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Results for “Connective Tissue Growth Factor”

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PubMed2007

Transforming growth factor-beta2 and connective tissue growth factor in proliferative vitreoretinal diseases: possible involvement of hyalocytes and therapeutic potential of Rho kinase inhibitor.

The critical association of connective tissue growth factor (CTGF), which is thought to be one of the downstream mediators of transforming growth factor-beta (TGF-beta), with vitreoretinal diseases remains to be clarified. In the current study, we first demonstrated the correlation between the concentrations of TGF-beta2 as well as CTGF in the vitreous and CTGF gene regulation in cultured hyalocytes. Concentrations of TGF-beta2 and CTGF in the vitreous from patients with proliferative vitreoretinal diseases were significantly higher than in those with nonproliferative diseases, and there was a positive correlation between their concentrations (r = 0.320, P < 0.01). Cultured hyalocytes expressed CTGF mRNA, which was enhanced in the presence of TGF-beta2, associated with nuclear accumulation of Smad4. TGF-beta2-dependent Smad4 translocation and CTGF gene expression were mediated through Rho kinase and at least partially via p38 mitogen-activated protein kinase. Finally, fasudil, a Rho kinase inhibitor already in clinical use, inhibited both Smad4 translocation and CTGF gene expression. In conclusion, combined effects of TGF-beta2 and CTGF appear to be involved in the pathogenesis of proliferative vitreoretinal diseases. Hyalocytes may be a possible source of CTGF and thus might play a role in vitreoretinal interface diseases. Furthermore, Rho kinase inhibitors might have therapeutic potential to control fibrotic disorders in the eye.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2004

Integrated actions of transforming growth factor-beta1 and connective tissue growth factor in renal fibrosis.

Matrix accumulation in the renal tubulointerstitium is predictive of a progressive decline in renal function. Transforming growth factor-beta(1) (TGF-beta(1)) and, more recently, connective tissue growth factor (CTGF) are recognized to play key roles in mediating the fibrogenic response, independently of the primary renal insult. Further definition of the independent and interrelated effects of CTGF and TGF-beta(1) is critical for the development of effective antifibrotic strategies. CTGF (20 ng/ml) induced fibronectin and collagen IV secretion in primary cultures of human proximal tubule cells (PTC) and cortical fibroblasts (CF) compared with control values (P < 0.005 in all cases). This effect was inhibited by neutralizing antibodies to either TGF-beta or to the TGF-beta type II receptor (TbetaRII). TGF-beta(1) induced a greater increase in fibronectin and collagen IV secretion in both PTC (P < 0.01) and CF (P < 0.01) compared with that observed with CTGF alone. The combination of TGF-beta(1) and CTGF was additive in their effects on both PTC and CF fibronectin and collagen IV secretion. TGF-beta(1) (2 ng/ml) stimulated CTGF mRNA expression within 30 min, which was sustained for up to 24 h, with a consequent increase in CTGF protein (P < 0.05), whereas CTGF had no effect on TGF-beta(1) mRNA or protein expression. TGF-beta(1) (2 ng/ml) induced phosphorylated (p)Smad-2 within 15 min, which was sustained for up to 24 h. CTGF had a delayed effect on increasing pSmad-2 expression, which was evident at 24 h. In conclusion, this study has demonstrated the key dependence of the fibrogenic actions of CTGF on TGF-beta. It has further uniquely demonstrated that CTGF requires TGF-beta, signaling through the TbetaRII in both PTCs and CFs, to exert its fibrogenic response in this in vitro model.

Antibodies↗

PubMed2005

Smad3 and extracellular signal-regulated kinase 1/2 coordinately mediate transforming growth factor-beta-induced expression of connective tissue growth factor in human fibroblasts.

Connective tissue growth factor (CTGF) is secreted by fibroblasts stimulated with transforming growth factor-beta (TGF-beta). CTGF is a potent enhancer of fibroblast proliferation, chemotaxis, and extracellular matrix deposition, and it is thought to mediate some of the fibrogenic effects of TGF-beta. Here, we have elucidated signaling pathways involved in regulating the TGF-beta-induced production of CTGF in primary fibroblasts. TGF-beta induced the expression of CTGF messenger RNA and protein in human gingival fibroblasts after 2 h of treatment. Adenoviral overexpression of Smad3 enhanced the TGF-beta-elicited expression of CTGF, whereas Smad7 and dominant-negative Smad3 suppressed the effects of TGF-beta on CTGF and Cyr61 expression. Pre-treatment of cells with PD98059, an inhibitor for extracellular signal-regulated kinase (ERK)1/2-activator mitogen-activated protein kinase (MAPK)/ERK kinase (MEK)1, potently inhibited the TGF-beta-induced expression of CTGF. Furthermore, co-expression of Smad3 with constitutively active MEK1 resulted in potent induction of CTGF production without exogenous TGF-beta stimulation. Together, these results demonstrate that Smad3 and ERK1/2 coordinately mediate TGF-beta-induced release of CTGF by fibroblasts. It is conceivable that the crosstalk between Smad3 and ERK1/2 signaling cascades plays an important role in regulating CTGF expression, e.g., in wound repair and tissue fibrosis and could be exploited in therapeutic targeting of fibrotic conditions.

Cells, Cultured↗

PubMed2005

Transforming growth factor-beta enhances connective tissue growth factor expression in L6 rat skeletal myotubes.

Transforming growth factor (TGF)-beta plays an important role in fibrosis of various organs and tissues. TGF-beta1 stimulates fibroblastic cells to form extracellular matrix (ECM), and regulates all critical events in wound healing. Connective tissue growth factor (CTGF), a TGF-beta-inducible molecule, has recently been reported to promote fibroblast proliferation, migration, adhesion and extracellular matrix formation, both in vivo and in vitro. In this study, we demonstrated that TGF-beta1 enhances CTGF mRNA and protein levels in L6 rat skeletal muscle myotubes. TGF-beta might, therefore, play a role in fibrosis of skeletal muscle by stimulating CTGF expression in the muscle tissue itself.

Animals↗

PubMed2007

Actin-dependent regulation of connective tissue growth factor.

Expression of connective tissue growth factor (CTGF) in endothelial cells is modulated by shear stress affecting the organization of the cytoskeleton. The molecular connection between alterations of actin and CTGF expression was investigated in human umbilical vein endothelial cells (HUVEC) and a microvascular endothelial cell line. Overexpression of nonpolymerizable monomeric actin R62D interfered with stress fiber formation in HUVEC and concomitantly reduced immunoreactive CTGF. In microvascular endothelial cells, flow-dependent upregulation of CTGF was prevented by this actin mutant. In contrast, overexpression of actin S14C strengthened filamentous actin and increased CTGF expression. These data indicated an inverse relationship between CTGF expression and monomeric actin. Coexpression of the mutant actins and different CTGF promoter constructs revealed an actin-sensitive site between 3 and 4.5 kb of the CTGF promoter. A CArG-like box at -3791 bp was responsible for actin-dependent CTGF induction as shown by mutagenesis. Overexpression of actin S14C activated the nonmutated promoter significantly more strongly than the mutated promoter. Actin polymerization is regulated by the small GTPase RhoA and activation of serum response factor (SRF). Overexpression of constitutively active RhoA or SRF significantly increased CTGF protein synthesis. The 4.5-kb promoter construct, but not the construct with a mutation in the CArG box, was activated by SRF or RhoA, providing evidence for a functional role of this site in CTGF induction. These findings provide novel evidence that monomeric actin is the connecting link between alterations in the cytoskeleton and CTGF gene expression and demonstrate the importance of SRF in regulating CTGF transcription.

Actins↗

PubMed2000

Regulation of connective tissue growth factor gene expression in retinal vascular endothelial cells by angiogenic growth factors.

BACKGROUND: Connective tissue growth factor (CTGF) is a novel, cysteine-rich secreted protein, which is implicated in fibrotic disorders and atherosclerosis. To elucidate the role of CTGF in fibrovascular proliferative retinopathy, we investigated the regulation of CTGF gene expression in a cell line of retinal vascular endothelial cells (RVEC) stimulated with fetal calf serum (FCS) and angiogenic growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-BB (PDGF-BB), endothelial growth factor (EGF), transforming growth factor-beta 1 and -beta 3 (TGF-beta 1, TGF-beta 3), and insulin-like growth factor-I (IGF-I). METHODS: RVEC derived from Macaca mulatta (CRL-1780; ATCC) were stimulated with 10% FCS as well as with VEGF, bFGF, PDGF-BB, TGF-beta 1, TGF-beta 3, EGF, or IGF-I. Time-dependent CTGF gene expression was assessed by northern blot analysis. RESULTS: FCS, TGF-beta 1, TGF-beta 3, bFGF, and EGF induced an upregulation of CTGF gene expression in RVEC in a time-dependent manner. Highest expression was induced with TGF-beta 1. No response on CTGF gene expression could be detected to VEGF, PDGF-BB, or IGF-I. CONCLUSION: The present study demonstrates for the first time that CTGF mRNA is expressed at high levels in RVEC, and that the level of the temporal pattern of its expression is differentially regulated by angiogenic growth factors, indicating a significant role of CTGF in the pathological course of uncontrolled retinal angiogenesis.

Animals↗

PubMedDate not supplied

Down-regulation of connective tissue growth factor and type I collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental liver fibrosis.

Transforming growth factor (TGF)-beta 1 is a major mediator of liver fibrosis. Connective tissue growth factor (CTGF) mediates TGF-beta 1 pro-fibrogenic effects in vitro, but its in vivo role is unknown. Both TGF-beta 1 and CTGF are overexpressed in hepatic stellate cells during liver fibrosis. We have used antisense oligonucleotides to examine the role of CTGF in carbon tetrachloride-induced liver fibrosis in mice. Mice received carbon tetrachloride together with CTGF or TGF-beta 1 antisense oligonucleotides for 2 weeks (preventive model), or carbon tetrachloride for 2 weeks followed by carbon tetrachloride and oligonucleotides for 2 more weeks (curative model). In both models, CTGF and TGF-beta 1 oligonucleotides decreased by more than 50 percent the mRNA expression of their targets. Type I collagen mRNA was also decreased by about 40 percent in the preventive experiment. Tissue inhibitor of matrix metalloproteinase-1 mRNA expression and fibrotic deposition evaluated by Sirius red staining were not modified in any group. In summary, our results suggest that hepatic stellate cells can be targeted in vivo with oligonucleotides, and that reducing CTGF levels can lead to a decrease in fibrogenesis as shown by the reduction in type I collagen expression. The lack of effect on fibrosis may be due to the persistence of high tissue inhibitor of matrix metalloproteinase-1 expression.

Animals↗

PubMed2001

Transforming growth factor-beta and connective tissue growth factor: key cytokines in scleroderma pathogenesis.

Evidence for a role for members of the transforming growth factor beta (TGF-beta) family of cytokines in the pathogensis of systemic sclerosis and other fibrotic conditions is provided from studies of TGF-beta protein and gene expression in lesional biopsy specimens, from altered responses of explanted fibroblasts to TGF-beta stimulation which are associated with increased receptor expression on these cells and from genetic data linking TGF-beta gene loci to the disease. Of the many effects of TGF-beta on fibroblast properties induction of the connective tissue growth factor/Cyr61/NOV (CCN) family members, connective tissue growth factor (CTGF) may be particularly relevant to fibrosis. Moreover, systemic sclerosis (SSc) fibroblasts demonstrate constitutive over expression of CTGF that promotes migration, proliferation and matrix production. Studies of mechanisms regulating constitutive expression of CTGF by SSc fibroblasts are currently being undertaken and indicate that a TGF-beta responsive element in the CTGF promoter is involved, although this appears to function independent of the Smad proteins, suggesting that other TGF-beta-regulated pathways may be involved. TGF-neutralizing strategies have now been shown to abrogate many animal models of fibrosis, and will soon reach the clinical arena for SSc. These agents will further clarify the role of this ligand in initiating or sustaining fibrosis and offer the exciting possibility of targeted therapy for this disease.

Connective Tissue Growth Factor↗

PubMed2003

Comparison of prostaglandin F2alpha, bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression.

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.

Alprostadil↗

PubMed2004

[Transforming growth factor beta 1 modulates connective tissue growth factor expression via Smad2 signaling pathway in podocyte in vitro].

OBJECTIVE: To assess the expression of connective tissue growth factor (CTGF), and the signaling pathway for the regulation of CTGF by transforming growth factor beta1 (TGF beta(1)) in podocytes. METHODS: In this study, we observed the effects of three potent profibrotic growth factors-TGF beta(1), Platelet-derived growth factor (PDGF), and Angiotensin II (AngII) on the expression of CTGF protein by Western blot analysis in cultured mouse podocytes, which is one of the most important cell construction of glomerular filter barrier, and we also investigated the underlying ERK and Smads signaling pathway through which TGF beta(1) regulates CTGF expression. The levels of CTGF mRNA were assayed by RT-PCR. RESULTS: Basal levels of CTGF protein were observed in cultured podocytes, treatment with 20 ng/ml PDGF and 10(-6) mol/L Ang. II for 24 h did not stimulate the expression of CTGF protein compared with control (P > 0.05), but significantly increase in levels of CTGF protein were seen in 1 ng/ml TGF beta(1) treated cells compared with with control (P < 0.05), and the levels of CTGF were up-regulated in a TGF beta(1) dose-dependent manner; The level of CTGF mRNA was also stimulated by 1 ng/ml TGF beta(1) at 12 h. 1 ng/ml TGF beta(1) induced phosphorylation of Smad(2) and ERK(1/2), and both reached the peak at 30 min; suppression of phosphorylation of Smad(2) with Staurosporine, a Serine/Threonine kinase inhibitor, diminished TGF beta(1)-triggered expression of CTGF protein, while blockade of phosphorylation of ERK(1/2) with PD98059, a specific ERK(1/2) activation inhibitor, did not decrease the TGF beta(1)-triggered expression of CTGF protein. CONCLUSION: TGF beta(1) stimulated the expression of CTGF protein via Smad(2)-dependent and ERK(1/2)-independent signaling pathway in podocyte in vitro.

Angiotensin II↗

PubMed2000

Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells.

Fibroblastic proliferation accompanies many angiogenesis-related retinal and systemic diseases. Since connective tissue growth factor (CTGF) is a potent mitogen for fibrosis, extracellular matrix production, and angiogenesis, we have studied the effects and mechanism by which vascular endothelial growth factor (VEGF) regulates CTGF gene expression in retinal capillary cells. In our study, VEGF increased CTGF mRNA levels in a time- and concentration-dependent manner in bovine retinal endothelial cells and pericytes, without the need of new protein synthesis and without altering mRNA stability. VEGF activated the tyrosine receptor phosphorylation of KDR and Flt1 and increased the binding of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit to KDR and Flt1, both of which could mediate CTGF gene induction. VEGF-induced CTGF expression was mediated primarily by PI3-kinase activation, whereas PKC and ERK pathways made only minimal contributions. Furthermore, overexpression of constitutive active Akt was sufficient to induce CTGF gene expression, and inhibition of Akt activation by overexpressing dominant negative mutant of Akt abolished the VEGF-induced CTGF expression. These data suggest that VEGF can increase CTGF gene expression in bovine retinal capillary cells via KDR or Flt receptors and the activation of PI3-kinase-Akt pathway independently of PKC or Ras-ERK pathway, possibly inducing the fibrosis observed in retinal neovascular diseases.

Animals↗

PubMed2001

Peroxisome proliferator-activated receptor gamma inhibits transforming growth factor beta-induced connective tissue growth factor expression in human aortic smooth muscle cells by interfering with Smad3.

Activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) after balloon injury significantly inhibits VSMC proliferation and neointima formation. However, the precise mechanisms of this inhibition have not been determined. We hypothesized that activation of PPAR gamma in vascular injury could attenuate VSMC growth and matrix production during vascular lesion formation. Since connective tissue growth factor (CTGF) is a key factor regulating extracellular matrix production, abrogation of transforming growth factor beta (TGF-beta)-induced CTGF production by PPAR gamma activation may be one of the mechanisms through which PPAR gamma agonists inhibit neointima formation after vascular injury. In this study, we demonstrate that the PPAR gamma natural ligand (15-deoxyprostaglandin J(2)) and a synthetic ligand (GW7845) significantly inhibit TGF-beta-induced CTGF production in a dose-dependent manner in HASMCs. In addition, suppression of CTGF mRNA expression is relieved by pretreatment with an antagonist of PPAR gamma (GW9662), suggesting that the inhibition of CTGF expression is mediated by PPAR gamma. To elucidate further the molecular mechanism by which PPAR gamma inhibits CTGF expression, an approximately 2-kilobase pair CTGF promoter was cloned. We found that PPAR gamma activation inhibits TGF-beta-induced CTGF promoter activity in a dose-dependent manner, and suppression of CTGF promoter activity by PPAR gamma activation is completely rescued by overexpression of Smad3, but not by Smad4. Furthermore, PPAR gamma physically interacts with Smad3 but not Smad4 in vitro in glutathione S-transferase pull-down experiments. Taken together, the data suggest that PPAR gamma inhibits TGF-beta-induced CTGF expression in HASMCs by directly interfering with the Smad3 signaling pathway.

Aorta↗

PubMed2002

Neoplastic cells and proliferating endothelial cells express connective tissue growth factor (CTGF) in glioblastoma.

Connective tissue growth factor (CTGF) was recently described as a cysteine-rich mitogenic peptide that is the prototypic member of the CCN family of protein including CTGF/fisp12, cef/10/cyr61 and nov. Recently CTGF was reported to have a function of mediating endothelial cell adhesion and migration through integrin alpha(v)beta3, promoting endothelial cell survival, and inducing angiogenesis in vivo. To clarify involvement of CTGF in tumor angiogenesis, using brain tissues with glioblastoma, we examined CTGF expression and localization by immunohistochemical staining and reverse transcription-polymerase chain reaction. The result showed that both glioblastoma tumor cells and proliferating endothelial cells are positive for CTGF by immunohistochemical staining, and CTGF mRNA expression was evaluated by RT-PCR in the brain tissues of glioblastoma. These findings suggest that CTGF may have the potential to stimulate angiogenesis in glioblastoma and function as an angiogenic factor in the process of tumor growth.

Adolescent↗

PubMed2001

Identification of human ccn2 (connective tissue growth factor) promoter polymorphisms.

BACKGROUND: Connective tissue growth factor (CCN2; CTGF) is a newly identified growth factor, which is involved in the regulation of wound repair and fibrosis. Because there is variation among individuals with respect to tissue response to injury, genetic factors might be involved in the final outcome of tissue repair or scarring. For example, polymorphisms in the promoter region of genes, such as those encoding transforming growth factor beta1 (TGF-beta1), interleukin 10 (IL-10), and tumour necrosis factor alpha (TNF-alpha), influence transcriptional responses and are thought to contribute to the dysregulation of these genes in pathological conditions. AIM: To investigate whether the promoter region of the ccn2 (ctgf) gene contains polymorphic sequences that might account for differential expression. MATERIALS/METHODS: Seventy seven human DNA samples were sequenced-45 were from healthy controls and 32 were from patients with ischaemic heart disease (IHD)-using M13 tailed sequence specific ccn2 (ctgf) primers for amplification of a 600 bp fragment upstream of the transcription start site. Amplicons were bidirectionally sequenced with a dye primer M13 forward and reverse sequencing kit. RESULTS: A C to G substitution was identified at position -132 in one of the patients with IHD. Moreover, in five of the 32 patients with IHD and in six of the 45 healthy controls, a G to C polymorphism was found at position -447. These substitutions at -132 and -447 are thought to lie within predicted binding domains for the transcription factors Pbx-1 and MZF1, respectively. In addition, insertions at position -43 (G), -47 (C), -71 (G) and a C to T substitution at position -198 were found in all DNA samples compared with the published ccn2 (ctgf) promoter sequence. These corrections do not involve sequences predicted to function as transcription factor binding sites. CONCLUSION: Sequence analysis of the ccn2 (ctgf) promoter of 77 human DNA samples has revealed corrections and polymorphic sites. The latter lie within putative regulatory elements.

Alleles↗

PubMed2003

Angiotensin II increases connective tissue growth factor in the kidney.

Connective tissue growth factor (CTGF) has been described as a novel fibrotic mediator. CTGF is overexpressed in several kidney diseases and is induced by different factors involved in renal injury. Angiotensin II (AngII) participates in the pathogenesis of kidney damage, contributing to fibrosis; however, whether AngII regulates CTGF in the kidney has not been explored. Systemic infusion of AngII into normal rats for 3 days increased renal CTGF mRNA and protein levels. At day 7, AngII-infused rats presented overexpression of CTGF in glomeruli, tubuli, and renal arteries, as well as tubular injury and elevated fibronectin deposition. Only treatment with an AT(1) receptor antagonist, but not an AT(2), diminished CTGF and fibronectin overexpression and ameliorated tubular damage. In rats with immune complex nephritis, renal overexpression of CTGF was diminished by the ACE inhibitor quinapril, correlated with a diminution in fibrosis. In cultured renal cells (mesangial and tubular epithelial cells) AngII, via AT(1), increased CTGF mRNA and protein production, and a CTGF antisense oligonucleotide decreased AngII-induced fibronectin synthesis. Our data show that AngII regulates CTGF in the kidney and cultured in mesangial and tubular cells. This novel finding suggests that CTGF could be a mediator of the profibrogenic effects of AngII in the kidney.

Angiotensin II↗

PubMed1997

Connective tissue growth factor. Friend or foe?

Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted peptide, which is implicated in human atherosclerosis and fibrotic disorders such as systemic scleroderma. CTGF is a member of the peptide family that includes serum-induced immediate early gene products, a v-src-induced peptide, and a putative proto-oncogene. The CTGF gene family is a modular protein and is conserved throughout evolution. CTGF mRNA has been found in the human, mouse, chicken, frog, and fly. The functions of the CTGF gene family include embryogenesis, wound healing, and regulation of extracellular matrix production. Human CTGF is undetectable in normal blood vessels but overexpressed in atherosclerotic lesions, suggesting an important role in atherogenesis.

Connective Tissue Growth Factor↗

PubMed2003

Role of connective tissue growth factor in mediating hypertrophy of human proximal tubular cells induced by angiotensin II.

BACKGROUND/AIMS: Cellular hypertrophy is an early important pathological feature of renal diseases such as diabetic nephropathy and remnant kidney. Recent studies have demonstrated that angiotensin II (Ang II) might be a major contributor in regulating cell hypertrophy. However, the exact mechanism is still unclear. The aim of our present work was to investigate the possible role of a newly clarified fibrogenic factor, connective tissue growth factor (CTGF), in mediating the effect of Ang II-induced tubular cell hypertrophy. METHODS: The cell line HK2 was grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum. After the cells were rested in serum-free medium for 24 h, the dose and time response of CTGF mRNA expression to Ang II stimulation was observed by RT-PCR, and protein synthesis was observed by Western blotting. The effect of anti-CTGF on Ang II (10(-7)M)-induced [(3)H]-leucine incorporation, total protein content (Coomassie brilliant blue G250 method) and change in cellular size [determined by a scanning electronic microscope (SEM)] was also observed. The influence of anti-CTGF antibody on the cell cycle was analyzed by using a fluorescence-activated cell sorter flow cytometer. RESULTS: The results showed that Ang II induced expression of CTGF mRNA in a time- and dose-dependent manner (p < 0.05 and 0.01, respectively). Stimulation of cells with Ang II (10(-7)M) for 48 h resulted in a 92% increase in [(3)H]-leucine incorporation (5,584 cpm/10(5) cells at 0 h vs. 10,741 cpm/10(5) cells at 48 h; p = 0.01), which was significantly abolished by treatment with anti-CTGF antibody. Ang II (10(-7)M) significantly increased the total protein content in HK2 cells (control: 0.169 +/- 0.011 mg/10(5) cells vs. Ang II: 0.202 +/- 0.010 mg/10(5) cells; p = 0.03), which was markedly inhibited by cotreatment with anti-CTGF antibody. The average cellular diameter determined by SEM showed that the increase in cell size induced by Ang II could be significantly inhibited by anti-CTGF antibody (control: 11.92 +/- 1.62 microm, Ang II group: 20.63 +/- 3.83 microm, Ang II + anti-CTGF group: 16.43 +/- 3.23 microm; p < 0.01, respectively). Furthermore, the flow cytometer study showed that Ang II arrested the cell cycle at G0-G1 phase, which was significantly reversed by treatment with anti-CTGF antibody (G0-G1 percentage: in Ang II group: 76 +/- 1.8%, in Ang II + anti-CTGF group: 71 +/- 1.78%; p = 0.04). CONCLUSION: Our data are the first to clearly demonstrate that CTGF might be an important mediator of Ang II-induced renal hypertrophy, which suggests that inhibiting the production of CTGF might be the new strategy in early prevention of renal fibrosis.

Angiotensin II↗

PubMed2002

Expression of connective tissue growth factor in intra-abdominal adhesions.

PURPOSE: Connective tissue growth factor stimulates fibroblast proliferation and extracellular matrix deposition in many fibrotic disorders. The aim of our study was to determine the expression pattern of connective tissue growth factor in postoperative intra-abdominal adhesions. METHODS: Adhesions were created in 46 Sprague-Dawley rats by complete dissection and resuturing of a peritoneal patch 2 cm in diameter, lateral from the midline incision. Animals were killed at postoperative Days 3, 6, 9, 12, 15, 18, and 21 and the adhesions scored on a scale of 0 to 5. Tissue samples from adhesion areas and from uninvolved peritoneum were evaluated by Northern and Western blotting for temporal connective tissue growth factor mRNA and protein expression, respectively. Immunohistochemical analysis was performed for connective tissue growth factor localization. RESULTS: Adhesions formed in all animals after surgery and were confined to the peritoneal patches. Adhesion formation increased across time, with significant correlation between adhesion scores and postoperative days (r = 0.329, P = 0.026). Connective tissue growth factor mRNA concentrations were significantly elevated in adhesion tissue throughout the three-week period when compared with normal peritoneum (P = 0.012); peak levels occurred between Days 6 and 15. Western blots demonstrated connective tissue growth factor protein expression in adhesions from Days 6 to 21, in contrast to negligible bands in normal peritoneum. Fibroblasts within the adhesive tissue, but not in uninjured peritoneum, stained positive for connective tissue growth factor by immunohistochemistry. CONCLUSIONS: We have demonstrated a specific temporal and spatial expression pattern for connective tissue growth factor in intra-abdominal adhesions during a three-week postoperative time course. According to what is known about the functional role of connective tissue growth factor in fibrogenesis, our findings warrant further investigations addressing a causal relationship between this growth factor and fibrous adhesion formation.

Animals↗