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OBJECTIVE: To delineate the constitutive pulmonary vascular phenotype of the TßRIIΔk-fib mouse model of scleroderma, and to selectively induce pulmonary endothelial cell injury using vascular endothelial growth factor (VEGF) inhibition to develop a model with features characteristic of pulmonary arterial hypertension (PAH). METHODS: The TßRIIΔk-fib mouse strain expresses a kinase-deficient transforming growth factor ß (TGFß) receptor type II driven by a fibroblast-specific promoter, leading to ligand-dependent up-regulation of TGFß signaling, and replicates key fibrotic features of scleroderma. Structural, biochemical, and functional assessments of pulmonary vessels, including in vivo hemodynamic studies, were performed before and following VEGF inhibition, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGFß and VEGF signaling axes in tissue sections and explanted smooth muscle cells. RESULTS: In the TßRIIΔk-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly elevated pulmonary artery pressure resembled the well-described chronic hypoxia model of pulmonary hypertension. Following administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells. These changes resulted in right ventricular hypertrophy, confirming hemodynamically significant PAH. Altered expression of TGFß and VEGF ligand and receptor was consistent with a scleroderma phenotype. CONCLUSION: In this study, we replicated key features of systemic sclerosis-related PAH in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support the underlying role of functional interplay between TGFß and VEGF, which provides insight into the pathogenesis of this disease.
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Endotelio Vascular/fisiopatología , Hipertensión Pulmonar/fisiopatología , Circulación Pulmonar/fisiología , Esclerodermia Sistémica/fisiopatología , Factor de Crecimiento Transformador beta/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Modelos Animales de Enfermedad , Hipertensión Pulmonar Primaria Familiar , Femenino , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/genética , Hipoxia/genética , Hipoxia/fisiopatología , Indoles/farmacología , Operón Lac , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pirroles/farmacología , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/genética , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVES: To assess whether the discrepancy between the strong antifibrotic effects of tyrosine kinase inhibitors (TKIs) in animal models and the inconsistent results in clinical studies might be related to the activation levels of drug targets. METHODS: Skin sections of bleomycin, TSK1, Fra-2 transgenic mice, SSc patients and controls were analysed by histology and immunohistochemistry. Subgroups of mice were treated with the TKIs nilotinib or imatinib. Differences in the activation levels of the TKI targets p-PDGFRß (platelet derived growth factor ß) and p-c-abl were assessed. RESULTS: In bleomycin and TSK1 mice, expression of activated p-PDGFRß (platelet derived growth factor receptor ß) and p-c-abl was ubiquitous with strong upregulation compared with controls. Treatment with TKIs resulted in successful target inhibition and consequently reduced dermal fibrosis. In the Fra-2 model, the activation levels of p-PDGFRß and p-c-abl were much lower than in the bleomycin and the TSK1 models. Accordingly, nilotinib did not prevent dermal fibrosis and target inhibition was unsuccessful. Notably, in skin biopsies of SSc patients, the mean activation levels of TKI targets were only moderate and in the majority of patients resembled those of the non-responsive Fra-2 model. CONCLUSIONS: Animal models for proof-of-concept studies should be selected based on a similar activation level and expression pattern of drug targets as in human SSc.
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Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Esclerodermia Sistémica/tratamiento farmacológico , Piel/patología , Adulto , Animales , Benzamidas/uso terapéutico , Biopsia , Bleomicina , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Femenino , Fibrosis , Antígeno 2 Relacionado con Fos/genética , Humanos , Mesilato de Imatinib , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Piperazinas/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-sis/metabolismo , Pirimidinas/uso terapéutico , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Piel/metabolismo , Resultado del TratamientoRESUMEN
BACKGROUND: Enhanced cell expression of MAdCAM-1 is critical in tissue recruitment of lymphocytes in response to stimuli expressing the α4ß7 integrin. MAdCAM-1 is well characterized in gut mucosa with emerging evidence of hepatic expression. AIMS: (i) Compare quantitative/semi-quantitatively MAdCAM-1 expression in relation to early and advanced liver diseases (ii) Define the fine structure of vascular plexuses/lymphatics in the portal tract on which MAdCAM-1 is expressed. METHODS: Using alkaline phosphatase anti-alkaline phosphatase methodology on paraffin embedded tissue sections (n=28) from cirrhotic individuals who underwent orthotopic liver transplant, we evaluated MAdCAM-1 expression and compared with pre-cirrhotic, fulminant hepatitis B, and non-cirrhotic portal hypertension tissue sections. The positive controls included normal colon tissue with negative controls without primary antibody and isotype-matched purified IgG. We developed a real time PCR to quantify levels of MAdCAM-1 mRNA in our samples. RESULTS: MAdCAM-1 was expressed in 27/28 of the cirrhotic sections, localized primarily to septal areas within (i) endothelium of the peribiliary vascular plexus (PBP) (ii) lymphoid aggregates, with absence from normal, non-cirrhotic portal hypertension and pre-cirrhotic livers. There was significant upregulation of MAdCAM-1 mRNA in cirrhosis (p<0.011), consistent with immunohistochemical analysis. CONCLUSIONS: MAdCAM-1 is up-regulated in cirrhosis with expression on PBP and lymphoid aggregates. MAdCAM-1 is likely to contribute to the localization and recruitment of α4ß7 lymphocytes during the pathogenesis of cirrhosis. MAdCAM-1 could be a useful marker of advanced liver disease. Further studies with respect to the expression of MAdCAM-1 in the presence of reversible and non-reversible stages of liver disease may be of merit.
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Inmunoglobulinas/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Mucoproteínas/metabolismo , Adulto , Moléculas de Adhesión Celular , Colon/metabolismo , Endotelio Linfático/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hígado/irrigación sanguínea , Tejido Linfoide/metabolismo , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: To determine the potential clinical and pathological significance of altered expression of interleukin 6 (IL-6) in systemic sclerosis (SSc). METHODS: Serum IL-6 and soluble IL-6 receptor levels were measured in patients with SSc (n=68) and healthy controls (n=15). Associations between serum IL-6 level and C reactive protein, platelet count and key clinical outcomes in SSc were explored. Expression of IL-6 in skin biopsies was also examined and western blot and reverse transcription PCRanalysis were performed using cultured dermal fibroblasts. The effect of IL-6 trans-signalling on production of extracellular matrix proteins was assessed and downstream signalling pathways were examined using pharmacological inhibitors. RESULTS: Serum IL-6 level was frequently elevated in patients with SSc, particularly in those with diffuse cutaneous SSc (dcSSc) with thrombocytosis and elevated acute phase markers. Prominent expression in the skin was observed in dermal fibroblasts, mononuclear cells and endothelial cells in patients with early dcSSc. In vitro experiments supported a potent profibrotic effect of IL-6 trans-signalling via the JAK2/STAT3 and ERK pathways. High IL-6 expression early in dcSSc appears to be associated with more severe skin involvement at 3 years and worse long-term survival than in those without elevated IL-6 levels. CONCLUSION: Our results confirm the overexpression of IL-6 in dcSSc and support the potential of IL-6 as a surrogate marker for clinical outcome in this disease. The data also provide rationale for clinical studies targeting IL-6 trans-signalling as a potential antifibrotic therapy for SSc.
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Interleucina-6/sangre , Esclerodermia Difusa/sangre , Esclerodermia Limitada/sangre , Piel/metabolismo , Biomarcadores/sangre , Proteína C-Reactiva/análisis , Células Cultivadas , Dermis/efectos de los fármacos , Dermis/metabolismo , Dermis/patología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Humanos , Interleucina-6/genética , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Recuento de Plaquetas , Receptores de Interleucina-6/sangre , Receptores de Interleucina-6/genética , Esclerodermia Difusa/mortalidad , Esclerodermia Difusa/patología , Esclerodermia Limitada/mortalidad , Esclerodermia Limitada/patología , Transducción de Señal , Piel/patología , Tasa de Supervivencia , Reino Unido/epidemiologíaRESUMEN
OBJECTIVE: Fibrotic diseases such as SSc (systemic sclerosis, scleroderma) are characterized by the abnormal presence of the myofibroblast, a specialized type of fibroblast that overexpresses the highly contractile protein α-smooth muscle actin. Myofibroblasts display excessive adhesive properties and hence exert a potent mechanical force. We aim to identify the precise contribution of adhesive signalling, which requires integrin-mediated activation of focal adhesion kinase (FAK)/src, to fibrogenic gene expression in normal and fibrotic SSc fibroblasts. METHODS: We subject either FAK wild-type and knockout fibroblasts or normal and SSc fibroblasts treated with FAK/src inhibitors to real-time polymerase chain, western blot, cell migration and collagen gel contraction analyses. RESULTS: FAK operates downstream of both integrin ß1 and reactive oxygen species (ROS) to promote the expression of genes involved in matrix production and remodelling, including CCN2, α-smooth muscle actin and type I collagen. Blocking either FAK/src with PP2 or ROS with N-acetyl cysteine alleviates the elevated contractile and migratory capability of lesional SSc dermal fibroblasts. CONCLUSIONS: Excessive adhesive signalling is intimately involved with the fibrotic phenotype of lesional SSc fibroblasts; blocking adhesive signalling or ROS generation may be beneficial in controlling the fibrosis observed in SSc.
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Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Miofibroblastos/fisiología , Especies Reactivas de Oxígeno/metabolismo , Esclerodermia Sistémica/etiología , Animales , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Fosforilación/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Esclerodermia Sistémica/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: Gastrointestinal involvement occurs in up to 90% of patients with SSc. Animal models of SSc mimic some of the pathophysiological disease processes of SSc. The transgenic (TG) mouse strain TßRIIΔk-fib is characterized by ligand-dependent up-regulation of TGF-ß signalling and has been shown to develop skin fibrosis, lung fibrosis and diminished aortic ring contractility and adventitial fibrosis. We investigated if similar changes are observed in the gut tissue in this mouse model. METHODS: Colonic tissue was examined using histology and immunohistochemistry analyses. Tissue architecture was examined by haematoxylin and eosin (H&E), picrosirius red and immunohistochemical markers for α-smooth muscle actin (α-SMA), phospho-Smad 2/3 (pSmad2/3), Ki-67, protein gene product 9.5 and S-100. Fibrosis was quantified using the NIS Elements BR 2.30 system and by Sircol assay. Colonic strip contractile responses to potassium chloride (KCl) and carbachol were assessed in isolated organ baths. Confirmatory gut fibroblast and intestinal tissue biochemical assays, including cellular signalling mechanisms, were performed. RESULTS: H&E staining and staining for α-SMA, Ki-67, pSmad2/3 or neural tissue staining showed no differences between TG and wild-type (WT) mice gut tissue. There was increased collagen deposition in the gut of TG mice. Quantitative PCR results of TG gut fibroblasts showed evidence of up-regulated collagen and CTGF transcription, and non-canonical TGF-ß signalling pathways were also up-regulated. The organ bath studies showed diminished colonic strip contractility in TG mice compared with WT control mice to KCl and carbachol. CONCLUSION: We have shown that this TG mouse model, previously shown to develop skin and lung, develops colonic fibrosis with associated effects on colonic tissue contractility. This may offer further insight in pathological processes leading to the development of gut fibrosis.
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Colon/patología , Enfermedades del Colon/patología , Esclerodermia Sistémica/patología , Animales , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Enfermedades del Colon/fisiopatología , Femenino , Fibroblastos/fisiología , Fibrosis/patología , Fibrosis/fisiopatología , Motilidad Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/fisiología , Motilidad Gastrointestinal/efectos de la radiación , Masculino , Ratones , Ratones Transgénicos , Contracción Muscular/fisiología , Músculo Liso/fisiología , Esclerodermia Sistémica/fisiopatologíaRESUMEN
RATIONALE: Fibrotic response to lung injury depends on development of a fibrogenic population of myofibroblasts. The importance of resident interstitial fibroblasts and role of transforming growth factor ß (TGFß) in this process is unclear. OBJECTIVES: To define the importance of TGFß signaling in resident lung fibroblasts in the development of experimental pulmonary fibrosis. METHODS: A compound genetic strategy in which mice homozygous for a floxed high-affinity type II TGFß receptor (TßRII) allele were crossed with a transgenic strain harboring a fibroblast-specific transgene encoding ligand-dependent Cre-recombinase was used. TßRII was deleted by postnatal administration of tamoxifen over 5 days to compound mutant mice with appropriate littermate controls. Illumina microarray gene profiling and quantitative reverse transcriptase-polymerase chain reaction were used to confirm anergy to TGFß in explanted lung fibroblasts. Bleomycin lung injury was used to induce lung fibrosis, which was analyzed by histology and biochemical methods. Immunofluorescence was used to define cell populations after lung injury. MEASUREMENTS AND MAIN RESULTS: There was significant attenuation of fibrosis in mice after deletion of TßRII in resident fibroblasts. At 7 days after injury the number of fibrocytes and myofibroblasts was substantially reduced. Potential regulators of fibrosis were suggested by gene expression profiles that identified key candidate profibrotic genes, including connective tissue growth factor and endothelin-1 expressed by wild-type but not mutant lung fibroblasts. CONCLUSIONS: Intact TGFß signaling in resident pulmonary fibroblasts is essential for pulmonary fibrosis to develop. Our data support a key regulatory role of these cells in determining fibrocyte recruitment and myofibroblast differentiation.
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Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Eliminación de Secuencia/genética , Animales , Bleomicina , Western Blotting , Linaje de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis por Matrices de Proteínas/métodos , Receptor Tipo II de Factor de Crecimiento Transformador beta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transducción de Señal/genética , Tamoxifeno , Transcripción Genética/genéticaRESUMEN
Connective tissue growth factor (CTGF, CCN2) is a matricellular protein which plays key roles in normal mammalian development and in tissue homeostasis and repair. In pathological conditions, dysregulated CCN2 has been associated with cancer, cardiovascular disease, and tissue fibrosis. In this study, genetic manipulation of the CCN2 gene was employed to investigate the role of CCN2 expression in vitro and in experimentally-induced models of pulmonary fibrosis and pulmonary arterial hypertension (PAH). Knocking down CCN2 using siRNA reduced expression of pro-fibrotic markers (fibronectin p < 0.01, collagen type I p < 0.05, α-SMA p < 0.0001, TIMP-1 p < 0.05 and IL-6 p < 0.05) in TGF-ß-treated lung fibroblasts derived from systemic sclerosis patients. In vivo studies were performed in mice using a conditional gene deletion strategy targeting CCN2 in a fibroblast-specific and time-dependent manner in two models of lung disease. CCN2 deletion significantly reduced pulmonary interstitial scarring and fibrosis following bleomycin-instillation, as assessed by fibrotic scores (wildtype bleomycin 3.733 ± 0.2667 vs CCN2 knockout (KO) bleomycin 4.917 ± 0.3436, p < 0.05) and micro-CT. In the well-established chronic hypoxia/Sugen model of pulmonary hypertension, CCN2 gene deletion resulted in a significant decrease in pulmonary vessel remodelling, less right ventricular hypertrophy and a reduction in the haemodynamic measurements characteristic of PAH (RVSP and RV/LV + S were significantly reduced (p < 0.05) in CCN2 KO compared to WT mice in hypoxic/SU5416 conditions). These results support a prominent role for CCN2 in pulmonary fibrosis and in vessel remodelling associated with PAH. Therefore, therapeutics aimed at blocking CCN2 function are likely to benefit several forms of severe lung disease.
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Factor de Crecimiento del Tejido Conjuntivo/deficiencia , Hipertensión Arterial Pulmonar/terapia , Fibrosis Pulmonar/terapia , Animales , Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Células Cultivadas , Colágeno Tipo I/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Modelos Animales de Enfermedad , Eliminación de Gen , Humanos , Ratones , Ratones Noqueados , Hipertensión Arterial Pulmonar/inducido químicamente , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Recent studies have demonstrated upregulation of monocyte chemoattractant protein-3 (MCP-3/CCL7) in fibrosis and have suggested that in addition to a major role in regulating leucocyte recruitment this chemokine may also promote extracellular matrix (ECM) overproduction by fibroblasts. In the present study we explore interplay between MCP-3 and transforming growth factor beta (TGFbeta), a potent profibrotic cytokine. We demonstrate that MCP-3 promotes activation of TGFbeta signalling pathways leading to increased type I collagen secretion. In addition we show that MCP-3 gene expression is stimulated by recombinant TGFbeta1, raising the possibility for synergy between these two mediators in the fibrotic microenvironment. Comparison of downstream signalling pathways that regulate collagen gene activation by both cytokines confirms the central role of MAPK pathway activation in mediating the effects of both factors. An additive effect of these two agonists was demonstrated by comparative microarray analysis for key TGFbeta regulated transcripts including PAI-1, OSF2 and IGFBP6. Together, our results confirm cross-talk between MCP-3 and TGFbeta that may be critical in the development of fibrosis.
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Quimiocina CCL7/metabolismo , Colágeno/biosíntesis , Fibroblastos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Quimiocina CCL7/genética , Quimiocina CCL7/farmacología , Colágeno/genética , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pteridinas/farmacología , ARN Interferente Pequeño/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína smad3/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc) strongly associated with anti-RNA polymerase III antibody (ARA) autoantibodies. We investigated genetic susceptibility and altered protein expression in renal biopsy specimens in ARA-positive patients with SRC. METHODS: ARA-positive patients (n = 99) with at least 5 years' follow-up (49% with a history of SRC) were selected from a well characterized SSc cohort (n = 2254). Cases were genotyped using the Illumina Human Omni-express chip. Based on initial regression analysis, 9 single-nucleotide polymorphisms (SNP) were chosen for validation in a separate cohort of 256 ARA-positive patients (40 with SRC). Immunostaining of tissue sections from SRC or control kidney was used to quantify expression of candidate proteins based upon genetic analysis of the discovery cohort. RESULTS: Analysis of 641,489 SNP suggested association of POU2F1 (rs2093658; P = 1.98 × 10-5), CTNND2 (rs1859082; P = 5.58 × 10-5), HECW2 (rs16849716; P = 1.2 × 10-4), and GPATCH2L (rs935332; P = 4.92 × 10-5) with SRC. Further, the validation cohort showed an association between rs935332 within the GPATCH2L region, with SRC (P = 0.025). Immunostaining of renal biopsy sections showed increased tubular expression of GPATCH2L (P = 0.026) and glomerular expression of CTNND2 (P = 0.026) in SRC samples (n = 8) compared with normal human kidney controls (n = 8), despite absence of any genetic replication for the associated SNP. CONCLUSION: Increased expression of 2 candidate proteins, GPATCH2L and CTNND2, in SRC compared with control kidney suggests a potential role in pathogenesis of SRC. For GPATCH2L, this may reflect genetic susceptibility in ARA-positive patients with SSc based upon 2 independent cohorts.
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Lesión Renal Aguda , Esclerodermia Localizada , Esclerodermia Sistémica , Autoanticuerpos , Humanos , ARN Polimerasa III/inmunología , Esclerodermia Localizada/inmunología , Esclerodermia Sistémica/inmunología , Ubiquitina-Proteína LigasasAsunto(s)
Infecciones por Citomegalovirus/complicaciones , Esclerodermia Difusa/virología , Antiinflamatorios no Esteroideos/uso terapéutico , Infecciones por Citomegalovirus/inmunología , Femenino , Humanos , Persona de Mediana Edad , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/uso terapéutico , Remisión Espontánea , Esclerodermia Difusa/tratamiento farmacológico , Esclerodermia Difusa/inmunologíaRESUMEN
BACKGROUND: Systemic sclerosis (SSc; scleroderma) is an uncommon autoimmune rheumatic disease characterised by autoimmunity, vasculopathy and fibrosis. Gene expression profiling distinguishes scleroderma from normal skin, and can detect different subsets of disease, with potential to identify prognostic biomarkers of organ involvement or response to therapy. We have performed gene expression profiling in skin samples from patients with limited cutaneous SSc (lcSSc). METHODS: Total RNA was extracted from clinically uninvolved skin biopsies of 15 patients with lcSSc and 8 healthy controls (HC). Gene expression profiling was performed on a DNA oligonucleotide microarray chip. Differentially expressed genes (DEG) were identified using significance analysis of microarrays (SAM). Functional enrichment analysis of gene signatures was done via g:Profiler. RESULTS: There were 218 DEG between lcSSc and HC samples (false discovery rate <10%): 181/218 DEG were upregulated in lcSSc samples. Hierarchical clustering of DEG suggested the presence of two separate groups of lcSSc samples: "limited 1" and "limited 2". The limited-1 group (13 samples, 10 unique patients) showed upregulation of genes involved in cell adhesion, cardiovascular system (CVS) development, extracellular matrix and immune and inflammatory response. The CVS development signature was of particular interest as its genes showed very strong enrichment in response to wounding, response to transforming growth factor (TGF)-ß and kinase cascade. Neither limited-2 samples (six samples, five unique patients) nor HC samples showed functional enrichment. There were no significant differences in demographic or clinical parameters between these two groups. These results were confirmed using a second independent cohort. CONCLUSIONS: Our study suggests the presence of molecular subsets in lcSSc based on gene expression profiling of biopsies from uninvolved skin. This may reflect important differences in pathogenesis within these patient groups. We identify differential expression of a subset of genes that relate to CVS and are enriched in fibrotic signalling. This may shed light on mechanisms of vascular disease in SSc. The enrichment in profibrotic profile suggests that dysregulated gene expression may contribute to vasculopathy and fibrosis in different disease subsets.
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Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/genética , Perfilación de la Expresión Génica/métodos , Esclerodermia Sistémica/diagnóstico , Esclerodermia Sistémica/genética , Piel/patología , Adulto , Anciano , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Gastrin is a growth factor for both gastrointestinal and non-gastrointestinal tumours. Endocytosis of gastrin has been demonstrated in tumour cell lines expressing cholecystokinin-B/gastrin receptor (CCK-BR); this has raised the possibility of receptor targeted therapy. The aim of this study was to examine endocytosis of gastrin and CCK-BR in tumour cell lines. A small gastrin analogue, RG-G7, and the anti-CCK-BR antibody, anti-GRE1, were fluorescently labelled and uptake by cancer cell lines including AR42J, HepG2, and C170HM2 as well as transfected NIH3T3 fibroblast cells was assessed using standard and confocal fluorescence microscopy. CCK-BR expression of cell lines was assayed by reverse transcription-polymerase chain reaction and Western blotting. Apoptosis was detected using a fluorescent TUNEL method. RG-G7 and anti-GRE1 antibody were specifically taken up by all cell lines expressing CCK-BR. In addition to cytoplasmic uptake with RG-G7 and anti-GRE1 the latter also showed specific uptake into the nucleus. A coincidence of anti-GRE1 and apoptosis was seen. Targeting CCK-BR by peptide or antibody may offer therapeutic opportunities for some cancers.
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Carcinoma Hepatocelular/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptor de Colecistoquinina B/metabolismo , Animales , Apoptosis/fisiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Endocitosis , Humanos , Immunoblotting/métodos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Microscopía Confocal/métodos , Células 3T3 NIH , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Receptor de Colecistoquinina B/biosíntesis , Receptor de Colecistoquinina B/genéticaRESUMEN
This data article contains complementary figures related to the research article entitled, "Transforming growth factor-ß-induced CUX1 isoforms are associated with fibrosis in systemic sclerosis lung fibroblasts" (Ikeda et al. (2016) [2], http://dx.doi.org/10.1016/j.bbrep.2016.06.022), which presents that TGF-ß increased CUX1 binding in the proximal promoter and enhancer of the COL1A2 and regulated COL1. Further, in the scleroderma (SSc) lung and diffuse alveolar damage lung sections, CUX1 localized within the α- smooth muscle actin (α-SMA) positive cells (Fragiadaki et al., 2011) [1], "High doses of TGF-beta potently suppress type I collagen via the transcription factor CUX1" (Ikeda et al., 2016) [2]. Here we show that CUX1 isoforms are localized within α-smooth muscle actin-positive cells in SSc skin and idiopathic pulmonary fibrosis (IPF) lung tissue sections. In particular, at the granular and prickle cell layers in the SSc skin sections, CUX1 and α-SMA are co-localized. In addition, at the fibrotic loci in the IPF lung tissue sections, CUX1 localized within the α-smooth muscle actin (α-SMA) positive cells.
RESUMEN
In the enhancer region of the human type I collagen alpha 2 (COL1A2) gene, we identified cis-elements for the transcription factor CUX1. However, the role of CUX1 in fibrosis remains unclear. Here we investigated the role of CUX1 in the regulation of COL1 expression and delineated the mechanisms underlying the regulation of COL1A2 expression by CUX1 in systemic sclerosis (SSc) lung fibroblasts. The binding of CUX1 to the COL1A2 enhancer region was assessed using electrophoretic mobility shift assays after treatment with transforming growth factor (TGF)-ß. Subsequently, the protein expression levels of CUX1 isoforms were determined using Western blotting. Finally, the expression levels of COL1 and fibrosis-related cytokines, including CTGF, ET-1, Wnt1 and ß-catenin were determined. The binding of CUX1 isoforms to the COL1A2 enhancer region increased after TGF-ß treatment. TGF-ß also increased the protein levels of the CUX1 isoforms p200, p150, p110, p75, p30 and p28. Moreover, SSc lung fibroblasts showed higher levels of CUX1 isoforms than normal lung fibroblasts, and treatment of SSc lung fibroblasts with a cathepsin L inhibitor (IW-CHO) decreased COL1 protein expression and reduced cell size, as measured using immunocytochemistry. In SSc and diffuse alveolar damage lung tissue sections, CUX1 localised within α-smooth muscle actin-positive cells. Our results suggested that CUX1 isoforms play vital roles in connective tissue deposition during wound repair and fibrosis.
RESUMEN
C-kit is a tyrosine kinase receptor which is expressed in a wide variety of tumour cells such as gastrointestinal stromal tumours (GISTs), germ cell tumours, malignant trans-formation of mast cells, breast adenocarcinomas, malignant melanomas and small cell lung cancers. Imatinib mesylate is a tyrosine kinase inhibitor initially developed against the bcr-abl fusion protein of CML, but also shows therapeutic inhibitory activity against c-Kit expressed in GISTs. Treatment of patients with neuroendocrine tumours (NETs) at present is limited. Our aim was to test NETs for c-Kit expression and hence identify patients for the consideration of therapy with imatinib mesylate. NET patient specimens (n=85) were assessed for expression of c-KIT proto-oncogene (CD117) by immunohistochemistry using two antibodies, a polyclonal antibody and a monoclonal. Of the samples 24% stained positive with the polyclonal antibody and 64% with the monoclonal antibody. This study highlights problems related to screening using c-kit antibodies for immunocytochemistry. It is possible that the polyclonal antibody is less specific. Studies need to be performed to determine if c-kit expression by NETs can be translated into therapeutic benefit by agents such as imatinib mesylate.
Asunto(s)
Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/terapia , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Adenosina Trifosfato/química , Anticuerpos Monoclonales/química , Antineoplásicos/farmacología , Benzamidas , Sitios de Unión , Epítopos/química , Tumores del Estroma Gastrointestinal/metabolismo , Humanos , Mesilato de Imatinib , Inmunohistoquímica , Piperazinas/farmacología , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proto-Oncogenes Mas , Pirimidinas/farmacologíaRESUMEN
OBJECTIVE: The excessive deposition of extracellular matrix, including type I collagen, is a key aspect in the pathogenesis of connective tissue diseases such as systemic sclerosis (SSc; scleroderma). To further our understanding of the mechanisms governing the dysregulation of type I collagen production in SSc, we investigated the role of the activator protein 1 (AP-1) family of transcription factors in regulating COL1A2 transcription. METHODS: The expression and nuclear localization of AP-1 family members (c-Jun, JunB, JunD, Fra-1, Fra-2, and c-Fos) were examined by immunohistochemistry and Western blotting in dermal biopsy specimens and explanted skin fibroblasts from patients with diffuse cutaneous SSc and healthy controls. Gene activation was determined by assessing the interaction of transcription factors with the COL1A2 enhancer using transient transfection of reporter gene constructs, electrophoretic mobility shift assays, chromatin immunoprecipitation analysis, and RNA interference involving knockdown of individual AP-1 family members. Inhibition of fibroblast mammalian target of rapamycin (mTOR), Akt, and glycogen synthase kinase 3ß (GSK-3ß) signaling pathways was achieved using small-molecule pharmacologic inhibitors. RESULTS: Binding of JunB to the COL1A2 enhancer was observed, with its coalescence directed by activation of gene transcription through the proximal promoter. Knockdown of JunB reduced enhancer activation and COL1A2 expression in response to transforming growth factor ß. In SSc dermal fibroblasts, increased mTOR/Akt signaling was associated with inactivation of GSK-3ß, leading to blockade of JunB degradation and, thus, constitutively high expression of JunB. CONCLUSION: In patients with SSc, the accumulation of JunB resulting from altered mTOR/Akt signaling and a failure of proteolytic degradation underpins the aberrant overexpression of type I collagen. These findings identify JunB as a potential target for antifibrotic therapy in SSc.
Asunto(s)
Colágeno Tipo I/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Piel/metabolismo , Piel/patología , Factores de Transcripción/metabolismo , Biopsia , Estudios de Casos y Controles , Células Cultivadas , Fibrosis/metabolismo , Fibrosis/patología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esclerodermia Sistémica/fisiopatología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
INTRODUCTION: Clinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients. METHODS: Dermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors. RESULTS: Luminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator. CONCLUSIONS: Our data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.
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Citocinas/análisis , Líquido Extracelular/inmunología , Esclerodermia Sistémica/inmunología , Vesícula , Análisis por Conglomerados , Femenino , Humanos , Masculino , Persona de Mediana Edad , PielRESUMEN
Immunotherapy has considerable potential in the treatment of cancer. Here we report on the uptake of an antibody raised against the CCK-B/Gastrin receptor (CCK-BR) by liver embryonic and liver tumor cell lines. In all five cell lines studied, expression of CCK-BR and uptake of labeled anti-CCK-BR antibody was observed. The labeled anti-CCK-BR antibody was localized in both the cytoplasm and nucleus of cells. In addition, we found a coincidence between the uptake of the labeled antibody by cells and the occurrence of apoptosis (cell death). The results suggest that antibodies directed against CCK-BR have potential for targeting and possibly destroying tumor cells bearing the receptor.
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Anticuerpos/metabolismo , Endocitosis , Receptores de Colecistoquinina/inmunología , Animales , Apoptosis , Carcinoma Hepatocelular , Humanos , Immunoblotting , Microscopía Confocal , Microscopía Fluorescente , Receptor de Colecistoquinina B , Células Tumorales CultivadasRESUMEN
Vascular involvement is frequent in systemic sclerosis, but the role of the lymphatic vasculature is poorly known. Our aim was to evaluate lymphatic vessels in systemic sclerosis skin lesions. We studied skin forearm biopsies of 9 patients with systemic sclerosis and 7 age-matched controls. Lymphatic vessels were labeled with the monoclonal antibody D2-40 and blood vessels with a polyclonal antibody to von Willebrand Factor. All blood and lymphatic vessels present in each section were counted and total area, inner luminal area, and shape factors were measured. The number of blood and lymphatic vessels in papillary dermis was greater and their diameter lower than in reticular dermis both in systemic sclerosis and controls. In the reticular dermis, the number of lymphatic vessels was markedly reduced in systemic sclerosis (4.9 +/- 1.1 microm(-2) versus 8.9 +/- 1.2 microm(-2)P = .03), and a similar trend was observed in papillary dermis (8.4 +/- 3.7 microm(-2) versus 8.1 +/- 5.3 microm(-2)). Interestingly, the number of periglandular lymphatics in systemic sclerosis was not different from controls. The inner luminal area (possibly indicating compensatory dilation) of lymphatic vessels, particularly the periglandular ones, was greater in systemic sclerosis than in controls. No differences were observed in the number of blood vessels, but the percentage of blood vessel profiles (without lumen) was significantly less in systemic sclerosis both in papillary and in reticular dermis. In conclusion, our data show that skin lesions in systemic sclerosis are characterized by a selective rarefaction of lymphatic vasculature that spares periglandular vessels and that might have a pathogenic role in the evolution and in the clinical manifestations of the disease.