Mechanical Stress-induced Connective Tissue Growth Factor Plays a Critical Role in Intestinal Fibrosis in Crohn's-like Colitis.

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 pro-fibrotic 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 (SMC). 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 towards normalizing expression of collagen mRNAs. In conclusion, our studies suggest that mechanical stress, by up-regulating pro-fibrotic mediators i.e. CTGF, may play a critical role in fibrosis in CD.

Connective-Tissue Growth Factor Contributes to TGF-ß1-induced Lung Fibrosis.

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.

Driving fibrosis in neuromuscular diseases: Role and regulation of Connective tissue growth factor (CCN2/CTGF).

Connective tissue growth factor or cellular communication network 2 (CCN2/CTGF) is a matricellular protein member of the CCN family involved in several crucial biological processes. In skeletal muscle, CCN2/CTGF abundance is elevated in human muscle biopsies and/or animal models for diverse neuromuscular pathologies, including muscular dystrophies, neurodegenerative disorders, muscle denervation, and muscle overuse. In this context, CCN2/CTGF is deeply involved in extracellular matrix (ECM) modulation, acting as a strong pro-fibrotic factor that promotes excessive ECM accumulation. Reducing CCN2/CTGF levels or biological activity in pathological conditions can decrease fibrosis, improve muscle architecture and function. In this work, we summarize information about the role of CCN2/CTGF in fibrosis associated with neuromuscular pathologies and the mechanisms and signaling pathways that regulate their expression in skeletal muscle.

Role of hypoxia in skeletal muscle fibrosis: Synergism between hypoxia and TGF-ß signaling upregulates CCN2/CTGF expression specifically in muscle fibers.

Several skeletal muscle diseases are characterized by fibrosis, the excessive accumulation of extracellular matrix. Transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CCN2/CTGF) are two profibrotic factors augmented in fibrotic skeletal muscle, together with signs of reduced vasculature that implies a decrease in oxygen supply. We observed that fibrotic muscles are characterized by the presence of positive nuclei for hypoxia-inducible factor-1α (HIF-1α), a key mediator of the hypoxia response. However, it is not clear how a hypoxic environment could contribute to the fibrotic phenotype in skeletal muscle. We evaluated the role of hypoxia and TGF-ß on CCN2 expression in vitro. Fibroblasts, myoblasts and differentiated myotubes were incubated with TGF-ß1 under hypoxic conditions. Hypoxia and TGF-ß1 induced CCN2 expression synergistically in myotubes but not in fibroblasts or undifferentiated muscle progenitors. This induction requires HIF-1α and the Smad-independent TGF-ß signaling pathway. We performed in vivo experiments using pharmacological stabilization of HIF-1α or hypoxia-induced via hindlimb ischemia together with intramuscular injections of TGF-ß1, and we found increased CCN2 expression. These observations suggest that hypoxic signaling together with TGF-ß signaling, which are both characteristics of a fibrotic skeletal muscle environment, induce the expression of CCN2 in skeletal muscle fibers and myotubes.

Connective Tissue Growth Factor Inhibition Enhances Cardiac Repair and Limits Fibrosis After Myocardial Infarction.

Myocardial infarction (MI)-induced cardiac fibrosis attenuates cardiac contractile function, and predisposes to arrhythmias and sudden cardiac death. Expression of connective tissue growth factor (CTGF) is elevated in affected organs in virtually every fibrotic disorder and in the diseased human myocardium. Mice were subjected to treatment with a CTGF monoclonal antibody (mAb) during infarct repair, post-MI left ventricular (LV) remodeling, or acute ischemia-reperfusion injury. CTGF mAb therapy during infarct repair improved survival and reduced LV dysfunction, and reduced post-MI LV hypertrophy and fibrosis. Mechanistically, CTGF mAb therapy induced expression of cardiac developmental and/or repair genes and attenuated expression of inflammatory and/or fibrotic genes.

Denervation-induced skeletal muscle fibrosis is mediated by CTGF/CCN2 independently of TGF-ß.

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.

Towards better definition, quantification and treatment of fibrosis in heart failure. A scientific roadmap by the Committee of Translational Research of the Heart Failure Association (HFA) of the European Society of Cardiology.

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.

Analysis of longitudinal semicontinuous data using marginalized two-part model.

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.

The inhibition of CTGF/CCN2 activity improves muscle and locomotor function in a murine ALS model.

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.

Role of ADAMTS-12 in Protecting Against Inflammatory Arthritis in Mice By Interacting With and Inactivating Proinflammatory Connective Tissue Growth Factor.

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.

Connective tissue growth factor-specific monoclonal antibody inhibits growth of malignant mesothelioma in an orthotopic mouse model.

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.

Vascular Endothelial Cell-Specific Connective Tissue Growth Factor (CTGF) Is Necessary for Development of Chronic Hypoxia-Induced Pulmonary Hypertension.

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.

Plasma Connective Tissue Growth Factor (CTGF/CCN2) Levels Predict Myocardial Infarction in the Veterans Affairs Diabetes Trial (VADT) Cohort.

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.

Oncogene addiction and radiation oncology: effect of radiotherapy with photons and carbon ions in ALK-EML4 translocated NSCLC.

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.

Radiation-induced pulmonary gene expression changes are attenuated by the CTGF antibody Pamrevlumab.

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.

Inhibition of CTGF ameliorates peritoneal fibrosis through suppression of fibroblast and myofibroblast accumulation and angiogenesis.

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.

Anti-connective tissue growth factor (CTGF/CCN2) monoclonal antibody attenuates skin fibrosis in mice models of systemic sclerosis.

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.

Effects of CTGF Blockade on Attenuation and Reversal of Radiation-Induced Pulmonary Fibrosis.

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.

ERK1/2 directly acts on CTGF/CCN2 expression to mediate myocardial fibrosis in cardiomyopathy caused by mutations in the lamin A/C gene.

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.

FG-3019, a Human Monoclonal Antibody Recognizing Connective Tissue Growth Factor, is Subject to Target-Mediated Drug Disposition.

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.