Role Of Connective Tissue Growth Factor Research Articles

The Role of Increased Connective Tissue Growth Factor in the Pathogenesis of Oral Submucous Fibrosis and its Malignant Transformation-An Immunohistochemical Study.

Connective tissue growth factor (CTGF), a matricellular protein of the CCN family of extracellular matrix-associated heparin-binding proteins, is highly expressed in various organ fibrosis and several malignant tumors. Although a few studies have been conducted using CTGF in oral submucous fibrosis (OSF) and oral squamous cell carcinoma, no study has demonstrated its relation with various stages of OSF and its malignant transformation. The present study investigated the possible role of CTGF in the pathogenesis of OSF and its malignant transformation by using immunohistochemistry.Ten formalin-fixed paraffin-embedded tissue blocks, each of Stage 1 OSF, Stage 2 OSF, Stage 3 OSF, Stage 4 OSF, well- differentiated squamous cell carcinoma (WDSCC) with OSF and WDSCC without OSF were stained for CTGF by immunohistochemistry. Ten cases of healthy buccal mucosa (NOM) were included as controls.The present study demonstrated a statistically significant expression of CTGF in the epithelium and connective tissue of OSF and WDSCC with and without OSF cases against its complete absence in NOM. We observed an upregulation of CTGF expression from NOM to various stages of OSF to WDSCC with or without OSF.A gradual upregulation of the CTGF expression in various stages of OSF to WDSCC (with and without OSF) against its complete absence in NOM suggests that CTGF plays an important role in the pathogenesis of OSF and its malignant transformation.

Connective tissue growth factor as an unfavorable prognostic marker promotes the proliferation, migration, and invasion of gliomas.

BackgroundIn consideration of the difficulty in diagnosing high heterogeneous glioma, valuable prognostic markers are urgent to be investigated. This study aimed to verify that connective tissue growth factor (CTGF) is associated with the clinical prognosis of glioma, also to analyze the effect of CTGF on the biological function.MethodsIn this study, glioma and non-tumor tissue samples were obtained in 2012 to 2014 from the Department of Neurosurgery of Nanfang Hospital of Southern Medical University, Guangzhou, China. Based on messenger RNA (mRNA) data from the Cancer Genome Atlas (TCGA) and CCGA dataset, combined with related clinical information, we detected the expression of CTGF mRNA in glioma and assessed its effect on the prognosis of glioma patients. High expression of CTGF mRNA and protein in glioma were verified by reverse transcription-polymerase chain reaction, immunohistochemistry, and Western blotting. The role of CTGF in the proliferation, migration, and invasion of gliomas were respectively identified by methylthiazoletetrazolium assay, Transwell and Boyden assay in vitro. The effect on glioma cell circle was assessed by flow cytometry. For higher expression of CTGF in glioblastoma (GBM), the biological function of CTGF in GBM was investigated by gene ontology (GO) analysis.ResultsIn depth analysis of TCGA data revealed that CTGF mRNA was highly expressed in glioma (GBM, n = 163; lowly proliferative glioma [LGG], n = 518; non-tumor brain tissue, n = 207; LGG, t = 2.410, GBM, t = 2.364, P < 0.05). CTGF mRNA and protein expression in glioma (86%) was significantly higher than that in non-tumor tissues (18%) verified by collected samples. Glioma patients with higher expression of CTGF showed an obviously poorer overall survival (35.4 and 27.0 months compared to 63.3 and 55.1 months in TCGA and Chinese Glioma Genome Atlas (CGGA) databases separately, CGGA: χ2 = 7.596, P = 0.0059; TCGA: χ2 = 10.46, P = 0.0012). Inhibiting CTGF expression could significantly suppress the proliferation, migration, and invasion of gliomas. CTGF higher expression had been observed in GBM, and GO analysis demonstrated that the function of CTGF in GBM was mainly associated with metabolism and energy pathways (P < 0.001).ConclusionsCTGF is highly expressed in glioma, especially GBM, as an unfavorable and independent prognostic marker for glioma patients and facilitates the progress of glioma.

The Role of CTGF in Inflammatory Responses Induced by Silica Particles in Human Bronchial Epithelial Cells.

Prolonged exposure to crystalline silica leads to persistent pulmonary inflammation and progressive fibrosis. Connective tissue growth factor (CTGF) has emerged as a potent proinflammatory and profibrotic regulator to participate in a variety of chronic inflammatory diseases. However, the role of CTGF in silica-induced pulmonary inflammation remains poorly understood. To explore the effect of CTGF on inflammatory responses caused by silica particles, human bronchial epithelial cells (16HBE) were transfected with CTGF siRNA and exposed to silica particles at concentrations of 0, 12.5, 25, 50, 100μg/ml for 48h. Intracellular CTGF mRNA and protein expressions were determined by RT-PCR and Western blotting, respectively. The levels of inflammatory cytokines including IL-8, TNF-α, IL-6, IL-1β, IL-17A and TGF-β1 were measured by ELISA kits. Silica particles induce significantly elevated intracellular CTGF mRNA expression in 16HBE cells in a dose-dependent manner when compared with blank control group (P < 0.05). The secretions of IL-8, TNF-α, IL-6 and IL-17A were also significantly increased by silica particles (P < 0.05). After exposure to 25 or 50μg/ml silica particles, the expression of intracellular CTGF mRNA was significantly inhibited in 16HBE cells when transfected with CTGF siRNA (P < 0.05). The secreted levels of IL-8, TNF-α, IL-6 and IL-17A induced by silica particles were also significantly lower from CTGF siRNA-transfected cells than that from normal 16HBE cells (P < 0.05). Inhibition of CTGF gene attenuates silica-induced inflammatory responses in bronchial epithelial cells, suggesting that CTGF could be a pivotal regulator in the development of silica-induced inflammation.

Abstract 3047: Connective tissue growth factor: Novel therapeutic target for non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer cases, includes adenocarcinoma, squamous cell lung carcinoma and large-cell lung carcinoma. Despite recent advances in targeted therapies for adenocarcinoma, they are effective in only a small percentage of patients while there are no targeted therapies available for squamous cell lung cancer. Therefore, it is critical to identify novel intervention strategies against NSCLC. In the present study, we have identified connective tissue growth factor (CTGF) as a possible therapeutic target for NSCLC. CTGF is an extracellular-matrix (ECM) associated protein, which is involved in cellular processes like cell adhesion, migration, proliferation, differentiation, survival, apoptosis and senescence. CTGF has been associated with both oncogenic as well as tumor suppressive activities in context of different cancer types. The function of CTGF in lung cancer pathogenesis remains controversial. We performed immunohistochemistry on tissue microarray containing 60 cases, which clearly suggested that CTGF expression increases in lung carcinoma. More specifically, among other subtypes, squamous cell lung cancer cases expressed more CTGF compared to their matched normal tissues. To determine the functional role of CTGF in NSCLC, we employed both genetic and pharmacologic approaches to inhibit CTGF activity. Our results clearly demonstrated that CTGF inhibition by both lentiviral knockdown as well as monoclonal antibody against CTGF (FG-3019) caused marked inhibition in cell proliferation, colony formation and xenograft growth in immunodeficient mice. We have also found that CTGF knockdown causes cell cycle arrest and apoptosis. Furthermore, we tested the efficacy of FG-3019 along with drugs like cisplatin and gemcitabine in patient-derived xenograft (PDX) model of squamous cell lung cancer. Our results demonstrated that CTGF inhibition enhances the effects of gemcitabine chemotherapy in PDX model of squamous cell lung carcinoma. These results clearly suggested that CTGF has a profound effect on lung tumorigenesis, as its inhibition not only causes attenuation in tumor growth but also potentiates the chemotherapeutic sensitivity of commonly used drugs for NSCLC. We have also elucidated that tumor hypoxia and Yap signaling are responsible for CTGF up-regulation in lung carcinoma. Overall, our study underscores the significance of CTGF inhibition in non-small cell lung carcinoma and offers the potential use of FG-3019 for its treatment. Citation Format: Priyanka Chaudhary, Marie-Liesse Labat, Guangfang Wang, Mark Onaitis. Connective tissue growth factor: Novel therapeutic target for non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3047.

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

BackgroundFibrosis 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.MethodsTo 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.ResultsAt 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.ConclusionsCTGF 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.

TGF-β1 Inhibits Human Trophoblast Cell Invasion by Upregulating Connective Tissue Growth Factor Expression.

Appropriate trophoblast invasion into the maternal endometrium is essential for successful human implantation and placentation. Connective tissue growth factor (CTGF), also known as CCN2, is a matricellular protein that is expressed in the placenta. Interestingly, the CTGF expression levels in the placenta and serum from patients with severe preeclampsia or fetal growth restriction are higher than those from healthy controls. However, to date, the role of CTGF in the regulation of trophoblast cell invasion remains unclear. Transforming growth factor-β1 (TGF-β1) is a potent stimulator of CTGF expression and has been shown to inhibit trophoblast cell invasiveness. However, whether CTGF mediates TGF-β1-inhibited human trophoblast cell invasion is unknown. In the present study, we show that treatment with TGF-β1 upregulates CTGF expression in a human trophoblast cell line, HTR-8/SVneo, and in primary human trophoblast cells. Our results also demonstrate that the SMAD2/3 signaling pathways are required for TGF-β1-induced upregulation of CTGF. Importantly, CTGF knockdown attenuates TGF-β1-inhibited cell invasion. Furthermore, cell invasiveness is decreased by treatment with recombinant CTGF. These results provide evidence that CTGF mediates TGF-β1-inhibited human trophoblast cell invasion.

Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson’s trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

Abstract 9809: Upregulation of Aldosterone by Steroidogenesis Acute Regulatory Protein (StAR) is Associated With Connective Tissue Growth Factor-Mediated Pulmonary Vascular Fibrosis in Pulmonary Arterial Hypertension In Vivo

Hyperaldosteronsim is associated with increased pulmonary vascular remodeling in patients with pulmonary arterial hypertension (PAH). In cardiomyocytes, aldosterone (ALDO) promotes collagen deposition by increasing levels of connective tissue growth factor (CTGF). The role of CTGF in ALDO-mediated vascular remodeling in PAH, however, is not known. We hypothesized that upregulation of CTGF by ALDO is a novel signaling mechanism that modulates pulmonary vascular fibrosis in PAH. To test this hypothesis, cultured human pulmonary artery endothelial cells were exposed to ALDO (10 -9 -10 -7 mol/l) for 24 hr and changes in CTGF levels were assessed by immunoblotting. Compared to vehicle control, ALDO induced a dose-dependent increase in CTGF expression (3.4 ± 0.2 vs. 6.7 ± 1.9 vs. 10.4 ± 1.6 vs. 17.6 ± 1.9 units, P=0.02). We next investigated the effect of ALDO on CTGF in PAH in vivo. Compared to controls, rats exposed to SU-5416 (20 mg/kg) and hypoxia (10% O 2 ) (Su/Hyp) for 21 d demonstrated increased pulmonary artery systolic pressure (PASP) (26 ± 2 vs. 81 ± 5 mmHg, P&lt;0.05), which was associated with a 397% (P&lt;0.02) increase in plasma ALDO levels. Next, we investigated changes in levels of steroidogenic acute regulatory protein (StAR) as a mechanism by which to account for hyperALDO in PAH. Compared to controls, immunohistochemistry revealed increased StAR in the pulmonary arterioles of Su/Hyp-PAH rats (19.2 ± 0.9 vs. 30.2 ± 0.5 units, P&lt;0.01) and in patients with PAH (14.3 ± 1.4 vs. 19.3 ± 0.9 units, P&lt;0.02). Immunohistochemistry revealed that compared to controls, Su/Hyp-PAH rats expressed increased levels of CTGF (88.1 ± 11.5 vs. 171.8 ± 5.9 units, P&lt;0.01) and collagen III (61.5 ± 18.7 vs. 126.5 ± 6.3 units, P&lt;0.01) in pulmonary arterioles. ALDO antagonism with eplerenone (0.6 mg/g chow) decreased CTGF and collagen III levels in SU/Hyp-PAH by 81% (p&lt;0.01) and 38% (P&lt;0.01), respectively, to decrease PASP (81 ± 5 vs. 62 ± 7 mmHg, P&lt;0.05). These data demonstrate that upregulation of StAR is associated with hyperALDO in PAH, which promotes vascular fibrosis through a mechanism involving CTGF. Identifying StAR and/or CTGF as novel treatment target(s) to attenuate pulmonary vascular fibrosis has potential therapeutic implications for patients with PAH.

Abstract B58: Connective tissue growth factor (CTGF) as a novel therapeutic target in high grade serous ovarian cancer

Abstract The tumor microenvironment plays an active role in promoting proliferation, invasion and metastasis, thereby making soluble factors secreted by cancer-associated fibroblasts promising therapeutic targets. As a result, secreted tumor stromal factors are increasingly being targeted by newly developed anti-cancer therapies. To identify such stromal factors in ovarian cancer, we performed gene expression profiling on microdissected fibroblasts from normal ovary and from high-grade, late stage serous ovarian tumors. We identified a signature of TGFb-regulated genes, over-expressed in cancer-associated fibroblasts, which encode for secreted factors that may serve as potential therapeutic targets. One of these genes, connective tissue growth factor (CTGF), is a secreted protein that promotes proliferation, motility and extracellular matrix production in a number of cancer types. CTGF is currently being pursued as a therapeutic target by FibroGen Inc., who has developed a therapeutic anti-CTGF monoclonal antibody FG-3019. FG-3019 is currently under clinical investigation in pancreatic cancer and fibrotic diseases. To determine the role of CTGF and its potential as a therapeutic target in high-grade serous ovarian cancer (HGSOC), we examined its effect on tumor promotion in ovarian cancer cell lines and associations between CTGF expression and clinico-pathologic characteristics in HGSOC patients. We performed functional studies in SKOV3 and OVCAR3 cell lines using recombinant protein, demonstrating that CTGF promotes anchorage-independent proliferation and migration. These effects of CTGF are blocked by FG-3019. During cancer progression, HGSOC cells from the ovary disseminate and adhere throughout the peritoneal cavity, posing a major challenge in treatment. We and others have shown that mesothelial cells in peritoneal tissue, peritoneal fibroblasts and ovarian cancer omental metastases express CTGF, suggesting that it may be targeted to help prevent recurrence following debulking surgery. To address this, we performed ex-vivo peritoneal adhesion assays, and demonstrate that recombinant CTGF increases a5b1 integrin-mediated adhesion of ovarian cancer cells to peritoneal tissue. In addition, enhancement of peritoneal adhesion by CTGF was blocked upon addition of FG-3019, further supporting its potential as a therapeutic target. In investigating clinico-pathologic characteristics in HGSOC patients, we confirmed over-expression of CTGF in cancer-associated fibroblasts by immunohistochemistry. Furthermore, we found that survival was correlated with CTGF expression, with HGSOC patients expressing the highest levels of tumor stromal CTGF harboring the poorest prognosis. Together, our findings identify and characterize CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in HGSOC. Further studies should investigate the therapeutic efficacy of FG-3019 in combination with platinum/taxane, in HGSOC. Citation Format: Kim Moran-Jones, Rajmohan Murali, David K. Chang, Carol W. Wong, Suzanne M. Spong, Neville F. Hacker, Samuel C. Mok, Michael J. Birrer, Goli Samimi. Connective tissue growth factor (CTGF) as a novel therapeutic target in high grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B58.

Connective tissue growth factor induces osteogenic differentiation of vascular smooth muscle cells through ERK signaling

Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of atherosclerosis by promoting vascular smooth muscle cell (VSMC) growth, migration, apoptosis, adhesion and the secretion of matrix components. The osteogenic differentiation of VSMCs is essential in the development of vascular calcification. However, the role of CTGF in the transdifferentiation and calcification of VSMCs is unclear. In the present study, we examined whether CTGF stimulates VSMC transdifferentiation. Primary VSMCs were obtained from mouse thoracic aortas by enzymatic digestion and identified by immunostaining for smooth muscle specific α-actin antibody (α-SMA). VSMC calcification was induced by the addition of CTGF to the osteogenic mediaum containing 5-10% FBS in the presence of 0.25 mM ascorbic acid and 10 mM β-glycerophosphate for 14 days. Calcified cells were determined by Alizarin Red S staining. Our results revealed that CTGF induced the expression of several bone markers, including alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG) and core-binding factor subunit α1 (Cbfα1)/runt-related transcription factor 2 (Runx2), as well as calcification. However, the inhibition of extracellular signal-regulated kinase (ERK) activity using the ERK-specific inhibitor, PD98059, blocked the induction of these proteins and VSMC calcification. Based on these data, we conclude that CTGF stimulates the transdifferentiation of VSMCs into osteoblasts and that the ERK signaling pathway appears to play a critical role in this process.

EFFECT OF HIGH GLUCOSE ON THE EXPRESSION OF CONNECTIVE TISSUE GROWTH FACTOR IN THE CULTURED CARDIOMYOCYTES OF NEONATAL RATS

ObjectivesTo investigate the effect of high glucose on the expression of connective tissue growth factor (CTGF) in the cultured cardiomyocytes of neonatal rats and the role of CTGF in the...

Abstract 2971: Human extramedullary bone and bone marrow in mice: First in vivo model of a genetically controlled hematopoietic environment - Role of CTGF and HIF1-α

Abstract The microenvironment contributes and may regulate cancer development, progression and resistance to treatment. Our group reported first the contribution of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) for tumor development and metastasis. Accumulating evidence has shown that the BM microenvironment also plays a pivotal role in the pathophysiology and propagation of leukemia. We here describe a novel, genetically controlled bone and bone marrow model using human BM derived MSCs and endothelial colony-forming cells (ECFCs) subcutaneously injected into the flanks of NOD/SCID/IL-2rαnull mice, where they developed into bone-like tissues with high osteoblast activity after 10 weeks, with bone structures and typical bone marrow cavities constituting a robust hematopoietic environment. In vivo imaging with Osteosense confirmed the presence of hydroxylapatite, and luciferase imaging of firefly-luciferase labeled human leukemic cells demonstrated the engraftment of human MOLM13/Luc/GFP leukemic cells. The extramedullary leukemic BM was markedly hypoxic, as shown by Pimonidazole staining. Factors critical for MSC to support normal and leukemic hematopoiesis are largely unknown and cannot readily be studied since human MSC do not engraft reliably in xenograft models. We therefore investigated the possibility of genetically modifying MSC in this system and found a significant reduction (50 ± 6%, p&amp;lt;0.001) in leukemial engraftment in extramedullary BM generated with HIF1α knockdown MSCs (1449 ± 194 cells/mm2), compared to vector controls (3037 ± 496 cells/mm2). This finding indicates that HIF1α expression in stromal cells is critical for the engraftment of leukemic cells. Surprisingly, shRNA knockdown of connective tissue growth factor (CTGF) in MSC resulted in the 2-3 fold increased expression of embryonic stem cell markers oct-4, nanog and sox2, increased adipogenic differentiation (PPAR-γ and C/EBβ) and in formation of a more spongy bone. Human leukemic Nalm-6 cells showed 3-fold higher migration to CTGF-KD as compared to control BM, a surprising finding that could mechanistically be related to the 3-fold increased expression of SDF-1 in CTGF-KD-MSC. Conclusion: We have developed a genetically controlled human extra-medullary bone marrow system that allows investigations of the role of specific genes on the homing and expansion of normal and leukemic cells/stem cells in vivo. HIF-1α and CTGF were shown to have distinct and unexpected functions in this system. Results gain further relevance by the recent demonstration of genetic abnormalities in human leukemic bone marrow cells (Andreeff 2008, Blau 2011). The system has allows us to established an in vivo bone and bone marrow model with a genetically controlled human microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2971. doi:1538-7445.AM2012-2971

Connective tissue growth factor antagonizes transforming growth factor-β1/Smad signalling in renal mesangial cells

The critical involvement of TGF-β1 (transforming growth factor-β1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-β1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-β1 and its physiological significance. CTGF was determined to bind directly to the TβRIII (TGF-β typeIII receptor) and antagonize TGF-β1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-β1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-β1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-β1-treated mesangial cells. Decreased levels of Smad2/3 phosphorylation were evident in STZ (streptozotocin)-induced diabetic mice, concomitant with increased levels of CTGF. Knockdown of TβRIII restored TGF-β1-mediated Smad signalling and cell contractility, suggesting that TβRIII is key for CTGF-mediated regulation of TGF-β1. Comparison of gene expression profiles from CTGF/TGF-β1-treated mesangial cells and human renal biopsy material with histological diagnosis of DN revealed significant correlation among gene clusters. In summary, mesangial cell responses to TGF-β1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.

Abstract P224: Connective Tissue Growth Factor Plays a Key Role in Myocardial Fibrosis After Angiotensin II Exposure

Background: AngiotensinII (AngII) exposure to rodents is a common model of fibrosis, characterized by hypertension, hypertrophy and eventual deposition of excess extracellular matrix (ECM) proteins resulting in organ dysfunction. We have previously shown that cellular infiltration of bone marrow derived progenitor cells (fibrocytes) occurs prior to ECM deposition and is associated with production of the connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-β). The objective of this project was to characterize the role of CTGF in promoting fibrocyte recruitment and subsequent fibrosis after AngII exposure. Methods: Mice were treated with AngII or saline using an osmotic mini-pump at 2.8mg/kg/day. After 6hr to 7d hearts were excised and embedded in paraffin or prepared for mRNA isolation. Immunohistochemistry was used to determine extent and localization of CTGF protein. Quantitative RT-PCR was used to determine relative CTGF and TGF-β mRNA levels. Primary cardiomyocyte and fibrocyte cultures were isolated from neonatal or 3d AngII exposed animals respectively. Primary cultures were stimulated to determine the source (qRT-PCR) and function (proliferation, migration and differentiation) of CTGF. Results: In animals exposed to AngII, CTGF mRNA peaked the earliest at 6hr (21-fold; p&lt;0.01) when compared to TGFbeta, which peaked at 3d (5-fold; p&lt;0.05). Concurrent CTGF protein expression was evident by 3d of AngII exposure and appeared localized to the cardiomyocytes. Findings were confirmed using isolated cardiomyocytes, which significantly increased expression of CTGF in response to AngII (2-fold; p&lt;0.05). While CTGF did not promote fibrocyte migration in transwell chamber assay it promoted significant fibrocyte proliferation in vitro (2-fold; p&lt;0.05). Conclusion: We provide strong evidence that AngII exposure first results in the production of CTGF by cardiomyocytes. Furthermore, we have shown that CTGF does not promote migration as a chemokine but instead contributes to proliferation of fibrocytes once recruited from the bone marrow into the myocardium.

Abstract 509: The CTGF antibody FG-3019 blocks CTGF-stimulated migration in ovarian cancer cells

Abstract Ovarian tumors generally arise from the surface epithelium of the ovary, and require interactions with host stroma to promote tumor cell proliferation and expansion. The signaling mechanisms between tumor-associated stroma and adjacent ovarian epithelial tumor cells remain unclear. We have previously analyzed gene expression profiles of laser-capture microdissected stromal cells from normal ovary and ovarian tumors and have demonstrated that normal ovarian stroma undergoes significant gene expression changes in response to the epithelial tumor microenvironment. Connective Tissue Growth Factor (CTGF), a TGF-β-regulated gene, was identified as specifically up-regulated in tumor-associated versus normal stroma. CTGF is a component of the tumor microenvironment with a demonstrated role in tumor progression and metastasis. FG-3019, a fully human monoclonal antibody against CTGF, is currently under clinical investigation in pancreatic cancer. The aim of this current study was to further elucidate the role of CTGF in ovarian cancer and examine its potential as a therapeutic target. We demonstrate that normal ovarian fibroblasts and ovarian cancer-associated fibroblasts treated with TGF-β secreted high levels of CTGF, whereas ovarian cancer epithelial cells secreted significantly lower levels. We next assessed the paracrine effect of CTGF on three different ovarian cancer cell lines, A224, OVCAR3, and SKOV3. Expression of endogenous CTGF is low in these cells which is consistent with the level found in primary ovarian tumor cells. Addition of recombinant CTGF for 6-hr stimulated migration of A224 (443 ± 20.5 vs. 85 ± 24.4 cells), OVCAR3 (104 ± 11.7 vs. 68 ± 30.1 cells) and SKOV3 (334 ± 45.6 vs. 82 ± 16.6 cells) in a transwell migration assay. Addition of the CTGF-blocking antibody FG-3019 significantly inhibited transwell migration in the presence of recombinant CTGF in all three cell lines (from 443 ± 20.5 to 197 ± 65.4 cells in A224, 334 ± 45.6 to 119 ± 29.0 cells in SKOV3 and 104 ± 11.7 to 36 ± 2.5 cells in OVCAR3). However, recombinant CTGF did not promote proliferation of these cell lines. We demonstrate that CTGF may contribute to ovarian tumor biology and that its function can be blocked by FG-3019, thus determining CTGF as a potential therapeutic target in ovarian tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 509. doi:10.1158/1538-7445.AM2011-509

Abstract 3474: Connective tissue growth factor promotes tumor angiogenesis by modulating multiple angiogenic factors via binding to cystine knot motifs

Abstract Introduction Connective tissue growth factor (CTGF), a member of the CCN (Cyr61/CTGF/Nov) protein family, may function as both a positive and negative regulator of angiogenesis. However, the mechanism(s) underlying the regulatory behavior of CTGF is poorly understood. Here, we have undertaken a multipronged approach to provide insight into how CTGF enhances tumor angiogenesis. Methods Lewis lung carcinomas (LLC) cells were subcutaneously injected in syngeneic transgenic mice expressing green fluorescent protein (GFP) under the control of CTGF promoter (CTGFp-GFP). Recombinant lentivirus was generated to overexpress FLAG tagged CTGF while recombinant adenovirus expressing CTGF shRNA was used to knock down CTGF expression in LLCs. The levels of CTGF mRNA were examined by RT-PCR analysis. Tumor sizes were measured within two weeks and intratumoral microvessel density was quantified by immunochemical detection of Meca-32+ blood vessels. In addition, CTGF binding proteins were identified using the yeast two-hybrid approach. Immunoprecipitation assay was performed to verify these interactions. In vitro angiogenesis activity was indicated by tubule length formation by human umbilical vein endothelial cells (HUVECs). The migratory activity was measured in Boyden chamber assay. Activation of downstream signal effectors including cell division cycle 42 GTP binding protein (Cdc42) and extracellular signal-regulated kinase (ERK)1/2 was detected in Western blotting analysis. Results CTGF was expressed in Meca-32+ endothelial cells of intratumoral vessels growing in CTGFp-GFP transgenic mice. CTGF expression was also highly induced in hypoxic cultured LLC cells. Ectopic expression of CTGF promoted LLC tumor growth (n=3) and the microvessel density. Knocking down CTGF expression in LLC tumors (n=3) inhibited the growth of LLC tumors and decreased the microvessel density compared to those with scramble shRNA. Several proangiogenic factors were identified and shown to bind CTGF via cystine knot motifs. The factors include vascular endothelial growth factor (VEGF)-A, von Willebrand factor (vWF), platelet derived growth factor (PDGF)-B and Slit3. The combination of CTGF and Slit3 together resulted in an increased tubule length and an enhanced activation of Cdc42. Elevated cell migration and enhanced activation of ERK1/2 were observed when NIH3T3 fibroblasts were cultured with both CTGF and PDGF-B proteins. Although CTGF has been shown to bind to VEGF-A and inhibits VEGF-A mediated angiogenesis, our findings demonstrate that CTGF functions in concert with angiogenic factors Slit3 and PDGF-B to promote angiogenesis. Conclusion This study shows a critical role for CTGF in LLC tumor angiogenesis. CTGF binding to cystine knot motifs of key angiogenic factors represent a novel mechanism to regulate angiogenesis through PDGF-B and Slit3 signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3474. doi:10.1158/1538-7445.AM2011-3474

The Role of Connective Tissue Growth Factor (CTGF/CCN2) in Skeletogenesis

Connective tissue growth factor (CTGF) is a 38 kDa, cysteine rich, extracellular matrix protein composed of 4 domains or modules. CTGF has been shown to regulate a diverse array of cellular functions and has been implicated in more complex biological processes such as angiogenesis, chondrogenesis, and osteogenesis. A role for CTGF in the development and maintenance of skeletal tissues first came to light in studies demonstrating its expression in cartilage and bone cells, which was dramatically increased during skeletal repair or regeneration. The physiological significance of CTGF in skeletogenesis was confirmed in CTGF-null mice, which exhibited multiple skeletal dysmorphisms as a result of impaired growth plate chondrogenesis, angiogenesis, and bone formation/mineralization. Given the emerging importance of CTGF in osteogenesis and chondrogenesis, this review will focus on its expression in skeletal tissues, its effects on osteoblast and chondrocyte differentiation and function, and the skeletal implications of ablation or over-expression of CTGF in knockout or transgenic mouse models, respectively. In addition, this review will examine the role of integrin-mediated signaling and the regulation of CTGF expression as it relates to skeletogenesis. We will emphasize CTGF studies in bone or bone cells, and will identify opportunities for future investigations concerning CTGF and chondrogenesis/osteogenesis.

Elevated Expression of Connective Tissue Growth Factor in Human Atrial Fibrillation and Angiotensin II-Treated Cardiomyocytes

Atrial fibrosis is a feature of structural remodeling in atrial fibrillation (AF). Connective tissue growth factor (CTGF) is a potent profibrotic factor, but its role of CTGF in AF is not yet fully understood. Right atrial appendages were obtained from 20 patients who underwent cardiac surgery (10 with sinus rhythm, 10 with AF). The mRNA level, protein level and immunohistochemical staining of CTGF were significantly increased in AF patients. In a porcine AF model, tissue angiotensin II (Ang II) and CTGF levels were significantly upregulated in both atria. In perfused rat hearts, Ang II stimulation increased CTGF expression, which could be inhibited by Ang II type I receptor antagonist. In a cell culture system, both atrial fibroblasts and myocytes were responsible for the increased CTGF expression under Ang II treatment. Ang II type I receptor antagonist could inhibit the Ang II-induced CTGF expression. Treating with recombinant CTGF, atrial fibroblasts expressed an increased level of collagen I. Furthermore, the CTGF level was highly correlated with tissue Ang II content in AF pigs. AF patients and animals exhibited a significantly increased expression of CTGF. Ang II stimulation upregulated CTGF expression in both atrial fibroblasts and myocytes. Ang II-induced CTGF expression might be involved in atrial substrate remodeling.

The role of CTGF in the diabetic rat retina and its relationship with VEGF and TGF‐β2, elucidated by treatment with CTGFsiRNA

The critical association of connective tissue growth factor (CTGF) with diabetic retinopathy (DR) remains to be clarified. We detected alterations in the gene and protein expression of CTGF and related cytokines, including vascular endothelial growth factor (VEGF) and transforming growth factor-β(2) (TGF-β(2) ), and their response to small interfering RNA (siRNA) targeting the CTGF (CTGFsiRNA) in the retina of diabetic rats. The relationships between CTGF, VEGF and TGF-β(2) levels, as well as the degree of apoptosis in the diabetic retina, were also investigated. Diabetes was induced in rats by the β-cell toxin streptozotocin (STZ). Retinas were obtained from control and diabetic rats and similar animals treated with CTGFsiRNA by intravitreal injection. mRNA level and protein expression of CTGF, VEGF and TGF-β(2) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and located by immunohistochemistry. Retinal apoptosis was detected by TUNEL staining. The levels of CTGF, VEGF and TGF-β(2) and the number of TUNEL-positive nuclei were significantly higher in diabetic retinas than in control retinas (p<0.01). The level of CTGF rose at 8weeks, earlier than levels of VEGF and TGF-β(2) , which rose at 12weeks after the onset of diabetes. The difference was significant (p<0.05). siRNA-mediated inhibition of CTGF mRNA inhibited retinal VEGF and TGF-β(2) and also resulted in a significant decrease in apoptosis. Significant correlations were found between CTGF and VEGF (p=0.009), CTGF and TGF-β(2) (p=0.01), and apoptosis and these three cytokines (p<0.01) in the rat retina early in diabetes. These results suggest that the diabetes-mediated increase in CTGF upregulates VEGF and TGF-β(2) expression and induces apoptosis in the retina. This elevation may be inhibited by treatment with CTGFsiRNA. Connective tissue growth factor may serve as a potential target for the prevention and treatment of DR.

Significance of Serum Connective Tissue Growth Factor in Patients with Hepatocellular Carcinoma and Relationship with Angiogenesis

Connective tissue growth factor (CTGF) expression changes variously in different human malignancies. The role of CTGF in hepatocellular carcinoma (HCC) is still not clear. The purpose of this study was to investigate the role of serum CTGF in patients with HCC and its correlation with HCC angiogenesis. CTGF, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) were measured by ELISA in preoperative sera of 88 patients with HCC with tumor resection and 39 healthy subjects. The relationship between CTGF and HCC clinicopathological parameters was observed. Prognostic significance of CTGF for survival of patients with HCC was assessed by Kaplan-Meier analysis. Preoperative serum CTGF level was significantly higher in patients with HCC than in healthy subjects (median, 63.5 vs. 11.4 ng/ml; P < 0.001). Serum CTGF correlated significantly with a series of clinicopathological parameters (big tumor size, advanced pathological tumor-node-metastasis stage, absence of tumor capsule, portal vein invasion). Serum CTGF showed a significant correlation with disease-free survival and overall survival of patients with HCC. Patients with high serum CTGF (>63.5 ng/ml) had a poorer disease-free survival time than the others (CTGF < or = 63.5 ng/ml; median disease-free survival time, 9.6 vs. 19.3 months). Patients with high serum CTGF had poorer overall survival time (median, 13.1 months) than the others (median, 21.7 months). In multivariate Cox analysis, CTGF was identified as an independent and significant prognostic factor of survival of HCC patients. Serum CTGF plays an important role in the progression of HCC. Serum CTGF may be a potential indicator of angiogenesis of HCC.