CTGF mRNA Levels Research Articles

Quercetin Treatment Ameliorates Inflammation and Fibrosis in Mice with Nonalcoholic Steatohepatitis1–3

We investigated whether quercetin protects from steatosis and limits the expression of proinflammatory and fibrogenic genes in C57BL/6J mice with nonalcoholic steatohepatitis (NASH) induced by feeding a methionine-choline–deficient (MCD) diet. Quercetin (50 mg/kg) was given by oral route daily. Mice were randomly divided into 4 groups that received for 2 or 4 wk: the control diet plus vehicle, control diet plus quercetin, MCD diet plus vehicle, and MCD diet plus quercetin. At both 2 and 4 wk, feeding the MCD diet resulted in liver steatosis, inflammatory cell accumulation, oxidative stress evaluated by the concentration of TBARS, and fibrosis evidenced by the staining of a-smooth muscle actin-positive cells in the liver. At both 2 and 4 wk, the MCD diet induced an increase in the mRNA levels of Il6, Tnf, Ptgs2, and Hmgb1 and increased the protein concentrations of Toll-like receptor-4, c-Jun terminal kinase, and p65 NF?B subunit compared with control rats. Feeding the mice the MCD diet also triggered an increase of Col1a1, Col3a1, Plod3, Tgfb1, Smad3, Smad7, Pdgfb, Ctgf, Areg, Mmp9, and Timp1 mRNA levels. These effects were totally or partially prevented by treatment with quercetin. The data obtained suggest that attenuation of multiple profibrotic and proinflammatory gene pathways contributes to the beneficial effects of quercetin in mice with MCD diet-induced steatohepatitis.

Oncostatin M inhibits TGF-β1-induced CTGF expression via STAT3 in human proximal tubular cells

Matricellular proteins play a critical role in the development of tubulointerstitial fibrosis and renal disease progression. Connective tissue growth factor (CTGF/CCN2), a CCN family member of matricellular proteins, represents an important mediator during development of glomerular and tubulointerstitial fibrosis in progressive kidney disease. We have recently reported that oncostatin M (OSM) is a potent inhibitor of TGF-β1-induced CTGF expression in human proximal tubular cells (PTC). In the present study we examined the role of TGF-β1- and OSM-induced signaling mechanisms in the regulation of CTGF mRNA expression in human proximal tubular HK-2 cells. Utilizing siRNA-mediated gene silencing we found that TGF-β1-induced expression of CTGF mRNA after 2h of stimulation at least partially depends on SMAD3 but not on SMAD2. In contrast to TGF-β1, OSM seems to exert a time-dependent dual effect on CTGF mRNA expression in these cells. While OSM led to a rapid and transient induction of CTGF mRNA expression between 15min and 1h of stimulation it markedly suppressed basal and TGF-β1-induced CTGF mRNA levels thereafter. Silencing of STAT1 or STAT3 attenuated basal CTGF mRNA levels indicating that both STAT isoforms may be involved in the regulation of basal CTGF mRNA expression. However, knockdown of STAT3 but not STAT1 prevented OSM-mediated suppression of basal and TGF-β1-induced upregulation of CTGF mRNA expression. Together these results suggest that the inhibitory effect of OSM on TGF-β1-induced CTGF mRNA expression is mainly driven by STAT3, thereby providing a signaling mechanism whereby OSM may contribute to tubulointerstitial protection.

Chronic inhibition of nuclear factor kappa B attenuates aldosterone/salt-induced renal injury

AimsRecent studies suggested that nuclear factor kappa B (NF-κB) plays a key role in the pathogenesis of renal injury. This study investigated whether NF-κB inhibition attenuates progressive renal damage in aldosterone/salt-induced renal injury and its mechanisms. Main methodsAdult male rats were uninephrectomized and treated with one of the following for 4weeks: vehicle (0.5% ethanol, subcutaneously); vehicle/1% NaCl (1% NaCl in drinking solution); aldosterone/1% NaCl (1% NaCl in drinking solution and aldosterone, 0.75μg/h, subcutaneously); or aldosterone/1%NaCl+pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB (100mg/kg/day, by gavage). The activity of NF-κB was measured by EMSA and immunohistochemistry, CTGF and ICAM-1 were measured by Western blot and real-time PCR, and TGF-β and CTGF were measured by immunohistochemistry. Key findingsRats that received aldosterone/1% NaCl exhibited hypertension and severe renal injury. Renal cortical mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, protein expression of CTGF and ICAM-1, and NF-κB–DNA binding activity were significantly upregulated in rats that received aldosterone/1% NaCl. Treatment with PDTC significantly decreased the percentage of cells positive for CTGF and TGF-β; mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, and protein levels of CTGF and ICAM-1 were also inhibited by PDTC. SignificanceThese data suggest that the NF-κB signal pathway plays a role in the progression of aldosterone/salt-induced renal injury.

Induction of Hypoxia Inducible Factor Rather than Modulation of Collagen Metabolism Improves Cardiac Function and Reduces Left Ventricular Hypertrophy after Aortocaval Shunt in Rats

Introduction: Matrix reorganization and collagen formation have been suggested to be responsible for cardiac remodeling. Recently, Hypoxia Inducible Factors (HIF) has been shown to be also involved. To differentiate the pathogenesis of cardiac remodeling we used two different prolyl 4-hydroxylase (P4H) inhibitors, FG 0041 known for its potential in reducing collagen formation, FG 2216 for its capability to induce HIF. This study was performed to analyze whether the beneficial effects of P4-HI are mediated by induction of HIF or by effects on collagen metabolism. Methods: 8-week-old Wistar rats either had sham operations (n=14) or received Aortocaval Shunt (ACS). From day 2 rats received P4-HI (n=10 for each P4-HI) or vehicle (n=15) by oral gavage. Echocardiography was performed 28 days after ACS; hemodynamic measurements were done after 30 days. Activity of the metalloproteinase 2 and its inhibitor (TIMP), mRNA levels of CTGF, TGFs1, ITGs1 and Collagen I and III protein were measured. Results: After 30 days both HIF was induced in ACS. Treatment with FG 2216 led to an increase of HIF after 30 days compared to FG 0041 and vehicle. Only FG 2216 prevented left ventricular end-diastolic (7.9 mm vs. 9.8 mm; p<0.001) and end-systolic (4.1 vs. 6.0 mm, p<0.001) dilatation and improved left ventricular ejection fraction (85% vs. 74%, p<0.05). Heart weight (1416 ± 71 vs. 1871 ± 51 mg, p<0.001) and lung weight, as a marker for heart failure, (1958 ± 102 mg vs. 2276 ± 105 mg, p<0.05) were lower in the group receiving FG 2216. Collagen protein and metalloproteinase activity accompanying ACS were significantly decreased by FG 0041, but not by FG 2216. Conclusion: Induction of HIF improves cardiac function and reduces cardiac hypertrophy independent of changes in mRNA, collagen protein or metalloproteinase activity in this model of hypertrophy. Thus FG 2216 affects remodeling by directly targeting cardiomyocytes and has negligible effect on collagen production or extracellular matrix composition.

Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis

IntroductionSystemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by fibrosis of the skin and internal organs. Ang II (angiotensin II), a vasoconstrictive peptide, is a well-known inducer of kidney, heart, and liver fibrosis. The goal of this study was to investigate the profibrotic potential of Ang II in the mouse skin.MethodsAng II was administered by subcutaneous osmotic mini pumps to C57BL/6 male mice. Collagen-content measurements were performed with Gomori Trichrome staining and hydroxyproline assay. The mRNA expression level of collagens, TGF-β1, TGF-β2, TGF-β3, CTGF, αSMA, CD3, Emr1, CD45/B220, MCP1, and FSP1 were quantified with real-time polymerase chain reaction (PCR). Immunostaining was performed for markers of inflammation and fibrosis, including, phospho-Smad2, αSMA, CD3, Mac3, CD45/B220, and CD163B. Fibrocytes were identified by double staining with CD45/FSP1 and CD45/PH4. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT) were identified by double staining with VE-cadherin/FSP1.ResultsAng II-infused mice develop prominent dermal fibrosis in the area proximal to the pump, as shown by increased collagen and CTGF mRNA levels, increased hydroxyproline content, and more tightly packed collagen fibers. In addition, elevated mRNA levels of TGF-β2 and TGF-β3 along with increased expression of pSmad2 were observed in the skin of Ang II-treated mice. Dermal fibrosis was accompanied by an increased number of infiltrating fibrocytes, and an increased number of αSMA-positive cells, as well as CD163B+ macrophages in the upper dermis. This correlated with significantly increased mRNA levels of αSMA, Emr1, and MCP1. Infiltration of CD3-, CD45/B220-, and Mac3-positive cells was observed mainly in the hypodermis. Furthermore, an increased number of double-positive VE-cadherin/FSP1 cells were detected in the hypodermis only.ConclusionsThis work demonstrates that Ang II induces both inflammation and fibrosis in the skin via MCP1 upregulation and accumulation of activated fibroblasts. Additionally, our data suggest that populations of these fibroblasts originate from circulating blood cells. Ang II infusion via osmotic minipumps could serve as a useful mouse model of skin fibrosis to gain new insights into pathogenic mechanisms and to test new antifibrotic therapies.

Nuclear Plakoglobin Is Essential for Differentiation of Cardiac Progenitor Cells to Adipocytes in Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disease of desmosome proteins characterized by fibroadipogenesis in the myocardium. We have implicated signaling properties of junction protein plakoglobin (PG) in the pathogenesis of ARVC. To delineate the pathogenic role of PG in adipogenesis in ARVC. We generated mice overexpressing PG, either a wildtype (PG(WT)) or a truncated (PG(TR)), known to cause ARVC, in the heart; and PG null (PG⁻/⁻) embryos. PG(WT) and PG(TR) mice exhibited fibro-adiposis, cardiac dysfunction, and premature death. Subcellular protein fractionation and immunofluorescence showed nuclear localization of PG(WT) and PG(TR) and reduced membrane localization of PG(TR). Coimmunoprecipitation showed reduced binding of PG(TR) but not PG(WT) to desmosome proteins DSP and DSG2. Transgene PG(WT) and PG(TR) were expressed in c-Kit+:Sca1+ cardiac progenitor cells (CPCs) isolated from the hearts of PG(WT) and PG(TR) by fluorescence activated cell sorting. CPCs isolated from the transgenic hearts showed enhanced adipogenesis, increased levels of adipogenic factors KLF15, C/EBP-α and noncanonical Wnt5b, and reduced level of CTGF, an inhibitor of adipogenesis. Treatment with BIO activated the canonical Wnt signaling, reversed the proadipogenic transcriptional switch and prevented adipogenesis in a dose-dependent manner. Moreover, c-Kit+ CPCs, isolated from PG⁻/⁻ embryos, were resistant to adipogenesis, expressed high mRNA levels of CTGF and other canonical Wnt signaling targets. Nuclear PG provokes adipogenesis in c-Kit+ CPCs by repressing the canonical Wnt signaling and inducing a proadipogenic gene expression. The findings suggest that adipocytes in ARVC, at least in part, originate from c-Kit+ CPCs.

Gene expression in different regions of rat tendons submitted to load exercise associated with nandrolone decanoate administration

The use of anabolic androgenic steroids (AAS) increases risk for tendon injury, however, little is known about the effect of AAS in tendon gene expression. This study evaluated the expression of genes involved on tendon remodeling of the proximal and distal regions of calcaneal tendon, intermediate and distal region of superficial flexor tendon and proximal, intermediate and distal region of deep flexor tendon submitted to jumping water load exercise associated with AAS administration. Wistar male rats were grouped as follows: sedentary (S), trained (T), AAS-treated (10 mg/kg/week) sedentary rats (AAS) and AAS-treated and trained rats (AAST). mRNA levels of COL1A1, COL3A1, TIMP-1, TIMP-2, MMP-2, IGF-Ia, GAPDH, CTGF and TGF-β were evaluated by real-time PCR. Training did not alter the expression of COL1A1, COL3A1 and MMP-2 genes, while AAS administration or its association with training reduced theses gene expression. IGF-Ia enhanced with exercise, while AAS and AAST decreased their expression. In conclusion, the training did not alter the collagen expression and main growth factors in different regions of rat tendon. However, AAS or AAST showed harmful effects in the tendon ECM remodeling. The gene expression was distinct in each tendon region of sedentary animals, indicating that these regions possess a variance in expression of key genes responsible for the tendon adaptation. Grant from FAPESP-06/50986-6.

Connective tissue growth factor expression in precancerous lesions of human esophageal epithelium and prognostic significance in esophageal squamous cell carcinoma

Connective tissue growth factor (CTGF, CCN2), a secreted protein, is involved in the development and progression of esophageal squamous cell carcinoma (ESCC). However, it remains unclear how CTGF expression affects the progression of ESCC. Our study implicated differences of CTGF protein status in precancerous lesions, and retrospectively examined the associations of CTGF mRNA and protein levels with clinical prognosis in ESCC patients. Here immunohistochemistry and the quantitative real-time real-time reverse transcription polymerase were performed for predicting the CTGF protein status and mRNA levels in ESCC patients, respectively. Different degrees of CTGF protein status presented in normal human esophageal epithelium and precancerous lesions, and CTGF protein was highly expressed in ESCCs. Survival analysis showed that CTGF protein status was significantly related to poor survival of ESCC patients (P= 0.024), while no significant difference was observed between CTGF mRNA levels and the survival of ESCC patients (P= 0.196). Multivariate Cox analysis demonstrated that CTGF protein status was the independent factor in prognosis of ESCC patients. In that way, CTGF protein status might elevate the progression of ESCC, and would be significant for the diagnosis of precancerous lesions or early ESCC.

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.

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

Expression of Cyr61, CTGF, and WISP-1 Correlates with Clinical Features of Lung Cancer

BackgroundCCN family, comprising six members (Cyr61, CTGF, Nov, WISP-1, WISP-2, WISP-3), is involved in the stimulation of cell proliferation, migration, adhesion, angiogenesis, and tumorigenesis. Several studies have shown that expression of Cyr61, CTGF, and WISP-1 affects the tumorigenic potential of lung cancer cells in vitro. However, the correlation of expression of CCN family proteins and clinical features of lung cancer remains unknown.Methodology and Principal FindingsIn the present work, we quantified the mRNA levels of Cyr61, CTGF, and WISP-1 in samples from 60 primary lung cancers and their matched normal lung tissues by quantitative real-time PCR assay. Downregulation of the Cyr61 and CTGF genes and upregulation of the WISP-1 gene were found in primary lung cancers compared to the paired normal lung tissues. Immunohistochemistry analysis also disclosed a similar expression pattern of Cyr61, CTGF, and WISP-1 protein in paired lung cancer tissues. Statistical analysis revealed significant associations between expression of either Cyr61 or CTGF with tumor stage, tumor histology, metastasis, smoking, and family history at diagnosis. A significant correlation also existed between WISP-1 expression with tumor histology, and patient age. Moreover, expression levels of Cyr61 and CTGF correlated with survival of the lung-cancer patients.ConclusionsOur results suggest that Cyr61, CTGF, and WISP-1 might be implicated in the development and progression of primary lung cancers, and their levels might serve as valuable prognostic markers, as well as potential targets for therapeutic intervention.

Increased Connective Tissue Growth Factor Relative to Brain Natriuretic Peptide as a Determinant of Myocardial Fibrosis

Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was to investigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF, and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in the development of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsy samples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiac myocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. In a rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportion to one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP was positively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression. Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cells demonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, G protein-coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereas aldosterone and transforming growth factor-beta preferentially induced expression of the CTGF gene. Finally, exogenous BNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase in CTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and diastolic heart failure.

Connective tissue growth factor is a key regulator of fibrosis in pressure overloaded rat heart

The myocardial fibrillar collagen deposition increases myocardial stiffness and exacerbates cardiac function in the pressure overloaded heart. Connective tissue growth factor (CTGF/CCN2/HCS24) is a profibrotic cytokine that promotes fibroblast proliferation and extracellular matrix production in connective tissues. Although, up-regulation of CTGF gene in human failing heart has been demonstrated, precise role of CTGF in maladaptive ventricular remodeling remains to be elucidate. In this study, we determined temporal changes in CTGF mRNA expression level and the correlation between markers of cardiac function during the cardiac hypertrophy induced by abdominal aortic banding in rat. Northern blot analysis showed that CTGF mRNA expression levels were up-regulated even 24 hours after banding. CTGF expression levels were closely correlated with levels of BNP expression (n = 45, r = 0.781, P < 0.001). At day7, the %area of myocardial fibrosis evaluated Masson-Trichrome staining and the abnormal left ventricular diastolic function evaluated by echocardiography strongly correlated with CTGF mRNA levels. Immunohistochemical analysis showed that CTGF protein expressed in cardiac myocytes and fibroblasts around myocardial fibrosis. In conclusion, we have demonstrated that CTGF was upregulated in pressure overloaded rat heart. Our results suggested that CTGF regulates collagen metabolism in myocardium and plays an important role in the development of diastolic dysfunction.

BNP and connective tissue growth factor interact and co-regulate myocardial fibrosis

Connective tissue growth factor (CTGF/CCN2/HCS24) is a profibrotic cytokine induced by TGF-beta and promotes proliferation and extracellular matrix production in connective tissues. Recently, up-regulation of CTGF gene in human failing heart has been demonstrated. On the other hand, BNP has been shown to have antifibrotic effects and block the action of TGF-beta. Therefore, we hypothesized that BNP interacts directly with CTGF as well as TGF-beta in cardiac myocytes (CM) and non-myocytes (CNM). Immunohistochemical staining using specific antibody for CTGF revealed that CTGF was produced not only in CNM but also CM. Recombinant CTGF protein markedly increased mRNA expression levels of procollagen type I and type III in CNM. TGF-beta, norepinephrine and angiotensin II increased CTGF mRNA levels in CM. Importantly, overexpression of CTGF mRNA induced by these stimuli was suppressed by the simultaneous administration of recombinant BNP protein in a dose dependent manner. Next, we examined CTGF mRNA expression levels in the rat cardiac hypertrophy model induced by abdominal aortic banding. CTGF mRNA expression levels were elevated in the hypertrophied hearts and the ratio of CTGF mRNA expression against BNP mRNA were significantly correlated with the % area of myocardial fibrosis. In conclusion, our study suggest that CTGF and BNP genes are synergistically regulated under hypertrophic stimuli, even though BNP may counteract the profibrotic process driven by CTGF.

Levels of expression of CYR61 and CTGF are prognostic for tumor progression and survival of individuals with gliomas.

The biological properties of CCN proteins include stimulation of cell proliferation, migration, and adhesion, as well as angiogenesis and tumorigenesis. We quantified CYR61, CTGF, WISP-1, and NOV mRNA expression levels in samples from sixty-six primary gliomas and five normal brain samples using quantitative real-time PCR assay. Statistical analysis was performed to explore the links between expression of the CCN genes and clinical and pathological parameters. Overexpression of CYR61, CTGF, WISP-1, and NOV occurred in 48% (32 of 66), 58% (38 of 66), 36% (24 of 66), and 15% (10 of 66) of primary gliomas, respectively. Interestingly, significant associations were found between CYR61 expression versus tumor grade, pathology, gender, and age at diagnosis. Also, a significant correlation existed between CTGF mRNA levels versus tumor grade, gender, and pathology. In contrast to CYR61 and CTGF, no significant association was found between expression of either WISP-1 or NOV versus any of the pathological features. Furthermore, Cox regression analysis showed that CYR61 and CTGF expression had a significant correlation with patient survival. These results suggest that CYR61 and CTGF may play a role in the progression of gliomas; their levels at diagnosis may have prognostic significance; and these proteins might serve as valuable targets for therapeutic intervention.

Connective tissue growth factor is up-regulated in the diabetic retina: amelioration by angiotensin-converting enzyme inhibition.

Connective tissue growth factor (CTGF) has been postulated to have prosclerotic and angiogenic properties. The aim of this present study was to characterize retinal CTGF expression in the absence and presence of diabetes and in the context of treatment with the angiotensin-converting enzyme (ACE) inhibitor, perindopril. Retinas were obtained from control, diabetic, and diabetic plus perindopril-treated (3 mg/d) rats. CTGF gene expression was quantitated by RT-PCR and localized by in situ hybridization. CTGF protein expression was analyzed by Western blotting and localized by immunohistochemistry. Diabetes was associated with a greater than 2-fold increase in CTGF mRNA levels, which was attenuated by perindopril treatment. CTGF immunoreactivity was increased almost 2-fold in diabetes and was ameliorated by the ACE inhibitor perindopril. By in situ hybridization and immunohistochemistry, the major site of CTGF gene expression in the retina of diabetic rats was the ganglion cell layer. Based on the known in vivo effects of CTGF, it is postulated that this growth factor plays a pivotal role in mediating diabetes-associated retinal pathology. Furthermore, the protective effects of ACE inhibitors on retinal pathology may partly be mediated via effects on retinal CTGF expression.

Connective tissue growth factor gene expression alters tumor progression in esophageal cancer.

The ability of cancer cells to initiate specific fibroblast reactions may subsequently determine tumor evolution. In the present study we examined the coordinated expression of transforming growth factor-beta-1 (TGF-beta1), its signaling receptors, and its downstream mediator-connective tissue growth factor (CTGF)--and their impact on tumor progression and fibrogenesis in esophageal carcinomas. Messenger ribonucleic acid (mRNA) expression of TGF-beta1, CTGF, TGF-beta receptor subtype I ALK5 (TbetaR-IALK5), and TGF-beta receptor type II (TbetaR-II) was studied by Northern blot analysis in esophageal cancer and the normal esophagus. By means of immunohistochemistry and Western blot analysis, the respective proteins were localized in the tissue samples and the protein content was quantitated. Northern blot analysis revealed 3-fold and 4-fold increases (p < 0.05) in TGF-beta1 and CTGF mRNA levels, respectively, in esophageal cancer in comparison with normal controls, whereas TbetaR-I mRNA levels were significantly decreased and TbetaR-II mRNA levels were unchanged in the cancer samples. Immunostaining revealed results similar to those seen on the RNA level. TGF-beta1 and CTGF immunoreactivity were increased, TbetaR-II was unchanged, and TbetaR-IALK5 immunoreactivity was decreased. CTGF immunoreactivity was mainly present in the stroma surrounding the cancer cells but was also present in the cancer cells. The degree of fibrosis was different in squamous and adenocarcinomas and was significantly related to CTGF mRNA expression levels. The presence of CTGF in squamous cell carcinomas was associated with longer survival, whereas in adenocarcinomas it influenced survival negatively. The findings indicate that TGF-beta signaling is disturbed in esophageal cancer. CTGF, a downstream effector of TGF-beta action, differentially influences the composition of tumor microenvironment and distinct cell-matrix interactions in the two histological types of esophageal carcinoma, resulting in differences in tumor progression and patient survival.