Connective Tissue Growth Factor Protein Expression Research Articles

IL-13 induces liver fibrosis via TGF-β-dependent and independent pathways

IL-13 is now well recognized as a crucial pro-fibrogenic cytokine, especially in Schistosomiasis associated chronic liver disease. However, the molecular/cellular mechanism of IL-13 in liver fibrosis is unknown. We hypothesized that IL-13 may exert its pro-fibrogenic effects through both, TGF-b-dependent and independent pathways in liver. IL-13, but not TGF-b treatment remarkably induced connective tissue growth factor (CTGF) protein expression in quiescent and activated rat hepatic stellate cells (HSCs), the key cells for collagen production during liver fibrogenesis. The data suggest that IL-13 induces pro-fibrogenic protein independent of TGF-b in HSCs. Alone, IL-13 treatment does not induce CTGF protein expression, however, IL-13 enhances TGF-b-induced CTGF expression in CFSC-2G, a cell line from CCl4-induced cirrhotic rat HSCs. Both, IL-13 and TGF-b induce a-smooth muscle actin (a-SMA) protein expression in CFSC-2G cells. However, IL-13-dependent a-SMA protein expression is completely abrogated with SB431542, a specific TGF-b receptor I (ALK5) inhibitor, suggesting that IL-13-induced a-SMA expression is TGF-b-dependent. Furthermore, IL-13 treatment directly up-regulated TGF-b protein expression STAT6-independent in CFSC-2G cells.

Role of connective tissue growth factor in experimental radiation nephropathy in rats

Connective tissue growth factor (CTGF) is a potent fibrogenic cytokine which has been associated with progressive glomerulosclerosis and tubulointerstitial fibrosis. We investigated the role of CTGF on the progression of a rat model of radiation nephropathy. The model of radiation nephropathy in rats was established as follows: control group (n = 12), underwent only laparotomy; irradiated group (n = 20), underwent a laparotomy, then the rats were subjected to a single dose 25 Gy X-ray to the kidneys. The rats were followed up one, three, six and nine months after renal exposure to radiation. Renal dysfunction was noted early in irradiated rats. Histological analysis showed focal glomerular sclerotic lesions at an early stage after irradiation. Radiation-induced glomerular and tubulointerstitial injuries were particularly severe the sixth month after irradiation as compared to the control group (P < 0.01). By immunohistochemistry, increased expression of CTGF was noted in the irradiated kidneys, which began to increase from the first month after irradiation, and remained significantly higher at the sixth and ninth month after irradiation (P < 0.01). Upon Western blot analysis CTGF protein expression showed an increase in the radiation treated kidneys compared with the control rats. The expression of CTGF closely correlated with the progression of radiation nephropathy. The expression of alpha-smooth muscle actin, vimentin, type III collagen and type IV collagen was also high in the irradiated kidney as compared to control rat kidneys (P < 0.05), and was most severe at the sixth and ninth month after irradiation (P < 0.01). By double immunostaining, CTGF expressing cells were found to be alpha-SMA-positive myofibroblasts and vimentin-positive tubular epithelial cells. Glomerular expression of CTGF closely correlated with the glomerular expression of alpha-SMA (r = 0.628, P < 0.01), vimentin (r = 0.462, P < 0.05) and accumulation of type IV collagen (r = 0.584, P < 0.01) in the irradiated kidney. Similarly, the expression of CTGF was positively correlated with the expression of alpha-SMA (r = 0.613, P < 0.01), vimentin (r = 0.629, P < 0.01), deposition of type III collagen (r = 0.741, P < 0.001) and type IV collagen (r = 0.799, P < 0.0001) in the tubulointerstitium of the irradiated kidney. Finally the expression of CTGF after the irradiation of the kidney positively correlated with the levels of blood urea nitrogen and serum creatinine. Overexpression of CTGF may play an important role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in radiation nephropathy.

Intracrine signalling of activin A in hepatocytes upregulates connective tissue growth factor (CTGF/CCN2) expression

Up to now, the effect of activin A on the expression of the important transforming growth factor (TGF)-beta downstream modulator connective tissue growth factor (CTGF) is not known, but might be of relevance for the functional effects of this cytokine on several liver cell types. In this study, activin A-dependent CTGF expression in hepatocytes (PC) primed by exogenous activin A and in PC maintained under complete activin-free culture conditions was analysed by Western blots, metabolic labelling, gene silencing, reverse transcriptase-polymerase chain reaction (RT-PCR) and CTGF reporter gene assays. This study was supplemented by immunocytochemical staining of activin A and CTGF in PC of injured liver. Using alkaline phosphatase alpha-alkaline phosphatase staining, it is demonstrated that activin A becomes increasingly detectable during the course of CCl(4)-liver damage. Addition of activin A to cultured PC induced CTGF protein expression via phosphorylation of Smad2 and Smad3. This induction can be inhibited by the antagonist follistatin and alpha-activin A antibody respectively. When PC were cultured under serum(i.e. activin A)-free culture conditions, a time-dependent increase of activin expression during the course of the culture was proven by RT-PCR. Silencing of inhibin beta(A) gene expression under serum-free conditions by small interfering RNAs greatly suppressed CTGF synthesis and the phosphorylations of Smad2 and Smad3. However, both the extracellularly acting follistatin and the alpha-activin A antibody could not inhibit spontaneous CTGF expression, which, however, was achieved by the cell-permeable TGF-beta Alk4/Alk5 receptor-kinase-inhibitor SB431542. In conclusion, the results point to activin A as an inducer of CTGF synthesis in PC. Intracellular activin A contributes to spontaneous CTGF expression in PC independent of exogenous activin A, which is proposed to occur via Alk4/Alk5-receptors. The findings might be important for many actions of activin A on the liver.

SGK1-Dependent Upregulation of Connective Tissue Growth Factor by Angiotensin II

Angiotensin II has previously been shown to trigger fibrosis, an effect involving connective tissue growth factor (CTGF). The signaling pathways linking angiotensin II to CTGF formation are, however, incompletely understood. A gene highly expressed in fibrosing tissue is the serum- and glucocorticoid-inducible kinase SGK1. The present study explored whether SGK1 is transcriptionally regulated by angiotensin II and participates in the angiotensin II-dependent regulation of CTGF expression. To this end, experiments have been performed in human kidney fibroblasts and mouse lung fibroblasts from gene-targeted mice lacking SGK1 (sgk1^–/–) and their wild-type littermates (sgk1^+/+). In human renal fibroblasts, SGK1 and CTGF protein expression were enhanced by angiotensin II (10 nM) within 4 h. In sgk1^+/+ mouse fibroblasts, SGK1 transcript levels were significantly increased after 4 h of angiotensin II treatment. Angiotensin II stimulated both transcript and protein abundance of CTGF in fibroblasts from sgk1^+/+ mice, effects significantly blunted in fibroblasts of sgk1^–/– mice. In conclusion, angiotensin II stimulates the expression of SGK1, which is in turn required for the stimulating effect of angiotensin II on the expression of CTGF. Thus, SGK1 presumably contributes to the profibrotic effect of angiotensin II.

Angiotensin II-Stimulated Collagen Synthesis in Aortic Adventitial Fibroblasts Is Mediated by Connective Tissue Growth Factor

Angiotensin II (Ang II), a potent mediator of vascular remodeling, can stimulate the synthesis of extracellular matrix in vascular cells. Recent studies indicate that connective tissue growth factor (CTGF) is involved in collagen synthesis. There is also increasing evidence that adventitial fibroblasts (AFs) are actively involved in vascular remodeling. However, whether collagen synthesis by AFs is mediated by CTGF, or whether it is relevant to Ang II, has not been studied. The present study was conducted to determine whether CTGF is expressed in AFs, and if so, whether the CTGF produced by AFs participates in collagen synthesis. The AFs were isolated from thoracic aorta of Wistar-Kyoto rats (WKY). The expression of CTGF was measured by Western blot or real-time PCR. Collagen synthesis was assessed by [(3)H]proline incorporation. Our results suggested that CTGF was expressed in AFs and secreted into medium. Ang II increased CTGF mRNA and protein expression in a time- and dose-dependent manner, with the maximal protein increase occurring at 24 h with an Ang II dose of 10(-7) mol/L, and this increase was inhibited by the Ang II receptor type 1 (AT(1)-R) antagonist losartan, but not by the Ang II receptor type 2 (AT(2)-R) antagonist PD123319. Ang II dose-dependently stimulated the incorporation of [(3)H]proline into cultured AFs, and this effect was inhibited by a CTGF antisense oligodeoxynucleotide. Overexpression of CTGF by pcDNA3.1(+)/CTGF increased [(3)H]proline incorporation in cultured AFs. The results demonstrated that, in cultured AFs, Ang II increased CTGF production via AT(1)-R, which could be mediators of collagen synthesis by Ang II. This finding suggests that CTGF might be a novel target for antifibrotic therapy in vascular diseases.

Increased Expression of Connective Tissue Growth Factor in Patients with Urethral Stricture

Urethral stricture (US) is a urologic disorder with a high morbidity and recurrence rate, and may arise due to external trauma, surgical procedures or urethritis. The pathogenesis of US is closely associated with scar formation, including excessive collagen-rich connective tissues. Previous studies have shown that connective tissue growth factor (CTGF) plays a key role in the fibrosis process of many tissues and organs, such as kidney and lung. We investigated the variation of CTGF expression in urethral tissues from 12 patients with US and normal urethral tissues. The stricture length range was 1 to 2.5 cm, and 4 patients underwent repeated urethral dilatation. The normal urethral tissues were obtained from 6 patients with heart disease, who were suffering from brain death. The expression of CTGF mRNA and protein was analyzed by real-time PCR, immunohistochemistry, and western blotting analyses. Results showed that the expression level of CTGF mRNA was significantly increased in US patients compared with the control. In addition, US patients who have longer history and more repeated surgical procedures have extremely high CTGF mRNA levels and its protein expression, while the age of patients, position of injury and stricture length were not found obvious relation with CTGF expression. Moreover, the degree of collagen deposition and muscle fiber proliferation in the submucosa is consistent with the increase in CTGF expression. In conclusion, our data suggest that the up-regulation of CTGF expression may be responsible for the fibrosis process of urethral tissues in US patients.

Homocysteine induces connective tissue growth factor expression in vascular smooth muscle cells.

Increased homocysteine levels in blood might be an important risk factor for the development of cardiovascular diseases. Connective tissue growth factor (CTGF) was found to be involved in atherosclerotic plaque progression. So far, the possible connection between homocysteine and CTGF has not been studied. This study was designed to test whether homocysteine could induce CTGF expression in vascular smooth muscle cells (VSMC). Hyperhomocysteinemia was induced in Sprague-Dawley rats after 4 weeks of a high-methionine diet. CTGF mRNA and protein expression was detected in the aortas isolated from hyperhomocysteinemic rats, but not in the controls. The underlying mechanism of homocysteine-induced CTGF expression was investigated in cultured human umbilical vein smooth muscle cells (HUVSMC). CTGF mRNA expression was induced after treatment with dl-homocysteine (50 micromol L(-1)) for 1 h, which remained at the elevated level for up to 8 h. CTGF protein level increased after homocysteine treatment for 8 h, and the elevated status was maintained for up to 48 h. Several intracellular signals elicited by homocysteine are involved in CTGF synthesis, including protein kinase C (PKC) activation and reactive oxygen species (ROS). Transfection HUVSMCs with a CTGF small interference RNA (siRNA) plasmid, which specifically inhibited the expression of CTGF, decreased extracellular matrix (ECM) accumulation caused by homocysteine. Our results demonstrate that homocysteine could increase the expression of CTGF in VSMC both in vivo and in vitro. The novel findings suggest that homocysteine might contribute to accelerated progression of atherosclerotic lesions by inducing CTGF expression.

Connective tissue growth factor: a crucial cytokine-mediating cardiac fibrosis in ongoing enterovirus myocarditis

Dilated cardiomyopathy (DCM) as a consequence of viral myocarditis is a worldwide cause of morbidity and death. The deposition of matrix proteins, such as collagen, in the course of ongoing viral myocarditis results in cardiac remodeling and finally in cardiac fibrosis, the hallmark of DCM. To identify mediators of virus-induced cardiac fibrosis, microarray analysis was conducted in a murine model of chronic coxsackievirus B3 (CVB3) myocarditis. By this attempt, we identified connective tissue growth factor (CTGF) as a novel factor highly expressed in infected hearts. Further investigations by quantitative reverse transcription polymerase chain reaction and Western blot analysis confirmed a strong induction of cardiac CTGF expression in the course of CVB3 myocarditis. By in situ hybridization and immunohistochemistry, basal CTGF messenger ribonucleic acid (mRNA) and protein expression were confined in noninfected control hearts mainly to endothelial cells, whereas in CVB3-infected hearts, also numerous fibroblasts were found to express CTGF. Regulation of CTGF is known to be basically mediated by transforming growth factor (TGF)-beta. In the course of CVB3 myocarditis, CTGF upregulation coincided with increased cardiac TGF-beta and procollagen type I mRNA expression, preceding the formation of fibrotic lesions. In in vitro experiments, we found that downregulation of CVB3 replication by means of small interfering RNAs (siRNAs) reverses the upregulation of CTGF mRNA expression. In contrast, downregulation of CTGF by siRNA molecules did not significantly reduce viral load, indicating that CTGF is not essential for CVB3 life cycle. The significantly enhanced transcript levels of TGF-beta, CTGF, and procollagen type I in cultivated CVB3-infected primary cardiac fibroblasts substantiate the role of fibroblasts as a relevant cell population in cardiac remodeling processes. We conclude that CTGF is a crucial molecule in the development of fibrosis in ongoing enteroviral myocarditis. Thus, downregulation of cardiac CTGF expression may open novel therapeutic approaches counteracting the development of cardiac fibrosis and subsequent heart muscle dysfunction.

Up-regulation of Connective Tissue Growth Factor in Hyperoxia-Induced Lung Fibrosis

Pulmonary oxygen toxicity plays an important role in the lung injury process that leads to the development of bronchopulmonary dysplasia. Connective tissue growth factor (CTGF) is a fibroblast mitogen and promoter of collagen deposition. We investigated the effects of postnatal hyperoxia on lung collagen and CTGF expression in rats. Rat pups were exposed to 7 d of >95% O2 and a further 3 wk of 60% O2. CTGF mRNA and protein expression increased after hyperoxia treatment, and the values were significantly higher in hyperoxia-exposed rats on postnatal d 7 and 14. Lung collagen levels increased as rats aged, and the values were comparable between room air-exposed and hyperoxia-exposed rats on postnatal d 7 and 14 and were significantly higher in hyperoxia-exposed rats on postnatal d 21 and 28. Increases in CTGF mRNA and protein expressions preceded the onset of increased lung collagen. These data demonstrate that CTGF is up-regulated at time points preceding the fibrotic phase of the lung injury adding credence to the hypothesis that CTGF seems to be involved in the pathogenesis of hyperoxia-induced lung fibrosis and an anti-CTGF strategy might attenuate hyperoxia-induced lung fibrosis.

Glucocorticoids activate TGF-β induced PAI-1 and CTGF expression in rat hepatocytes

BackgroundIn addition to the activation of hepatic stellate cells TGF-β govern apoptosis and growth control of hepatocytes in liver injury. In non-parenchymal cells, TGF-β induces plasminogen activator inhibitor 1 (PAI-1) and connective tissue growth factor (CTGF) expression, which are involved in extra cellular matrix formation. Both genes were also regulated by glucocorticoids, which in certain cases showed antagonistic effects to the TGF-β-Smad 3 pathway. The purpose of our work was to investigate the influence of TGF-β and dexamethasone on PAI-1 and CTGF expression and secretion in primary hepatocytes.ResultsBy examining PAI-1 and CTGF mRNA and protein expression in cell lysates and cell-conditioned media under the influence of TGF-β and dexamethasone, we analysed signalling pathways controlling their expression. TGF-β and dexamethasone significantly co-induce PAI-1 and CTGF protein expression. On the other hand, we showed that TGF-β diminished a glucocorticoid receptor dependent luciferase reporter signal in Hep-G2. Inhibition of Erk downstream activation decreased TGF-β induced CTGF and PAI-1 expression to a basal level. PAI-1 was directly secreted by hepatocytes, whereas secretion of CTGF was retarded.ConclusionThe data provide evidence that beside the TGF-β-Smad 3 pathway CTGF and PAI-1 expression is additionally dependent on Erk activity in hepatocytes giving new insights into regulation of the profibrogenic proteins.

Connective tissue growth factor expression is regulated by histamine in lung fibroblasts: Potential role of histamine in airway remodeling

In the inflamed lung of allergic asthma, an aberrant injury-repair response is accompanied by structural changes in the airway, known as airway remodeling. TGF-beta and its downstream mediator connective tissue growth factor (CTGF) are playing a key role in these processes, resulting in irreversible airway remodelling. As histamine is a key mediator of allergic reactions, we investigated whether histamine is involved in airway remodeling. The effect of histamine and TGF-beta1 on proliferation of lung fibroblast cells IMR-90 was studied by [(3)H]-thymidine proliferation assay. The regulation of CTGF by histamine and TGF-beta1 in lung fibroblasts was analyzed by RT-PCR, real-time PCR, Western blot analysis, and promoter analysis and characterized by specific histamine-receptor antagonists. Histamine and TGF-beta1 enhanced proliferation of lung fibroblast cells IMR-90. Both induced CTGF mRNA and protein expression with different time kinetics. Whereas TGF-beta1 induced maximal CTGF expression after 12 hours (347% +/- 23%), histamine-induced maximal CTGF expression was lower and delayed (maximum expression of 204% +/- 11% after 48 hours). Histamine and TGF-beta1 stimulated the CTGF promoter and the TGF-beta-response element in the CTGF promoter. The histamine-induced CTGF expression was mediated through the histamine receptor (HR1) and could be completely abolished by TNF-alpha. These findings demonstrate that histamine plays a potential role in the induction of airway remodeling mediated by the induction of lung fibroblasts proliferation and CTGF expression. This mechanism could be important for prophylactic strategies aiming at airway remodeling and could be a new indication for antihistamine treatment.

Amelioration of inflammation, angiogenesis and CTGF expression in an arthritis model by a TSP1‐derived peptide treatment

To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.

Oestrogen formation and connective tissue growth factor expression in rat granulosa cells

Ovarian follicular development involves continual remodelling of the extracellular matrix (ECM) forming the basement membrane and intercellular framework that support granulosa cell (GC) growth and differentiation. Insight into the molecular regulation of ovarian ECM remodelling is potentially translatable to tissue remodelling elsewhere in the body. We therefore studied the link between a gene marker of ECM remodelling (connective tissue growth factor (CTGF)) and oestrogen biosynthesis (cytochrome P450(aromatase) (P450(arom))) in rat granulosa cells. To determine if a cause-effect interaction exists, we used semi-quantitative in situ hybridisation to analyse patterns of CTGF and P450(arom) mRNA expression and immunohistochemistry to detect CTGF protein localisation throughout follicular development, and tested the actions of CTGF on oestrogen biosynthesis and oestradiol on CTGF mRNA expression in isolated GC in vitro. CTGF mRNA levels in GC rose gradually through small preantral (SP) and small antral (SA) stages of development to a maximum (fivefold higher) in large antral (LA) follicles. In preovulatory (PO) follicles, the CTGF mRNA level fell to 30% of that in SP follicles. P450(arom) mRNA also increased (threefold in LA relative to SP) throughout antral development follicles, but in contrast to CTGF continued to increase (12-fold) in PO follicles. In the cumulus oophorus of PO follicles, the residual GC CTGF mRNA expression increased with proximity to the oocyte, being inversely related to P450(arom). Elsewhere in the follicle wall, there was a mural-to-antral gradient of CTGF mRNA expression, again inversely related to P450(arom). Immunohistochemistry showed CTGF protein localisation broadly followed mRNA expression during follicular development, although the protein was also present in the theca interna and ovarian surface epithelium. Gradients in CTGF expression across the cumulus oophorus and follicle wall were similar to those observed for mRNA with CTGF protein expression being greatest in proximity to the oocyte. Treatment of isolated GC from preantral and SA follicles with recombinant human CTGF (1-100 ng/ml) did not affect basal or FSH-stimulated GC aromatase activity. However, in the absence of FSH, oestradiol (10(-7)-10(-5) M) stimulated CTGF mRNA expression up to twofold. Conversely, FSH (10 ng/ml) alone reduced CTGF mRNA expression by 40% and combined treatment with FSH and oestradiol further suppressed CTGF to 10% of the control value. The oestrogen receptor (ER) antagonist, ICI 182 780 blocked the stimulatory and inhibitory effects of oestradiol, suggesting a specific ER-mediated mode of action on CTGF. Therefore, CTGF gene expression in GC is under local control by oestrogen whose effect (positive or negative) is modulated by FSH. This helps explain why gene expression of CTGF and P450(arom) diverge in FSH-induced PO follicles and has implications for oestrogenic regulation of CTGF formation elsewhere in the body.

ALK-5 Mediates Endogenous and TGF-β1–Induced Expression of Connective Tissue Growth Factor in Embryonic Lung

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

Effect of N-acetylcysteine on plasma adiponectin and renal adiponectin receptors in streptozotocin-induced diabetic rats

This study investigated the effect of N-acetylcysteine on plasma adiponectin, renal adiponectin receptors, lipid metabolism and oxidative stress in streptozotocin-induced diabetic rats. Metabolic parameters, plasma adiponectin level, renal protein expression of adiponectin receptors were analyzed in controls and diabetic rats treated with or without N-acetylcysteine in drinking water for 8 weeks. Plasma lipid, creatinine and free 5-F 2t-isoprostane levels, urine protein excretion rate, mesangial matrix expansion index, and protein expression of renal connective tissue growth factor (CTGF) were increased in diabetic rats. The decreased plasma adiponectin levels and renal protein expression of adiponectin receptor 1 were accompanied by the decreased renal phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK)-alpha (Thr172) and protein expression of phospho-acetyl coenzyme A carboxylase (ACC) (Ser79) which led to the increased renal triglyceride levels in diabetic rats. There was no difference in the protein expression of renal adiponectin receptor 2 between control and diabetic rats. N-acetylcysteine treatment attenuated the increased oxidative stress, plasma and renal lipids, urine protein excretion rate, mesangial matrix expansion index, and protein expression of renal CTGF, but did not affect plasma adiponectin levels, renal protein expression of adiponectin receptor 1, phosphorylation of AMPK-alpha (Thr172) and renal protein expression of phospho-ACC (Ser79) in diabetic rats. These results suggested that the decreased plasma adiponectin and renal adiponectin receptor 1 result in the increased renal triglyceride that stimulates renal CTGF expression leading to the renal hypertrophy and the deteriorated renal function in the diabetic rats. N-acetylcysteine treatment attenuates the increased oxidative stress, but has no effect on the decreased plasma adiponectin and renal adiponectin receptor 1 in diabetic rats, indicating that oxidative stress may not contribute to the decreased plasma adiponectin and renal adiponectin receptor 1 protein expression in diabetic rats.

Thrombospondin-1 (TSP1) Promotes Thrombin Generation on the Surface of Fibroblasts (HS-68) and Induces Up-Regulation of Connective Tissue Growth Factor (CTGF) Gene and Protein Expression.

TSP1 has the ability to bind to human fibroblasts, to form a complex with coagulation factor V/Va (Thrombosis Research 116:533, 2005), to promote thrombin generation on the surface of a monocytic cell line and to neutralize tissue factor pathway inhibitor (TFPI) (J Biol Chem 275:31715, 2000). Disruption of TSP1 binding to neutrophils was associated with beneficial effects in an experimental animal model of inflammation, in part, by down regulating CTGF gene and protein expression (Arthritis Rheum 54:2415, 2006). CTGF is a novel potent cysteine-rich heparin-binding growth factor and is highly expressed by fibroblasts. CTGF plays a major role in angiogenesis and fibrosis. There is also growing evidence that CTGF may be the downstream autocrine mediator responsible for some of the cellular effects of TGF-beta. Since fibroblasts express tissue factor (TF) on their surface, and purified thrombin and TF-VIIa complex have been shown to up-regulate the gene expression of CTGF (J Biol Chem 275:14632, 2000) experiments were conducted to evaluate the ability of HS-68 to support assembly of the prothrombinase complex, TF-FVIIa, thrombin generation and the effect of thrombin generation on CTGF expression. The role of TSP1 in these reactions was assessed as well. Thrombin generation was measured by the chromogenic substrate S-2238. Although the initial rates of the reactions are available we are presenting the end-point values of the reaction expressed in umol/L of pNA released per minute. All reaction mixtures were performed in the presence of 2mM Ca++. When HS-68 cells were preincubated with FVII (5 nM) prior to the addition of activated factor V (FVa, 45nM)), FX (5nM) and prothrombin (FII, 1.4 uM), thrombin was efficiently generated (282 umol/L pNA/min), indicating that FVII was activated by TF expressed by the cell and that the HS-68 cell membrane provided an ideal surface for the reaction to occur. The addition of FII, FV, FVII and FX to the reaction mixtures was an absolute requirement. When the reaction mixture was evaluated in the presence of FII, FV, FVII, FX and TFPI (8nM), there was a 70% reduction in thrombin production (86 umol/L pNA released) confirming the important role of TFPI in regulating the activity of the TF-FVIIa complex. The addition of TSP1 to the reaction mixture containing FII, FV, FVII and FX at concentrations found in plasma during the inflammatory response (20nM) enhanced the production of thrombin (327 umol/L pNA released per min) and neutralized the inhibitory effect of TFPI by 50% (171 umol/L pNA released per min). Therefore, TSP1 promotes thrombin generation by participating in the assembly of the prothrombinase complex on the surface of HS-68 cells and by neutralizing, in part, the inhibitory effect of TFPI on TF-VIIa complex. Finally, thrombin generation on the surface of HS-68 cells was associated with up-regulation of CTGF gene expression from the baseline value by 67% at 1hr and 72% by 2 hrs. In summary, we have identified on human fibroblasts a pathway previously shown to play an important role on human neutrophils and in an experimental model of inflammation. Our laboratory is currently characterizing the binding of TSP1 to this cell line and silencing the gene for TSP1 to test its potential therapeutic benefit in an experimental model of erosive arthritis and to further determine the role of TSP1 in this pathway.

Amelioration of Inflammation, Angiogenesis and CTGF Expression in an Arthritis Model by a TSP1-Derived Peptide Treatment.

Objective. To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis.Methods. Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-APS). Animals were divided into four groups:negative control and groups receivingno treatment,treatment with a TSP1-derived peptide andtreatment with a scrambled peptide.Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR.Results. Histological data indicated that TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunohistochemical and RT-PCR analyses showed an increase of the CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to TSP1-derived peptide treated animals. By immunohistochemistry, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals.Conclusion. Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.Vasculature measurements at the perisynovial tissue of the TSP1-derived peptide treated (DT) and scramble peptide treated (DS) animals after day 16 PG-APS-induced erosive arthritis.Vascular MeasurementsDisease TSP1-derived peptide treated (DT)Disease scrambled peptide treated (DS)P ValueVascular area mean±SEM (μm2)77.41 ± 7.96 μm2336 ± 49.21 μm2p=0.0008Vessel wall thickness (μm)3.53 ± 0.21μm8.92 ± 0.48;μmp<0.0001Vascular index (VI) ± SEM (VI=total number of vessels/mm2)5.6x10−3 ± 0.37x10−3 vessels/mm217.9x10−3 ± 1.33x10−3 vessels/mm2p=0.006

Connective Tissue Growth Factor Plays an Important Role in Advanced Glycation End Product–Induced Tubular Epithelial-to-Mesenchymal Transition

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.

A peptide from thrombospondin 1 modulates experimental erosive arthritis by regulating connective tissue growth factor

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with leukocyte adhesion to and extravasation through vascular endothelium into synovial tissue. Recent evidence indicates that the thrombospondin 1 gene is up-regulated in patients with RA. We have identified a region within the TSP-1 type 3 repeats that inhibits human neutrophil elastase (HNE) and binds to human neutrophils. The present study was undertaken to investigate the therapeutic benefit of this TSP-1-derived peptide sequence and its effect on connective tissue growth factor (CTGF), a protein involved in fibrotic disorders and in neovascularization, which is a hallmark of RA. CTGF gene and protein expression, as well as protein levels of CTGF in the synovium, after treatment with the TSP-1-derived peptide were studied in the peptidoglycan-polysaccharide animal model of erosive arthritis. Peptide treatment prevented joint infiltration and inflammation and was associated with reduced circulating antigen levels of HNE and TSP-1. Additionally, CTGF was up-regulated in this experimental model of RA. Treatment with the TSP-1-derived peptide was associated with down-regulation of the message and protein levels of CTGF. Immunofluorescence studies showed that the mean area fraction of CTGF immunoreactivity in the peptide-treated group of animals was significantly less than that in the untreated group. These results document a role for TSP-1 in regulating CTGF gene and protein expression in synovial tissue, suggesting a link with the disease course in this model of RA. This TSP-1-derived synthetic peptide may represent an important template for drug development in RA and other inflammatory conditions associated with neutrophil activation.

Effect of Connective Tissue Growth Factor on Hypoxia-Inducible Factor 1α Degradation and Tumor Angiogenesis

Connective tissue growth factor (CTGF) inhibits the metastatic activity of human lung cancer cells in a mouse model; however, the mechanism of this modulation is unclear. We investigated the role of angiogenesis in this process. CL1-5 and A549 human lung adenocarcinoma cells were stably transfected with vectors containing CTGF or hypoxia-inducible factor (HIF) 1alpha or with vector controls. Transfected cells were injected into nude mice (n = 10 per group), and tumor growth, metastasis, and mouse survival were measured. Excised xenograft tumors and primary human lung adenocarcinomas (n = 24) were subjected to immunohistochemistry with antibodies to the endothelial cell marker CD31 and to CTGF. Expression of HIF-1alpha and vascular endothelial growth factor (VEGF) A was assessed in vitro by using reporter gene assays. Cells were transiently transfected with HIF-1alpha mutant and antisense arrest-defective 1 protein (ARD-1), and HIF-1alpha acetylation was assayed by immunoprecipitation. All statistical tests were two-sided. Xenograft tumors derived from CTGF transfectants grew more slowly than those from control-transfected cells and had reduced expression of HIF-1alpha and VEGF-A, vascularization (as assessed by CD31 expression), and metastasis (all P<.001). Xenograft tumors derived from CTGF-overexpressing cells that were transfected with HIF-1alpha had higher VEGF-A expression than CTGF-overexpressing xenografts. Mice with CTGF/HIF-1alpha xenografts had lower survival than mice carrying CTGF-overexpressing xenografts (CL1-5/Neo, mean = 69.6 days, 95% confidence interval [CI] = 53.9 to 85.3 days versus CL1-5/CTGF, mean = 102.1 days, 95% CI = 92.1 to 112.1 days; P = .001, CL1-5/CTGF, mean = 102.1 days, 95% CI = 92.1 to 112.1 days versus CL1-5/CTGF/HIF-1alpha, mean = 81.7 days, 95% CI = 66.5 to 96.9 days; P = .011, CL1-5/Neo, mean = 69.6 days, 95% CI = 53.9 to 85.3 days versus CL1-5/CTGF/HIF-1alpha, mean = 81.7 days, 95% CI = 66.5 to 96.9 days; P = .122). Tumors of patients with the same disease stage but with high CTGF protein expression had reduced microvessel density compared with tumors with low expression. Transfection with antisense-ARD1 decreased the level of acetylated HIF-1alpha and restored HIF-1alpha and VEGF-A expression in CTGF-overexpressing cells. CTGF inhibition of metastasis involves the inhibition of VEGF-A-dependent angiogenesis, possibly by promoting HIF-1alpha protein degradation.