Transforming growth factor-β pathway is activated in cholecystolithiasis

Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)-beta were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas. Human corneal fibroblasts were incubated with TGF-beta1, -beta2, and -beta3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF-beta and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established. All three TGF-beta isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF-beta and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF-beta-stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas. These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-beta, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF-beta induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring.

To analyze the involvement of connective tissue growth factor (CTGF) in the transforming growth factor-beta (TGF-beta) pathway during acute necrotizing pancreatitis (ANP) in humans and rats. Connective tissue growth factor is involved in several fibrotic diseases and has a critical role in fibrogenesis and tissue remodeling after injury. Normal human pancreas tissue samples were obtained through an organ donor program from five individuals without a history of pancreatic disease. Human ANP tissues were obtained from eight persons undergoing surgery for this disease. In rats, ANP was induced by intraductal infusion of taurocholate. The expression of CTGF was studied by Northern blot analysis, in situ hybridization, and immunohistochemistry in both human and rat pancreatic tissue samples. Northern blot analysis revealed enhanced CTGF mRNA expression in human ANP tissue samples compared with normal controls. In addition, a concomitant increase in TGF-beta1 was present. By in situ hybridization, CTGF mRNA was localized in the remaining acinar and ductal cells and in fibroblasts. In regions of intense damage adjacent to areas of necrosis, CTGF mRNA signals were most intense. Inflammatory cells were devoid of any CTGF mRNA signals. By immunohistochemistry, CTGF protein was localized at high levels in the same cell types as CTGF mRNA. In ANP in rats, concomitantly enhanced mRNA levels of CTGF, TGF-beta1, and collagen type 1 were present, with a biphasic peak pattern on days 2 to 3 and day 7 after induction of ANP. These data indicate that CTGF participates in tissue remodeling in ANP. The expression of CTGF predominantly in the remaining acinar and ductal cells indicates that extracellular matrix synthesis after necrosis is at least partly regulated by the remaining pancreatic parenchyma and only to a minor extent by inflammatory cells. Blockage of CTGF, a downstream mediator of TGF-beta in fibrogenesis, might be useful as a target to influence and reduce fibrogenesis in this disorder.

Connective tissue growth factor (CTGF) belongs to a family of factors that regulate fibrogenesis and wound healing. While the significance of transforming growth factor beta (TGF-beta) in liver fibrosis is well established, the role of CTGF in fibrosing hepatopathy is still unknown. CTGF was analyzed in 10 normal and in 16 cirrhotic liver tissue samples. Northern blot analysis was used to examine the concomitant expression of CTGF and TGF-beta1 mRNAs, and the cellular localization of CTGF mRNA was studied by in situ hybridization. For identification of myofibroblasts and activated hepatic stellate cells, alpha-smooth muscle actin (alpha-SMA) immunohistochemistry was used. Northern blot analysis showed 6.5-fold enhanced expression of CTGF mRNA and 7.8-fold enhanced expression of TGF-beta1 mRNA in liver cirrhosis in comparison with normal controls (p<0.01). By in situ hybridization, CTGF mRNA was detectable in only a few spindle cells in the portal tracts in normal liver samples. In contrast, there was strong expression of CTGF mRNA in fibroblasts and myofibroblast-like cells present in fibrous septa surrounding the cirrhotic nodules, in stellate cells, in endothelial cells and in mesenchymal cells around ductular proliferations, and in ductular epithelial cells. There was a strong correlation between CTGF mRNA and TGF-beta1 mRNA as well as the degree of fibrosis (p<0.01). Overexpression of CTGF in liver cirrhosis, especially in fibroblasts/myofibroblasts and stellate cells, suggests that this novel factor may play an important role in hepatic fibrosis.

Objective To observe the effects of dual targets intervention on the expression of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in diabetic rat retina.Methods Forty-eight Sprague-Dawley rats were randomly divided into control group (CON1 group) and diabetes mellitus group (DM group).The rats of DM group were induced with streptozotocin injection creating a diabetic model.Retinas were obtained at eight,10,12 weeks after DM induction from both groups.CTGF and VEGF mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR).Based on the results of above experiments,60 rats with same conditions were selected.Fifty rats were induced with streptozotocin injection creating a diabetic model,and 10 rats comprised the control group (CON2 group).Then the 50 diabetic rats were randomly divided into ranibizumab and CTGF shRNA dual targets intervention group,ranibizumab single-target intervention group,CTGF shRNA singletarget intervention group and non-intervention group.Retinas were obtained at one week after intervention from all the groups.CTGF and VEGF mRNA levels were examined by RT-PCR.Results The levels of CTGF mRNA were significantly higher in DM group than that in CON1 group at the 8th weeks after DM induction,and this up-regulation was maintained through the 12th week (t=-2.49,-2.67,-2.42;P＜0.05).There was no difference on VEGF mRNA levels between DM group and CON1 group at the 8th weeks after DM induction (t=-0.443,P =0.669).VEGF mRNA levels of DM group started to be significantly elevated over those in the CON1 group at the 10th week,and remained to be higher at the 12th week (t=-2.35,-2.57; P＜0.05).The VEGF mRNA of ranibizumab single-target intervention group was significantly lower than that in non-intervention group (t=-3.44,P=0.014),which was similar to CON2 group (t =-1.37,P＞ 0.05) ; however,the CTGF mRNA level was significantly increased as compared to the non-intervention group (t =2.48,P ＜ 0.05).In the CTGF shRNA single-target intervention group,the levels of CTGF and VEGF mRNA were decreased as compared to the nonintervention group (t =0.23,-2.92; P＜ 0.05).In the ranibizumab and CTGF shRNA dual targets intervention group,the levels of CTGF and VEGF mRNA were decreased as compared to the nonintervention group (t=-6.09,-5.11; P＜0.001),which was similar to CON2 group (t=-1.16,1.139; P＞0.05).Conclusions Both CTGF and VEGF gene expression are up-regulated in early diabetic rat retina,and the level of CTGF increased earlier than VEGF.Ranibizumab combined with CTGF shRNA could simultaneously reduce the level of CTGF and VEGF mRNA in diabetic rat retina. Key words: Diabetic retinopathy/therapy; Antibodies, monoclonal/therapeutic use; Vascular endothelial growth factors; Animal experimentation

Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells

To investigate the role of connective tissue growth factor (CTGF) after trabeculectomy associated with wound healing and to identify the role of CTGF in this process. It was a experimental study. Forty-nine rabbits were used and divided into 5 groups: normal eyes without trabeculectomy group (group A), ocular hypertension (OHT) model without trabeculectomy group (group B), OHT model with trabeculectomy group (group C), normal eyes with trabeculectomy group (group D) and normal eyes with sham operation group (group E). Group A and B were as control. CTGF mRNA was detected by RT-PCR using blebs and tissues harvested at day 2, 5, 7, and 14. Three replicates of three blebs per time point in the right eyes were collected. The expression of CTGF protein was detected by immunohistochemistry and inflammatory histopathology was inspected by HE staining using the whole eyeball harvested in the left eyes. Compared to group A and B, the expression of CTGF was significantly increased at day 5 after surgery (F = 19.54, P < 0.05) in group C, D, and E. The expression of CTGF mRNA in group C is significantly higher than that in group D at day 2 and 5 (t = 2.300, 5.140, P < 0.05), while group D is significantly higher than that in group E at day 2, 5, 7, and 14 (t = -2.927, -6.424, -4.176, -4.997, P < 0.05). The expression of CTGF protein in group C is significantly higher than that in group D (t = -7.147, -10.955, -9.900, -6.385, P < 0.05), and group D is higher than that in group E (F = 68.33, P < 0.05) at day 2, 5, 7, and 14, respectively. Inflammatory reaction reached peak at day 5 after surgery in group C, D, and E showing an infiltration of neutrophil, monocytes, macrophages, and the proliferation of fibroblast. Overexpression of CTGF in the blebs after trabeculectomy demonstrates that CTGF may play an important role in the process of wound healing. Furthermore, ocular hypertension may be involved in the upregulation of CTGF expression.

Transforming growth factor-beta (TGF-beta) stimulates alpha(1)(I) collagen mRNA synthesis in human lung fibroblasts through a mechanism that is partially sensitive to cycloheximide and that may involve synthesis of connective tissue growth factor (CTGF). Northern blot analyses indicate that TGF-beta stimulates time- and dose-dependent increases in CTGF mRNA. In TGF-beta-stimulated fibroblasts, maximal levels of CTGF mRNA (3.7-fold above baseline) occur at 6 h. The TGF-beta-stimulated increase in CTGF mRNA was not blocked by cycloheximide. Nuclear run-on analysis indicates that TGF-beta increases the CTGF transcription rate. The TGF-beta-stimulated increases in CTGF transcription and steady-state levels of CTGF mRNA are attenuated in prostaglandin E(2) (PGE(2))-treated fibroblasts. PGE(2) fails to attenuate luciferase activity induced by TGF-beta in fibroblasts transfected with the TGF-beta-responsive luciferase reporter construct p3TP-LUX. In amino acid-deprived fibroblasts, PGE(2) and insulin regulate alpha(1)(I) collagen mRNA levels without affecting CTGF mRNA levels. The data suggest that the regulation of alpha(1)(I) collagen mRNA levels by TGF-beta and PGE(2) may function through both CTGF-dependent and CTGF-independent mechanisms.

Objective: To investigate the effect of advanced glycation end products (AGEs) on the expression of connective tissue growth factor (CTGF) and fibronectin (FN) in human peritoneal mesothelial cells (HPMC). To observe the effect of genistein (Gen) on the expression of CTGF and FN in HPMC induced by AGEs. Methods: First, HPMC were stimulated with different concentrations of AGEs (0, 200, 600 and 1,000 mg/l) for 48 h; the expression of FN was detected by reverse transcription-polymerase chain reaction (RT-PCR). Second, HPMC were divided into the following groups: (1) control group, (2) AGE-treated group (600 mg/l AGEs) and (3) Gen-treated groups with 600 mg/l AGEs and 25, 50 and 100 µMGen, respectively. The expression of messenger RNA (mRNA) for FN and CTGF was measured by RT-PCR; the expression of FN and CTGF protein was detected by enzyme-linked immunosorbent assay (ELISA) after 48 h. Results: The expression of FN mRNA in HPMC increased in a dose-dependent manner after induction with AGEs. Compared with controls, 600 mg/l AGEs markedly promoted the expression of mRNA and protein for FN and CTGF. Compared with the AGE-treated group (600 mg/l), 25, 50, and 100 µM Gen significantly inhibited the expression of mRNA and protein for FN and CTGF. Conclusion: AGEs can markedly increase the expression of mRNA and protein for FN and CTGF; however, Gen can inhibit the expression of FN and CTGF mRNA and protein stimulated by AGEs, which implies that Gen probably decreases the accumulation of extracellular matrix through inhibiting the expression of CTGF, and it may play a role in anti-peritoneal fibrosis.

We characterized a rabbit polyclonal antibody raised against human recombinant connective tissue growth factor (CTGF). The antibody recognised a higher molecular mass form (approx. 56kDa) of CTGF in mesangial cell lysates as well as the monomeric (36–38kDa) and lower molecular mass forms (&amp;lt; 30kDa) reported previously. Immunohistochemistry detected CTGF protein in glomeruli of kidneys of non-obese diabetic mice 14 days after the onset of diabetes, and this was prominent by 70 days. CTGF protein is also present in glomeruli of human patients with diabetic nephropathy. No CTGF was detected in either normal murine or human glomeruli. Transient transfection of a transformed human mesangial cell line with a CTGF–V5 epitope fusion protein markedly increased fibronectin and plasminogen activator inhibitor-1 synthesis in cultures maintained in normal glucose (4mM) conditions; a CTGF-antisense construct reduced the elevated synthesis of these proteins in high glucose (30mM) cultures. Culture of primary human mesangial cells for 14 days in high glucose, or in low glucose supplemented with recombinant CTGF or transforming growth factor β1, markedly increased CTGF mRNA levels and fibronectin synthesis. However, whilst co-culture with a CTGF-antisense oligonucleotide reduced the CTGF mRNA pool by greater than 90% in high glucose, it only partially reduced fibronectin mRNA levels and synthesis. A chick anti-CTGF neutralizing antibody had a similar effect on fibronectin synthesis. Thus both CTGF and CTGF-independent pathways mediate increased fibronectin synthesis in high glucose. Nevertheless CTGF expression in diabetic kidneys is likely to be a key event in the development of glomerulosclerosis by affecting both matrix synthesis and, potentially through plasminogen activator inhibitor-1, its turnover.

Objective To observe the changes of follistatin-like protein 1 (FSTL1) in serum of patients with proliferative diabetic retinopathy (PDR). Methods Twenty PDR patients confirmed by clinical examination and 20 normal people were included in the study. Human retinal vascular endothelial cells (HRCEC) were divided into HRCEC blank control group, 3 h hypoxia group, 6 h hypoxia group. Human umbilical vein endothelial cell (HUVEC) were divided into HUVEC blank control group, 3h hypoxia group, 6h hypoxia group. Real-time quantitative PCR (RT-PCR) and ELISA were used to determine the expression of FSTL1, TGF-β, VEGF, connective tissue growth factor (CTGF) mRNA and protein in peripheral blood and cells of all groups from all subjects. Results The expressions of FSTL1, TGF-β1, CTGF, VEGF mRNA in blood samples of patients with PDR were 1.79±0.58, 0.97±0.21, 1.85±0.69 and 1.38±0.44. The expressions of FSTL1, TGF-β1 protein were 1.19±0.50, 0.71±0.24 ng/ml and 734.03±116.45, 649.36±44.23 ng/L. Compared with normal people, the differences were statistically significant (tmRNA=0.90, 0.21, 2.85, 1.77; P=0.00, 0.00, 0.04, 0.02. tprotein=1.88, 7.68; P=0.00, 0.02). The cell viability of HRCEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.66±0.05 and 0.64±0.04, respectively. Compared with the blank control group, the difference was statistically significant (F=13.02, P=0.00). The cell viability of HUVEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.63±0.06 and 0.68±0.06, respectively. Compared with the blank control group, the difference was statistically significant (F=26.52, P=0.00). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HRCEC blank control group and 3 h hypoxia group, the differences were statistically significant (F=14.75, 44.93, 85.54, 6.23; P=0.01, 0.00, 0.00, 0.03). Compared with the HRCEC blank control and 3 h hypoxia group, the expressions of FSTL1 and TGF-β1 protein were statistically significant (P<0.05). There was a statistically significant difference in TGF-β1 protein expression in the hypoxic 6 h group (P=0.03) and no significant difference in FSTL1 protein expression (P=0.68). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HUVEC blank control group and 3h hypoxia group, the differences were statistically significant (F=19.08, 25.12, 22.89, 13.07; P=0.00, 0.00, 0.00, 0.01). Immunofluorescence staining results showed that FSTL1, TGF-β1, CTGF, and VEGF proteins were positively expressed in cells in the 3h hypoxia and 6h hypoxia groups. Conclusion The expression of FSTL1 gene and protein in serum of PDR patients was significantly higher than that of normal people. Key words: Follistatin-related proteins; Human umbilical vein endothelial cells; Diabetic retinopathy

To investigate the effect of somatostatin analogue-octreotide (OCT) on expression of connective tissue growth factor (CTGF) gene of murine hepatic stellate cells (HSCs) in vitro. HSCs separated from Sprague Dawley rats by in situ perfusion and Nycodenz gradient were divided into 5 groups. HSCs in 4 out of 5 groups were co-cultured with octreotide at different dosages, and the remaining group served as control. The expression of CTGF and TGF-beta mRNA were assessed by reverse transcription-polymerase chain reaction (RT-PCR). OCT down-regulates the expression of CTGF and TGF-beta mRNA in HSCs. The effect is increased with a dose dependent manner. OCT could exert the inhibitory effect on HSCs by down-regulating the expression of CTGF and TGF-beta. This provides a potential for the prevention and management of hepatic fibrosis.

This month in Gastroenterology

Background:This study aims to investigate the expression profile, molecular mechanisms, and biological functions of Connective Tissue Growth Factor (CTGF) in bladder cancer (BLCA).Methods:For accurate CTGF mRNA expression assessment, 1728 samples were collected. Additionally, for uncovering potential signaling pathways, differentially co-expressed CTGF genes were employed. For exploring CTGF’s effects, its impact on immune microenvironment and drug sensitivity was studied.Results:CTGF mRNA was significantly underexpressed in BLCA (standardized mean difference [SMD] = −1.06, 95% CI: −1.89–0.23), and this downregulation was confirmed at the protein level (p < 0.0001). CTGF was mainly involved in immune microenvironment-related pathways and biological processes (BPs) associated with stromal remodeling and extracellular matrix dynamics. Moreover, A statistically significant correlation was identified between the expression levels of CTGF and the infiltration degrees of variety of immune cells. Notably, CTGF expression was positively correlated with sensitivity to EGFR inhibitors (e.g., Afatinib) and negatively correlated with resistance to PARP inhibitors (e.g., Olaparib).Conclusions:This study elucidated the low expression of CTGF in BLCA. CTGF may promotes tumor progression by remodeling the immune microenvironment and extracellular matrix. Additionally, its expression is positively correlated with sensitivity to EGFR inhibitors and negatively correlated with resistance to PARP inhibitors.

We characterized a rabbit polyclonal antibody raised against human recombinant connective tissue growth factor (CTGF). The antibody recognised a higher molecular mass form (approx. 56 kDa) of CTGF in mesangial cell lysates as well as the monomeric (36-38 kDa) and lower molecular mass forms (<30 kDa) reported previously. Immunohistochemistry detected CTGF protein in glomeruli of kidneys of non-obese diabetic mice 14 days after the onset of diabetes, and this was prominent by 70 days. CTGF protein is also present in glomeruli of human patients with diabetic nephropathy. No CTGF was detected in either normal murine or human glomeruli. Transient transfection of a transformed human mesangial cell line with a CTGF-V5 epitope fusion protein markedly increased fibronectin and plasminogen activator inhibitor-1 synthesis in cultures maintained in normal glucose (4 mM) conditions; a CTGF-antisense construct reduced the elevated synthesis of these proteins in high glucose (30 mM) cultures. Culture of primary human mesangial cells for 14 days in high glucose, or in low glucose supplemented with recombinant CTGF or transforming growth factor beta1, markedly increased CTGF mRNA levels and fibronectin synthesis. However, whilst co-culture with a CTGF-antisense oligonucleotide reduced the CTGF mRNA pool by greater than 90% in high glucose, it only partially reduced fibronectin mRNA levels and synthesis. A chick anti-CTGF neutralizing antibody had a similar effect on fibronectin synthesis. Thus both CTGF and CTGF-independent pathways mediate increased fibronectin synthesis in high glucose. Nevertheless CTGF expression in diabetic kidneys is likely to be a key event in the development of glomerulosclerosis by affecting both matrix synthesis and, potentially through plasminogen activator inhibitor-1, its turnover.

Transforming growth factor-beta1 (TGF-beta1) has been implicated as a major negative regulator of lung branching morphogenesis. Since connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta1 effects on mesenchymal cells, we hypothesized that TGF-beta1 induces CTGF expression in mouse embryonic lung explants and that CTGF mediates TGF-beta1 inhibition of branching morphogenesis. We show that addition of TGF-beta1 to the serum-free medium of embryonic day (E)12.5 lung explant cultures inhibited branching morphogenesis and induced CTGF mRNA expression in time- and dose-dependent manners. In contrast to basal endogenous CTGF protein, which was exclusively localized in the distal airway epithelium, TGF-beta1-induced CTGF protein was localized in both the epithelium and the mesenchyme. Addition of exogenous CTGF to culture medium directly inhibited branching morphogenesis. To identify the signal transduction pathway through which TGF-beta1 induces CTGF, we used SB431542, a specific inhibitor for TGF-beta type I receptor (TbetaRI)/ALK-5 to block TGF-beta1-induced Smad2/3 phosphorylation. Consequently, SB431542 stimulated normal branching morphogenesis and blocked TGF-beta1 inhibition of branching. Furthermore, SB-431542 blocked both endogenous and TGF-beta1-induced expression of CTGF mRNA and protein. These results demonstrate for the first time that TGF-beta1 induces CTGF expression in mouse embryonic lung explants, that CTGF inhibits branching morphogenesis, and that both endogenous and TGF-beta1-induced CTGF expression are mediated by the TbetaRI/ALK-5-dependent Smad2 signaling pathway.