Serum and glucocorticoid-regulated kinase 1 regulates transforming growth factor ß1-connective tissue growth factor pathway in chronic rhinosinusitis.

PURPOSE: Serum/glucocorticoid-regulated kinase 1 (SGK1) has been identified as a crucial regulator in fibrotic disorders. Herein, we explored SGK1 role in tissue remodeling of chronic rhinosinusitis (CRS). METHODS: Lentivirus was employed to generate an SGK1-overexpressing human bronchial epithelial cell (16HBE) line. To screen SGK1 downstream genes, RNA sequencing was performed on SGK1-overexpressing and control cell lines. To determine protein and gene expression levels, immunohistochemistry, western blotting, and quantitative real-time polymerase chain reaction were employed. Correlation analysis was performed using mRNA expression levels of SGK1, transforming growth factor ß1 (TGF-ß1), and connective tissue growth factor (CTGF) derived from CRS mucosal tissue and GEO database. Gene set enrichment analysis was conducted using gene sets from Molecular Signatures Database. The severity of symptoms in CRS patients was assessed using the 22-Item Sinonasal Outcome Test. RESULTS: SGK1 overexpression significantly increased the expression of connective tissue growth factor (CTGF) in 16HBE cells (P < 0.01). Consistently, CTGF protein level was considerably greater in mucosal tissue of CRS without nasal polyps (CRSsNP) than in CRS with nasal polyps (CRSwNP) (P < 0.05) or in control subjects (P < 0.01). TGF-ß1 protein level was higher in mucosal tissue of CRSsNP patients than in CRSwNP patients (P < 0.001) or in the control group (P < 0.01). mRNA levels of SGK1 and CTGF (P < 0.05, r = 0.668; P = 0.001, r = 0.630), TGF-ß1 and CTGF (P < 0.05, r = 0.560; P < 0.05, r = 0.420), as well as SGK1 and TGF-ß1(P < 0.05, r = 0.612; P < 0.05, r = 0.524) were significantly correlated in CRS mucosal tissue and GSE36830 dataset, respectively. TGF-ß1-induced upregulated genes were significantly enriched in SGK1 overexpression group. In vitro assays, TGF-ß1 promoted SGK1 and CTGF expression in a concentration- and time-dependent manner. Administrating an SGK1 inhibitor, GSK650394, significantly inhibited TGF-ß1-induced CTGF expression in 16HBE and dispersed primary nasal polyp cells. CONCLUSIONS: TGF-ß1 stimulation significantly increases SGK1 and CTGF expression. By regulating TGF-ß1-CTGF pathway, SGK1 may participate in tissue remodeling in the pathological mechanism of CRS.

Electrochemical immuno determination of connective tissue growth factor levels on nitrogen-doped graphene.

Connective tissue growth factor (CTGF) is a disease marker of rheumatoid arthritis (RA), and its rapid and sensitive detection is essential for the diagnosis of RA. In this work, a three-dimensional pore structure of alkali-activated nitrogen-doped graphene (aN-G) was used as an electrode modification material, and a label-free electrochemical immunosensor for the sensitive detection of CTGF was successfully constructed by the formation of an amide bond between amino groups in protein and carboxyl groups on the carbon surface. Under optimized conditions, the sensor achieved accurate detection of CTGF in the wide range of 0.0625 ~ 2000 pg mL-1. It had good accuracy (95.0 ~ 100.1%), repeatability (1.2 ~ 2.2%), stability, selectivity, and a low limit of detection (0.0424 pg mL-1, S/N = 3). The sensor was used in serum samples of patients with RA, and CTGF was also successfully detected. Based on this, the electrochemical sensor is expected to become an effective method for RA diagnosis and treatment effect evaluation.

Cmah-dystrophin deficient mdx mice display an accelerated cardiac phenotype that is improved following peptide-PMO exon skipping treatment.

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids-the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah-/-;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah-/-;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah-/-;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah-/-;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah-/-;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics.

Autologous bone marrow-derived mesenchymal stem cells for interstitial fibrosis and tubular atrophy: a pilot study.

BACKGROUND: Mesenchymal stem cells (MSCs)-based therapy has shown promising results for renal injury. In this study, the efficacy and safety of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) in treating nonspecific interstitial fibrosis and tubular atrophy (IFTA) were evaluated. METHODS: From March 2011 to January 2013, 11 renal transplanted patients with IFTA were recruited. At baseline, patients were given one intra-arterial infusion of BM-MSCs; 7 days and 1 month later, another two intravenous infusions of cells were followed. Serum creatinine, creatinine clearance rate, and serum cystatin-C at baseline and 7 days, 1 month, 3 months, 6 months, and 12 months after the intra-arterial infusion of BM-MSCs were used to assess renal function. At baseline and 6 months, histological examination based on hematoxylin-eosin, Masson's trichrome and periodic acid-Schiff staining and immunohistochemistry for transforming growth factor ß1 (TGF-ß1) and connective tissue growth factor (CTGF) was performed. Adverse events were recorded to evaluate the safety of BM-MSCs treatment. RESULTS: At 12 months, the renal function of 6 patients (54.5%) was improved, 3 (27.3%) were stable and 2 (18.2%) were worsened. At 6 months, the mean IFTA scores of all participators were similar with the baseline (1.73 ± 0.41 vs.1.50 ± 0.0.77, p = 0.242); however, it was significantly decreased when only 6 patients with improved renal function were analyzed (1.67 ± 0.41 vs. 1.08 ± 0.20, p = 0.013). Besides, decreased expression of TGF-ß1 and CTGF were also observed at 6 months. During 1 year follow-up period, only two minor complications including infection and allergy were observed. CONCLUSION: Our results demonstrated that autologous BM-MSCs are safe and beneficial for IFTA patients. Abbreviations: MSCs: mesenchymal stem cells; BM-MSCs: marrow-derived mesenchymal stem cells; IFTA: interstitial fibrosis and tubular atrophy; CAN: chronic allograft nephropathy; CNIs: calcineurin inhibitors; Scr: serum creatinine; CCr: creatinine clearance rate; Cys-C: cystatin-C; TGF-ß1: transforming growth factor ß1; CTGF: connective tissue growth factor.

Establishing a Link between Endothelial Cell Metabolism and Vascular Behaviour in a Type 1 Diabetes Mouse Model.

BACKGROUND/AIMS: Vascular complications contribute significantly to the extensive morbidity and mortality rates observed in people with diabetes. Despite well known that the diabetic kidney and heart exhibit imbalanced angiogenesis, the mechanisms implicated in this angiogenic paradox remain unknown. In this study, we examined the angiogenic and metabolic gene expression profile (GEP) of endothelial cells (ECs) isolated from a mouse model with type1 diabetes mellitus (T1DM). METHODS: ECs were isolated from kidneys and hearts of healthy and streptozocin (STZ)-treated mice. RNA was then extracted for molecular studies. GEP of 84 angiogenic and 84 AMP-activated Protein Kinase (AMPK)-dependent genes were examined by microarrays. Real time PCR confirmed the changes observed in significantly altered genes. Microvessel density (MVD) was analysed by immunohistochemistry, fibrosis was assessed by the Sirius red histological staining and connective tissue growth factor (CTGF) was quantified by ELISA. RESULTS: The relative percentage of ECs and MVD were increased in the kidneys of T1DM animals whereas the opposite trend was observed in the hearts of diabetic mice. Accordingly, the majority of AMPK-associated genes were upregulated in kidneys and downregulated in hearts of these animals. Angiogenic GEP revealed significant differences in Tgfß, Notch signaling and Timp2 in both diabetic organs. These findings were in agreement with the angiogenesis histological assays. Fibrosis was augmented in both organs in diabetic as compared to healthy animals. CONCLUSION: Altogether, our findings indicate, for the first time, that T1DM heart and kidney ECs present opposite metabolic cues, which are accompanied by distinct angiogenic patterns. These findings enable the development of innovative organ-specific therapeutic strategies targeting diabetic-associated vascular disorders.

Association between AXL, Hippo Transducers, and Survival Outcomes in Male Breast Cancer.

Male breast cancer (MBC) is an uncommon malignancy. We have previously reported that the expression of the Hippo transducers TAZ/YAP and their target CTGF was associated with inferior survival in MBC patients. Preclinical evidence demonstrated that Axl is a transcriptional target of TAZ/YAP. Thus, we herein assessed AXL expression to further investigate the significance of active TAZ/YAP-driven transcription in MBC. For this study, 255 MBC samples represented in tissue microarrays were screened for AXL expression, and 116 patients were included. The association between categorical variables was verified by the Pearson's Chi-squared test of independence (2-tailed) or the Fisher Exact test. The relationship between continuous variables was tested with the Pearson's correlation coefficient. The Kaplan-Meier method was used for estimating survival curves, which were compared by log-rank test. Factors potentially impacting 10-year and overall survival were verified in Cox proportional regression models. AXL was positively associated with the TAZ/CTGF and YAP/CTGF phenotypes (P = 0.001 and P = 0.002, respectively). Patients with TAZ/CTGF/AXL- or YAP/CTGF/AXL-expressing tumors had inferior survival compared with non-triple-positive patients (log rank P = 0.042 and P = 0.048, respectively). The variables TAZ/CTGF/AXL and YAP/CTGF/AXL were adverse factors for 10-year survival in the multivariate Cox models (HR 2.31, 95%CI:1.02-5.22, P = 0.045, and HR 2.27, 95%CI:1.00-5.13, P = 0.050). Nearly comparable results were obtained from multivariate analyses of overall survival. The expression pattern of AXL corroborates the idea of the detrimental role of TAZ/YAP activation in MBC. Overall, Hippo-linked biomarkers deserve increased attention in this rare disease. J. Cell. Physiol. 232: 2246-2252, 2017. © 2016 Wiley Periodicals, Inc.

Anti-connective tissue growth factor detects and reduces plaque inflammation in early-stage carotid atherosclerotic lesions.

This study explored connective tissue growth factor (CTGF)-targeted ultrasmall superparamagnetic iron oxides (USPIOs) for noninvasive MRI of CTGF within carotid atherosclerotic lesions in apoE-deficient (apoE-/-) mice. Anti-CTGF polyclonal and nonspecific IgG antibodies were conjugated to polyethylene glycol-coated USPIOs, and apoE-/- carotid partial ligation-model mice were imaged via MRI before and after contrast administration. ApoE-/- mice were treated with CTGF-neutralizing antibodies for 3 weeks. Carotid artery diameter and plaque volume were measured via MRI in IgG and CTGF antibody-treated groups. Anti-CTGF-USPIO-treated macrophages showed the greatest iron uptake. MRI signal loss was observed in carotid atherosclerotic lesions 24 h after anti-CTGF-USPIO administration, consistent with the presence of nanoparticles, as indicated by pathological examinations. Atheromata in anti-CTGF-treated mice showed reduced macrophage deposition, CTGF expression, and plaque volume. Anti-CTGF-USPIOs can be used for the direct detection of CTGF and imaging of atherosclerotic lesions in vivo. CTGF is a potential therapeutic target for treating atherosclerosis.

[High glucose dialysate enhances peritoneal fibrosis through upregulating glucose transporters GLUT1 and SGLT1].

Objective: To investigate the role of glucose transporter 1 (GLUT1) and sodium-glucose cotransporter 1 (SGLT1) in high glucose dialysate-induced peritoneal fibrosis. Methods: Thirty six male SD rats were randomly divided into 6 groups (6 in each):normal control group, sham operation group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorizin group (PD+Z group), PD+phloretin+phlorizin group (PD+T+Z group). Rat model of uraemia was established using 5/6 nephrotomy, and 2.5% dextrose peritoneal dialysis solution was used in peritoneal dialysis. Peritoneal equilibration test was performed 24 h after dialysis to evaluate transport function of peritoneum in rats; HE staining was used to observe the morphology of peritoneal tissue; and immunohistochemistry was used to detect the expression of GLUT1, SGLT1, TGF-ß1 and connective tissue growth factor (CTGF) in peritoneum. Human peritoneal microvascular endothelial cells (HPECs) were divided into 5 groups:normal control group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorezin group (PD+Z group), and PD+phloretin+phlorezin group (PD+T+Z group). Real time PCR and Western blotting were used to detect mRNA and protein expressions of GLUT1, SGLT1, TGF-ß1, CTGF in peritoneal membrane and HPECs. Results:In vivo, compared with sham operation group, rats in PD group had thickened peritoneum, higher ultrafiltration volume, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-ß1 were significantly increased (all P<0.05); compared with PD group, thickened peritoneum was attenuated, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-ß1 were significantly decreased in PD+T, PD+Z and PD+T+Z groups (all P<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in peritoneum were positively correlated with the expressions of TGF-ß1 and CTGF (all P<0.05). In vitro, the mRNA and protein expressions of GLUT1, SGLT1, TGF-ß1, CTGF were significantly increased in HPECs of peritoneal dialysis group (all P<0.05), and those in PD+T, PD+Z, and PD+T+Z groups were decreased (all P<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in HPECs were positively correlated with the expressions of TGF-ß1 and CTGF (all P<0.05). Conclusion: High glucose peritoneal dialysis fluid may promote peritoneal fibrosis by upregulating the expressions of GLUT1 and SGLT1.

Rapamycin increases CCN2 expression of lung fibroblasts via phosphoinositide 3-kinase.

Excessive production of connective tissue growth factor (CTGF, CCN2) and increased motor ability of the activated fibroblast phenotype contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, molecules and signal pathways regulating CCN2 expression and migration of lung fibroblasts are still elusive. We hypothesize that rapamycin, via binding and blocking mammalian target of rapamycin (mTOR) complex (mTORC), affects CCN2 expression and migration of lung fibroblasts in vitro. Primary normal and fibrotic human lung fibroblasts were isolated from lung tissues of three patients with primary spontaneous pneumothorax and three with IPF. Cells were incubated with regular medium, or medium containing rapamycin, human recombinant transforming growth factor (TGF)-ß1, or both. CCN2 and tissue inhibitor of metalloproteinase (TIMP)-1 expression in cells or supernatant was detected. Wound healing and migration assay was used to measure the migratory potential. TGF-ß type I receptor (TßRI)/Smad inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. We demonstrated that rapamycin amplified basal or TGF-ß1-induced CCN2 mRNA and protein expression in normal or fibrotic fibroblasts by Smad-independent but PI3K-dependent pathway. Additionally, rapamycin also enhanced TIMP-1 expression as indicated by ELISA. However, wound healing and migrating assay showed rapamycin did not affect the mobility of fibroblasts. Collectively, this study implies a significant fibrogenic induction activity of rapamycin by activating AKT and inducing CCN2 expression in vitro and provides the possible mechanisms for the in vivo findings which previously showed no antifibrotic effect of rapamycin on lung fibrosis.

In vitro selection and characterization of deoxyribonucleic acid aptamers against connective tissue growth factor.

Connective tissue growth factor (CTGF) is a secreted matricellular protein possessing complex biological functions. CTGF modulates a number of signaling pathways that are involved in cell adhesion, migration, angiogenesis, myofibroblast activation, extracellular matrix deposition and tissue remodeling. Aptamers are oligonucleic acid chains or polypeptides that bind with specific target molecules hence have the potential to be used in the detection and blockade of the targets. In this study, we selected CTGF-targeting DNA aptamers by using systematic evolution of ligands by exponential enrichment (SELEX). After 8 iterative rounds of selection, cloning, DNA sequencing and affinity determination, six aptamers with high affinities to CTGF were obtained. Among them, one (C-ap17P) binds with the N-terminal region (aa 1-190) and the other five (C-ap11, 12, 14, 15 and 18) bind with the C-terminal region (aa 191-350) of hCTGF specifically. The biological stability assay indicated that a representative aptamer, C-ap17P, could keep its integrity at a rather high level for at least 24 h in complete DMEM cell culture medium. These CTGF aptamers might be used as a easy and fast detection tool for CTGF and be developed as CTGF-specific inhibitors for both research works and clinical applications.

Increased and correlated expression of connective tissue growth factor and transforming growth factor beta 1 in surgically removed periodontal tissues with chronic periodontitis.

BACKGROUND AND OBJECTIVE: Both gingival tissue destruction and regeneration are associated with chronic periodontitis, although the former overwhelms the latter. Studies have shown that transforming growth factor beta 1 (TGF-ß1), a growth factor largely involved in tissue regeneration and remodeling, is upregulated in chronic periodontitis. However, the gingival expression of connective tissue growth factor (CTGF or CCN2), a TGF-ß1-upregulated gene, in patients with periodontitis remains undetermined. Although both CTGF/CCN2 and TGF-b1 increase the production of extracellular matrix, they have many different biological functions. Therefore, it is important to delineate the impact of periodontitis on gingival CTGF/CCN2 expression. MATERIAL AND METHODS: Periodontal tissue specimens were collected from seven individuals without periodontitis (group 1) and from 14 with periodontitis (group 2). The expression of CTGF and TGFß1 mRNAs were quantified using real-time PCR. RESULTS: Analysis using the nonparametric Mann-Whitney U-test showed that the levels of expression of both CTGF/CCN2 and TGFß1 mRNAs were significantly increased in individuals with periodontitis compared with individuals without periodontitis. Furthermore, analysis using a nonparametric correlation (Spearman r) test showed a positive correlation between TGFß1 and CTGF/CCN2 mRNAs. CONCLUSION: The gingival expression levels of CTGF/CCN2 and TGFß1 mRNAs in individuals with periodontitis are upregulated and correlated.

Cigarette smoke condensate inhibits collagen gel contraction and prostaglandin E2 production in human gingival fibroblasts.

BACKGROUND: Granulation tissue remodeling and myofibroblastic differentiation are critically important events during wound healing. Tobacco smoking has a detrimental effect in gingival tissue repair. However, studies evaluating the effects of cigarette smoke on these events are lacking. MATERIAL AND METHODS: We used gingival fibroblasts cultured within free-floating and restrained collagen gels to simulate the initial and final steps of the granulation tissue phase during tissue repair. Collagen gel contraction was stimulated with serum or transforming growth factor-ß1. Cigarette smoke condensate (CSC) was used to evaluate the effects of tobacco smoke on gel contraction. Protein levels of alpha-smooth muscle actin, ß1 integrin, matrix metalloproteinase-3 and connective tissue growth factor were evaluated through Western blot. Prostaglandin E(2) (PGE(2)) levels were determined through ELISA. Actin organization was evaluated through confocal microscopy. RESULTS: CSC reduced collagen gel contraction induced by serum and transforming growth factor-ß1 in restrained collagen gels. CSC also altered the development of actin stress fibers in fibroblasts cultured within restrained collagen gels. PGE(2) levels were strongly diminished by CSC in three-dimensional cell cultures. However, other proteins involved in granulation tissue remodeling and myofibroblastic differentiation such as alpha-smooth muscle actin, ß1 integrin, matrix metalloproteinase-3 and connective tissue growth factor, were unmodified by CSC. CONCLUSIONS: CSC may alter the capacity of gingival fibroblasts to remodel and contract a collagen matrix. Inhibition of PGE(2) production and alterations of actin stress fibers in these cells may impair proper tissue maturation during wound healing in smokers.

CCN2/CTGF expression via cellular uptake of BMP-1 is associated with reparative dentinogenesis.

OBJECTIVE: CCN family member 2/connective tissue growth factor (CCN2/CTGF) is known as an osteogenesis-related molecule and is thought to be implicated in tooth growth. Bone morphogenetic protein-1 (BMP-1) contributes to tooth development by the degradation of dentin-specific substrates as a metalloprotease. In this study, we demonstrated the correlations between CCN2/CTGF and BMP-1 in human carious teeth and the subcellular dynamics of BMP-1 in human dental pulp cells. MATERIALS AND METHODS: Expression of CCN2/CTGF and BMP-1 in human carious teeth was analyzed by immunohistochemistry. BMP-1-induced CCN2/CTGF protein expression in primary cultures of human dental pulp cells was observed by immunoblotting. Intracellular dynamics of exogenously administered fluorescence-labeled BMP-1 were observed using confocal microscope. RESULTS: Immunoreactivities for CCN2/CTGF and BMP-1 were increased in odontoblast-like cells and reparative dentin-subjacent dental caries. BMP-1 induced the expression of CCN2/CTGF independently of protease activity in the cells but not that of dentin sialophosphoprotein (DSPP) or dentin matrix protein-1 (DMP-1). Exogenously added BMP-1 was internalized into the cytoplasm, and the potent dynamin inhibitor dynasore clearly suppressed the BMP-1-induced CCN2/CTGF expression in the cells. CONCLUSION: CCN2/CTGF and BMP-1 coexist beneath caries lesion and CCN2/CTGF expression is regulated by dynamin-related cellular uptake of BMP-1, which suggests a novel property of metalloprotease in reparative dentinogenesis.

High doses of (-)-epigallocatechin-3-gallate from green tea induces cardiac fibrosis in mice.

OBJECTIVE: Tea is the most consumed beverage in the world. (-)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, is effective in the prevention of several chronic diseases, and is marketed as part of many dietary supplements. We have now examined the myocardiotoxic effect of high doses of EGCG in mice. RESULTS: EGCG (500 and 1000 mg/kg·d) induced cardiac collagen synthesis and fibrosis-related protein expression, such as connective tissue growth factor (CTGF) and fibronectin (FN) in mice. Moreover, EGCG decreased the protein expression of p-AMPK and increased the levels of p-p70S6 K and p-S6. CONCLUSION: This is the first evidence that high oral doses of EGCG could induce cardiac fibrosis, and shed new light on the understanding of EGCG-mediated myocardiotoxicity.

Immunohistochemical localization of connective tissue growth factor, transforming growth factor-beta1 and phosphorylated-smad2/3 in the developing periodontium of rats.

BACKGROUND AND OBJECTIVE: Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta1 (TGF-ß1), and TGF-ß1-induced CTGF expression is regulated through the SMAD pathway. CTGF is implicated in the development of cartilage, bone and tooth. However, its expression in the developing periodontium is unclear. Therefore, we aimed to investigate the immunolocalization of CTGF, TGF-ß1 and phosphorylated SMAD2/3 (pSMAD2/3) in the developing periodontium of rats. MATERIAL AND METHODS: The maxillaries of Wistar rats, 2, 3, 7 and 12 wk of age, were used and the localization of CTGF, TGF-ß1 and pSMAD2/3 was detected using immunoperoxidase techniques. RESULTS: Hertwig' s epithelial root sheath (HERS) cells were strongly positive for CTGF and TGF-ß1, but not for pSMAD2/3. Positive staining for CTGF, TGF-ß1 and pSMAD2/3 was found in bone and periodontal ligament. In cementum, most cementoblasts associated with cellular cementum and some cementocytes stained strongly for CTGF, whereas cementoblasts associated with acellular cementum did not express CTGF. No signal for TGF-ß1 was observed in cellular and acellular cementum. In addition, most cementocytes were strongly positive for pSMAD2/3. CONCLUSION: CTGF, TGF-ß1 and pSMAD2/3 are localized in bone and periodontal ligament, but are differentially expressed in HERS and cementum. The results of our study indicate that the regulation of CTGF expression by TGF-ß1 might be cell-type specific in periodontium.

Epigallocatechin-3-gallate inhibits lysophosphatidic acid-stimulated connective tissue growth factor via JNK and Smad3 suppression in human gingival fibroblasts.

BACKGROUND/PURPOSE: Connective tissue growth factor (CTGF/CCN2) is involved in the development and progression of fibrotic diseases, including gingival overgrowth (GO). Recent studies indicate that lysophosphatidic acid (LPA) is also significantly involved in wound healing and the development of fibrosis. This study investigated whether epigallocatechin-3-gallate (EGCG) can inhibit LPA-induced CCN2 expression in human gingival fibroblast (GF) and its mechanism. METHODS: Western blot analyses were used to study the signaling pathways of LPA-induced CCN2 expression in human GFs and the effects of EGCG on this pathway. RESULTS: LPA stimulated CCN2 synthesis in human GFs. This effect can be significantly inhibited bytransforming growth factor-ß type I receptor/ALK5, Smad3, and JNK inhibitors but not ERK, P38, and MAPK inhibitors. EGCG completely inhibited LPA-induced CCN2 expression through attenuating the LPA-induced JNK and Smad3 phosphorylation in human GFs. CONCLUSION: LPA produced at the surgical wound may contribute to the recurrence of GO by upregulating CCN2 expression in human GFs. This effect was mediated by Smad3 and JNK activation and ALK5 transactivation. EGCG could be a useful agent for reducing the recurrence of GO after surgery through suppression of JNK and Smad3 activations.

Engineering three-dimensional constructs of the periodontal ligament in hyaluronan-gelatin hydrogel films and a mechanically active environment.

BACKGROUND AND OBJECTIVE: Periodontal ligament (PDL) cells in stationary two-dimensional culture systems are in a double default state. Our aim therefore was to engineer and characterize three-dimensional constructs, by seeding PDL cells into hyaluronan-gelatin hydrogel films (80-100 µm) in a format capable of being mechanically deformed. MATERIAL AND METHODS: Human PDL constructs were cultured with and without connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 in (i) stationary cultures, and (ii) mechanically active cultures subjected to cyclic strains of 12% at 0.2 Hz each min, 6 h/d, in a Flexercell FX-4000 Strain Unit. The following parameters were measured: cell number and viability by laser scanning confocal microscopy; cell proliferation with the MTS assay; the expression of a panel of 18 genes using real-time RT-PCR; matrix metalloproteinases (MMPs) 1-3, TIMP-1, CTGF and FGF-2 protein levels in supernatants from mechanically activated cultures with Enzyme-linked immunosorbent assays. Constructs from stationary cultures were also examined by scanning electron microscopy and immunostained for actin and vinculin. RESULTS: Although initially randomly distributed, the cells became organized into a bilayer by day 7; apoptotic cells remained constant at approximately 5% of the total. CTGF/FGF-2 stimulated cell proliferation in stationary cultures, but relative quantity values suggested modest effects on gene expression. Two transcription factors (RUNX2 and PPARG), two collagens (COL1A1, COL3A1), four MMPs (MMP-1-3, TIMP-1), TGFB1, RANKL, OPG and P4HB were detected by gel electrophoresis and Ct values < 35. In mechanically active cultures, with the exception of P4HB, TGFB1 and RANKL, each was upregulated at some point in the time scale, as was the synthesis of MMPs and TIMP-1. SOX9, MYOD, SP7, BMP2, BGLAP or COL2A1 were not detected in either stationary or mechanically active cultures. CONCLUSION: Three-dimensional tissue constructs provide additional complexity to monolayer culture systems, and suggest some of the assumptions regarding cell growth, differentiation and matrix turnover based on two-dimensional cultures may not apply to cells in three-dimensional matrices. Primarily developed as a transitional in vitro model for studying cell-cell and cell-matrix interactions in tooth support, the system is also suitable for investigating the pathogenesis of periodontal diseases, and importantly from the clinical point of view, in a mechanically active environment.

The expression of glutathione peroxidase-1 and the anabolism of collagen regulation pathway transforming growth factor-beta1-connective tissue growth factor in women with uterine prolapse and the clinic significance.

OBJECTIVES: To investigate the expression of the anabolism of collagen regulation pathways connective tissue growth factor (CTGF) -transforming growth factor-betal (TGF-beta1) and glutathione peroxidase-1 (GPx1) in women with uterine prolapse and a study of the clinic significance. MATERIALS AND METHODS: The expression of TGF-beta1, CTGF, and GPx1 was detected by immunohistochemical staining in pubocervical fascia tissue of 30 women with uterine prolapse, including ten cases of POP-QII, ten cases of POP-QIII, ten cases of POP-QIV, and 20 cases were control group with non-prolapse and non-malignant lesions. RESULTS: There was a negative correlation between the POP-Q and expression of TGF-beta1. With the increase of POP-Q degree, the expression degree of TGF-beta1 decreased correspondingly, which also applied to CTGF and GPx1. On the other hand, there was a positive correlation between TGF-beta1 and CTGF. The synergistic change trend was found between TGF-beta1 and CTGF. It could also be seen between CTGF and GPx1 and betweenTGF-beta1 and GPx1. CONCLUSION: The expression of the antioxidase GPx1 in pelvic support structure of POP women was decreased, which resulted in the antioxidation reduced. It could break the balance of oxidation and antioxidation in pelvic support structure, and may induce an increase of ROS level and the down-regulation of TGF-beta1-CTGF pathway. It could inhibit the anabolism of collagen and injury the pelvic support structure, thus promoting the occurrence and development of POP.

The presence and regulation of connective tissue growth factor in the human endometrium.

BACKGROUND: The human endometrium efficiently repairs each month after menstruation. The mechanisms involved in this repair process remain undefined. Aberrations in endometrial repair may lead to the common disorder of heavy menstrual bleeding. We hypothesized that connective tissue growth factor (CTGF) is increased at the time of endometrial repair post-menses and that this increase is regulated by prostaglandins (PGs) and hypoxic conditions present during menstruation. METHODS AND RESULTS: Examination of 41 endometrial biopsies from 5 stages of the menstrual cycle revealed maximal CTGF mRNA expression (using quantitative RT-PCR) at menstruation and peak protein levels during the proliferative phase. CTGF was immunolocalized to epithelial and stromal cells, with intense staining of occasional stromal cells during the proliferative phase. Dual immunohistochemistry identified these cells as macrophages. Treatment of endometrial epithelial cells with 100 nM PGE(2), PGF(2α) or hypoxia (0.5% O(2)) revealed a significant increase in CTGF mRNA expression (P < 0.01 for all, versus vehicle control). Cells treated simultaneously with PGE(2) and hypoxia revealed a synergistic increase in CTGF expression (P < 0.05 versus PGE(2) or hypoxia alone) and maximal secreted CTGF protein levels (P < 0.05 versus control). CONCLUSIONS: CTGF is increased in the human endometrium at the time of endometrial repair post-menses. The increase in CTGF may be mediated by PG production and the transient hypoxic episode observed in the endometrium at menstruation.

Inverse expression of cystein-rich 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2) in borderline tumors and carcinomas of the ovary.

Members of the CCN [cystein-rich 61 (Cyr61)/connective tissue growth factor (CTGF)/nephroblastoma (NOV)] protein family are involved in the regulation of cellular proliferation, apoptosis, and migration and are also assumed to play a role in carcinogenesis. Therefore, we performed a retrospective study to investigate the immunohistochemical expression of both Cyr61 and CTGF in 92 borderline tumors (BOTs) and 107 invasive carcinomas of the ovary (IOCs). To determine their diagnostic and prognostic value, we correlated protein expression with clinicopathologic factors including overall and disease-free survival. Cyr61 and CTGF were found to be inversely expressed in both BOTs and IOCs, with a stronger expression of Cyr61 in IOCs. Moreover, Cyr61 was found to be preferentially expressed in high-grade serous carcinomas, whereas CTGF was found more frequently in low-grade serous carcinomas. Weak Cyr61 levels correlated with both low estrogen receptor and p53 expression (P=0.038, P=0.04, respectively). However, no association was observed between CTGF, estrogen receptor, and p53 expression levels in IOCs. Regarding prognosis, Cyr61 was found to be of no value, but the loss of CTGF was found to be associated with a poor prognosis in multivariate analysis of overall (relative risk 2.8; P=0.050) and disease-free (relative risk 2.3; P=0.031) survival. Cyr61 and CTGF are inversely expressed in BOTs and IOCs, and loss of CTGF independently indicates poor prognosis in IOCs.