction in keloids involves the expression of the TGF-beta 1 gene by the neovascular endothelial cells, thus activating the adjacent fibroblasts to express markedly elevated levels of TGF-beta 1, as well as type I and VI collagen genes.

Topics: Adolescent; Adult; Collagen; Gene Expression Regulation; Humans; Immunoenzyme Techniques; Keloid; Nucleic Acid Hybridization; RNA, Messenger; Transforming Growth Factor beta