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 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.

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.

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.

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.

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.

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.

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.

Connective tissue growth factor regulates cardiac function and tissue remodeling in a mouse model of dilated cardiomyopathy.

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.

Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves cell therapy.

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.

Connective tissue growth factor antibody therapy attenuates hyperoxia-induced lung injury in neonatal rats.

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.

Inhibition of platelet-derived growth factor signaling attenuates pulmonary fibrosis.

Pulmonary fibrosis is the consequence of a variety of diseases with no satisfying treatment option. Therapy-induced fibrosis also limits the efficacy of chemotherapy and radiotherapy in numerous cancers. Here, we studied the potential of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors (RTKIs) to attenuate radiation-induced pulmonary fibrosis. Thoraces of C57BL/6 mice were irradiated (20 Gy), and mice were treated with three distinct PDGF RTKIs (SU9518, SU11657, or Imatinib). Irradiation was found to induce severe lung fibrosis resulting in dramatically reduced mouse survival. Treatment with PDGF RTKIs markedly attenuated the development of pulmonary fibrosis in excellent correlation with clinical, histological, and computed tomography results. Importantly, RTKIs also prolonged the life span of irradiated mice. We found that radiation up-regulated expression of PDGF (A-D) isoforms leading to phosphorylation of PDGF receptor, which was strongly inhibited by RTKIs. Our findings suggest a pivotal role of PDGF signaling in the pathogenesis of pulmonary fibrosis and indicate that inhibition of fibrogenesis, rather than inflammation, is critical to antifibrotic treatment. This study points the way to a potential new approach for treating idiopathic or therapy-related forms of lung 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.

NK1.1+ cells mediate the antitumor effects of a dual Toll-like receptor 7/8 agonist in the disseminated B16-F10 melanoma model.

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.

Combination of vascular endothelial growth factor receptor/platelet-derived growth factor receptor inhibition markedly improves radiation tumor therapy.

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.

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.

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.

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.

Comparison of human B cell activation by TLR7 and TLR9 agonists.

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.

TLR7 agonist 852A inhibition of tumor cell proliferation is dependent on plasmacytoid dendritic cells and type I IFN.

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.