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1.
Matrix Biol ; 82: 20-37, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30716392

RESUMO

Muscular fibrosis is caused by excessive accumulation of extracellular matrix (ECM) that replaces functional tissue, and it is a feature of several myopathies and neuropathies. Knowledge of the biology and regulation of pro-fibrotic factors is critical for the development of new therapeutic strategies. Upon unilateral sciatic nerve transection, we observed accumulation of ECM proteins such as collagen and fibronectin in the denervated hindlimb, together with increased levels of the profibrotic factors transforming growth factor type ß (TGF-ß) and connective tissue growth factor (CTGF/CCN2). In mice hemizygous for CTGF/CCN2 or in mice treated with a blocking antibody against CTGF/CCN2, we observed reduced accumulation of ECM proteins after denervation as compared to control mice, with no changes in fibro/adipogenic progenitors (FAPs), suggesting a direct role of CTGF/CCN2 on denervation-induced fibrosis. During time course experiments, we observed that ECM proteins and CTGF/CCN2 levels are increased early after denervation (2-4 days), while TGF-ß signaling shows a delayed kinetics of appearance (1-2 weeks). Furthermore, blockade of TGF-ß signaling does not decrease fibronectin or CTGF levels after 4 days of denervation. These results suggest that in our model CTGF/CCN2 is not up-regulated by canonical TGF-ß signaling early after denervation and that other factors are likely involved in the early fibrotic response following skeletal muscle denervation.

2.
Eur J Heart Fail ; 21(3): 272-285, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30714667

RESUMO

Fibrosis is a pivotal player in heart failure development and progression. Measurements of (markers of) fibrosis in tissue and blood may help to diagnose and risk stratify patients with heart failure, and its treatment may be effective in preventing heart failure and its progression. A lack of pathophysiological insights and uniform definitions has hampered the research in fibrosis and heart failure. The Translational Research Committee of the Heart Failure Association discussed several aspects of fibrosis in their workshop. Early insidious perturbations such as subclinical hypertension or inflammation may trigger first fibrotic events, while more dramatic triggers such as myocardial infarction and myocarditis give rise to full blown scar formation and ongoing fibrosis in diseased hearts. Aging itself is also associated with a cardiac phenotype that includes fibrosis. Fibrosis is an extremely heterogeneous phenomenon, as several stages of the fibrotic process exist, each with different fibrosis subtypes and a different composition of various cells and proteins - resulting in a very complex pathophysiology. As a result, detection of fibrosis, e.g. using current cardiac imaging modalities or plasma biomarkers, will detect only specific subforms of fibrosis, but cannot capture all aspects of the complex fibrotic process. Furthermore, several anti-fibrotic therapies are under investigation, but such therapies generally target aspecific aspects of the fibrotic process and suffer from a lack of precision. This review discusses the mechanisms and the caveats and proposes a roadmap for future research.

3.
J Transl Med ; 16(1): 301, 2018 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-30400798

RESUMO

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.

4.
Hum Mol Genet ; 27(16): 2913-2926, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-29860398

RESUMO

Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset progressive neurodegenerative disease characterized by upper and lower motoneuron degeneration. A total of 20% of familial ALS (fALS) cases are explained by mutations in the superoxide dismutase 1 (SOD1) enzyme. Although more than 20 years have passed since the generation of the first ALS mouse model, the precise molecular mechanisms of ALS pathogenesis remain unknown. CTGF/CCN2 is a matricellular protein with associated fibrotic activity that is up-regulated in several chronic diseases. The inhibition of CTGF/CCN2 with the monoclonal neutralizing antibody FG-3019 reduces fibrosis in several chronic disorders including the mdx mice, a murine model for Duchenne muscular dystrophy (DMD). In this work, we show that there are increased levels of CTGF/CCN2 in skeletal muscle and spinal cord of hSOD1G93A mice. In this scenario, we show evidence that FG-3019 not only reduces fibrosis in skeletal muscle of hSOD1G93A mice, but also improves muscle and locomotor performance. We demonstrate that treatment with FG-3019 reduces muscle atrophy in hSOD1G93A mice. We also found improvement of neuromuscular junction (NMJ) innervation together with a reduction in myelin degeneration in the sciatic nerve, suggesting that alterations in nerve-muscle communication are partially improved in FG-3019-treated hSOD1G93A mice. Moreover, we also found that CTGF/CCN2 is expressed in astrocytes and neurons, predominantly in dorsal areas of spinal cord from symptomatic hSOD1G93A mice. Together, these results reveal that CTGF/CCN2 might be a novel therapeutic target to ameliorate symptoms and improve the quality of life of ALS patients.

5.
Oncotarget ; 9(26): 18494-18509, 2018 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-29719620

RESUMO

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.

6.
Arthritis Rheumatol ; 70(11): 1745-1756, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29750395

RESUMO

OBJECTIVE: It has been reported that ADAMTS-12 is a susceptibility gene for rheumatoid arthritis (RA) development, and its level is significantly increased in RA patients. In addition, ADAMTS-12 is reported to be required for inflammation in otherwise healthy subjects. This study was undertaken to determine the role of ADAMTS-12 and the underlying mechanisms in the pathogenesis of inflammatory arthritis. METHODS: The collagen-induced arthritis (CIA) model was established in ADAMTS-12-deficient mice and their control littermates to determine the role of ADAMTS-12 in vivo. Micro-computed tomography scanning was used to demonstrate the destruction of the ankle joint; histologic analysis illustrated synovitis, pannus formation, and bone and cartilage destruction; enzyme-linked immunosorbent assay was performed to measure serum levels of inflammatory cytokines; and protein-protein interaction assays were performed to detect the interactions of ADAMTS-12 and its various deletion mutants with connective tissue growth factor (CTGF). RESULTS: Deficiency of ADAMTS-12 led to accelerated inflammatory arthritis in the CIA mouse model. Loss of ADAMTS-12 caused enhanced osteoclastogenesis. In vitro and in vivo protein-protein interaction assays demonstrated that ADAMTS-12 bound and processed CTGF, a previously unrecognized substrate of ADAMTS-12. In addition, deletion of ADAMTS-12 enhanced, while overexpression of ADMATS-12 reduced, CTGF-mediated inflammation. Furthermore, ADAMTS-12 regulation of inflammation was largely lost in CTGF-deficient macrophages. Importantly, blocking of CTGF attenuated elevated inflammatory arthritis seen in the ADAMTS-12-deficient CIA mouse model. CONCLUSION: This study provides evidence that ADAMTS-12 is a critical regulator of inflammatory arthritis and that this is mediated, at least in part, through control of CTGF turnover.

7.
Front Physiol ; 9: 138, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29535639

RESUMO

Chronic hypoxia frequently complicates the care of patients with interstitial lung disease, contributing to the development of pulmonary hypertension (PH), and premature death. Connective tissue growth factor (CTGF), a matricellular protein of the Cyr61/CTGF/Nov (CCN) family, is known to exacerbate vascular remodeling within the lung. We have previously demonstrated that vascular endothelial-cell specific down-regulation of CTGF is associated with protection against the development of PH associated with hypoxia, though the mechanism for this effect is unknown. In this study, we generated a transgenic mouse line in which the Ctgf gene was floxed and deleted in vascular endothelial cells that expressed Cre recombinase under the control of VE-Cadherin promoter (eCTGF KO mice). Lack of vascular endothelial-derived CTGF protected against the development of PH secondary to chronic hypoxia, as well as in another model of bleomycin-induced pulmonary hypertension. Importantly, attenuation of PH was associated with a decrease in infiltrating inflammatory cells expressing CD11b or integrin αM (ITGAM), a known adhesion receptor for CTGF, in the lungs of hypoxia-exposed eCTGF KO mice. Moreover, these pathological changes were associated with activation of-Rho GTPase family member-cell division control protein 42 homolog (Cdc42) signaling, known to be associated with alteration in endothelial barrier function. These data indicate that endothelial-specific deletion of CTGF results in protection against development of chronic-hypoxia induced PH. This protection is conferred by both a decrease in inflammatory cell recruitment to the lung, and a reduction in lung Cdc42 activity. Based on our studies, CTGF inhibitor treatment should be investigated in patients with PH associated with chronic hypoxia secondary to chronic lung disease.

8.
Diabetes Care ; 41(4): 840-846, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29382658

RESUMO

OBJECTIVE: Connective tissue growth factor (CTGF), also known as CCN2, is a potent chemotactic and extracellular matrix-inducing matricellular protein that has been implicated in progression of inflammatory and fibroproliferative disorders. An emerging role of CTGF/CCN2 is that of a prosclerotic factor implicated in the development of cardiac disease. Our objective was to determine the role of CTGF/CCN2 as a predictor of cardiovascular events in type 2 diabetes in the Veterans Affairs Diabetes Trial (VADT) cohort. RESEARCH DESIGN AND METHODS: Levels of CTGF/CCN2 were measured in 952 VADT patients a median of 1.9 years after entry into the study. Participants were followed for an average of 3.3 years for vascular outcomes. CTGF/CCN2 categories were defined as below the detectable limit (referent, 54.5%), lower half of detectable values (22.8%), and upper half of detectable values (22.7%). Hazard ratios (HRs) for cardiovascular end points in relation to CTGF/CCN2 categories were calculated by Cox proportional hazards models. RESULTS: During follow-up, 4.8% had a myocardial infarction (MI), 6.9% had an MI or cardiovascular death, and 6.9% died. After adjustments by conventional risk factors, individuals in the highest category of CTGF/CCN2 were at higher risk of MI (HR 2.43 [95% CI 1.15, 5.14]), MI or cardiovascular death (HR 2.71 [95% CI 1.44, 5.08]), and all-cause mortality (HR 2.70 [95% CI 1.43, 5.08]) relative to individuals with CTGF below the detectable limit. CONCLUSIONS: Our study indicates that high levels of CTGF/CCN2 predict future MI and cardiovascular death in patients with type 2 diabetes.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/sangue , Diabetes Mellitus Tipo 2/sangue , Infarto do Miocárdio/sangue , Estudos de Coortes , Diabetes Mellitus Tipo 2/complicações , Determinação de Ponto Final , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/mortalidade , Modelos de Riscos Proporcionais , Estudos Prospectivos , Fatores de Risco , Veteranos
9.
Respir Res ; 19(1): 14, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29347981

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 , 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 farmacológico
10.
Radiat Oncol ; 13(1): 1, 2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-29304828

RESUMO

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/genética
11.
Sci Rep ; 7(1): 5392, 2017 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-28710437

RESUMO

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.

12.
Arthritis Res Ther ; 19(1): 134, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28610597

RESUMO

BACKGROUND: Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. Although the involvement of connective tissue growth factor (CTGF/CCN2) has been well-documented in SSc fibrosis, the therapeutic potential of targeting CTGF in SSc has not been fully investigated. Our aim was to examine the therapeutic potential of CTGF blockade in a preclinical model of SSc using two approaches: smooth muscle cell fibroblast-specific deletion of CTGF (CTGF knockout (KO)) or a human anti-CTGF monoclonal antibody, FG-3019. METHODS: Angiotensin II (Ang II) was administered for 14 days by subcutaneous osmotic pump to CTGF KO or C57BL/6 J mice. FG-3019 was administered intraperitoneally three times per week for 2 weeks. Skin fibrosis was evaluated by histology and hydroxyproline assay. Immunohistochemistry staining was used for alpha smooth muscle actin (αSMA), platelet-derived growth factor receptor ß (PDGFRß), pSmad2, CD45, von Willebrand factor (vWF), and immunofluorescence staining was utilized for procollagen and Fsp1. RESULTS: Ang II-induced skin fibrosis was mitigated in both CTGF KO and FG-3019-treated mice. The blockade of CTGF reduced the number of cells expressing PDGFRß, procollagen, αSMA, pSmad2, CD45, and Fsp1 in the dermis. In addition, inhibition of CTGF attenuated vascular injury as measured by the presence of vWF-positive cells. CONCLUSIONS: Our data indicate that inhibition of CTGF signaling presents an attractive therapeutic approach in SSc.


Assuntos
Anticorpos Monoclonais/farmacologia , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Escleroderma Sistêmico/prevenção & controle , Pele/efeitos dos fármacos , Actinas/metabolismo , Angiotensina II , Animais , Anticorpos Monoclonais/imunologia , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/imunologia , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose/induzido quimicamente , Fibrose/prevenção & controle , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Escleroderma Sistêmico/imunologia , Pele/metabolismo , Pele/patologia
13.
J Natl Cancer Inst ; 109(8)2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28376190

RESUMO

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 (∼50%-80%) or reversed (∼50%) 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 , 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 X
14.
Hum Mol Genet ; 25(11): 2220-2233, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27131347

RESUMO

Cardiomyopathy caused by lamin A/C gene mutations (LMNA cardiomyopathy) is characterized by increased myocardial fibrosis, which impairs left ventricular relaxation and predisposes to heart failure, and cardiac conduction abnormalities. While we previously discovered abnormally elevated extracellular signal-regulated kinase 1/2 (ERK1/2) activities in heart in LMNA cardiomyopathy, its role on the development of myocardial fibrosis remains unclear. We now showed that transforming growth factor (TGF)-ß/Smad signaling participates in the activation of ERK1/2 signaling in LMNA cardiomyopathy. ERK1/2 acts on connective tissue growth factor (CTGF/CCN2) expression to mediate the myocardial fibrosis and left ventricular dysfunction. Studies in vivo demonstrate that inhibiting CTGF/CCN2 using a specific antibody decreases myocardial fibrosis and improves the left ventricular dysfunction. Together, these findings show that cardiac ERK1/2 activity is modulated in part by TGF-ß/Smad signaling, leading to altered activation of CTGF/CCN2 to mediate fibrosis and alter cardiac function. This identifies a novel mechanism in the development of LMNA cardiomyopathy.


Assuntos
Cardiomiopatias/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Fibrose/genética , Lamina Tipo A/genética , Fator de Crescimento Transformador beta/genética , Animais , Cardiomiopatias/patologia , Fibrose/patologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Smad/genética , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia
15.
Pharm Res ; 33(8): 1833-49, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27059922

RESUMO

PURPOSE: To evaluate and model the pharmacokinetic and pharmacodynamic behavior in rats of FG-3019, a human monoclonal antibody targeting connective tissue growth factor (CTGF). METHODS: FG-3019, human CTGF (rhCTGF), or the N-terminal domain of rhCTGF were administered intravenously to rats and concentrations of these proteins as well as endogenous CTGF were determined by immunoassays. FG-3019, or (125)I-labeled FG-3019, and human CTGF (rhCTGF) were co-administered to assess the impact of CTGF on the elimination rate and tissue localization of FG-3019, which was further characterized by immunohistochemical analysis. A PK/PD model for target-mediated elimination of FG-3019 was developed to fit the kinetic data. RESULTS: FG-3019 exhibited non-linear pharmacokinetics in rats. Circulating concentrations of the N-terminal half of CTGF increased after dosing with FG-3019, reached maximal levels after 1-5 days, and returned toward baseline levels as FG-3019 cleared from the circulation, whereas the concentration of intact CTGF was unaffected by administration of FG-3019. Co-administration of rhCTGF dramatically enhanced the rate of FG-3019 elimination, redistributing the majority of (125)I-labeled FG-3019 from the blood to the liver, kidney, spleen and adrenal gland. FG-3019 co-administered with CTGF was found along the sinusoids of the liver and adrenal glands, the capillaries of the kidney glomeruli and in the spleen. A pharmacokinetic model for target-mediated elimination of FG-3019 was used to fit the time courses of FG-3019 and endogenous CTGF plasma concentrations, as well as time courses of rhCTGF and rhCTGF N-fragment after intravenous administration of these species. CONCLUSIONS: FG-3019 is subject to target mediated elimination in rats.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Fator de Crescimento do Tecido Conjuntivo/administração & dosagem , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Administração Intravenosa , Animais , Anticorpos Monoclonais/metabolismo , Relação Dose-Resposta a Droga , Interações de Medicamentos/fisiologia , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologia
16.
J Mol Cell Cardiol ; 89(Pt B): 214-22, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26549358

RESUMO

Cardiac structural changes associated with dilated cardiomyopathy (DCM) include cardiomyocyte hypertrophy and myocardial fibrosis. Connective tissue growth factor (CTGF) has been associated with tissue remodeling and is highly expressed in failing hearts. Our aim was to test if inhibition of CTGF would alter the course of cardiac remodeling and preserve cardiac function in the protein kinase Cε (PKCε) mouse model of DCM. Transgenic mice expressing constitutively active PKCε in cardiomyocytes develop cardiac dysfunction that was evident by 3 months of age, and that progressed to cardiac fibrosis, heart failure, and increased mortality. Beginning at 3 months of age, PKCε mice were treated with a neutralizing monoclonal antibody to CTGF (FG-3149) for an additional 3 months. CTGF inhibition significantly improved left ventricular (LV) systolic and diastolic functions in PKCε mice, and slowed the progression of LV dilatation. Using gene arrays and quantitative PCR, the expression of many genes associated with tissue remodeling was elevated in PKCε mice, but significantly decreased by CTGF inhibition. However total collagen deposition was not attenuated. The observation of significantly improved LV function by CTGF inhibition in PKCε mice suggests that CTGF inhibition may benefit patients with DCM. Additional studies to explore this potential are warranted.


Assuntos
Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Envelhecimento/patologia , Animais , Anticorpos Neutralizantes/farmacologia , Cardiomiopatia Dilatada/patologia , Colágeno/metabolismo , Diástole/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibrose , Perfilação da Expressão Gênica , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Proteína Quinase C-épsilon/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sístole/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos
17.
Radiother Oncol ; 114(2): 173-81, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25592111

RESUMO

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 Xenoenxerto
18.
Oncotarget ; 6(7): 4663-76, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25609199

RESUMO

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 Xenoenxerto
19.
Hypertension ; 63(6): 1235-40, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24688123

RESUMO

Connective tissue growth factor (CTGF) is involved in the pathogenesis of various fibrotic disorders. However, its role in the heart is not clear. To investigate the role of CTGF in regulating the development of cardiac fibrosis and heart failure, we subjected mice to thoracic aortic constriction (TAC) or angiotensin II infusion, and antagonized the function of CTGF with CTGF monoclonal antibody (mAb). After 8 weeks of TAC, mice treated with CTGF mAb had significantly better preserved left ventricular (LV) systolic function and reduced LV dilatation compared with mice treated with control immunoglobulin G. CTGF mAb-treated mice exhibited significantly smaller cardiomyocyte cross-sectional area and reduced expression of hypertrophic marker genes. CTGF mAb treatment reduced the TAC-induced production of collagen 1 but did not significantly attenuate TAC-induced accumulation of interstitial fibrosis. Analysis of genes regulating extracellular matrix proteolysis showed decreased expression of plasminogen activator inhibitor-1 and matrix metalloproteinase-2 in mice treated with CTGF mAb. In contrast to TAC, antagonizing the function of CTGF had no effect on LV dysfunction or LV hypertrophy in mice subjected to 4-week angiotensin II infusion. Further analysis showed that angiotensin II-induced expression of hypertrophic marker genes or collagens was not affected by treatment with CTGF mAb. In conclusion, CTGF mAb protects from adverse LV remodeling and LV dysfunction in hearts subjected to pressure overload by TAC. Antagonizing the function of CTGF may offer protection from cardiac end-organ damage in patients with hypertension.


Assuntos
Anticorpos Monoclonais/farmacologia , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Insuficiência Cardíaca/complicações , Disfunção Ventricular Esquerda/prevenção & controle , Remodelação Ventricular/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Colágeno Tipo I/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/imunologia , Constrição Patológica/fisiopatologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Pressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Disfunção Ventricular Esquerda/etiologia , Suporte de Carga/fisiologia
20.
Invest Ophthalmol Vis Sci ; 54(13): 7836-48, 2013 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-24204045

RESUMO

PURPOSE: We have previously demonstrated elevated levels of connective tissue growth factor (CTGF/CCN2) in the aqueous humor (AqH) of pseudoexfoliation glaucoma (PXFG) patients when compared with cataract controls. Furthermore, there is a significant trabecular meshwork (TM) and lamina cribrosa (LC) fibrotic phenotype associated with glaucoma, possibly driven by CTGF. The purpose of this study was to investigate the potential of anti-CTGF immunotherapy in glaucoma. METHODS: Primary TM and LC cells were cultured from human donors with (GTM/GLC) and without (NTM/NLC) primary open angle glaucoma (POAG). Aqueous humor samples from PXFG, POAG, and control cataract patients were applied to N/GTM and N/GLC cells in the presence or absence of a therapeutic, humanized monoclonal anti-CTGF antibody FG-3019 (10 µg/mL). Hydrogen peroxide (H2O2) was also used as a stimulus. Expression of fibrotic genes (fibronectin-1, fibrillin-1, CTGF, collagen type I α1, and α-smooth muscle actin) was assessed by q-PCR. Protein expression of collagen 1A1 and α-smooth muscle actin was examined in N/G TM cells by SDS-PAGE. The modulatory effect of FG-3019 (10 µg/mL) and IgG (10 µg/mL) were also assessed. RESULTS: Treatment of cells with AqH from PXFG and POAG patients and H2O2 induced a significant (P < 0.05) increase in expression of profibrotic genes, which was significantly reduced by pretreatment with FG-3019 (P < 0.05). FG-3019 also reduced expression of α-smooth muscle actin and collagen 1A1 protein expression in N/GTM cells. CONCLUSIONS: FG-3019 is effective in blocking extracellular matrix production in TM and LC cells, thus supporting a role for the use of anti-CTGF immunotherapy in the treatment of glaucoma.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Matriz Extracelular/metabolismo , Glaucoma de Ângulo Aberto/tratamento farmacológico , Malha Trabecular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/imunologia , Feminino , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/patologia , Humanos , Masculino , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/patologia
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