CTGF mRNA Research Articles

Involvement of Connective Tissue Growth Factor in Human and Experimental Hypertensive Nephrosclerosis

Background/Aims: Connective tissue growth factor (CTGF; CCN2) has been implicated as a marker and mediator of fibrosis in human and experimental renal disease. Methods: We performed a comparative analysis of CTGF expression in hypertensive patients with and without nephrosclerosis, and in uninephrectomized and sham-operated spontaneously hypertensive rats (UNX-SHR and 2K-SHR). Results: Urinary and plasma CTGF were elevated in patients with hypertensive nephrosclerosis, and increased renal CTGF expression was mainly localized in podocytes. Accordingly, elevation of urinary, plasma, and tissue CTGF in UNX-SHR coincided and correlated with proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis. Thirty-two weeks after uninephrectomy, mean glomerular CTGF mRNA expression was increased 1.3-fold over baseline, mainly due to 1.7-fold higher expression in glomeruli undergoing sclerosis. In parallel, tubulointerstitial CTGF and α-smooth muscle actin were upregulated in UNX-SHR. CTGF was increased in the media of arcuate and interlobar arteries, while arterioles remained negative. Conclusions: Glomerulosclerosis, tubulointerstitial fibrosis, and arterial media hypertrophy lesions of hypertensive nephrosclerosis are all characterized by increased CTGF tissue expression, which is associated with a concomitant increase in CTGF in blood and urine. These findings identify CTGF as a promising biomarker for progression of hypertensive nephrosclerosis, and as a likely key factor in the pathogenesis of this disease.

Biologic effect of transforming growth factor-β1 on urethra cells cultured in vitro

To investigate the effects of transforming growth factor-β1 (TGF-β1) on growth controlling and the expression of connective tissue growth factor mRNA(CTGF mRNA) in urethra epithelium cells and fibroblasts cultured in vitro. Urethra epithelial cells and fibroblasts were cultured in vitro and identified. The fourth generation cells were divided into control group (cultured by cell medium without TGF-β1) and experimental groups(cultured by cell medium containing TGF-β1 1, 2, 4 and 8 µg/L), the vital force of cells were examined by MTT and cell counting, the expression of CTGF mRNA were examined by RT-PCR after 24 hours. The optical density and cell count decreased in experimental groups of urethra epithelium cells and increased in experimental groups of fibroblasts with the concentration of TGF-β1 being heightened compared with the control group (P < 0.05). The expression of CTGF mRNA increased with the heightening concentration of TGF-β1 in all experimental groups of urethra epithelium cells and fibroblasts by RT-PCR (P < 0.05). TGF-β1 can inhibit the growth of urethra epithelium cells and promote the growth of fibroblasts in vitro, it can induce the expression of CTGF mRNA in two cells above-mentioned.

Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin-1α and β

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)-beta and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF-beta-induced expression of CTGF in fibroblasts by an interleukin (IL)-1 alpha-dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL-1alpha and beta. Human dermal fibroblasts and NIH 3T3 cells were treated with IL-1alpha or beta in presence or absence of TGF-beta1. IL-1 suppressed basal and TGF-beta-induced CTGF mRNA and protein expression. IL-1alpha and beta inhibited TGF-beta-stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3-binding CAGA elements. Furthermore, IL-1alpha and beta inhibited TGF-beta-stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF-beta activated kinase1 (TAK1) is necessary for IL-1 inhibition of TGF-beta-stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin.

Regulative effects of Erk and p38 signal pathway on expression of CTGF in rat muscle-derived stem cells

Objective To investigate the role of Erk and p38 pathway in the process of CTGF expression in TGF-β1-induced muscle-derived stem cells(MDSCs).Methods MDSCs were isolated from the skeletal muscle of neonatal rats.After phenotyping,these MDSCs were cultured under 8 conditions according to group assignment:normal control group(MDSCs+basic culture),negative control group(MDSCs+basic culture+TGF-β1),experimental group 1-3(MDSCs pretreated with 20,40 and 80 μM of PD98059 respectively+basic culture+TCF-β1),experimental group 4-6(NDSCs pretreated with 0.2,0.5 and 1.0 μM of SB203580+basic culture+TGF-β1).After 48 hours,the MDSCs CTGF mRNA expression and protein level were quantified with Real Time-PCR and Western Blot.Results There were statistically significant differences of CTCF expression levels among normal control group,negative control group and experimental group 1-3(P 0.05).Conclusion Erk plays a role in TGF-β1-CTCF signal transduction of rat NDSCs,whereas p38 does not. Key words: Transforming growth factor beta1; Stem cells; CTGF; signal pathway

A phosphodiesterase 4 inhibitor, roflumilast N-oxide, inhibits human lung fibroblast functions in vitro

The PDE4 inhibitor roflumilast mitigates bleomycin-induced lung fibrotic remodeling in rodents. In the current study it was explored whether roflumilast N-oxide, the active metabolite of roflumilast influences functions of cultured lung fibroblasts. Cells of the human foetal lung fibroblast strain GM06114 were stimulated with TNF-α (5 ng ml −1) and cell surface ICAM-1 and eotaxin release were assessed. [ methyl- 3H] thymidine incorporation was measured following stimulation with bFGF (10 ng ml −1). α-Smooth muscle actin (protein), CTGF (mRNA) and fibronectin (mRNA) were determined secondary to TGFß1 (1 ng ml −1). In the presence of PGE 2 (1 nM), roflumilast N-oxide reduced TNF-α-induced ICAM-1 and eotaxin by about 70% and >90% with half-maximum inhibition at 0.9 nM and 0.5 nM, respectively. Roflumilast N-oxide also attenuated [ methyl- 3H] thymidine incorporation secondary to bFGF by about 75% with half-maximum inhibition at 0.7 nM when cells were co-incubated with IL-1ß (10 pg ml −1). In the presence of this cytokine roflumilast N-oxide (1 μM) diminished TGFß1-induced expression of α-smooth muscle actin and transcripts of CTGF and fibronectin. In addition, IL-1ß up-regulated PDE4 activity in the lung fibroblasts. Taken together, these findings indicate that roflumilast N-oxide directly targets human lung fibroblasts, which may at least partially explain the efficacy of roflumilast to mitigate a pulmonary fibrotic response in vivo.

Connective tissue growth factor associated with oncogenic activities and drug resistance in glioblastoma multiforme

Connective tissue growth factor (CTGF or CCN2) is a secreted protein that belongs to the CCN [cysteine-rich CYR61/CTGF/nephroblastoma-overexpressed gene] family. These proteins have been implicated in various biological processes, including stimulation of cell proliferation, migration, angiogenesis and tumorigenesis. In a previous study, we found that CTGF mRNA was elevated in primary gliomas, and a significant correlation existed between CTGF mRNA levels versus tumor grade, histology and patient survival. In this study, the role of CTGF in glioma tumorigenesis was explored. Forced expression of CTGF in glioblastoma multiforme (GBM) cells accelerated their growth in liquid culture and soft agar, stimulated cells migration in Boyden chamber assays and significantly increased their ability to form large, vascularized tumors in nude mice. CTGF induced the expression of the antiapoptotic proteins, Bcl-xl, Survivin and Flip. Overexpression of CTGF caused the U343 GBM cells to survive for longer than 40 days in serum-free medium and resist antitumor drugs including tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand, VELCADE (bortezomib, proteasome inhibitor) and temozolomide. Our data suggest that CTGF plays an important role in glioma progression, by supporting tumor cells survival and drug resistance.

FoxO Proteins Mediate Hypoxic Induction of Connective Tissue Growth Factor in Endothelial Cells

Hypoxia, a driving force in neovascularization, promotes alterations in gene expression mediated by hypoxia-inducible factor (HIF)-1alpha. Connective tissue growth factor (CTGF, CCN2) is a modulator of endothelial cell growth and migration, but its regulation by hypoxia is poorly understood. Therefore, we analyzed signaling pathways involved in the regulation of CTGF by hypoxia in endothelial cells. Exposure to low oxygen tension or treatment with the hypoxia-mimetic dimethyloxalyl glycine (DMOG) stabilized HIF-1alpha and up-regulated CTGF in human umbilical vein endothelial cells and in a murine microvascular endothelial cell line. Induction of CTGF correlated with a HIF-dependent increase in protein and mRNA levels, and nuclear accumulation of the transcription factor FoxO3a. By contrast, gene expression and cellular localization of FoxO1 were not significantly altered by hypoxia. Expression of CTGF was strongly reduced by siRNA silencing of FoxO1 or FoxO3a. Furthermore, nuclear exclusion of FoxO1/3a transcription factors by inhibition of serine/threonine protein phosphatases by okadaic acid inhibited CTGF expression, providing evidence for both FoxO proteins as regulators of CTGF expression. The DMOG-stimulated induction of CTGF was further increased when endothelial cells were co-incubated with transforming growth factor-beta, an activator of Smad signaling. Activation of RhoA-Rho kinase signaling by the microtubule-disrupting drug combretastatin A4 also enhanced the DMOG-induced CTGF expression, thus placing CTGF induction by hypoxia in a network of interacting signaling pathways. Our findings provide evidence that FoxO1, hypoxia-stimulated expression of FoxO3a and its nuclear accumulation are required for the induction of CTGF by hypoxia in endothelial cells.

Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia

Tubular epithelial cells secrete connective tissue growth factor (CTGF, CCN2), which contributes to tubulointerstitial fibrosis. However, the molecular regulation of CTGF in human primary tubular epithelial cells (hPTECs) is not well defined. Therefore, CTGF expression was characterized in hPTECs isolated from healthy parts of tumor nephrectomies, with special emphasis on the regulation by transforming growth factor-beta (TGF-beta) and hypoxia, essential factors in the development of fibrosis. CTGF synthesis was strongly dependent on cell density. High CTGF levels were detected in sparse cells, whereas CTGF expression was reduced in confluent cells. Concomitantly, stimulation of CTGF by TGF-beta or the histone deacetylase inhibitor trichostatin was prevented in dense cells. Exposure of hPTECs to low oxygen tension (1% O2) or the hypoxia mimetic dimethyl-oxalylglycine for 24 h reduced CTGF gene expression in most of the 17 preparations analyzed. Preincubation of the cells under hypoxic conditions significantly reduced TGF-beta-mediated upregulation of CTGF. In line with these data, CTGF mRNA was only induced in interstitial cells, but not in tubular cells in kidneys of mice exposed to hypoxia. Longer exposure to hypoxia or TGF-beta (up to 72 h) did not induce hPTECs to adopt a mesenchymal phenotype characterized by upregulation of alpha-smooth muscle actin, downregulation of E-cadherin, or increased sensitivity of the cells in terms of CTGF expression. Sensitivity was restored by inhibition of DNA methylation. Taken together, our data provide evidence that exposure to hypoxia decreased CTGF gene expression. Furthermore, hypoxia per se was not sufficient to induce a mesenchymal phenotype in primary tubular epithelial cells.

Differential TGF-β Signaling in Retinal Vascular Cells: A Role in Diabetic Retinopathy?

Purpose. An early hallmark of preclinical diabetic retinopathy is thickening of the capillary basal lamina (BL). TGF-beta, a multipotent cytokine acting through its receptors ALK5 and -1, has been postulated to be involved in this phenomenon. In light of this possible role, TGF-beta signaling and its downstream molecular effects were characterized in cultured vascular endothelial cells and pericytes of the retina. Methods. Bovine retinal endothelial cells and pericytes were stimulated with TGF-beta1 in the presence or absence of SD-208, a specific inhibitor of the TGF-beta type I receptor ALK5, or ALK5 small interfering (si)RNA. TGF-beta-signaling pathways were characterized by analysis of phosphorylated Smad2 or -1/5/8 proteins and TGF-beta target genes (PAI-1, fibronectin, CTGF, Smad7, and Id1) and protein (fibronectin). Results. ALK5 was expressed in both cell types, whereas ALK1 was exclusively expressed in endothelial cells. In endothelial cells, TGF-beta induced Smad2 phosphorylation at high concentrations, which was efficiently blocked by ALK5 inhibition. In contrast, in pericytes, Smad2 phosphorylation was rapidly induced at low concentrations of TGF-beta. The ALK1-Smad1/5/8 pathway was activated by TGF-beta in endothelial cells only. TGF-beta caused ALK5-mediated upregulation of PAI-1, Smad7, and fibronectin and in pericytes at lower TGF-beta concentrations than in endothelial cells. CTGF mRNA expression was induced only in pericytes. Fibronectin protein was confirmed to be regulated by TGF-beta in both cell types. Conclusions. TGF-beta signaling in retinal endothelial cells and pericytes show that these cells, and in particular the pericytes, have the essential characteristics to allow for a role of TGF-beta in BL thickening in preclinical diabetic retinopathy.

Effect and mechanism of connective tissue growth factor on myocardium fibrosis of renal hypertensive rat

Objective To investigate the roles of CTGF in the development of myocardial fibrosis of renovascular hypertensive rats from molecular level. Methods Renovascular hypertensive rat ( RHR ) animal models were constructed. Sham operational rats were used as control group. CTGF, TGF-β1were evaluated by qualitative and semiquantitative immunohistoehemical staining in the sixth and the twelfth week after operation to explore their distribution and expression in left ventrieular tissue. CTGF mRNA, TGF-β1 mRNA were evaluated by RT-PCR, and the total collagen in left ventrieular interstitial tissue were observed, CVF, PIP and Coll were also measured. Results Compared with sham operated rat, CVF, PIP and Coll carboxypropetide of precollagen Ⅰ and collagen concentration in renovaseular hypertensive rat were higher(P<0.01). The expression of CTGF, TGF-β1 and CTGF mRNA, TGFβ1 mRNA in myocardium of left ventricle of RHR were higher than those in SOR, CTGF primarily reacted with myocardial interstitium and artery. TGF-β1 mainly expressed in myocardial cells. The expression of CTGF,TGF-β1 and CTGF mRNA, TGFβ1mRNA in myocarclium of left ventricle of the twelfth week RHR or SHR were higher than that in the sixth RHR or SHR( P <0. 05). Correlative analysis revealed that CTGF was positively correlated with ( r =0.815, P < 0.01) ,TGF-β1 ( r = 0. 626, P < 0. 05 ), CVF( r = 0. 742, P < 0. 01 ), Coll ( r = O. 793, P < 0. 01 ) and PICP( r = 0. 675, P <0. 01 ). Conclusions CTGF may play an important role in the development of myocardial interstitial fibrosis. Key words: Intercellular signaling peptides and proteins/PD ; Hypertension; renaL/DT; Endomyocardial fibrosis/DT

Deep dermal fibroblasts contribute to hypertrophic scarring

Hypertrophic scar (HTS) following thermal injury is a dermal fibroproliferative disorder that leads to considerable morbidity. The development of HTS involves numerous cell types and cytokines with dermal fibroblasts being a key cell. We have previously reported that the phenotype of fibroblasts isolated from HTS was altered compared to fibroblasts from normal skin. In this study, normal skin was horizontally sectioned into five layers using a dermatome from which fibroblasts were isolated and cultured. Cells from the deeper layers were observed to proliferate at a slow rate, but were morphologically larger. In ELISA and FACS assays, cells from the deeper layers produced more TGF-β1 and TGF-β1 producing cells were higher. In quantitative RT-PCR, the cells from the deeper layers had higher CTGF and HSP47 mRNA levels compared to those from superficial layers. In western blot, FACS and collagen gel assays, fibroblasts from the deeper layers produced more α-smooth muscle actin (α-SMA), had higher α-SMA positive cells and contracted collagen gels more. Fibroblasts from the deeper layers were also found to produce more collagen, but less collagenase by mass spectrometry and collagenase assay. Interestingly, cells from the deeper layers also produced more of the proteoglycan, versican, but less decorin. Taken together, these data strongly demonstrate that fibroblasts from the deeper layers of the dermis resemble HTS fibroblasts, suggesting that the deeper layer fibroblasts may be critical in the formation of HTS.

Effect of all-trans retinoic acid on liver fibrosis induced by common bile duct ligation in rats

The aim of this study was to investigate the effect and possible mechanism of all-trans retinoic acid (ATRA) on liver fibrosis induced by common bile duct ligation (CBDL) in rats. Fifty-three female Wistar rats were randomly divided into 5 groups: sham operation group (group J, 5 animals) and groups A, B, C and D (12 animals in each group). The rats in groups A, B, C and D were subjected to CBDL to induce liver fibrosis, while those in group J to sham operation. From the 3rd week the rats in groups B, C and D respectively received daily administration of ATRA via gastric tube at three different doses [0.1, 1.5 and 7.5 mg/kg body weight (BW)]. Animals were sacrificed at 6th week. Rats' liver tissues were observed for pathologic changes under a light microscope. The protein levels of type I collagen (COL I), matrix metalloproteinase-2 (MMP2), MMP13 and tissue inhibitors of metalloproteinase-1 (TIMP-1) in liver tissues were determined by immunohistochemical techniques. The expression levels of TGF-beta1 and CTGF mRNA in liver tissues were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that loss of normal hepatic architecture and formation of obvious fibrosis were observed in group A, while ATRA treatment for 4 weeks notably alleviated the pathological changes of hepatocytes. The expression of COL I and TIMP-1 proteins in group A was increased, while decreased in ATRA-treated CBDL groups (P<0.05). ATRA (1.5 and 7.5 mg/kg BW) reduced the expression levels of COL I protein more greatly than that of 0.1 mg/kg BW (P<0.05). ATRA treatment increased the protein levels of MMP2 and MMP13. The expression levels of TGF-beta1 and CTGF mRNA in group A were increased. In comparison with group A, the mRNA levels of TGF-beta1 and CTGF in ATRA-treated CBDL groups were significantly decreased (P<0.05). It was concluded that ATRA could inhibit CBDL-induced liver fibrosis in rats by suppressing the expression of TGF-beta1 and CTGF so as to diminish the inhibition of TIMP-1 on MMP2 and MMP13 and increase the activity of MMP2 and MMP13.

A10 TRANSFORMING GROWTH FACTOR-BETA INDUCES PERSISTENT SYNOVIAL FIBROSIS WHILE CONNECTIVE TISSUE GROWTH FACTOR-INDUCED FIBROSIS IS REVERSIBLE. ROLE FOR LYSYS HYDROXYLASE

Purpose: The main characteristics of osteoarthritis are cartilage damage, osteophyte formation and synovial fibrosis. Synovial fibrosis can contribute to the loss of the function of an OA joint. TGF-b is considered one of the main players in fibrotic diseases. However, CTGF, which can be induced by TGF-b, has been reported to be an important player as well, stated to contribute to maintenance of fibrotic tissue. We investigated the underlying mechanism in that leads to the observed difference in fibrosis persistence between TGF-band CTGF. Methods: We injected C57Bl/6 mice intra-articularly with Ad-TGF-b or Ad-CTGF. After 3, 7 and 21 days knee joint synovial tissue was isolated for RNA isolation or whole knee joints for histology. With Q-RT-PCR relative mRNA levels were determined of matrix molecules, proteases, protease inhibitors, and growth factors. To analyze the enzymes involved in collagen cross link formation, mRNA expression of lysyl hydroxylase (LH) was determined. CTGF-levels induced by TGF-b were measured by ELISA in 24 hour patella-synovial wash-outs of C57Bl/6 mice intra-articularly injected with Ad-TGF-b (3, 7 and 14 days). Results: TGF-b and CTGF induced synovial fibrosis in murine knee joints as shown by histology (as observed by an increase in synovial width). However, TGF-b-induced fibrosis was very persistent, whereas CTGFinduced fibrosis resorbed by day 28. TGF-b induced elevated collagen type I and very high levels of aggrecan expression and hardly any changes in collagen type II and III. CTGF induced no clear changes in collagen type I, II and III. CTGF even decreased aggrecan expression (day 21 only). CTGF induced no changes in MMP3, -9, -13, ADAMTS-4 or -5, whereas TGF-b increased MMP-3, -13 and ADAMTS4. Thus elevated levels of matrix degrading enzyme expression cannot explain the less persistent CTGF-induced fibrosis. TIMP1 (associated with matrix accumulation in fibrotic disorders), was highly up regulated by TGF-b (all days) and only slightly up regulated by CTGF (day 7). TGF-b also induced elevated mRNA levels of TGF-b1 and CTGF. Only TGF-b induced high levels of LH expression, especially LH2b, which has been implicated in hard-to-degrade collagen linkage and was found up regulated in fibrotic lesions. As TGF-b can induce CTGF and up regulated CTGF mRNA in our experiments, we measured CTGF levels in 24 hour patella-synovial wash outs and found that TGF-b induced 64 ng CTGF/ml after 3 days and that CTGF levels were still elevated by day 14. Conclusions: TGF-b induced increased collagen type I and aggrecan mRNA levels in synovial tissue, but also high levels of degrading enzymes. The latter does not explain the persistent nature of TGF-b versus CTGFinduced synovial fibrosis. Lack of collagen type I mRNA induced by CTGF corresponds to findings of other groups. Despite this fact, fibrosis is found. Only TGF-b induced high levels of TIMP1 expression, which was previously found associated with strain dependent fibrosis sensitivity in mice and implicated in accumulation of ECM in fibrotic disorders. TGF-b, not CTGF, induced high levels of LH which are crucial for ECM cross linking. Moreover, LH2b was reported by van der Slot et al. to induce harder-to-degrade crosslinks compared to LH2a. The 2b splicing variant was highly expressed upon stimulation with TGF-b. Since TGF-b clearly up regulated MMP expression a dominant role for TIMP appears unlikely. The strong induction of LH2b by TGF-b compared to CTGF, and consequently harder to degrade cross links, appears to be the most likely cause of the induction of irreversible fibrosis by TGF-b. A11 EFFICACY OF A SINGLE ULTRASOUND GUIDED INJECTION IN HIP OSTEOARTHRITIS

Antisense inhibition of connective tissue growth factor (CTGF/CCN2) mRNA limits hypertrophic scarring without affecting wound healing in vivo

Augmented expression of connective tissue growth factor (CTGF/CCN2) is observed in healing wounds and in a variety of fibrotic disorders. It appears to enhance many of the effects of transforming growth factor-beta and has been shown to have independent fibrogenic functions. Despite these observations, its importance to dermal wound healing and the transition from wound to scar remains poorly defined. In this study, we use established rabbit models to evaluate the roles of CTGF in dermal wound healing and hypertrophic scarring. We show that CTGF mRNA demonstrates persistent up-regulation in hypertrophic scars. Treatment of wounds with antisense oligonucleotides to CTGF has no measurable effect on early wound closure. However, antisense therapy significantly limits subsequent hypertrophic scarring. Inhibition of CTGF is associated with a marked reduction in the number of myofibroblasts in scars and decreased transcription of TIMP-1 and types I and III collagen. These findings confirm CTGF to be a key mediator of hypertrophic scarring in this model. Its effect on myofibroblasts in this setting suggests a mechanism whereby it plays this role. Its limited participation in early healing implies that it may be a useful and specific target for modulating hypertrophic scarring following injury.

Role of transforming growth factor-beta signaling pathway in pathogenesis of benign biliary stricture

To characterize the expression of members of the transforming growth factor-beta (TGF-beta)/Smad/connective tissue growth factor (CTGF) signaling pathway in the tissue of benign biliary stricture, and to investigate the effect of TGF-beta signaling pathway in the pathogenesis of benign biliary stricture. Paraffin embedded materials from 23 cases of benign biliary stricture were analyzed for members of the TGF-beta/Smad/CTGF signaling pathway. TGF-beta (1), TbetaR I , TbetaR II , Smad4, Smad7 and CTGF protein were detected by immunohistochemical strepto-advidinbiotin complex method, and CTGF mRNA was evaluated by hybridization in situ, while 6 cases of normal bile duct served as controls. The percentages of positive cells were counted. The correlation between TGF-beta (1), Smad4 and CTGF was analyzed. The positive expression ratios of TGF-beta (1), TbetaR I , TbetaR II , Smad4, CTGF and CTGF mRNA in 23 cases with benign biliary stricture were 91.3%, 82.6%, 87.0%, 78.3%, 82.6% and 65.2%, respectively, significantly higher than that in 6 cases of normal bile duct respectively (vs 33.3%, 16.7%, 50.0%, 33.3%, 50.0%, 16.7%, respectively, P < 0.05). The positive expression ratio of Smad7 in cases with benign biliary stricture was 70.0%, higher than that in normal bile duct, but this difference is not statistically significant 70.0% vs 50%, P > 0.05). There was a positive correlation between positive expression of TGF-beta (1), Smad4 and CTGF in cases with benign biliary stricture. The high expression of TGF-beta/Smad/CTGF signaling pathway plays an important role in the pathogenesis of benign biliary stricture.

Bacteria induce CTGF and CYR61 expression in epithelial cells in a lysophosphatidic acid receptor-dependent manner

Cysteine-rich protein 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2) are members of the CCN (CYR61, CTGF, nephroblastoma overexpressed gene) family and exert pleiotropic functions such as regulation of adhesion, migration, extracellular matrix deposition, or cell differentiation, and play an important role in wound healing. This study focused on the nature of the so far unknown CTGF and CYR61 mRNA expression of epithelial cells after infection with bacteria. We demonstrate that infection of epithelial cells with attenuated Yersinia enterocolitica lacking the virulence plasmid pYV leads to the expression of CYR61 and CTGF. Virulent Y. enterocolitica bearing the pYV virulence plasmid suppressed the mRNA expression of these genes. Yersinia-mediated inhibition of CTGF and CYR61 mRNA expression is partially mediated by the cysteine protease YopT. Further characterization of the Yersinia factors, which trigger CTGF and CYR61 mRNA expression, demonstrated that these factors were secreted and could be enriched in lipid extracts. Beside Yersinia, several other bacteria such as Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, or Staphylococcus aureus, as well as supernatants of these bacteria induced CTGF and CYR61 expression. Blocking experiments with the lysophosphatidic acid (LPA) receptor-specific inhibitor Ki16425 suggest a general involvement of LPA receptors in bacteria-triggered CTGF and CYR61 expression. These data suggest that LPA receptor-dependent expression of CTGF and CYR61 represents a common host response after interaction with bacteria.

Promoter Hypermethylation Contributes to Frequent Inactivation of a Putative Conditional Tumor Suppressor Gene Connective Tissue Growth Factor in Ovarian Cancer

Connective tissue growth factor (CTGF) is a secreted protein belonging to the CCN family, members of which are implicated in various biological processes. We identified a homozygous loss of CTGF (6q23.2) in the course of screening a panel of ovarian cancer cell lines for genomic copy number aberrations using in-house array-based comparative genomic hybridization. CTGF mRNA expression was observed in normal ovarian tissue and immortalized ovarian epithelial cells but was reduced in many ovarian cancer cell lines without its homozygous deletion (12 of 23 lines) and restored after treatment with 5-aza 2'-deoxycytidine. The methylation status around the CTGF CpG island correlated inversely with the expression, and a putative target region for methylation showed promoter activity. CTGF methylation was frequently observed in primary ovarian cancer tissues (39 of 66, 59%) and inversely correlated with CTGF mRNA expression. In an immunohistochemical analysis of primary ovarian cancers, CTGF protein expression was frequently reduced (84 of 103 cases, 82%). Ovarian cancer tended to lack CTGF expression more frequently in the earlier stages (stages I and II) than the advanced stages (stages III and IV). CTGF protein was also differentially expressed among histologic subtypes. Exogenous restoration of CTGF expression or treatment with recombinant CTGF inhibited the growth of ovarian cancer cells lacking its expression, whereas knockdown of endogenous CTGF accelerated growth of ovarian cancer cells with expression of this gene. These results suggest that epigenetic silencing by hypermethylation of the CTGF promoter leads to a loss of CTGF function, which may be a factor in the carcinogenesis of ovarian cancer in a stage-dependent and/or histologic subtype-dependent manner.

46 ACUTE RESPIRATORY DISTRESS SYNDROME ALVEOLAR FLUID INDUCES MYOFIBROBLAST GENES BUT SUPPRESSES KERATINOCYTE GROWTH FACTOR IN NORMAL HUMAN LUNG FIBROBLASTS.

<h3>Rationale</h3> Alveolar repair is critical to ARDS outcome. Myofibroblast induction is integral to healing by facilitating wound apposition (α-smooth muscle actin (α-SMA), secreting matrix (collagens 1 and 3), cytokines (TGF-β1 and connective tissue growth factor [CTGF]), and epithelial mitogens (keratinocyte growth factor [KGF]). We hypothesized that bronchoalveolar lavage fluid (BALF) from ARDS patients would induce multiple features of myofibroblasts. <h3>Methods</h3> ARDS BALF was collected within 48 hours of intubation. Patients with cardiogenic pulmonary edema were included as negative controls. ARDS and control BALF were incubated with normal human lung fibroblasts for 24 hours, and α-SMA, collagens 1 and 3, CTGF, TGF-β1, and KGF mRNA was measured by real-time RT-PCR. <h3>Results</h3> Ten ARDS patients and five negative controls were enrolled. The average age for ARDS patients was 53 ± 16 years. The average PaO₂/ FiO₂ ratio, compliance, and APACHE 2 scores were 118 ± 70, 21 ± 10, and 28 ± 8, respectively. ARDS BALF induced 110% more α-SMA (<i>p</i> &lt; .05) and 85% more CTGF (<i>p</i> &lt; .05) than controls. In addition, ARDS BALF induced 43% more collagen 1 (<i>p</i> = .11) and 52% more collagen 3 (<i>p</i> = .08) than controls. In contrast, control BALF induced 70% more KGF (<i>p</i> &lt; .03) than ARDS BALF. There was a significant inverse correlation between ARDS induction of α-SMA and KGF (<i>r</i> = −.78, <i>p</i> &lt; .01). The TGF-β1 receptor inhibitor SB431542 significantly reduced ARDS BALF induction of α-SMA, CTGF, and collagens 1 and 3 (<i>p</i> &lt; .05 for all) but had no impact on KGF expression (<i>p</i> = .33). <h3>Conclusion</h3> Early ALI/ARDS BALF has predominant profibrotic characteristics and suppresses epithelial mitogen induction.

High glucose upregulates connective tissue growth factor expression in human vascular smooth muscle cells

BackgroundConnective tissue growth factor (CTGF) is a potent profibrotic factor, which is implicated in fibroblast proliferation, angiogenesis and extracellular matrix (ECM) synthesis. It is a downstream mediator of some of the effects of transforming growth factor β (TGFβ) and is potentially induced by hyperglycemia in human renal mesangial cells. However, whether high glucose could induce the CTGF expression in vascular smooth muscle cells (VSMCs) remains unknown. Therefore, this study was designed to test whether high glucose could regulate CTGF expression in human VSMC. The effect of modulating CTGF expression on VSMC proliferation and migration was further investigated.ResultsExpression of CTGF mRNA was up-regulated as early as 6 hours in cultured human VSMCs after exposed to high glucose condition, followed by ECM components (collagen type I and fibronectin) accumulation. The upregulation of CTGF mRNA appears to be TGFβ-dependent since anti-TGFβ antibody blocks the effect of high glucose on CTGF gene expression. A small interference RNA (siRNA) targeting CTGF mRNA (CTGF-siRNA) effectively suppressed CTGF up-regulation stimulated by high glucose up to 79% inhibition. As a consequence of decreased expression of CTGF gene, the deposition of ECM proteins in the VSMC was also declined. Moreover, CTGF-siRNA expressing vector partially inhibited the high glucose-induced VSMC proliferation and migration.ConclusionOur data suggest that in the development of macrovascular complications in diabetes, CTGF might be an important factor involved in the patho-physiological responses to high glucose in human VSMCs. In addition, the modulatory effects of CTGF-siRNA during this process suggest that specific targeting CTGF by RNA interference could be useful in preventing intimal hyperplasia in diabetic macrovascular complications.

The Effect of Estradiol on the Expression of TGF-β1、 CTGF in rat Hepatic Stellate Cells

目的 探讨雌二醇对肝星状细胞(HSC)的影响及其可能的作用途径.方法 培养大鼠HSC,初次传代后,HSC随机分为4组,分别加入不同浓度的雌二醇,加药后72小时,噻唑蓝(MTT)法检测雌二醇对HSC增殖的影响;逆转录-聚合酶链式反应(RT-PCR)分析其对HSC的转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)的mRNA表达的影响;免疫细胞化学分析其对HSC的α-SMA蛋白的表达的影响.结果 MTT法发现雌二醇抑制HSC的增殖;RT-PCR分析雌二醇干预组HSC TGF-β1mRNA、CTGFmRNA的表达明显下降;免疫细胞化学分析雌二醇干预组HSC α-SMA蛋白表达明显下降;并且都随浓度增加抑制作用增强,差异有显著性(P＜0.05).结论 雌二醇具有抑制肝纤维化作用是因为:①可以抑制HSC的增殖、活化;②抑制了致纤维化生长因子TGF-β1及其下游信号CTGFmRNA表达。