Blockade Of Connective Tissue Growth Factor Research Articles

M2-polarized macrophages mediate wound healing by regulating connective tissue growth factor via AKT, ERK1/2, and STAT3 signaling pathways.

Timely and sufficient M1 recruitment and M2 polarization are necessary for fibrosis during wound healing. The mechanism of how M2 mediates wound healing is worth exploring. Abnormally up-regulated connective tissue growth factor (CTGF) influences multiple organ fibrosis, including cardiac, pulmonary, hepatic, renal, and cutaneous fibrosis. Previous studies reported that M2 contributed to hepatic and renal fibrosis by secreting CTGF. It is worth discussing if M2 regulates fibrosis through secreting CTGF in wound healing. We established the murine wound model and inhibited macrophages during proliferation phase with clodronate liposomes in vivo. Macrophages depletion led to down-regulation of wound healing rates, collagen deposition, as well as expression of collagen 1/3 and Ki67. M2 was induced by interleukin-4 (IL-4) and measured by flow cytometry in vitro. Secreted pro-fibrotic and anti-fibrotic factors were tested by enzyme-linked immunosorbent assay (ELISA). M2 was polarized, which producing more CTGF, transforming growth factor-beta1 (TGF-β1), and IL-6, as well as less tumor necrosis factor-α (TNF-α) and IL-10. M2 CTGF gene was blocked using siCTGF. Effects of M2 on fibroblasts activities were detected by cell counting kit 8 (CCK8) and cellular wound healing assay. Expressions of related signaling pathway were assessed by western blotting. Blockade of CTGF in M2 deactivated fibroblasts proliferation and migration by regulating AKT, ERK1/2, and STAT3 pathway. Recombinant CTGF restored these effects. Our research, for the first time, indicated that M2 promoted wound healing by secreting CTGF, which further mediating proliferation and migration of fibroblasts via AKT, ERK1/2, and STAT3 pathway.

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-β1-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.

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

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. 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. 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. 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.

TGF-β2 induces transdifferentiation and fibrosis in human lens epithelial cells via regulating gremlin and CTGF

Transforming growth factor (TGF)-β2, gremlin and connective tissue growth factor (CTGF) are known to play important roles in the induction of epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) synthesis. However, the complex functional relationship among gremlin, CTGF and TGF-β2 in the induction of EMT and ECM synthesis in human lens epithelial cells (HLECs) has not been reported. In this study, we found that TGF-β2, CTGF and gremlin can individually induce the expression of α-smooth muscle actin (α-SMA), fibronectin (Fn), collagen type I (COL-I), Smad2 and Smad3 in HLECs. Blockade of CTGF and gremlin effectively inhibited TGF-β2-induced expression of α-SMA, Fn, COL-I, Smad2, and Smad3 in HLECs. Furthermore blockade of Smad2 and Smad3 effectively inhibited CTGF and gremlin induced expression of α-SMA, Fn, COL-I in HLECs. In conclusion, TGF-β2, CTGF and gremlin are all involved in EMT and ECM synthesis via activation of Smad signaling pathway in HLECs. Specifically silencing CTGF and gremlin can effectively block the TGF-β2-induced EMT, ECM synthesis due to failure in activation of Smad signaling pathway in HLECs.

SAT0062 Blockade of connective tissue growth factor (CTGF) ameliorates murine model of rheumatoid arthritis

BackgroundOur previous study demonstrated changes in the profiles of serum protein biomarkers in infliximab-treated rheumatoid arthritis (RA) patients using a novel approach to proteomic research (1). Several proteins exhibited vast...

Inhibition of Connective Tissue Growth Factor Ameliorates Disease in a Murine Model of Rheumatoid Arthritis

We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to evaluate the effects of blockade of the CTGF pathway on the development of collagen-induced arthritis (CIA) in mice. Arthritis was induced in DBA/1J mice by immunization with a combination of type II collagen (CII) and Freund's complete adjuvant. We evaluated the development of arthritis in mice with CIA left untreated versus treated with neutralizing anti-CTGF monoclonal antibody (mAb). Inhibition of CTGF in mice treated with neutralizing anti-CTGF mAb significantly ameliorated arthritis compared to the untreated mice with CIA. Serum levels of matrix metalloproteinase 3 were reduced by anti-CTGF mAb treatment. Moreover, blockade of CTGF decreased interleukin-17 expression on purified CD4+ T lymphocytes. Although the expression of the retinoic acid receptor-related orphan receptor γt gene was not suppressed by anti-CTGF mAb treatment, that of interferon regulatory factor 4 (IRF-4) and IκBζ (Nfkbiz), which are other important molecules for the differentiation of Th17 cells, was suppressed. In addition, blockade of CTGF inhibited pathologic proliferation of T lymphocytes in response to CII restimulation in vitro. Moreover, aberrant osteoclastogenesis in mice with CIA was restored by anti-CTGF mAb treatment. Our findings indicate that blockade of CTGF prevents the progression of arthritis in mice with CIA. Anti-CTGF mAb treatment suppresses pathologic T cell function and restores aberrant osteoclastogenesis in mice with CIA. CTGF may become a new target for the treatment of RA.

Inhibition of connective tissue growth factor suppresses hepatic stellate cell activation in vitro and prevents liver fibrosis in vivo

Activation of hepatic stellate cells (HSC) represents a critical event in fibrosis, and connective tissue growth factor (CTGF) plays a profibrotic activity and a key factor in the pathogenesis of tissue fibrosis. The current study aimed to determine whether lentivirus-mediated short hairpin RNA (shRNA)-targeted CTGF downregulates the CTGF expression and furthermore whether it suppresses the activation and proliferation of HSC in vitro and prevents liver fibrosis in vivo. HSC-T6 cells were treated with recombinant lentivirus carrying CTGF siRNA. Real-time PCR, Western blotting, MTT, and flow cytometry were performed to investigate the activation and proliferation of HSC-T6 cells in response to CTGF silence. CCl4-induced rats were received lentivirus containing CTGF siRNA by intraportal vein injection. Levels of liver fibrosis were assessed by biochemical and histopathologic examinations. Recombinant lentivirus containing CTGF siRNA could effectively and specifically downregulate the expression of CTGF in both HSC-T6 cells and CCl4-induced rats with liver fibrosis. Blockade of CTGF resulted in significant inhibition of HSC activation and proliferation with decrease in TIMPs, MMP2, MMP9, and collagen I, as well as increase in cells in S phase. Silencing CTGF expression with siRNA prevented liver fibrosis in CCl4-induced rat model. These findings indicated that CTGF plays a key role in the pathogenesis of liver fibrosis and lentiviral-mediated CTGF siRNA has the potential to be an effective treatment for liver fibrosis.

High Glucose Induces Connective Tissue Growth Factor Expression and Extracellular Matrix Accumulation in Rat Aorta Vascular Smooth Muscle Cells Via Extracellular Signal-Regulated Kinase 1/2

Connective tissue growth factor (CTGF) is a potent pro-fibrotic factor, which is implicated in fibrosis through extracellular matrix (ECM) induction in diabetic cardiovascular complications. It is an important downstream mediator in the fibrotic action of transforming growth factor β (TGFβ) and is potentially induced by hyperglycemia in human vascular smooth muscle cells (VSMCs). Therefore, the goal of this study is to identify the signaling pathways of CTGF effects on ECM accumulation and cell proliferation in VSMCs under hyperglycemia. We found that high glucose stimulated the levels of CTGF mRNA and protein and followed by VSMC proliferation and ECM components accumulation such as collagen type 1, collagen type 3 and fibronectin. By depleting endogenous CTGF we showed that CTGF is indispensable for the cell proliferation and ECM components accumulation in high glucose-stimulated VSMCs. In addition, pretreatment with the MEK1/2 specific inhibitors, PD98059 or U0126 potently inhibited the CTGF production and ECM components accumulation in high glucose-stimulated VSMCs. Furthermore, knockdown with ERK1/2 MAPK siRNA resulted in significantly down regulated of CTGF production, ECM components accumulation and cell proliferation in high glucose-stimulated VSMCs. Finally, ERK1/2 signaling regulated Egr-1 protein expression and treatment with recombinant CTGF reversed the Egr-1 expression in high glucose-induced VSMCs. It is conceivable that ERK1/2 MAPK signaling pathway plays an important role in regulating CTGF expression and suggests that blockade of CTGF through ERK1/2 MAPK signaling may be beneficial for therapeutic target of diabetic cardiovascular complication such as atherosclerosis.

Connective Tissue Growth Factor Is Responsible for Transforming Growth Factor-Beta-Induced Peritoneal Mesothelial Cell Apoptosis

Background: Previous studies found that transforming growth factor-β (TGF-β) induces mesothelial production of connective tissue growth factor (CTGF), which may be downstream mediators of TGF-β. Since high dose TGF-β induces apoptosis of peritoneal mesothelial cells (PMC), we study the effect of CTGF blockade in the system of TGF-β-induced PMC apoptosis. Method: We examined the effect of TGF-W in primary culture of rat peritoneal mesothelial cells (PMC). PMC apoptosis was studied by flow cytometry. The effect of CTGF was blocked by antibody and short-interfering RNA (siRNA). Expression of apoptotic gene was studied by real-time polymerase chain reaction. Result: In cultured unstimulated rat PMC, there is a low but definite incidence of spontaneous apoptosis. Stimulation with TGF-β 50 pg/ml induces an upregulation of apoptotic gene BAX expression and a downregulation of anti-apoptotic gene BCL-2L expression, and a 4-fold increase in PMC apoptosis. The effect of TGF-β-induced PMC apoptosis was partly prevented by antibody against CTGF, and completely abolished by CTGF-specific siRNA, while CTGF-blockade by siRNA had no effect on PMC necrosis. CTGF silencing by siRNA prevented the down-regulation of BCL-2L expression induced by TGF-β, had no effect on the BAX expression. Conclusion: Our results indicate that CTGF is an important downstream mediator of TGF-β-induced PMC apoptosis.

Role of connective tissue growth factor in fibronectin synthesis in cultured human prostate stromal cells

Objectives To clarify the correlation of connective tissue growth factor (CTGF) expression and fibronectin (FN) synthesis after transforming growth factor-beta-1 (TGF-β1) stimulation in human prostate stromal cells. Methods Primary cultures of human prostate stromal cells were established from nine normal prostates by the explant method. The gene and protein expressions of CTGF and FN after TGF-β1 treatment were examined. We also investigated the effect of CTGF blockade on TGF-β1-induced FN synthesis by CTGF antisense oligodeoxynucleotide treatment. Results CTGF expression was detected in all nine prostate stromal cells by reverse transcriptase-polymerase chain reaction and immunoblotting. In prostatic tissues, CTGF expression was observed more strongly in epithelial cells than in the stromal area by immunohistochemistry. The upregulation of both CTGF and FN protein by TGF-β1 treatment was demonstrated in a dose-dependent manner. CTGF antisense oligodeoxynucleotide inhibited TGF-β1-stimulated FN synthesis. Conclusions CTGF plays a crucial role in extracellular matrix production as a TGF-β1 downstream mediator in human prostate stromal cells, suggesting that CTGF blockade is likely to be a therapeutic target against benign prostatic hyperplasia.

Role of connective tissue growth factor in human renal tubular epithelial cell transdifferentiation in vitro

To observe the effect of connective tissue growth factor (CTGF) on the transdifferentiation of human renal tubular epithelial cells and to explore the influence of CTGF antisense oligodeoxynucleotide (ASODN) transfection on the transdifferentiation process induced by transforming growth factor-beta1 (TGF-beta1). Human renal tubular epithelial cells of the strain HKC were cultured and divided into 3 groups: (1) negative control group, (2) low dose CTGF group, treated with recombinant human CTGF (rhCTGF) with the terminal concentration of 2.5 microg/L, and (3) high dose CTGF group, treated with rhCTGF with the terminal concentration of 5.0 microg/L). To evaluate the contribution of CTGF to the transdifferentiation induced by TGF-beta1, Another HKC cells were divided into 4 groups: (1) untreated control group (Group C), (2) Group T, stimulated by TGF-beta1 (10.0 microg/L), (3) Group S, stimulated by sense ODN transfection + TGF-beta1 (10.0 microg/L), and (4) Group A, stimulated by antisense ODN transfection + TGF-beta1 (10.0 microg/L). RT-PCR was used to detect the mRNA expression of alpha-smooth muscle actin (alpha-SMA) and collagen type IV (col IV) mRNA. Indirect immunofluorescence assay and flow cytometry were used to assess the level of intracellular alpha-SMA protein. ELISA was used to determine the concentration of col IV in the media. The normal HKC cells were round and the HKC cells stimulated with rhCTGF became elongated. Upon the stimulation of different concentrations of rhCTGF, the expression of alpha-SMA mRNA increased markedly (both P < 0.01), while the mRNA expression of collagen type IV gene was down-regulated significantly (both P < 0.01). The percentage of alpha-SMA positive cells was significantly higher in the stimulated groups than that in negative control with significant difference among any 2 groups (38.9%, 65.5% vs. 2.4% respectively, all P < 0.01). Under this condition, collagen type IV secreted into the culture medium was lowered markedly upon the induction of CTGF (P < 0.01). RT-PCR analysis showed that the CTGF gene expression was upregulated by TGF-beta1 stimulation and peaked in 3 hours, and the alpha-SMA expression was upregulated by TGF-beta1 stimulation, however, peaked in 6 hours. The CTGF mRNA expression of the HKC cells transfected with CTGF ASODN that was stimulated by TGF-beta1 10 microg/L was significantly suppressed (P < 0.01) and the alpha-SMA mRNA expression induced by TGF-beta1 10 microg/L was significantly inhibited by CTGF ASODN transfection (P < 0.01). Indirect immunofluorescence assay showed that normal HKC cells did not express alpha-SMA, 48 hours after stimulation of TGF-beta1 10 microg/L the HKC cells showed expression of alpha-SMA in the cytoplasm, and the intracytoplasmic alpha-SMA expression was significantly down-regulated by the transfection of CTGF ASODN (P < 0.01). CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (MyoF) in vitro, and CTGF blockade results in a dramatic inhibition of TGF-beta-induced transdifferentiation of renal tubular cells. So CTGF may be a crucial factor in promoting tubular-epithelial myofibroblast transdifferentiation.

Endothelin-1, via ET A Receptor and Independently of Transforming Growth Factor-β, Increases the Connective Tissue Growth Factor in Vascular Smooth Muscle Cells

Endothelin (ET)-1 is a potent vasoconstrictor that participates in cardiovascular diseases. Connective tissue growth factor (CTGF) is a novel fibrotic mediator that is overexpressed in human atherosclerotic lesions, myocardial infarction, and experimental models of hypertension. In vascular smooth muscle cells (VSMCs), CTGF regulates cell proliferation/apoptosis, migration, and extracellular matrix (ECM) accumulation. Our aim was to investigate whether ET-1 could regulate CTGF and to investigate the potential role of ET-1 in vascular fibrosis. In growth-arrested rat VSMCs, ET-1 upregulated CTGF mRNA expression, promoter activity, and protein production. The blockade of CTGF by a CTGF antisense oligonucleotide decreased FN and type I collagen expression in ET-1-treated cells, showing that CTGF participates in ET-1-induced ECM accumulation. The ETA, but not ETB, antagonist diminished ET-1-induced CTGF expression gene and production. Several intracellular signals elicited by ET-1, via ETA receptors, are involved in CTGF synthesis, including activation of RhoA/Rho-kinase and mitogen-activated protein kinase and production of reactive oxygen species. CTGF is a mediator of TGF-beta- and angiotensin (Ang) II-induced fibrosis. In VSMCs, ET-1 did not upregulate TGF-beta gene or protein. The presence of neutralizing transforming growth factor (TGF)-beta antibody did not modify ET-1-induced CTGF production, showing a TGF-beta-independent regulation. We have also found an interrelationship between Ang II and ET-1 because the ETA antagonist diminished CTGF upregulation caused by Ang II. Collectively, our results show that, in cultured VSMCs, ET-1, independently of TGF-beta and through the activation of several intracellular signals via ETA receptors, regulates CTGF. This novel finding suggests that CTGF could be a mediator of the profibrotic effects of ET-1 in vascular diseases.

Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro

Connective tissue growth factor (CTGF) has been reported to play an important role in mediating the profibrotic effects of transforming growth factor-beta (TGF-beta) in various renal diseases. To elucidate the role of CTGF in renal tubular epithelial-myofibroblast transdifferentiation, we examined the expression of alpha-smooth muscle actin (alpha-SMA), vimentin, tenascin-C, and collagen IV expression upon the stimulation of CTGF in cultured human proximal tubular epithelial cell line (HKC), and further investigated the effects of endogenous CTGF blockade on the transdifferentiation process induced by TGF-beta. It is revealed that upon the stimulation of recombinant human CTGF (rhCTGF, 2.5 or 5.0 microg/L), the expression of alpha-SMA and tenascin-C mRNA increased significantly (p<0.01), while collagen IV gene expression decreased significantly (p<0.01), all in a dose-dependent manner. The percentage of alpha-SMA-positive cells was significantly larger in the rhCTGF-stimulated groups than that in negative control (38.9%, 65.5% vs. 2.4%, respectively, p<0.01) as confirmed by flow cytometry. Both cytoplasmic and secretory tenascin-C expression was upregulated by the stimulation of rhCTGF (p<0.01). Under this condition, collagen IV secreted into the culture media was lowered markedly (p<0.01). On RT-PCR analysis, TGF-beta1 upregulated CTGF gene expression, preceding that of alpha-SMA. The alpha-SMA mRNA expression induced by TGF-beta1 was significantly inhibited by CTGF antisense oligodeoxynucleotide (ODN) transfection (p<0.01). With prolonged incubation time, CTGF antisense ODN also inhibited intracellular alpha-SMA protein synthesis, as demonstrated by indirect immuno-fluorescence. So it is concluded that CTGF could promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblasts in vitro, both directly and as a downstream mediator of TGF-beta, and CTGF blockade would be a possible therapeutic target against tubulointerstitial fibrosis.

Role of connective tissue growth factor in renal tubular epithelial-myofibroblast transdifferentiation and extracellular matrix accumulation in vitro

Connective tissue growth factor (CTGF) has been reported to play an important role in mediating the profibrotic effects of transforming growth factor-β (TGF-β) in the pathogenesis of renal fibrosis. To further elucidate the role of CTGF in renal tubular transdifferentiation and extracellular matrix (ECM) metabolism, we examined the time-course of CTGF, α-smooth muscle actin (α-SMA), fibronectin and plasminogen activator inhibitor-1(PAI-1) gene expression upon the stimulation of TGF-β1 (5 μg/L) in cultured human proximal tubular epithelial cell line (HKC), and further investigated the effects of endogenous CTGF blockade. On reverse transcriptional-polymerase chain reaction (RT-PCR) analysis, TGF-β1 upregulated CTGF gene expression, preceding that of α-SMA, fibronectin and PAI-1. The α-SMA, fibronectin and PAI-1 mRNA expression induced by TGF-β1 were significantly inhibited by CTGF antisense oligodeoxynucleotide (ODN) transfection. With prolonged incubation time, CTGF antisense ODN also inhibited intracellular α-SMA and PAI-1 protein synthesis, lowered the level of fibronectin and PAI-1 protein secreted into the media, as confirmed by indirect immuno-fluorescence, flow cytometry, enzyme-linked immunosorbent assay (ELISA) and Western blot methods respectively. These results suggested that CTGF may play a crucial role in the renal tubular epithelial-transdifferentiation and the following deposition/degradation process of ECM during tubulointerstitial fibrosis.

Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.

Connective tissue growth factor (CTGF) is one of the candidate factors mediating downstream events of transforming growth factor-beta (TGF-beta), but its role in fibrogenic properties of TGF-beta and in tubulointerstitial fibrosis has not yet been clarified. Using unilateral ureteral obstruction (UUO) in rats, we analyzed gene expression of TGF-beta1, CTGF, and fibronectin. We further investigated the effect of blockade of endogenous CTGF on TGF-beta-induced fibronectin expression in cultured rat renal fibroblasts by antisense oligodeoxynucleotide (ODN) treatment. After UUO, CTGF mRNA expression in the obstructed kidney was significantly upregulated subsequent to TGF-beta1, followed by marked induction of fibronectin mRNA. By in situ hybridization, CTGF mRNA was detected mainly in the interstitial fibrotic areas and tubular epithelial cells as well as in parietal glomerular epithelial cells in the obstructed kidney. The interstitial cells expressing CTGF mRNA were also positive for alpha-smooth muscle actin. CTGF antisense ODN transfected into cultured renal fibroblasts significantly attenuated TGF-beta-stimulated upregulation of fibronectin mRNA and protein compared with control ODN transfection, together with inhibited synthesis of type I collagen. With the use of a reporter assay, rat fibronectin promoter activity was increased by 2.5-fold with stimulation by TGF-beta1, and this increase was abolished with antisense CTGF treatment. Thus CTGF plays a crucial role in fibronectin synthesis induced by TGF-beta, suggesting that CTGF blockade could be a possible therapeutic target against tubulointerstitial fibrosis.