RESUMO
Crohn's disease (CD) is an inflammatory bowel disease characterized by transmural inflammation and intestinal fibrosis. Mechanisms of fibrosis in CD are not well understood. Transmural inflammation is associated with inflammatory cell infiltration, stenosis, and distention, which present mechanical stress (MS) to the bowel wall. We hypothesize that MS induces gene expression of profibrotic mediators such as connective tissue growth factor (CTGF), which may contribute to fibrosis in CD. A rodent model of CD was induced by intracolonic instillation of TNBS to the distal colon. TNBS instillation induced a localized transmural inflammation (site I), with a distended colon segment (site P) proximal to site I. We detected significant fibrosis and collagen content not only in site I but also in site P in CD rats by day 7. CTGF expression increased significantly in sites P and I, but not in the segment distal to the inflammation site. Increased CTGF expression was detected mainly in the smooth muscle cells (SMCs). When rats were fed exclusively with clear liquid diet to prevent mechanical distention in colitis, expression of CTGF in sites P and I was blocked. Direct stretch led to robust expression of CTGF in colonic SMC. Treatment of CD rats with anti-CTGF antibody FG-3149 reduced fibrosis and collagen content in both sites P and I and exhibited consistent trends toward normalizing expression of collagen mRNAs. In conclusion, our studies suggest that mechanical stress, by upregulating profibrotic mediators, i.e., CTGF, may play a critical role in fibrosis in CD.NEW & NOTEWORTHY We found that CTGF expression increased significantly not only in the inflammation site but in the distended segment proximal to inflammation in a rodent model of CD-like colitis. Release of mechanical distention prevented CTGF expression in CD rats, whereas direct stretch induced CTGF expression. Treatment with anti-CTGF antibody reduced fibrosis and collagen contents in CD rats. Thus, mechanical stress, via upregulating profibrotic mediators, i.e., CTGF, may play a critical role in fibrosis in CD.
Assuntos
Fator de Crescimento do Tecido Conjuntivo , Doença de Crohn , Fibrose , Ratos Sprague-Dawley , Estresse Mecânico , Animais , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Ratos , Masculino , Colite/metabolismo , Colite/induzido quimicamente , Colite/patologia , Colo/metabolismo , Colo/patologia , Modelos Animais de Doenças , Ácido Trinitrobenzenossulfônico , Colágeno/metabolismoRESUMO
Idiopathic pulmonary fibrosis is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and in the lung. Connective-tissue growth factor (CTGF) exacerbates pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we demonstrate upregulation of CTGF in a rat lung fibrosis model induced by an adenovirus vector encoding active TGF-ß1 (AdTGF-ß1). We show that CTGF is also upregulated in patients with idiopathic pulmonary fibrosis. Expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on Days 7 and 14 as well as endothelial cells sorted from fibrotic lungs on Days 14 and 28. These findings suggest contributions of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-ß increases profibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-ß in inducing pulmonary fibrosis. Also, the fibrotic extracellular matrix upregulated CTGF expression, compared with the normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for treatment of pulmonary fibrosis.
Assuntos
Fator de Crescimento do Tecido Conjuntivo , Fibrose Pulmonar Idiopática , Fator de Crescimento Transformador beta1 , Animais , Anticorpos Monoclonais Humanizados , Fator de Crescimento do Tecido Conjuntivo/genética , Células Endoteliais/metabolismo , Fibrose , Fibrose Pulmonar Idiopática/genética , Ratos , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND: Connective tissue growth factor (CTGF), is a secreted matricellular factor that has been linked to increased risk of cardiovascular disease in diabetic subjects. Despite the biological role of CTGF in diabetes, it still remains unclear how CTGF expression is regulated. In this study, we aim to identify the clinical parameters that modulate plasma CTGF levels measured longitudinally in type 1 diabetic patients over a period of 10 years. A number of patients had negligible measured values of plasma CTGF that formed a point mass at zero, whereas others had high positive values of CTGF that were measured on a continuous scale. The observed combination of excessive zero and continuous positively distributed non-zero values in the CTGF outcome is referred to as semicontinuous data. METHODS: We propose a novel application of a marginalized two-part model (mTP) extended to accommodate longitudinal semicontinuous data in which the marginal mean is expressed in terms of the covariates and estimates of their effect on the mean responses are generated. The continuous component is assumed to follow distributions that stem from the generalized gamma family whereas the binary measure is analyzed using logistic model and both have correlated random effects. Other approaches including the one- and two-part with uncorrelated and correlated random effects models were also applied and their estimates were all compared. RESULTS: Our results using the mTP model identified intensive glucose control treatment and smoking as clinical factors that were associated with decreased and increased odds of observing non-zero CTGF values respectively. In addition, hemoglobin A1c, systolic blood pressure, and high density lipoprotein were all shown to be significant risk factors that contribute to increasing CTGF levels. These findings were consistently observed under the mTP model but varied with the distributions for the other models. Accuracy and precision of the mTP model was further validated using simulation studies. CONCLUSION: The mTP model identified new clinical determinants that modulate the levels of CTGF in diabetic subjects. Applicability of this approach can be extended to other biomarkers measured in patient populations that display a combination of negligible zero and non-zero values.
Assuntos
Análise de Dados , Modelos Estatísticos , Simulação por Computador , Fator de Crescimento do Tecido Conjuntivo/sangue , Diabetes Mellitus Tipo 1/sangue , HumanosRESUMO
Malignant mesothelioma is an aggressive neoplasm with no particularly effective treatments. We previously reported that overexpression of connective tissue growth factor (CTGF/CCN2) promotes mesothelioma growth, thus suggesting it as a novel molecular target. A human monoclonal antibody that antagonizes CTGF (FG-3019, pamrevlumab) attenuates malignant properties of different kinds of human cancers and is currently under clinical trial for the treatment of pancreatic cancer. This study reports the effects of FG-3019 on human mesothelioma in vitro and in vivo. We analyzed the effects of FG-3019 on the proliferation, apoptosis, migration/invasion, adhesion and anchorage-independent growth in three human mesothelioma cell lines, among which ACC-MESO-4 was most efficiently blocked with FG-3019 and was chosen for in vivo experiments. We also evaluated the coexistent effects of fibroblasts on mesothelioma in vitro, which are also known to produce CTGF in various pathologic situations. Coexistent fibroblasts in transwell systems remarkably promoted the proliferation and migration/invasion of mesothelioma cells. In orthotopic nude mice model, FG-3019 significantly inhibited mesothelioma growth. Histological analyses revealed that FG-3019 not only inhibited the proliferation but also induced apoptosis in both mesothelioma cells and fibroblasts. Our data suggest that FG-3019 antibody therapy could be a novel additional choice for the treatment of mesothelioma.
RESUMO
BACKGROUND: Fibrosis is a delayed side effect of radiation therapy (RT). Connective tissue growth factor (CTGF) promotes the development of fibrosis in multiple settings, including pulmonary radiation injury. METHODS: To better understand the cellular interactions involved in RT-induced lung injury and the role of CTGF in these responses, microarray expression profiling was performed on lungs of irradiated and non-irradiated mice, including mice treated with the anti-CTGF antibody pamrevlumab (FG-3019). Between group comparisons (Welch's t-tests) and principal components analyses were performed in Genespring. RESULTS: At the mRNA level, the ability of pamrevlumab to prolong survival and ameliorate RT-induced radiologic, histologic and functional lung deficits was correlated with the reversal of a clear enrichment in mast cell, macrophage, dendritic cell and mesenchymal gene signatures. Cytokine, growth factor and matrix remodeling genes that are likely to contribute to RT pneumonitis and fibrosis were elevated by RT and attenuated by pamrevlumab, and likely contribute to the cross-talk between enriched cell-types in injured lung. CONCLUSIONS: CTGF inhibition had a normalizing effect on select cell-types, including immune cells not typically regarded as being regulated by CTGF. These results suggest that interactions between RT-recruited cell-types are critical to maintaining the injured state; that CTGF plays a key role in this process; and that pamrevlumab can ameliorate RT-induced lung injury in mice and may provide therapeutic benefit in other immune and fibrotic disorders.
Assuntos
Anticorpos Monoclonais/farmacologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Pulmão/metabolismo , Pulmão/efeitos da radiação , Fibrose Pulmonar/metabolismo , Lesões Experimentais por Radiação/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Feminino , Expressão Gênica , Pulmão/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Mastócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/etiologia , Lesões Experimentais por Radiação/tratamento farmacológicoRESUMO
BACKGROUND: Patients with Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) positive lung cancer are sensitive to ALK-kinase inhibitors. TAE684 is a potent second generation ALK inhibitor that overcomes Crizotinib resistance. Radiotherapy is an integral therapeutic component of locally advanced lung cancer. Therefore, we sought to investigate the effects of combined radiotherapy and ALK-inhibition via TAE684 in ALK-positive vs. wild type lung cancer cells. METHODS: Human non-small cell lung cancer (NSCLC) cell lines harboring wild-type ALK (A549), EML4-ALK translocation (H3122) and murine Lewis Lung Cancer (LLC) cells were investigated. Cells were irradiated with 1-4 Gy X-Rays (320 keV) and carbon ions (Spread-out Bragg Peak, SOBP (245.4-257.0 MeV/u)) at Heidelberg Ion Therapy center. TAE684 was administered at the dose range 0-100 nM. Clonogenic survival, proliferation and apoptosis via caspase 3/7 expression level were assessed in all three cell lines using time-lapse live microscopy. RESULTS: TAE684 inhibited the proliferation of H3122 cells in a dose-dependent manner with a half maximal inhibitory concentration (IC50) of ~ 8.2 nM. However, A549 and LLC cells were relatively resistant to TAE684 and IC50 was not reached at concentrations tested (up to 100 nM) in proliferation assay. The antiproliferative effect of TAE684 was augmented by radiotherapy in H3122 cells. TAE684 significantly sensitized H3122 cells to particle therapy with carbon ions (sensitizer enhancement ratio ~1.61, p < 0.05). Caspase 3/7 activity was evidently enhanced after combination therapy in H3122 cells. CONCLUSIONS: This is the first report demonstrating synergistic effects of combined TAE684 and radiotherapy in EML4-ALK positive lung cancer cells. In addition to conventional photon radiotherapy, ALK-inhibition also enhanced the effects of particle irradiation using carbon ions. Our data indicate beneficial effects of combined ALK-inhibition and radiotherapy in treatment of this distinct subpopulation of NSCLC that warrant further evaluation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Quimiorradioterapia/métodos , Neoplasias Pulmonares , Vício Oncogênico , Pirimidinas/farmacologia , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinoma Pulmonar de Lewis , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Radioterapia com Íons Pesados , Humanos , Neoplasias Pulmonares/genética , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Fusão Oncogênica , Fótons , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/genética , Serina Endopeptidases/genéticaRESUMO
Peritoneal fibrosis (PF) is a serious complication in various clinical settings, but the mechanisms driving it remain to be fully determined. Connective tissue growth factor (CTGF) is known to regulate fibroblast activities. We therefore examined if CTGF inhibition has anti-fibrotic effects in PF. PF was induced by repetitive intraperitoneal injections of chlorhexidine gluconate (CG) in mice with type I pro-collagen promoter-driven green fluorescent protein (GFP) expression to identify fibroblasts. FG-3019, an anti-CTGF monoclonal antibody, was used to inhibit CTGF. CG-induced PF was significantly attenuated in FG-3019-treated mice. CG challenges induced marked accumulations of proliferating fibroblasts and of myofibroblasts, which were both reduced by FG-3019. Levels of peritoneal CTGF expression were increased by CG challenges, and suppressed in FG-3019-treated mice. FG-3019 treatment also reduced the number of CD31+ vessels and VEGF-A-positive cells in fibrotic peritoneum. In vitro studies using NIH 3T3 fibroblasts and peritoneal mesothelial cells (PMCs) showed that CTGF blockade suppressed TGF-ß1-induced fibroblast proliferation and myofibroblast differentiation, PMC mesothelial-to-mesenchymal transition, and VEGF-A production. These findings suggest that the inhibition of CTGF by FG-3019 might be a novel treatment for PF through the regulation of fibroblast and myofibroblast accumulation and angiogenesis.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Fator de Crescimento do Tecido Conjuntivo/genética , Neovascularização Patológica/prevenção & controle , Fibrose Peritoneal/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Anticorpos Monoclonais Humanizados , Diferenciação Celular/efeitos dos fármacos , Clorexidina/análogos & derivados , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Células NIH 3T3 , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Fibrose Peritoneal/induzido quimicamente , Fibrose Peritoneal/genética , Fibrose Peritoneal/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Background: Radiotherapy is a mainstay for the treatment of lung cancer that can induce pneumonitis or pulmonary fibrosis. The matricellular protein connective tissue growth factor (CTGF) is a central mediator of tissue remodeling. Methods: A radiation-induced mouse model of pulmonary fibrosis was used to determine if transient administration of a human antibody to CTGF (FG-3019) started at different times before or after 20 Gy thoracic irradiation reduced acute and chronic radiation toxicity. Mice (25 mice/group; 10 mice/group in a confirmation study) were examined by computed tomography, histology, gene expression changes, and for survival. In vitro experiments were performed to directly study the interaction of CTGF blockade and radiation. All statistical tests were two-sided. Results: Administration of FG-3019 prevented (â¼50%-80%) or reversed (â¼50%) lung remodeling, improved lung function, improved mouse health, and rescued mice from lethal irradiation ( P < .01). Importantly, when antibody treatment was initiated at 16 weeks after thoracic irradiation, FG-3019 reversed established lung remodeling and restored lung function. CTGF blockade abrogated M2 polarized macrophage influx, normalized radiation-induced gene expression changes, and reduced myofibroblast abundance and Osteopontin expression. Conclusion: These results indicate that blocking CTGF attenuates radiation-induced pulmonary remodeling and can reverse the process after initiation. CTGF has a central role in radiation-induced fibrogenesis, and FG-3019 may benefit patients with radiation-induced pulmonary fibrosis or patients with other forms or origin of chronic fibrotic diseases.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Fibrose Pulmonar/tratamento farmacológico , Lesões por Radiação/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Fibroblastos , Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Macrófagos/efeitos dos fármacos , Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C57BL , Edema Pulmonar/prevenção & controle , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Troca Gasosa Pulmonar/efeitos dos fármacos , Troca Gasosa Pulmonar/efeitos da radiação , Lesões por Radiação/etiologia , Lesões por Radiação/genética , Lesões por Radiação/patologia , Pneumonite por Radiação/prevenção & controle , Radioterapia/efeitos adversos , Tomografia Computadorizada por Raios XRESUMO
In addition to local cytotoxic activity, radiotherapy may also elicit local and systemic antitumor immunity, which may be augmented by immunotherapeutic agents including Toll-like receptor (TLR) 7/8 agonists. Here, we investigated the ability of 3M-011 (854A), a TLR7/8 agonist, to boost the antigen-presenting activity of dendritic cells (DC) as an adjuvant to radiotherapy. The combined treatment induced marked local and systemic responses in subcutaneous and orthotopic mouse models of colorectal and pancreatic cancer. In vitro cytotoxicity assays as well as in vivo depletion experiments with monoclonal antibodies identified NK and CD8 T cells as the cell populations mediating the cytotoxic effects of the treatment, while in vivo depletion of CD11c+ dendritic cells (DC) in CD11c-DTR transgenic mice revealed DC as the pivotal immune hub in this setting. The specificity of the immune reaction was confirmed by ELISPOT assays. TLR7/8 agonists therefore seem to be potent adjuvants to radiotherapy, inducing strong local and profound systemic immune responses to tumor antigens released by conventional therapy.
Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/terapia , Radiação Ionizante , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Neoplasias Colorretais/metabolismo , Terapia Combinada , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/efeitos da radiação , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Imidazóis/farmacologia , Técnicas Imunoenzimáticas , Imunoterapia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/efeitos da radiação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/metabolismo , Quinolinas/farmacologia , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND PURPOSE: Non-small cell lung cancer (NSCLC) patients with chromosomal rearrangements of the anaplastic lymphoma kinase gene (ALK) are sensitive to the tyrosine kinase inhibitor crizotinib. We aimed to investigate the effects of combined radiotherapy and crizotinib in ALK-positive vs. wild type NSCLC models. MATERIAL AND METHODS: Clonogenic survival, proliferation and apoptosis of cells exposed to crizotinib and radiotherapy (photon and carbon ions) were evaluated in ALK mutation positive (ALK+; H3122) and negative (ALK-; A549 and LLC) NSCLC lines. The syngeneic mouse (LLC) and human (H3122) xenograft tumor models were further studied in vivo. Tumor growth kinetics, microvascular density (MVD), perfusion and proliferation were assessed. RESULTS: Crizotinib exerted potent and selective anti-proliferative and pro-apoptotic effects in ALK+ H3122 cells which were augmented by radiotherapy. The synergistic effect of this combination in ALK+ NSCLC was confirmed by isobologram analysis. Crizotinib also sensitized H3122 cells to particle therapy with carbon ions. In H3122 xenografts, dual combination was most effective in reducing tumor proliferation, MVD and perfusion. In contrast, in the LLC model, crizotinib led only to a transient tumor growth inhibition and combined treatment was inferior to radiotherapy alone. CONCLUSIONS: Crizotinib elicits beneficial effects in combination with radiotherapy only in ALK-positive NSCLC.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Proteínas de Fusão Oncogênica/biossíntese , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Lewis/radioterapia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Linhagem Celular Tumoral , Quimiorradioterapia , Crizotinibe , Sinergismo Farmacológico , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/farmacologia , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In Duchenne muscular dystrophy (DMD) and the mdx mouse model, the absence of the cytoskeletal protein dystrophin causes defective anchoring of myofibres to the basal lamina. The resultant myofibre degeneration and necrosis lead to a progressive loss of muscle mass, increased fibrosis and ultimately fatal weakness. Connective tissue growth factor (CTGF/CCN-2) is critically involved in several chronic fibro-degenerative diseases. In DMD, the role of CTGF might extend well beyond replacement fibrosis secondary to loss of muscle fibres, since its overexpression in skeletal muscle could by itself induce a dystrophic phenotype. Using two independent approaches, we here show that mdx mice with reduced CTGF availability do indeed have less severe muscular dystrophy. Mdx mice with hemizygous CTGF deletion (mdx-Ctgf+/-), and mdx mice treated with a neutralizing anti-CTGF monoclonal antibody (FG-3019), performed better in an exercise endurance test, had better muscle strength in isolated muscles and reduced skeletal muscle impairment, apoptotic damage and fibrosis. Transforming growth factor type-ß (TGF-ß), pERK1/2 and p38 signalling remained unaffected during CTGF suppression. Moreover, both mdx-Ctgf+/- and FG-3019 treated mdx mice had improved grafting upon intramuscular injection of dystrophin-positive satellite cells. These findings reveal the potential of targeting CTGF to reduce disease progression and to improve cell therapy in DMD.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Apoptose/genética , Terapia Baseada em Transplante de Células e Tecidos , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibrose/genética , Genótipo , Sobrevivência de Enxerto/genética , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Força Muscular/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/terapia , Transdução de Sinais , Proteínas Smad/metabolismo , Transplante de Células-Tronco , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
CTGF is a secreted matricellular protein with very complex biology. It has been shown to modulate many signaling pathways leading to cell adhesion and migration, angiogenesis, myofibroblast activation, and extracellular matrix deposition and remodeling, which together lead to tissue remodeling and fibrosis. It has been reported in the literature that inhibition of CTGF expression by siRNA prevents CCl4-induced liver fibrosis and can reverse fibrosis when administered after significant collagen deposition is observed. A monoclonal antibody to CTGF that is currently in clinical development (FG-3019) has demonstrated the ability to reverse vascular stiffening and improve cardiac function in a rat model of diabetic complications. FG-3019 has also exhibited activity in a murine radiation-induced pulmonary fibrosis model. When FG-3019 was administered to mice after a significant radiation-induced increase in lung density could be observed by CT imaging, the density of the lungs was observed to decrease over the period during which the antibody was administered and to remain stable after therapy had ceased. When considered together, these data indicate that inhibition of CTGF can prevent and reverse the process of fibrosis.
RESUMO
Despite recent advances in neonatal intensive care and surfactant therapy, bronchopulmonary dysplasia (BPD) continues to be one of the most common long-term pulmonary complications associated with preterm birth. Clinical efforts to prevent and treat BPD have been largely unsuccessful due to its multifactorial nature and poorly understood disease process. Connective tissue growth factor (CTGF) is a matricellular protein that plays an important role in tissue development and remodeling. Previous studies have demonstrated that hyperoxia exposure up-regulates CTGF expression in neonatal rat lungs. Whether CTGF overexpression plays a role in the pathogenesis of BPD, and whether CTGF antagonism has a therapeutic potential for BPD, are unknown. In the present study, we examined CTGF expression in lung autopsy specimens from patients with BPD and control subjects with no BPD. We assessed the effect of a CTGF-neutralizing monoclonal antibody (CTGF Ab) on preventing hyperoxia-induced lung injury in neonatal rats. Our study demonstrates that CTGF expression is increased in BPD lungs. In newborn rats, exposure to 90% oxygen for 14 days resulted in activation of ß-catenin signaling, decreased alveolarization and vascular development, and physiological and histological evidence of pulmonary hypertension (PH). However, treatment with CTGF Ab prevented ß-catenin signaling activation, improved alveolarization and vascular development, and attenuated PH during hyperoxia. These data indicate that CTGF-ß-catenin signaling plays a critical role in the pathogenesis of experimental BPD. CTGF antagonism may offer a novel therapeutic strategy to alleviate BPD and PH in neonates.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Neutralizantes/farmacologia , Displasia Broncopulmonar/tratamento farmacológico , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Hiperóxia/tratamento farmacológico , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patologia , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Humanos , Hiperóxia/metabolismo , Hiperóxia/patologia , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Recém-Nascido , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
The aim of the current study was to evaluate the influence of chemotherapeutic drugs on immunotherapy with Imidazoquinoline Toll-like Receptor (TLR) agonists in cancer. First, the previously described antitumor efficacy of TLR agonists [i.e. a TLR7 agonist (852A) and a dual TLR7/8 agonist (3M-011)] was confirmed in additional cancer models, and second the therapeutic potential of TLR agonists in combination with cyclophosphamide was investigated. The antitumor potential was evaluated against a panel of syngeneic tumor models; namely B16-F10 melanoma, M3 melanoma and MC-26 colon carcinoma. Systemic administration of either 3M-011 or 852A in these various syngeneic models induced significant antitumor activity as evidenced by delays in tumor growth curves. Combination of cyclophosphamide with either 3M-011 or 852A demonstrated that cyclophosphamide does not negatively interfere with the TLR agonist's antitumor effects, but may, depending on the dosing schedule, to actually potentiate the effect. These findings suggest that the immunomodulatory TLR agonists may be used in combination with cytotoxic agents in the treatment of cancer.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Imunomodulação/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Quinolinas/uso terapêutico , Sulfonamidas/uso terapêutico , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Carga Tumoral , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Células HEK293 , Humanos , Interferon Tipo I/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Modelos Animais , NF-kappa B/imunologia , Quinolinas/administração & dosagem , Estatísticas não Paramétricas , Sulfonamidas/administração & dosagem , Células Tumorais CultivadasRESUMO
BACKGROUND: We have previously shown that small molecule PDGF receptor tyrosine kinase inhibitors (RTKI) can drastically attenuate radiation-induced pulmonary fibrosis if the drug administration starts at the time of radiation during acute inflammation with present but limited effects against acute inflammation. To rule out interactions of the drug with acute inflammation, we investigated here in an interventive trial if a later drug administration start at a time when the acute inflammation has subsided--has also beneficial antifibrotic effects. METHODS: Whole thoraces of C57BL/6 mice were irradiated with 20 Gy and treated with the RTKI imatinib starting either 3 days after radiation (during acute inflammation) or two weeks after radiation (after the acute inflammation has subsided as demonstrated by leucocyte count). Lungs were monitored and analyzed by clinical, histological and in vivo non-invasive computed tomography as a quantitative measure for lung density and lung fibrosis. RESULTS: Irradiation induced severe lung fibrosis resulting in markedly reduced mouse survival vs. non-irradiated controls. Both early start of imatinib treatment during inflammation and late imatinib start markedly attenuated the development of pulmonary fibrosis as demonstrated by clinical, histological and qualitative and quantitative computed tomography results such as reduced lung density. Both administration schedules resulted in prolonged lifespans. The earlier drug treatment start resulted in slightly stronger beneficial antifibrotic effects along all measured endpoints than the later start. CONCLUSIONS: Our findings show that imatinib, even when administered after the acute inflammation has subsided, attenuates radiation-induced lung fibrosis in mice. Our data also indicate that the fibrotic fate is not only determined by the early inflammatory events but rather a complex process in which secondary events at later time points are important. Because of the clinical availability of imatinib or similar compounds, a meaningful attenuation of radiation-induced lung fibrosis in patients seems possible.
Assuntos
Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Pneumonite por Radiação/prevenção & controle , Animais , Benzamidas , Feminino , Mesilato de Imatinib , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Innate immune stimulation with Toll-like receptor (TLR) agonists is a proposed modality for immunotherapy of melanoma. Here, a TLR7/8 agonist, 3M-011, was used effectively as a single systemic agent against disseminated mouse B16-F10 melanoma. The investigation of the mechanism of antitumor action revealed that the agonist had no direct cytotoxic effects on tumor cells tested in vitro. In addition, 3M-011 retained its effectiveness in scid/B6 mice and scid/NOD mice, eliminating the requirement for T and B cells, but lost its activity in beige (bg/bg) and NK1.1-immunodepleted mice, suggesting a critical role for natural killer (NK) cells in the antitumor response. NK cytotoxicity was enhanced in vivo by the TLR7/8 agonist; this activation was long lasting, as determined by sustained expression of the activation marker CD69. Also, in human in vitro studies, 3M-011 potentiated NK cytotoxicity. TLR7/8-mediated NK-dependent antitumor activity was retained in IFN-alpha/beta receptor-deficient as well as perforin-deficient mice, while depletion of IFN-gamma significantly decreased the ability of 3M-011 to delay tumor growth. Thus, IFN-gamma-dependent functions of NK cell populations appear essential for cancer immunotherapy with TLR7/8 agonists.
Assuntos
Antineoplásicos/uso terapêutico , Imidazóis/uso terapêutico , Células Matadoras Naturais/imunologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Quinolinas/uso terapêutico , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Animais , Feminino , Humanos , Interferon gama/imunologia , Camundongos , NF-kappa B/imunologiaRESUMO
BACKGROUND: Human B cells and plasmacytoid dendritic cells (pDC) are the only cells known to express both TLR7 and TLR9. Plasmacytoid dendritic cells are the primary IFN-alpha producing cells in response to TLR7 and TLR9 agonists. The direct effects of TLR7 stimulation on human B cells is less understood. The objective of this study was to compare the effects of TLR7 and TLR9 stimulation on human B cell function. RESULTS: Gene expression and protein production of cytokines, chemokines, various B cell activation markers, and immunoglobulins were evaluated. Purified human CD19+ B cells (99.9%, containing both naïve and memory populations) from peripheral blood were stimulated with a TLR7-selective agonist (852A), TLR7/8 agonist (3M-003), or TLR9 selective agonist CpG ODN (CpG2006). TLR7 and TLR9 agonists similarly modulated the expression of cytokine and chemokine genes (IL-6, MIP1 alpha, MIP1 beta, TNF alpha and LTA), co-stimulatory molecules (CD80, CD40 and CD58), Fc receptors (CD23, CD32), anti-apoptotic genes (BCL2L1), certain transcription factors (MYC, TCFL5), and genes critical for B cell proliferation and differentiation (CD72, IL21R). Both agonists also induced protein expression of the above cytokines and chemokines. Additionally, TLR7 and TLR9 agonists induced the production of IgM and IgG. A TLR8-selective agonist was comparatively ineffective at stimulating purified human B cells. CONCLUSION: These results demonstrate that despite their molecular differences, the TLR7 and TLR9 agonists induce similar genes and proteins in purified human B cells.
Assuntos
Linfócitos B/imunologia , Ativação Linfocitária/efeitos dos fármacos , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Memória Imunológica , Ativação Linfocitária/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Quinolinas/farmacologia , Sulfonamidas/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Receptor Toll-Like 9/agonistasRESUMO
Antitumor effects of the toll-like receptor 7 (TLR7) agonist, 852A, were evaluated. Supernatants from human peripheral blood mononuclear cells (PBMC) stimulated with 852A inhibited the proliferation of tumor cell lines Hs294T and 769-P but had no effect on others (786-O and Caki-1). Because addition of 852A directly to the Hs294T cells did not inhibit their proliferation, the mechanism(s) of inhibition of tumor cell proliferation was investigated. Low nanomolar concentrations of 852A stimulated the production of interferon-alpha (IFN-alpha), IFN-inducible protein-10 (IP-10), interleukin-1 receptor antagonist (IL-1Ra), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from human PBMCs. Cytokines stimulated by submicromolar concentrations of 852A were sufficient to inhibit Hs294T proliferation. At higher concentrations (3-30 microM), 852A induced the production of IL-12p70, IL-18, and IFN-gamma. PBMC cultures depleted of plasmacytoid dendritic cells (pDC) did not produce IFN-alpha, and their conditioned medium did not inhibit Hs294T proliferation. Anti-IFN-alpha/beta receptor (IFNAR) and anti-IFN-alpha antibodies partially abrogated Hs294T proliferation inhibition by 852A-stimulated PBMC supernatants, whereas separate neutralization of TRAIL, tumor necrosis factor-alpha (TNF-alpha, IFN-gamma, IFN-beta, or IFN-omega had no effect. In vivo, six doses of 852A administration significantly delayed the onset of lung colonies in a B16 melanoma model. Thus, the results demonstrate that the TLR7 agonist 852A inhibits in vitro proliferation of some tumor cells in a pDC-dependent and IFN-alpha-dependent manner and can delay tumor growth in vivo.
Assuntos
Células Dendríticas/imunologia , Interferon Tipo I/imunologia , Neoplasias/patologia , Quinolinas/farmacologia , Sulfonamidas/farmacologia , Receptor 7 Toll-Like/agonistas , Aminoquinolinas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Imiquimode , Leucócitos Mononucleares/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Melanoma/patologia , Camundongos , Oligodesoxirribonucleotídeos/farmacologia , Frações Subcelulares/efeitos dos fármacos , Receptor Toll-Like 9/agonistasRESUMO
PURPOSE: Investigations on the combination of radiotherapy with vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) antiangiogenic agents, which has the potential to improve the clinical outcome in cancer patients. EXPERIMENTAL DESIGN: Here, we analyze the combined VEGF (SU5416) and PDGF (SU6668) receptor tyrosine kinase inhibition with irradiation in human endothelium (HUVEC), prostate cancer (PC3), and glioblastoma (U87) in vitro and in vivo. RESULTS: Combined inhibition of VEGF and PDGF signaling resulted in enhanced apoptosis, reduced cell proliferation, and clonogenic survival as well as reduced endothelial cell migration and tube formation compared with single pathway inhibition. These effects were further enhanced by additional irradiation. Likewise, in PC3 and U87 tumors growing s.c. on BALB/c nu/nu mice, dual inhibition of VEGF and PDGF signaling significantly increased tumor growth delay versus each monotherapy. Interestingly, radiation at approximately 20% of the dose necessary to induce local tumor control exerts similar tumor growth-inhibitory effects as the antiangiogenic drugs given at their maximum effective dose. Addition of radiotherapy to both mono- as well as dual-antiangiogenic treatment markedly increased tumor growth delay. With respect to tumor angiogenesis, radiation further decreased microvessel density (CD31 count) and tumor cell proliferation (Ki-67 index) in all drug-treated groups. Of note, the slowly growing PC3 tumor responded better to the antiangiogenic drug treatments than the faster-growing U87 tumor. In addition to the beneficial effect of abrogating VEGF survival signaling when combined with radiation, we identified here a novel mechanism for the tumor escape from radiation damage. We found that radiation induced up-regulation of all four isoforms of PDGF (A-D) in endothelial cells supporting adjacent smooth muscle cells resulting in a prosurvival effect of radiation. The addition of SU6668 attenuated this undesirable paracrine radiation effect, which may rationalize the combined application of radiation with PDGF signaling inhibition to increase antitumor effects. CONCLUSION: A relative low radiation dose markedly enhances local antitumor effects of combined VEGF and PDGF signaling inhibition, suggesting a promising combination regimen for local tumor treatment with radiotherapy remaining an essential element.