RESUMEN
Hypertrophic scars (HS) are fibro-hyperproliferative dermal lesions with effusive continuous accumulation of extracellular matrix components, particularly collagen. They usually occur after dermal injury in genetically susceptible individuals and cause both physical and psychological distress for the affected individuals. Transforming growth factor-ß1 (TGF-ß1) is known to mediate wound healing process by regulating cell differentiation, collagen production and extracellular matrix degradation. The sustained high expression of TGF-ß1 is believed to result in the formation of hypertrophic scars. Inhibition of TGF-ß1 signaling pathway may represent one of effective strategies for limiting excessive scarring. LY2109761, an orally active TßRI/II kinase dual inhibitor, has been previously reported that it had inhibitory effects on carcinomas and attenuates Radiation-induced pulmonary murine fibrosis. Our results revealed that LY2109761 reduced TGF-ß1-induced collagen production and α-smooth muscle actin (α-SMA) expression, and attenuated TGF-ß1-induced cell contraction in hypertrophic scar fibroblasts. The data from this study provide evidence supporting the potential use of LY2109761 as a novel treatment for hypertrophic scars.
Asunto(s)
Cicatriz Hipertrófica/tratamiento farmacológico , Colágeno/metabolismo , Fibroblastos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirroles/farmacología , Piel/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Actinas/metabolismo , Células Cultivadas , Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/patología , Fibroblastos/enzimología , Fibroblastos/patología , Fibrosis , Humanos , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal/efectos de los fármacos , Piel/enzimología , Piel/patología , Proteína smad3/metabolismoRESUMEN
Hypertrophic scar (HS) is an abnormally proliferative disorder characterized by excessive proliferation of fibroblasts and redundant deposition of extracellular matrix. An unbalance between fibroblast proliferation and apoptosis has been assumed to play an important role in HS formation. To explore the regulative effects of dracorhodin perchlorate (Dp), one of the derivants of dracorhodin that is a major constituent in the traditional Chinese medicine, on primary fibroblasts from human skin hypertrophic scars, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis were respectively used to evaluate the inhibitory effect of Dp on the cells and to determine cell cycle distribution. Additionally, cellular apoptosis was separately detected with Hoechst 33258 staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. The expression levels of caspase-3 mRNA and protein were respectively measured with reverse transcription-polymerase chain reaction and western blot analysis, and caspase-3 activity were determined using a colorimetric assay kit. The results showed that Dp significantly inhibited cell growth, and induced apoptosis in fibroblasts in a dose-and time-dependent manner, arresting cell cycle at G1 phase. Additionally, Dp slightly up-regulated caspase-3 mRNA expression in fibroblasts, but significantly down-regulated caspase-3 protein expression in a dose- and time-dependent manner, and concurrently elevated caspase-3 activity. Taken together, these data indicated that Dp could effectively inhibit cell proliferation, and induced cell cycle arrest and apoptosis in fibroblasts, at least partially via modulation of caspase-3 expression and its activity, which suggests that Dp is an effective and potential candidate to develop for HS treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzopiranos/farmacología , Caspasa 3/metabolismo , Cicatriz Hipertrófica/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Fibroblastos/efectos de los fármacos , Adolescente , Adulto , Caspasa 3/genética , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Niño , Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/patología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Femenino , Fibroblastos/enzimología , Fibroblastos/patología , Citometría de Flujo , Humanos , Etiquetado Corte-Fin in Situ , Estructura Molecular , Cultivo Primario de Células , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Adulto JovenRESUMEN
BACKGROUND: Pressure therapy improves hypertrophic scar healing, but the mechanisms for this process are not well understood. We sought to investigate the differential expression of matrix metalloproteinases (Mmps) and collagen in posttraumatic hypertrophic scar tissue with mechanical pressure and delineate the molecular mechanisms of pressure therapy for hypertrophic scars. METHODS: Fibroblast lines of normal skin and scar tissue were established and a mechanical pressure system was devised to simulate pressure therapy. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assays were used to compare differences in the mRNA and protein expression of Mmps and collagen in scar fibroblasts before and after pressure therapy. RESULTS: The expression differed between the hypertrophic scar cell line and the normal cell line. RT-PCR assays showed that Collagen I, highly expressed in the hypertrophic scar cell line, decreased significantly after pressure therapy. Mmp2, Mmp9, and Mmp12 expression in the hypertrophic scar tissue increased significantly after pressure therapy (P < 0.05). Western blotting assays further revealed that Mmp9 and Mmp12 expression increased significantly in the hypertrophic scar tissue after pressure therapy (P < 0.05) but not Mmp2 expression (P > 0.05). CONCLUSION: Mechanical pressure induces degradation of Collagen I in hypertrophic scar tissue by affecting the expression of Mmp9 and Mmp12.
Asunto(s)
Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/metabolismo , Colágeno Tipo I/metabolismo , Metaloproteinasa 12 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Línea Celular , Colágeno Tipo I/genética , Humanos , Metaloproteinasa 12 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genéticaRESUMEN
CCN2 plays an important role in the pathogenesis of hypertrophic scars (HTSs). Although CCN2 is involved in many fibroproliferative events, the CCN2 induction signaling pathway in HTSs is yet to be elucidated. Here, we first investigated the effect of the mitogen-activated protein kinases (MAPKs) on CCN2-induced α-smooth muscle actin (α-SMA) and collagen I expression in human HTS fibroblasts (HTSFs). Then, we established HTSs in a rabbit ear model and determined the effect of MAPKs on the pathogenesis of HTSs. MAPK pathways were activated by CCN2 in HTSFs. Extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors significantly inhibited CCN2-induced expression of α-SMA and collagen I in HTSFs. In the rabbit ear model of the HTS, JNK and ERK inhibitors significantly improved the architecture of the rabbit ear scar and reduced scar formation on the rabbit ear. Our results indicate that ERK and JNK mediate collagen I expression and scarring of the rabbit ear, and may be considered for specific drug therapy targets for HTSs.
Asunto(s)
Cicatriz Hipertrófica/enzimología , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/enzimología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Piel/enzimología , Actinas/metabolismo , Animales , Células Cultivadas , Cicatriz Hipertrófica/tratamiento farmacológico , Cicatriz Hipertrófica/genética , Cicatriz Hipertrófica/patología , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática , Proteínas de la Matriz Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Regulación de la Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Conejos , Proteínas Recombinantes/metabolismo , Piel/efectos de los fármacos , Piel/patología , Factores de Tiempo , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de HeridasRESUMEN
OBJECTIVE: To investigate the effects and regulatory mechanism of ILK on angiogenesis in hypertrophic scar. METHODS: The human scar microvascular endothelial cells (HSMECs) were isolated from 6 patients' hypertrophic scar in vitro. The HSMECs with good condition in 2nd to 4th generation were selected as experimental objectives. (1) HSMECs were divided into the blank control group (treated with routine culture), negative control group (treated with only Lipofectamine 2000), LY294002 group (incubated with 50 nmol/L LY294002), ILK siRNA group (incubated with 20 nmol/L ILK siRNA). RT-PCR and Western Blot were used to detect the expression of ILK mRNA and its protein after transfecion for 48 h. (2) The digested HSMECs of four groups were resuspended with DMEM without serum and then seeded onto the upper compartment of transwell insert which contained complete medium in its lower compartment. The cell migration experiment was stopped in 10 h and then the migrated cells were counted to analyze the effects of different interventions on the migration ability of HSMECs. (3) The thawed ECMatrix was put into each well of pre-colled 48-well tissue culture plate, and then the plate was put into the incubator at 37 degrees C to make it to become gel. The HSMECs of four groups were seeded onto the surface of the ECMatrix gel and were put into incubator. Eight random view-fields per well should be valued by the sheet of pattern recognition about angiogenesis after 8 hours to evaluate the ability of angiogenesis in vitro between four groups. RESULTS: (1) The expression of ILK mRNA (ILK mRNA = 0.829 +/- 0.109, t = 13.151, P = 0.006) and protein (ILK protein = 0.096 +/- 0.049, t = 36.656, P = 0.000) were both inhibited obviously in ILK siRNA group compared with the blank control group (ILK mRNA = 0.829 +/- 0.109, ILK protein = 1). And, the expression of ILK in LY294002 group was slightly lower than that of black control group, but there was no statistical difference. (2) The number of migrated cells in ILK siRNA group (88.111 +/- 3.079) and LY294002 group (138. 667 +/- 2.404) were respectively lower than that in blank control group (322.333 +/- 3.712, P < 0. 05) in 10th hour. (3) Compared to blank control group (4.333 +/- 0.191), the ability of angiogenesis in vitro decreased significantly ILK siRNA group (2.625 +/- 0.125) and LY294002 group (3.125 +/- 0.250), in which, the vascular network structures were not formed perfectly in 8th hour (P < 0.05). CONCLUSIONS: The ability of HSMECs' migration and angiogenesis in vitro are inhibited significantly when the expression of ILK is down-regulated. It reveals that ILK may play an role in the regulation of scar angiogenesis.
Asunto(s)
Movimiento Celular , Cicatriz Hipertrófica , Células Endoteliales/efectos de los fármacos , Neovascularización Patológica/etiología , Proteínas Serina-Treonina Quinasas/fisiología , Proliferación Celular , Cromonas/farmacología , Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/patología , Células Endoteliales/citología , Humanos , Lípidos/farmacología , Morfolinas/farmacología , Neovascularización Patológica/patología , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/análisis , ARN Interferente Pequeño/metabolismoRESUMEN
OBJECTIVE: To explore the binding domain of hydroxypyruvate isomerase homologues (HYI) in the interaction with protein P311 in hypertrophic scar fibroblasts (Fb). METHODS: (1) P 311 was amplified by PCR using plasmid pMD18-T-P 311 as template. The total RNA of hypertrophic scar Fb was extracted by Trizol to amplify HYI with RT-PCR. Recombinant vectors pGADT7-P 311 and pGBKT7-HYI were constructed by double-enzyme digestion, and they were verified by PCR and sequencing. The secondary structure of protein HYI was analyzed with software Prot Seale and HNN. Fragments of HYI-1 (1-447 bp), HYI-2 (247-447 bp), HYI-3 (1-279 bp), and HYI-4 (247-654 bp) were amplified based on the result of software analysis. And then the recombinant vectors pGBKT7-HYI-1, 2, 3, and 4 were constructed by double-enzyme digestion and verified by PCR and sequencing. (2) AH109 yeast cells were transformed into competent cells by lithium acetate method and divided into 7 groups roughly in the same amount, including HYI full length, HYI-1, HYI-2, HYI-3, and HYI-4 hybrid groups, positive control group, and negative control group. Cells in the first five groups were respectively transformed with recombinant vector pGBKT7-HYI full length, pGBKT7-HYI-1, pGBKT7-HYI-2, pGBKT7-HYI-3, pGBKT7-HYI-4 and recombinant vector pGADT7-P 311, and that in the rest two groups were transformed with recombinant vectors pGBKT7-53 and pGADT7-RecT, pGADT7-RecT and pGBKT7-Lam by polyethyleneglycol/lithium acetate method. Immediately after transformation, a part of the transformed cells in each group was spread onto the medium lacking leucine, tryptophan, adenine, and histidine (briefly called four-factor lacking medium), and another portion of the cells was spread onto the medium lacking leucine and tryptophan (briefly called two-factor lacking medium). After 3 to 6 days' culture, the growth of yeast was observed, and the expression of ß-galactosidase of yeast was detected by color reaction with 5-bromo-4-chloro-indolyl-ß-D-galactopyranoside. RESULTS: (1) Cloned P 311 and the reported P 311 (GenBank ID hsu36189) had the same sequence. The A base at 496 bp in reported HYI (GenBank ID AY775560) was replaced by G base as found in cloned HYI. It was verified that the insert segment of each recombinant vector was correct. (2) Among those 216 amino acids which composed the protein HYI, 101 amino acids might form α helices, 90 amino acids might form random coils, 25 amino acids might form extended-chains as revealed in the simulated structure analysis by computer. (3) Cloned segments HYI-1, 2, 3, 4 showed expected lengths. It was verified that the insert segment of each recombinant vector was correct. (4) Except for strains in negative control group which did not show growth on four-factor lacking medium, all strains in other groups grew on both kinds of media, and growth of colonies was less in HYI-2 (with the fewest number of α helices) and HYI-3 hybrid groups. (5) Positive expression of ß-galactosidase was observed in strains of all groups growing on four-factor lacking medium except for the HYI-2 hybrid group. No expression of ß-galactosidase was observed in strains of negative control group which grew on two-factor lacking medium. CONCLUSIONS: Protein HYI may closely bind with protein P311 by α helix, which plays an important role in fibroblast-to-myofibroblast transdifferentiation in hypertrophic scar.
Asunto(s)
Isomerasas Aldosa-Cetosa/genética , Cicatriz Hipertrófica/genética , Fibroblastos/enzimología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Oncogénicas/metabolismo , Cicatriz Hipertrófica/enzimología , Clonación Molecular , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Plásmidos , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
Hypertrophic scarring, a common proliferative disorder of dermal fibroblasts, results from an overproduction of fibroblasts and excessive deposition of collagen. Although treatment with surgical excision or steroid hormones can modify the symptoms, numerous treatment-related complications have been described. In view of this, we investigated the therapeutic effects of essential oil (EO) from rhizomes of Ligusticum chuanxiong Hort. (Umbelliferae) on formed hypertrophic scars in a rabbit ear model. EO was prepared as a liposomal formulation (liposome-enveloped essential oil, LEO) and a rabbit ear model with hypertrophic scars was established. LEO (2.5, 5, and 10%) was applied once daily to the scars for 28 days. On postoperative day 56, the scar tissue was excised for masson's trichrome staining, detection of fibroblast apoptosis, assays of the levels of collagens I and III, and analysis of the mRNA expression of matrix metalloproteinase-1 (MMP-1), caspase-3 and -9, and transforming growth factor beta 1 (TGF-ß(1)). In addition, the scar elevation index (SEI) was also determined. As a result, LEO treatment significantly alleviated formed hypertrophic scars on rabbit ears. The levels of TGF-ß(1), MMP-1, collagen I, and collagen III were evidently decreased, and caspase -3 and -9 levels and apoptosis cells were markedly increased in the scar tissue. SEI was also significantly reduced. Histological findings exhibited significant amelioration of the collagen tissue. These results suggest that LEO possesses the favorable therapeutic effects on formed hypertrophic scars in the rabbit ear model and may be an effective cure for human hypertrophic scars.
Asunto(s)
Cicatriz Hipertrófica/tratamiento farmacológico , Oído/patología , Ligusticum/química , Aceites Volátiles/uso terapéutico , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Química Farmacéutica , Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Etiquetado Corte-Fin in Situ , Liposomas , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Aceites Volátiles/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Coloración y Etiquetado , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Hypertrophic scars are a pathological process characterized by an excessive deposition of extracellular matrix components. Using a tissue-engineered reconstructed human skin (RHS) method, we previously reported that pathological keratinocytes induce formation of a fibrotic dermal matrix. We further investigated keratinocyte action using conditioned media. Results showed that conditioned media induce a similar action on dermal thickness similar to when an epidermis is present. Using a two-dimensional electrophoresis technique, we then compared conditioned media from normal or hypertrophic scar keratinocytes and determined that TIMP-1 was increased in conditioned media from hypertrophic scar keratinocytes. This differential profile was confirmed using ELISA, assaying TIMP-1 presence on media from monolayer cultured keratinocytes and from RHS. The dermal matrix of these RHS was recreated using mesenchymal cells from three different origins (skin, wound and hypertrophic scar). The effect of increased TIMP-1 levels on dermal fibrosis was also validated independently from the mesenchymal cell origin. Immunodetection of TIMP-1 showed that this protein was increased in the epidermis of hypertrophic scar biopsies. The findings of this study represent an important advance in understanding the role of keratinocytes as a direct potent modulator for matrix degradation and scar tissue remodeling, possibly through inactivation of MMPs.
Asunto(s)
Cicatriz Hipertrófica/enzimología , Queratinocitos/enzimología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Células Cultivadas , Cicatriz Hipertrófica/patología , Medios de Cultivo Condicionados , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Fibrosis , Humanos , Inmunohistoquímica , Queratinocitos/patología , Cicatrización de Heridas/fisiologíaRESUMEN
Hypertrophic scar (HS) remains a major problem in plastic surgery. In order to explore the regulative effect of phosphatase and tensin homolog (PTEN) on HS, PTEN and AKT expression was detected by reverse transcription PCR, immunohistochemistry and western blot. Adenovirus-mediated PTEN overexpression in cultured hypertrophic scar fibroblasts (HSFBs) and normal skin fibroblasts was also introduced to evaluate its biological function. Our results showed that PTEN expression was significantly decreased in HS whereas p-Akt level was significantly higher in HS compared with normal skin (P < 0.01). Furthermore, we found that adenovirus-mediated PTEN overexpression led to decreased AKT activation, and significantly reduced cell proliferation and collagen synthesis of HSFBs, while increased the apoptosis. Taken together, these data suggest that PTEN inhibits proliferation and function of HSFBs through AKT pathway. Our results reveal a novel biological role for PTEN/AKT pathway in HS and suggest PTEN as a potential therapeutic target for HS treatment.
Asunto(s)
Proliferación Celular , Cicatriz Hipertrófica/patología , Fibroblastos/enzimología , Fosfohidrolasa PTEN/metabolismo , Adolescente , Adulto , Apoptosis , Células Cultivadas , Niño , Cicatriz Hipertrófica/enzimología , Colágeno/biosíntesis , Femenino , Fibroblastos/patología , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Fosfohidrolasa PTEN/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Adulto JovenRESUMEN
Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK-ERK-MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.
Asunto(s)
Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Mecanotransducción Celular , Piel/enzimología , Piel/patología , Animales , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/patología , Fibroblastos/metabolismo , Fibrosis , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Noqueados , Ratones Transgénicos , Transducción de SeñalRESUMEN
Although numerous factors are implicated in skin fibrosis, the exact pathophysiology of hypertrophic scarring remains unknown. We recently demonstrated that mechanical force initiates hypertrophic scar formation in a murine model, potentially enhancing cellular survival through Akt. Here, we specifically examined Akt-mediated mechanotransduction in fibroblasts using both strain culture systems and our murine scar model. In vitro, static strain increased fibroblast motility, an effect blocked by wortmannin (a phosphoinositide-3-kinase/Akt inhibitor). We also demonstrated that high-frequency cyclic strain was more effective at inducing Akt phosphorylation than low frequency or static strain. In vivo, Akt phosphorylation was induced by mechanical loading of dermal fibroblasts in both unwounded and wounded murine skin. Mechanically loaded scars also exhibited strong expression of α-smooth muscle actin, a putative marker of pathologic scar formation. In vivo inhibition of Akt increased apoptosis but did not significantly abrogate hypertrophic scar development. These data suggest that although Akt signaling is activated in fibroblasts during mechanical loading of skin, this is not the critical pathway in hypertrophic scar formation. Future studies are needed to fully elucidate the critical mechanotransduction components and pathways which activate skin fibrosis.
Asunto(s)
Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/etiología , Fibroblastos/fisiología , Mecanotransducción Celular/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Androstadienos/farmacología , Animales , Movimiento Celular , Supervivencia Celular , Cicatriz Hipertrófica/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Estrés Mecánico , WortmaninaRESUMEN
BACKGROUND: Hypertrophic scars and keloids are fibroproliferative skin disorders characterised by progressive deposition of collagen. Our study is designed to investigate the expression and concentration of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in different types of scars and keloids. METHODS: Total RNA from 19 proliferative hypertrophic scar samples of patients with extended burns (total body surface area (TBSA): 21+/-12%), 18 mature hypertrophic scar samples from patients after elective surgery, 14 keloid samples and 18 normotrophic scar samples was, respectively, extracted, and then mRNA was isolated. Besides, biopsies were obtained from non-scarred skin of the patients and extraction of total RNA performed. Relative mRNA expression of MMP 2, MMP 9, TIMP 1 and TIMP 2 was measured with reverse transcriptase polymerase chain reaction (RT-PCR). Serum concentrations of MMP-1, -2, -9, TIMP-1, and -2 were determined using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Patients with extended hypertrophic scars after burn trauma presented a significantly higher TIMP-1 concentration (p<0.05) in their sera than the other patients. The relative expression of MMP 2 was significantly higher in samples of proliferative hypertrophic scars after burn injury. The relative expression of TIMP 1 and TIMP 2 was significantly higher in scar tissue of patients with proliferative and mature hypertrophic scars and keloids than in their regular skin and in scar samples of patients with normotrophic scars. The expression of TIMP 1 was significantly higher in samples of patients with keloids than in patients with hypertrophic scars. CONCLUSIONS: The concentration of TIMP-1 in sera of patients varies depending on the size of the involved fibrotic scar tissue. A decrease in MMP-to-TIMP expression in scar tissue may contribute to increased synthesis and deposition of collagen, leading to a severe fibrotic reaction with pathologic scar formation. The results implicate non-operative therapy options in these patients that not only down-regulate TIMPs but also increase the activity of MMPs.
Asunto(s)
Quemaduras/enzimología , Cicatriz Hipertrófica/enzimología , Queloide/enzimología , Metaloproteinasas de la Matriz Secretadas/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Adolescente , Adulto , Quemaduras/sangre , Quemaduras/patología , Estudios de Casos y Controles , Cicatriz Hipertrófica/sangre , Cicatriz Hipertrófica/etiología , Estudios de Cohortes , Femenino , Humanos , Queloide/sangre , Queloide/etiología , Masculino , Metaloproteinasas de la Matriz Secretadas/genética , Persona de Mediana Edad , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidores Tisulares de Metaloproteinasas/genética , Adulto JovenRESUMEN
Cell-matrix interactions are of significant importance for tissue homeostasis of the skin and, if disturbed, may lead to ageing and hyperplastic scar formation. We have studied fibroblasts stably overexpressing manganese superoxide dismutase (MnSOD) with a defined capacity for the removal of superoxide anions and concomitant accumulation of hydrogen peroxide to evaluate the role of enhanced MnSOD activity on the dynamics of cell-matrix interactions in the three-dimensional collagen lattice contraction assay. MnSOD overexpressing fibroblast populated collagen lattices revealed a significantly enhanced contraction compared to collagen lattices populated with vector control cells. The enhanced collagen lattice contraction was in part due to an increase in active TGF-beta1 and the accumulation of H2O2 in MnSOD overexpressing fibroblasts populated collagen lattices. Inhibition of TGF-beta1 signalling by the ALK4,5,7 kinases' inhibitor SB431542 at least partly inhibited the enhanced collagen lattice contraction of MnSOD overexpressing fibroblasts populated lattices. In addition, supplementation of vector control fibroblast populated collagen lattices with recombinant TGF-beta1 concentration dependently enhanced the collagen lattice contraction. In the presence of the antioxidant Ebselen, a mimic of H2O2 and other hydroperoxides/peroxynitrite-detoxifying glutathione peroxidase, collagen lattice contraction and the activation of TGF-beta1 were significantly reduced in collagen lattices populated with MnSOD overexpressing fibroblasts. Collectively, these data suggest that H2O2 or other hydroperoxides or peroxynitrite or a combination thereof may function as important second messengers in collagen lattice contraction and act at least in part via TGF-beta1 activation.
Asunto(s)
Envejecimiento/metabolismo , Cicatriz Hipertrófica/enzimología , Fibroblastos/metabolismo , Superóxido Dismutasa/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Envejecimiento/genética , Envejecimiento/patología , Azoles/farmacología , Benzamidas/farmacología , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Uniones Célula-Matriz/efectos de los fármacos , Uniones Célula-Matriz/genética , Cicatriz Hipertrófica/genética , Cicatriz Hipertrófica/patología , Colágeno/metabolismo , Dermis/patología , Dioxoles/farmacología , Fibroblastos/patología , Glutatión Peroxidasa/antagonistas & inhibidores , Humanos , Peróxido de Hidrógeno/metabolismo , Isoindoles , Compuestos de Organoselenio/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Superóxido Dismutasa/genética , Transgenes , Regulación hacia ArribaRESUMEN
Proteolytic degradation of extracellular matrix is one of the principal features of cutaneous wound healing but little is known about the activities of gelatinases; matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) on abnormal scar formation. The aim of this study is to determine collagen levels and the gelatinase activities in tissue from hypertrophic scars, atrophic scars, keloids and donor skin in 36 patients and 14 donors. Gelatinase levels (proenzyme + active enzyme) were determined by ELISA and their activities by gelatin zymography. MMP-9 activity was undetectable in gelatin zymography analysis. Pro-MMP-2 levels (median) were highest in normal skin group 53.58 (36.40-75.11) OD microg(-1) protein, while active MMP-2 levels were highest in keloid group 52.53 (42.47-61.51) OD microg(-1) protein. The active/pro ratio was the highest in keloid group 0.97 followed by hypertrophic scar, normal skin and atrophic scar groups 0.69 > 0.54 > 0.48, respectively. According to results of our study, the two-phase theory of the duration of hypertrophic scar and keloid formation can be supported by the data of tissue collagen and gelatinase analysis. This study is the first to relate scar formation relationship in regard to gelatinase activation ratio in a keloid, hypertrophic and atrophic scar patient group which is chosen appropriate in age and sex.
Asunto(s)
Cicatriz Hipertrófica/enzimología , Queloide/enzimología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Adolescente , Adulto , Cicatriz Hipertrófica/patología , Colágeno/metabolismo , Precursores Enzimáticos/metabolismo , Femenino , Gelatinasas/metabolismo , Humanos , Queloide/patología , Masculino , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 9 de la Matriz/química , Persona de Mediana Edad , Proyectos Piloto , Piel/enzimología , Piel/patología , Adulto JovenRESUMEN
OBJECTIVE: To study the change of xanthine oxidase in pathological scars. METHODS: The tissues of hypertrophic scar (10 cases), keloid (10 cases) and normal skin (8 cases) were collected for this project, in which the content of malonaldehyde, the activity of xanthine oxidase and total of antioxidant capacity were detected by spectrophotometric method. The expression of xanthine oxidase was evaluated by immunohistochemistry technique. RESULTS: Compared with normal skin tissue, the content of malonaldehyde and xanthine oxidase activity were significantly higher in pathological scars (P < 0.05), but no significant difference was observed in the decrease of total antioxidant capacity. Differences in above-mentioned indexes all were not remarkable between hypertrophic scars and keloids. Immunohistochemical study indicated that the expressions of xanthine oxidase in the epidermal keratinocytes and dermal fibroblasts of pathological scars increased markedly in contrast with normal skin (P < 0.05). CONCLUSION: The xanthine oxidase activity shows to be increased in pathological scars, and the xanthine oxidase expressions are enlarged in epidermal keratinocytes and dermal fibroblasts of pathological scars, which may be a reason resulting in the increase of free excessive growth of scar tissues. radical level, and may play a role in pathogenesis of excessive growth of scar tissues.
Asunto(s)
Cicatriz Hipertrófica/enzimología , Queloide/enzimología , Xantina Oxidasa/metabolismo , Adolescente , Adulto , Antioxidantes/metabolismo , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patología , Femenino , Humanos , Inmunohistoquímica , Queloide/metabolismo , Queloide/patología , Masculino , Malondialdehído/metabolismo , Adulto JovenRESUMEN
Hypertrophic scarring, which results from excessive collagen deposition at sites of dermal wound repair, can be functionally and cosmetically debilitating to the surgical patient. Pharmacological regulation of collagen synthesis and deposition is a direct approach to the control of scar tissue formation. One of the key steps in collagen stabilization is the cleavage of the C-terminal propeptide from the precursor molecule to form collagen fibrils, a reaction catalyzed by procollagen C-proteinase (PCP). We tested the ability of a PCP inhibitor to reduce hypertrophic scar formation in a rabbit ear model. After the placement of four, 7-mm dermal wounds on each ear, New Zealand white rabbits received PCP inhibitor subcutaneously in the left ear at four time points postwounding: days 7, 9, 11, 13 (early treatment; n=20 wounds) or days 11, 13, 15, 17 (late treatment; n=20 wounds). The right ear of each animal served as a control (vehicle alone). Wounds were harvested on postoperative day 28 and scar hypertrophy quantified by measurement of the scar elevation index. Early treatment of wounds with PCP inhibitor did not reduce scar formation compared with controls (p>0.05). However, late treatment resulted in a statistically significant reduction in the scar elevation index (p<0.01). Our results point not only to the potential use of PCP inhibitors to mitigate hypertrophic scarring but also to the temporal importance of drug delivery for antiscarring therapy.
Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Cicatriz Hipertrófica/prevención & control , Metaloendopeptidasas/antagonistas & inhibidores , Cicatrización de Heridas/fisiología , Análisis de Varianza , Animales , Proteína Morfogenética Ósea 1 , Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/fisiopatología , Modelos Animales de Enfermedad , Oído/lesiones , Femenino , ConejosRESUMEN
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.
Asunto(s)
Cicatriz Hipertrófica/tratamiento farmacológico , Queloide/tratamiento farmacológico , Piel/efectos de los fármacos , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Bleomicina/farmacología , Bleomicina/uso terapéutico , Proliferación Celular , Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/patología , Colágeno/metabolismo , Citocinas/farmacología , Citocinas/uso terapéutico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/metabolismo , Sustancias de Crecimiento/farmacología , Sustancias de Crecimiento/uso terapéutico , Humanos , Interferón gamma/farmacología , Interferón gamma/uso terapéutico , Queloide/enzimología , Queloide/patología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Ensayos Clínicos Controlados Aleatorios como Asunto , Piel/metabolismo , Piel/patología , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/uso terapéutico , Factor de Crecimiento Transformador beta3 , Transglutaminasas/antagonistas & inhibidores , Transglutaminasas/metabolismoRESUMEN
Failure of apoptosis has been postulated to cause the hypercellularity and thus excess scar-tissue formation of hypertrophic scars (HTS). Here, we have examined the susceptibility of fibroblasts derived from normal or HTS to apoptosis induced during collagen-gel contraction, a wound-healing model. Normal scar (NS) fibroblasts underwent significant apoptosis (>40% total) in contractile collagen, whereas apoptosis was not detected in HTS cells. This inability was specific to apoptosis induced by contractile collagen because apoptosis could be induced using diverse modalities. Since chronic fibrotic tissue is known to be excessively cross-linked, we next examined whether collagen matrix that had been conditioned by HTS fibroblasts became refractory to enzymatic breakdown and indeed, found that it is resistant to breakdown by both collagenase D and matrix metalloproteinase-2. Newly formed extracellular matrix is stabilized by the enzyme, tissue transglutaminase, which we demonstrated to be overexpressed by HTS fibroblasts in vivo and in vitro. Reducing tissue transglutaminase activity in collagen gels containing HTS fibroblasts permitted induction of apoptosis on gel contraction, whereas increasing enzymic activity in NS cell-containing gels completely abrogated collagen-contraction-induced-apoptosis. Together, these observations show that HTS fibroblasts exhibit resistance to a specific form of apoptosis elicited by contraction of collagen gels, and that this phenomenon is dependent on excess activity of cell surface tissue transglutaminase.
Asunto(s)
Apoptosis , Cicatriz Hipertrófica/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Transglutaminasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Técnicas de Cultivo de Célula , Niño , Cicatriz Hipertrófica/enzimología , Colagenasas/metabolismo , Femenino , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Persona de Mediana Edad , Regulación hacia Arriba , Cicatrización de HeridasRESUMEN
Hypertrophic scarring is a pathological process characterized by fibroblastic hyperproliferation and by excessive deposition of extracellular matrix components. It has been hypothesized that abnormalities in epidermal-dermal crosstalk explain this pathology. To test this hypothesis, a tissue-engineered model of self-assembled reconstructed skin was used in this study to mimic interactions between dermal and epidermal cells in normal or pathological skin. These skin equivalents were constructed using three dermal cell types: normal wound (Wmyo) or hypertrophic wound (Hmyo) myofibroblasts and normal skin fibroblasts (Fb). Epidermis was reconstructed with normal skin keratinocytes (NK) or hypertrophic scar keratinocytes (HK). In the absence of keratinocytes, Hmyo formed a thicker dermis than Wmyo. When seeded with NK, the dermal thickness of Hmyo (121.2 +/- 31.4 microm vs 196.2 +/- 27.8 microm) and Fb (43.7 +/- 7.1 microm vs 83.6 +/- 16.3 microm) dermis was significantly (p < 0.05) reduced, while that of Wmyo (201.5 +/- 15.7 microm vs 160.7 +/- 21.1 microm) was increased. However, the presence of HK always induced significantly thicker dermis formation than observed with NK (Wmyo: 238.8 +/- 25.9 microm; Hmyo: 145.5 +/- 22.4 microm; Fb: 74.2 +/- 11.2 microm). These results correlated with collagen and MMP-1 secretion and with cell proliferation, which were increased when keratinocytes were added, except for the collagen secretion of Hmyo and Fb in the presence of NK. The level of dermal apoptosis was not different when epidermis was added to the dermis (<1% in each category). These observations strongly suggest that hypertrophic scar keratinocytes play a role in the development of pathological fibrosis by influencing the behaviour of dermal cells.
Asunto(s)
Cicatriz Hipertrófica/patología , Piel/patología , Cicatrización de Heridas , Apoptosis , Estudios de Casos y Controles , Comunicación Celular , Diferenciación Celular , Cicatriz Hipertrófica/enzimología , Dermis/enzimología , Dermis/patología , Epidermis/enzimología , Epidermis/patología , Fibroblastos/patología , Fibrosis , Humanos , Queratinocitos/patología , Metaloproteinasa 1 de la Matriz/metabolismo , Modelos Biológicos , Piel/enzimología , Ingeniería de Tejidos/métodosRESUMEN
OBJECTIVE: To explore the change of gene expression of extracellular-signal regulated protein kinase 5 (ERK5) and its upstream signaling molecule (MEK5) in fetal skin of differentially developmental stages and hypertrophic scars. METHODS: After morphological characteristics of skin of different developmental stages and hypertrophic scars were detected with pathological methods, gene expression of ERK5 and MEK5 was examined with reverse transcription-polymerase chain reaction analysis (RT-PCR). RESULTS: In early gestational fetal skin, genes of ERK5 and MEK5 were strongly expressed, while in late gestational skin and children skin, the expression of ERK5 and MEK5 was apparently decreased (P < 0.05). In normal skin, the level of gene expression of ERK5 was lower. In proliferative hypertrophic scars, mRNA content of this gene was apparently increased. In mature scars, the content of this gene transcript was 3.2 times the normal skin. In contrast, the levels of MEK5 transcript in normal skin and hypertrophic scars of various phases showed no substantial changes (P > 0.05). CONCLUSION: ERKS medicating signaling pathway might be involved in regulating cutaneous development at the embryonic stage and determining cutaneous structure ad function. The increase of gene transcription of ERK5 and MEK5 in younger fetal skin might be a reason for rapid proliferation of the skin cells and scraless healing of skin. The activation of ERK5 gene expression in hypertrophic scars versus normal skin might be one of the mechanisms controlling the formation of hypertrophic scars, in which the role of MEK5 needed to be further studied.