RESUMEN
OBJECTIVES: Systemic sclerosis (SSc) is characterised by extensive tissue fibrosis maintained by mechanotranductive/proadhesive signalling. Drugs targeting this pathway are therefore of likely therapeutic benefit. The mechanosensitive transcriptional co-activator, yes activated protein-1 (YAP1), is activated in SSc fibroblasts. The terpenoid celastrol is a YAP1 inhibitor; however, if celastrol can alleviate SSc fibrosis is unknown. Moreover, the cell niches required for skin fibrosis are unknown. METHODS: Human dermal fibroblasts from healthy individuals and patients with diffuse cutaneous SSc were treated with or without transforming growth factor ß1 (TGFß1), with or without celastrol. Mice were subjected to the bleomycin-induced model of skin SSc, in the presence or absence of celastrol. Fibrosis was assessed using RNA Sequencing, real-time PCR, spatial transcriptomic analyses, Western blot, ELISA and histological analyses. RESULTS: In dermal fibroblasts, celastrol impaired the ability of TGFß1 to induce an SSc-like pattern of gene expression, including that of cellular communication network factor 2, collagen I and TGFß1. Celastrol alleviated the persistent fibrotic phenotype of dermal fibroblasts cultured from lesions of SSc patients. In the bleomycin-induced model of skin SSc, increased expression of genes associated with reticular fibroblast and hippo/YAP clusters was observed; conversely, celastrol inhibited these bleomycin-induced changes and blocked nuclear localisation of YAP. CONCLUSIONS: Our data clarify niches within the skin activated in fibrosis and suggest that compounds, such as celastrol, that antagonise the YAP pathway may be potential treatments for SSc skin fibrosis.
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Esclerodermia Sistémica , Enfermedades de la Piel , Humanos , Animales , Ratones , Tripterygium , Esclerodermia Sistémica/patología , Fibrosis , Enfermedades de la Piel/patología , Piel/patología , Bleomicina/farmacología , Fibroblastos/metabolismo , Factores de Transcripción/metabolismo , Células Cultivadas , Modelos Animales de EnfermedadRESUMEN
Although cancer cells are located within a microenvironment consisting of immune cells, endothelial cells, fibroblasts and extracellular matrix (ECM), the role of the cancer-associated fibroblasts (CAFs) in driving tumorigenesis is relatively underinvestigated. Recent data suggest that a stiff ECM, generated by CAFs, and associated integrin-dependent signaling underlies the development of drug resistance to BRAF inhibitors in melanoma. Drugs targeting the matricellular protein CCN2 (centralized communication network 2, formerly termed connective tissue growth factor), are in clinical development for cancers; for example, FG-3019, an antibody targeting CCN2 has recently entered Phase III trials for pancreatic cancer. Recent data show that fibroblast-specific production of CCN2, which signals through integrins and whose overexpression in human melanomas is independent of BRAF mutational status, is essential for neovascularization, including vasculogenic mimicry, in melanoma. In clinical melanoma samples, a FAP/ITGA11/COL1A1/CCN2-expressing CAF population negatively correlates with disease-free survival. These data emphasize the essential role for a CCN2-expressing subset of CAFs in cancer progression and suggest that targeting the CAFs in the tumor microenvironment, for example by blocking the action of CCN2, may be useful in combination therapies to treat cancers.
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Antineoplásicos/farmacología , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Tumor stroma resembles a fibrotic microenvironment, being characterized by the presence of myofibroblast-like cancer-associated fibroblasts (CAFs). In wild-type mice injected with melanoma cells, we show that the stem cell transcription factor Sox2 is expressed by tumor cells and induced in CAFs derived from synthetic fibroblasts. These fibroblasts were labeled postnatally with green fluorescent protein using mice expressing a tamoxifen-dependent Cre recombinase under the control of a fibroblast-specific promoter/enhancer. Conversely, fibroblast activation was impaired in mice with a fibroblast-specific deletion of cellular communication network 2 (Ccn2), associated with reduced expression of α-smooth muscle actin and Sox2. Multipotent Sox2-expressing skin-derived precursor (SKP) spheroids were cultured from murine back skin. Using lineage tracing and flow cytometry, approximately 40% of SKPs were found to be derived from type I collagen-lineage cells and acquired multipotency in culture. Inhibition of mechanotransduction pathways prevented myofibroblast differentiation of SKPs and expression of Ccn2. In SKPs deleted for Ccn2, differentiation into a myofibroblast, but not an adipocyte or neuronal phenotype, was also impaired. In human melanoma, CCN2 expression was associated with a profibrotic integrin alpha (ITGA) 11-expressing subset of CAFs that negatively associated with survival. These results suggest that synthetic dermal fibroblasts are plastic, and that CCN2 is required for the differentiation of dermal progenitor cells into a myofibroblast/CAF phenotype and is, therefore, a therapeutic target in melanoma.
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Fibroblastos Asociados al Cáncer/patología , Factor de Crecimiento del Tejido Conjuntivo/fisiología , Fibroblastos/patología , Fibrosis/patología , Melanoma Experimental/patología , Piel/patología , Células Madre/patología , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Diferenciación Celular , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Fibrosis/metabolismo , Humanos , Mecanotransducción Celular , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones , Ratones Noqueados , Pronóstico , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Piel/metabolismo , Células Madre/metabolismo , Tasa de Supervivencia , Microambiente TumoralRESUMEN
The effector cells ultimately responsible for fibrosis are myofibroblasts. In the fibrotic autoimmune connective tissue disease scleroderma, myofibroblasts are autonomously activated, and retain their phenotype upon culturing. Since the 1990s, researchers have exploited this fact to use scleroderma fibroblasts as a model system to uncover the fundamental mechanisms underlying myofibroblast persistence in fibrotic conditions. These studies have suggested that an autocrine transforming growth factor (TGF)beta signaling loop is insufficient to explain the persistent myofibroblast phenotype but instead support the hypothesis that fibrotic myofibroblasts possess an intrinsically activated pro-adhesive signaling pathway, and that this contributes to the perpetuation of pathological fibrosis. This review focuses on these observations.
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Mecanotransducción Celular , Miofibroblastos , Diferenciación Celular , Fibroblastos , Fibrosis , Humanos , Miofibroblastos/patología , Fenotipo , Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta1 , Cicatrización de HeridasRESUMEN
BACKGROUND: Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys. METHODS: We treated Pkd1 gene knockout (Pkd1KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated Pkd1KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP). RESULTS: V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation in vitro, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of CCN2 significantly reduced cyst growth and myofibroblasts in Pkd1KO mouse kidneys. We found that YAP regulates CCN2, and YAP inhibition or gene deletion reduces renal fibrosis in Pkd1KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in Pkd1KO kidneys. Further in vitro studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells. CONCLUSIONS: Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.
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Proteínas de Ciclo Celular/fisiología , Factor de Crecimiento del Tejido Conjuntivo/fisiología , Riñón/patología , Miofibroblastos/fisiología , Riñón Poliquístico Autosómico Dominante/patología , Receptores de Vasopresinas/fisiología , Factores de Transcripción/fisiología , Animales , Desamino Arginina Vasopresina/farmacología , Matriz Extracelular/metabolismo , Fibrosis , Humanos , Ratones , Canales Catiónicos TRPP/fisiologíaRESUMEN
Cellular communication network (CCN) proteins are matricellular proteins that coordinate signaling among extracellular matrix, secreted proteins, and cell surface receptors. Their specific in vivo function is context-dependent, but they play profound roles in pathological conditions, such as fibrosis and cancers. Anti-CCN therapies are in clinical consideration. Only recently, however, has the function of these complex molecules begun to emerge. This review summarizes and interprets our current knowledge regarding these fascinating molecules and provides experimental evidence for their utility as therapeutic targets.
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Proteínas CCN de Señalización Intercelular/metabolismo , Comunicación Celular , Microambiente Celular , Matriz Extracelular/metabolismo , Uniones Intercelulares/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Animales , Proteínas CCN de Señalización Intercelular/genética , Matriz Extracelular/genética , Matriz Extracelular/patología , Fibrosis , Regulación Neoplásica de la Expresión Génica , Humanos , Uniones Intercelulares/genética , Uniones Intercelulares/patología , Neoplasias/genética , Neoplasias/patología , Microambiente TumoralRESUMEN
In skeletal muscle, extracellular matrix (ECM) remodeling can either support the complete regeneration of injured muscle or facilitate pathologic fibrosis and muscle degeneration. Muscular dystrophy (MD) is a group of genetic disorders that results in a progressive decline in muscle function and is characterized by the abundant deposition of fibrotic tissue. Unlike acute injury, where ECM remodeling is acute and transient, in MD, remodeling persists until fibrosis obstructs the regenerative efforts of diseased muscles. Thus, understanding how ECM is deposited and organized is critical in the context of muscle repair. Connective tissue growth factor (CTGF or CCN2) is a matricellular protein expressed by multiple cell types in response to tissue injury. Although used as a general marker of fibrosis, the cell type-dependent role of CTGF in dystrophic muscle has not been elucidated. To address this question, a conditional Ctgf myofiber and fibroblast-knockout mouse lines were generated and crossed to a dystrophic background. Only myofiber-selective inhibition of CTGF protected δ-sarcoglycan-null ( Sgcd-/-) mice from the dystrophic phenotype, and it did so by affecting collagen organization in a way that allowed for improvements in dystrophic muscle regeneration and function. To confirm that muscle-specific CTGF functions to mediate collagen organization, we generated mice with transgenic muscle-specific overexpression of CTGF. Again, genetic modulation of CTGF in muscle was not sufficient to drive fibrosis, but altered collagen content and organization after injury. Our results show that the myofibers are critical mediators of the deleterious effects associated with CTGF in MD and acutely injured skeletal muscle.-Petrosino, J. M., Leask, A., Accornero, F. Genetic manipulation of CCN2/CTGF unveils cell-specific ECM-remodeling effects in injured skeletal muscle.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo , Matriz Extracelular , Regulación de la Expresión Génica , Fibras Musculares Esqueléticas , Distrofia Muscular Animal , Animales , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Factor de Crecimiento del Tejido Conjuntivo/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fibrosis , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Sarcoglicanos/deficienciaRESUMEN
Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway that coactivate large tumor suppressor homolog (LATS) kinases. Mob1a/1b double deficiency in mouse liver (LMob1DKO) results in hyperplasia of oval cells and immature cholangiocytes accompanied by inflammatory cell infiltration and fibrosis. More than half of mutant mice die within 3 wk of birth. All survivors eventually develop liver cancers, particularly combined hepatocellular and cholangiocarcinomas (cHC-CCs) and intrahepatic cholangiocellular carcinomas (ICCs), and die by age 60 wk. Because this phenotype is the most severe among mutant mice lacking a Hippo signaling component, MOB1A/1B constitute the critical hub of Hippo signaling in mammalian liver. LMob1DKO liver cells show hyperproliferation, increased cell saturation density, hepatocyte dedifferentiation, enhanced epithelial-mesenchymal transition and cell migration, and elevated transforming growth factor beta(TGF-ß)2/3 production. These changes are strongly dependent on Yes-Associated Protein-1 (Yap1) and partially dependent on PDZ-binding motif (Taz) and Tgfbr2, but independent of connective tissue growth factor (Ctgf). In human liver cancers, YAP1 activation is frequent in cHC-CCs and ICCs and correlates with SMAD family member 2 activation. Drug screening revealed that antiparasitic macrocyclic lactones inhibit YAP1 activation in vitro and in vivo. Targeting YAP1/TAZ with these drugs in combination with inhibition of the TGF-ß pathway may be effective treatment for cHC-CCs and ICCs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de los Conductos Biliares/patología , Carcinogénesis/metabolismo , Colangiocarcinoma/patología , Neoplasias Hepáticas/patología , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Aciltransferasas , Animales , Neoplasias de los Conductos Biliares/metabolismo , Línea Celular Tumoral , Colangiocarcinoma/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Transición Epitelial-Mesenquimal , Genes Supresores de Tumor , Humanos , Hiperplasia/genética , Hiperplasia/patología , Péptidos y Proteínas de Señalización Intracelular , Hígado/patología , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Noqueados , Ratones Desnudos , Fosfoproteínas/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAPRESUMEN
OBJECTIVES: One mechanism by which cartilage responds to mechanical load is by releasing heparin-bound growth factors from the pericellular matrix (PCM). By proteomic analysis of the PCM, we identified connective tissue growth factor (CTGF) and here investigate its function and mechanism of action. METHODS: Recombinant CTGF (rCTGF) was used to stimulate human chondrocytes for microarray analysis. Endogenous CTGF was investigated by in vitro binding assays and confocal microscopy. Its release from cut cartilage (injury CM) was analysed by Western blot under reducing and non-reducing conditions. A postnatal, conditional CtgfcKO mouse was generated for cartilage injury experiments and to explore the course of osteoarthritis (OA) by destabilisation of the medial meniscus. siRNA knockdown was performed on isolated human chondrocytes. RESULTS: The biological responses of rCTGF were TGFß dependent. CTGF displaced latent TGFß from cartilage and both were released on cartilage injury. CTGF and latent TGFß migrated as a single high molecular weight band under non-reducing conditions, suggesting that they were in a covalent (disulfide) complex. This was confirmed by immunoprecipitation. Using CtgfcKO mice, CTGF was required for sequestration of latent TGFß in the matrix and activation of the latent complex at the cell surface through TGFßR3. In vivo deletion of CTGF increased the thickness of the articular cartilage and protected mice from OA. CONCLUSIONS: CTGF is a latent TGFß binding protein that controls the matrix sequestration and activation of TGFß in cartilage. Deletion of CTGF in vivo caused a paradoxical increase in Smad2 phosphorylation resulting in thicker cartilage that was protected from OA.
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Artritis Experimental/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/fisiología , Osteoartritis/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Artritis Experimental/patología , Artritis Experimental/prevención & control , Cartílago Articular/lesiones , Cartílago Articular/metabolismo , Cartílago Articular/patología , Células Cultivadas , Condrocitos/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/deficiencia , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/farmacología , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Ratones Noqueados , Osteoartritis/patología , Osteoartritis/prevención & control , Proteoglicanos/metabolismo , Proteómica , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Recombinantes/farmacología , Proteína Smad2/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Lymphangiogenesis is correlated with the degree of renal interstitial fibrosis. Pro-fibrotic transforming growth factor ß induces VEGF-C production, the main driver of lymphangiogenesis. Connective tissue growth factor (CTGF) is an important determinant of fibrotic tissue remodeling, but its possible involvement in lymphangiogenesis has not been explored. We found prominent lymphangiogenesis during tubulointerstitial fibrosis to be associated with increased expression of CTGF and VEGF-C in human obstructed nephropathy as well as in diabetic kidney disease. Using CTGF knockout mice, we investigated the involvement of CTGF in development of fibrosis and associated lymphangiogenesis in obstructive nephropathy. The increase of lymphatic vessels and VEGF-C in obstructed kidneys was significantly reduced in CTGF knockout compared to wild-type mice. Also in mouse kidneys subjected to ischemia-reperfusion injury, CTGF knockdown was associated with reduced lymphangiogenesis. In vitro, CTGF induced VEGF-C production in HK-2 cells, while CTGF siRNA suppressed transforming growth factor ß1-induced VEGF-C upregulation. Furthermore, surface plasmon resonance analysis showed that CTGF and VEGF-C directly interact. Interestingly, VEGF-C-induced capillary-like tube formation by human lymphatic endothelial cells was suppressed by full-length CTGF but not by naturally occurring proteolytic CTGF fragments. Thus, CTGF is significantly involved in fibrosis-associated renal lymphangiogenesis through regulation of, and direct interaction with, VEGF-C.
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Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Enfermedades Renales/patología , Túbulos Renales/patología , Linfangiogénesis , Factor C de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular , Factor de Crecimiento del Tejido Conjuntivo/genética , Modelos Animales de Enfermedad , Fibrosis , Humanos , Enfermedades Renales/etiología , Enfermedades Renales/cirugía , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Unión Proteica , ARN Interferente Pequeño/metabolismo , Daño por Reperfusión/complicaciones , Transducción de Señal , Resonancia por Plasmón de Superficie , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia ArribaRESUMEN
Fibrotic diseases are a significant global burden for which there are limited treatment options. The effector cells of fibrosis are activated fibroblasts called myofibroblasts, a highly contractile cell type characterized by the appearance of α-smooth muscle actin stress fibers. The underlying mechanism behind myofibroblast differentiation and persistence has been under much investigation and is known to involve a complex signaling network involving transforming growth factor-ß, endothelin-1, angiotensin II, CCN2 (connective tissue growth factor), and platelet-derived growth factor. This review addresses the contribution of these signaling molecules to cardiac fibrosis.
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Miocardio/patología , Angiotensina II/fisiología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Animales , Antiinflamatorios/uso terapéutico , Arritmias Cardíacas/etiología , Atrofia , Cicatriz/patología , Factor de Crecimiento del Tejido Conjuntivo/fisiología , Antagonistas de los Receptores de Endotelina/uso terapéutico , Endotelina-1/fisiología , Fibrosis , Humanos , Hipoxia/etiología , Modelos Cardiovasculares , Terapia Molecular Dirigida , Miofibroblastos/fisiología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Piridonas/uso terapéutico , Ratas , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Wnts are required for cardiogenesis but the role of specific Wnts in cardiac repair remains unknown. In this report, we show that a dynamic Wnt1/ßcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. Acute ischaemic cardiac injury upregulates Wnt1 that is initially expressed in the epicardium and subsequently by cardiac fibroblasts in the region of injury. Following cardiac injury, the epicardium is activated organ-wide in a Wnt-dependent manner, expands, undergoes epithelial-mesenchymal transition (EMT) to generate cardiac fibroblasts, which localize in the subepicardial space. The injured regions in the heart are Wnt responsive as well and Wnt1 induces cardiac fibroblasts to proliferate and express pro-fibrotic genes. Disruption of downstream Wnt signalling in epicardial cells decreases epicardial expansion, EMT and leads to impaired cardiac function and ventricular dilatation after cardiac injury. Furthermore, disruption of Wnt/ßcatenin signalling in cardiac fibroblasts impairs wound healing and decreases cardiac performance as well. These findings reveal that a pro-fibrotic Wnt1/ßcatenin injury response is critically required for preserving cardiac function after acute ischaemic cardiac injury.
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Fibroblastos/metabolismo , Corazón/fisiología , Infarto del Miocardio/patología , Pericardio/metabolismo , Regeneración/fisiología , Transducción de Señal/fisiología , Proteína Wnt1/fisiología , beta Catenina/fisiología , Animales , División Celular , Transición Epitelial-Mesenquimal , Fibrosis , Regulación de la Expresión Génica , Hibridación in Situ , Ratones , Ratones Transgénicos , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Pericardio/patología , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/fisiología , Regulación hacia Arriba , Proteína Wnt1/biosíntesis , Proteína Wnt1/genética , Cicatrización de Heridas/fisiologíaRESUMEN
UNLABELLED: Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvß6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvß6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter-driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvß6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen-inducible Cre-loxP system down-regulated integrin αvß6 in DDC-damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvß6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis-related genes, smaller areas of alpha-smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvß6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-ß1 activation in vitro. CONCLUSIONS: CTGF and integrin αvß6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-ß1. CTGF and integrin αvß6 are potential therapeutic targets to control DRs and fibrosis in related liver disease.
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Antígenos de Neoplasias/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Integrinas/metabolismo , Cirrosis Hepática/metabolismo , Células Madre Adultas/metabolismo , Animales , Neoplasias de los Conductos Biliares/metabolismo , Conductos Biliares Intrahepáticos , Adhesión Celular , Colangiocarcinoma/metabolismo , Femenino , Fibronectinas/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Piridinas , Conejos , Ratas , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: One of the main contributors to maladaptive cardiac remodeling is fibrosis. Connective tissue growth factor (CTGF), a matricellular protein that is secreted into the cardiac extracellular matrix by both cardiomyocytes and fibroblasts, is often associated with development of fibrosis. However, recent studies have questioned the role of CTGF as a pro-fibrotic factor. Therefore, we aimed to investigate the effect of CTGF on cardiac fibrosis, and on functional, structural, and electrophysiological parameters in a mouse model of CTGF knockout (KO) and chronic pressure overload. METHODS AND RESULTS: A new mouse model of global conditional CTGF KO induced by tamoxifen-driven deletion of CTGF, was subjected to 16weeks of chronic pressure overload via transverse aortic constriction (TAC, control was sham surgery). CTGF KO TAC mice presented with hypertrophic hearts, and echocardiography revealed a decrease in contractility on a similar level as control TAC mice. Ex vivo epicardial mapping showed a low incidence of pacing-induced ventricular arrhythmias (2/12 in control TAC vs. 0/10 in CTGF KO TAC, n.s.) and a tendency towards recovery of the longitudinal conduction velocity of CTGF KO TAC hearts. Picrosirius Red staining on these hearts unveiled increased fibrosis at a similar level as control TAC hearts. Furthermore, genes related to fibrogenesis were also similarly upregulated in both TAC groups. Histological analysis revealed an increase in fibronectin and vimentin protein expression, a significant reduction in connexin43 (Cx43) protein expression, and no difference in NaV1.5 expression of CTGF KO ventricles as compared with sham treated animals. CONCLUSION: Conditional CTGF inhibition failed to prevent TAC-induced cardiac fibrosis and hypertrophy. Additionally, no large differences were found in other parameters between CTGF KO and control TAC mice. With no profound effect of CTGF on fibrosis formation, other factors or pathways are likely responsible for fibrosis development.
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Síndrome de Brugada/genética , Cardiomegalia/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Miocardio/metabolismo , Remodelación Ventricular , Animales , Aorta/cirugía , Compuestos Azo , Síndrome de Brugada/etiología , Síndrome de Brugada/metabolismo , Síndrome de Brugada/patología , Trastorno del Sistema de Conducción Cardíaco , Cardiomegalia/etiología , Cardiomegalia/metabolismo , Cardiomegalia/patología , Factor de Crecimiento del Tejido Conjuntivo/deficiencia , Conexina 43/genética , Conexina 43/metabolismo , Constricción Patológica/complicaciones , Constricción Patológica/cirugía , Ecocardiografía , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrosis , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patología , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Técnicas de Cultivo de Órganos , Presión , Transducción de Señal , Vimentina/genética , Vimentina/metabolismoRESUMEN
There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET-based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti-fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells.
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Colágeno/biosíntesis , Fibroblastos/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Microscopía Confocal , ARN de Transferencia de Glicerina/metabolismo , ARN de Transferencia de Prolina/metabolismo , Animales , Carbocianinas/metabolismo , Células Cultivadas , Fibroblastos/patología , Fibrosis , Colorantes Fluorescentes/metabolismo , Humanos , Cinética , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , ARN de Transferencia de Glicerina/genética , ARN de Transferencia de Prolina/genética , TransfecciónRESUMEN
The matricellular protein periostin is expressed in the skin. Although periostin has been hypothesized to contribute to dermal homeostasis and repair, this has not been directly tested. To assess the contribution of periostin to dermal healing, 6 mm full-thickness excisional wounds were created in the skin of periostin-knockout and wild-type, sex-matched control mice. In wild-type mice, periostin was potently induced 5-7 days after wounding. In the absence of periostin, day 7 wounds showed a significant reduction in myofibroblasts, as visualized by expression of α-smooth muscle actin (α-SMA) within the granulation tissue. Delivery of recombinant human periostin by electrospun collagen scaffolds restored α-SMA expression. Isolated wild-type and knockout dermal fibroblasts did not differ in in vitro assays of adhesion or migration; however, in 3D culture, periostin-knockout fibroblasts showed a significantly reduced ability to contract a collagen matrix, and adopted a dendritic phenotype. Recombinant periostin restored the defects in cell morphology and matrix contraction displayed by periostin-deficient fibroblasts in a manner that was sensitive to a neutralizing anti-ß1-integrin and to the FAK and Src inhibitor PP2. We propose that periostin promotes wound contraction by facilitating myofibroblast differentiation and contraction.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Miofibroblastos/citología , Piel/metabolismo , Cicatrización de Heridas , Actinas/metabolismo , Animales , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Forma de la Célula , Colágeno/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Tejido de Granulación/metabolismo , Integrina beta1/metabolismo , Cinética , Ratones , Ratones Noqueados , Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Transducción de Señal , Piel/citología , Piel/patología , Factor de Crecimiento Transformador beta/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
The CCN family of matricellular proteins, which includes CCN2 and CCN1, is believed to have a major in vivo role in controlling tissue morphogenesis and repair. In adult skin, the proadhesive matricellular protein connective tissue growth factor (CTGF/CCN2) is specifically up-regulated in fibrosis and wound healing. In mice, CCN2 is required for dermal fibrogenesis, but whether CCN2 is required for cutaneous tissue repair is unknown. To address this question, in this report we subjected adult mice bearing a fibroblast-specific deletion of CCN2 to the dermal punch model of cutaneous tissue repair. Loss of CCN2 did not appreciably affect the kinetics of tissue repair, collagen content, or the number of α-smooth muscle actin-positive cells. CCN1 (cyr61), which has in vitro effect similar to CCN2, is also induced in cutaneous tissue repair. Fibroblast-specific CCN1/CCN2 double knockout mice were also generated; loss of both CCN1 and CCN2 together did not appreciably affect cutaneous tissue repair. However, loss of CCN2 resulted in impaired recruitment of NG2-positive pericyte-like cells to the wound area. Collectively, these results indicate that neither CCN2 nor CCN1 is essential for cutaneous tissue repair; CCN2 appears to be required for recruitment of pericyte-like cells and may represent a specific antifibrotic target.
Asunto(s)
Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Piel/metabolismo , Piel/patología , Cicatrización de Heridas , Animales , Western Blotting , Adhesión Celular , Células Cultivadas , Regulación de la Expresión Génica , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Noqueados , Piel/lesiones , Regulación hacia ArribaRESUMEN
OBJECTIVE: Protein phosphatase and tensin homolog (PTEN) expression is reduced in dermal fibroblasts isolated from patients with diffuse cutaneous systemic sclerosis, a fibrotic autoimmune disease. In support of this finding, deletion of the PTEN gene in the dermal fibroblasts of mice has been shown to result in skin fibrosis and in vivo overexpression of connective tissue growth factor (CTGF; CCN2), a proadhesive matricellular protein; however, whether CCN2 is required for the fibrosis caused by loss of PTEN is unclear. This study was undertaken to investigate the role of CCN2 in fibrosis caused by reduced PTEN expression. METHODS: We generated conditional knockout mice in which PTEN was deleted in fibroblasts, either alone or in combination with CCN2. Skin samples were collected for histologic examination, immunohistochemical analysis, and collagen assay. RESULTS: Loss of CCN2 resulted in resistance to the increases in collagen production and myofibroblast recruitment that are caused by loss of PTEN. CCN2 deficiency did not impair Akt phosphorylation or the increases in the intensity of proliferating cell nuclear antigen staining that were caused by loss of PTEN. CONCLUSION: These data are consistent with the notion that CCN2 is required for particular aspects of the fibroproliferative response; therapeutic strategies blocking CCN2 may be of clinical benefit in combating fibrotic disease.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/genética , Dermis/patología , Fibroblastos/patología , Fosfohidrolasa PTEN/genética , Esclerodermia Difusa/genética , Esclerodermia Difusa/patología , Animales , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Dermis/metabolismo , Fibroblastos/metabolismo , Fibrosis/genética , Fibrosis/metabolismo , Fibrosis/patología , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esclerodermia Difusa/metabolismoRESUMEN
OBJECTIVE: Currently, our ability to treat intervertebral disc (IVD) degeneration is hampered by an incomplete understanding of disc development and aging. The specific function of matricellular proteins, including CCN2, during these processes remains an enigma. The aim of this study was to determine the tissue-specific localization of CCN proteins and to characterize their role in IVD tissues during embryonic development and age-related degeneration by using a mouse model of notochord-specific CCN2 deletion. METHODS: Expression of CCN proteins was assessed in IVD tissues from wild-type mice beginning on embryonic day 15.5 to 17 months of age. Given the enrichment of CCN2 in notochord-derived tissues, we generated notochord-specific CCN2-null mice to assess the impact on the IVD structure and extracellular matrix composition. Using a combination of histologic evaluation and magnetic resonance imaging (MRI), IVD health was assessed. RESULTS: Loss of the CCN2 gene in notochord-derived cells disrupted the formation of IVDs in embryonic and newborn mice, resulting in decreased levels of aggrecan and type II collagen and concomitantly increased levels of type I collagen within the nucleus pulposus. CCN2-knockout mice also had altered expression of CCN1 (Cyr61) and CCN3 (Nov). Mirroring its role during early development, notochord-specific CCN2 deletion accelerated age-associated degeneration of IVDs. CONCLUSION: Using a notochord-specific gene targeting strategy, this study demonstrates that CCN2 expression by nucleus pulposus cells is essential to the regulation of IVD development and age-associated tissue maintenance. The ability of CCN2 to regulate the composition of the intervertebral disc suggests that it may represent an intriguing clinical target for the treatment of disc degeneration.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/deficiencia , Degeneración del Disco Intervertebral/fisiopatología , Disco Intervertebral/embriología , Disco Intervertebral/fisiopatología , Notocorda/embriología , Notocorda/fisiopatología , Agrecanos/fisiología , Envejecimiento/fisiología , Animales , Colágeno Tipo I/fisiología , Colágeno Tipo II/fisiología , Factor de Crecimiento del Tejido Conjuntivo/genética , Modelos Animales de Enfermedad , Desarrollo Embrionario/fisiología , Femenino , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/patología , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Notocorda/patologíaRESUMEN
The largest mammalian organ, skin, consisting of a dermal connective tissue layer that underlies and supports the epidermis, acts as a protective barrier that excludes external pathogens and disseminates sensory signals emanating from the local microenvironment. Dermal connective tissue is comprised of a collagen-rich extracellular matrix (ECM) that is produced by connective tissue fibroblasts resident within the dermis. When wounded, a tissue repair program is induced whereby fibroblasts, in response to alterations in the microenvironment, produce new ECM components, resulting in the formation of a scar. Failure to terminate the normal tissue repair program causes fibrotic conditions including: hypertrophic scars, keloids, and the systemic autoimmune connective tissue disease scleroderma (systemic sclerosis, SSc). Histological and single-cell RNA sequencing (scRNAseq) studies have revealed that fibroblasts are heterogeneous and highly plastic. Understanding how this diversity contributes to dermal homeostasis, wounding, fibrosis, and cancer may ultimately result in novel anti-fibrotic therapies and personalized medicine. This review summarizes studies supporting this concept.