RESUMO
Human umbilical vein endothelial (HUVE) cells have been previously reported to express the genes for the A and B chains of PDGF and to secrete PDGF-related factors into culture media. Antihuman PDGF IgG affinity chromatography was used to purify PDGF-related activity from HUVE cell-conditioned media. Immunoblot analysis of the affinity-purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD and that less than 10% of the purified biologically active molecules are PDGF A or B chain peptides. Screening of an HUVE cell cDNA library in the expression vector lambda gtl 1 with the anti-PDGF antibody resulted in the cloning and sequencing of a cDNA with an open reading frame encoding a 38-kD cysteine-rich secreted protein which we show to be the major PDGF-related mitogen secreted by human vascular endothelial cells. The protein has a 45% overall homology to the translation product of the v-src-induced CEF-10 mRNA from chick embryo fibroblasts. We have termed this new mitogen connective tissue growth factor.
Assuntos
Endotélio Vascular/fisiologia , Genes src , Substâncias de Crescimento/genética , Proteínas Imediatamente Precoces , Peptídeos e Proteínas de Sinalização Intercelular , Fator de Crescimento Derivado de Plaquetas/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Bioensaio , Linhagem Celular , Clonagem Molecular , Fator de Crescimento do Tecido Conjuntivo , Meios de Cultura , Endotélio Vascular/metabolismo , Biblioteca Gênica , Substâncias de Crescimento/isolamento & purificação , Substâncias de Crescimento/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Fator de Crescimento Derivado de Plaquetas/isolamento & purificação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Biossíntese de Proteínas , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Transcrição Gênica , Veias UmbilicaisRESUMO
Chemotaxis assays in modified Boyden chambers were used to detect fibroblast chemoattractants in materials released from early-stage inflammatory cells, namely, mast cells, platelets, and neutrophils. Strong attractant activity was found in substances released from platelets. This activity was accounted for mainly by the platelet-derived growth factor (PDGF), which is released from the platelets and which was active as a chemoattractant at 0.5-1.0 mitogenic units/ml. The mitogenic activity of purified PDGF, measured by [3H]thymidine incorporation, occurs at a similar concentration range. By varying the gradient of PDGF, we demonstrated that PDGF stimulates chemotaxis rather than random motility. Preincubation of suspensions of fibroblasts in the presence of PDGF decreased the subsequent migration of cells to a gradient of PDGF as well as to a gradient of fibronectin, which is also in attractant for fibroblasts. The chemotactic response of fibroblasts to PDGF was not inhibited by hydroxyurea or azidocytidine but was inhibited by actinomycin D and cycloheximide, suggesting that synthesis of RNA and proteins but not of DNA is required for the chemotactic response to occur. Fibroblast growth factor, epidermal growth factor, nerve growth factor, and insulin were not chemotactic for human skin fibroblasts, suggesting that the chemoattractant activity of PDGF for fibroblasts is not a general property of growth factors and mitogens. These results suggest that PDGF could have two functions in wound healing: to attract fibroblasts to migrate into the clot and then to induce their proliferation.
Assuntos
Plaquetas/fisiologia , Quimiotaxia , Fibroblastos/fisiologia , Substâncias de Crescimento/fisiologia , Peptídeos/fisiologia , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Humanos , Hidroxiureia/farmacologia , Masculino , Mastócitos/fisiologia , Neutrófilos/fisiologia , Fator de Crescimento Derivado de PlaquetasRESUMO
Subcutaneous implantation of Hunt-Schilling wound chambers in rats induces a wound repair response causing the chamber first to fill with fluid and subsequently with connective tissue. The presence of a type I collagen gel encouraged a more rapid dispersion of cells throughout the chamber but had no effect on the rate of new collagen deposition. Addition of platelet-derived growth factor (PDGF; 50 ng/chamber) to the collagen-filled chambers caused an earlier influx of connective tissue cells, a marked increase in DNA synthesis, and a greater collagen deposition in the chamber during the first 2 wk after implantation. After 3 wk, however, the levels of collagen were similar in PDGF-supplemented and control chambers. Diabetic animals exhibited a decreased rate of repair which was restored to normal by addition of PDGF to the wound chamber. Combinations of PDGF and insulin caused an even more rapid increase in collagen deposition. These results suggest that the levels of various growth factors, particularly PDGF, may be limiting at wound sites and that supplementation of wounds with these factors can accelerate the rate of new tissue formation.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Cicatrização , Animais , Colágeno/metabolismo , DNA/biossíntese , Fator de Crescimento Epidérmico/farmacologia , Géis , Insulina/farmacologia , Ratos , Cicatrização/efeitos dos fármacosRESUMO
Alveolar macrophages from normal individuals and patients with interstitial lung diseases spontaneously expressed a 4.2-kilobase mRNA complementary to the c-sis gene, a proto-oncogene coding for one of the chains of platelet-derived growth factor (PDGF). Concomitantly, these cells released a mediator with the properties of PDGF, including: chemotactic factor for smooth muscle cells whose activity was resistant to heat and acid, but sensitive to reduction; mitogenic (competence) activity for fibroblasts; ability to compete with PDGF for its receptor; and precipitated by an anti-PDGF antibody. While blood monocytes did not contain c-sis mRNA transcripts, monocytes matured in vitro expressed c-sis, consistent with the concept that expression of c-sis occurs during the differentiation of monocytes into alveolar macrophages. Together with the known actions of PDGF, these observations suggest that the c-sis proto-oncogene and its PDGF product are part of the armamentarium available to the alveolar macrophages for normal lung defense and participation in lung inflammation.
Assuntos
Regulação da Expressão Gênica , Pneumopatias/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proto-Oncogenes , Alvéolos Pulmonares/metabolismo , Quimiotaxia , Cicloeximida/farmacologia , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Pneumopatias/genética , Fator de Crescimento Derivado de Plaquetas/genética , Proto-Oncogene Mas , RNA Mensageiro/metabolismo , Radioimunoensaio , Transcrição Gênica , Tripsina/metabolismoRESUMO
Using cDNA cloning, we have identified a new member of the gro/KC inflammatory protein superfamily (gro-beta) produced by activated human monocytes and neutrophils and expressed at sites of inflammation in vivo. The open reading frame encodes a protein with 91% sequence homology to the human gro protein. Interestingly, some of the amino acid substitutions are located at regions where proteolytic cleavage occurs in the gro protein generating biologically active peptides (neutrophil activating peptide III). These changes would likely alter the processing of the gro-beta peptide and suggest that peptides with biological activities different from those produced by gro-alpha are encoded by this transcript.
Assuntos
Quimiocinas CXC , Peptídeos e Proteínas de Sinalização Intercelular , Leucócitos/fisiologia , Monócitos/fisiologia , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Northern Blotting , Quimiocina CXCL1 , Quimiocina CXCL2 , Clonagem Molecular , Humanos , Macrófagos/fisiologia , Dados de Sequência Molecular , Neutrófilos/fisiologia , CicatrizaçãoRESUMO
Connective tissue growth factor (CTGF) is a cysteine-rich mitogenic peptide that binds heparin and is secreted by fibroblasts after activation with transforming growth factor beta (TGF-beta). CTGF is a member of a highly conserved family of peptides that include immediate early gene products cef10, cyr61, fisp12; a putative avian proto-oncogene, nov; and a drosophila gene, twisted gastrulation, tsg, that controls medial mesoderm induction during dorsal-ventral axis pattern formation, a process also controlled by TGF-beta related peptides (dpp, scw). In the adult mammal, CTGF functions as a downstream mediator of TGF-beta action on connective tissue cells, where it stimulates cell proliferation and extracellular matrix synthesis. CTGF does not appear to act on epithelial cells or immune cells. Because the biological actions of TGF-beta are complex and affect many different cell types, CTGF may serve as a more specific target for selective intervention in processes involving connective tissue formation during wound repair or fibrotic disorders. Northern blot and in situ hybridization studies have demonstrated that CTGF is coordinately expressed with TGF-beta in every fibrotic disorder examined to date. Agents that inhibit CTGF production or action could lead to the development of new therapeutic approaches for the control of fibrotic disorders in humans.
Assuntos
Substâncias de Crescimento/genética , Substâncias de Crescimento/fisiologia , Proteínas Imediatamente Precoces , Peptídeos e Proteínas de Sinalização Intercelular , Mitógenos/genética , Mitógenos/fisiologia , Animais , Fator de Crescimento do Tecido Conjuntivo , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Proteína Sobre-Expressa em Nefroblastoma , Proto-Oncogene Mas , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Connective tissue growth factor (CTGF) is a cysteine-rich peptide that exhibits platelet-derived growth factor (PDGF)-like biological and immunological activities. CTGF is a member of a family of peptides that include serum-induced immediate early gene products, a v-src-induced peptide, and a putative avian transforming gene, nov. In the present study, we demonstrate that human foreskin fibroblasts produce high levels of CTGF mRNA and protein after activation with transforming growth factor beta (TGF-beta) but not other growth factors including PDGF, epidermal growth factor, and basic fibroblast growth factor. Because of the high level selective induction of CTGF by TGF-beta, it appears that CTGF is a major autocrine growth factor produced by TGF-beta-treated human skin fibroblasts. Cycloheximide did not block the large TGF-beta stimulation of CTGF gene expression, indicating that it is directly regulated by TGF-beta. Similar regulatory mechanisms appear to function in vivo during wound repair where there is a coordinate expression of TGF-beta 1 before CTGF in regenerating tissue, suggesting a cascade process for control of tissue regeneration and repair.
Assuntos
Regulação da Expressão Gênica , Substâncias de Crescimento/genética , Proteínas Imediatamente Precoces , Peptídeos e Proteínas de Sinalização Intercelular , Pele/metabolismo , Cicatrização , Células 3T3 , Animais , Sequência de Bases , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo , DNA/química , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , Proteína Sobre-Expressa em Nefroblastoma , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344 , Pele/citologia , Pele/efeitos dos fármacos , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The mortality rate induced by 3 doses of iv doxorubicin was evaluated in F344 rats, and a dose of 8 mg doxorubicin/kg body weight was the maximum dose tolerated with an acceptable mortality rate. Rats treated with 8 mg doxorubicin/kg prior to or on the day of wounding demonstrated decreased wound breaking strength in incisional wounds at all intervals after wounding. Decreased amounts of collagen and DNA and less cellularity were noted in wound chambers from rats treated in the same manner. In both the incisional wound and wound chamber models, rats treated with doxorubicin 7 days after wounding showed a less dramatic healing impairment. No difference in collagen types was noted between chambers from the doxorubicin-treated and untreated rats. Doxorubicin also produced a significant reduction in platelet and white blood cell counts 1 week after it was administered. The data indicate that doxorubicin impedes healing by decreasing wound cellularity and collagen synthesis.
Assuntos
Doxorrubicina/toxicidade , Cicatrização/efeitos dos fármacos , Animais , Colágeno/biossíntese , DNA/análise , Hidroxiprolina/análise , Masculino , Ratos , Ratos Endogâmicos F344 , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
The Twisted Gastrulation (TSG) protein is one of five secreted proteins required to pattern the dorsal part of the early Drosophila embryo. Unlike the Decapentaplegic (DPP) protein that is required to pattern the entire dorsal half of the embryo, TSG is needed only to specify the fate of the dorsal midline cells. Here we have misexpressed the tsg gene with different promoters to address its mechanism of action and relationship to DPP. When expressed in a ventral stripe of cells, TSG protein can diffuse to the dorsalmost cells and can rescue the dorsal midline cells in tsg mutant embryos. Despite elevated levels that exceed that exceed those needed for biological activity, there was no change in dorsal midline or lateral cell fates under any conditions tested. We conclude that TSG does not modulate an activity gradient of DPP. Instead, it functions in a permissive rather than instructive role to elaborate cell fates along the dorsal midline after peak levels of DPP activity have 'primed' cells to respond to TSG. The interaction between TSG and DPP defines a novel type of combinatorial synergism.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Drosophila/metabolismo , Proteínas Imediatamente Precoces , Proteínas de Insetos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Animais , Animais Geneticamente Modificados , Fator de Crescimento do Tecido Conjuntivo , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Substâncias de Crescimento/metabolismo , Heparina/metabolismo , Hibridização In Situ , Proteínas de Insetos/genética , Mutação , Transdução de SinaisRESUMO
Immunoelectron microscopy was utilized to detect type V collagen in human amnion. Monospecific antibodies to type V collagen were detected with protein A-gold conjugates in tissue sections and epoxy-embedded sections of human amnion. Type V collagen was localized to the immediate vicinity of the basal lamina, but was distinct from laminin and type IV collagen, which localized only to the lamina lucida and lamina densa, respectively, of the basal lamina. At high magnification, 12 nm unbanded fibrils were seen to be labelled by anti-type V collagen antibody; these fibrils extended from the lamina densa of the basal lamina well into the interstitial matrix. In comparison, only the amorphous matrix of the lamina densa showed labelling with anti-type IV collagen antibodies. Anti-laminin antibodies labelled the lamina lucida. Quantitative analysis of grain distribution revealed the laminin labelling to be centered over the distal half of the lamina lucida (mean distance from the cell surface = 70 nm). In contrast, type IV collagen was centered over the lamina densa (mean = 115 nm). Both distributions were essentially Gaussian and distinct from the broad distribution of type V collagen. Type I collagen fibers with characteristic 67 nm periodicity were unlabelled with antibodies to type V collagen, although labelled type V fibrils were frequently enmeshed among the type I fibers. Antibodies to type I collagen labelled these fibers but not the type V fibrils. The results indicate that in human amnion, type V collagen is a 12 nm diameter, unbanded fibril which extends from the lamina densa of the basal lamina into the adjacent interstitial matrix. We hypothesize that type V collagen functions as a network of anchoring fibrils between the cell basal lamina and the extracellular matrix, especially type I collagen fibres. Type V collagen thus appears to be a unique interstitial collagen.
Assuntos
Âmnio/ultraestrutura , Colágeno/análise , Citoesqueleto/ultraestrutura , Membrana Basal/ultraestrutura , Feminino , Humanos , Soros Imunes , Técnicas Imunoenzimáticas , Laminina/análise , Microscopia Eletrônica , GravidezRESUMO
We have detected and partially characterized factors that promote the directed migration of mouse epidermal cells in a modified Boyden chamber assay. Smooth muscle cells grown in culture were found to secrete a potent chemoattractant for epidermal cells. This activity was further characterized and compared to the chemotactic activities found in wound fluid and conditioned medium from 3T3 L1 cells and with interleukin 1. The migration of epidermal cells during wound healing in vivo might be regulated by such factors.
Assuntos
Fatores Quimiotáticos/análise , Células Epidérmicas , Músculo Liso/análise , Animais , Movimento Celular , Células Cultivadas , Meios de Cultura/análise , Fibronectinas/farmacologia , Interleucina-1/fisiologia , Masculino , Camundongos , Ratos , Ratos Endogâmicos , CicatrizaçãoRESUMO
Despite a number of studies, the etiology of keloids remains unknown. We have investigated the response of fibroblasts derived from keloid tissue and normal adult skin to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Keloid fibroblasts were more responsive in both chemotactic and mitogenic assays to all three isoforms of PDGF than fibroblasts from normal skin. No enhanced response of the cells to either EGF or FGF was detected. The enhanced PDGF response of keloid fibroblasts appears to be mediated by elevated levels of PDGF alpha receptors, which are 4-5 times higher than those in normal human skin fibroblasts.
Assuntos
Fibroblastos/química , Queloide/patologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Receptores do Fator de Crescimento Derivado de Plaquetas/análise , Adolescente , Adulto , Western Blotting , Quimiotaxia , DNA/biossíntese , Fator de Crescimento Epidérmico/fisiologia , Feminino , Fatores de Crescimento de Fibroblastos/fisiologia , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Connective tissue growth factor (CTGF) is a 36-to 38-kDa peptide that is selectively induced by transforming growth factor-beta (TGF-beta) in fibroblastic cell types. We compared the biologic activities of CTGF with TGF-beta on fibroblasts in culture and in animal models of fibroplasia. CTGF was active as a mitogen in monolayer cultures of normal rat kidney fibroblasts. CTGF did not stimulate anchorage-independent growth of NRK fibroblasts, however, or inhibit the growth of mink lung epithelial cells, distinguishing CTGF's growth-regulatory activities from those of TGF-beta. In NRK fibroblasts, both TGF-beta and CTGF significantly increased the transcripts encoding alpha 1 type I collagen, alpha 5 integrin, and fibronectin. Stimulation of type I collagen and fibronectin protein synthesis by TGF-beta and CTGF was confirmed by pulse labeling of cells with [35S]methionine. Subcutaneous injection of TGF-beta and CTGF into neonatal NIH Swiss mice resulted in a large stimulation of granulation tissue and fibrosis at the site of injection. In situ hybridization studies revealed that TGF-beta injection induced high levels of CTGF mRNA in the dermal fibroblasts at the injection site, demonstrating that TGF-beta can induce the expression of CTGF in connective tissue cells in vivo. No CTGF transcripts were detected in the epidermal cells in either control or TGF-beta-injected skin or in fibroblasts in control (saline-injected) skin. These results demonstrate that, like TGF-beta, CTGF can induce connective tissue cell proliferation and extracellular matrix synthesis.
Assuntos
Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Tecido de Granulação/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Proteínas Imediatamente Precoces , Peptídeos e Proteínas de Sinalização Intercelular , Animais , Divisão Celular/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo , Células Epiteliais , Epitélio/efeitos dos fármacos , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Fibroblastos/citologia , Tecido de Granulação/crescimento & desenvolvimento , Inibidores do Crescimento/farmacologia , Substâncias de Crescimento/genética , Camundongos , Camundongos Endogâmicos , Mitógenos/farmacologia , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes , Pele/efeitos dos fármacos , Pele/patologia , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The role of some growth factors and cytokines in the pathogenesis of systemic sclerosis (SSc) has been suggested. In particular, the contribution of transforming growth factor beta in the progression of skin sclerosis is suspected. Connective tissue growth factor (CTGF) was originally identified in human umbilical vein endothelial cells, and a recent study has revealed that human skin fibroblasts produce CTGF after stimulation with transforming growth factor beta. In the present study, the distribution of CTGF gene expression in tissue sections from patients with SSc was investigated by digoxigenin-labeled in situ hybridization. Strong CTGF mRNA signals were observed in the fibroblasts in sclerotic lesions, especially in the deep dermis, of the skin specimens from patients with SSc, whereas there was no expression in the skin from normal controls. The number of fibroblasts with positive hybridization signals was more abundant in the dermis from the sclerotic stage than in that from the inflammatory stage. Our findings indicate a correlation between CTGF gene expression and skin sclerosis and support the hypothesis that transforming growth factor-beta plays an important role in the pathogenesis of SSc, because transforming growth factor beta is the only inducer for CTGF identified to date.
Assuntos
Expressão Gênica , Substâncias de Crescimento/genética , Proteínas Imediatamente Precoces , Peptídeos e Proteínas de Sinalização Intercelular , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Pele/patologia , Adolescente , Adulto , Atrofia , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo , Dermatite/metabolismo , Dermatite/patologia , Progressão da Doença , Feminino , Fibroblastos/metabolismo , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Valores de Referência , Esclerose , Pele/metabolismoRESUMO
Adult human skin was separated at the dermal-epidermal junction (DEJ) by 4 published methods that involved different mechanisms of action: cold 1 M salt (tissue extraction), cold trypsinization (enzymatic), induction of a suction blister (mechanical), and warm phosphate-buffered saline (protease activation). The localization of DEJ macromolecules was studied after each separation method. By all of the methods tested, bullous pemphigoid antigen remained closely associated with the epidermis while laminin, the basement membrane heparan sulfate proteoglycan, and collagen types IV and V remained with the dermal side of the separation. The bullous pemphigoid antigen is, then, the DEJ component most closely associated with the epidermal basal cell. Of the basement membrane components tested, only the basement membrane heparan sulfate proteoglycan was trypsin-sensitive.
Assuntos
Pele/ultraestrutura , Adulto , Antígenos/análise , Membrana Basal/análise , Colágeno/análise , Imunofluorescência , Heparitina Sulfato/análise , Humanos , Penfigoide Bolhoso/imunologiaRESUMO
Connective tissue growth factor (CTGF) is a novel peptide that exhibits platelet-derived growth factor-like activities and is produced by skin fibroblasts after activation with transforming growth factor-beta. Coordinate expression of transforming growth factor-beta followed by CTGF during wound repair suggests a cascade process for control of tissue regeneration. We recently reported a significant correlation between CTGF mRNA expression and histologic sclerosis in systemic sclerosis. To confirm the relation between CTGF and skin fibrosis, we investigated CTGF gene expression in tissue expression in tissue sections from patients with localized scleroderma, keloid, other sclerotic skin disorders using nonradioactive in situ hybridization. In localized scleroderma, the fibroblasts with positive signals for CTGF mRNA were scattered throughout the sclerotic lesions with no preferential distribution around the inflammatory cells or perivascular regions, whereas the adjacent nonaffected dermis was negative for CTGF mRNA. In keloid tissue, the fibroblasts positive for CTGF mRNA were diffusely distributed, especially in the peripheral expanding lesions. In scar tissue, however, the fibroblasts in the fibrotic lesions showed partially positive signals for CTGF mRNA. In eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture, CTGF mRNA was also expressed partially in the fibroblasts of the fibrotic lesions. Our findings reinforce a correlation between CTGF gene expression and skin sclerosis and support the hypothesis that transforming growth factor-beta plays an important role in the pathogenesis of fibrosis, as it is the only inducer for CTGF identified to date.
Assuntos
Substâncias de Crescimento/genética , Proteínas Imediatamente Precoces , Peptídeos e Proteínas de Sinalização Intercelular , Queloide/metabolismo , RNA Mensageiro/análise , Esclerodermia Localizada/metabolismo , Pele/patologia , Adolescente , Adulto , Criança , Fator de Crescimento do Tecido Conjuntivo , Fibrose , Expressão Gênica , Substâncias de Crescimento/fisiologia , Humanos , Pessoa de Meia-IdadeRESUMO
Desmoplasia, the formation of highly cellular, excessive connective tissue stroma associated with some cancers, shares many features with the wound healing response. Since connective tissue growth factor (CTGF) has previously been demonstrated to play a role in wound repair, we wanted to determine if it might be involved in the pathogenesis of stromal demoplasia in mammary cancer. We assayed 11 human invasive mammary ductal carcinomas by Northern blot and 7 out of 11 were positive for both CTGF expression and transforming growth factor-beta 1 (TGF-beta 1, a principal CTGF inducer). One specimen was positive only for TGF-beta 1. The remaining 3 tumors lacked significant stromal involvement and were negative for either factor. In every case we assayed, in which there was marked connective tissue involvement, both CTGF and TGF-beta 1 messages were found. We also assayed 3 murine mammary tumor models. The GI-101 xenograft model had marked stroma and was positive for both factors in-vivo, but positive for only TGF-beta 1 mRNA expression in culture where fibroblasts were absent. The DMBA murine tumor lacked significant stroma and was negative for CTGF and TGF-beta 1 expression by Northern blot, while the stromal rich DMBA-MMTV tumor contained multifocal desmoplasia and was positive for both factors. We performed in-situ hybridization for CTGF and TGF-beta 1 on the GI-101 and DMBA-MMTV tumors. CTGF message was observed only in the fibroblasts of the stroma, while TGF-beta 1 mRNA hybridization was present in tumor epithelial cells and leukocytes. These results suggest that cancer stroma formation involves induction of similar fibroproliferative growth factors (TGF-beta 1 and CTGF) as wound repair.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Substâncias de Crescimento/genética , Proteínas Imediatamente Precoces , Peptídeos e Proteínas de Sinalização Intercelular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Animais , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Fator de Crescimento do Tecido Conjuntivo , Feminino , Expressão Gênica , Humanos , Hibridização In Situ , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Nus , Fator de Crescimento Transformador beta/genética , Células Tumorais CultivadasRESUMO
Inflammatory bowel disease (IBD) is a multifactorial disorder which is characterized by massive damage of the epithelium and the underlying mesenchyme of the intestine. Due to the potent effect of connective tissue growth factor (CTGF) on fibroblast proliferation and connective tissue deposition we speculated about a possible role of this mitogen in IBD. Here we demonstrate a strikingly increased expression of CTGF mRNA in surgical specimens of patients suffering from two forms of IBD, Crohn's disease and ulcerative colitis. In most specimens, the levels of CTGF mRNA correlated with the degree of inflammation as assessed by histological analysis of adjacent tissue samples and by expression analysis of the pro-inflammatory cytokine interleukin-1 beta. However, areas of little inflammation which were characterized by severe fibrosis also revealed high levels of CTGF mRNA. Expression of transforming growth factor beta-1 (TGF-beta 1), the only known inducer of CTGF so far, as well as of the CTGF target genes collagen I alpha 1, fibronectin and integrin alpha 5 revealed a strong correlation with the expression of CTGF. These data suggest a prominent role of CTGF in the repair of mucosal injury in IBD and in the aberrant deposition of extracellular matrix leading to fibrosis and stenosis, one major complication in IBD, especially in Crohn's disease.
Assuntos
Substâncias de Crescimento/fisiologia , Proteínas Imediatamente Precoces , Doenças Inflamatórias Intestinais/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Mucosa Intestinal/metabolismo , Animais , Fator de Crescimento do Tecido Conjuntivo , Fibrose , Expressão Gênica , Substâncias de Crescimento/genética , Humanos , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Intestinos/patologia , Coelhos , Fator de Crescimento Transformador beta/metabolismoRESUMO
The method described here for the quantitation of cell migration in Boyden chambers is applicable to many cell types, especially connective tissue cells and endothelial cells. Currently, there are many groups working toward the isolation of chemotactic factors for these cells and this method would be useful for the large numbers of repetitive assays done during a purification. In our hands the assay is very reproducible and can be learned within a short time. The major problem with the assay is that the upper surface cells must be completely removed as well as any cellular debris containing dye. However, if the proper care is used in removing these cells, the results vary less than those obtained by counting cells directly.
Assuntos
Movimento Celular , Quimiotaxia , Substâncias de Crescimento/farmacologia , Músculo Liso Vascular/fisiologia , Animais , Aorta/fisiologia , Bovinos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento Derivado de Plaquetas/farmacologia , Espectrofotometria/métodosRESUMO
PURPOSE: Transforming growth factor beta1 (TGF-beta) stimulates the differentiation of myofibroblasts as indicated by the nascent expression of alpha-smooth muscle (alpha-SM) actin protein and its organization into stress fibers. Downstream messengers of TGF-beta in the conversion from the fibroblast to the myofibroblast phenotype were investigated. Whether TGF-beta increases the transcription of a second growth factor, connective tissue growth factor 1 (CTGF), which could mediate myofibroblast differentiation, was evaluated. CTGF, a newly identified growth factor, is highly expressed in dermal granulation tissue. METHODS: In this study, primary cultures of rabbit corneal fibroblasts were exposed to growth factors to investigate CTGF mRNA and protein expression during myofibroblast differentiation. Statistical analysis was used to evaluate the impact of growth factor treatment on myofibroblast differentiation. RESULTS: . TGF-beta treatment induced both CTGF mRNA and protein in rabbit corneal fibroblasts; in contrast, fibroblast growth factor-2 (FGF) and heparin led to a decrease in CTGF mRNA. Addition of recombinant CTGF to rabbit corneal fibroblast cultures did not significantly increase alpha-SM actin mRNA or protein nor did it appear to affect assembly of alpha-SM actin stress fibers. CONCLUSIONS: This is the first study to present evidence for the induction of CTGF by TGF-beta treatment of corneal fibroblasts. It is doubtful that CTGF is the TGF-beta mediator of the corneal fibroblast to myofibroblast transition because CTGF does not induce alpha-SM actin in subconfluent fibroblast cultures. CTGF may play a supporting role in myofibroblast differentiation.