Increase of CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells.

PubMed

Kunzmann, Steffen; Krempl, Christine; Seidenspinner, Silvia; Glaser, Kirsten; Speer, Christian P; Fehrholz, Markus

2018-04-16

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV-infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV-mediated increase of CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

[Expression of connective tissue growth factor (CTGF), osteopontin (OPN) and clinical significances in the laryngeal squamous cell carcinoma tissues].

PubMed

Li, Youzhong; Lu, Yongde; Ceng, Yici; Yang, Xinming

2007-02-01

To study the expressions of CTGF and OPN and detect their clinical value and correlation in the laryngeal squamous cell carcinoma tissues and paracancerous tissues. SP immunohistochemical method was used for the assays of CTGF and OPN on the routinely paraffin-embedded sections of surgical operated specimens of 41 cases with laryngeal squamous cell carcinoma and 20 ones with paracancerous tissues. The positive rate of CTGF and the score were significantly lower in cancer tissues than those in paracancerous tissues (61.0% vs 90.0%, P < 0.05; 2.41 +/- 1.60 vs 4.24 +/- 1.42, P < 0.01), but those of OPN were opposite (61.0% vs 15.0%, P < 0.01; 3. 10 +/- 1.63 vs 1.12 +/- 0.84, P < 0.01). The positive rates and scores of CTGF were significantly higher in the cases without-metastasis of lymph node and clinical stage T1 than those in the ones with-metastasis of lymph node and clinical stage T3 (P < 0.01) . The positive rates and scores of OPN were significantly lower in the cases without-metastasis of lymph node, clinical stage T1 and histological grade I those that in the ones with-metastasis of lymph node, Clinical stage T3 and histological grade III (P < 0.01). The closely negative correlation was found between the score of CTGF and that of OPN. The expression of CTGF and/or OPN might be important biological markers in reflecting the progression, biological behaviors, metastatic potential and prognosis of the laryngeal squamous cell carcinoma.

Cyclical cell stretching of skin-derived fibroblasts downregulates connective tissue growth factor (CTGF) production.

PubMed

Kanazawa, Yuichiro; Nomura, Jun; Yoshimoto, Shinya; Suzuki, Toshikazu; Kita, Kazuko; Suzuki, Nobuo; Ichinose, Masaharu

2009-01-01

Delayed healing of skin wounds can be caused by wound instability, whereas appropriate massage or exercise prevents sclerosis and scar contracture. However, the mechanism by which wound healing is related to mechanical stress has not been fully elucidated. The present study aimed to identify whether mechanical stretching of fibroblasts reduces their production of extracellular matrix. We transferred skin fibroblasts into collagen-coated elastic silicone chambers, cultured them on a stretching apparatus, and used RT-PCR to examine the effects of mechanical stretching on the expression levels of 17 genes related to extracellular matrix production and growth factor secretion. We found that connective tissue growth factor (CTGF) was downregulated after 24 hr of cell stretching. Specifically, the CTGF mRNA and protein levels were 50% and 48% of the control levels, respectively. These findings suggest that cyclic stretching of fibroblasts contributes to anti-fibrotic processes by reducing CTGF production.

Laser-induced thermotherapy (LITT) elevates mRNA expression of connective tissue growth factor (CTGF) associated with reduced tumor growth of liver metastases compared to hepatic resection.

PubMed

Isbert, Christoph; Ritz, Jörg-Peter; Roggan, André; Schuppan, Detlef; Ajubi, Navid; Buhr, Heinz Johannes; Hohenberger, Werner; Germer, Christoph-Thomas

2007-01-01

Proliferation and synthesis of hepatocellular tissue after tissue damage are promoted by specific growth factors such as hepatic tissue growth factor (HGF) and connective growth factor (CTGF). Laser-induced thermotherapy (LITT) for the treatment of liver metastases is deemed to be a parenchyma-saving procedure compared to hepatic resection. The aim of this study was to compare the impact of LITT and hepatic resection on intrahepatic residual tumor tissue and expression levels of mRNA HGF/CTGF within liver and tumor tissue. Two independent adenocarcinomas (CC531) were implanted into 75 WAG rats, one in the right (untreated tumor) and one in the left liver lobe (treated tumor). The left lobe tumor was treated either by LITT or partial hepatectomy. The control tumor was submitted to in-situ hybridization of HGF and CTGF 24-96 hours and 14 days after intervention. Volumes of the untreated tumors prior to intervention were 38+/-8 mm(3) in group I (laser), 39 +/- 7 mm(3) in group II (resection), and 42 +/- 12 mm(3) in group III (control) and did not differ significantly (P > 0.05). Fourteen days after the intervention the mean tumor+/-SEM volume of untreated tumor in group I (laser) [223 +/- 36] was smaller than in group II (resection) [1233.28 +/- 181.52; P < 0.001], and in group III (control) [978.92 +/- 87.57; P < 0.003]. Forty-eight hours after the intervention intrahepatic mRNA expression level of HGF in group II (resection) was almost twofold higher than in group I (laser) [7.2 +/- 1.0 c/mf vs. 3.9 +/- 0.4 c/mf; P<0.01]. Fourteen days after the intervention intrahepatic mRNA expression level of CTGF in group I (laser) was higher than in group II (resection) [13.89 +/- 0.77 c/mf vs. 9.09 +/- 0.78 c/mf; P < 0.003]. LITT leads to a decrease of residual tumor growth in comparison to hepatic resection. Accelerated tumor growth after hepatic resection is associated with higher mRNA level of HGF and reduced tumor growth after LITT with higher mRNA level of CTGF. The

Expression of connective tissue growth factor (CTGF/CCN2) in breast cancer cells is associated with increased migration and angiogenesis.

PubMed

Chien, Wenwen; O'Kelly, James; Lu, Daning; Leiter, Amanda; Sohn, Julia; Yin, Dong; Karlan, Beth; Vadgama, Jay; Lyons, Karen M; Koeffler, H Phillip

2011-06-01

Connective tissue growth factor (CTGF/CCN2) belongs to the CCN family of matricellular proteins, comprising Cyr61, CTGF, NovH and WISP1-3. The CCN proteins contain an N-terminal signal peptide followed by four conserved domains sharing sequence similarities with the insulin-like growth factor binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a C-terminal growth factor cysteine knot domain. To investigate the role of CCN2 in breast cancer, we transfected MCF-7 cells with full-length CCN2, and with four mutant constructs in which one of the domains had been deleted. MCF-7 cells stably expressing full-length CCN2 demonstrated reduced cell proliferation, increased migration in Boyden chamber assays and promoted angiogenesis in chorioallantoic membrane assays compared to control cells. Deletion of the C-terminal cysteine knot domain, but not of any other domain-deleted mutants, abolished activities mediated by full-length CCN2. We have dissected the role of CCN2 in breast tumorigenesis on a structural basis.

Regulation of CCN2/CTGF Expression in the Nucleus Pulposus of the Intervertebral Disc: Role of Smad and AP1 Signaling

PubMed Central

Tran, Cassie M.; Markova, Dessislava; Smith, Harvey E.; Susarla, Bala; Ponnappan, Ravi Kumar; Anderson, D Greg; Symes, Aviva; Shapiro, Irving M.; Risbud, Makarand V.

2011-01-01

Objective To investigate TGFβ regulation of CTGF expression in cells of the nucleus pulposus. Methods Real Time RT-PCR and Western blot analysis was used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effect of Smad2/3/7 and AP1on TGFβ mediated CTGF promoter activity. Results CTGF expression was lower in the neonatal disc compared with the skeletally mature rat disc. An increase in CTGF expression and promoter activity was observed in nucleus pulposus cells after TGFβ treatment. Deletion analysis indicated that promoter constructs lacking smad and AP1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3-MH2 domain alone increased CTGF activity. Further evidence of Smad3 and AP1 involvement was seen when DN-Smad3, SiRNA-Smad3, smad7 and DN-AP1 suppressed TGFβ mediated activation of the CTGF promoter. When either Smad3 or AP1 sites were mutated, CTGF promoter induction by TGFβ was suppressed. We also observed a decrease in expression of CTGF in discs of Smad3 null mice compared to the wild type. Analysis of human nucleus pulposus indicated a trend of increasing CTGF and TGFβ expression in the degenerate state. Conclusion TGFβ, through Smad3 and AP1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerate disc. PMID:20222112

Evaluation of genistein ability to modulate CTGF mRNA/protein expression, genes expression of TGFβ isoforms and expression of selected genes regulating cell cycle in keloid fibroblasts in vitro.

PubMed

Jurzak, Magdalena; Adamczyk, Katarzyna; Antończak, Paweł; Garncarczyk, Agnieszka; Kuśmierz, Dariusz; Latocha, Małgorzata

2014-01-01

Keloids are characterized by overgrowth of connective tissue in the skin that arises as a consequence of abnormal wound healing. Normal wound healing is regulated by a complex set of interactions within a network of profibrotic and antifibrotic cytokines that regulate new extracellular matrix (ECM) synthesis and remodeling. These proteins include transforming growth factor β (TGFβ) isoforms and connective tissue growth factor (CTGF). TGFβ1 stimulates fibroblasts to synthesize and contract ECM and acts as a central mediator of profibrotic response. CTGF is induced by TGFβ1 and is considered a downstream mediator of TGFβ1action in fibroblasts. CTGF plays a crucial role in keloid pathogenesis by promoting prolonged collagen synthesis and deposition and as a consequence sustained fibrotic response. During keloids formation, besides imbalanced ECM synthesis and degradation, fibroblast proliferation and it's resistance to apoptosis is observed. Key genes that may play a role in keloid formation and growth involve: suppressor gene p53.,cyclin-depend- ent kinase inhibitor CDKN1A (p21) and BCL2 family genes: antiapoptotic BCL-2 and proapoptotic BAX. Genistein (4',5,7-trihydroxyisoflavone) exhibits multidirectional biological action. The concentration of genistein is relatively high in soybean. Genistein has been shown as effective antioxidant and chemopreventive agent. Genistein can bind to estrogen receptors (ERs) and modulate estrogen action due to its structure similarity to human estrogens. Genistein also inhibits transcription factors NFκB. Akt and AP-l signaling pathways, that are important for cytokines expression and cell proliferation, differentiation, survival and apoptosis. The aim of the study was to investigate genistein as a potential inhibitor of CTGF and TGFβ1, β2 and β3 isoforms expression and a potential regulator of p53. CDKN1A(p21), BAX and BCL-2 expression in normal fibroblasts and fibroblasts derived from keloids cultured in vitro. Real time

BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor, CTGF) expression.

PubMed

Ren, Wei; Sun, Xiaoxiao; Wang, Ke; Feng, Honglei; Liu, Yuehong; Fei, Chang; Wan, Shaoheng; Wang, Wei; Luo, Jinyong; Shi, Qiong; Tang, Min; Zuo, Guowei; Weng, Yaguang; He, Tongchuan; Zhang, Yan

2014-03-01

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, regulate a wide range of cellular responses including cell proliferation, differentiation, adhesion, migration, and apoptosis. BMP9, the latest BMP to be discovered, is reportedly expressed in a variety of human carcinoma cell lines, but the role of BMP9 in breast cancer has not been fully clarified. In a previous study, BMP9 was found to inhibit the growth, migration, and invasiveness of MDA-MB-231 breast cancer cells. In the current study, the effect of BMP9 on the bone metastasis of breast cancer cells was investigated. After absent or low expression of BMP9 was detected in the MDA-MB-231 breast cancer cells and breast non-tumor adjacent tissues using Western blot and immunohistochemistry, In our previous study, BMP9 could inhibit the proliferation and invasiveness of breast cancer cells MDA-MB-231 in vitro and in vivo. This paper shows that BMP9 inhibit the bone metastasis of breast cancer cells by activating the BMP/Smad signaling pathway and downregulating connective tissue growth factor (CTGF); however, when CTGF expression was maintained, the inhibitory effect of BMP9 on the MDA-MB-231 cells was abolished. Together, these observations indicate that BMP9 is an important mediator of breast cancer bone metastasis and a potential therapeutic target for treating this deadly disease.

[Expression of connective tissue growth factor in colorectal cancer and its association with prognosis].

PubMed

Sun, Zheng; Yang, Ping; Liang, Li-yuan; Zhang, Tong; Zhang, Wei-jian; Cao, Jie

2012-11-01

To investigate the expression of connective tissue growth factor (CTGF) in colorectal cancer(CRC) and its association with clinicopathologic parameters and overall survival rate. Fresh tumor tissues and matched distal normal colon tissues were collected from 92 patients diagnosed as CRC by surgical operation. The expression level of CTGF mRNA was quantified by quantitative reverse transcription PCR. Thirty out of 92 pairs of tissue specimens were selected randomly to detect CTGF protein by immunohistochemistry. All the cases were followed up to identify prognostic factors for survival. CTGF mRNA expression was up-regulated in CRC. The positive rate of CTGF protein expression tissues (73.3%) was significantly higher than that in the corresponding normal tissues (23.3%, P<0.01). CTGF expression was lower in patients with lymphatic metastasis or stage III/IIII disease (all P<0.05). A negative association was also observed between the CTGF protein positive rate and tumor infiltration depth (P<0.05). The relative expression of CTGF mRNA in tumor tissues was classified into high and low expression groups. The 5-year cumulative survival rate was lower in patients with low CTGF expression (29.3%) as compared to those with high CTGF expressions (68.3%) (P<0.01). Cox regression analysis revealed that the relative expression level of CTGF was independent factor of overall survival (RR=2.960, 95%CI:1.491-1.587, P<0.01). ROC curve analysis showed that sensitivity and specificity of CTGF mRNA expression for prediction of 5-year survival were 64.9% and 74.5%, respectively. The aberrant expression of CTGF is associated with the malignant biological behaviors of CRC. Low expression of CTGF is associated with worse prognosis of CRC.

Vascular Endothelial Cell-Specific Connective Tissue Growth Factor (CTGF) Is Necessary for Development of Chronic Hypoxia-Induced Pulmonary Hypertension.

PubMed

Pi, Liya; Fu, Chunhua; Lu, Yuanquing; Zhou, Junmei; Jorgensen, Marda; Shenoy, Vinayak; Lipson, Kenneth E; Scott, Edward W; Bryant, Andrew J

2018-01-01

Chronic hypoxia frequently complicates the care of patients with interstitial lung disease, contributing to the development of pulmonary hypertension (PH), and premature death. Connective tissue growth factor (CTGF), a matricellular protein of the Cyr61/CTGF/Nov (CCN) family, is known to exacerbate vascular remodeling within the lung. We have previously demonstrated that vascular endothelial-cell specific down-regulation of CTGF is associated with protection against the development of PH associated with hypoxia, though the mechanism for this effect is unknown. In this study, we generated a transgenic mouse line in which the Ctgf gene was floxed and deleted in vascular endothelial cells that expressed Cre recombinase under the control of VE-Cadherin promoter (eCTGF KO mice). Lack of vascular endothelial-derived CTGF protected against the development of PH secondary to chronic hypoxia, as well as in another model of bleomycin-induced pulmonary hypertension. Importantly, attenuation of PH was associated with a decrease in infiltrating inflammatory cells expressing CD11b or integrin α M (ITGAM), a known adhesion receptor for CTGF, in the lungs of hypoxia-exposed eCTGF KO mice. Moreover, these pathological changes were associated with activation of-Rho GTPase family member-cell division control protein 42 homolog (Cdc42) signaling, known to be associated with alteration in endothelial barrier function. These data indicate that endothelial-specific deletion of CTGF results in protection against development of chronic-hypoxia induced PH. This protection is conferred by both a decrease in inflammatory cell recruitment to the lung, and a reduction in lung Cdc42 activity. Based on our studies, CTGF inhibitor treatment should be investigated in patients with PH associated with chronic hypoxia secondary to chronic lung disease.

Relationship between hepatic CTGF expression and routine blood tests at the time of liver transplantation for biliary atresia: hope or hype for a biomarker of hepatic fibrosis.

PubMed

Haafiz, Allah; Farrington, Christian; Andres, Joel; Islam, Saleem

2011-01-01

Progressive hepatic fibrosis (HF) is a prominent feature of biliary atresia (BA), the most common indication for liver transplantation (LT) in children. Despite its importance in BA, HF is not evaluated in routine patient care because the invasiveness of liver biopsy makes histologic monitoring of fibrosis unfeasible. Therefore, the identification of noninvasive markers to assess HF is desirable especially in children. The main goal of this pilot project was to establish an investigational framework correlating hepatic expression of fibrogenic markers with routine blood tests in BA. Using liver explants from patients with BA (n = 26), immune-expression of connective tissue growth factor (CTGF), a key fibrogenic cytokine was determined using horseradish-labeled antibodies. Expression intensities of lobular (L-CTGF) and portal (P-CTGF) CTGF were determined by using ImageJ software. These CTGF intensities were correlated with blood tests performed at the time of LT. Correlation coefficients were determined for each blood test variable versus mean L-CTGF and P-CTGF expression intensities. A P-value of less than 0.05 was considered statistically significant. All patients had end-stage liver disease and persistent cholestasis at the time of LT. Kendall tau (τ) rank correlation coefficient for L-CTGF and white blood cell (WBC) was inversed (-0.52; P ≤ 0.02). Similar but statistically nonsignificant inverse relationships were noted between L-CTGF and prothrombin time (PT) (-0.15; P ≤ 0.4), international normalized ratio (INR) (-0.14; P ≤ 0.5), and platelet count (-0.36; P ≤ 0.09). Inversed (τ) rank correlation coefficients were also evident between P-CTGF expression and gamma-glutamyl transpeptidase (GGT), PT, INR, and platelet count. Pearson correlation coefficients for combinational analysis of standardized total bilirubin (TB), alkaline phosphatase, GGT, and platelet count with L-CTGF (0.33; P = 0.3) and P-CTGF (0.06; P = 0.8), were not significant. Similar

Involvement of CTGF, a hypertrophic chondrocyte-specific gene product, in tumor angiogenesis.

PubMed

Shimo, T; Nakanishi, T; Nishida, T; Asano, M; Sasaki, A; Kanyama, M; Kuboki, T; Matsumura, T; Takigawa, M

2001-01-01

Connective tissue growth factor (CTGF) is a potent secreted signaling factor which functions in multiple stages of angiogenesis. In the present study, we examined the role of CTGF in tumor angiogenesis and made the following observations: (1) Histological analysis of human breast cancer (MDA231) cell and human fibrosarcoma (HT1080) cell xenografts in BALB/c nude mice showed a high level of neovascularization. Human squamous cell carcinoma (A431) xenografts induced only a low level of neovascularization. (2) CTGF mRNA was strongly expressed in MDA231 and in HT1080 cells in vivo and in vitro, but not in A431 cells. (3) CTGF protein was markedly produced in MDA231 cells and HT1080 cells and secreted into culture medium, and its production was greater during phases of growth rather than confluency. (4) Production of CTGF in bovine aorta endothelial cells was induced by CTGF, VEGF, bFGF and TGF-beta. (5) Neovascularization induced by HT1080 cells or MDA231 cells on chicken chorioallantoic membrane was suppressed in the presence of neutralizing CTGF-specific polyclonal antibody. These results suggest that CTGF regulates progression in tumor angiogenesis and the release or secretion of CTGF from tumor cells is essential for the angiogenesis. Copyright 2001 S. Karger AG, Basel

MicroRNA-145 Inhibits Cell Migration and Invasion and Regulates Epithelial-Mesenchymal Transition (EMT) by Targeting Connective Tissue Growth Factor (CTGF) in Esophageal Squamous Cell Carcinoma.

PubMed

Han, Qiang; Zhang, Hua-Yong; Zhong, Bei-Long; Wang, Xiao-Jing; Zhang, Bing; Chen, Hua

2016-10-23

BACKGROUND This study investigated the mechanism of miR-145 in targeting connective tissue growth factor (CTGF), which affects the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of ESCC cells. MATERIAL AND METHODS A total of 50 ESCC tissues and their corresponding normal adjacent esophageal tissue samples were collected. Then, miR-145 expression in both ESCC clinical specimens and cell lines was detected using quantitative real-time PCR. CTGF protein was detected using immunohistochemistry. Dual luciferase reporter gene assay was employed to assess the effect of miR-145 on the 3'UTR luciferase activity of CTGF. Eca109 cells were transfected with miR-145 mimics and CTGF siRNA, respectively, and changes in cellular proliferation, migration, and invasion were detected via MTT assay, wound-healing assay, and Transwell assay, respectively. Western blotting assay was used to detect the expression of marker genes related to EMT. RESULTS MiR-145 was significantly down-regulated in ESCC tissues and cell lines compared with normal tissues and cell lines (P<0.05). We found significantly more positively expressed CTGF protein in ESCC tissues was than in normal adjacent esophageal tissues (P<0.01). Dual luciferase reporter gene assay showed that miR-145 can specifically bind with the 3'UTR of CTGF and significantly inhibit the luciferase activity by 55% (P<0.01). Up-regulation of miR-145 or down-regulation of CTGF can suppress the proliferation, migration, invasion, and EMT process of ESCC cells. CONCLUSIONS MiR-145 was significantly down-regulated in ESCC tissues and cell lines, while the protein expression of CTGF exhibited the opposite trend. MiR-145 inhibited the proliferation, migration, invasiveness, and the EMT process of ESCC cells through targeted regulation of CTGF expression.

Expression and clinical significance of connective tissue growth factor in thyroid carcinomas.

PubMed

Wang, Guimin; Zhang, Wei; Meng, Wei; Liu, Jia; Wang, Peisong; Lin, Shan; Xu, Liyan; Li, Enmin; Chen, Guang

2013-08-01

To examine expression of the connective tissue growth factor (CTGF) gene in human thyroid cancer and establish whether a correlation exists between the presence of CTGF protein and clinicopathological parameters of the disease. CTGF protein expression was investigated retrospectively by immunohistochemical analysis of CTGF protein levels in thyroid tumour tissue. Associations between immunohistochemical score and several clinicopathological parameters were examined. In total, 131 thyroid tissue specimens were included. High levels of CTGF protein were observed in papillary thyroid carcinoma tissue; benign thyroid tumour tissue scored negatively for CTGF protein. In papillary thyroid carcinoma, there was a significant relationship between high CTGF protein levels and Union for International Cancer Control disease stage III-IV, and presence of lymph node metastasis. In papillary thyroid carcinomas, CTGF protein levels were not significantly associated with sex or age. These findings suggest that the CTGF protein level is increased in papillary thyroid carcinoma cells compared with benign thyroid tumours. CTGF expression might play a role in the development of malignant tumours in the thyroid.

Caffeine and rolipram affect Smad signalling and TGF-β1 stimulated CTGF and transgelin expression in lung epithelial cells.

PubMed

Fehrholz, Markus; Speer, Christian P; Kunzmann, Steffen

2014-01-01

Caffeine administration is an important part of the therapeutic treatment of bronchopulmonary dysplasia (BPD) in preterm infants. However, caffeine mediated effects on airway remodelling are still undefined. The TGF-β/Smad signalling pathway is one of the key pathways involved in airway remodelling. Connective tissue growth factor (CTGF), a downstream mediator of TGF-β, and transgelin, a binding and stabilising protein of the cytoskeleton, are both regulated by TGF-β1 and play an important role in airway remodelling. Both have also been implicated in the pathogenesis of BPD. The aim of the present study was to clarify whether caffeine, an unspecific phosphodiesterase (PDE) inhibitor, and rolipram, a prototypical PDE-4 selective inhibitor, were both able to affect TGF-β1-induced Smad signalling and CTGF/transgelin expression in lung epithelial cells. Furthermore, the effect of transgelin knock-down on Smad signalling was studied. The pharmacological effect of caffeine and rolipram on Smad signalling was investigated by means of a luciferase assay via transfection of a TGF-β1-inducible reporter plasmid in A549 cells. The regulation of CTGF and transgelin expression by caffeine and rolipram were studied by promoter analysis, real-time PCR and Western blot. Endogenous transgelin expression was down-regulated by lentiviral transduction mediating transgelin-specific shRNA expression. The addition of caffeine and rolipram inhibited TGF-β1 induced reporter gene activity in a concentration-related manner. They also antagonized the TGF-β1 induced up-regulation of CTGF and transgelin on the promoter-, the mRNA-, and the protein-level. Functional analysis showed that transgelin silencing reduced TGF-β1 induced Smad-signalling and CTGF induction in lung epithelial cells. The present study highlights possible new molecular mechanisms of caffeine and rolipram including an inhibition of Smad signalling and of TGF-β1 regulated genes involved in airway remodelling. An

Expression of connective tissue growth factor in tumor tissues is an independent predictor of poor prognosis in patients with gastric cancer.

PubMed

Liu, Lu-Ying; Han, Yan-Chun; Wu, Shu-Hua; Lv, Zeng-Hua

2008-04-07

To examine the expression of connective tissue growth factor (CTGF), also known as CCN2, in gastric carcinoma (GC), and the correlation between the expression of CTGF, clinicopathologic features and clinical outcomes of patients with GC. One hundred and twenty-two GC patients were included in the present study. All patients were followed up for at least 5 years. Proteins of CTGF were detected using the Powervision two-step immunostaining method. Of the specimens from 122 GC patients analyzed for CTGF expression, 58 (58/122, 47.5%) had a high CTGF expression in cytoplasm of gastric carcinoma cells and 64 (64/122, 52.5%) had a low CTGF expression. Patients with a high CTGF expression showed a higher incidence of lymph node metastasis than those with a low CTGF expression (P = 0.032). Patients with a high CTGF expression had significantly lower 5-year survival rate than those with a low CTGF expression (27.6% vs 46.9%, P = 0.0178), especially those staging I + II + III (35.7% vs 65.2%, P = 0.0027). GC patients with an elevated CTGF expression have more lymph node metastases and a shorter survival time. CTGF seems to be an independent prognostic factor for the successful differentiation of high-risk GC patients staging I + II + III. Over-expression of CTGF in human GC cells results in an increased aggressive ability.

Expression of connective tissue growth factor in the livers of non-viral hepatocellular carcinoma patients with metabolic risk factors.

PubMed

Akahoshi, Keiichi; Tanaka, Shinji; Mogushi, Kaoru; Shimada, Shu; Matsumura, Satoshi; Akiyama, Yoshimitsu; Aihara, Arihiro; Mitsunori, Yusuke; Ban, Daisuke; Ochiai, Takanori; Kudo, Atsushi; Arii, Shigeki; Tanabe, Minoru

2016-09-01

The incidence of hepatocellular carcinoma (HCC) associated with metabolic risk factors, such as diabetes and obesity, has been increasing. However, the underlying mechanism that links these diseases remains unclear. We performed genome-wide expression analysis of human liver tissues of non-viral HCC patients with or without metabolic risk factors. The upregulated genes that associated with diabetes and obesity were investigated by in vitro and in vivo experiments, and immunohistochemistry of human liver tissues was performed. Among the upregulated genes, connective tissue growth factor (CTGF) expression was induced to a greater extent by combined glucose and insulin administration to human hepatoma cells. Genome-wide expression analysis revealed upregulation of a chemokine network in CTGF-overexpressing hepatoma cells, which displayed an increased ability to induce in vitro activation of macrophages, and in vivo infiltration of liver macrophages. Immunohistochemistry of human liver tissues validated the correlations between CTGF expression and diabetes or obesity as well as activation of liver macrophages in patients with non-viral HCC. Recurrence-free survival was significantly poorer in the CTGF-positive patients compared with the CTGF-negative patients (p = 0.002). Multivariate analysis determined that CTGF expression (HR 2.361; 95 % CI 1.195-4.665; p = 0.013) and vascular invasion (HR 2.367; 95 % CI 1.270-4.410; p = 0.007) were independent prognostic factors for recurrence of non-viral HCC. Our data suggest that CTGF could be involved in oncogenic pathways promoting non-viral HCC associated with metabolic risk factors via induction of liver inflammation and is expected to be a novel HCC risk biomarker and potential therapeutic target.

BMP signaling and podocyte markers are decreased in human diabetic nephropathy in association with CTGF overexpression.

PubMed

Turk, Tamara; Leeuwis, Jan Willem; Gray, Julia; Torti, Suzy V; Lyons, Karen M; Nguyen, Tri Q; Goldschmeding, Roel

2009-07-01

Diabetic nephropathy is characterized by decreased expression of bone morphogenetic protein-7 (BMP-7) and decreased podocyte number and differentiation. Extracellular antagonists such as connective tissue growth factor (CTGF; CCN-2) and sclerostin domain-containing-1 (SOSTDC1; USAG-1) are important determinants of BMP signaling activity in glomeruli. We studied BMP signaling activity in glomeruli from diabetic patients and non-diabetic individuals and from control and diabetic CTGF(+/+) and CTGF(+/-) mice. BMP signaling activity was visualized by phosphorylated Smad1, -5, and -8 (pSmad1/5/8) immunostaining, and related to expression of CTGF, SOSTDC1, and the podocyte differentiation markers WT1, synaptopodin, and nephrin. In control and diabetic glomeruli, pSmad1/5/8 was mainly localized in podocytes, but both number of positive cells and staining intensity were decreased in diabetes. Nephrin and synaptopodin were decreased in diabetic glomeruli. Decrease of pSmad1/5/8 was only partially explained by decrease in podocyte number. SOSTDC1 and CTGF were expressed exclusively in podocytes. In diabetic glomeruli, SOSTDC1 decreased in parallel with podocyte number, whereas CTGF was strongly increased. In diabetic CTGF(+/-) mice, pSmad1/5/8 was preserved, compared with diabetic CTGF(+/+) mice. In conclusion, in human diabetic nephropathy, BMP signaling activity is diminished, together with reduction of podocyte markers. This might relate to concomitant overexpression of CTGF but not SOSTDC1.

Microrna-199a-5p Functions as a Tumor Suppressor via Suppressing Connective Tissue Growth Factor (CTGF) in Follicular Thyroid Carcinoma.

PubMed

Sun, Dawei; Han, Shen; Liu, Chao; Zhou, Rui; Sun, Weihai; Zhang, Zhijun; Qu, Jianjun

2016-04-11

BACKGROUND The objective of this study was to explore the role of miR-199a-5p in the development of thyroid cancer, including its anti-proliferation effect and downstream signaling pathway. MATERIAL AND METHODS We conducted qRT-PCR analysis to detect the expressions of several microRNAs in 42 follicular thyroid carcinoma patients and 42 controls. We identified CTGF as target of miR-491, and viability and cell cycle status were determined in FTC-133 cells transfected with CTGF siRNA, miR-199a mimics, or inhibitors. RESULTS We identified an underexpression of miR-199a-5p in follicular thyroid carcinoma tissue samples compared with controls. Then we confirmed CTGF as a target of miR-199a-5p thyroid cells by using informatics analysis and luciferase reporter assay. Additionally, we found that mRNA and protein expression levels of CTGF were both clearly higher in malignant tissues than in benign tissues. miR-199a-5p mimics and CTGF siRNA similarly downregulated the expression of CTGF, and reduced the viability of FTC-133 cells by arresting the cell cycle in G0 phase. Transfection of miR-199a-5p inhibitors increased the expression of CTGF and promoted the viability of the cells by increasing the fraction of cells in G2/M and S phases. CONCLUSIONS Our study proves that the CTGF gene is a target of miR-199a-5p, demonstrating the negatively related association between CTGF and miR-199a. These findings suggest that miR-199a-5p might be a novel therapeutic target in the treatment of follicular thyroid carcinoma.

BMP Signaling and Podocyte Markers Are Decreased in Human Diabetic Nephropathy in Association With CTGF Overexpression

PubMed Central

Turk, Tamara; Leeuwis, Jan Willem; Gray, Julia; Torti, Suzy V.; Lyons, Karen M.; Nguyen, Tri Q.; Goldschmeding, Roel

2009-01-01

Diabetic nephropathy is characterized by decreased expression of bone morphogenetic protein-7 (BMP-7) and decreased podocyte number and differentiation. Extracellular antagonists such as connective tissue growth factor (CTGF; CCN-2) and sclerostin domain-containing-1 (SOSTDC1; USAG-1) are important determinants of BMP signaling activity in glomeruli. We studied BMP signaling activity in glomeruli from diabetic patients and non-diabetic individuals and from control and diabetic CTGF+/+ and CTGF+/− mice. BMP signaling activity was visualized by phosphorylated Smad1, -5, and -8 (pSmad1/5/8) immunostaining, and related to expression of CTGF, SOSTDC1, and the podocyte differentiation markers WT1, synaptopodin, and nephrin. In control and diabetic glomeruli, pSmad1/5/8 was mainly localized in podocytes, but both number of positive cells and staining intensity were decreased in diabetes. Nephrin and synaptopodin were decreased in diabetic glomeruli. Decrease of pSmad1/5/8 was only partially explained by decrease in podocyte number. SOSTDC1 and CTGF were expressed exclusively in podocytes. In diabetic glomeruli, SOSTDC1 decreased in parallel with podocyte number, whereas CTGF was strongly increased. In diabetic CTGF+/− mice, pSmad1/5/8 was preserved, compared with diabetic CTGF+/+ mice. In conclusion, in human diabetic nephropathy, BMP signaling activity is diminished, together with reduction of podocyte markers. This might relate to concomitant overexpression of CTGF but not SOSTDC1. (J Histochem Cytochem 57:623–631, 2009) PMID:19255250

Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.

PubMed

Riser, B L; Denichilo, M; Cortes, P; Baker, C; Grondin, J M; Yee, J; Narins, R G

2000-01-01

Connective tissue growth factor (CTGF) is a peptide secreted by cultured endothelial cells and fibroblasts when stimulated by transforming growth factor-beta (TGF-beta), and is overexpressed during fibrotic processes in coronary arteries and in skin. To determine whether CTGF is implicated in the pathogenesis of diabetic glomerulosclerosis, cultured rat mesangial cells (MC) as well as kidney cortex and microdissected glomeruli were examined from obese, diabetic db/db mice and their normal counterparts. Exposure of MC to recombinant human CTGF significantly increased fibronectin and collagen type I production. Furthermore, unstimulated MC expressed low levels of CTGF message and secreted minimal amounts of CTGF protein (36 to 38 kD) into the media. However, sodium heparin treatment resulted in a greater than fourfold increase in media-associated CTGF, suggesting that the majority of CTGF produced was cell- or matrix-bound. Exposure of MC to TGF-beta, increased glucose concentrations, or cyclic mechanical strain, all causal factors in diabetic glomerulosclerosis, markedly induced the expression of CTGF transcripts, while recombinant human CTGF was able to autoinduce its own expression. TGF-, and high glucose, but not mechanical strain, stimulated the concomitant secretion of CTGF protein, the former also inducing abundant quantities of a small molecular weight form of CTGF (18 kD) containing the heparin-binding domain. The induction of CTGF protein by a high glucose concentration was mediated by TGF-beta, since a TGF-beta-neutralizing antibody blocked this stimulation. In vivo studies using quantitative reverse transcription-PCR demonstrated that although CTGF transcripts were low in the glomeruli of control mice, expression was increased 28-fold after approximately 3.5 mo of diabetes. This change occurred early in the course of diabetic nephropathy when mesangial expansion was mild, and interstitial disease and proteinuria were absent. A substantially reduced

Connective tissue growth factor immunohistochemical expression is associated with gallbladder cancer progression.

PubMed

Garcia, Patricia; Leal, Pamela; Alvarez, Hector; Brebi, Priscilla; Ili, Carmen; Tapia, Oscar; Roa, Juan C

2013-02-01

Gallbladder cancer (GBC) is an aggressive neoplasia associated with late diagnosis, unsatisfactory treatment, and poor prognosis. Molecular mechanisms involved in GBC pathogenesis remain poorly understood. Connective tissue growth factor (CTGF) is thought to play a role in the pathologic processes and is overexpressed in several human cancers, including GBC. No information is available about CTGF expression in early stages of gallbladder carcinogenesis. Objective.- To evaluate the expression level of CTGF in benign and malignant lesions of gallbladder and its correlation with clinicopathologic features and GBC prognosis. Connective tissue growth factor protein was examined by immunohistochemistry on tissue microarrays containing tissue samples of chronic cholecystitis (n = 51), dysplasia (n = 15), and GBC (n = 169). The samples were scored according to intensity of staining as low/absent and high CTGF expressers. Statistical analysis was performed using the χ(2) test or Fisher exact probability test with a significance level of P < .05. Survival analysis was assessed by the Kaplan-Meier method and the log-rank test. Connective tissue growth factor expression showed a progressive increase from chronic cholecystitis to dysplasia and then to early and advanced carcinoma. Immunohistochemical expression (score ≥2) was significantly higher in advanced tumors, in comparison with chronic cholecystitis (P < .001) and dysplasia (P = .03). High levels of CTGF expression correlated with better survival (P = .04). Our results suggest a role for CTGF in GBC progression and a positive association with better prognosis. In addition, they underscore the importance of considering the involvement of inflammation on GBC development.

Expression of connective tissue growth factor in male breast cancer: clinicopathologic correlations and prognostic value.

PubMed

Lacle, Miangela M; van Diest, Paul J; Goldschmeding, Roel; van der Wall, Elsken; Nguyen, Tri Q

2015-01-01

Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of secreted proteins that are believed to play an important role in the development of neoplasia. In particular, CTGF has been reported to play an important role in mammary tumorigenesis and to have prognostic value in female breast cancer (FBC). The aim of the present study was to investigate clinicopathologic correlations and prognostic value of CTGF in male breast cancer (MBC) and to compare these findings with FBC. For this, we studied CTGF protein expression by immunohistochemistry in 109 MBC cases and 75 FBC cases. In MBC, stromal CTGF expression was seen in the majority of the cases 78% (85/109) with high expression in 31/109 cases (28.4%), but expression in tumor cells was only seen in 9.2% (10/109) of cases. High stromal CTGF expression correlated with high grade and high proliferation index (>15%) assessed by MIB-1 immunohistochemical staining. CTGF expression in tumor epithelial cells did not correlate with any of the clinicopathologic features. In FBC, stromal CTGF expression positively correlated with mitotic count and tumor CTGF expression was associated with triple negative status of the tumor (p = 0.002). Neither stromal nor tumor epithelial cell CTGF expression had prognostic value in MBC and FBC. In conclusion, stromal CTGF expression was seen in a high percentage of MBC and was correlated with high grade and high proliferation index. In view of the important role of the microenvironment in cancer progression, this might suggest that stromal CTGF could be an interesting target for novel therapies and molecular imaging. However, the lack of association with prognosis warrants caution. The potential role of CTGF as a therapeutic target for triple negative FBC deserves to be further studied.

Expression and clinical significance of connective tissue growth factor in advanced head and neck squamous cell cancer.

PubMed

Kikuchi, Ryoko; Kikuchi, Yoshihiro; Tsuda, Hitoshi; Maekawa, Hitoshi; Kozaki, Ken-Ichi; Imoto, Issei; Tamai, Seiichi; Shiotani, Akihiro; Iwaya, Keiichi; Sakamoto, Masaru; Sekiya, Takao; Matsubara, Osamu

2014-07-01

Connective tissue growth factor (CTGF) has been reported to play critical roles in the tumorigenesis of several human malignancies. This study was performed to evaluate CTGF protein expression in head and neck squamous cell carcinoma (HNSCC). Surgical specimens from 76 primary HNSCC were obtained with written informed consents and the expression level of CTGF was immunohistochemically evaluated. The cytoplasmic immunoreactivity of CTGF in cancer cells was semiquantitatively classified into low and high expression. Among all 76 cases with or without neoadjuvant therapy, low CTGF showed significantly longer (P = 0.0282) overall survival (OS), but not disease-free survival (DFS) than high CTGF. Although low CTGF in patients with stage I, II and III did not result in any significant difference of the OS and DFS, stage IV HNSCC patients with low CTGF showed significantly longer OS (P = 0.032) and DFS (P = 0.0107) than those with high CTGF. These differences in stage IV cases were also confirmed using multivariate analyses. These results suggest that low CTGF in stage IV HNSCC is an independent prognostic factor, despite with or without neoadjuvant therapy.

Connective tissue growth factor (CTGF) from basics to clinics.

PubMed

Ramazani, Yasaman; Knops, Noël; Elmonem, Mohamed A; Nguyen, Tri Q; Arcolino, Fanny Oliveira; van den Heuvel, Lambert; Levtchenko, Elena; Kuypers, Dirk; Goldschmeding, Roel

2018-03-21

Connective tissue growth factor, also known as CCN2, is a cysteine-rich matricellular protein involved in the control of biological processes, such as cell proliferation, differentiation, adhesion and angiogenesis, as well as multiple pathologies, such as tumor development and tissue fibrosis. Here, we describe the molecular and biological characteristics of CTGF, its regulation and various functions in the spectrum of development and regeneration to fibrosis. We further outline the preclinical and clinical studies concerning compounds targeting CTGF in various pathologies with the focus on heart, lung, liver, kidney and solid organ transplantation. Finally, we address the advances and pitfalls of translational fibrosis research and provide suggestions to move towards a better management of fibrosis. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

mTOR Complexes Repress Hypertrophic Agonist-Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells.

PubMed

Sundararaj, Kamala; Pleasant, Dorea L; Moschella, Phillip C; Panneerselvam, Kavin; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2016-02-01

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 μM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium.

MicroRNA-143-3p inhibits hyperplastic scar formation by targeting connective tissue growth factor CTGF/CCN2 via the Akt/mTOR pathway.

PubMed

Mu, Shengzhi; Kang, Bei; Zeng, Weihui; Sun, Yaowen; Yang, Fan

2016-05-01

Post-traumatic hypertrophic scar (HS) is a fibrotic disease with excessive extracellular matrix (ECM) production, which is a response to tissue injury by fibroblasts. Although emerging evidence has indicated that miRNA contributes to hypertrophic scarring, the role of miRNA in HS formation remains unclear. In this study, we found that miR-143-3p was markedly downregulated in HS tissues and fibroblasts (HSFs) using qRT-PCR. The expression of connective tissue growth factor (CTGF/CCN2) was upregulated both in HS tissues and HSFs, which is proposed to play a key role in ECM deposition in HS. The protein expression of collagen I (Col I), collagen III (Col III), and α-smooth muscle actin (α-SMA) was obviously inhibited after treatment with miR-143-3p in HSFs. The CCK-8 assay showed that miR-143-3p transfection reduced the proliferation ability of HSFs, and flow cytometry showed that either early or late apoptosis of HSFs was upregulated by miR-143-3p. In addition, the activity of caspase 3 and caspase 9 was increased after miR-143-3p transfection. On the contrary, the miR-143-3p inhibitor was demonstrated to increase cell proliferation and inhibit apoptosis of HSFs. Moreover, miR-143-3p targeted the 3'-UTR of CTGF and caused a significant decrease of CTGF. Western blot demonstrated that Akt/mTOR phosphorylation and the expression of CTGF, Col I, Col III, and α-SMA were inhibited by miR-143-3p, but increased by CTGF overexpression. In conclusion, we found that miR-143-3p inhibits hypertrophic scarring by regulating the proliferation and apoptosis of human HSFs, inhibiting ECM production-associated protein expression by targeting CTGF, and restraining the Akt/mTOR pathway.

miR-483 Targeting of CTGF Suppresses Endothelial-to-Mesenchymal Transition: Therapeutic Implications in Kawasaki Disease

PubMed Central

He, Ming; Chen, Zhen; Martin, Marcy; Zhang, Jin; Sangwung, Panjamaporn; Woo, Brian; Tremoulet, Adriana H.; Shimizu, Chisato; Jain, Mukesh K.; Burns, Jane C.; Shyy, John Y-J.

2016-01-01

Rationale Endothelial-to-mesenchymal transition (EndoMT) is implicated in myofibroblast-like cell-mediated damage to the coronary arterial wall in acute Kawasaki disease (KD) patients, as evidenced by positive staining for connective tissue growth factor (CTGF) and EndoMT markers in KD autopsy tissues. However, little is known about the molecular basis of EndoMT involved in KD. Objective We investigated the microRNA (miRNA) regulation of CTGF and the consequent EndoMT in KD pathogenesis. As well, the modulation of this process by statin therapy was studied. Methods and Results Sera from healthy children and KD subjects were incubated with human umbilical vein endothelial cells (HUVECs). Cardiovascular disease-related miRNAs, CTGF, and EndoMT markers were quantified using RT-qPCR, ELISA, and Western blotting. Compared to healthy controls, HUVEC incubated with sera from acute KD patients had decreased miR-483, increased CTGF, and increased EndoMT markers. Bioinformatics analysis followed by functional validation demonstrated that Krüppel-like factor 4 (KLF4) transactivates miR-483, which in turn targets the 3′ untranslated region of CTGF mRNA. Overexpression of KLF4 or pre-miR-483 suppressed, whereas knockdown of KLF4 or anti-miR-483 enhanced, CTGF expression in ECs in vitro and in vivo. Furthermore, atorvastatin, currently being tested in a Phase I/IIa clinical trial in KD children, induced KLF4-miR-483, which suppressed CTGF and EndoMT in ECs. Conclusions KD sera suppress the KLF4-miR-483 axis in ECs leading to increased expression of CTGF and induction of EndoMT. This detrimental process in the endothelium may contribute to coronary artery abnormalities in KD patients. Statin therapy may benefit acute KD patients, in part through the restoration of KLF4-miR-483 expression. Clinical Trial Registration NCT01431105 PMID:27923814

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

PubMed

Mize, T W; Sundararaj, K P; Leite, R S; Huang, Y

2015-06-01

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-β1), 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-β1-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. Periodontal tissue specimens were collected from seven individuals without periodontitis (group 1) and from 14 with periodontitis (group 2). The expression of CTGF and TGFβ1 mRNAs were quantified using real-time PCR. Analysis using the nonparametric Mann-Whitney U-test showed that the levels of expression of both CTGF/CCN2 and TGFβ1 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 TGFβ1 and CTGF/CCN2 mRNAs. The gingival expression levels of CTGF/CCN2 and TGFβ1 mRNAs in individuals with periodontitis are upregulated and correlated. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

PubMed

Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

2010-03-01

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

Expression of connective tissue growth factor and interleukin-11 in intratumoral tissue is associated with poor survival after curative resection of hepatocellular carcinoma.

PubMed

Xiang, Zuo-Lin; Zeng, Zhao-Chong; Fan, Jia; Tang, Zhao-You; Zeng, Hai-Ying

2012-05-01

In the present study, we evaluated the prognostic value of intratumoral and peritumoral expression of connective tissue growth factor (CTGF), transforming growth factor-beta 1 (TGF-β1), and interleukin-11 (IL-11) in patients with hepatocellular carcinoma (HCC) after curative resection. Expression of CTGF, TGF-β1, and IL-11 was assessed by immunohistochemical staining of tissue microarrays containing paired tumor and peritumoral liver tissue from 290 patients who had undergone hepatectomy for histologically proven HCC. The prognostic value of these and other clinicopathologic factors were evaluated. The median follow-up time was 54.3 months (range, 4.3-118.3 months). High intratumoral CTGF expression was associated with vascular invasion (P = 0.015), intratumoral IL-11 expression correlated with higher tumor node metastasis (TNM) stage (P = 0.009), and peritumoral CTGF overexpression correlated with lack of tumor encapsulation (P = 0.031). Correlation analysis of these proteins revealed that intratumoral CTGF and IL-11 correlated with high intratumoral TGF-β1 expression (r = 0.325, P < 0.001; and r = 0.273, P < 0.001, respectively). TNM stage (P < 0.001), high intratumoral CTGF levels (P = 0.010), and intratumoral IL-11 expression (P = 0.015) were independent prognostic factors for progression-free survival (PFS). Vascular invasion (P = 0.032), TNM stage (P < 0.001), high intratumoral CTGF levels (P = 0.036), and intratumoral IL-11 expression (P = 0.013) were independent prognostic factors for overall survival (OS). High intratumoral CTGF and intratumoral IL-11 expression were associated with PFS and OS after hepatectomy, and the combination of intratumoral CTGF with IL-11 may be predictive of survival.

Connective Tissue Growth Factor (CTGF/CCN2) mediates angiogenic effect of S1P in human dermal microvascular endothelial cells

PubMed Central

MARKIEWICZ, MAGARET; NAKERAKANTI, SASHIDHAR S.; KAPANADZE, BAGRAT; GHATNEKAR, ANGELA; TROJANOWSKA, MARIA

2010-01-01

Objective The primary objective of this study was to examine the potential interaction between sphingosine-1-phosphate (S1P), a pleiotropic lipid mediator, and CTGF/CCN2 a secreted multimodular protein, in the process of endothelial cell migration. The second objective was to determine whether C- and N-terminal domains of CTGF/CCN2 have specific function in cell migration. Materials and Methods Migration of human dermal microvascular endothelial cells (HDMECs) was examined in monolayer wound healing “scratch” assay, while capillary-like tube formation was examined in 3 dimensional collagen co-culture assays. Results We observed that S1P stimulates HDMECs migration concomitant with upregulation of CTGF/CCN2 expression. Furthermore, the blockade of endogenous CTGF/CCN2 via siRNA abrogated S1P induced HDMECs migration and capillary-like tube formation. Full length CTGF induced cell migration and capillary-like tube formation with potency similar to that of S1P, while C-terminal domain of CTGF was slightly less effective. However; N-terminal domain had only a residual activity in inducing capillary-like tube formation. Conclusions This study revealed that CTGF/CCN2 is required for the S1P induced endothelial cell migration, which suggests that CTGF/CCN2 may be an important mediator of S1P induced physiological and pathological angiogenesis. Moreover, this study shows that the pro-migratory activity of CTGF/CCN2 is located in the C-terminal domain. PMID:21166920

Connective tissue growth factor (CTGF/CCN2) mediates angiogenic effect of S1P in human dermal microvascular endothelial cells.

PubMed

Markiewicz, Margaret; Nakerakanti, Sashidhar S; Kapanadze, Bagrat; Ghatnekar, Angela; Trojanowska, Maria

2011-01-01

The primary objective of this study was to examine the potential interaction between S1P, a pleiotropic lipid mediator, and CTGF/CCN2, a secreted multimodular protein, in the process of endothelial cell migration. The secondary objective was to determine whether C- and N-terminal domains of CTGF/CCN2 have a specific function in cell migration. Migration of HDMECs was examined in monolayer wound healing "scratch" assay, whereas capillary-like tube formation was examined in three-dimensional collagen co-culture assays. We observed that S1P stimulates migration of HDMECs concomitant with upregulation of CTGF/CCN2 expression. Furthermore, the blockade of endogenous CTGF/CCN2 via siRNA abrogated S1P-induced HDMEC migration and capillary-like tube formation. Full-length CTGF induced cell migration and capillary-like tube formation with a potency similar to that of S1P, while C-terminal domain of CTGF was slightly less effective. However, N-terminal domain had only a residual activity in inducing capillary-like tube formation. This study revealed that CTGF/CCN2 is required for the S1P-induced endothelial cell migration, which suggests that CTGF/CCN2 may be an important mediator of S1P-induced physiological and pathological angiogenesis. Moreover, this study shows that the pro-migratory activity of CTGF/CCN2 is located in the C-terminal domain. © 2010 John Wiley & Sons Ltd.

GPER in CAFs regulates hypoxia-driven breast cancer invasion in a CTGF-dependent manner.

PubMed

Ren, Juan; Guo, Hui; Wu, Huili; Tian, Tao; Dong, Danfeng; Zhang, Yuelang; Sui, Yanxia; Zhang, Yong; Zhao, Dongli; Wang, Shufeng; Li, Zongfang; Zhang, Xiaozhi; Liu, Rui; Qian, Jianshneg; Wei, Hongxia; Jiang, Wenjun; Liu, Ya; Li, Yi

2015-04-01

Recent advances indicate that cancer‑associated fibroblasts (CAFs) play a key role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors often experience low oxygen tension environments, which induce gene expression changes and biological features leading to poor outcomes. The G-protein estrogen receptor (GPER) exhibits a stimulatory role in diverse types of cancer cells and in CAFs under hypoxic conditions. We investigated the role of CAFs and hypoxia in breast cancer aggressiveness, and examined the effect of GPER in CAFs on hypoxia-driven breast cancer progression. The results showed that hypoxia upregulated HIF-1α, GPER and α-SMA expression in CAFs, and induced the secretion of Interleukin-6 (IL-6), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in CAFs. However, GPER silencing abrogated the above hypoxia-driven cytokine expression in CAFs. Moreover, knockdown of GPER in CAFs suppressed breast cancer cell invasion induced by CAF conditioned media (CM). Furthermore, GPER silencing in CAFs inhibited hypoxia-increased CTGF expression in CAFs and breast cancer cells cultured with CM from CAFs under hypoxic conditions. In addition, CTGF is responsible for the observed effects of GPER on CAFs activation and breast cancer invasion. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression.

Influence of Expression Plasmid of Connective Tissue Growth Factor and Tissue Inhibitor of Metalloproteinase-1 shRNA on Hepatic Precancerous Fibrosis in Rats.

PubMed

Zhang, Qun; Shu, Fu-Li; Jiang, Yu-Feng; Huang, Xin-En

2015-01-01

In this study, influence caused by expression plasmids of connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) short hairpin RNA (shRNA) on mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII in hepatic tissue with hepatic fibrosis, a precancerous condition, in rats is analyzed. To screen and construct shRNA expression plasimid which effectively interferes RNA targets of CTGF and TIMP-1 in rats. 50 cleaning Wistar male rats are allocated randomly at 5 different groups after precancerous fibrosis models and then injection of shRNA expression plasimids. Plasmid psiRNA-GFP-Com (CTGF and TIMP-1 included), psiRNA-GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA- DUO-GFPzeo of blank plasmid are injected at group A, B, C and D, respectively, and as model control group that none plasimid is injected at group E. In 2 weeks after last injection, to hepatic tissue at different groups, protein expression of CTGF, TIMP-1, procol-α1and PC III is tested by immunohistochemical method and,mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII is measured by real-time PCR. One-way ANOVA is used to comparison between-groups. Compared with model group, there is no obvious difference of mRNA expression among CTGF,TIMP-1,procol-α1,PC III and of protein expression among CTGF, TIMP-1, procol-α1, PC III in hepatic tissue at group injected with blank plasmid. Expression quantity of mRNA of CTGF, TIMP-1, procol-α1 and PCIII at group A, B and C decreases, protein expression of CTGF, TIMP-1, procol-α1, PC III in hepatic tissue is lower, where the inhibition of combination RNA interference group (group A) on procol-α1 mRNA transcription and procol-α1 protein expression is superior to that of single interference group (group B and C) (P<0.01 or P<0.05). RNA interference on CTGF and/or TIMP-1 is obviously a inhibiting factor for mRNA and protein expression of CTGF, TIMP-1, procol-α1 and PCIII. Combination RNA interference on genes of CTGF and TIMP-1 is superior

Effects of Antiproteinuric Intervention on Elevated Connective Tissue Growth Factor (CTGF/CCN-2) Plasma and Urine Levels in Nondiabetic Nephropathy

PubMed Central

Slagman, Maartje C.J.; Nguyen, Tri Q.; Waanders, Femke; Vogt, Liffert; Hemmelder, Marc H.; Goldschmeding, Roel; Navis, Gerjan

2011-01-01

Summary Background and objectives Connective Tissue Growth Factor (CTGF/CCN-2) is a key player in fibrosis. Plasma CTGF levels predict end-stage renal disease and mortality in diabetic chronic kidney disease (CKD), supporting roles in intra- and extrarenal fibrosis. Few data are available on CTGF in nondiabetic CKD. We investigated CTGF levels and effects of antiproteinuric interventions in nondiabetic proteinuric CKD. Design, setting, participants, & measurements In a crossover randomized controlled trial, 33 nondiabetic CKD patients (3.2 [2.5 to 4.0] g/24 h proteinuria) were treated during 6-week periods with placebo, ARB (100 mg/d losartan), and ARB plus diuretics (100 mg/d losartan plus 25 mg/d hydrochlorothiazide) combined with consecutively regular and low sodium diets (193 ± 62 versus 93 ± 52 mmol Na+/d). Results CTGF was elevated in plasma (464 [387 to 556] pmol/L) and urine (205 [135 to 311] pmol/24 h) of patients compared with healthy controls (n = 21; 96 [86 to 108] pmol/L and 73 [55 to 98] pmol/24 h). Urinary CTGF was lowered by antiproteinuric intervention, in proportion to the reduction of proteinuria, with normalization during triple therapy (CTGF 99 [67 to 146] in CKD versus 73 [55 to 98] pmol/24 h in controls). In contrast, plasma CTGF was not affected. Conclusions Urinary and plasma CTGF are elevated in nondiabetic CKD. Only urinary CTGF is normalized by antiproteinuric intervention, consistent with amelioration of tubular dysfunction. The lack of effect on plasma CTGF suggests that its driving force might be independent of proteinuria and that short-term antiproteinuric interventions are not sufficient to correct the systemic profibrotic state in CKD. PMID:21784839

SHIP, a novel factor to ameliorate extracellular matrix accumulation via suppressing PI3K/Akt/CTGF signaling in diabetic kidney disease.

PubMed

Li, Fan; Li, Lisha; Cheng, Meijuan; Wang, Xiumin; Hao, Jun; Liu, Shuxia; Duan, Huijun

2017-01-22

Tubular interstitial extracellular matrix accumulation, which plays a key role in the pathogenesis and progression of diabetic kidney disease (DKD), is believed to be mediated by activation of PI3K/Akt signal pathway. However, it is still not clear whether SH2 domain-containing inositol 5'-phosphatase (SHIP), known as a negative regulator of PI3K/Akt pathway is also involved in extracellular matrix metabolism of diabetic kidney. In the present study, decreased SHIP and increased phospho-Akt (Ser 473, Thr 308) were found in renal tubular cells of diabetic mice accompanied by overexpression of connective tissue growth factor (CTGF) and extracellular matrix deposition versus normal mice. Again, high glucose attenuated SHIP expression in a time-dependent manner, concomitant with activation of PI3K/Akt signaling and extracellular matrix production in human renal proximal tubular epithelial cells (HK2) cultured in vitro, which was significantly prevented by transfection of M90-SHIP vector. Furthermore, in vivo delivery of rAd-INPP5D vector (SHIP expression vector) via intraperitoneal injection in diabetic mice increased SHIP expression by 3.36 times followed by 65.26%, 70.38% and 46.71% decreases of phospho-Akt (Ser 473), phospho-Akt (Thr 308) and CTGF expression versus diabetic mice receiving rAd-EGFP vector. Meanwhile, increased renal extracellular matrix accumulation of diabetic mice was also inhibited with intraperitoneal injection of rAd-INPP5D vector. These above data suggested that overexpression of SHIP might be a potent method to lessen renal extracellular matrix accumulation via inactivation of PI3K/Akt pathway and suppression of CTGF expression in DKD. Copyright © 2016 Elsevier Inc. All rights reserved.

Induction of antiproliferative connective tissue growth factor expression in Wilms' tumor cells by sphingosine-1-phosphate receptor 2.

PubMed

Li, Mei-Hong; Sanchez, Teresa; Pappalardo, Anna; Lynch, Kevin R; Hla, Timothy; Ferrer, Fernando

2008-10-01

Connective tissue growth factor (CTGF), a member of the CCN family of secreted matricellular proteins, regulates fibrosis, angiogenesis, cell proliferation, apoptosis, tumor growth, and metastasis. However, the role of CTGF and its regulation mechanism in Wilms' tumor remains largely unknown. We found that the bioactive lipid sphingosine-1-phosphate (S1P) induced CTGF expression in a concentration- and time-dependent manner in a Wilms' tumor cell line (WiT49), whereas FTY720-phosphate, an S1P analogue that binds all S1P receptors except S1P2, did not. Further, the specific S1P2 antagonist JTE-013 completely inhibited S1P-induced CTGF expression, whereas the S1P1 antagonist VPC44116 did not, indicating that this effect was mediated by S1P2. This was confirmed by adenoviral transduction of S1P2 in WiT49 cells, which showed that overexpression of S1P2 increased the expression of CTGF. Induction of CTGF by S1P was sensitive to ROCK inhibitor Y-27632 and c-Jun NH2-terminal kinase inhibitor SP600125, suggesting the requirement of RhoA/ROCK and c-Jun NH2-terminal kinase pathways for S1P-induced CTGF expression. Interestingly, the expression levels of CTGF were decreased in 8 of 10 Wilms' tumor tissues compared with matched normal tissues by quantitative real-time PCR and Western blot analysis. In vitro, human recombinant CTGF significantly inhibited the proliferation of WiT49 cells. In addition, overexpression of CTGF resulted in significant inhibition of WiT49 cell growth. Taken together, these data suggest that CTGF protein induced by S1P2 might act as a growth inhibitor in Wilms' tumor.

Losartan improves resistance artery lesions and prevents CTGF and TGF-beta production in mild hypertensive patients.

PubMed

Gómez-Garre, D; Martín-Ventura, J L; Granados, R; Sancho, T; Torres, R; Ruano, M; García-Puig, J; Egido, J

2006-04-01

Although structural and functional changes of resistance arteries have been proposed to participate in arterial hypertension (HTA) outcome, not all therapies may correct these alterations, even if they normalize the blood pressure (BP). The aim of this study was to investigate the mechanisms of the protection afforded by the angiotensin receptor antagonist losartan in resistance arteries from patients with essential HTA. In all, 22 untreated hypertensive patients were randomized to receive losartan or amlodipine for 1 year and the morphological characteristics of resistance vessels from subcutaneous biopsies were evaluated. Protein expression of connective tissue growth factor (CTGF), transforming growth factor beta (TGF-beta), and collagens III and IV was detected by immunohistochemistry. In comparison with normotensive subjects, resistance arteries from hypertensive patients showed a significant media:lumen (M/L) ratio increment and a higher protein expression of CTGF, TGF-beta, and collagens. After 1 year of treatment, both losartan and amlodipine similarly controlled BP. However, M/L only decreased in patients under losartan treatment, whereas in the amlodipine-treated group this ratio continued to increase significantly. The administration of losartan prevented significant increments in CTGF, TGF-beta, and collagens in resistance arteries. By contrast, amlodipine-treated patients showed a higher vascular CTGF, TGF-beta, and collagen IV staining than before treatment. Our results show that the administration of losartan, but not amlodipine, to hypertensive patients improves structural abnormalities and prevents the production of CTGF and TGF-beta in small arteries, despite similar BP lowering. These data may explain the molecular mechanisms of the better vascular protection afforded by drugs interfering with the renin-angiotensin system.

Curcumin inhibits TGF-β1-induced connective tissue growth factor expression through the interruption of Smad2 signaling in human gingival fibroblasts.

PubMed

Chen, Jung-Tsu; Wang, Chen-Ying; Chen, Min-Huey

2018-01-13

Many fibrotic processes are associated with an increased level of transforming growth factor-β1 (TGF-β1). TGF-β1 can increase synthesis of matrix proteins and enhance secretion of protease inhibitors, resulting in matrix accumulation. Connective tissue growth factor (CTGF) is a downstream profibrotic effector of TGF-β1 and is associated with the fibrosis in several human organs. Curcumin has been applied to reduce matrix accumulation in fibrotic diseases. This study was aimed to evaluate whether curcumin could suppress TGF-β1-induced CTGF expression and its related signaling pathway involving in this inhibitory action in primary human gingival fibroblasts. The differences in CTGF expression among three types of gingival overgrowth and normal gingival tissues were assessed by immunohistochemistry. Gingival fibroblast viability in cultured media with different concentrations of curcumin was studied by MTT assay. The effect of curcumin on TGF-β1-induced CTGF expression in primary human gingival fibroblasts was examined by immunoblotting. Moreover, the proteins involved in TGF-β1 signaling pathways including TGF-β1 receptors and Smad2 were also analyzed by immunoblotting. CTGF was highly expressed in fibroblasts, epithelial cells and some of endothelial cells, smooth muscle cells, and inflammatory cells in phenytoin-induced gingival overgrowth tissues rather than in those of hereditary and inflammatory gingival overgrowth tissues. Moreover, CTGF expression in the epithelial and connective tissue layers was higher in phenytoin-induced gingival overgrowth tissues than in normal gingival tissues. Curcumin was nontoxic and could reduce TGF-β1-induced CTGF expression by attenuating the phosphorylation and nuclear translocation of Smad2. Curcumin can suppress TGF-β1-induced CTGF expression through the interruption of Smad2 signaling. Copyright © 2018. Published by Elsevier B.V.

Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves cell therapy.

PubMed

Morales, Maria Gabriela; Gutierrez, Jaime; Cabello-Verrugio, Claudio; Cabrera, Daniel; Lipson, Kenneth E; Goldschmeding, Roel; Brandan, Enrique

2013-12-15

In Duchenne muscular dystrophy (DMD) and the mdx mouse model, the absence of the cytoskeletal protein dystrophin causes defective anchoring of myofibres to the basal lamina. The resultant myofibre degeneration and necrosis lead to a progressive loss of muscle mass, increased fibrosis and ultimately fatal weakness. Connective tissue growth factor (CTGF/CCN-2) is critically involved in several chronic fibro-degenerative diseases. In DMD, the role of CTGF might extend well beyond replacement fibrosis secondary to loss of muscle fibres, since its overexpression in skeletal muscle could by itself induce a dystrophic phenotype. Using two independent approaches, we here show that mdx mice with reduced CTGF availability do indeed have less severe muscular dystrophy. Mdx mice with hemizygous CTGF deletion (mdx-Ctgf+/-), and mdx mice treated with a neutralizing anti-CTGF monoclonal antibody (FG-3019), performed better in an exercise endurance test, had better muscle strength in isolated muscles and reduced skeletal muscle impairment, apoptotic damage and fibrosis. Transforming growth factor type-β (TGF-β), pERK1/2 and p38 signalling remained unaffected during CTGF suppression. Moreover, both mdx-Ctgf+/- and FG-3019 treated mdx mice had improved grafting upon intramuscular injection of dystrophin-positive satellite cells. These findings reveal the potential of targeting CTGF to reduce disease progression and to improve cell therapy in DMD.

Src is a major signaling component for CTGF induction by TGF-β1 in osteoblasts

PubMed Central

X, Zhang; JA, Arnott; S, Rehman; WG, DeLong; A, Sanjay; FF, Safadi; SN, Popoff

2010-01-01

Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor beta 1(TGF-β1) where it acts as a downstream mediator of TGF-β1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk and Smad signaling for CTGF induction by TGF-β1 in osteoblasts, however the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-β1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-β1. Additionally, inhibiting Src activation prevented Erk activation, Smad 2 & 3 activation and nuclear translocation by TGF-β1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway through directly mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059 it inhibited TGF-β1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) on the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. This data demonstrates that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-β1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. PMID:20432467

The Angio-Fibrotic Switch of VEGF and CTGF in Proliferative Diabetic Retinopathy

PubMed Central

Kuiper, Esther J.; Van Nieuwenhoven, Frans A.; de Smet, Marc D.; van Meurs, Jan C.; Tanck, Michael W.; Oliver, Noelynn; Klaassen, Ingeborg; Van Noorden, Cornelis J. F.; Goldschmeding, Roel; Schlingemann, Reinier O.

2008-01-01

Background In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) cause blindness by neovascularization and subsequent fibrosis, but their relative contribution to both processes is unknown. We hypothesize that the balance between levels of pro-angiogenic VEGF and pro-fibrotic CTGF regulates angiogenesis, the angio-fibrotic switch, and the resulting fibrosis and scarring. Methods/Principal Findings VEGF and CTGF were measured by ELISA in 68 vitreous samples of patients with proliferative DR (PDR, N = 32), macular hole (N = 13) or macular pucker (N = 23) and were related to clinical data, including degree of intra-ocular neovascularization and fibrosis. In addition, clinical cases of PDR (n = 4) were studied before and after pan-retinal photocoagulation and intra-vitreal injections with bevacizumab, an antibody against VEGF. Neovascularization and fibrosis in various degrees occurred almost exclusively in PDR patients. In PDR patients, vitreous CTGF levels were significantly associated with degree of fibrosis and with VEGF levels, but not with neovascularization, whereas VEGF levels were associated only with neovascularization. The ratio of CTGF and VEGF was the strongest predictor of degree of fibrosis. As predicted by these findings, patients with PDR demonstrated a temporary increase in intra-ocular fibrosis after anti-VEGF treatment or laser treatment. Conclusions/Significance CTGF is primarily a pro-fibrotic factor in the eye, and a shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy. PMID:18628999

Inhibition effect of small interfering RNA of connective tissue growth factor on the expression of extracellular matrix molecules in cultured human renal proximal tubular cells.

PubMed

Liu, Yuyuan; Li, Weiwei; Liu, Hong; Peng, Youming; Yang, Qiu; Xiao, Li; Liu, Yinghong; Liu, Fuyou

2014-03-01

In this study, we investigated the effect of small interfering RNA (siRNA) of connective tissue growth factor (CTGF) by pRetro-Super (PRS) retrovirus vector on the expression of CTGF and related extracellular matrix molecules in human renal proximal tubular cells (HKCs) induced by high glucose, to provide help for renal tubulointerstitial fibrosis therapy. HKCs were exposed to d-glucose to observe their dose and time effect, while the mannitol as osmotic control. Retrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect HKC. The expression of CTGF, fibronectin (FN) and collagen-type I (col1) were measured by semi-quantitative RT-PCR and Western blot. In response to high glucose, CTGF expression in HKCs was increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Introduction of PRS-CTGF-siRNA resulted in the significant reduction of CTGF, FN, col1 mRNA (p < 0.01, respectively) and CTGF, col1 protein (p < 0.05, respectively) expression, while PRS void vector group did not have these effects (p > 0.05). CTGF siRNA therapy can effectively reduce the levels of CTGF, FN and col1 induced by high glucose in cultured HKCs, which suggested that it may be a potential therapeutic strategy to prevent the renal interstitial fibrosis in the future.

Decreased expression of connective tissue growth factor in non-small cell lung cancer is associated with clinicopathological variables and can be restored by epigenetic modifiers.

PubMed

Drzewiecka, Hanna; Gałęcki, Bartłomiej; Jarmołowska-Jurczyszyn, Donata; Kluk, Andrzej; Dyszkiewicz, Wojciech; Jagodziński, Paweł P

2016-09-01

Recent studies indicated undisputed contribution of connective tissue growth factor (CTGF) in the development of many cancers, including non-small cell lung cancer (NSCLC). However, the functional role and regulation of CTGF expression during tumorigenesis remain elusive. Our goal was to determine CTGF transcript and protein levels in tumoral and matched control tissues from 98 NSCLC patients, to correlate the results with clinicopathological features and to investigate whether the CTGF expression can be epigenetically regulated in NSCLC. We used quantitative PCR, Western blotting and immunohistochemistry to evaluate CTGF expression in lung cancerous and histopathologically unchanged tissues. We tested the impact of 5-Aza-2'-deoxycytidine (5-dAzaC) and trichostatin A (TSA) on CTGF transcript and protein levels in NSCLC cells (A549, Calu-1). DNA methylation status of the CTGF regulatory region was evaluated by bisulfite sequencing. The influence of 5-dAzaC and TSA on NSCLC cells viability and proliferation was monitored by the trypan blue assay. We found significantly decreased levels of CTGF mRNA and protein (both p < 0.0000001) in cancerous tissues of NSCLC patients. Down-regulation of CTGF occurred regardless of gender in all histological subtypes of NSCLC. Moreover, we showed that 5-dAzaC and TSA were able to restore CTGF mRNA and protein contents in NSCLC cells. However, no methylation within CTGF regulatory region was detected. Both compounds significantly reduced NSCLC cells proliferation. Decreased expression of CTGF is a common feature in NSCLC; however, it can be restored by the chromatin-modifying agents such as 5-dAzaC or TSA and consequently restrain cancer development.

The physiological role of CTGF/CCN2 in zebrafish notochond development and biological analysis of the proximal promoter region.

PubMed

Chiou, Ming-Jyun; Chao, Tsung-Tai; Wu, Jen-Leih; Kuo, Ching-Ming; Chen, Jyh-Yih

2006-10-20

During mouse embryogenesis, CTGF/CCN2 is expressed in zones containing hypertrophic chondroctyes and calcifying cartilage such as long bones, ribs, vertebral column, and phalanges. But in fish, its expression is yet unclear. Development of the vertebrae is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process is highly conserved during evolution. Analysis of 3.2kb of the CTGF/CCN2 proximal promoter sequence revealed a consensus TATAA box, putative AP1, Brn-2, CdxA, C/EBP alpha, C/EBP beta, C-Ets-, delta E, HFH-2, and HSF2 binding sites. Transient expression experiments with a 5'-deletion revealed at least 4 regulatory regions in the zebrafish CTGF/CCN2 gene, 2 with a stimulatory effect on transcription and 2 with an apparent inhibitory effect after IGF-I treatment in the ZFL cell line. To study the promoter-specific expression, we constructed a series of CTGF/CCN2 (3.0-, 2.5-, 2.0-, 1.5-, 1.0-, and 0.4-kb) promoter-driven green fluorescent protein (GFP) fragments encoding the GFP cDNA transgene which was microinjected into zebrafish embryos. Morphological studies of transgenic zebrafish indicated that the CTGF/CCN2 promoter-driven GFP transcripts appeared in the notochord. Targeted knockdown of the CTGF/CCN2 gene by two antisense morpholino oligonucleotides resulted in disruptions to notochord development. From a comparative point of view, this study of the CTGF/CCN2 gene in zebrafish may correlate well with those previously published on the mouse. These molecular results suggest that CTGF/CCN2 plays an important role in notochord development and is required for general embryonic development.

Efficient delivery of connective tissue growth factor shRNA using PAMAM nanoparticles.

PubMed

Huang, Z J; Yi, B; Yuan, H; Yang, G P

2014-08-28

The aim of this study was to detect the anti-fibrosis activity of connective tissue growth factor (CTGF) small hairpin RNA (shRNA) mediated by polyamidoamine dendrimer nanoparticles in rat myocardial cell lines and myocardium. CTGF shRNAs were constructed from inverted oligonucleotides and a polyamidoamine nanoparticle vector was used to transfer shRNA into H9c2 myocardial cells and spontaneously hypertensive rats. The expression of CTGF, transforming growth factor-b1, and laminin were measured by semi-quantitative reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. pCTGF-shRNA significantly reduced CTGF upregulation induced by angiotensin II in H9c2 myocardial cells. The mRNA and protein expression of CTGF and laminin in pCTGF-shRNA-transferred spontaneously hypertensive rats decreased significantly compared to the control group and pHK-shRNA group (P < 0.05). The expression of transforming growth factor-b1 showed no significant difference among the 3 groups (P > 0.05). pCTGF-shRNA mediated by polyamidoamine can be used to successfully reduce myocardial CTGF and laminin expression, suggesting that this system can be used to improve myocardial fibrosis therapy.

Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

PubMed

Gao, Wei; Cai, Liting; Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

2014-01-01

Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

Anti-CTGF Single-Chain Variable Fragment Dimers Inhibit Human Airway Smooth Muscle (ASM) Cell Proliferation by Down-Regulating p-Akt and p-mTOR Levels

PubMed Central

Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

2014-01-01

Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma. PMID:25478966

Increased expression of Interleukin-13 and connective tissue growth factor, and their potential roles during foreign body encapsulation of subcutaneous implants.

PubMed

Ward, W Kenneth; Li, Allen G; Siddiqui, Yasmin; Federiuk, Isaac F; Wang, Xiao-Jing

2008-01-01

The purpose of this study was to better understand whether interleukin-13 (IL-13) and connective tissue growth factor (CTGF) are highly expressed during foreign body encapsulation of subcutaneous devices. Mock biosensors were implanted into rats for three lengths of time (7-, 21- and 48-55 days) to address different stages of the foreign body response. Using quantitative real-time PCR and immunofluorescence, the expression of IL13, CTGF, collagen 1, decorin and fibronectin were measured in this tissue. IL-13, a product of Th2 cells, was highly expressed at all time points, with greatest expression at day 21. The IL-13 expression was paralleled by increased presence of T-cells at all time points. CTGF was also found to be more highly expressed in foreign body tissue than in controls. Collagen and decorin were highly expressed at the middle and later stages. Given the increased expression of IL-13 and CTGF in foreign body tissue, and their roles in other fibrotic disorders, these cytokines may well contribute to the formation of the foreign body capsule. Since the peak gene expression of IL-13 occurred later than the previously-reported TGFbeta expression peak, IL-13 is probably not the major stimulus to TGFbeta expression during foreign body encapsulation and may contribute to fibrosis independently.

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

PubMed

Liang, Yanbin; Li, Chen; Guzman, Victor M; Evinger, Albert J; Protzman, Charles E; Krauss, Achim H-P; Woodward, David F

2003-07-18

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

Expression variations of connective tissue growth factor in pulmonary arteries from smokers with and without chronic obstructive pulmonary disease

PubMed Central

Zhou, Si-jing; Li, Min; Zeng, Da-xiong; Zhu, Zhong-ming; Hu, Xian-Wei; Li, Yong-huai; Wang, Ran; Sun, Geng-yun

2015-01-01

Cigarette smoking contributes to the development of pulmonary hypertension (PH) complicated with chronic obstructive pulmonary disease (COPD), and the pulmonary vascular remodeling, the structural basis of PH, could be attributed to abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs).In this study, morphometrical analysis showed that the pulmonary vessel wall thickness in smoker group and COPD group was significantly greater than in nonsmokers. In addition, we determined the expression patterns of connective tissue growth factor (CTGF) and cyclin D1 in PASMCs harvested from smokers with normal lung function or mild to moderate COPD, finding that the expression levels of CTGF and cyclin D1 were significantly increased in smoker group and COPD group. In vitro experiment showed that the expression of CTGF, cyclin D1 and E2F were significantly increased in human PASMCs (HPASMCs) treated with 2% cigarette smoke extract (CSE), and two CTGF siRNAs with different mRNA hits successfully attenuated the upregulated cyclin D1 and E2F, and significantly restored the CSE-induced proliferation of HPASMCs by causing cell cycle arrest in G0. These findings suggest that CTGF may contribute to the pathogenesis of abnormal proliferation of HPASMCs by promoting the expression of its downstream effectors in smokers with or without COPD. PMID:25708588

Loss of connective tissue growth factor as an unfavorable prognosis factor activates miR-18b by PI3K/AKT/C-Jun and C-Myc and promotes cell growth in nasopharyngeal carcinoma.

PubMed

Yu, X; Zhen, Y; Yang, H; Wang, H; Zhou, Y; Wang, E; Marincola, F M; Mai, C; Chen, Y; Wei, H; Song, Y; Lyu, X; Ye, Y; Cai, L; Wu, Q; Zhao, M; Hua, S; Fu, Q; Zhang, Y; Yao, K; Liu, Z; Li, X; Fang, W

2013-05-16

Connective tissue growth factor (CTGF) has different roles in different types of cancer. However, the involvement and molecular basis of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC) have almost never been reported. In this study, we observed that downregulated CTGF expression was significantly associated with NPC progression and poor prognosis. Knockdown of CTGF markedly elevated the ability of cell proliferation in vivo and in vitro. Subsequently, we discovered that the reduction of CTGF increased the expression of miR-18b, an oncomir-promoting cell proliferation. Further, we discovered that attenuated CTGF-mediated upregulation of miR-18b was dependent on the increased binding of transcription factors Jun proto-oncogene (C-Jun) and v-Myc myelocytomatosis viral oncogene homolog (C-Myc) to miR-18b promoter region via phosphoinositide 3-kinase (PI3K)/AKT pathway. Finally, we further found that miR-18b directly suppressed the expression of CTGF in NPC. In clinical fresh specimens, miR-18b was widely overexpressed and inversely correlated with CTGF expression in NPC. Our studies are the first to demonstrate that reduced CTGF as an unfavorable prognosis factor mediates the activation of miR-18b, an oncomir directly suppresses CTGF expression, by PI3K/AKT/C-Jun and C-Myc and promotes cell growth of NPC.

Members of the Cyr61/CTGF/NOV Protein Family: Emerging Players in Hepatic Progenitor Cell Activation and Intrahepatic Cholangiocarcinoma

PubMed Central

Jorgensen, Marda; Song, Joanna; Zhou, Junmei; Liu, Chen

2016-01-01

Hepatic stem/progenitor cells (HPC) reside quiescently in normal biliary trees and are activated in the form of ductular reactions during severe liver damage when the replicative ability of hepatocytes is inhibited. HPC niches are full of profibrotic stimuli favoring scarring and hepatocarcinogenesis. The Cyr61/CTGF/NOV (CCN) protein family consists of six members, CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3, which function as extracellular signaling modulators to mediate cell-matrix interaction during angiogenesis, wound healing, fibrosis, and tumorigenesis. This study investigated expression patterns of CCN proteins in HPC and cholangiocarcinoma (CCA). Mouse HPC were induced by the biliary toxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Differential expression patterns of CCN proteins were found in HPC from DDC damaged mice and in human CCA tumors. In addition, we utilized reporter mice that carried Ccn2/Ctgf promoter driven GFP and detected strong Ccn2/Ctgf expression in epithelial cell adhesion molecule (EpCAM)+ HPC under normal conditions and in DDC-induced liver damage. Abundant CCN2/CTGF protein was also found in cytokeratin 19 (CK19)+ human HPC that were surrounded by α-smooth muscle actin (α-SMA)+ myofibroblast cells in intrahepatic CCA tumors. These results suggest that CCN proteins, particularly CCN2/CTGF, function in HPC activation and CCA development. PMID:27829832

Transforming growth factor-β (TGF-β) expression is increased in the subsynovial connective tissues of patients with idiopathic carpal tunnel syndrome.

PubMed

Chikenji, Takako; Gingery, Anne; Zhao, Chunfeng; Passe, Sandra M; Ozasa, Yasuhiro; Larson, Dirk; An, Kai-Nan; Amadio, Peter C

2014-01-01

Non-inflammatory fibrosis of the subsynovial connective tissue (SSCT) is a hallmark of carpal tunnel syndrome (CTS). The etiology of this finding and its relationship to the development of CTS remain poorly understood. Recent studies have found that transforming growth factor-β (TGF-β) plays a central role in fibrosis. The purpose of this study was to investigate the expression of TGF-β and connective tissue growth factor (CTGF), a downstream mediator of TGF-β, in the pathogenesis of CTS. We compared SSCT specimens from 26 idiopathic CTS patients with specimens from 10 human cadaver controls with no previous diagnosis of CTS. Immunohistochemistry was performed to determine levels TGF-β1, CTGF, collagen 1(Col1) and collagen 3 (Col3) expression. TGF-β1 (p < 0.01), CTGF (p < 0.01), and Col3 (p < 0.01) were increased in SSCT of CTS patients compared with control tissue. In addition, a strong positive correlation was found between TGF-β1 and CTGF, (R(2) = 0.80, p < 0.01) and a moderate positive correlation between Col3 and TGF-β1 (R(2) = 0.49, p < 0.01). These finding suggest that there is an increased expression of TGF-β and CTGF, a TGF-β regulated protein, and that this TGF-β activation may be responsible for SSCT fibrosis in CTS patients. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Connective tissue growth factor and its regulation: a new element in diabetic glomerulosclerosis.

PubMed

Riser, B L; Cortes, P

2001-01-01

Connective tissue growth factor (CTGF), a member of the closely related CCN family of cytokines appears to be fibrotic in skin. To determine whether CTGF is implicated in diabetic glomerulosclerosis we studied cultured rat mesangial cells (MC) as well as kidney cortex and microdissected glomeruli from obese, diabetic db/db mice and their normal counterparts. Exposure of MC to rhCTGF significantly increased fibronectin and collagen type I secretion. Further, unstimulated MC expressed low levels of CTGF message and secreted minimal amounts of CTGF protein (36-38 kDa). However, exposure to TGF-beta, increased glucose concentrations, or cyclic mechanical strain, all causal factors in glomerulosclerosis, markedly induced the expression of CTGF transcripts. With all but mechanical strain there was a concomitant stimulation of CTGF protein secretion. TGF-beta also induced abundant quantities of a small molecular weight form of CTGF (18 kDa). The induction of CTGF protein by a high glucose concentration was mediated by TGF-beta, since a TGF-beta neutralizing antibody blocked this stimulation. In vivo studies using quantitative RT-PCR demonstrated that while CTGF transcripts were low in the glomeruli of control mice, expression was increased 27-fold after approximately 3.5 months of diabetes. These changes occurred early in diabetic nephropathy when mesangial expansion was mild, and interstitial disease and proteinuria were absent. A substantially reduced elevation of CTGF mRNA (2-fold) observed in whole kidney cortices indicted that the primary alteration of CTGF expression was in the glomerulus. These results suggest that CTGF upregulation is an important factor in the pathogenesis of mesangial matrix accumulation in both diabetic and non-diabetic glomerulosclerosis, acting downstream of TGF-beta.

CONNECTIVE TISSUE GROWTH FACTOR IS A TARGET OF NOTCH SIGNALING IN CELLS OF THE OSTEOBLASTIC LINEAGE

PubMed Central

Canalis, Ernesto; Zanotti, Stefano; Smerdel-Ramoya, Anna

2014-01-01

Connective tissue growth factor (Ctgf) or CCN2 is a protein synthesized by osteoblasts necessary for skeletal homeostasis, although its overexpression inhibits osteogenic signals and bone formation. Ctgf is induced by bone morphogenetic proteins, transforming growth factor β and Wnt; and in the present studies, we explored whether Notch regulated Ctgf expression in osteoblasts. We employed RosaNotch mice, where the Notch intracellular domain (NICD) is expressed following the excision of a STOP cassette, placed between the Rosa26 promoter and NICD. Notch was activated by transduction of adenoviral vectors expressing Cre recombinase (Ad-CMV-Cre). Notch induced Ctgf mRNA levels in a time dependent manner and increased Ctgf heterogeneous nuclear RNA. Notch also destabilized Ctgf mRNA shortening its half-life from 13 h to 3 h. The effect of Notch on Ctgf expression was lost following Rbpjκ downregulation, demonstrating that it was mediated by Notch canonical signaling. However, downregulation of the classic Notch target genes Hes1, Hey1 and Hey2 did not modify the effect of Notch on Ctgf expression. Wild type osteoblasts exposed to immobilized Delta-like 1 displayed enhanced Notch signaling and increased Ctgf expression. In addition to the effects of Notch in vitro, Notch induced Ctgf in vivo, and calvariae and femurs from RosaNotch mice mated with transgenics expressing the Cre recombinase in cells of the osteoblastic lineage exhibited increased expression of Ctgf. In conclusion, Ctgf is a target of Notch canonical signaling in osteoblasts, and may act in concert with Notch to regulate skeletal homeostasis. PMID:24792956

Nanolayered siRNA delivery platforms for local silencing of CTGF reduce cutaneous scar contraction in third-degree burns

PubMed Central

Castleberry, Steven A.; Golberg, Alexander; Sharkh, Malak Abu; Khan, Saiqa; Almquist, Benjamin D.; Austen, William G.; Yarmush, Martin L.; Hammond, Paula T.

2017-01-01

Wound healing is an incredibly complex biological process that often results in thickened collagen-enriched healed tissue called scar. Cutaneous scars lack many functional structures of the skin such as hair follicles, sweat glands, and papillae. The absence of these structures contributes to a number of the long-term morbidities of wound healing, including loss of function for tissues, increased risk of re-injury, and aesthetic complications. Scar formation is a pervasive factor in our daily lives; however, in the case of serious traumatic injury, scars can create long-lasting complications due to contraction and poor tissue remodeling. Within this report we target the expression of connective tissue growth factor (CTGF), a key mediator of TGFβ pro-fibrotic response in cutaneous wound healing, with controlled local delivery of RNA interference. Through this work we describe both a thorough in vitro analysis of nanolayer coated sutures for the controlled delivery of siRNA and its application to improve scar outcomes in a third-degree burn induced scar model in rats. We demonstrate that the knockdown of CTGF significantly altered the local expression of αSMA, TIMP1, and Col1a1, which are known to play roles in scar formation. The knockdown of CTGF within the healing burn wounds resulted in improved tissue remodeling, reduced scar contraction, and the regeneration of papillary structures within the healing tissue. This work adds support to a number of previous reports that indicate CTGF as a potential therapeutic target for fibrosis. Additionally, we believe that the controlled local delivery of siRNA from ultrathin polymer coatings described within this work is a promising approach in RNA interference that could be applied in developing improved cancer therapies, regenerative medicine, and fundamental scientific research. PMID:27108403

Nelumbo nucifera Gaertn leaves extract inhibits the angiogenesis and metastasis of breast cancer cells by downregulation connective tissue growth factor (CTGF) mediated PI3K/AKT/ERK signaling.

PubMed

Chang, Chun-Hua; Ou, Ting-Tsz; Yang, Mon-Yuan; Huang, Chi-Chou; Wang, Chau-Jong

2016-07-21

Nelumbo nucifera Gaertn (Nymphaeaceae) has been recognized as a medicinal plant, which was distributed throughout the Asia. The aqueous extract of Nelumbo nucifera leaves extract (NLE) has various biologically active components such as polyphenols, flavonoids, oligomeric procyanidines. However, the role of NLE in breast cancer therapy is poorly understood. The purpose of this study was to identify the hypothesis that NLE can suppress tumor angiogenesis and metastasis through CTGF (connective tissue growth factor), which has been implicated in tumor angiogenesis and progression in breast cancer MDA-MB-231 cells. We examined the effects of NLE on angiogenesis in the chicken chorioallantoic membrane (CAM) model. The data showed that NLE could reduce the chorionic plexus at day 17 in CAM and the duration of this inhibition was dose-dependent. In Xenograft model, NLE treatment significantly reduced tumor weight and CD31 (capillary density) over control, respectively. We examined the role of angiogenesis involved restructuring of endothelium using human umbilical vein endothelial cell (HUVEC) in Matrigel angiogenesis model. The results indicated that vascular-like structure formation was further blocked by NLE treatment. Moreover, knockdown of CTGF expression markedly reduced the expression of MMP2 as well as VEGF, and attenuated PI3K-AKT-ERK activation, indication that these signaling pathways are crucial in mediating CTGF function. The present results suggest that NLE might be useful for treatment in therapy-resistance triple negative breast cancer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Connective tissue growth factor is a substrate of ADAM28

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mochizuki, Satsuki; Tanaka, Rena; Shimoda, Masayuki

2010-11-26

Research highlights: {yields} The hyper-variable region in the cysteine-rich domain of ADAM28 binds to C-terminal domain of CTGF. {yields} ADAM28 cleaves CTGF alone and CTGF in the CTGF/VEGF{sub 165} complex. {yields} CTGF digestion by ADAM28 releases biologically active VEGF{sub 165} from the complex. {yields} ADAM28, CTGF and VEGF{sub 165} are commonly co-expressed by carcinoma cells in human breast carcinoma tissues. {yields} These suggest that ADAM28 promotes VEGF{sub 165}-induced angiogenesis in the breast carcinomas by selective CTGF digestion in the CTGF/VEGF{sub 165} complex. -- Abstract: ADAM28, a member of the ADAM (a disintegrin and metalloproteinase) gene family, is over-expressed by carcinomamore » cells and the expression correlates with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, information about substrates of ADAM28 is limited. We screened interacting molecules of ADAM28 in human lung cDNA library by yeast two-hybrid system and identified connective tissue growth factor (CTGF). Binding of CTGF to proADAM28 was demonstrated by yeast two-hybrid assay and protein binding assay. ADAM28 cleaved CTGF in dose- and time-dependent manners at the Ala{sup 181}-Tyr{sup 182} and Asp{sup 191}-Pro{sup 192} bonds in the hinge region of the molecule. ADAM28 selectively digested CTGF in the complex of CTGF and vascular endothelial growth factor{sub 165} (VEGF{sub 165}), releasing biologically active VEGF{sub 165} from the complex. RT-PCR and immunohistochemical analyses demonstrated that ADAM28, CTGF and VEGF are commonly co-expressed in the breast carcinoma tissues. These data provide the first evidence that CTGF is a novel substrate of ADAM28 and suggest that ADAM28 may promote VEGF{sub 165}-induced angiogenesis in the breast carcinomas by the CTGF digestion in the CTGF/VEGF{sub 165} complex.« less

Promoter hypermethylation contributes to frequent inactivation of a putative conditional tumor suppressor gene connective tissue growth factor in ovarian cancer.

PubMed

Kikuchi, Ryoko; Tsuda, Hitoshi; Kanai, Yae; Kasamatsu, Takahiro; Sengoku, Kazuo; Hirohashi, Setsuo; Inazawa, Johji; Imoto, Issei

2007-08-01

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

Serial analysis of gene expression identifies connective tissue growth factor expression as a prognostic biomarker in gallbladder cancer.

PubMed

Alvarez, Hector; Corvalan, Alejandro; Roa, Juan C; Argani, Pedram; Murillo, Francisco; Edwards, Jennifer; Beaty, Robert; Feldmann, Georg; Hong, Seung-Mo; Mullendore, Michael; Roa, Ivan; Ibañez, Luis; Pimentel, Fernando; Diaz, Alfonso; Riggins, Gregory J; Maitra, Anirban

2008-05-01

Gallbladder cancer (GBC) is an uncommon neoplasm in the United States, but one with high mortality rates. This malignancy remains largely understudied at the molecular level such that few targeted therapies or predictive biomarkers exist. We built the first series of serial analysis of gene expression (SAGE) libraries from GBC and nonneoplastic gallbladder mucosa, composed of 21-bp long-SAGE tags. SAGE libraries were generated from three stage-matched GBC patients (representing Hispanic/Latino, Native American, and Caucasian ethnicities, respectively) and one histologically alithiasic gallbladder. Real-time quantitative PCR was done on microdissected epithelium from five matched GBC and corresponding nonneoplastic gallbladder mucosa. Immunohistochemical analysis was done on a panel of 182 archival GBC in high-throughput tissue microarray format. SAGE tags corresponding to connective tissue growth factor (CTGF) transcripts were identified as differentially overexpressed in all pairwise comparisons of GBC (P < 0.001). Real-time quantitative PCR confirmed significant overexpression of CTGF transcripts in microdissected primary GBC (P < 0.05), but not in metastatic GBC, compared with nonneoplastic gallbladder epithelium. By immunohistochemistry, 66 of 182 (36%) GBC had high CTGF antigen labeling, which was significantly associated with better survival on univariate analysis (P = 0.0069, log-rank test). An unbiased analysis of the GBC transcriptome by SAGE has identified CTGF expression as a predictive biomarker of favorable prognosis in this malignancy. The SAGE libraries from GBC and nonneoplastic gallbladder mucosa are publicly available at the Cancer Genome Anatomy Project web site and should facilitate much needed research into this lethal neoplasm.

Crucial Role of Mesangial Cell-derived Connective Tissue Growth Factor in a Mouse Model of Anti-Glomerular Basement Membrane Glomerulonephritis

PubMed Central

Toda, Naohiro; Mori, Kiyoshi; Kasahara, Masato; Ishii, Akira; Koga, Kenichi; Ohno, Shoko; Mori, Keita P.; Kato, Yukiko; Osaki, Keisuke; Kuwabara, Takashige; Kojima, Katsutoshi; Taura, Daisuke; Sone, Masakatsu; Matsusaka, Taiji; Nakao, Kazuwa; Mukoyama, Masashi; Yanagita, Motoko; Yokoi, Hideki

2017-01-01

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis. PMID:28191821

Crucial Role of Mesangial Cell-derived Connective Tissue Growth Factor in a Mouse Model of Anti-Glomerular Basement Membrane Glomerulonephritis.

PubMed

Toda, Naohiro; Mori, Kiyoshi; Kasahara, Masato; Ishii, Akira; Koga, Kenichi; Ohno, Shoko; Mori, Keita P; Kato, Yukiko; Osaki, Keisuke; Kuwabara, Takashige; Kojima, Katsutoshi; Taura, Daisuke; Sone, Masakatsu; Matsusaka, Taiji; Nakao, Kazuwa; Mukoyama, Masashi; Yanagita, Motoko; Yokoi, Hideki

2017-02-13

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis.

Advanced Glycation End-Products Induce Connective Tissue Growth Factor-Mediated Renal Fibrosis Predominantly through Transforming Growth Factor β-Independent Pathway

PubMed Central

Zhou, Guihua; Li, Cai; Cai, Lu

2004-01-01

Advanced glycation end-products (AGEs) play a critical role in diabetic nephropathy by stimulating extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF) is a potent inducer of ECM synthesis and increases in the diabetic kidneys. To determine the critical role of CTGF in AGE-induced ECM accumulation leading to diabetic nephropathy, rats were given AGEs by intravenous injection for 6 weeks. AGE treatment induced a significant renal ECM accumulation, as shown by increases in periodic acid-Schiff-positive materials, fibronectin, and type IV collagen (Col IV) accumulation in glomeruli, and a mild renal dysfunction, as shown by increases in urinary volume and protein content. AGE treatment also caused significant increases in renal CTGF and transforming growth factor (TGF)-β1 mRNA and protein expression. Direct exposure of rat mesangial cells to AGEs in vitro significantly induced increases in fibronectin and Col IV production, which could be completely prevented by pretreatment with anti-CTGF antibody. AGE treatment also significantly increased both TGF-β1 and CTGF mRNA expression; however, inhibition of TGF-β1 mRNA expression by shRNA or neutralization of TGF-β1 protein by anti-TGF-β1 antibody did not significantly prevent AGE-increased expression of CTGF mRNA and protein. These results suggest that AGE-induced CTGF expression, predominantly through a TGF-β1-independent pathway, plays a critical role in renal ECM accumulation leading to diabetic nephropathy. PMID:15579446

Topically applied connective tissue growth factor/CCN2 improves diabetic preclinical cutaneous wound healing: potential role for CTGF in human diabetic foot ulcer healing.

PubMed

Henshaw, F R; Boughton, P; Lo, L; McLennan, S V; Twigg, S M

2015-01-01

Topical application of CTGF/CCN2 to rodent diabetic and control wounds was examined. In parallel research, correlation of CTGF wound fluid levels with healing rate in human diabetic foot ulcers was undertaken. Full thickness cutaneous wounds in diabetic and nondiabetic control rats were treated topically with 1 μg rhCTGF or vehicle alone, on 2 consecutive days. Wound healing rate was observed on day 14 and wound sites were examined for breaking strength and granulation tissue. In the human study across 32 subjects, serial CTGF regulation was analyzed longitudinally in postdebridement diabetic wound fluid. CTGF treated diabetic wounds had an accelerated closure rate compared with vehicle treated diabetic wounds. Healed skin withstood more strain before breaking in CTGF treated rat wounds. Granulation tissue from CTGF treatment in diabetic wounds showed collagen IV accumulation compared with nondiabetic animals. Wound α-smooth muscle actin was increased in CTGF treated diabetic wounds compared with untreated diabetic wounds, as was macrophage infiltration. Endogenous wound fluid CTGF protein rate of increase in human diabetic foot ulcers correlated positively with foot ulcer healing rate (r = 0.406; P < 0.001). These data collectively increasingly substantiate a functional role for CTGF in human diabetic foot ulcers.

PLGA-PEG-PLGA microspheres as a delivery vehicle for antisense oligonucleotides to CTGF: Implications on post-surgical peritoneal adhesion prevention

NASA Astrophysics Data System (ADS)

Azeke, John Imuetinyan-Jesu, Jr.

Abdominal adhesions are the aberrant result of peritoneal wound healing commonly associated with surgery and inflammation. A subject of a large number of studies since the first half of the last century, peritoneal adhesion prevention has, for the most part, evaded the scientific community and continues to cost Americans an estimated $2-4 billion annually. It is known that transforming growth factor-beta (TGF-beta) plays a key role in the wound healing cascade; however, suppression of this multifunctional growth factor's activity may have more harmful consequences than can be tolerated. As a result, much attention has fallen on connective tissue growth factor (CTGF), a downstream mediator of TGF-beta's fibrotic action. It has been demonstrated in several in vitro models, that the suppression of CTGF hinders fibroblast proliferation, a necessary condition for fibrosis. Furthermore, antisense oligonucleotides (antisense oligos, AO) to CTGF have been shown to knock down CTGF mRNA levels by specifically hindering the translation of CTGF protein. Antisense technologies have met with a great deal of excitement as a viable means of preventing diseases such as adhesions by hindering protein translation at the mRNA level. However, the great challenge associated with the use of these drugs lies in the short circulation time when administered "naked". Viral delivery systems, although excellent platforms in metabolic studies, are not ideal for diagnostic use because of the inherent danger associated with viral vectors. Microparticles made of biodegradable polymers have therefore presented themselves as a viable means of delivering these drugs to target cells over extended periods. Herein, we present two in vivo studies confirming the up-regulation of TGF-beta protein and CTGF mRNA following injury to the uterine tissues of female rats. We were able to selectively knockdown post-operative CTGF protein levels following surgery, however, our observations led us to conclude that

The Skeletal Site-Specific Role of Connective Tissue Growth Factor in Prenatal Osteogenesis

PubMed Central

Lambi, Alex G.; Pankratz, Talia L.; Mundy, Christina; Gannon, Maureen; Barbe, Mary F.; Richtsmeier, Joan T.; Popoff, Steven N.

2013-01-01

Background Connective tissue growth factor (CTGF/CCN2) is a matricellular protein that is highly expressed during bone development. Mice with global CTGF ablation (knockout, KO) have multiple skeletal dysmorphisms and perinatal lethality. A quantitative analysis of the bone phenotype has not been conducted. Results We demonstrated skeletal site-specific changes in growth plate organization, bone microarchitecture, and shape and gene expression levels in CTGF KO compared with wild-type mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and nonallometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. Dysregulation of the transforming growth factor-β-CTGF axis coupled with unique morphologic traits provides a potential mechanistic explanation for the skull phenotype. Conclusions We present novel data on a skeletal phenotype in CTGF KO mice, in which ablation of CTGF causes site-specific aberrations in bone formation. PMID:23073844

Can insulin-like growth factor 1 (IGF-1), IGF-1 receptor connective tissue growth factor and Ki-67 labelling index have a prognostic role in pulmonary carcinoids?

PubMed

Kanakis, Georgios A; Grimelius, Lars; Papaioannou, Dimitrios; Kaltsas, Gregory; Tsolakis, Apostolos V

2018-04-27

Altered expression of Insulin-like Growth Factor-1 (IGF-1), its receptor (IGF-1R), Connective Tissue Growth Factor (CTGF) and Hypoxia Inducible Factor-1 (HIF-1), has been implicated in tumorigenesis. So far, these factors have not been studied systematically in Pulmonary Carcinoids (PCs). To examine IGF-1, IGF-1R, CTGF and HIF-1 expression in PCs, and assess their prognostic value over established factors. Retrospective study of 121 PCs (104 Typical and 17 Atypical). The expression of growth factors was studied immunohistochemically and tumors were considered positive if immunoreactivity appeared in >50% of cells. All studied parameters were expressed in the majority of tumors (IGF-1, IGF-1R, CTGF and HIF-1, in 78.5%, 67%, 72% and 78%, respectively). Their expression tended to be more frequent in TCs and in tumors with Ki-67≤2% (significant only for HIF-1; 82 vs. 53%; p=0.023 and 83 vs. 63%; p=0.025 respectively). CTGF was the only factor correlated with more extensive disease (larger size; presence of lymph node and distant metastases). According to logistic regression analysis, only advanced age, Ki-67≥3.4% and lymph node involvement could predict the development of distant metastases. IGF-1, IGF-1R, CTGF and HIF-1 are avidly expressed in PCs; however, their presence did not appear to be of statistically significant value over established prognostic factors.

Downregulation of connective tissue growth factor inhibits the growth and invasion of gastric cancer cells and attenuates peritoneal dissemination

PubMed Central

2011-01-01

Background Connective tissue growth factor (CTGF) has been shown to be implicated in tumor development and progression. However, the role of CTGF in gastric cancer remains largely unknown. Results In this study, we showed that CTGF was highly expressed in gastric cancer tissues compared with matched normal gastric tissues. The CTGF expression in tumor tissue was associated with histologic grade, lymph node metastasis and peritoneal dissemination (P < 0.05). Patients with positive CTGF expression had significantly lower cumulative postoperative 5 year survival rate than those with negative CTGF expression (22.9% versus 48.1%, P < 0.001). We demonstrated that knockdown of CTGF expression significantly inhibited cell growth of gastric cancer cells and decreased cyclin D1 expression. Moreover, knockdown of CTGF expression also markedly reduced the migration and invasion of gastric cancer cells and decreased the expression of matrix metalloproteinase (MMP)-2 and MMP-9. Animal studies revealed that nude mice injected with the CTGF knockdown stable cell lines featured a smaller number of peritoneal seeding nodules than the control cell lines. Conclusions These data suggest that CTGF plays an important role in cell growth and invasion in human gastric cancer and it appears to be a potential prognostic marker for patients with gastric cancer. PMID:21955589

Downregulation of connective tissue growth factor inhibits the growth and invasion of gastric cancer cells and attenuates peritoneal dissemination.

PubMed

Jiang, Cheng-Gang; Lv, Ling; Liu, Fu-Rong; Wang, Zhen-Ning; Liu, Fu-Nan; Li, Yan-Shu; Wang, Chun-Yu; Zhang, Hong-Yan; Sun, Zhe; Xu, Hui-Mian

2011-09-28

Connective tissue growth factor (CTGF) has been shown to be implicated in tumor development and progression. However, the role of CTGF in gastric cancer remains largely unknown. In this study, we showed that CTGF was highly expressed in gastric cancer tissues compared with matched normal gastric tissues. The CTGF expression in tumor tissue was associated with histologic grade, lymph node metastasis and peritoneal dissemination (P < 0.05). Patients with positive CTGF expression had significantly lower cumulative postoperative 5 year survival rate than those with negative CTGF expression (22.9% versus 48.1%, P < 0.001). We demonstrated that knockdown of CTGF expression significantly inhibited cell growth of gastric cancer cells and decreased cyclin D1 expression. Moreover, knockdown of CTGF expression also markedly reduced the migration and invasion of gastric cancer cells and decreased the expression of matrix metalloproteinase (MMP)-2 and MMP-9. Animal studies revealed that nude mice injected with the CTGF knockdown stable cell lines featured a smaller number of peritoneal seeding nodules than the control cell lines. These data suggest that CTGF plays an important role in cell growth and invasion in human gastric cancer and it appears to be a potential prognostic marker for patients with gastric cancer.

Connective tissue growth factor mediates TGF-β1-induced low-grade serous ovarian tumor cell apoptosis.

PubMed

Cheng, Jung-Chien; Chang, Hsun-Ming; Leung, Peter C K

2017-10-17

Ovarian low-grade serous carcinoma (LGSC) is a rare disease and is now considered to be a distinct entity from high-grade serous carcinoma (HGSC), which is the most common and malignant form of epithelial ovarian cancer. Connective tissue growth factor (CTGF) is a secreted matricellular protein that has been shown to modulate many biological functions by interacting with multiple molecules in the microenvironment. Increasing evidence indicates that aberrant expression of CTGF is associated with cancer development and progression. Transforming growth factor-β1 (TGF-β1) is a well-known molecule that can strongly up-regulate CTGF expression in different types of normal and cancer cells. Our previous study demonstrated that TGF-β1 induces apoptosis of LGSC cells. However, the effect of TGF-β1 on CTGF expression in LGSC needs to be defined. In addition, whether CTGF mediates TGF-β1-induced LGSC cell apoptosis remains unknown. In the present study, we show that TGF-β1 treatment up-regulates CTGF expression by activating SMAD3 signaling in two human LGSC cell lines. Additionally, siRNA-mediated CTGF knockdown attenuates TGF-β1-induced cell apoptosis. Moreover, our results show that the inhibitory effect of the CTGF knockdown on TGF-β1-induced cell apoptosis is mediated by down-regulating SMAD3 expression. This study demonstrates an important role for CTGF in mediating the pro-apoptotic effects of TGF-β1 on LGCS.

Connective tissue growth factor induces cardiac hypertrophy through Akt signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayata, Nozomi; Fujio, Yasushi; Yamamoto, Yasuhiro

2008-05-30

In the process of cardiac remodeling, connective tissue growth factor (CTGF/CCN2) is secreted from cardiac myocytes. Though CTGF is well known to promote fibroblast proliferation, its pathophysiological effects in cardiac myocytes remain to be elucidated. In this study, we examined the biological effects of CTGF in rat neonatal cardiomyocytes. Cardiac myocytes stimulated with full length CTGF and its C-terminal region peptide showed the increase in cell surface area. Similar to hypertrophic ligands for G-protein coupled receptors, such as endothelin-1, CTGF activated amino acid uptake; however, CTGF-induced hypertrophy is not associated with the increased expression of skeletal actin or BNP, analyzedmore » by Northern-blotting. CTGF treatment activated ERK1/2, p38 MAPK, JNK and Akt. The inhibition of Akt by transducing dominant-negative Akt abrogated CTGF-mediated increase in cell size, while the inhibition of MAP kinases did not affect the cardiac hypertrophy. These findings indicate that CTGF is a novel hypertrophic factor in cardiac myocytes.« less

Role of CTGF in White Matter Development in Tuberous Sclerosis

DTIC Science & Technology

2015-02-01

previously shown to affect CTGF expression. Our preliminary results show that SRF is downregulated in Tsc1 mutant brains and this can be rescued by rapamycin ...expression. Our preliminary results show that SRF is downregulated in Tsc1 mutant brains and this can be rescued by rapamycin treatment suggesting a...on SRF pathway in our previous report, here we show that SRF levels are decreased in vivo in mutant mice, and this can be rescued by rapamycin

Connective tissue growth factor enhances the migration of gastric cancer through downregulation of E-cadherin via the NF-κB pathway.

PubMed

Mao, Zhengfa; Ma, Xiaoyan; Rong, Yefei; Cui, Lei; Wang, Xuqing; Wu, Wenchuan; Zhang, Jianxin; Jin, Dayong

2011-01-01

Local invasion and distant metastasis are difficult problems for surgical intervention and treatment in gastric cancer. Connective tissue growth factor (CTGF/CCN2) was considered to have an important role in this process. In this study, we demonstrated that expression of CTGF was significantly upregulated in clinical tissue samples of gastric carcinoma (GC) samples. Forced expression of CTGF in AGS GC cells promoted their migration in culture and significantly increased tumor metastasis in nude mice, whereas RNA interference-mediated knockdown of CTGF in GC cells significantly inhibited cell migration in vitro. We disclose that CTGF downregulated the expression of E-cadherin through activation of the nuclear factor-κappa B (NF-κB) pathway. The effects of CTGF in GC cells were abolished by dominant negative IκappaB. Collectively, these data reported here demonstrate CTGF could modulate the NF-κappaB pathway and perhaps be a promising therapeutic target for gastric cancer invasion and metastasis. © 2010 Japanese Cancer Association.

TGF-β-independent CTGF induction regulates cell adhesion mediated drug resistance by increasing collagen I in HCC.

PubMed

Song, Yeonhwa; Kim, Jin-Sun; Choi, Eun Kyung; Kim, Joon; Kim, Kang Mo; Seo, Haeng Ran

2017-03-28

Hepatocellular carcinoma (HCC) is resistant to conventional chemotherapeutic agents and remains an unmet medical need. Here, we demonstrate a mechanism of cell adhesion-mediated drug resistance using a variety of HCC spheroid models to overcome environment-mediated drug resistance in HCC. We classified spheroids into two groups, tightly compacted and loosely compacted aggregates, based on investigation of dynamics of spheroid formation. Our results show that compactness of HCC spheroids correlated with fibroblast-like characteristics, collagen 1A1 (COL1A1) content, and capacity for chemoresistance. We also showed that ablation of COL1A1 attenuated not only the capacity for compact-spheroid formation, but also chemoresistance. Generally, connective tissue growth factor (CTGF) acts downstream of transforming growth factor (TGF)-β and promotes collagen I fiber deposition in the tumor microenvironment. Importantly, we found that TGF-β-independent CTGF is upregulated and regulates cell adhesion-mediated drug resistance by inducing COL1A1 in tightly compacted HCC spheroids. Furthermore, losartan, which inhibits collagen I synthesis, impaired the compactness of spheroids via disruption of cell-cell contacts and increased the efficacy of anticancer therapeutics in HCC cell line- and HCC patient-derived tumor spheroids. These results strongly suggest functional roles for CTGF-induced collagen I expression in formation of compact spheroids and in evading anticancer therapies in HCC, and suggest that losartan, administered in combination with conventional chemotherapy, might be an effective treatment for liver cancer.

Connective tissue growth factor is activated by gastrin and involved in gastrin-induced migration and invasion.

PubMed

Bhandari, Sabin; Bakke, Ingunn; Kumar, J; Beisvag, Vidar; Sandvik, Arne K; Thommesen, Liv; Varro, Andrea; Nørsett, Kristin G

2016-06-17

Connective tissue growth factor (CTGF) has been reported in gastric adenocarcinoma and in carcinoid tumors. The aim of this study was to explore a possible link between CTGF and gastrin in gastric epithelial cells and to study the role of CTGF in gastrin induced migration and invasion of AGS-GR cells. The effects of gastrin were studied using RT-qPCR, Western blot and assays for migration and invasion. We report an association between serum gastrin concentrations and CTGF abundancy in the gastric corpus mucosa of hypergastrinemic subjects and mice. We found a higher expression of CTGF in gastric mucosa tissue adjacent to tumor compared to normal control tissue. We showed that gastrin induced expression of CTGF in gastric epithelial AGS-GR cells via MEK, PKC and PKB/AKT pathways. CTGF inhibited gastrin induced migration and invasion of AGS-GR cells. We conclude that CTGF expression is stimulated by gastrin and involved in remodeling of the gastric epithelium. Copyright © 2016 Elsevier Inc. All rights reserved.

Deletion of connective tissue growth factor ameliorates peritoneal fibrosis by inhibiting angiogenesis and inflammation.

PubMed

Toda, Naohiro; Mori, Kiyoshi; Kasahara, Masato; Koga, Kenichi; Ishii, Akira; Mori, Keita P; Osaki, Keisuke; Mukoyama, Masashi; Yanagita, Motoko; Yokoi, Hideki

2018-06-01

Connective tissue growth factor (CTGF/CCN2) regulates the signalling of other growth factors and promotes fibrosis. CTGF is increased in mice and humans with peritoneal fibrosis. Inhibition of CTGF has not been examined as a potential therapeutic target for peritoneal fibrosis because systemic CTGF knockout mice die at the perinatal stage. To study the role of CTGF in peritoneal fibrosis of adult mice, we generated CTGF conditional knockout (cKO) mice by crossing CTGF floxed mice with RosaCreERT2 mice. We administered tamoxifen to Rosa-CTGF cKO mice to delete the CTGF gene throughout the body. We induced peritoneal fibrosis by intraperitoneal injection of chlorhexidine gluconate (CG) in wild-type and Rosa-CTGF cKO mice. Induction of peritoneal fibrosis in wild-type mice increased CTGF expression and produced severe thickening of the peritoneum. In contrast, CG-treated Rosa-CTGF cKO mice exhibited reduced thickening of the peritoneum. Peritoneal equilibration test revealed that the excessive peritoneal small-solute transport in CG-treated wild-type mice was normalized by CTGF deletion. CG-treated Rosa-CTGF cKO mice exhibited a reduced number of αSMA-, Ki67-, CD31- and MAC-2-positive cells in the peritoneum. Analyses of peritoneal mRNA showed that CG-treated Rosa-CTGF cKO mice exhibited reduced expression of Cd68, Acta2 (αSMA), Pecam1 (CD31) and Vegfa. These results indicate that a deficiency of CTGF can reduce peritoneal thickening and help to maintain peritoneal function by reducing angiogenesis and inflammation in peritoneal fibrosis. These results suggest that CTGF plays an important role in the progression of peritoneal fibrosis.

Role of Connective Tissue Growth Factor in the Retinal Vasculature during Development and Ischemia

PubMed Central

Pi, Liya; Xia, Huiming; Liu, Jianwen; Shenoy, Anitha K.; Hauswirth, William W.; Scott, Edward W.

2011-01-01

Purpose. To investigate the function of connective tissue growth factor (CTGF), a matricellular protein of the CCN (Cyr61/CTGF/Nov) family, in retinal vasculature during development and ischemia. Methods. CTGF expression was determined using RT-PCR, immunohistochemistry, and transgenic mice carrying CTGF promoter-driven-GFP. CTGF antibody was intraocularly injected into neonates at postnatal day (P)2, and its effect on retinal angiogenesis was analyzed at P4. Transgenic animals expressing GFP regulated by the glial fibrillary acidic protein promoter were used for astrocyte visualization. Retinal vascular occlusion was introduced by rose Bengal and laser photocoagulation on chimeric mice that were reconstituted with GFP+ bone marrow cells. Vascular repair in response to VEGF-A and CTGF was analyzed. Results. A temporal increase in CTGF at both mRNA and protein levels was observed in the ganglion cell layer and inner nuclear layer during development. Endothelial cells and pericytes were identified as the main cellular sources of CTGF during retinal angiogenesis. CTGF stimulated the migration of astrocytes, retinal endothelial cells, and pericytes in vitro. Inhibition of CTGF by specific antibody affected vascular filopodial extension, growth of the superficial vascular plexus, and astrocyte remodeling. In adult mice, CTGF was prominently expressed in the perivascular cells of arteries. CTGF activated bone marrow-derived perivascular cells and promoted fibrovascular membrane formation in the laser-induced adult retinopathy model. Conclusions. CTGF is expressed in vascular beds and acts on multiple cell types. It is important for vessel growth during early retinal development and promotes the fibrovascular reaction in murine retinal ischemia after laser injury. PMID:21969300

Connective Tissue Growth Factor reporter mice label a subpopulation of mesenchymal progenitor cells that reside in the trabecular bone region.

PubMed

Wang, Wen; Strecker, Sara; Liu, Yaling; Wang, Liping; Assanah, Fayekah; Smith, Spenser; Maye, Peter

2015-02-01

Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously had been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into osteoblast, adipocyte, and stromal cell lineages after transplantation into a parietal bone defect. In vivo examination of CTGF reporter expression in bone tissue sections revealed that it marked cells highly localized to the trabecular bone region and was not expressed in the perichondrium or periosteum. Mesenchymal cells retaining high CTGF reporter expression were adjacent to, but distinct from mature osteoblasts lining bone surfaces and endothelial cells forming the vascular sinuses. Comparison of CTGF and Osterix reporter expression in bone tissue sections indicated an inverse correlation between the strength of CTGF expression and osteoblast maturation. Down-regulation of CTGF reporter expression also occurred during in vitro osteogenic differentiation. Collectively, our studies indicate that CTGF reporter mice selectively identify a subpopulation of bone marrow mesenchymal progenitor cells that reside in the trabecular bone region. Copyright © 2014 Elsevier Inc. All rights reserved.

Connective Tissue Growth Factor Reporter Mice Label a Subpopulation of Mesenchymal Progenitor Cells that Reside in the Trabecular Bone Region

PubMed Central

Wang, Wen; Strecker, Sara; Liu, Yaling; Wang, Liping; Assanah, Fayekah; Smith, Spenser; Maye, Peter

2014-01-01

Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously has been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into osteoblast, adipocyte, and stromal cell lineages after transplantation into a parietal bone defect. In vivo examination of CTGF reporter expression in bone tissue sections revealed it marked cells highly localized to the trabecular bone region and was not expressed in the perichondrium or periosteum. Mesenchymal cells retaining high CTGF reporter expression were adjacent to, but distinct from mature osteoblasts lining bone surfaces and endothelial cells forming the vascular sinuses. Comparison of CTGF and Osterix reporter expression in bone tissue sections indicated an inverse correlation between the strength of CTGF expression and osteoblast maturation. Down-regulation of CTGF reporter expression also occurred during in vitro osteogenic differentiation. Collectively, our studies indicate that CTGF reporter mice selectively identify a subpopulation of bone marrow mesenchymal progenitor cells that reside in the trabecular bone region. PMID:25464947

Connective tissue growth factor acts as a therapeutic agent and predictor for peritoneal carcinomatosis of colorectal cancer.

PubMed

Lin, Been-Ren; Chang, Cheng-Chi; Chen, Robert Jeen-Chen; Jeng, Yung-Ming; Liang, Jin-Tung; Lee, Po-Huang; Chang, King-Jen; Kuo, Min-Liang

2011-05-15

Here, we aimed to investigate the role of connective tissue growth factor (CTGF) in peritoneal carcinomatosis (PC) associated with colorectal cancer (CRC) and to characterize the underlying mechanism of CTGF mediating adhesion. A cohort of 136 CRC patient specimens was analyzed in this study. CRC cell lines were used for in vitro adhesion assay and in vivo peritoneal dissemination experiment. Recombinant CTGF protein treatment, transfection of CTGF expression plasmids, and knockdown of CTGF expression in CRC cells were utilized to evaluate the integrin α5, which served as a target of CTGF in inhibiting peritoneal seeding. The analysis of CRC tissues revealed an inverse correlation between CTGF expression and prevalence of PC. Lower CTGF level in CRC patients was associated with higher peritoneal recurrence rate after surgery. Inducing CTGF expression in cancer cells resulted in decreased incidence of PC and increased rate of mice survival. The mice received intraperitoneal injection of recombinant CTGF protein simultaneously with cancer cells or following tumor formation; in both cases, peritoneal tumor dissemination was found to be effectively inhibited in the mouse model. Functional assay revealed that CTGF significantly decreased the CRC cell adhesion ability, and integrin α5 was confirmed by reverse transcriptase PCR and functional blocking assay as a downstream effector in the CTGF-mediated inhibition of CRC cell adhesion. CTGF acts as a molecular predictor of PC and could be a potential therapeutic target for the chemoprevention and treatment of PC in CRC patients. ©2011 AACR.

Connective tissue growth factor as a novel therapeutic target in high grade serous ovarian cancer.

PubMed

Moran-Jones, Kim; Gloss, Brian S; Murali, Rajmohan; Chang, David K; Colvin, Emily K; Jones, Marc D; Yuen, Samuel; Howell, Viive M; Brown, Laura M; Wong, Carol W; Spong, Suzanne M; Scarlett, Christopher J; Hacker, Neville F; Ghosh, Sue; Mok, Samuel C; Birrer, Michael J; Samimi, Goli

2015-12-29

Ovarian cancer is the most common cause of death among women with gynecologic cancer. We examined molecular profiles of fibroblasts from normal ovary and high-grade serous ovarian tumors to identify novel therapeutic targets involved in tumor progression. We identified 2,300 genes that are significantly differentially expressed in tumor-associated fibroblasts. Fibroblast expression of one of these genes, connective tissue growth factor (CTGF), was confirmed by immunohistochemistry. CTGF protein expression in ovarian tumor fibroblasts significantly correlated with gene expression levels. CTGF is a secreted component of the tumor microenvironment and is being pursued as a therapeutic target in pancreatic cancer. We examined its effect in in vitro and ex vivo ovarian cancer models, and examined associations between CTGF expression and clinico-pathologic characteristics in patients. CTGF promotes migration and peritoneal adhesion of ovarian cancer cells. These effects are abrogated by FG-3019, a human monoclonal antibody against CTGF, currently under clinical investigation as a therapeutic agent. Immunohistochemical analyses of high-grade serous ovarian tumors reveal that the highest level of tumor stromal CTGF expression was correlated with the poorest prognosis. Our findings identify CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in high-grade serous ovarian cancer. These results warrant further studies into the therapeutic efficacy of FG-3019 in high-grade serous ovarian cancer.

Connective tissue growth factor as a novel therapeutic target in high grade serous ovarian cancer

PubMed Central

Moran-Jones, Kim; Gloss, Brian S.; Murali, Rajmohan; Chang, David K.; Colvin, Emily K.; Jones, Marc D.; Yuen, Samuel; Howell, Viive M.; Brown, Laura M.; Wong, Carol W.; Spong, Suzanne M.; Scarlett, Christopher J.; Hacker, Neville F.; Ghosh, Sue; Mok, Samuel C.; Birrer, Michael J.; Samimi, Goli

2015-01-01

Ovarian cancer is the most common cause of death among women with gynecologic cancer. We examined molecular profiles of fibroblasts from normal ovary and high-grade serous ovarian tumors to identify novel therapeutic targets involved in tumor progression. We identified 2,300 genes that are significantly differentially expressed in tumor-associated fibroblasts. Fibroblast expression of one of these genes, connective tissue growth factor (CTGF), was confirmed by immunohistochemistry. CTGF protein expression in ovarian tumor fibroblasts significantly correlated with gene expression levels. CTGF is a secreted component of the tumor microenvironment and is being pursued as a therapeutic target in pancreatic cancer. We examined its effect in in vitro and ex vivo ovarian cancer models, and examined associations between CTGF expression and clinico-pathologic characteristics in patients. CTGF promotes migration and peritoneal adhesion of ovarian cancer cells. These effects are abrogated by FG-3019, a human monoclonal antibody against CTGF, currently under clinical investigation as a therapeutic agent. Immunohistochemical analyses of high-grade serous ovarian tumors reveal that the highest level of tumor stromal CTGF expression was correlated with the poorest prognosis. Our findings identify CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in high-grade serous ovarian cancer. These results warrant further studies into the therapeutic efficacy of FG-3019 in high-grade serous ovarian cancer. PMID:26575166

Induction of chemokine receptor CXCR4 expression by transforming growth factor-β1 in human basal cell carcinoma cells.

PubMed

Chu, Chia-Yu; Sheen, Yi-Shuan; Cha, Shih-Ting; Hu, Yeh-Fang; Tan, Ching-Ting; Chiu, Hsien-Ching; Chang, Cheng-Chi; Chen, Min-Wei; Kuo, Min-Liang; Jee, Shiou-Hwa

2013-11-01

Higher CXCR4 expression enhances basal cell carcinoma (BCC) invasion and angiogenesis. The underlying mechanism of increased CXCR4 expression in invasive BCC is still not well understood. To investigate the mechanisms involved in the regulation of CXCR4 expression in invasive BCC. We used qRT-PCR, RT-PCR, Western blot, and flow cytometric analyses to examine different CXCR4 levels among the clinical samples, co-cultured BCC cells and BCC cells treated with recombinant transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF). Immunohistochemical studies were used to demonstrate the correlation between TGF-β1 and CXCR4 expressions. The signal transduction pathway and transcriptional regulation were confirmed by treatments with chemical inhibitors, neutralizing antibodies, or short interfering RNAs, as well as luciferase reporter activity. Invasive BCC has higher TGF-β1 and CTGF levels compared to non-invasive BCC. Non-contact dermal fibroblasts co-culture with human BCC cells also increases the expression of CXCR4 in BCC cells. Treatment with recombinant human TGF-β1, but not CTGF, enhanced the CXCR4 levels in time- and dose-dependent manners. The protein level and surface expression of CXCR4 in human BCC cells was increased by TGF-β1 treatment. TGF-β1 was intensely expressed in the surrounding fibroblasts of invasive BCC and was positively correlated with the CXCR4 expression of BCC cells. The transcriptional regulation of CXCR4 by TGF-β1 is mediated by its binding to the TGF-β receptor II and phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2)-ETS-1 pathway. TGF-β1 induces upregulation of CXCR4 in human BCC cells by phosphorylation of ERK1/2-ETS-1 pathway. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Prenatal administration of retinoic acid upregulates connective tissue growth factor in the nitrofen CDH model.

PubMed

Ruttenstock, Elke Maria; Doi, Takashi; Dingemann, Jens; Puri, Prem

2011-06-01

Recent studies have suggested that retinoids may be involved in the molecular mechanisms of pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH). Connective tissue growth factor (CTGF) plays a key role in foetal lung development and remodelling during later gestation. CTGF knockout mice exhibit PH with similar characteristics to the human and nitrofen-induced PH. Prenatal administration of retinoic acid (RA) has been shown to stimulate alveologenesis in nitrofen-induced PH. In vitro studies have revealed that RA can induce CTGF gene expression. We hypothesized that pulmonary gene expression of CTGF is downregulated during the later stages of lung development, and that prenatal administration of RA upregulates CTGF in the nitrofen CDH model. Pregnant rats were exposed to either olive oil or nitrofen on day 9 (D9) of gestation. RA was given intraperitoneally on D18, D19 and D20. Foetuses were harvested on D21 and divided into control, CDH, control + RA and CDH + RA group. Pulmonary CTGF gene and protein expression levels were determined using RT-PCR and immunohistochemistry. On D21, CTGF relative mRNA expression levels were significantly downregulated in CDH group compared to controls. After RA treatment, expression levels of CTGF were significantly upregulated in CDH + RA and control + RA compared to the CDH group. Immunohistochemical studies confirmed these results. Downregulation of pulmonary CTGF gene and protein expression during later stages of lung development may interfere with normal alveologenesis in the nitrofen CDH model. Upregulation of CTGF pulmonary gene expression after prenatal RA treatment may promote lung growth by promoting alveologenesis in the nitrofen-induced CDH model.

Connective Tissue Growth Factor Is Involved in Structural Retinal Vascular Changes in Long-Term Experimental Diabetes

PubMed Central

Van Geest, Rob J.; Leeuwis, Jan Willem; Dendooven, Amélie; Pfister, Frederick; Bosch, Klazien; Hoeben, Kees A.; Vogels, Ilse M.C.; Van der Giezen, Dionne M.; Dietrich, Nadine; Hammes, Hans-Peter; Goldschmeding, Roel; Klaassen, Ingeborg; Van Noorden, Cornelis J.F.

2014-01-01

Early retinal vascular changes in the development of diabetic retinopathy (DR) include capillary basal lamina (BL) thickening, pericyte loss and the development of acellular capillaries. Expression of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family member CCN2 or connective tissue growth factor (CTGF), a potent inducer of the expression of BL components, is upregulated early in diabetes. Diabetic mice lacking one functional CTGF allele (CTGF+/−) do not show this BL thickening. As early events in DR may be interrelated, we hypothesized that CTGF plays a role in the pathological changes of retinal capillaries other than BL thickening. We studied the effects of long-term (6-8 months) streptozotocin-induced diabetes on retinal capillary BL thickness, numbers of pericytes and the development of acellular capillaries in wild type and CTGF+/− mice. Our results show that an absence of BL thickening of retinal capillaries in long-term diabetic CTGF+/− mice is associated with reduced pericyte dropout and reduced formation of acellular capillaries. We conclude that CTGF is involved in structural retinal vascular changes in diabetic rodents. Inhibition of CTGF in the eye may therefore be protective against the development of DR. PMID:24217924

Down-regulation of connective tissue growth factor by inhibition of transforming growth factor beta blocks the tumor-stroma cross-talk and tumor progression in hepatocellular carcinoma.

PubMed

Mazzocca, Antonio; Fransvea, Emilia; Dituri, Francesco; Lupo, Luigi; Antonaci, Salvatore; Giannelli, Gianluigi

2010-02-01

Tumor-stroma interactions in hepatocellular carcinoma (HCC) are of key importance to tumor progression. In this study, we show that HCC invasive cells produce high levels of connective tissue growth factor (CTGF) and generate tumors with a high stromal component in a xenograft model. A transforming growth factor beta (TGF-beta) receptor inhibitor, LY2109761, inhibited the synthesis and release of CTGF, as well as reducing the stromal component of the tumors. In addition, the TGF-beta-dependent down-regulation of CTGF diminished tumor growth, intravasation, and metastatic dissemination of HCC cells by inhibiting cancer-associated fibroblast proliferation. By contrast, noninvasive HCC cells were found to produce low levels of CTGF. Upon TGF-beta1 stimulation, noninvasive HCC cells form tumors with a high stromal content and CTGF expression, which is inhibited by treatment with LY2109761. In addition, the acquired intravasation and metastatic spread of noninvasive HCC cells after TGF-beta1 stimulation was blocked by LY2109761. LY2109761 interrupts the cross-talk between cancer cells and cancer-associated fibroblasts, leading to a significant reduction of HCC growth and dissemination. Interestingly, patients with high CTGF expression had poor prognosis, suggesting that treatment aimed at reducing TGF-beta-dependent CTGF expression may offer clinical benefits. Taken together, our preclinical results indicate that LY2109761 targets the cross-talk between HCC and the stroma and provide a rationale for future clinical trials.

Inhibition of connective tissue growth factor overexpression decreases growth of hepatocellular carcinoma cells in vitro and in vivo.

PubMed

Jia, Xiao-Qin; Cheng, Hai-Qing; Li, Hong; Zhu, Yan; Li, Yu-Hua; Feng, Zhen-Qing; Zhang, Jian-Ping

2011-11-01

We have previously found that connective tissue growth factor (CTGF) is highly expressed in a rat model of liver cancer. Here, we examined expression of CTGF in human hepatocellular carcinoma (HCC) cells and its effect on cell growth. Real-time PCR was used to observe expression of CTGF in human HCC cell lines HepG2, SMMC-7721, MHCC-97H and LO2. siRNA for the CTGF gene was designed, synthesized and cloned into a Plk0.1-GFP-SP6 vector to construct a lentivirus-mediated shRNA/CTGF. CTGF mRNA and protein expression in HepG2 cells treated by CTGF-specific shRNA was evaluated by real-time PCR and Western blotting. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to evaluate the growth effect, and a colony formation assay was used for observing clonogenic growth. In vivo, tumor cell proliferation was evaluated in a nude mouse model of xenotransplantation. Statistical significance was determined by t test for comparison between two groups, or analysis of variance (ANOVA) for multiple groups. Immunohistochemical staining of CTGF was seen in 35 of 40 HCC samples (87.5%). CTGF was overexpressed 5-fold in 20 HCC tissues, compared with surrounding non-tumor liver tissue. CTGF mRNA level was 5 - 8-fold higher in HepG2, SMMC-7721 and MHCC-97H than in LO2 cells. This indicated that the inhibition rate of cell growth was 43% after knockdown of CTGF expression (P < 0.05). Soft agar colony formation assay showed that siRNA mediated knockdown of CTGF inhibited colony formation in soft agar of HepG2 cells (P < 0.05). The volume of tumors from CTGF-shRNA-expressing cells only accounted for 35% of the tumors from the scrambled control-infected HepG2 cells (P < 0.05). CTGF was overexpressed in human HCC cells and downregulation of CTGF inhibited HCC growth in vitro and in vivo. Knockdown of CTGF may be a potential therapeutic strategy for treatment of HCC.

Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis

PubMed Central

YANG, ZHIZHOU; SUN, ZHAORUI; LIU, HONGMEI; REN, YI; SHAO, DANBING; ZHANG, WEI; LIN, JINFENG; WOLFRAM, JOY; WANG, FENG; NIE, SHINAN

2015-01-01

It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson’s trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury. PMID:25815693

Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

PubMed

Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

2015-07-01

It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

Connective tissue growth factor regulates adipocyte differentiation of mesenchymal stromal cells and facilitates leukemia bone marrow engraftment

PubMed Central

Battula, V. Lokesh; Chen, Ye; Cabreira, Maria da Graca; Ruvolo, Vivian; Wang, Zhiqiang; Ma, Wencai; Konoplev, Sergej; Shpall, Elizabeth; Lyons, Karen; Strunk, Dirk; Bueso-Ramos, Carlos; Davis, Richard Eric; Konopleva, Marina

2013-01-01

Mesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM) microenvironment. Connective tissue growth factor (CTGF) is highly expressed in MSCs, but its role in the BM stroma is unknown. Therefore, we knocked down (KD) CTGF expression in human BM-derived MSCs by CTGF short hairpin RNA. CTGF KD MSCs exhibited fivefold lower proliferation compared with control MSCs and had markedly fewer S-phase cells. CTGF KD MSCs differentiated into adipocytes at a sixfold higher rate than controls in vitro and in vivo. To study the effect of CTGF on engraftment of leukemia cells into BM, an in vivo model of humanized extramedullary BM (EXM-BM) was developed in NOD/SCID/IL-2rgnull mice. Transplanted Nalm-6 or Molm-13 human leukemia cells engrafted at a threefold higher rate in adipocyte-rich CTGF KD MSC-derived EXM-BM than in control EXM-BM. Leptin was found to be highly expressed in CTGF KD EXM-BM and in BM samples of patients with acute myeloid and acute lymphoblastic leukemia, whereas it was not expressed in normal controls. Given the established role of the leptin receptor in leukemia cells, the data suggest an important role of CTGF in MSC differentiation into adipocytes and of leptin in homing and progression of leukemia. PMID:23741006

Mechanisms of bradykinin-induced expression of connective tissue growth factor and nephrin in podocytes

PubMed Central

Msallem, J. Abou; Chalhoub, H.; Al-Hariri, M.; Saad, L.; Jaffa, M. A.; Ziyadeh, F. N.

2015-01-01

Diabetic nephropathy (DN) is the main cause of morbidity and mortality in diabetes and is characterized by mesangial matrix deposition and podocytopathy, including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the present study, we aimed to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in ROS generation, and this was associated with a significant increase in NADPH oxidase (NOX)1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt, and this effect was inhibited in the presence of NOX1 and Nox4 small interfering (si)RNA. Furthermore, podocytes stimulated with BK resulted in a significant increase in protein and mRNA levels of connective tissue growth factor (CTGF) and, at the same time, a significant decrease in protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies. PMID:26447218

Acellular dermal matrix scaffolds coated with connective tissue growth factor accelerate diabetic wound healing by increasing fibronectin through PKC signalling pathway.

PubMed

Yan, Wenxia; Liu, Hanping; Deng, Xiaoyuan; Jin, Ying; Wang, Ning; Chu, Jing

2018-03-01

The regional injection of connective tissue growth factor (CTGF) for diabetic wound healing requires multiple components and results in a substantial loss of its biological activity. Acellular dermal matrix (ADM) scaffolds are optimal candidates for delivering these factors to local ischaemic environments. In this study, we explored whether CTGF loaded on ADM scaffolds can enhance fibronectin (FN) expression to accelerate diabetic wound healing via the protein kinase C (PKC) signalling pathway. The performance of CTGF and CTGF + PKC inhibitor, which were loaded on ADM scaffolds to treat dorsal skin wounds in streptozotocin-induced diabetic mice, was evaluated with naked ADM as a control. Wound closure showed that ADM scaffolds loaded with CTGF induced greater diabetic wound healing in the early stage of the wound in diabetic mice. Moreover, ADM scaffolds loaded with CTGF obviously increased the expression of FN both at the mRNA and protein levels, whereas the expression of FN was significantly reduced in the inhibitor group. Furthermore, the ADM + CTGF group, which produce FN, obviously promoted alpha-smooth muscle actin and transforming growth factor-beta expression and enhanced neovasculature and collagen synthesis at the wound sites. ADM scaffolds loaded with CTGF + PKC inhibitor delayed diabetic wound healing, indicating that FN expression was mediated by the PKC signalling pathway. Our findings offer new perspectives for the treatment of diabetic wound healing and suggest a rationale for the clinical evaluation of CTGF use in diabetic wound healing. Copyright © 2017 John Wiley & Sons, Ltd.

Decorin alleviated chronic hydrocephalus via inhibiting TGF-β1/Smad/CTGF pathway after subarachnoid hemorrhage in rats.

PubMed

Yan, Hui; Chen, Yujie; Li, Lingyong; Jiang, Jiaode; Wu, Guangyong; Zuo, Yuchun; Zhang, John H; Feng, Hua; Yan, Xiaoxin; Liu, Fei

2016-01-01

Chronic hydrocephalus is one of the severe complications after subarachnoid hemorrhage (SAH). However, there is no efficient treatment for the prevention of chronic hydrocephalus, partially due to poor understanding of underlying pathogenesis, subarachnoid fibrosis. Transforming growth factor-β1(TGF-β1) is a potent fibrogenic factor implicated in wide range of fibrotic diseases. To investigate whether decorin, a natural antagonist for TGF-β1, protects against subarachnoid fibrosis and chronic hydrocephalus after SAH, two-hemorrhage-injection SAH model was conducted in 6-week-old rats. Recombinant human decorin(rhDecorin) (30ug/2ul) was administered before blood injection and on the 10th day after SAH. TGF-β1, p-Smad2/3, connective tissue growth factor (CTGF), collagen I and pro-collagen I c-terminal propeptide were assessed via western blotting, enzyme-linked immunosorbent assay, radioimmunoassay and immunofluorescence. And neurobehavioral tests and Morris water maze were employed to evaluate long-term neurological functions after SAH. We found that SAH induced heightened activation of TGF-β1/Smad/CTGF axis, presenting as a two peak response of TGF-β1 in cerebrospinal fluid, elevation of TGF-β1, p-Smad2/3, CTGF, collagen I in brain parenchyma and pro-collagen I c-terminal propeptide in cerebrospinal fluid, and increased lateral ventricle index. rhDecorin treatment effectively inhibited up-regulation of TGF-β1, p-Smad2/3, CTGF, collagen I and pro-collagen I c-terminal propeptide after SAH. Moreover, rhDecorin treatment significantly reduced lateral ventricular index and incidence of chronic hydrocephalus after SAH. Importantly, rhDecorin improved neurocognitive deficits after SAH. In conclusion, rhDecorin suppresses extracellular matrix accumulation and following subarachnoid fibrosis via inhibiting TGF-β1/Smad/CTGF pathway, preventing development of hydrocephalus and attenuating long-term neurocognitive defects after SAH. Copyright © 2015 Elsevier B

Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

PubMed

Jang, Jun-Ho; Chand, Hitendra S; Bruse, Shannon; Doyle-Eisele, Melanie; Royer, Christopher; McDonald, Jacob; Qualls, Clifford; Klingelhutz, Aloysius J; Lin, Yong; Mallampalli, Rama; Tesfaigzi, Yohannes; Nyunoya, Toru

2017-04-01

The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

Role of CTGF in sensitivity to hyperthermia in ovarian and uterine cancers

DOE PAGES

Hatakeyama, Hiroto; Wu, Sherry Y.; Lyons, Yasmin A.; ...

2016-11-01

Even though hyperthermia is a promising treatment for cancer, the relationship between specific temperatures and clinical benefits and predictors of sensitivity of cancer to hyperthermia is poorly understood. Ovarian and uterine tumors have diverse hyperthermia sensitivities. Integrative analyses of the specific gene signatures and the differences in response to hyperthermia between hyperthermia-sensitive and -resistant cancer cells identified CTGF as a key regulator of sensitivity. CTGF silencing sensitized resistant cells to hyperthermia. CTGF small interfering RNA (siRNA) treatment also sensitized resistant cancers to localized hyperthermia induced by copper sulfide nanoparticles and near-infrared laser in orthotopic ovarian cancer models. Lastly, CTGF silencingmore » aggravated energy stress induced by hyperthermia and enhanced apoptosis of hyperthermia-resistant cancers.« less

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

PubMed

Muñoz, M; Martin, D; Carrocera, S; Alonso-Guervos, M; Mora, M I; Corrales, F J; Peynot, N; Giraud-Delville, C; Duranthon, V; Sandra, O; Gómez, E

2017-10-01

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

The role of tumor cell-derived connective tissue growth factor (CTGF/CCN2) in pancreatic tumor growth.

PubMed

Bennewith, Kevin L; Huang, Xin; Ham, Christine M; Graves, Edward E; Erler, Janine T; Kambham, Neeraja; Feazell, Jonathan; Yang, George P; Koong, Albert; Giaccia, Amato J

2009-02-01

Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2 derived from either tumor cells or stromal cells as it affects the growth of pancreatic tumors is unknown. Using genetic inhibition of CCN2, we have discovered that CCN2 derived from tumor cells is a critical regulator of pancreatic tumor growth. Pancreatic tumor cells derived from CCN2 shRNA-expressing clones showed dramatically reduced growth in soft agar and when implanted s.c. We also observed a role for CCN2 in the growth of pancreatic tumors implanted orthotopically, with tumor volume measurements obtained by positron emission tomography imaging. Mechanistically, CCN2 protects cells from hypoxia-mediated apoptosis, providing an in vivo selection for tumor cells that express high levels of CCN2. We found that CCN2 expression and secretion was increased in hypoxic pancreatic tumor cells in vitro, and we observed colocalization of CCN2 and hypoxia in pancreatic tumor xenografts and clinical pancreatic adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms responsible for pancreatic tumor growth and progression, and also indicate that CCN2 produced by tumor cells represents a viable therapeutic target for the treatment of pancreatic cancer.

Connective tissue growth factor and β-catenin constitute an autocrine loop for activation in rat sarcomatoid mesothelioma.

PubMed

Jiang, Li; Yamashita, Yoriko; Chew, Shan-Hwu; Akatsuka, Shinya; Ukai, Shun; Wang, Shenqi; Nagai, Hirotaka; Okazaki, Yasumasa; Takahashi, Takashi; Toyokuni, Shinya

2014-08-01

Due to the formerly widespread use of asbestos, malignant mesothelioma (MM) is increasingly frequent worldwide. MM is classified into epithelioid (EM), sarcomatoid (SM), and biphasic subtypes. SM is less common than EM but is recognized as the most aggressive type of MM, and these patients have a poor prognosis. To identify genes responsible for the aggressiveness of SM, we induced EM and SM in rats, using asbestos, and compared their transcriptomes. Based on the results, we focused on connective tissue growth factor (Ctgf), whose expression was significantly increased in SM compared with EM; EM itself exhibited an increased expression of Ctgf compared with normal mesothelium. Particularly in SM, Ctgf was a major regulator of MM proliferation and invasion through activation of the β-catenin-TCF-LEF signalling pathway, which is autocrine and formed a positive feedback loop via LRP6 as a receptor for secreted Ctgf. High Ctgf expression also played a role in the epithelial-mesenchymal transition in MM. Furthermore, Ctgf is a novel serum biomarker for both early diagnosis and determining the MM prognosis in rats. These data link Ctgf to SM through the LRP6-GSK3β-β-catenin-TCF-Ctgf autocrine axis and suggest Ctgf as a therapeutic target. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The roles of connective tissue growth factor and integrin-linked kinase in high glucose-induced phenotypic alterations of podocytes.

PubMed

Dai, Hou-Yong; Zheng, Min; Lv, Lin-Li; Tang, Ri-Ning; Ma, Kun-Ling; Liu, Dan; Wu, Min; Liu, Bi-Cheng

2012-01-01

Emerging evidence has suggested that podocytes undergo epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN). Connective tissue growth factor (CTGF) and integrin-linked kinase (ILK) are involved in the progression of DN. However, the underlying mechanisms of EMT are not well understood. The study aimed to investigate the roles of CTGF and ILK in high glucose-induced phenotypic alterations of podocytes and determine whether ILK signaling is downstream of CTGF. The epithelial marker of nephrin and the mesenchymal marker of desmin were investigated by real-time RT-PCR and Western blotting. The results demonstrated that podocytes displayed a spreading, arborized morphology in normal glucose, whereas they had a cobblestone morphology in high glucose conditions, accompanied by decreased nephrin expression and increased desmin expression, suggesting podocytes underwent EMT. In response to high glucose, CTGF and ILK expression in podocytes were increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Furthermore, the inhibition of CTGF with anti-CTGF antibody prevented the phenotypic transition, as demonstrated by the preservation of epithelial morphology, the suppression of high glucose-induced desmin overexpression and the restoration of nephrin. Of note, the upregulation of ILK induced by high glucose was partially blocked by the inhibition of CTGF. In summary, these findings suggested that CTGF and ILK were involved in high glucose-induced phenotypic alterations of podocytes. ILK acted as a downstream kinase of CTGF and high glucose-induced ILK expression might occur through CTGF-dependent and -independent pathways. Copyright © 2011 Wiley Periodicals, Inc.

Connective tissue growth factor confers drug resistance in breast cancer through concomitant up-regulation of Bcl-xL and cIAP1.

PubMed

Wang, Ming-Yang; Chen, Pai-Sheng; Prakash, Ekambaranellore; Hsu, Hsing-Chih; Huang, Hsin-Yi; Lin, Ming-Tsan; Chang, King-Jen; Kuo, Min-Liang

2009-04-15

Connective tissue growth factor (CTGF) expression is elevated in advanced breast cancer and promotes metastasis. Chemotherapy response is only transient in most metastatic diseases. In the present study, we examined whether CTGF expression could confer drug resistance in human breast cancer. In breast cancer patients who received neoadjuvant chemotherapy, CTGF expression was inversely associated with chemotherapy response. Overexpression of CTGF in MCF7 cells (MCF7/CTGF) enhanced clonogenic ability, cell viability, and resistance to apoptosis on exposure to doxorubicin and paclitaxel. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) mitigated this drug resistance capacity. CTGF overexpression resulted in resistance to doxorubicin- and paclitaxel-induced apoptosis by up-regulation of Bcl-xL and cellular inhibitor of apoptosis protein 1 (cIAP1). Knockdown of Bcl-xL or cIAP1 with specific small interfering RNAs abolished the CTGF-mediated resistance to apoptosis induced by the chemotherapeutic agents in MCF7/CTGF cells. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 effectively reversed the resistance to apoptosis as well as the up-regulation of Bcl-xL and cIAP1 in MCF7/CTGF cells. A neutralizing antibody against integrin alpha(v)beta(3) significantly attenuated CTGF-mediated ERK1/2 activation and up-regulation of Bcl-xL and cIAP1, indicating that the integrin alpha(v)beta(3)/ERK1/2 signaling pathway is essential for CTGF functions. The Bcl-xL level also correlated with the CTGF level in breast cancer patients. We also found that a COOH-terminal domain peptide from CTGF could exert activities similar to full-length CTGF, in activation of ERK1/2, up-regulation of Bcl-xL/cIAP1, and resistance to apoptosis. We conclude that CTGF expression could confer resistance to chemotherapeutic agents through augmenting a survival pathway through ERK1/2-dependent Bcl-xL/cIAP1 up-regulation.

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

PubMed Central

Maybin, J.A.; Barcroft, J.; Thiruchelvam, U.; Hirani, N.; Jabbour, H.N.; Critchley, H.O.D.

2012-01-01

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 PGE2, PGF2α or hypoxia (0.5% O2) revealed a significant increase in CTGF mRNA expression (P < 0.01 for all, versus vehicle control). Cells treated simultaneously with PGE2 and hypoxia revealed a synergistic increase in CTGF expression (P < 0.05 versus PGE2 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. PMID:22328559

Supplementation with CTGF, SDF1, NGF, and HGF promotes ovine in vitro oocyte maturation and early embryo development.

PubMed

Wang, D H; Ren, J; Zhou, C J; Han, Z; Wang, L; Liang, C G

2018-05-17

The strategies for improving the in vitro maturation (IVM) of domestic animal oocytes focus on promoting nuclear and cytoplasmic maturation. The identification of paracrine factors and their supplementation in the culture medium represent effective approaches for oocyte maturation and embryo development. This study investigated the effects of paracrine factor supplementation including connective tissue growth factor (CTGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and stromal derived factor 1 (SDF1) on ovine oocytes and early parthenogenetic embryos using an in vitro culture system. First, we identified the optimal concentrations of CTGF (30 ng/mL), SDF1 (10 ng/mL), NGF (3 ng/mL), and HGF (100 ng/mL) for promoting oocyte maturation, which combined, induced nuclear maturation in 94.19% of oocytes. This combination also promoted cumulus cell expansion and inhibited oocyte/cumulus apoptosis, while enabling a larger proportion (33.04%) of embryos to develop into blastocysts than in the controls and prevented embryo apoptosis. These novel findings demonstrate that the paracrine factors CTGF, SDF1, NGF, and HGF facilitate ovine oocyte and early parthenogenetic embryo development in vitro. Thus, supplementation with these factors may help optimize the IVM of ovine oocytes and early parthenogenetic embryo development strategies. Copyright © 2018 Elsevier Inc. All rights reserved.

miR-133b Regulation of Connective Tissue Growth Factor

PubMed Central

Gjymishka, Altin; Pi, Liya; Oh, Seh-Hoon; Jorgensen, Marda; Liu, Chen; Protopapadakis, Yianni; Patel, Ashnee; Petersen, Bryon E.

2017-01-01

miRNAs are involved in liver regeneration, and their expression is dysregulated in hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF), a direct target of miR-133b, is crucial in the ductular reaction (DR)/oval cell (OC) response for generating new hepatocyte lineages during liver injury in the context of hepatotoxin-inhibited hepatocyte proliferation. Herein, we investigate whether miR-133b regulation of CTGF influences HCC cell proliferation and migration, and DR/OC response. We analyzed miR-133b expression and found it to be down-regulated in HCC patient samples and induced in the rat DR/OC activation model of 2-acetylaminofluorene with partial hepatectomy. Furthermore, overexpression of miR-133b via adenoviral system in vitro led to decreased CTGF expression and reduced proliferation and Transwell migration of both HepG2 HCC cells and WBF-344 rat OCs. In vivo, overexpression of miR-133b in DR/OC activation models of 2-acetylaminofluorene with partial hepatectomy in rats, and 3,5-diethoxycarbonyl-1,4-dihydrocollidine in mice, led to down-regulation of CTGF expression and OC proliferation. Collectively, these results show that miR-133b regulation of CTGF is a novel mechanism critical for the proliferation and migration of HCC cells and OC response. PMID:26945106

Fell-Muir lecture: connective tissue growth factor (CCN2) – a pernicious and pleiotropic player in the development of kidney fibrosis

PubMed Central

Mason, Roger M

2013-01-01

Connective tissue growth factor (CTGF, CCN2) is a member of the CCN family of matricellular proteins. It interacts with many other proteins, including plasma membrane proteins, modulating cell function. It is expressed at low levels in normal adult kidney cells but is increased in kidney diseases, playing important roles in inflammation and in the development of glomerular and interstitial fibrosis in chronic disease. This review reports the evidence for its expression in human and animal models of chronic kidney disease and summarizes data showing that anti-CTGF therapy can successfully attenuate fibrotic changes in several such models, suggesting that therapies targeting CTGF and events downstream of it in renal cells may be useful for the treatment of human kidney fibrosis. Connective tissue growth factor stimulates the development of fibrosis in the kidney in many ways including activating cells to increase extracellular matrix synthesis, inducing cell cycle arrest and hypertrophy, and prolonging survival of activated cells. The relationship between CTGF and the pro-fibrotic factor TGFβ is examined and mechanisms by which CTGF promotes signalling by the latter are discussed. No specific cellular receptors for CTGF have been discovered but it interacts with and activates several plasma membrane proteins including low-density lipoprotein receptor-related protein (LRP)-1, LRP-6, tropomyosin-related kinase A, integrins and heparan sulphate proteoglycans. Intracellular signalling and downstream events triggered by such interactions are reviewed. Finally, the relationships between CTGF and several anti-fibrotic factors, such as bone morphogenetic factor-4 (BMP4), BMP7, hepatocyte growth factor, CCN3 and Oncostatin M, are discussed. These may determine whether injured tissue heals or progresses to fibrosis. PMID:23110747

Connective tissue growth factor activates pluripotency genes and mesenchymal-epithelial transition in head and neck cancer cells.

PubMed

Chang, Cheng-Chi; Hsu, Wen-Hao; Wang, Chen-Chien; Chou, Chun-Hung; Kuo, Mark Yen-Ping; Lin, Been-Ren; Chen, Szu-Ta; Tai, Shyh-Kuan; Kuo, Min-Liang; Yang, Muh-Hwa

2013-07-01

The epithelial-mesenchymal transition (EMT) is a key mechanism in both embryonic development and cancer metastasis. The EMT introduces stem-like properties to cancer cells. However, during somatic cell reprogramming, mesenchymal-epithelial transition (MET), the reverse process of EMT, is a crucial step toward pluripotency. Connective tissue growth factor (CTGF) is a multifunctional secreted protein that acts as either an oncoprotein or a tumor suppressor among different cancers. Here, we show that in head and neck squamous cell carcinoma (HNSCC), CTGF promotes the MET and reduces invasiveness. Moreover, we found that CTGF enhances the stem-like properties of HNSCC cells and increases the expression of multiple pluripotency genes. Mechanistic studies showed that CTGF induces c-Jun expression through αvβ3 integrin and that c-Jun directly activates the transcription of the pluripotency genes NANOG, SOX2, and POU5F1. Knockdown of CTGF in TW2.6 cells was shown to reduce tumor formation and attenuate E-cadherin expression in xenotransplanted tumors. In HNSCC patient samples, CTGF expression was positively correlated with the levels of CDH1, NANOG, SOX2, and POU5F1. Coexpression of CTGF and the pluripotency genes was found to be associated with a worse prognosis. These findings are valuable in elucidating the interplay between epithelial plasticity and stem-like properties during cancer progression and provide useful information for developing a novel classification system and therapeutic strategies for HNSCC. ©2013 AACR.

Hypoxia enhances the interaction between pancreatic stellate cells and cancer cells via increased secretion of connective tissue growth factor.

PubMed

Eguchi, Daiki; Ikenaga, Naoki; Ohuchida, Kenoki; Kozono, Shingo; Cui, Lin; Fujiwara, Kenji; Fujino, Minoru; Ohtsuka, Takao; Mizumoto, Kazuhiro; Tanaka, Masao

2013-05-01

Pancreatic cancer (PC), a hypovascular tumor, thrives under hypoxic conditions. Pancreatic stellate cells (PSCs) promote PC progression by secreting soluble factors, but their functions in hypoxia are poorly understood. This study aimed to clarify the effects of hypoxic conditions on the interaction between PC cells and PSCs. We isolated human PSCs from fresh pancreatic ductal adenocarcinomas and analyzed functional differences in PSCs between normoxia (21% O2) and hypoxia (1% O2), including expression of various factors related to tumor-stromal interactions. We particularly analyzed effects on PC invasiveness of an overexpressed molecule-connective tissue growth factor (CTGF)-in PSCs under hypoxic conditions, using RNA interference techniques. Conditioned media from hypoxic PSCs enhanced PC cell invasiveness more intensely than that from normoxic PSCs (P < 0.01). When co-cultured with PSCs, PC cell invasion was more enhanced under hypoxia than under normoxia (P < 0.05). Among various soluble factors, which were related to invasiveness, CTGF was one of the overexpressed molecules in hypoxic PSCs. A higher level of CTGF expression was also found in supernatant of hypoxic PSCs than in supernatant of normoxic PSCs. PC cell invasiveness was reduced by CTGF knockdown in hypoxic PSCs co-cultured with PC cells (P < 0.05). Hypoxia induces PSCs' secretion of CTGF, leading to enhancement of PC invasiveness. CTGF derived from hypoxia-stimulated PSCs may be a new therapeutic target for pancreatic cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Transforming Growth Factor β1 Promotes Migration and Invasion of Human Hepatocellular Carcinoma Cells Via Up-Regulation of Connective Tissue Growth Factor.

PubMed

Liu, Haizhou; Wang, Shaoyang; Ma, Weimin; Lu, Youguang

2015-12-01

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a poor patient survival. Expression of TGF-β1 is up-regulated in HCC and is thought to play a crucial role in the occurrence and development of HCC. However, the mechanism of TGF-β1-mediated facilitation of malignant growth and invasion remains unclear, although some previous studies highlighted a potential involvement of the connective tissue growth factor (CTGF). Here we demonstrate that the in vitro migration of the HCC cell line SMMC-7721 is increased in the presence of recombinant TGF-β1, and that this effect is reversed by the specific inhibitor SB431542. Furthermore, TGF-β1 treatment up-regulated the expression of its own mRNA as well as the expression of CTGF mRNA. The TGF-β1-stimulated migration of SMMC-7721 cells was diminished by siRNA silencing of CTGF. These in vitro observations were validated in a murine xenograft model. In particular, silencing of CTFG diminished the TGF-β1-induced tumorigenesis in experimental animals. In conclusion, TGF-β1 plays a critical role in HCC migration and invasion, and this effect is dependent on CTGF.

MicroRNA‑133b inhibits connective tissue growth factor in colorectal cancer and correlates with the clinical stage of the disease.

PubMed

Guo, Yihang; Li, Xiaorong; Lin, Changwei; Zhang, Yi; Hu, Gui; Zhou, Jianyu; Du, Juan; Gao, Kai; Gan, Yi; Deng, Hao

2015-04-01

Accumulating evidence indicates that dysregulation of microRNA‑133b (miR‑133b) is an important step in the development of certain types of human cancer and contributes to tumorigenesis. Altered expression of miR‑133b has been reported in colon carcinoma, but its association with clinical stage in colorectal cancer (CRC) has remained elusive. Connective tissue growth factor (CTGF), a potentially promising candidate gene for interaction with miR‑133b, was screened using microarray analysis. The expression of miR‑133b and CTGF was evaluated using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The regulatory effects of miR‑133b on CTGF were evaluated using a dual‑luciferase reporter assay. CTGF was identified as a functional target of miR‑133b. The results demonstrated low expression of miR‑133b in CRC specimens with poor cell differentiation (P=0.011), lymph node metastasis (P=0.037) and advanced clinical stages (stage III or IV vs. I or II; P=0.036). Furthermore, there was a significant association between a high level of expression of CTGF mRNA and an advanced clinical stage (stage III or IV vs. I or II; P=0.015) and lymph node metastasis (P=0.034). CTGF expression was negatively regulated by miR‑133b in the human colorectum, suggesting that miR‑133b and CTGF may be candidate therapeutic targets in colorectal cancer.

Connective tissue growth factor inhibits gastric cancer peritoneal metastasis by blocking integrin α3β1-dependent adhesion.

PubMed

Chen, Chiung-Nien; Chang, Cheng-Chi; Lai, Hong-Shiee; Jeng, Yung-Ming; Chen, Chia-I; Chang, King-Jeng; Lee, Po-Huang; Lee, Hsinyu

2015-07-01

Connective tissue growth factor (CTGF) plays important roles in normal and pathological conditions. The aim of this study was to investigate the role of CTGF in peritoneal metastasis as well as the underlying mechanism in gastric cancer progression. CTGF expression levels for wild-type and stable overexpression clones were determined by Western blotting and quantitative polymerase chain reaction (Q-PCR). Univariate and multivariate analyses, immunohistochemistry, and survival probability analyses were performed on gastric cancer patients. The extracellular matrix components involved in CTGF-regulated adhesion were determined. Recombinant CTGF was added to cells or coinoculated with gastric cancer cells into mice to evaluate its therapeutic potential. CTGF overexpression and treatment with the recombinant protein significantly inhibited cell adhesion. In vivo peritoneal metastasis demonstrated that CTGF-stable transfectants markedly decreased the number and size of tumor nodules in the mesentery. Statistical analysis of gastric cancer patient data showed that patients expressing higher CTGF levels had earlier TNM staging and a higher survival probability after the surgery. Integrin α3β1 was the cell adhesion molecule mediating gastric cancer cell adhesion to laminin, and blocking of integrin α3β1 prevented gastric cancer cell adhesion to recombinant CTGF. Coimmunoprecipitation results indicated that CTGF binds to integrin α3. Coinoculation of recombinant CTGF and gastric cancer cell lines in mice showed effective inhibition of peritoneal dissemination. Our results suggested that gastric cancer peritoneal metastasis is mediated through integrin α3β1 binding to laminin, and CTGF effectively blocks the interaction by binding to integrin α3β1, thus demonstrating the therapeutic potential of recombinant CTGF in gastric cancer patients.

MicroRNA-145 is downregulated in glial tumors and regulates glioma cell migration by targeting connective tissue growth factor.

PubMed

Lee, Hae Kyung; Bier, Ariel; Cazacu, Simona; Finniss, Susan; Xiang, Cunli; Twito, Hodaya; Poisson, Laila M; Mikkelsen, Tom; Slavin, Shimon; Jacoby, Elad; Yalon, Michal; Toren, Amos; Rempel, Sandra A; Brodie, Chaya

2013-01-01

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3'-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3'-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.

HBX Protein-Induced Downregulation of microRNA-18a is Responsible for Upregulation of Connective Tissue Growth Factor in HBV Infection-Associated Hepatocarcinoma.

PubMed

Liu, Xiaomin; Zhang, Yingjian; Wang, Ping; Wang, Hongyun; Su, Huanhuan; Zhou, Xin; Zhang, Lamei

2016-07-16

BACKGROUND This study was designed to improve our understanding of the role of miR-18a and its target (connective tissue growth factor (CTGF), which are mediators in HBX-induced hepatocellular carcinoma (HCC). MATERIAL AND METHODS We first investigated the expression of several candidate microRNAs (miRNAs) reported to have been aberrantly expressed between HepG2 and HepG2.2.15, which is characterized by stable HBV infection, while the CTGF is identified as a target of miR-18a. Furthermore, the expression of CTGF evaluated in HepG2 was transfected with HBX, while the HepG2.2.15 was transfected with miR-18a and CTGF siRNA. We examined the cell cycle at the same time. RESULTS We found that the expression of miR-18a was abnormally reduced in the HBV-positive HCC tissue samples compared with HBV-negative HCC samples. Through the use of a luciferase reporter system, we also identified CTGF 3'UTR (1046-1052 bp) as the exact binding site for miR-18a. We also observed a clear increase in CTGF mRNA and protein expression levels in HBV-positive HCC human tissue samples in comparison with the HBV-negative controls, indicating a possible negatively associated relationship between miR-18a and CTGF. Furthermore, we investigated the effect of HBX overexpression on miR-18a and CTGF, as well as the viability and cell cycle status of HepG2 cells. In addition, we found that HBX introduction downregulated miR-18a, upregulated CTGF, elevated the viability, and promoted cell cycle progression. We transfected HepG2.2.15 with miR-18a mimics and CTGF siRNA, finding that upregulated miR-18a and downregulated CTGF suppress the viability and cause cell cycle arrest. CONCLUSIONS Our study shows the role of the CTGF gene as a target of miR-18a, and identifies the function of HBV/HBX/miR-18a/CTGF as a key signaling pathway mediating HBV infection-induced HCC.

[The role of transforming growth factor-β1/connective tissue growth factor signaling pathway in paraquat-induced pulmonary fibrosis].

PubMed

Li, H H; Cai, Q; Wang, Y P; Liu, H R; Huang, M

2016-07-20

Objective: To investigate the effects of Paraquat on human embryonic lung fibroblasts (MRC5) and explore the role of transforming growth factor-β 1 /connective tissue growth factor signaling pathway in paraquat-induced pulmonary fibrosis. Methods: MRC5 cells were cultured with different concentration of PQ (0, 12.5, 25, 50, 100, 200, 400 μmol/L) for 24 h. The viability of cells was measured by MTT. The protein level of TGF-β 1 were analyzed by ELISA after PQ treatment (0, 25, 50, 100 μmol/L) . To examine whether TGF-β 1 /CTGF signaling pathway was involved in paraquat-induced cytotoxicity, cells was divided into 6 groups: (1) control; (2) 25 μmol/L PQ group; (3) 50 μmol/L PQ group; (4) 100 μmol/L PQ group; (5) TGF-β 1 positive control group (50 μmol/L rhTGF-β 1 ) ; (6) stimulate group (100 μmol/L PQ+50 μmol/L TGF-β 1 ) . The protein levels of p-Smad2, p-Smad3 and CTGF were assayed by western blot. The mRNA level of CTGF was assayed by real time RT-PCR. Results: MTT showed that cell viability decreased with increasing PQ concentration ( P <0.05) . The protein expression of TGF-β 1 treated with PQ (25, 50, 100 μmol/L) significantly increased compared with control in a dose-independent manner ( P <0.05) . Exposure to PQ (25, 50, 100 μmol/L) induced increase of protein levels of p-Smad2 and p-Smad3. Noteworthy, the expression of p-Smad2 and p-Smad3 were dramatically increased following PQ plus TGF-β 1 stimulation ( P <0.05) . Exposure to PQ (50, 100μmol/L) induced increase of CTGF protein expression and similar greatly increase following PQ plus TGF-β 1 stimulation ( P <0.05) . Real time RT-PCR showed CTGF mRNA in all groups also significantly up-regulated compared with control ( P <0.05) . Conclusion: TGF-β 1 regulates the expression of target gene CTGF to exhibit its pro-fibrogenic effects by activating TGF-β 1 /Smad signaling pathway in PQ-induced pulmonary fibrosis.

TAZ Expression as a Prognostic Indicator in Colorectal Cancer

PubMed Central

Tham, Jill M.; Zhang, Xiaoqian; Zeng, Qi; Zhang, Shu-Dong; Hong, WanJin

2013-01-01

The Hippo pathway restricts the activity of transcriptional coactivators TAZ (WWTR1) and YAP. TAZ and YAP are reported to be overexpressed in various cancers, however, their prognostic significance in colorectal cancers remains unstudied. The expression levels of TAZ and YAP, and their downstream transcriptional targets, AXL and CTGF, were extracted from two independent colon cancer patient datasets available in the Gene Expression Omnibus database, totaling 522 patients. We found that mRNA expressions of both TAZ and YAP were positively correlated with those of AXL and CTGF (p<0.05). High level mRNA expression of TAZ, AXL or CTGF significantly correlated with shorter survival. Importantly, patients co-overexpressing all 3 genes had a significantly shorter survival time, and combinatorial expression of these 3 genes was an independent predictor for survival. The downstream target genes for TAZ-AXL-CTGF overexpression were identified by Java application MyStats. Interestingly, genes that are associated with colon cancer progression (ANTXR1, EFEMP2, SULF1, TAGLN, VCAN, ZEB1 and ZEB2) were upregulated in patients co-overexpressing TAZ-AXL-CTGF. This TAZ-AXL-CTGF gene expression signature (GES) was then applied to Connectivity Map to identify small molecules that could potentially be utilized to reverse this GES. Of the top 20 small molecules identified by connectivity map, amiloride (a potassium sparing diuretic,) and tretinoin (all-trans retinoic acid) have shown therapeutic promise in inhibition of colon cancer cell growth. Using MyStats, we found that low level expression of either ANO1 or SQLE were associated with a better prognosis in patients who co-overexpressed TAZ-AXL-CTGF, and that ANO1 was an independent predictor of survival together with TAZ-AXL-CTGF. Finally, we confirmed that TAZ regulates Axl, and plays an important role in clonogenicity and non-adherent growth in vitro and tumor formation in vivo. These data suggest that TAZ could be a therapeutic

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuzaki, Shinichi; Ishizuka, Tamotsu, E-mail: tamotsui@showa.gunma-u.ac.jp; Yamada, Hidenori

Highlights: {yields} The involvement of extracellular acidification in airway remodeling was investigated. {yields} Extracellular acidification alone induced CTGF production in human ASMCs. {yields} Extracellular acidification enhanced TGF-{beta}-induced CTGF production in human ASMCs. {yields} Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. {yields} OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connectivemore » tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-{beta}-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G{sub q/11} protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP{sub 3}) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G{sub q/11} protein and inositol-1,4,5-trisphosphate-induced Ca{sup 2+} mobilization in human ASMCs.« less

miR-221 stimulates breast cancer cells and cancer-associated fibroblasts (CAFs) through selective interference with the A20/c-Rel/CTGF signaling.

PubMed

Santolla, Maria Francesca; Lappano, Rosamaria; Cirillo, Francesca; Rigiracciolo, Damiano Cosimo; Sebastiani, Anna; Abonante, Sergio; Tassone, Pierfrancesco; Tagliaferri, Pierosandro; Di Martino, Maria Teresa; Maggiolini, Marcello; Vivacqua, Adele

2018-05-02

MicroRNA (miRNAs) are non-coding small RNA molecules that regulate gene expression by inhibiting the translation of target mRNAs. Among several dysregulated miRNAs in human cancer, the up-regulation of miR-221 has been associated with development of a variety of hematologic and solid malignancies. In this study, we investigated the involvement of miR-221 in breast cancer. TaqMan microRNA assay was used to detect the miR-221 levels in normal cells and in MDA-MB 231 and SkBr3 breast cancer cells as well as in main players of the tumor microenvironment, namely cancer-associated fibroblasts (CAFs). miR-221 mimic sequence and locked nucleic acid (LNA)-i-miR-221 construct were used to induce or inhibit, respectively, the miR-221 expression in cells used. Quantitative PCR and western blotting analysis were performed to evaluate the levels of the miR-221 target gene A20 (TNFAIP3), as well as the member of the NF-kB complex namely c-Rel and the connective tissue growth factor (CTGF). Chromatin immunoprecipitation (ChIP) assay was performed to ascertain the recruitment of c-Rel to the CTFG promoter. Finally, the cell growth and migration in the presence of LNA-i-miR-221 or silencing c-Rel and CTGF by specific short hairpin were assessed by cell count, colony formation and boyden chambers assays. Statistical analysis was performed by ANOVA. We first demonstrated that LNA-i-miR-221 inhibits both endogenous and ectopic expression of miR-221 in our experimental models. Next, we found that the A20 down-regulation, as well as the up-regulation of c-Rel induced by miR-221 were no longer evident using LNA-i-miR-221. Moreover, we established that the miR-221 dependent recruitment of c-Rel to the NF-kB binding site located within the CTGF promoter region is prevented by using LNA-i-miR-221. Furthermore, we determined that the up-regulation of CTGF mRNA and protein levels by miR-221 is no longer evident using LNA-i-miR221 and silencing c-Rel. Finally, we assessed that cell growth and

Intermittent hypoxia causes histological kidney damage and increases growth factor expression in a mouse model of obstructive sleep apnea

PubMed Central

Ayas, Najib T.

2018-01-01

Epidemiological studies demonstrate an association between obstructive sleep apnea (OSA) and accelerated loss of kidney function. It is unclear whether the decline in function is due to OSA per se or to other confounding factors such as obesity. In addition, the structural kidney abnormalities associated with OSA are unclear. The objective of this study was to determine whether intermittent hypoxia (IH), a key pathological feature of OSA, induces renal histopathological damage using a mouse model. Ten 8-week old wild-type male CB57BL/6 mice were randomly assigned to receive either IH or intermittent air (IA) for 60 days. After euthanasia, one kidney per animal was paraformaldehyde-fixed and then sectioned for histopathological and immunohistochemical analysis. Measurements of glomerular hypertrophy and mesangial matrix expansion were made in periodic acid–Schiff stained kidney sections, while glomerular transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) and vascular endothelial growth factor-A (VEGF-A) proteins were semi-quantified by immunohistochemistry. The antigen-antibody reaction was detected by 3,3′-diaminobenzidine chromogen where the color intensity semi-quantified glomerular protein expression. To enhance the accuracy of protein semi-quantification, the percentage of only highly-positive staining was used for analysis. Levels of TGF-β, CTGF and VEGF-A proteins in the kidney cortex were further quantified by western blotting. Cellular apoptosis was also investigated by measuring cortical antiapoptotic B-cell lymphoma 2 (Bcl-2) and apoptotic Bcl-2-associated X (Bax) proteins by western blotting. Further investigation of cellular apoptosis was carried out by fluorometric terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining. Finally, the levels of serum creatinine and 24-hour urinary albumin were measured as a general index of renal function. Our results indicate that mice exposed to IH have an

Connective tissue growth factor is required for skeletal development and postnatal skeletal homeostasis in male mice.

PubMed

Canalis, Ernesto; Zanotti, Stefano; Beamer, Wesley G; Economides, Aris N; Smerdel-Ramoya, Anna

2010-08-01

Connective tissue growth factor (CTGF), a member of the cysteine-rich 61 (Cyr 61), CTGF, nephroblastoma overexpressed (NOV) (CCN) family of proteins, is synthesized by osteoblasts, and its overexpression inhibits osteoblastogenesis and causes osteopenia. The global inactivation of Ctgf leads to defective endochondral bone formation and perinatal lethality; therefore, the consequences of Ctgf inactivation on the postnatal skeleton are not known. To study the function of CTGF, we generated Ctgf(+/LacZ) heterozygous null mice and tissue-specific null Ctgf mice by mating Ctgf conditional mice, where Ctgf is flanked by lox sequences with mice expressing the Cre recombinase under the control of the paired-related homeobox gene 1 (Prx1) enhancer (Prx1-Cre) or the osteocalcin promoter (Oc-Cre). Ctgf(+/LacZ) heterozygous mice exhibited transient osteopenia at 1 month of age secondary to decreased trabecular number. A similar osteopenic phenotype was observed in 1-month-old Ctgf conditional null male mice generated with Prx1-Cre, suggesting that the decreased trabecular number was secondary to impaired endochondral bone formation. In contrast, when the conditional deletion of Ctgf was achieved by Oc-Cre, an osteopenic phenotype was observed only in 6-month-old male mice. Osteoblast and osteoclast number, bone formation, and eroded surface were not affected in Ctgf heterozygous or conditional null mice. In conclusion, CTGF is necessary for normal skeletal development but to a lesser extent for postnatal skeletal homeostasis.

Immunohistochemical Analysis of the Role Connective Tissue Growth Factor in Drug-induced Gingival Overgrowth in Response to Phenytoin, Cyclosporine, and Nifedipine

PubMed Central

Anand, A. J.; Gopalakrishnan, Sivaram; Karthikeyan, R.; Mishra, Debasish; Mohapatra, Shreeyam

2018-01-01

Objective: To evaluate for the presence of connective tissue growth factor (CTGF) in drug (phenytoin, cyclosporine, and nifedipine)-induced gingival overgrowth (DIGO) and to compare it with healthy controls in the absence of overgrowth. Materials and Methods: Thirty-five patients were chosen for the study and segregated into study (25) and control groups (10). The study group consisted of phenytoin-induced (10), cyclosporine-induced (10), and nifedipine-induced (5) gingival overgrowth. After completing necessary medical evaluations, biopsy was done. The tissue samples were fixed in 10% formalin and then immunohistochemically evaluated for the presence of CTGF. The statistical analysis of the values was done using statistical package SPSS PC+ (Statistical Package for the Social Sciences, version 4.01). Results: The outcome of immunohistochemistry shows that DIGO samples express more CTGF than control group and phenytoin expresses more CTGF followed by nifedipine and cyclosporine. Conclusion: The study shows that there is an increase in the levels of CTGF in patients with DIGO in comparison to the control group without any gingival overgrowth. In the study, we compared the levels of CTGF in DIGO induced by three most commonly used drugs phenytoin, cyclosporine, and nifedipine. By comparing the levels of CTGF, we find that cyclosporine induces the production of least amount of CTGF. Therefore, it might be a more viable drug choice with reduced side effects. PMID:29629324

Connective tissue growth factor is a positive regulator of epithelial-mesenchymal transition and promotes the adhesion with gastric cancer cells in human peritoneal mesothelial cells.

PubMed

Jiang, Cheng-Gang; Lv, Ling; Liu, Fu-Rong; Wang, Zhen-Ning; Na, Di; Li, Feng; Li, Jia-Bin; Sun, Zhe; Xu, Hui-Mian

2013-01-01

Connective tissue growth factor (CTGF) is involved in human cancer development and progression. Epithelial to mesenchymal transition (EMT) plays an important role in many biological processes. In this study, we wished to investigate the role of CTGF in EMT of peritoneal mesothelial cells and the effects of CTGF on adhesion of gastric cancer cells to mesothelial cells. Human peritoneal mesothelial cells (HPMCs) were cultured with TGF-β1 or various concentrations of CTGF for different time. The EMT process was monitored by morphology. Real-time RT-PCR and Western blot were used to evaluate the expression of vimentin, α-SMA , E-cadherin and β-catenin. RNA interference was used to achieve selective and specific knockdown of CTGF. We demonstrated that CTGF induced EMT of mesothelial cells in a dose- and time-dependent manner. HPMCs were exposed to TGF-β1 also underwent EMT which was associated with the induction of CTGF expression. Transfection with CTGF siRNA was able to reverse the EMT partially after treatment of TGF-β1. Moreover, the induced EMT of HPMCs was associated with an increased adhesion of gastric cancer cells to mesothelial cells. These findings suggest that CTGF is not only an important mediator but a potent activator of EMT in peritoneal mesothelial cells, which in turn promotes gastric cancer cell adhesion to peritoneum. Copyright © 2012 Elsevier Ltd. All rights reserved.

Connective tissue growth factor and integrin αvβ6: a new pair of regulators critical for ductular reaction and biliary fibrosis in mice.

PubMed

Pi, Liya; Robinson, Paulette M; Jorgensen, Marda; Oh, Seh-Hoon; Brown, Alicia R; Weinreb, Paul H; Trinh, Thu Le; Yianni, Protopapadakis; Liu, Chen; Leask, Andrew; Violette, Shelia M; Scott, Edward W; Schultz, Gregory S; Petersen, Bryon E

2015-02-01

Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvβ6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvβ6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter-driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvβ6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen-inducible Cre-loxP system down-regulated integrin αvβ6 in DDC-damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvβ6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis-related genes, smaller areas of alpha-smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvβ6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-β1 activation in vitro. CTGF and integrin αvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-β1. CTGF and integrin αvβ6 are potential therapeutic targets to control DRs and fibrosis in related liver

TGF-beta inhibits IL-1beta-activated PAR-2 expression through multiple pathways in human primary synovial cells.

PubMed

Tsai, Shin-Han; Sheu, Ming-Thau; Liang, Yu-Chih; Cheng, Hsiu-Tan; Fang, Sheng-Shiung; Chen, Chien-Ho

2009-10-23

To investigate the mechanism how Transforming growth factor-beta(TGF-beta) represses Interleukin-1beta (IL-1beta)-induced Proteinase-Activated Receptor-2 (PAR-2) expression in human primary synovial cells (hPSCs). Human chondrocytes and hPSCs isolated from cartilages and synovium of Osteoarthritis (OA) patients were cultured with 10% fetal bovine serum media or serum free media before treatment with IL-1beta, TGF-beta1, or Connective tissue growth factor (CTGF). The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Collagen zymography was performed to assess the activity of Matrix metalloproteinases-13 (MMP-13). It was demonstrated that IL-1beta induces PAR-2 expression via p38 pathway in hPSCs. This induction can be repressed by TGF-beta and was observed to persist for at least 48 hrs, suggesting that TGF-beta inhibits PAR-2 expression through multiple pathways. First of all, TGF-beta was able to inhibit PAR-2 activity by inhibiting IL-1beta-induced p38 signal transduction and secondly the inhibition was also indirectly due to MMP-13 inactivation. Finally, TGF-beta was able to induce CTGF, and in turn CTGF represses PAR-2 expression by inhibiting IL-1beta-induced phospho-p38 level. TGF-beta could prevent OA from progression with the anabolic ability to induce CTGF production to maintain extracellular matrix (ECM) integrity and to down regulate PAR-2 expression, and the anti-catabolic ability to induce Tissue inhibitors of metalloproteinase-3 (TIMP-3) production to inhibit MMPs leading to avoid PAR-2 over-expression. Because IL-1beta-induced PAR-2 expressed in hPSCs might play a significantly important role in early phase of OA, PAR-2 repression by exogenous TGF-beta or other agents might be an ideal therapeutic target to prevent OA from progression.

Downregulation of connective tissue growth factor by three-dimensional matrix enhances ovarian carcinoma cell invasion.

PubMed

Barbolina, Maria V; Adley, Brian P; Kelly, David L; Shepard, Jaclyn; Fought, Angela J; Scholtens, Denise; Penzes, Peter; Shea, Lonnie D; Stack, M Sharon

2009-08-15

Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancies, due mainly to the prevalence of undetected metastatic disease. The process of cell invasion during intraperitoneal anchoring of metastatic lesions requires concerted regulation of many processes, including modulation of adhesion to the extracellular matrix and localized invasion. Exploratory cDNA microarray analysis of early response genes (altered after 4 hr of 3D collagen culture) coupled with confirmatory real-time reverse-transcriptase polymerase chain reaction, multiple 3D cell culture matrices, Western blot, immunostaining, adhesion, migration and invasion assays were used to identify modulators of adhesion pertinent to EOC progression and metastasis. cDNA microarray analysis indicated a dramatic downregulation of connective tissue growth factor (CTGF) in EOC cells placed in invasion- mimicking conditions (3D Type I collagen). Examination of human EOC specimens revealed that CTGF expression was absent in 46% of the tested samples (n = 41), but was present in 100% of normal ovarian epithelium samples (n = 7). Reduced CTGF expression occurs in many types of cells and may be a general phenomenon displayed by cells encountering a 3D environment. CTGF levels were inversely correlated with invasion such that downregulation of CTGF increased, while its upregulation reduced collagen invasion. Cells adhered preferentially to a surface comprised of both collagen I and CTGF relative to either component alone using alpha6beta1 and alpha3beta1 integrins. Together these data suggest that downregulation of CTGF in EOC cells may be important for cell invasion through modulation of cell-matrix adhesion.

Downregulation of Connective Tissue Growth Factor by Three-Dimensional Matrix Enhances Ovarian Carcinoma Cell Invasion

PubMed Central

Barbolina, Maria V.; Adley, Brian P.; Kelly, David L.; Shepard, Jaclyn; Fought, Angela J.; Scholtens, Denise; Penzes, Peter; Shea, Lonnie D.; Sharon Stack, M

2010-01-01

Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancy, due mainly to the prevalence of undetected metastatic disease. The process of cell invasion during intra-peritoneal anchoring of metastatic lesions requires concerted regulation of many processes, including modulation of adhesion to the extracellular matrix and localized invasion. Exploratory cDNA microarray analysis of early response genes (altered after 4 hours of 3-dimensional collagen culture) coupled with confirmatory real-time RT-PCR, multiple three-dimensional cell culture matrices, Western blot, immunostaining, adhesion, migration, and invasion assays were used to identify modulators of adhesion pertinent to EOC progression and metastasis. cDNA microarray analysis indicated a dramatic downregulation of connective tissue growth factor (CTGF) in EOC cells placed in invasion-mimicking conditions (3-dimensional type I collagen). Examination of human EOC specimens revealed that CTGF expression was absent in 46% of the tested samples (n=41), but was present in 100% of normal ovarian epithelium samples (n=7). Reduced CTGF expression occurs in many types of cells and may be a general phenomenon displayed by cells encountering a 3D environment. CTGF levels were inversely correlated with invasion such that downregulation of CTGF increased, while its upregulation reduced, collagen invasion. Cells adhered preferentially to a surface comprised of both collagen I and CTGF relative to either component alone using α6β1 and α3β1 integrins. Together these data suggest that downregulation of CTGF in EOC cells may be important for cell invasion through modulation of cell-matrix adhesion. PMID:19382180

Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion.

PubMed

Edwards, Lincoln A; Woolard, Kevin; Son, Myung Jin; Li, Aiguo; Lee, Jeongwu; Ene, Chibawanye; Mantey, Samuel A; Maric, Dragan; Song, Hua; Belova, Galina; Jensen, Robert T; Zhang, Wei; Fine, Howard A

2011-08-03

Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin β1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-κB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF(200 ng/mL): 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-κB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF(200 ng/mL) + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ

Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types

PubMed Central

Heinemeier, K M; Olesen, J L; Haddad, F; Langberg, H; Kjaer, M; Baldwin, K M; Schjerling, P

2007-01-01

Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle–tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-β-1 (TGF-β-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague–Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7–9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-β-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-β-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-β-1 in loading-induced collagen synthesis in the muscle–tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus. PMID:17540706

Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types.

PubMed

Heinemeier, K M; Olesen, J L; Haddad, F; Langberg, H; Kjaer, M; Baldwin, K M; Schjerling, P

2007-08-01

Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle-tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-beta-1 (TGF-beta-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-beta-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-beta-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-beta-1 in loading-induced collagen synthesis in the muscle-tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus.

LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor

PubMed Central

Ying, Lihua; Lau, Agatha; Alvira, Cristina M.; West, Robert; Cann, Gordon M.; Zhou, Bin; Kinnear, Caroline; Jan, Eric; Sarnow, Peter; Van de Rijn, Matt; Rabinovitch, Marlene

2009-01-01

Summary Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3′ UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types. PMID:19366727

Connective Tissue Growth Factor Transgenic Mouse Develops Cardiac Hypertrophy, Lean Body Mass and Alopecia.

PubMed

Nuglozeh, Edem

2017-07-01

Connective Tissue Growth Factor (CTGF/CCN2) is one of the six members of cysteine-rich, heparin-binding proteins, secreted as modular protein and recognised to play a major function in cell processes such as adhesion, migration, proliferation and differentiation as well as chondrogenesis, skeletogenesis, angiogenesis and wound healing. The capacity of CTGF to interact with different growth factors lends an important role during early and late development, especially in the anterior region of the embryo. CTGF Knockout (KO) mice have several craniofacial defects and bone miss shaped due to an impairment of the vascular system development during chondrogenesis. The aim of the study was to establish an association between multiple modular functions of CTGF and the phenotype and cardiovascular functions in transgenic mouse. Bicistronic cassette was constructed using pIRES expressing vector (Clontech, Palo Alto, CA). The construct harbours mouse cDNA in tandem with LacZ cDNA as a reporter gene under the control of Cytomegalovirus (CMV) promoter. The plasmid was linearised with NotI restriction enzyme, and 50 ng of linearised plasmid was injected into mouse pronucleus for the chimaera production. Immunohistochemical methods were used to assess the colocalisation renin and CTGF as well as morphology and rheology of the cardiovascular system. The chimeric mice were backcrossed against the wild-type C57BL/6 to generate hemizygous (F1) mouse. Most of the offsprings died as a result of respiratory distress and those that survived have low CTGF gene copy number, approximately 40 molecules per mouse genome. The copy number assessment on the dead pups showed 5×10 3 molecules per mouse genome explaining the threshold of the gene in terms of toxicity. Interestingly, the result of this cross showed 85% of the progenies to be positive deviating from Mendelian first law. All F2 progenies died excluding the possibility of establishing the CTGF transgenic mouse line, situation that

Effect of Brain- and Tumor-Derived Connective Tissue Growth Factor on Glioma Invasion

PubMed Central

Edwards, Lincoln A.; Woolard, Kevin; Son, Myung Jin; Li, Aiguo; Lee, Jeongwu; Ene, Chibawanye; Mantey, Samuel A.; Maric, Dragan; Song, Hua; Belova, Galina; Jensen, Robert T.; Zhang, Wei

2011-01-01

Background Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. Methods Highly infiltrative patient-derived glioma tumor–initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin β1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. Results Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1–TrkA receptor complexes and nuclear factor kappa B (NF-κB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF200 ng/mL: 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-κB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF200 ng/mL + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most

Homologous peptide of connective tissue growth factor ameliorates epithelial to mesenchymal transition of tubular epithelial cells.

PubMed

Shi, Yujun; Tu, Zhidan; Wang, Wei; Li, Qing; Ye, Feng; Wang, Jinjing; Qiu, Jing; Zhang, Li; Bu, Hong; Li, Youping

2006-10-01

The hallmark of failing renal transplants is tubular atrophy and interstitial fibrosis. The cytokine connective tissue growth factor (CTGF or CCN2) plays an important role in epithelial-mesenchymal transition (EMT) of tubular epithelial cells (TECs). A unique domain within CTGF (IRTPKISKPIKFELSG) which binds to its potential receptor integrin alpha v beta3 has been identified. This study was carried out to further characterize a synthetic hexadeca-peptide (P2) homologous to this domain and to determine its effect on CTGF-mediated solid phase cell adhesion, EMT induction and fibrogenesis in rat renal NRK-52E cells. Results showed that both P2 and recombinant CTGF bound to NRK-52E cells. Unlike CTGF, P2 had little effect on EMT induction including cytoskeleton remodeling and expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin, nor did it have effect on fibrogenic induction including alternation of extracellular matrix (ECM) proteins, collagen type I and IV at gene and protein levels. All data showed that P2 bound preferably on the surface of NRK-52E cells and inhibited the effect of CTGF on EMT induction and cell fibrogenesis, probably by occupying the binding sites of CTGF within its potential receptors. Therefore, P2 may be used as a potential anti-fibrotic agent.

TAZ promotes epithelial to mesenchymal transition via the upregulation of connective tissue growth factor expression in neuroblastoma cells.

PubMed

Wang, Qiang; Xu, Zhilin; An, Qun; Jiang, Dapeng; Wang, Long; Liang, Bingxue; Li, Zhaozhu

2015-02-01

Neuroblastoma (NB) is a neuroendocrine cancer that occurs most commonly in infants and young children. The Hippo signaling pathway regulates cell proliferation and apoptosis, and its primary downstream effectors are TAZ and yes‑associated protein 1 (YAP). The effect of TAZ on the metastatic progression of neuroblastoma and the underlying mechanisms involved remain elusive. In the current study, it was determined by western blot analysis that the migratory and invasive properties of SK‑N‑BE(2) human neuroblastoma cells are associated with high expression levels of TAZ. Repressed expression of TAZ in SK‑N‑BE(2) cells was shown to result in a reduction in aggressiveness of the cell line, by Transwell migration and invasion assay. In contrast, overexpression of TAZ in SK‑N‑SH human neuroblastoma cells was shown by Transwell migration and invasion assays, and western blot analysis, to result in epithelial‑mesenchymal transition (EMT) and increased invasiveness. Mechanistically, the overexpression of TAZ was demonstrated to upregulate the expression levels of connective tissue growth factor (CTGF), by western blot analysis and chromatin immunoprecipitation assay, while the knockdown of TAZ downregulated it. Furthermore, TAZ was shown by luciferase assay to induce CTGF expression by modulating the activation of the TGF‑β/Smad3 signaling pathway. In conclusion, the present study is, to the best of our knowledge, the first to demonstrate that the overexpression of TAZ induces EMT, increasing the invasive abilities of neuroblastoma cells. This suggests that TAZ may serve as a potential target in the development of novel therapies for the treatment of neuroblastoma.

Pirfenidone prevents radiation-induced intestinal fibrosis in rats by inhibiting fibroblast proliferation and differentiation and suppressing the TGF-β1/Smad/CTGF signaling pathway.

PubMed

Sun, Yan-Wu; Zhang, Yi-Yi; Ke, Xin-Jie; Wu, Xue-Jing; Chen, Zhi-Fen; Chi, Pan

2018-03-05

Radiation-induced intestinal fibrosis (RIF) is a chronic toxicity following radiation, and can be very difficult to treat. Pirfenidone is a promising anti-fibrotic agent that inhibits fibrosis progression in various clinical and experimental studies. This study was aimed to explore whether pirfenidone could protect against RIF, and to evaluate the underlying mechanism. An animal model of RIF was induced by exposure of a single dose of 20 Gy to the pelvis. Rats were orally administered with pirfenidone (200, 400 md/kg/d) for 12 weeks. Primary rat intestinal fibroblasts were cultured to determine the effects of pirfenidone on TGF-β1-induced (5 ng/ml) proliferation and transdifferentiation of fibroblasts. The expression of collagen I, α-SMA, and TGF-β1/Smad/CTGF pathway proteins were analyzed by qRT-PCR and/or western blot analysis. The cell proliferation rate was determined by CCK-8 assay. The results indicated that pirfenidone significantly attenuated fibrotic lesion in irradiated intestines and reduced collagen deposition by inhibiting TGF-β1/Smad/CTGF pathway in rat models. Moreover, in primary rat intestinal fibroblasts, pirfenidone decreased the up-regulation of TGF-β1-induced collagen I and α-SMA by suppressing TGF-β1/Smad/CTGF signaling pathway. Altogether, our findings suggested that pirfenidone attenuated RIF by inhibiting the proliferation and differentiation of intestinal fibroblasts and suppressing the TGF-β1/Smad/CTGF signaling pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

Connective tissue growth factor linked to the E7 tumor antigen generates potent antitumor immune responses mediated by an antiapoptotic mechanism.

PubMed

Cheng, W-F; Chang, M-C; Sun, W-Z; Lee, C-N; Lin, H-W; Su, Y-N; Hsieh, C-Y; Chen, C-A

2008-07-01

A novel method for generating an antigen-specific cancer vaccine and immunotherapy has emerged using a DNA vaccine. However, antigen-presenting cells (APCs) have a limited life span, which hinders their long-term ability to prime antigen-specific T cells. Connective tissue growth factor (CTGF) has a role in cell survival. This study explored the intradermal administration of DNA encoding CTGF with a model tumor antigen, human papilloma virus type 16 E7. Mice vaccinated with CTGF/E7 DNA exhibited a dramatic increase in E7-specific CD4(+) and CD8(+) T-cell precursors. They also showed an impressive antitumor effect against E7-expressing tumors compared with mice vaccinated with the wild-type E7 DNA. The delivery of DNA encoding CTGF and E7 or CTGF alone could prolong the survival of transduced dendritic cells (DCs) in vivo. In addition, CTGF/E7-transduced DCs could enhance a higher number of E7-specific CD8(+) T cells than E7-transduced DCs. By prolonging the survival of APCs, DNA vaccine encoding CTGF linked to a tumor antigen represents an innovative approach to enhance DNA vaccine potency and holds promise for cancer prophylaxis and immunotherapy.

Stage-specific control of connective tissue growth factor (CTGF/CCN2) expression in chondrocytes by Sox9 and beta-catenin.

PubMed

Huang, Bau-Lin; Brugger, Sean M; Lyons, Karen M

2010-09-03

CCN2/connective tissue growth factor is highly expressed in hypertrophic chondrocytes and is required for chondrogenesis. However, the transcriptional mechanisms controlling its expression in cartilage are largely unknown. The activity of the Ccn2 promoter was, therefore, investigated in osteochondro-progenitor cells and hypertrophic chondrocytes to ascertain these mechanisms. Sox9 and T-cell factor (TCF) x lymphoid enhancer factor (LEF) factors contain HMG domains and bind to related consensus sites. TCF x LEF factors are normally repressive but when bound to DNA in a complex with beta-catenin become activators of gene expression. In silico analysis of the Ccn2 proximal promoter identified multiple consensus TCF x LEF elements, one of which was also a consensus binding site for Sox9. Using luciferase reporter constructs, the TCF x LEF x Sox9 site was found to be involved in stage-specific expression of Ccn2. Luciferase, electrophoretic mobility shift assay (EMSA), and ChIP analysis revealed that Sox9 represses Ccn2 expression by binding to the consensus TCF x LEF x Sox9 site. On the other hand, the same assays showed that in hypertrophic chondrocytes, TCF x LEF x beta-catenin complexes occupy the consensus TCF x LEF x Sox9 site and activate Ccn2 expression. Furthermore, transgenic mice in which lacZ expression is driven under the control of the proximal Ccn2 promoter revealed that the proximal Ccn2 promoter responded to Wnt signaling in cartilage. Hence, we propose that differential occupancy of the TCF x LEF x Sox9 site by Sox9 versus beta-catenin restricts high levels of Ccn2 expression to hypertrophic chondrocytes.

Overexpressed connective tissue growth factor in cardiomyocytes attenuates left ventricular remodeling induced by angiotensin II perfusion.

PubMed

Zhang, Ying; Yan, Hua; Guang, Gong-Chang; Deng, Zheng-Rong

2017-01-01

To evaluate the improving effects of specifically overexpressed connective tissue growth factor (CTGF) in cardiomyocytes on mice with hypertension induced by angiotensin II (AngII) perfusion, 24 transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) were divided into two equal groups that were perfused with acetic acid and AngII, respectively, for 7 days. Another 24 cage-control wild-type C57BL/6 mice (NLC) were divided and treated identically. Blood pressure was detected by caudal artery cannulation. Cardiac structural and functional changes were observed by echocardiography. Cardiac fibrosis was detected by Masson staining. After AngII perfusion, blood pressures of NLC and Tg-CTGF mice, especially those of the formers, significantly increased. Compared with NLC + AngII group, Tg-CTGF + AngII group had significantly lower left ventricular posterior wall thickness at end-diastole and left ventricular posterior wall thickness at end-systole as well as significantly higher left ventricular end-systolic diameter and left ventricular end-diastolic diameter (P < 0.05). Reverse transcription-polymerase chain reaction (RT-PCR) showed that Tg-CTGF + AngII group had significantly lower collagen I, α-SMA, and TGF-β mRNA expressions in cardiac tissues (P < 0.05). Tg-CTGF can protect AngII-induced cardiac remodeling of mice with hypertension by mitigating inflammatory response. CTGF may be a therapy target for hypertension-induced myocardial fibrosis, but the detailed mechanism still needs in-depth studies.

The roles of connective tissue growth factor in the development of anastomotic esophageal strictures.

PubMed

Zhao, Haibin; Zhao, Lingna; Zhou, Zhihua; Wu, Yaoyi

2015-08-12

The aim of this study was to investigate the roles of connective tissue growth factor (CTGF) in the development of anastomotic strictures after surgical repair of the esophagus. Tissues collected from the patients were divided into three groups based on the results of endoscopy and clinical grading. Patients without dysphagia after esophagectomy were used as the control population. The protein levels of CTGF, TGF-β1, Smad2, and Smad4 were determined by immunohistochemistry (IHC) and western blot analyses, while the mRNA levels of the two growth factors were evaluated by real-time polymerase chain reaction. Compared with the control group, significantly increased (p < 0.01) levels of CTGF and TGF-β1 protein were observed in the anastomotic stenosis (AS) group, and levels of the two proteins detected by the IHC and western blot analyses were also significantly increased with the increasing severity of stenosis (p < 0.05). The mRNA levels of CTGF and TGF-β1 in the tissues collected from the patients with stenosis were significantly up-regulated (p < 0.05) as compared with those from the control group. In addition, the levels of Smad2 and Smad4 protein were also significantly increased (p < 0.05) with the increasing severity of stenosis, and the protein levels were positively correlated with the levels of CTGF (r = 0.59, p < 0.05) and TGF-β1 (r = 0.63, p < 0.05). Inhibition of CTGF protein or mRNA expression may be a distinctive and effective therapy for the treatment of postoperative anastomotic strictures.

PCPA protects against monocrotaline-induced pulmonary arterial remodeling in rats: potential roles of connective tissue growth factor.

PubMed

Bai, Yang; Li, Zhong-Xia; Zhao, Yue-Tong; Liu, Mo; Wang, Yun; Lian, Guo-Chao; Zhao, Qi; Wang, Huai-Liang

2017-12-19

The purpose of this study was to investigate the mechanism of monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) and determine whether 4-chloro-DL-phenylalanine (PCPA) could inhibit pulmonary arterial remodeling associated with connective tissue growth factor (CTGF) expression and downstream signal pathway. MCT was administered to forty Sprague Dawley rats to establish the PAH model. PCPA was administered at doses of 50 and 100 mg/kg once daily for 3 weeks via intraperitoneal injection. On day 22, the pulmonary arterial pressure (PAP), right ventricle hypertrophy index (RVI) and pulmonary artery morphology were assessed and the serotonin receptor-1B (SR-1B), CTGF, p-ERK/ERK were measured by western blot or immunohistochemistry. The concentration of serotonin in plasma was checked by ELISA. Apoptosis and apoptosis-related indexes were detected by TUNEL and western blot. In the MCT-induced PAH models, the PAP, RVI, pulmonary vascular remodeling, SR-1B index, CTGF index, anti-apoptotic factors bcl-xl and bcl-2, serotonin concentration in plasma were all increased and the pro-apoptotic factor caspase-3 was reduced. PCPA significantly ameliorated pulmonary arterial remodeling induced by MCT, and this action was associated with accelerated apoptosis and down-regulation of CTGF, SR-1B and p-ERK/ERK. The present study suggests that PCPA protects against the pathogenesis of PAH by suppressing remodeling and inducing apoptosis, which are likely associated with CTGF and downstream ERK signaling pathway in rats.

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes

PubMed Central

Itoh, Shinsuke; Hattori, Takako; Tomita, Nao; Aoyama, Eriko; Yutani, Yasutaka; Yamashiro, Takashi; Takigawa, Masaharu

2013-01-01

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage. PMID:23951098

The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji

2014-01-17

Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains amore » highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.« less

Insulin-like growth factor-I regulates GPER expression and function in cancer cells.

PubMed

De Marco, P; Bartella, V; Vivacqua, A; Lappano, R; Santolla, M F; Morcavallo, A; Pezzi, V; Belfiore, A; Maggiolini, M

2013-02-07

Functional cross talk between insulin-like growth factor-I (IGF-I) system and estrogen signaling has been largely reported, although the underlying molecular mechanisms remain to be fully elucidated. As GPR30/GPER mediates rapid cell responses to estrogens, we evaluated the potential of IGF-I to regulate GPER expression and function in estrogen receptor (ER)α-positive breast (MCF-7) and endometrial (Ishikawa) cancer cells. We found that IGF-I transactivates the GPER promoter sequence and upregulates GPER mRNA and protein levels in both cells types. Similar data were found, at least in part, in carcinoma-associated fibroblasts. The upregulation of GPER expression by IGF-I involved the IGF-IR/PKCδ/ERK/c-fos/AP1 transduction pathway and required ERα, as ascertained by specific pharmacological inhibitors and gene-silencing. In both MCF-7 and Ishikawa cancer cells, the IGF-I-dependent cell migration required GPER and its main target gene CTGF, whereas the IGF-I-induced proliferation required both GPER and cyclin D1. Our data demonstrate that the IGF-I system regulates GPER expression and function, triggering the activation of a signaling network that leads to the migration and proliferation of cancer cells.

CCN2 (CTGF) gene polymorphism is a novel prognostic risk factor for cardiovascular outcomes in hemodialysis patients.

PubMed

Cozzolino, Mario; Biondi, Maria Luisa; Banfi, Elena; Riser, Bruce L; Mehmeti, Florjan; Cusi, Daniele; Gallieni, Maurizio

2010-01-01

The very high cardiovascular (CV) mortality and morbidity rates in hemodialysis (HD) patients are greatly related to atherosclerosis. CCN2 (connective tissue growth factor/CTGF) is a profibrotic factor that is secreted by endothelial cells, involved in atherogenesis, promoting fibroblast proliferation and matrix production. CCN2 protein is significantly increased in complicated fibrous plaques and enhances monocyte migration into atherosclerotic lesions. The aim of this study was to investigate a possible association between CCN2 gene polymorphism and CV morbidity and mortality in HD patients. 98 HD patients, followed for 24 months, were genotyped for the common polymorphism on the CCN2 gene (G-945C). HD patient characteristics were: age 64 ± 13 years, males 64%, diabetes 24%, hypertension 62%, smokers 38%, dyslipidemia 28%, all undergoing standard HD three times weekly. All-cause mortality was not associated with CCN2 polymorphism (G-945C). In contrast, however, the GG genotype was strongly associated with CV mortality: OR 13 (1.49-155), p = 0.0048. Interestingly, the GG genotype was also greatly associated with the serious CV events of stroke and myocardial infarction in surviving HD patients: OR 13.3 (2.5-87.08), p = 0.0001. We demonstrate for the first time that CCN2 gene polymorphism is a prognostic risk factor for CV morbidity and mortality in HD patients. These data may have important implications for better understanding the link between accelerated atherosclerosis and increased mortality in HD population. Copyright © 2010 S. Karger AG, Basel.

Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression.

PubMed

Eraky, Salma M; El-Mesery, Mohamed; El-Karef, Amro; Eissa, Laila A; El-Gayar, Amal M

2018-05-01

Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-β1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-β1, and CTGF. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Tissular growth factors profile after teduglutide administration on an animal model of intestinal anastomosis.

PubMed

Costa, Beatriz Pinto; Gonçalves, Ana Cristina; Abrantes, Ana Margarida; Alves, Raquel; Matafome, Paulo; Seiça, Raquel; Sarmento-Ribeiro, Ana Bela; Botelho, Maria Filomena; Castro-Sousa, Francisco

2018-01-16

Teduglutide is an enterotrophic analogue of glucagon-like peptide-2, with an indirect and poorly understood mechanism of action, approved for the rehabilitation of short-bowel syndrome. This study aims to analyze the response of tissue growth factors to surgical injury and teduglutide administration on an animal model of intestinal anastomosis. Wistar rats (n = 59) were distributed into four groups: "ileal resection" or "laparotomy", each one subdivided into "postoperative teduglutide administration" or "no treatment"; and sacrificed at the third or the seventh day, with ileal sample harvesting. Gene expression of insulin-like growth factor 1 (Igf1), vascular endothelial growth factor a (Vegfa), transforming growth factor β1 (Tgfβ1), connective tissue growth factor (Ctgf), fibroblast growth factor 2 (Fgf2), fibroblast growth factor 7 (Fgf7), epidermal growth factor (Egf), heparin-binding epidermal-like growth factor (Hbegf), platelet-derived growth factor b (Pdgfb) and glucagon-like peptide 2 receptor (Glp2r)was studied by real-time polymerase chain reaction. Upregulation of Fgf7, Fgf2, Egf, Vegfaand Glp2rat the third day and of Pdgfat the seventh day was verified in the perianastomotic segment. Teduglutide administration was associated with higher fold-change of relative gene expression of Vegfa(3.6 ± 1.3 vs.1.9 ± 2.0, p = 0.0001), Hbegf(2.2 ± 2.3 vs. 1.1 ± 0.9, p = 0.001), Igf1(1.6 ± 7.6 vs. 0.9 ± 0.7, p = 0.002) and Ctgf(1.1 ± 2.1 vs. 0.6 ± 2.0, p = 0.013); and lower fold-change of Tgfβ1, Fgf7and Glp2r. Those results underscore the recognized role of Igf1and Hbegfas molecular mediators of the effects of teduglutide and suggest that other humoral factors, like Vegfand Ctgf, may also be relevant in the perioperative context. Induction of Vegfa, Igf1and Ctgfgene expressions might indicate a favorable influence of teduglutide on the intestinal anastomotic healing.

hCG-dependent regulation of angiogenic factors in human granulosa lutein cells.

PubMed

Phan, B; Rakenius, A; Pietrowski, D; Bettendorf, H; Keck, C; Herr, D

2006-07-01

As prerequisite for development and maintenance of many diseases angiogenesis is of particular interest in medicine. Pathologic angiogenesis takes place in chronic arthritis, collagen diseases, arteriosclerosis, retinopathy associated with diabetes, and particularly in cancers. However, angiogenesis as a physiological process regularly occurs in the ovary. After ovulation the corpus luteum is formed by rapid vascularization of initially avascular granulosa lutein cell tissue. This process is regulated by gonadotropic hormones. In order to gain further insights in the regulatory mechanisms of angiogenesis in the ovary, we investigated these mechanisms in cell culture of human granulosa lutein cells. In particular, we determined the expression and production of several angiogenic factors including tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), Leptin, connective tissue growth factor (CTGF), meningioma-associated complimentary DNA (Mac25), basic fibroblast growth factor (bFGF), and Midkine. In addition, we showed that human chorionic gonadotropin (hCG) has distinct effects on their expression and production. hCG enhances the expression and production of TIMP-1, whereas it downregulates the expression of CTGF and Mac25. Furthermore it decreases the expression of Leptin. Our results provide evidence that hCG determines growth and development of the corpus luteum by mediating angiogenic pathways in human granulosa lutein cells. Hence we describe a further approach to understand the regulation of angiogenesis in the ovary.

The long noncoding RNA GAS5 negatively regulates the adipogenic differentiation of MSCs by modulating the miR-18a/CTGF axis as a ceRNA.

PubMed

Li, Ming; Xie, Zhongyu; Wang, Peng; Li, Jinteng; Liu, Wenjie; Tang, Su'an; Liu, Zhenhua; Wu, Xiaohua; Wu, Yanfeng; Shen, Huiyong

2018-05-10

Mesenchymal stem cells (MSCs) are important pluripotent stem cells and a major source of adipocytes in the body. However, the mechanism of adipogenic differentiation has not yet been completely elucidated. In this study, the long noncoding RNA GAS5 was found to be negatively correlated with MSC adipogenic differentiation. GAS5 overexpression negatively regulated adipocyte formation, whereas GAS5 knockdown had the opposite effect. Further mechanistic analyses using luciferase reporter assays revealed that GAS5 regulates the adipogenic differentiation of MSCs by acting as competing endogenous RNA (ceRNA) to sponge miR-18a, which promotes adipogenic differentiation. Mutation of the binding sites for GAS5 in miR-18a abolished the effect of the interaction. The miR-18a mimic and inhibitor reversed the negative regulatory effect of GAS5 on MSCs adipogenic differentiation. In addition, GAS5 inhibited miR-18a, which downregulates connective tissue growth factor (CTGF) expression, to negatively regulate the adipogenic differentiation of MSCs. Taken together, the results show that GAS5 serves as a sponge for miR-18a, inhibiting its capability to suppress CTGF protein translation and ultimately decreasing the adipogenic differentiation of MSCs. GAS5 is an important molecule involved in the adipogenic differentiation of MSCs and may contribute to the functional regulation and clinical applications of MSCs.

Inhibition by curcumin of multiple sites of the transforming growth factor-beta1 signalling pathway ameliorates the progression of liver fibrosis induced by carbon tetrachloride in rats

PubMed Central

2012-01-01

Background At present there is no effective and accepted therapy for hepatic fibrosis. Transforming growth factor (TGF)-β1 signaling pathway contributes greatly to hepatic fibrosis. Reducing TGF-β synthesis or inhibiting components of its complex signaling pathway represent important therapeutic targets. The aim of the study was to investigate the effect of curcumin on liver fibrosis and whether curcumin attenuates the TGF-β1 signaling pathway. Methods Sprague–Dawley rat was induced liver fibrosis by carbon tetrachloride (CCl4) for six weeks together with or without curcumin, and hepatic histopathology and collagen content were employed to quantify liver necro-inflammation and fibrosis. Moreover, the mRNA and protein expression levels of TGF-β1, Smad2, phosphorylated Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) were determined by quantitative real time-PCR, Western blot, or immunohistochemistry. Results Rats treated with curcumin improved liver necro-inflammation, and reduced liver fibrosis in association with decreased α-smooth muscle actin expression, and decreased collagen deposition. Furthermore, curcumin significantly attenuated expressions of TGFβ1, Smad2, phosphorylated Smad2, Smad3, and CTGF and induced expression of the Smad7. Conclusions Curcumin significantly attenuated the severity of CCl4-induced liver inflammation and fibrosis through inhibition of TGF-β1/Smad signalling pathway and CTGF expression. These data suggest that curcumin might be an effective antifibrotic drug in the prevention of liver disease progression. PMID:22978413

[Effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction].

PubMed

Tan, Wenqing; Wang, Wei; Zheng, Xuan; Chen, Jiying; Yuan, Xiangning; Zhang, Fangfang; Wang, Shuting; Tao, Lijian

2018-05-28

To investigate the effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction (UUO) and to observe the effect of fluorofenidone on the expressions of collagen type I (Col I), collagen type III (Col III), α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), platelet derived growth factor (PDGF) in the renal tissues of UUO rats.
 Methods: Male Sprague-Dawley (SD) rats were randomly divided into a sham-operated group, a UUO group, and a flurofenidone group (n=5). UUO model was induced by ligating the left ureter in rats. The rats were treated with 125 mg/(kg.d) fluorofenidone by gastric gavage in the fluorofenidone group at 24 h before the operation, and the rats were treated with the identical dose of 0.5% sodium carboxyl methyl cellulose (CMC-Na) in the other 2 groups. The rats were sacrificed at 14 days after UUO. Pathological changes of the renal tissue were observed by HE and Masson staining, the mRNA expressions of Col I, Col III, α-SMA, PDGF and CTGF were detected by real-time PCR, and the protein expressions of Col I, Col III, PDGF and CTGF were detected by immunohistochemical staining.
 Results: The renal interstitial damage index, relative collagen area and mRNA and protein expressions of Col I and Col III in the renal tissues of the rats in the UUO group significantly increased (P<0.05), and fluorofenidone could reduce these indexes (P<0.05). Compared with the sham-operated group, the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF in the renal tissues of the rats in the UUO group were increased, but fluorofenidone could decrease the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF (P<0.05).
 Conclusion: Fluorofenidone (125 mg/kg.d) could attenuate renal interstitial fibrosis through inhibition of fibroblast proliferation, myofibroblastic activation, PDGF and CTGF expression.

Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes.

PubMed

Wong, Marcy; Siegrist, Mark; Goodwin, Kelly

2003-10-01

Endochondral ossification is regulated by many factors, including mechanical stimuli, which can suppress or accelerate chondrocyte maturation. Mathematical models of endochondral ossification have suggested that tension (or shear stress) can accelerate the formation of endochondral bone, while hydrostatic stress preserves the cartilage phenotype. The goal of this study was to test this hypothesis by examining the expression of hypertrophic chondrocyte markers (transcription factor Cbfa1, MMP-13, type X collagen, VEGF, CTGF) and cartilage matrix proteins under cyclic tension and cyclic hydrostatic pressure. Chondrocyte-seeded alginate constructs were exposed to one of the two loading modes for a period of 3 h per day for 3 days. Gene expression was analyzed using real-time RT-PCR. Cyclic tension upregulated the expression of Cbfa1, MMP-13, CTGF, type X collagen and VEGF and downregulated the expression of TIMP-1. Cyclic tension also upregulated the expression of type 2 collagen, COMP and lubricin, but did not change the expression of SOX9 and aggrecan. Cyclic hydrostatic pressure downregulated the expression of MMP-13 and type I collagen and upregulated expression of TIMP-1 compared to the unloaded controls. Hydrostatic pressure may slow chondrocyte differentiation and have a chondroprotective, anti-angiogenic influence on cartilage tissue. Our results suggest that cyclic tension activates the Cbfa1/MMP-13 pathway and increases the expression of terminal differentiation hypertrophic markers. Mammalian chondrocytes appear to have evolved complex mechanoresponsive mechanisms, the effects of which can be observed in the histomorphologic establishment of the cartilaginous skeleton during development and maturation.

Phase 1 Study of Anti-CTGF Monoclonal Antibody in Patients with Diabetes and Microalbuminuria

PubMed Central

Schwartz, Sherwyn; Williams, Mark E.; Arauz-Pacheco, Carlos; Bolton, Warren K.; Lee, Tyson; Li, Dongxia; Neff, Thomas B.; Urquilla, Pedro R.; Sewell, K. Lea

2010-01-01

Background and objectives: This report summarizes the first phase 1 trial treating patients with microalbuminuric diabetic kidney disease (DKD) using FG-3019, a human monoclonal antibody to connective tissue growth factor (CTGF). CTGF is critically involved in processes of progressive fibrosis, including DKD. This phase 1, open-label, dose-escalation trial evaluated safety, pharmacokinetics, and possible therapeutic effects of FG-3019 on albuminuria, proteinuria, and tubular proteins. Design, setting, participants, and measurements: Microalbuminuric subjects (n = 24) with type 2 (79%) or type 1 (21%) diabetes received 3 or 10 mg/kg FG-3019 dosed intravenously every 14 days for four doses. Albuminuria and safety follow-up were to days 62 and 365, respectively. Results: No infusion was interrupted for symptoms, although 5 of 24 subjects had mild infusion-day adverse events thought to be possibly drug-related. No subject developed anti-FG-3019 antibodies. FG-3019 clearance was lower at 10 mg/kg than at 3 mg/kg, suggesting a saturable elimination pathway. Although this study was not designed for efficacy testing, it was notable that urinary albumin/creatinine ratio (ACR) decreased significantly from mean pretreatment ACR of 48 mg/g to mean post-treatment (day 56) ACR of 20 mg/g (P = 0.027) without evidence for a dose-response relationship. Conclusions: Treatment of microalbuminuric DKD subjects using FG-3019 was well tolerated and associated with a decrease in albuminuria. The data demonstrate a saturable pathway for drug elimination, minimal infusion adverse events, and no significant drug-attributable adverse effects over the year of follow-up. Changes in albuminuria were promising but require validation in a prospective, randomized, blinded study. PMID:20522536

miR-29b promotes skin wound healing and reduces excessive scar formation by inhibition of the TGF-β1/Smad/CTGF signaling pathway.

PubMed

Guo, Jingdong; Lin, Quan; Shao, Ying; Rong, Li; Zhang, Duo

2017-04-01

The hypertrophic scar is a medical difficulty of humans, which has caused great pain to patients. Here, we investigated the inhibitory effect of miR-29b on scar formation. The scalded model was established in mice and miR-29b mimics or a negative control was subcutaneously injected into the injury skin. Then various molecular biological experiments were performed to assess the effect of miR-29b on scar formation. According to our present study, first, the results demonstrated that miR-29b was down-regulated in thermal injury tissue and miR-29b treatment could promote wound healing, inhibit scar formation, and alleviate histopathological morphologic alteration in scald tissues. Additionally, miR-29b treatment suppressed collagen deposition and fibrotic gene expression in scar tissues. Finally, we found that miR-29b treatment inhibited the TGF-β1/Smad/CTGF signaling pathway. Taken together, our data suggest that miR-29b treatment has an inhibitory effect against scar formation via inhibition of the TGF-β1/Smad/CTGF signaling pathway and may provide a potential molecular basis for future treatments for hypertrophic scars.

Connective tissue growth factor promotes temozolomide resistance in glioblastoma through TGF-β1-dependent activation of Smad/ERK signaling.

PubMed

Zeng, Huijun; Yang, Zhao; Xu, Ningbo; Liu, Boyang; Fu, Zhao; Lian, Changlin; Guo, Hongbo

2017-06-15

Limited benefits and clinical utility of temozolomide (TMZ) for glioblastoma (GB) are frequently compromised by the development of acquired drug resistance. Overcoming TMZ resistance and uncovering the underlying mechanisms are challenges faced during GB chemotherapy. In this study, we reported that connective tissue growth factor (CTGF) was associated with GB chemoresistance and significantly upregulated in TMZ-treated GB cells. CTGF knockdown promoted TMZ-induced cell apoptosis and enhanced chemosensitivity, whereas its overexpression markedly conferred TMZ resistance in vitro and in vivo. Moreover, CTGF promoted TMZ resistance through stem-like properties acquisition and CD44 interference reversed the CTGF-induced TMZ resistance. Mechanistically, further investigation revealed that the TMZ-induced CTGF upregulation was tissue growth factor (TGF-β) dependent, and regulated by TGF-β1 activation through Smad and ERK1/2 signaling. Together, our results suggest a pivotal role of CTGF-mediated TMZ resistance through TGF-β1-dependent activation of Smad/ERK signaling pathways. These data provide us insights for identifying potential targets that are beneficial for overcoming TMZ resistance in GB.

Proteolytic processing of connective tissue growth factor in normal ocular tissues and during corneal wound healing.

PubMed

Robinson, Paulette M; Smith, Tyler S; Patel, Dilan; Dave, Meera; Lewin, Alfred S; Pi, Liya; Scott, Edward W; Tuli, Sonal S; Schultz, Gregory S

2012-12-13

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is up-regulated by TGF-β and mediates most key fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. This study addresses the role of proteolytic processing of CTGF in human corneal fibroblasts (HCF) stimulated with TGF-β, normal ocular tissues and wounded corneas. Proteolytic processing of CTGF in HCF cultures, normal animal eyes, and excimer laser wounded rat corneas were examined by Western blot. The identity of a 21-kDa band was determined by tandem mass spectrometry, and possible alternative splice variants of CTGF were assessed by 5' Rapid Amplification of cDNA Ends (RACE). HCF stimulated by TGF-β contained full length 38-kDa CTGF and fragments of 25, 21, 18, and 13 kDa, while conditioned medium contained full length 38- and a 21-kDa fragment of CTGF that contained the middle "hinge" region of CTGF. Fragmentation of recombinant CTGF incubated in HCF extracts was blocked by the aspartate protease inhibitor, pepstatin. Normal mouse, rat, and rabbit whole eyes and rabbit ocular tissues contained abundant amounts of C-terminal 25- and 21-kDa fragments and trace amounts of 38-kDa CTGF, although no alternative transcripts were detected. All forms of CTGF (38, 25, and 21 kDa) were detected during healing of excimer ablated rat corneas, peaking on day 11. Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing, which may have important implications in regulation of corneal scar formation.

Toll-like receptor 6 and connective tissue growth factor are significantly upregulated in mitomycin-C-treated urothelial carcinoma cells under hydrostatic pressure stimulation.

PubMed

Chen, Shao-Kuan; Chung, Chih-Ang; Cheng, Yu-Che; Huang, Chi-Jung; Chen, Wen-Yih; Ruaan, Ruoh-Chyu; Li, Chuan; Tsao, Chia-Wen; Hu, Wei-Wen; Chien, Chih-Cheng

2014-06-01

Urothelial carcinoma (UC) is the most common histologic subtype of bladder cancer. The administration of mitomycin C (MMC) into the bladder after transurethral resection of the bladder tumor (TURBT) is a common treatment strategy for preventing recurrence after surgery. We previously applied hydrostatic pressure combined with MMC in UC cells and found that hydrostatic pressure synergistically enhanced MMC-induced UC cell apoptosis through the Fas/FasL pathways. To understand the alteration of gene expressions in UC cells caused by hydrostatic pressure and MMC, oligonucleotide microarray was used to explore all the differentially expressed genes. After bioinformatics analysis and gene annotation, Toll-like receptor 6 (TLR6) and connective tissue growth factor (CTGF) showed significant upregulation among altered genes, and their gene and protein expressions with each treatment of UC cells were validated by quantitative real-time PCR and immunoblotting. Under treatment with MMC and hydrostatic pressure, UC cells showed increasing apoptosis using extrinsic pathways through upregulation of TLR6 and CTGF.

Serum connective tissue growth factor is a highly discriminatory biomarker for the diagnosis of rheumatoid arthritis.

PubMed

Yang, Xinyu; Lin, Ke; Ni, Shanmin; Wang, Jianmin; Tian, Qingqing; Chen, Huaijun; Brown, Matthew A; Zheng, Kaidi; Zhai, Weitao; Sun, Li; Jin, Shengwei; Wang, Jianguang

2017-11-22

Our previous proteomic study indicated that connective tissue growth factor (CTGF) may be a potential biomarker for rheumatoid arthritis (RA) diagnosis. The aim was to assess the performance of CTGF as a biomarker of RA. Serum and synovial fluid CTGF was detected using a direct high sensitivity sandwich ELISA kit. Serum CTGF levels were tested for discriminatory capacity and optimal assay cutoffs determined in a training cohort of 98 cases of RA with 103 healthy controls. The assay performance was then validated in a further cohort of 572 patients (with RA (n = 217), ankylosing spondylitis (n = 92), gout (n = 74), osteoarthritis (n = 52), systemic lupus erythematosus (n = 72), or primary Sjögren's syndrome (pSS) (n = 65)). Significant elevation of synovial fluid CTGF concentration was found in RA patients, demonstrating excellent diagnostic ability to predict RA (area under the curve (AUC) = 0.97). Similar results were found in serum CTGF detection. At the optimal cutoff value 88.66 pg/mL, the sensitivity, specificity, and the AUC was 0.86, 0.92, and 0.92, respectively, in the training cohort. Similar performance was observed in the validation cohort, with sensitivity, specificity, positive likelihood, and negative likelihood of 0.82, 0.91, 5.74, and 0.12, respectively. Stronger discriminatory capacity was seen with the combination of CTGF and anti-citrullinated protein antibody (ACPA) (AUC = 0.96) than with either ACPA or rheumatoid factor (RF) alone (AUC = 0.80 or 0.79, respectively). The discriminatory performance of serum CTGF was consistent across all inflammatory conditions tested (AUC >0.92 in all cases), with the sole exception of pSS. Serum CTGF did not vary with symptom duration or disease activity. Serum CTGF is a promising diagnostic biomarker for RA, with performance in the current study better than either ACPA or RF.

Dynamic Vibration Cooperates with Connective Tissue Growth Factor to Modulate Stem Cell Behaviors

PubMed Central

Tong, Zhixiang; Zerdoum, Aidan B.; Duncan, Randall L.

2014-01-01

Vocal fold disorders affect 3–9% of the U.S. population. Tissue engineering offers an alternative strategy for vocal fold repair. Successful engineering of vocal fold tissues requires a strategic combination of therapeutic cells, biomimetic scaffolds, and physiologically relevant mechanical and biochemical factors. Specifically, we aim to create a vocal fold-like microenvironment to coax stem cells to adopt the phenotype of vocal fold fibroblasts (VFFs). Herein, high frequency vibratory stimulations and soluble connective tissue growth factor (CTGF) were sequentially introduced to mesenchymal stem cells (MSCs) cultured on a poly(ɛ-caprolactone) (PCL)-derived microfibrous scaffold for a total of 6 days. The initial 3-day vibratory culture resulted in an increased production of hyaluronic acids (HA), tenascin-C (TNC), decorin (DCN), and matrix metalloproteinase-1 (MMP1). The subsequent 3-day CTGF treatment further enhanced the cellular production of TNC and DCN, whereas CTGF treatment alone without the vibratory preconditioning significantly promoted the synthesis of collagen I (Col 1) and sulfated glycosaminoglycans (sGAGs). The highest level of MMP1, TNC, Col III, and DCN production was found for cells being exposed to the combined vibration and CTGF treatment. Noteworthy, the vibration and CTGF elicited a differential stimulatory effect on elastin (ELN), HA synthase 1 (HAS1), and fibroblast-specific protein-1 (FSP-1). The mitogenic activity of CTGF was only elicited in naïve cells without the vibratory preconditioning. The combined treatment had profound, but opposite effects on mitogen-activated protein kinase (MAPK) pathways, Erk1/2 and p38, and the Erk1/2 pathway was critical for the observed mechano-biochemical responses. Collectively, vibratory stresses and CTGF signals cooperatively coaxed MSCs toward a VFF-like phenotype and accelerated the synthesis and remodeling of vocal fold matrices. PMID:24456068

P38 pathway as a key downstream signal of connective tissue growth factor to regulate metastatic potential in non-small-cell lung cancer.

PubMed

Kato, Shinichiro; Yokoyama, Satoru; Hayakawa, Yoshihiro; Li, Luhui; Iwakami, Yusuke; Sakurai, Hiroaki; Saiki, Ikuo

2016-10-01

Although the secretory matricellular protein connective tissue growth factor (CTGF) has been reported to be related to lung cancer metastasis, the precise mechanism by which CTGF regulates lung cancer metastasis has not been elucidated. In the present study, we show the molecular link between CTGF secretion and the p38 pathway in the invasive and metastatic potential of non-small-cell lung cancer (NSCLC). Among three different human NSCLC cell lines (PC-14, A549, and PC-9), their in vitro invasiveness was inversely correlated with the level of CTGF secretion. By supplementing or reducing CTGF secretion in NSCLC culture, dysregulation of the invasive and metastatic potential of NSCLC cell lines was largely compensated. By focusing on the protein kinases that are known to be regulated by CTGF, we found that the p38 pathway is a key downstream signal of CTGF to regulate the metastatic potential of NSCLC. Importantly, a negative correlation between CTGF and phosphorylation status of p38 was identified in The Cancer Genome Atlas lung adenocarcinoma dataset. In the context of the clinical importance of our findings, we showed that p38 inhibitor, SB203580, reduced the metastatic potential of NSCLC secreting low levels of CTGF. Collectively, our present findings indicate that the CTGF/p38 axis is a novel therapeutic target of NSCLC metastasis, particularly NSCLC secreting low levels of CTGF. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Glucose impairs tamoxifen responsiveness modulating connective tissue growth factor in breast cancer cells.

PubMed

Ambrosio, Maria Rosaria; D'Esposito, Vittoria; Costa, Valerio; Liguoro, Domenico; Collina, Francesca; Cantile, Monica; Prevete, Nella; Passaro, Carmela; Mosca, Giusy; De Laurentiis, Michelino; Di Bonito, Maurizio; Botti, Gerardo; Franco, Renato; Beguinot, Francesco; Ciccodicola, Alfredo; Formisano, Pietro

2017-12-12

Type 2 diabetes and obesity are negative prognostic factors in patients with breast cancer (BC). We found that sensitivity to tamoxifen was reduced by 2-fold by 25 mM glucose (High Glucose; HG) compared to 5.5 mM glucose (Low Glucose; LG) in MCF7 BC cells. Shifting from HG to LG ameliorated MCF7 cell responsiveness to tamoxifen. RNA-Sequencing of MCF7 BC cells revealed that cell cycle-related genes were mainly affected by glucose. Connective Tissue Growth Factor (CTGF) was identified as a glucose-induced modulator of cell sensitivity to tamoxifen. Co-culturing MCF7 cells with human adipocytes exposed to HG, enhanced CTGF mRNA levels and reduced tamoxifen responsiveness of BC cells. Inhibition of adipocyte-released IL8 reverted these effects. Interestingly, CTGF immuno-detection in bioptic specimens from women with estrogen receptor positive (ER + ) BC correlated with hormone therapy resistance, distant metastases, reduced overall and disease-free survival. Thus, glucose affects tamoxifen responsiveness directly modulating CTGF in BC cells, and indirectly promoting IL8 release by adipocytes.

The Role of the Rho/ROCK Pathway in Ang II and TGF-β1-Induced Atrial Remodeling

PubMed Central

Lu, Gui-Hua; Xu, Cheng-Gui; Xu, Zhe; Tang, Kai; Cheng, Yun-Jiu; Gao, Xiu-Ren; Wu, Su-Hua

2016-01-01

Objectives To study the role of the Rho/ROCK pathway in Ang II and TGF-β1-induced atrial remodeling. Methods and Results A canine atrial fibrillation (AF) model was established by rapid atrial pacing (RAP) of the left atrium. The roles of TGF-β1, the RhoA/ROCK signaling pathway and connective tissue growth factor (CTGF) in atrial remodeling were studied via both in vitro and in vivo experiments. Each of the dogs that received RAP developed persistent AF within 4 weeks. The mRNA expression levels of TGF-β1 (1.32±0.38), Collagen-I(1.33±0.91), CTGF(5.83±3.71), RhoA(1.23±0.57) and ROCK-1 (1.02±0.27) in the left atrium were significantly increased following 4 weeks of RAP. Angiotensin II (Ang II) induced the proliferation of atrial fibroblasts and up-regulated the expression of both CTGF and ROCK-1 in a dose-dependent manner. Simvastatin and Y27632 reversed Ang II-induced CFs proliferation, as well as ROCK-1(0.89±0.05 and 1.27±0.03, respectively) and CTGF (0.87±0.04 and 0.91±0.02, respectively) expression. The expression mRNA of ROCK-1(1.74±0.13) and CTGF (2.28±0.11) can upregulated by TGF-β1, and down-regulated by Simvastatin (1.22±0.03 vs 2.27±0.11), Y27632 (1.01±0.04 vs 1.64±0.03), Los (1.04±0.11 vs 1.26±0.05), respectively. Losartan and Simvastatin attenuated the effects of TGF-β1, inhibited RhoA activity as opposed to RhoA protein expression. Y27632 had no effect on either the expression or the activity of RhoA. Conclusions The increased expression of profibrotic factors (CTGF, ROCK1 and Smad2/3) played an important role in our RAP-induced AF model. Increased atrial profibrotic factors involve the activation of either the TGF-β1/RhoA/ROCK-1 or the TGF-β1/Smad2/3 signaling pathway. PMID:27611832

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration.

PubMed

Xu, Kang; Sun, Yanjun; Kh Al-Ani, Mohanad; Wang, Chunli; Sha, Yongqiang; Sung, Kl Paul; Dong, Nianguo; Qiu, Xuefeng; Yang, Li

2018-01-03

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80-90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young's were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect

Connective tissue growth factor is expressed in bone marrow stromal cells and promotes interleukin-7-dependent B lymphopoiesis.

PubMed

Cheung, Laurence C; Strickland, Deborah H; Howlett, Meegan; Ford, Jette; Charles, Adrian K; Lyons, Karen M; Brigstock, David R; Goldschmeding, Roel; Cole, Catherine H; Alexander, Warren S; Kees, Ursula R

2014-07-01

Hematopoiesis occurs in a complex bone marrow microenvironment in which bone marrow stromal cells provide critical support to the process through direct cell contact and indirectly through the secretion of cytokines and growth factors. We report that connective tissue growth factor (Ctgf, also known as Ccn2) is highly expressed in murine bone marrow stromal cells. In contrast, connective tissue growth factor is barely detectable in unfractionated adult bone marrow cells. While connective tissue growth factor has been implicated in hematopoietic malignancies, and is known to play critical roles in skeletogenesis and regulation of bone marrow stromal cells, its role in hematopoiesis has not been described. Here we demonstrate that the absence of connective tissue growth factor in mice results in impaired hematopoiesis. Using a chimeric fetal liver transplantation model, we show that absence of connective tissue growth factor has an impact on B-cell development, in particular from pro-B to more mature stages, which is linked to a requirement for connective tissue growth factor in bone marrow stromal cells. Using in vitro culture systems, we demonstrate that connective tissue growth factor potentiates B-cell proliferation and promotes pro-B to pre-B differentiation in the presence of interleukin-7. This study provides a better understanding of the functions of connective tissue growth factor within the bone marrow, showing the dual regulatory role of the growth factor in skeletogenesis and in stage-specific B lymphopoiesis. Copyright© Ferrata Storti Foundation.

Connective tissue growth factor is expressed in bone marrow stromal cells and promotes interleukin-7-dependent B lymphopoiesis

PubMed Central

Cheung, Laurence C.; Strickland, Deborah H.; Howlett, Meegan; Ford, Jette; Charles, Adrian K.; Lyons, Karen M.; Brigstock, David R.; Goldschmeding, Roel; Cole, Catherine H.; Alexander, Warren S.; Kees, Ursula R.

2014-01-01

Hematopoiesis occurs in a complex bone marrow microenvironment in which bone marrow stromal cells provide critical support to the process through direct cell contact and indirectly through the secretion of cytokines and growth factors. We report that connective tissue growth factor (Ctgf, also known as Ccn2) is highly expressed in murine bone marrow stromal cells. In contrast, connective tissue growth factor is barely detectable in unfractionated adult bone marrow cells. While connective tissue growth factor has been implicated in hematopoietic malignancies, and is known to play critical roles in skeletogenesis and regulation of bone marrow stromal cells, its role in hematopoiesis has not been described. Here we demonstrate that the absence of connective tissue growth factor in mice results in impaired hematopoiesis. Using a chimeric fetal liver transplantation model, we show that absence of connective tissue growth factor has an impact on B-cell development, in particular from pro-B to more mature stages, which is linked to a requirement for connective tissue growth factor in bone marrow stromal cells. Using in vitro culture systems, we demonstrate that connective tissue growth factor potentiates B-cell proliferation and promotes pro-B to pre-B differentiation in the presence of interleukin-7. This study provides a better understanding of the functions of connective tissue growth factor within the bone marrow, showing the dual regulatory role of the growth factor in skeletogenesis and in stage-specific B lymphopoiesis. PMID:24727816

Blockade of lysophosphatidic acid receptors LPAR1/3 ameliorates lung fibrosis induced by irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gan, Lu; Xue, Jian-Xin; Laboratory of Stem Cell Biology, West China Hospital, Sichuan University, Chengdu

2011-05-27

Highlights: {yields} Lysophosphatidic acid (LPA) levels and its receptors LPAR1/3 transcripts were elevated during the development of radiation-induced lung fibrosis. {yields} Lung fibrosis was obviously alleviated in mice treated with the dual LPAR1/3 antagonist, VPC12249. {yields} VPC12249 administration effectively inhibited radiation-induced fibroblast accumulation in vivo, and suppressed LPA-induced fibroblast proliferation in vitro. {yields} LPA-LPAR1/3 signaling regulated TGF{beta}1 and CTGF expressions in radiation-challenged lungs, but only influenced CTGF expression in cultured fibroblasts. {yields} LPA-LPAR1/3 signaling induced fibroblast proliferation through a CTGF-dependent pathway, rather than through TGF{beta}1 activation. -- Abstract: Lung fibrosis is a common and serious complication of radiation therapy formore » lung cancer, for which there are no efficient treatments. Emerging evidence indicates that lysophosphatidic acid (LPA) and its receptors (LPARs) are involved in the pathogenesis of fibrosis. Here, we reported that thoracic radiation with 16 Gy in mice induced development of radiation lung fibrosis (RLF) accompanied by obvious increases in LPA release and LPAR1 and LPAR3 (LPAR1/3) transcripts. RLF was significantly alleviated in mice treated with the dual LPAR1/3 antagonist, VPC12249. VPC12249 administration effectively prolonged animal survival, restored lung structure, inhibited fibroblast accumulation and reduced collagen deposition. Moreover, profibrotic cytokines in radiation-challenged lungs obviously decreased following administration of VPC12249, including transforming growth factor {beta}1 (TGF{beta}1) and connective tissue growth factor (CTGF). In vitro, LPA induced both fibroblast proliferation and CTGF expression in a dose-dependent manner, and both were suppressed by blockade of LPAR1/3. The pro-proliferative activity of LPA on fibroblasts was inhibited by siRNA directed against CTGF. Together, our data suggest that the LPA

Temporal and Spatial Expression of CCN Genes in Zebrafish

PubMed Central

Fernando, Carol A; Conrad, Patricia A; Bartels, Cynthia F; Marques, Tomas; To, Michael; Balow, Stephanie A; Nakamura, Yukio; Warman, Matthew L

2010-01-01

The six mammalian CCN genes (Cyr61, CTGF, Nov, WISP1, WISP2, WISP3) encode a family of secreted, cysteine-rich, multimodular proteins having roles in cell proliferation, adhesion, migration, and differentiation during embryogenesis, wound healing, and angiogenesis. We used bioinformatics to identify 9 CCN genes in zebrafish (zCCNs), 6 of which have not been previously described. When compared with mammalian CCN family members, 3 were paralogs of Cyr61, 2 of CTGF, 2 of WISP1, 1 of WISP2, and 1 of WISP3. No paralog of Nov was found. In situ hybridization was performed to characterize the sites of expression of the zCCNs during early zebrafish development. zCCNs demonstrated both unique and overlapping patterns of expression, suggesting potential division of labor between orthologous genes and providing an alternate approach to gene function studies that will complement studies in mammalian models. Developmental Dynamics 239:1755–1767, 2010. © 2010 Wiley-Liss, Inc. PMID:20503371

Adiponectin Is Involved in Connective Tissue Growth Factor-Induced Proliferation, Migration and Overproduction of the Extracellular Matrix in Keloid Fibroblasts.

PubMed

Luo, Limin; Li, Jun; Liu, Han; Jian, Xiaoqing; Zou, Qianlei; Zhao, Qing; Le, Qu; Chen, Hongdou; Gao, Xinghua; He, Chundi

2017-05-12

Adiponectin, an adipocyte-derived hormone, exerts pleiotropic biological effects on metabolism, inflammation, vascular homeostasis, apoptosis and immunity. Recently, adiponectin has been suggested to attenuate the progression of human dermal fibrosis. Connective tissue growth factor (CTGF) is induced in keloids and is thought to be participated in the formation of keloid fibrosis. However, the roles played by adiponectin in keloids remain unclear. In this study, we explored the effects of adiponectin on CTGF-induced cell proliferation, migration and the deposition of extracellular matrix (ECM) and their associated intracellular signalling pathways in keloid fibroblasts (KFs). We also explored possible mechanisms of keloid pathogenesis. Primary fibroblast cultures were established from foreskin biopsies and skin biopsies from patients with keloids. The expression of adiponectin and adiponectin receptors (adipoRs) was evaluated by reverse transcription-PCR (RT-PCR), quantitative real-time RT-PCR, immunofluorescence staining, and immunohistochemical analysis. Next, KFs and normal dermal fibroblasts (NFs) were treated with CTGF in the presence or absence of adiponectin. A cell counting kit-8 (CCK-8) and the Transwell assay were used to examine cell proliferation and migration. The level of the collagen I, fibronectin (FN) and α-smooth muscle actin (α-SMA) mRNAs and proteins were determined by quantitative real-time RT-PCR and western blotting. The effects of RNA interference (RNAi) targeting the adipoR genes were detected. Phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase-protein kinase (PI3K-Akt) were examined by western blotting to further investigate the signalling pathways. Furthermore, inhibitors of signal transduction pathways were investigated. The expression levels of adiponectin and adipoRs were significantly decreased in keloids compared with those

Connective Tissue Growth Factor Domain 4 Amplifies Fibrotic Kidney Disease through Activation of LDL Receptor-Related Protein 6.

PubMed

Johnson, Bryce G; Ren, Shuyu; Karaca, Gamze; Gomez, Ivan G; Fligny, Cécile; Smith, Benjamin; Ergun, Ayla; Locke, George; Gao, Benbo; Hayes, Sebastian; MacDonnell, Scott; Duffield, Jeremy S

2017-06-01

Connective tissue growth factor (CTGF), a matrix-associated protein with four distinct cytokine binding domains, has roles in vasculogenesis, wound healing responses, and fibrogenesis and is upregulated in fibroblasts and myofibroblasts in disease. Here, we investigated the role of CTGF in fibrogenic cells. In mice, tissue-specific inducible overexpression of CTGF by kidney pericytes and fibroblasts had no bearing on nephrogenesis or kidney homeostasis but exacerbated inflammation and fibrosis after ureteral obstruction. These effects required the WNT receptor LDL receptor-related protein 6 (LRP6). Additionally, pericytes isolated from these mice became hypermigratory and hyperproliferative on overexpression of CTGF. CTGF is cleaved in vivo into distinct domains. Treatment with recombinant domain 1, 1+2 (N terminus), or 4 (C terminus) independently activated myofibroblast differentiation and wound healing responses in cultured pericytes, but domain 4 showed the broadest profibrotic activity. Domain 4 exhibited low-affinity binding to LRP6 in in vitro binding assays, and inhibition of LRP6 or critical signaling cascades downstream of LRP6, including JNK and WNT/ β -catenin, inhibited the biologic activity of domain 4. Administration of blocking antibodies specifically against CTGF domain 4 or recombinant Dickkopf-related protein-1, an endogenous inhibitor of LRP6, effectively inhibited inflammation and fibrosis associated with ureteral obstruction in vivo Therefore, domain 4 of CTGF and the WNT signaling pathway are important new targets in fibrosis. Copyright © 2017 by the American Society of Nephrology.

Expression of cyclooxygenase-1 and cyclooxygenase-2, syndecan-1 and connective tissue growth factor in benign and malignant breast tissue from premenopausal women.

PubMed

Fahlén, M; Zhang, H; Löfgren, L; Masironi, B; von Schoultz, E; von Schoultz, B; Sahlin, L

2017-05-01

Stromal factors have been identified as important for tumorigenesis and metastases of breast cancer. From 49 premenopausal women, samples were collected from benign or malignant tumors and the seemingly normal tissue adjacent to the tumor. The factors studied, with real-time polymerase chain reaction (PCR) and immunohistochemistry, were cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), syndecan-1 (S-1) and connective tissue growth factor (CTGF). COX-1 and S-1 mRNA levels were higher in the malignant tumors than in normal and benign tissues. The COX-2 mRNA level was lower in the malignant tumor than in the normal tissue, while CTGF mRNA did not differ between the groups. COX-1 immunostaining was higher in stroma from malignant tumors than in benign tissues, whereas COX-2 immunostaining was higher in the malignant tissue. Glandular S-1 immunostaining was lower in malignant tumors compared to benign and normal tissues, and the opposite was found in stroma. Conclusively, mRNA levels of COX-1 and COX-2 were oppositely regulated, with COX-1 being increased in the malignant tumor while COX-2 was decreased. S-1 protein localization switched from glandular to stromal cells in malignant tissues. Thus, these markers are, in premenopausal women, localized and regulated differently in normal/benign breast tissue as compared to the malignant tumor.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

PubMed Central

2011-01-01

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life. PMID:21481241

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with conjugated linoleic acid during gestation and suckling.

PubMed

Selga, Elisabet; Pérez-Cano, Francisco J; Franch, Angels; Ramírez-Santana, Carolina; Rivero, Montserrat; Ciudad, Carlos J; Castellote, Cristina; Noé, Véronique

2011-04-11

Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip(®) Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Radiation-Induced Esophagitis In Vivo and In Vitro Reveals That Epidermal Growth Factor Is a Potential Candidate for Therapeutic Intervention Strategy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kyung Su; Jeon, Seong-Uk; Lee, Chan-Ju

Purpose: To establish and characterize radiation-induced esophagitis (RIE) in vivo and in vitro. Methods and Materials: Fractionated thoracic irradiation at 0, 8, 12, or 15 Gy was given daily for 5 days to Balb/c or C57Bl/6 mice. Changes in body weight gain and daily food intake were assessed. At the end of the study, we removed the esophagus and examined histology by hematoxylin and eosin staining, immune cell infiltration and apoptosis by fluorescence-activated cell sorting, and gene expression changes by quantitative real-time polymerase chain reaction. Het-1A human esophageal epithelial cells were irradiated at 6 Gy, treated with recombinant human growth factors, and examined for genemore » expression changes, apoptosis, proliferation, and signal transduction pathways. Results: We observed that irradiation at 12 Gy or 15 Gy per fraction produced significant reduction in body weight and decreased food intake in Balb/c mice but not as much in C57Bl/6 mice. Further analyses of Balb/c mice irradiated at 12 Gy/fraction revealed attenuated epithelium, inflamed mucosa, and increased numbers of infiltrating CD4+ helper T cells and apoptotic cells. Moreover, we found that expression of tissue inhibitor for metalloproteinase-1, plasminogen activator inhibitor-1, granulocyte macrophage-colony stimulating factor, vascular endothelial growth factor, and stromal-derived factor-1 were increased, whereas epidermal growth factor (EGF) was decreased. Irradiated Het-1A cells similarly showed a significant decrease in expression of EGF and connective tissue growth factor (CTGF). Treatment of EGF but not CTGF partially protected Het-1A cells from radiation-induced apoptosis and revealed phosphorylation of EGFR, AKT, and ERK signaling pathways. Conclusions: We established a mouse model of RIE in Balb/c mice with 12 Gy × 5 fractions, which showed reduced body weight gain, food intake, and histopathologic features similar to those of human esophagitis. Decreased EGF

CCN3 (NOV) Is a Negative Regulator of CCN2 (CTGF) and a Novel Endogenous Inhibitor of the Fibrotic Pathway in an in Vitro Model of Renal Disease

PubMed Central

Riser, Bruce L.; Najmabadi, Feridoon; Perbal, Bernard; Peterson, Darryl R.; Rambow, Jo Ann; Riser, Melisa L.; Sukowski, Ernest; Yeger, Herman; Riser, Sarah C.

2009-01-01

Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-β, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-β treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-β treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-β to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments. PMID:19359517

CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease.

PubMed

Riser, Bruce L; Najmabadi, Feridoon; Perbal, Bernard; Peterson, Darryl R; Rambow, Jo Ann; Riser, Melisa L; Sukowski, Ernest; Yeger, Herman; Riser, Sarah C

2009-05-01

Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-beta, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-beta treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-beta treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-beta to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments.

Downregulation of connective tissue growth factor reduces migration and invasiveness of osteosarcoma cells.

PubMed

Huang, Yinjun; Zhao, Shichang; Zhang, Changqing; Li, Xiaolin

2016-02-01

As one of the most serious types of primary bone tumor, osteosarcoma (OSA) features metastatic lesions, and resistance to chemotherapy is common. The underlying mechanisms of these characteristics may account for the failure of treatments and the poor prognosis of patients with OSA. It has been reported that inhibition of Cyr61 suppresses OSA cell proliferation as it represents a target of statins. In addition to cystein‑rich protein 61 (Cyr61) and nephroblastoma overexpression, connective tissue growth factor (CTGF) is a member of the CCN family and may therefore exhibit effects on human OSA cells similar to those of Cyr61. In the current study, acridine orange/ethidium bromide staining were used to determine the rate of apoptosis. The present study demonstrated that small interfering RNA‑mediated silencing of CTGF promoted cell death and suppressed OSA cell migration and invasion, as indicated by wound healing and Transwell assays, while lentivirus‑mediated overexpression of CTGF reversed these effects. Furthermore, a colorimetric caspase assay demonstrated that CTGF knockdown enhanced the efficacy of chemotherapeutic drugs. The results of the present study provided a novel molecular target which may be utilized for the treatment of metastatic OSA.

Chemical chaperon 4-phenylbutyrate protects against the endoplasmic reticulum stress-mediated renal fibrosis in vivo and in vitro.

PubMed

Liu, Shing-Hwa; Yang, Ching-Chin; Chan, Ding-Cheng; Wu, Cheng-Tien; Chen, Li-Ping; Huang, Jenq-Wen; Hung, Kuan-Yu; Chiang, Chih-Kang

2016-04-19

Renal tubulointerstitial fibrosis is the common and final pathologic change of kidney in end-stage renal disease. Interesting, endoplasmic reticulum (ER) stress is known to contribute to the pathophysiological mechanisms during the development of renal fibrosis. Here, we investigated the effects of chemical chaperon sodium 4-phenylbutyrate (4-PBA) on renal fibrosis in vivo and in vitro. In a rat unilateral ureteral obstruction (UUO) model, 4-PBA mimicked endogenous ER chaperon in the kidneys and significantly reduced glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), activating transcription factor 4 (ATF4), and phosphorylated JNK protein expressions as well as restored spliced X-box-binding protein 1 (XBP1) expressions in the kidneys of UUO rats. 4-PBA also attenuated the increases of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) protein expressions, tubulointerstitial fibrosis, and apoptosis in the kidneys of UUO rats. Moreover, transforming growth factor (TGF)-β markedly increased ER stress-associated molecules, profibrotic factors, and apoptotic markers in the renal tubular cells (NRK-52E), all of which could be significantly counteracted by 4-PBA treatment. 4-PBA also diminished TGF-β-increased CTGF promoter activity and CTGF mRNA expression in NRK-52E cells. Taken together, our results indicated that 4-PBA acts as an ER chaperone to ameliorate ER stress-induced renal tubular cell apoptosis and renal fibrosis.

* Hierarchically Structured Electrospun Scaffolds with Chemically Conjugated Growth Factor for Ligament Tissue Engineering.

PubMed

Pauly, Hannah M; Sathy, Binulal N; Olvera, Dinorath; McCarthy, Helen O; Kelly, Daniel J; Popat, Ketul C; Dunne, Nicholas J; Haut Donahue, Tammy Lynn

2017-08-01

The anterior cruciate ligament (ACL) of the knee is vital for proper joint function and is commonly ruptured during sports injuries or car accidents. Due to a lack of intrinsic healing capacity and drawbacks with allografts and autografts, there is a need for a tissue-engineered ACL replacement. Our group has previously used aligned sheets of electrospun polycaprolactone nanofibers to develop solid cylindrical bundles of longitudinally aligned nanofibers. We have shown that these nanofiber bundles support cell proliferation and elongation and the hierarchical structure and material properties are similar to the native human ACL. It is possible to combine multiple nanofiber bundles to create a scaffold that attempts to mimic the macroscale structure of the ACL. The goal of this work was to develop a hierarchical bioactive scaffold for ligament tissue engineering using connective tissue growth factor (CTGF)-conjugated nanofiber bundles and evaluate the behavior of mesenchymal stem cells (MSCs) on these scaffolds in vitro and in vivo. CTGF was immobilized onto the surface of individual nanofiber bundles or scaffolds consisting of multiple nanofiber bundles. The conjugation efficiency and the release of conjugated CTGF were assessed using X-ray photoelectron spectroscopy, assays, and immunofluorescence staining. Scaffolds were seeded with MSCs and maintained in vitro for 7 days (individual nanofiber bundles), in vitro for 21 days (scaled-up scaffolds of 20 nanofiber bundles), or in vivo for 6 weeks (small scaffolds of 4 nanofiber bundles), and ligament-specific tissue formation was assessed in comparison to non-CTGF-conjugated control scaffolds. Results showed that CTGF conjugation encouraged cell proliferation and ligament-specific tissue formation in vitro and in vivo. The results suggest that hierarchical electrospun nanofiber bundles conjugated with CTGF are a scalable and bioactive scaffold for ACL tissue engineering.

Investigating the association between polymorphisms in connective tissue growth factor and susceptibility to colon carcinoma.

PubMed

Ahmad, Abrar; Askari, Shlear; Befekadu, Rahel; Hahn-Strömberg, Victoria

2015-04-01

There have been numerous studies on the gene expression of connective tissue growth factor (CTGF) in colorectal cancer, however very few have investigated polymorphisms in this gene. The present study aimed to determine whether single nucleotide polymorphisms (SNPs) in the CTGF gene are associated with a higher susceptibility to colon cancer and/or an invasive tumor growth pattern. The CTGF gene was genotyped for seven SNPs (rs6918698, rs1931002, rs9493150, rs12526196, rs12527705, rs9399005 and rs12527379) by pyrosequencing. Formalin‑fixed paraffin‑embedded tissue samples (n=112) from patients diagnosed with colon carcinoma, and an equal number of blood samples from healthy controls, were selected for genomic DNA extraction. The complexity index was measured using images of tumor samples (n=64) stained for cytokeratin‑8. The images were analyzed and correlated with the identified CTGF SNPs and clinicopathological parameters of the patients, including age, gender, tumor penetration, lymph node metastasis, systemic metastasis, differentiation and localization of tumor. It was demonstrated that the frequency of the SNP rs6918698 GG genotype was significantly associated (P=0.05) with an increased risk of colon cancer, as compared with the GC and CC genotypes. The other six SNPs (rs1931002, rs9493150, rs12526196, rs12527705, rs9399005 and rs12527379) exhibited no significant difference in the genotype and allele frequencies between patients diagnosed with colon carcinoma and the normal healthy population. A trend was observed between genotype variation at rs6918698 and the complexity index (P=0.052). The complexity index and genotypes for any of the studied SNPs were not significantly correlated with clinical or pathological parameters of the patients. These results indicate that the rs6918698 GG genotype is associated with an increased risk of developing colon carcinoma, and genetic variations at the rs6918698 are associated with the growth pattern of the tumor

Molecular factors involved in the hypolipidemic- and insulin-sensitizing effects of a ginger (Zingiber officinale Roscoe) extract in rats fed a high-fat diet.

PubMed

de Las Heras, Natalia; Valero-Muñoz, María; Martín-Fernández, Beatriz; Ballesteros, Sandra; López-Farré, Antonio; Ruiz-Roso, Baltasar; Lahera, Vicente

2017-02-01

Hypolipidemic and hypoglycemic properties of ginger in animal models have been reported. However, information related to the mechanisms and factors involved in the metabolic effects of ginger at a hepatic level are limited. The aim of the present study was to investigate molecular factors involved in the hypoglycemic and hypolipidemic effects of a hydroethanolic ginger extract (GE) in the liver of rats fed a high-fat diet (HFD). The study was conducted in male Wistar rats divided into the following 3 groups: (i) Rats fed a standard diet (3.5% fat), the control group; (ii) rats fed an HFD (33.5% fat); and (iii) rats fed an HFD treated with GE (250 mg·kg -1 ·day -1 ) for 5 weeks (HFD+GE). Plasma levels of glucose, insulin, lipid profile, leptin, and adiponectin were measured. Liver expression of glycerol phosphate acyltransferase (GPAT), cholesterol 7 alpha-hydroxylase, peroxisome proliferator-activated receptors (PPAR), PPARα and PPARγ, glucose transporter 2 (GLUT-2), liver X receptor, sterol regulatory element-binding protein (SREBP1c), connective tissue growth factor (CTGF), and collagen I was measured. Data were analyzed using a 1-way ANOVA, followed by a Newman-Keuls test if differences were noted. The study showed that GE improved lipid profile and attenuated the increase of plasma levels of glucose, insulin, and leptin in HFD rats. This effect was associated with a higher liver expression of PPARα, PPARγ, and GLUT-2 and an enhancement of plasma adiponectin levels. Furthermore, GE reduced liver expression of GPAT, SREBP1c, CTGF, and collagen I. The results suggest that GE might be considered as an alternative therapeutic strategy in the management of overweight and hepatic and metabolic-related alterations.

Condyle and mandibular bone change after unilateral condylar neck fracture in growing rats.

PubMed

Hu, Y; Yang, H-f; Li, S; Chen, J-z; Luo, Y-w; Yang, C

2012-08-01

Unilateral fracture of the condylar neck in immature subjects might lead to mandible asymmetry and condyle remodelling. A rat model was used to investigate mandibular deviation and condylar remodelling associated with condyle fracture. 72 4-week-old male rats were randomly divided into three groups: an experimental group (unilateral transverse condylar fracture induced surgically), a sham operation group (surgical exposure but no fracture), and a non-operative control group (no operation). The rats were killed at intervals up to 9weeks after surgery, and outcomes were assessed using various measures of mandible deviation, histological and X-ray observation, and immunohistochemical measures of expression levels of connective tissue growth factor (CTGF) and type II collagen (Col II). The fracture led to the degeneration of mandibular size, associated with atrophy of fractured condylar process. Progressive remodelling of cartilage and increasing expression levels of CTGF and Col II were found. The authors conclude that condylar fracture can lead to asymmetries in mandible and condyle remodelling and expression of CTGF and Col II in condylar cartilage on both the ipsilateral and the contralateral sides. Copyright © 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Effect and Mechanism of QiShenYiQi Pill on Experimental Autoimmune Myocarditis Rats.

PubMed

Lv, Shichao; Wu, Meifang; Li, Meng; Wang, Qiang; Xu, Ling; Wang, Xiaojing; Zhang, Junping

2016-03-06

To observe the effect of QiShenYiQi pill (QSYQ) on experimental autoimmune myocarditis rats, and to explore its mechanism of action. Lewis rats underwent the injection of myocardial myosin mixed with Freund's complete adjuvant were randomized into 3 groups: model, valsartan, and QSYQ groups. Rats injected with phosphate-buffered saline (PBS) mixed with Freund's complete adjuvant were used as the control group. Rats were euthanized at 4 and 8 weeks, and we weighed rat body mass, heart mass, and left ventricular mass. Myocardium sections were stained with hematoxylin and eosin (H&E) and Masson trichrome. Myocardial TGF-β1 and CTGF protein expression was detected by immunohistochemistry, and myocardial TGF-β1 and CTGF mRNA expression was detected by real-time qPCR. QSYQ reduced HMI and LVMI, as well as the histological score of hearts and CVF, which further decreased over time, and its effect was significantly greater than that of valsartan at 4 and 8 weeks. After 4 weeks, QSYQ inhibited the protein and mRNA expression of TGF-β1 and CTGF, and its effect on lowering CTGF was significantly greater than that of valsartan. In addition, after 8 weeks, QSYQ also inhibited the protein and mRNA expression of CTGF, whereas there was no significant difference in the expression of myocardial TGF-β1. This study provides evidence that QSYQ can improve cardiac remodeling of experimental autoimmune myocarditis rats. It also effectively improved the degree of myocardial fibrosis, which is related to the mechanism of regulation of TGF-β1 CTGF.

Chlorogenic Acid Inhibits Liver Fibrosis by Blocking the miR-21-Regulated TGF-β1/Smad7 Signaling Pathway in Vitro and in Vivo.

PubMed

Yang, Fan; Luo, Lei; Zhu, Zhi-De; Zhou, Xuan; Wang, Yao; Xue, Juan; Zhang, Juan; Cai, Xin; Chen, Zhi-Lin; Ma, Qian; Chen, Yun-Fei; Wang, Yu-Jie; Luo, Ying-Ying; Liu, Pan; Zhao, Lei

2017-01-01

Aims: Chlorogenic acid (CGA) is a phenolic acid that has a wide range of pharmacological effects. However, the protective effects and mechanisms of CGA on liver fibrosis are not clear. This study explored the effects of CGA on miR-21-regulated TGF-β1/Smad7 liver fibrosis in the hepatic stellate LX2 cell line and in CCl4-induced liver fibrosis in Sprague-Dawley rats. Methods: The mRNA expression of miR-21, Smad7, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase 1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), and transforming growth factor-β1 (TGF-β1) and the protein levels of Smad2, p-Smad2, Smad3, p-Smad3, Smad2/3, p-Smad2/3, Smad7, CTGF, α-SMA, TIMP-1, MMP-9 and TGF-β1 were assayed in LX2 cells and liver tissue. The effects of CGA after miR-21 knockdown or overexpression were analyzed in LX2 cells. The liver tissue and serum were collected for histopathological examination, immunohistochemistry (IHC) and ELISA. Results: The mRNA expression of miR-21, CTGF, α-SMA, TIMP-1, and TGF-β1 and the protein expression of p-Smad2, p-Smad3, p-Smad2/3, CTGF, α-SMA, TIMP-1, and TGF-β1 were inhibited by CGA both in vitro and in vivo . Meanwhile, CGA elevated the mRNA and protein expression of Smad7 and MMP-9. After miR-21 knockdown and overexpression, the downstream molecules also changed accordingly. CGA also lessened the degree of liver fibrosis in the pathological manifestation and reduced α-SMA and collagen I expression in liver tissue and TGF-β1 in serum. Conclusion: CGA might relieve liver fibrosis through the miR-21-regulated TGF-β1/Smad7 signaling pathway, which suggests that CGA might be a new anti-fibrosis agent that improves liver fibrosis.

Effect and Mechanism of QiShenYiQi Pill on Experimental Autoimmune Myocarditis Rats

PubMed Central

Lv, Shichao; Wu, Meifang; Li, Meng; Wang, Qiang; Xu, Ling; Wang, Xiaojing; Zhang, Junping

2016-01-01

Background To observe the effect of QiShenYiQi pill (QSYQ) on experimental autoimmune myocarditis rats, and to explore its mechanism of action. Material/methods Lewis rats underwent the injection of myocardial myosin mixed with Freund’s complete adjuvant were randomized into 3 groups: model, valsartan, and QSYQ groups. Rats injected with phosphate-buffered saline (PBS) mixed with Freund’s complete adjuvant were used as the control group. Rats were euthanized at 4 and 8 weeks, and we weighed rat body mass, heart mass, and left ventricular mass. Myocardium sections were stained with hematoxylin and eosin (H&E) and Masson trichrome. Myocardial TGF-β1 and CTGF protein expression was detected by immunohistochemistry, and myocardial TGF-β1 and CTGF mRNA expression was detected by real-time qPCR. Results QSYQ reduced HMI and LVMI, as well as the histological score of hearts and CVF, which further decreased over time, and its effect was significantly greater than that of valsartan at 4 and 8 weeks. After 4 weeks, QSYQ inhibited the protein and mRNA expression of TGF-β1 and CTGF, and its effect on lowering CTGF was significantly greater than that of valsartan. In addition, after 8 weeks, QSYQ also inhibited the protein and mRNA expression of CTGF, whereas there was no significant difference in the expression of myocardial TGF-β1. Conclusions This study provides evidence that QSYQ can improve cardiac remodeling of experimental autoimmune myocarditis rats. It also effectively improved the degree of myocardial fibrosis, which is related to the mechanism of regulation of TGF-β1 CTGF. PMID:26946470

Eyeing the Cyr61/CTGF/NOV (CCN) group of genes in development and diseases: highlights of their structural likenesses and functional dissimilarities.

PubMed

Krupska, Izabela; Bruford, Elspeth A; Chaqour, Brahim

2015-09-23

"CCN" is an acronym referring to the first letter of each of the first three members of this original group of mammalian functionally and phylogenetically distinct extracellular matrix (ECM) proteins [i.e., cysteine-rich 61 (CYR61), connective tissue growth factor (CTGF), and nephroblastoma-overexpressed (NOV)]. Although "CCN" genes are unlikely to have arisen from a common ancestral gene, their encoded proteins share multimodular structures in which most cysteine residues are strictly conserved in their positions within several structural motifs. The CCN genes can be subdivided into members developmentally indispensable for embryonic viability (e.g., CCN1, 2 and 5), each assuming unique tissue-specific functions, and members not essential for embryonic development (e.g., CCN3, 4 and 6), probably due to a balance of functional redundancy and specialization during evolution. The temporo-spatial regulation of the CCN genes and the structural information contained within the sequences of their encoded proteins reflect diversity in their context and tissue-specific functions. Genetic association studies and experimental anomalies, replicated in various animal models, have shown that altered CCN gene structure or expression is associated with "injury" stimuli--whether mechanical (e.g., trauma, shear stress) or chemical (e.g., ischemia, hyperglycemia, hyperlipidemia, inflammation). Consequently, increased organ-specific susceptibility to structural damages ensues. These data underscore the critical functions of CCN proteins in the dynamics of tissue repair and regeneration and in the compensatory responses preceding organ failure. A better understanding of the regulation and mode of action of each CCN member will be useful in developing specific gain- or loss-of-function strategies for therapeutic purposes.

Engineering Human TMJ Discs with Protein-Releasing 3D-Printed Scaffolds.

PubMed

Legemate, K; Tarafder, S; Jun, Y; Lee, C H

2016-07-01

The temporomandibular joint (TMJ) disc is a heterogeneous fibrocartilaginous tissue positioned between the mandibular condyle and glenoid fossa of the temporal bone, with important roles in TMJ functions. Tissue engineering TMJ discs has emerged as an alternative approach to overcoming limitations of current treatments for TMJ disorders. However, the anisotropic collagen orientation and inhomogeneous fibrocartilaginous matrix distribution present challenges in the tissue engineering of functional TMJ discs. Here, we developed 3-dimensional (3D)-printed anatomically correct scaffolds with region-variant microstrand alignment, mimicking anisotropic collagen alignment in the TMJ disc and corresponding mechanical properties. Connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TGFβ3) were then delivered in the scaffolds by spatially embedding CTGF- or TGFβ3-encapsulated microspheres (µS) to reconstruct the regionally variant fibrocartilaginous matrix in the native TMJ disc. When cultured with human mesenchymal stem/progenitor cells (MSCs) for 6 wk, 3D-printed scaffolds with CTGF/TGFβ3-µS resulted in a heterogeneous fibrocartilaginous matrix with overall distribution of collagen-rich fibrous structure in the anterior/posterior (AP) bands and fibrocartilaginous matrix in the intermediate zone, reminiscent of the native TMJ disc. High dose of CTGF/TGFβ3-µS (100 mg µS/g of scaffold) showed significantly more collagen II and aggrecan in the intermediate zone than a low dose (50 mg µS/g of scaffold). Similarly, a high dose of CTGF/TGFβ3-µS yielded significantly higher collagen I expression in the AP bands compared with the low-dose and empty µS. From stress relaxation tests, the ratio of relaxation modulus to instantaneous modulus was significantly smaller with CTGF/TGFβ3-µS than empty µS. Similarly, a significantly higher coefficient of viscosity was achieved with the high dose of CTGF/TGFβ3-µS compared with the low-dose and empty

Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell‐Derived Choroidal Endothelium

PubMed Central

Songstad, Allison E.; Worthington, Kristan S.; Chirco, Kathleen R.; Giacalone, Joseph C.; Whitmore, S. Scott; Anfinson, Kristin R.; Ochoa, Dalyz; Cranston, Cathryn M.; Riker, Megan J.; Neiman, Maurine; Stone, Edwin M.; Mullins, Robert F.

2017-01-01

Abstract Age‐related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of retinal pigment epithelial (RPE) and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be lost in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient‐specific, stem cell‐derived CECs will be required. In this study, we report for the first time a stepwise differentiation protocol suitable for generating human iPSC‐derived CEC‐like cells. RNA‐seq analysis of the monkey CEC line, RF/6A, combined with two statistical screens allowed us to develop media comprised of various protein combinations. In both screens, connective tissue growth factor (CTGF) was identified as the key component required for driving CEC development. A second factor tumor necrosis factor (TNF)‐related weak inducer of apoptosis receptor was also found to promote iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF‐driven iPSC‐derived CEC‐like cells formed capillary tube‐like vascular networks, and expressed the EC‐specific markers CD31, ICAM1, PLVAP, vWF, and the CEC‐restricted marker CA4. In combination with RPE and photoreceptor cells, patient‐specific iPSC derived CEC‐like cells will enable scientists to accurately evaluate AMD pathophysiology and develop effective cell replacement therapies. Stem Cells Translational Medicine 2017;6:1533–1546 PMID:28474838

The Water Fraction of Calendula officinalis Hydroethanol Extract Stimulates In Vitro and In Vivo Proliferation of Dermal Fibroblasts in Wound Healing.

PubMed

Dinda, Manikarna; Mazumdar, Swagata; Das, Saurabh; Ganguly, Durba; Dasgupta, Uma B; Dutta, Ananya; Jana, Kuladip; Karmakar, Parimal

2016-10-01

The active fraction and/or compounds of Calendula officinalis responsible for wound healing are not known yet. In this work we studied the molecular target of C. officinalis hydroethanol extract (CEE) and its active fraction (water fraction of hydroethanol extract, WCEE) on primary human dermal fibroblasts (HDF). In vivo, CEE or WCEE were topically applied on excisional wounds of BALB/c mice and the rate of wound contraction and immunohistological studies were carried out. We found that CEE and only its WCEE significantly stimulated the proliferation as well as the migration of HDF cells. Also they up-regulate the expression of connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA) in vitro. In vivo, CEE or WCEE treated mice groups showed faster wound healing and increased expression of CTGF and α-SMA compared to placebo control group. The increased expression of both the proteins during granulation phase of wound repair demonstrated the potential role of C. officinalis in wound healing. In addition, HPLC-ESI MS analysis of the active water fraction revealed the presence of two major compounds, rutin and quercetin-3-O-glucoside. Thus, our results showed that C. officinalis potentiated wound healing by stimulating the expression of CTGF and α-SMA and further we identified active compounds. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Chinese herbal medicine Shenqi Detoxification Granule inhibits fibrosis in adenine induced chronic renal failure rats.

PubMed

Peng, Min; Cai, Pingping; Ma, Hongbo; Meng, Hongyan; Xu, Yuan; Zhang, Xiaoyi; Si, Guomin

2014-01-01

Progressive fibrosis accompanies all chronic renal disease, connective tissue growth factor (CTGF,) and platelet-derived growth factor-B, (PDGF-B,) play important roles in extra-cellular matrix abnormal accumulation, while endothelin-1 (ET-1) nitric oxide (NO,) are related to endothelial dysfunction, which mediates the progression of renal fibrosis. Shenqi Detoxification Granule (SDG), a traditional Chinese herbal formula, has been used for treatment of chronic renal failure in clinic for many years. In order to evaluate the efficacy, and explore the mechanism of SDG to inhibit the progression of renal fibrosis, study was carried out using the adenine-induced Wister rats as the CRF model, and losartan as postive control drug. Levels of serum creatinine [Scr], and blood urea nitrogen (BUN), albumin (ALB), 24hrs, urine protein (24hUP), triacylglycerol (TG), and cholesterol (CHO), together with ET-1, and NO were detected. Pathological changes of renal tissues were observed by HE, staining. In addition, CTGF and PDGF-B expression were analyzed by immuno-histo-chemistry. The results indicated that SDG can effectively reduce Scr, BUN, 24hUP, TG, and CHO levels, increase ALB levels, inhibit renal tissue damage in CRF rats, and the mechanism maybe reduce PDGF-B, CTGF expression and ET-1/NO. Shenqi Detoxification Granule is a beneficial treatment for chronic renal failure.

Connective Tissue Growth Factor (CTGF) as a Regulator of Lactogenic Differentiation

DTIC Science & Technology

2009-06-09

1 1.62 Myeloid leukemia factor 1, Mlf1 1.57 ADAMTS-l4 1.55 E2F transcription factor, E2F2 1.44 Tensin 4 -1.5 BCL2/adenovirus E1B interacting... Mlf1 1.57 ADAMTS-l4 1.55 Ras homolog gene family, member B, RhoB 1.48 Cell Differentiation-associated Wingless-type MMTV integration site family...B, relB 1.92 Myeloid leukemia factor 1, Mlf1 1.57 Growth Factor, Catalytic Activity-associated Dual specificity protein phosphatase 8, Dusp8

GPER mediates cardiotropic effects in spontaneously hypertensive rat hearts.

PubMed

De Francesco, Ernestina Marianna; Angelone, Tommaso; Pasqua, Teresa; Pupo, Marco; Cerra, Maria Carmela; Maggiolini, Marcello

2013-01-01

Estrogens promote beneficial effects in the cardiovascular system mainly through the estrogen receptor (ER)α and ERβ, which act as ligand-gated transcription factors. Recently, the G protein-coupled estrogen receptor (GPER) has been implicated in the estrogenic signaling in diverse tissues, including the cardiovascular system. In this study, we demonstrate that left ventricles of male Spontaneously Hypertensive Rats (SHR) express higher levels of GPER compared to normotensive Wistar Kyoto (WKY) rats. In addition, we show that the selective GPER agonist G-1 induces negative inotropic and lusitropic effects to a higher extent in isolated and Langendorff perfused hearts of male SHR compared to WKY rats. These cardiotropic effects elicited by G-1 involved the GPER/eNOS transduction signaling, as determined by using the GPER antagonist G15 and the eNOS inhibitor L-NIO. Similarly, the G-1 induced activation of ERK1/2, AKT, GSK3β, c-Jun and eNOS was abrogated by G15, while L-NIO prevented only the eNOS phosphorylation. In hypoxic Langendorff perfused WKY rat heart preparations, we also found an increased expression of GPER along with that of the hypoxic mediator HIF-1α and the fibrotic marker CTGF. Interestingly, G15 and L-NIO prevented the ability of G-1 to down-regulate the expression of both HIF-1α and CTGF, which were found expressed to a higher extent in SHR compared to WKY rat hearts. Collectively, the present study provides novel data into the potential role played by GPER in hypertensive disease on the basis of its involvement in myocardial inotropism and lusitropism as well as the expression of the apoptotic HIF-1α and fibrotic CTGF factors. Hence, GPER may be considered as a useful target in the treatment of some cardiac dysfunctions associated with stressful conditions like the essential hypertension.

An Analysis of Pathological Activities of CCN Proteins in Joint Disorders: Mechanical Stretch-Mediated CCN2 Expression in Cultured Meniscus Cells.

PubMed

Furumatsu, Takayuki; Ozaki, Toshifumi

2017-01-01

The multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, also known as connective tissue growth factor, regulates cellular proliferation, differentiation, and tissue regeneration. Recent literatures have described important roles of CCN2 in the meniscus metabolism. However, the mechanical stress-mediated transcriptional regulation of CCN2 in the meniscus remains unclear. The meniscus is a fibrocartilaginous tissue that controls complex biomechanics of the knee joint. Therefore, the injured unstable meniscus has a poor healing potential especially in the avascular inner region. In addition, dysfunction of the meniscus correlates with the progression of degenerative knee joint disorders and joint space narrowing. Here, we describe an experimental approach that investigates the distinct cellular behavior of inner and outer meniscus cells in response to mechanical stretch. Our experimental model can analyze the relationships between stretch-induced CCN2 expression and its functional role in the meniscus homeostasis.

REPAIR EFFECTS OF UMBILICAL CORD MESENCHYMAL STEM CELLS ON PODOCYTE DAMAGE OF IgA NEPHROPATHY.

PubMed

Zhang, D W; Qiu, H; Mei, Y M; Fu, H; Zheng, H G

2015-01-01

This study aimed to explore the influence of umbilical cord mesenchymal stem cells (UMSC) on stem cell homing and glomerular mesangial cell (GMC) after intravenous injection performed on mice tails with IgA nephropathy (IgAN) and its possible mechanism, which provide a new way and theoretical basis for the application of stem cell transplantation (SCT) in kidney disease treatment. Specific pathogen free (SPF) male Kunming mice were randomly divided into groups. A complex method applying bovine serum albumin (BSA) gavage, hypodermic injection of CCl4 and lipopolysaccharide (LPS) was used for building IgAN mice model. In addition, vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and cluster of differentiation (CD) 44 were observed by Masson staining and detected with immunohistochemistry (IHC) to confirm homing and location of mesenchymal stem cells (MSCs). Moreover, Western Blot was used for detecting VEGF and CTGF so as to explore the possible mechanism of applying UMSC in treating IgAN. Masson staining indicated that fibrosis degree of MSCs in treatment group was significantly lower than in negative control group after stem cell treatment. Routine urine test explained that proteinuria in treatment group were (7.15±0.31), (4.87±0.22), (2.95±0.16) g/24 h and (12.00±1.38) g/24 h in model group (P less than 0.05). MSCs were observed to be located in glomerulus and renal interstitium by IHC detection of CD44 and IHC qualitative observation of VEGF and CTGF had different positive expressions in three groups. Furthermore, different expressions of VEGF and CTGF were observed quantitatively by Western Blot. Fibrosis degree of renal tissue relieves, hematuresis and proteinuria eases and IgAN symptoms obviously improve after UMSC treatment, which hints that the treatment of HUMSC has protective effect on IgAN mice model.

Left and right ventricle late remodeling following myocardial infarction in rats.

PubMed

Stefanon, Ivanita; Valero-Muñoz, María; Fernandes, Aurélia Araújo; Ribeiro, Rogério Faustino; Rodríguez, Cristina; Miana, Maria; Martínez-González, José; Spalenza, Jessica S; Lahera, Vicente; Vassallo, Paula F; Cachofeiro, Victoria

2013-01-01

The mechanisms involved in cardiac remodeling in left (LV) and right ventricles (RV) after myocardial infarction (MI) are still unclear. We assayed factors involved in collagen turnover in both ventricles following MI in rats either presenting signs of heart failure (pulmonary congestion and increased LVEDP) or not (INF-HF or INF, respectively). MI was induced in male rats by ligation of the left coronary artery. Four weeks after MI gene expression of collagen I, connective tissue growth factor (CTGF), transforming growth factor β (TGF-β) and lysyl oxidase (LOX), metalloproteinase-2 (MMP2) and tissue inhibitor metalloproteinase-2 (TIMP2) as well as cardiac hemodynamic in both ventricles were evaluated. Ventricular dilatation, hypertrophy and an increase in interstitial fibrosis and myocyte size were observed in the RV and LV from INF-HF animals, whereas only LV dilatation and fibrosis in RV was present in INF. The LV fibrosis in INF-HF was associated with higher mRNA of collagen I, CTGF, TGF-β and LOX expressions than in INF and SHAM animals, while MMP2/TIMP2 mRNA ratio did not change. RV fibrosis in INF and INF-HF groups was associated with an increase in LOX mRNA and a reduction in MMP2/TIMP2 ratio. CTGF mRNA was increased only in the INF-HF group. INF and INF-HF animals presented different patterns of remodeling in both ventricles. In the INF-HF group, fibrosis seems to be consequence of collagen production in LV, and by reductions in collagen degradation in RV of both INF and INF-HF animals.

Renal Sympathetic Denervation in Rats Ameliorates Cardiac Dysfunction and Fibrosis Post-Myocardial Infarction Involving MicroRNAs

PubMed Central

Zheng, Xiaoxin; Li, Xiaoyan; Lyu, Yongnan; He, Yiyu; Wan, Weiguo; Jiang, Xuejun

2016-01-01

Background The role of renal sympathetic denervation (RSD) in ameliorating post-myocardial infarction (MI) left ventricular (LV) fibrosis via microRNA-dependent regulation of connective tissue growth factor (CTGF) remains unknown. Material/Methods MI and RSD were induced in Sprague–Dawley rats by ligating the left coronary artery and denervating the bilateral renal nerves, respectively. Norepinephrine, renin, angiotensin II and aldosterone in plasma, collagen, microRNA21, microRNA 101a, microRNA 133a and CTGF in heart tissue, as well as cardiac function were evaluated six weeks post-MI. Results In the RSD group, parameters of cardiac function were significantly improved as evidenced by increased LV ejection fraction (p<0.01), LV end-systolic diameter (p<0.01), end-diastolic diameter (p<0.05), LV systolic pressure (p<0.05), maximal rate of pressure rise and decline (dP/dtmax and dP/dtmin, p<0.05), and decreased LV end-diastolic pressure (p<0.05) when compared with MI rats. Further, reduced collagen deposition in peri-infarct myocardium was observed in RSD-treated rats along with higher microRNA101a and microRNA133a (p<0.05) and lower microRNA21 expression (p<0.01) than in MI rats. CTGF mRNA and protein levels were decreased in LV following RSD (p<0.01), accompanied by decreased expression of norepinephrine, renin, angiotensin II and aldosterone in plasma (p<0.05) compared with untreated MI rats. Conclusions The potential therapeutic effects of RSD on post-MI LV fibrosis may be partly mediated by inhibition of CTGF expression via upregulation of microRNA 101a and microRNA 133a and downregulation of microRNA21. PMID:27490896

TGF-β induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways.

PubMed

Strand, Douglas W; Liang, Yao-Yun; Yang, Feng; Barron, David A; Ressler, Steven J; Schauer, Isaiah G; Feng, Xin-Hua; Rowley, David R

2014-01-01

Transforming Growth Factor-β (TGF-β) regulates the reactive stroma microenvironment associated with most carcinomas and mediates expression of many stromal derived factors important for tumor progression, including FGF-2 and CTGF. TGF-β is over-expressed in most carcinomas, and FGF-2 action is important in tumor-induced angiogenesis. The signaling mechanisms of how TGF-β regulates FGF-2 expression in the reactive stroma microenvironment are not understood. Accordingly, we have assessed key signaling pathways that mediate TGF-β1-induced FGF-2 expression in prostate stromal fibroblasts and mouse embryo fibroblasts (MEFs) null for Smad2 and Smad3. TGF-β1 induced phosphorylation of Smad2, Smad3, p38 and ERK1/2 proteins in both control MEFs and prostate fibroblasts. Of these, Smad3, but not Smad2 was found to be required for TGF-β1 induction of FGF-2 expression in stromal cells. ChIP analysis revealed a Smad3/Smad4 complex was associated with the -1.9 to -2.3 kb upstream proximal promoter of the FGF-2 gene, further suggesting a Smad3-specific regulation. In addition, chemical inhibition of p38 or ERK1/2 MAPK activity also blocked TGF-β1-induced FGF-2 expression in a Smad3-independent manner. Conversely, inhibition of JNK signaling enhanced FGF-2 expression. Together, these data indicate that expression of FGF-2 in fibroblasts in the tumor stromal cell microenvironment is coordinately dependent on both intact Smad3 and MAP kinase signaling pathways. These pathways and key downstream mediators of TGF-β action in the tumor reactive stroma microenvironment, may evolve as putative targets for therapeutic intervention.

The regulation of trefoil factor 2 expression by the transcription factor Sp3.

PubMed

Liu, Jingjing; Wang, Xu; Cai, Yiling; Zhou, Jingping; Guleng, Bayasi; Shi, Huaxiu; Ren, Jianlin

2012-10-19

Trefoil factor family 2 (TFF2) participates in mucus stabilization and repair, apoptosis, and inflammatory responses. Previously published reports have indicated that several growth factors and basal transcription factors are associated with the expression of TFF2. However, the detailed mechanisms that regulate TFF2 expression are not fully understood. The present study was designed to assess the essential role of the transcription factor SP3 with respect to TFF2 expression. We first demonstrated that there was a negative correlation between the expression levels of SP3 and TFF2. Thus, in the examined cells, the overexpression of SP3 decreased the expression level of TFF2, whereas the inhibition of SP3 increased the expression level of TFF2. Moreover, we discovered two GC boxes in the TFF2 promoter and confirmed the specific binding of SP3 to this promoter. On the whole, this study indicated that Sp3 was a major regulator of TFF2 expression. This knowledge should contribute to our understanding of the role that is played by SP3 in the regulation of TFF2 expression. Copyright © 2012 Elsevier Inc. All rights reserved.

Physical training improves visceral adipose tissue health by remodelling extracellular matrix in rats with estrogen absence: a gene expression analysis.

PubMed

Duarte, Fernanda O; Gomes-Gatto, Camila do Valle; Oishi, Jorge C; Lino, Anderson Diogo de S; Stotzer, Uliana S; Rodrigues, Maria Fernanda C; Gatti da Silva, Guilherme H; Selistre-de-Araújo, Heloisa S

2017-08-01

Adipose tissue development is associated with modifications involving extracellular matrix remodelling, and metalloproteinases play a significant role in this process. Reduced circulating sexual hormones cause impacts on the size, morphology and functions of the adipose tissue, increasing susceptibility to diseases. This study investigated whether exercise training may be an alternative strategy to combat the effects promoted by estrogen decay through modulation in gene expression patterns in the extracellular matrix (ECM) of visceral adipose tissue of ovariectomized rats. Nulliparous rats (n = 40) were randomly distributed into four groups (n = 10/group): sham sedentary (Sh-S), sham resistance training (Sh-Rt), ovariectomized sedentary (Ovx-S) and ovariectomized resistance training (Ovx-Rt). The Sh-S animals did not have any type of training. The body mass and food intake, ECM gene expression, gelatinase MMP-2 activity and adipocyte area were measured. A lack of estrogen promoted an increase in body mass, food intake and the visceral, parametrial and subcutaneous adipocyte areas. The ovariectomy upregulated the expression of MMP-2, MMP-9, TGF-β, CTGF, VEGF-A and MMP-2 activity. On the other hand, resistance training decreased the body mass, food intake and the adipocyte area of the three fat depots analysed; upregulated TIMP-1, VEGF-A and MMP-2 gene expression; downregulated MMP-9, TGF-β and CTGF gene expression; and decreased the MMP-2 activity. We speculate that resistance training on a vertical ladder could play an important role in maintaining and remodelling ECM by modulation in the ECM gene expression and MMP-2 activity, avoiding its destabilization which is impaired by the lack of estrogen. © 2017 The Authors. International Journal of Experimental Pathology © 2017 International Journal of Experimental Pathology.

Targeting the Myofibroblast Genetic Switch: Inhibitors of Myocardin-Related Transcription Factor/Serum Response Factor–Regulated Gene Transcription Prevent Fibrosis in a Murine Model of Skin Injury

PubMed Central

Haak, Andrew J.; Tsou, Pei-Suen; Amin, Mohammad A.; Ruth, Jeffrey H.; Campbell, Phillip; Fox, David A.; Khanna, Dinesh; Larsen, Scott D.

2014-01-01

Systemic sclerosis (SSc), or scleroderma, similar to many fibrotic disorders, lacks effective therapies. Current trials focus on anti-inflammatory drugs or targeted approaches aimed at one of the many receptor mechanisms initiating fibrosis. In light of evidence that a myocardin-related transcription factor (MRTF)–and serum response factor (SRF)–regulated gene transcriptional program induced by Rho GTPases is essential for myofibroblast activation, we explored the hypothesis that inhibitors of this pathway may represent novel antifibrotics. MRTF/SRF-regulated genes show spontaneously increased expression in primary dermal fibroblasts from patients with diffuse cutaneous SSc. A novel small-molecule inhibitor of MRTF/SRF-regulated transcription (CCG-203971) inhibits expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and collagen 1 (COL1A2) in both SSc fibroblasts and in lysophosphatidic acid (LPA)–and transforming growth factor β (TGFβ)–stimulated fibroblasts. In vivo treatment with CCG-203971 also prevented bleomycin-induced skin thickening and collagen deposition. Thus, targeting the MRTF/SRF gene transcription pathway could provide an efficacious new approach to therapy for SSc and other fibrotic disorders. PMID:24706986

Expression of virulence factors by Staphylococcus aureus grown in serum.

PubMed

Oogai, Yuichi; Matsuo, Miki; Hashimoto, Masahito; Kato, Fuminori; Sugai, Motoyuki; Komatsuzawa, Hitoshi

2011-11-01

Staphylococcus aureus produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by in vitro experiments using bacterial medium. However, when S. aureus infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in S. aureus grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl₃ into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with agr inactivated grown in serum, the expression of RNA III, psm, and sec4 was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that S. aureus expresses virulence factors in adaptation to the host environment.

Left and Right Ventricle Late Remodeling Following Myocardial Infarction in Rats

PubMed Central

Stefanon, Ivanita; Valero-Muñoz, María; Fernandes, Aurélia Araújo; Ribeiro, Rogério Faustino; Rodríguez, Cristina; Miana, Maria; Martínez-González, José; Spalenza, Jessica S.; Lahera, Vicente; Vassallo, Paula F.; Cachofeiro, Victoria

2013-01-01

Background The mechanisms involved in cardiac remodeling in left (LV) and right ventricles (RV) after myocardial infarction (MI) are still unclear. We assayed factors involved in collagen turnover in both ventricles following MI in rats either presenting signs of heart failure (pulmonary congestion and increased LVEDP) or not (INF-HF or INF, respectively). Methods MI was induced in male rats by ligation of the left coronary artery. Four weeks after MI gene expression of collagen I, connective tissue growth factor (CTGF), transforming growth factor β (TGF-β) and lysyl oxidase (LOX), metalloproteinase-2 (MMP2) and tissue inhibitor metalloproteinase-2 (TIMP2) as well as cardiac hemodynamic in both ventricles were evaluated. Results Ventricular dilatation, hypertrophy and an increase in interstitial fibrosis and myocyte size were observed in the RV and LV from INF-HF animals, whereas only LV dilatation and fibrosis in RV was present in INF. The LV fibrosis in INF-HF was associated with higher mRNA of collagen I, CTGF, TGF-β and LOX expressions than in INF and SHAM animals, while MMP2/TIMP2 mRNA ratio did not change. RV fibrosis in INF and INF-HF groups was associated with an increase in LOX mRNA and a reduction in MMP2/TIMP2 ratio. CTGF mRNA was increased only in the INF-HF group. Conclusions INF and INF-HF animals presented different patterns of remodeling in both ventricles. In the INF-HF group, fibrosis seems to be consequence of collagen production in LV, and by reductions in collagen degradation in RV of both INF and INF-HF animals. PMID:23741440

Anterior-posterior regionalized gene expression in the Ciona notochord

PubMed Central

Veeman, Michael

2014-01-01

Background In the simple ascidian chordate Ciona the signaling pathways and gene regulatory networks giving rise to initial notochord induction are largely understood and the mechanisms of notochord morphogenesis are being systematically elucidated. The notochord has generally been thought of as a non-compartmentalized or regionalized organ that is not finely patterned at the level of gene expression. Quantitative imaging methods have recently shown, however, that notochord cell size, shape and behavior vary consistently along the anterior-posterior (AP) axis. Results Here we screen candidate genes by whole mount in situ hybridization for potential AP asymmetry. We identify 4 genes that show non-uniform expression in the notochord. Ezrin/radixin/moesin (ERM) is expressed more strongly in the secondary notochord lineage than the primary. CTGF is expressed stochastically in a subset of notochord cells. A novel calmodulin-like gene (BCamL) is expressed more strongly at both the anterior and posterior tips of the notochord. A TGF-β ortholog is expressed in a gradient from posterior to anterior. The asymmetries in ERM, BCamL and TGF-β expression are evident even before the notochord cells have intercalated into a single-file column. Conclusions We conclude that the Ciona notochord is not a homogeneous tissue but instead shows distinct patterns of regionalized gene expression. PMID:24288133

Uterine Wound Healing: A Complex Process Mediated by Proteins and Peptides.

PubMed

Lofrumento, Dario D; Di Nardo, Maria A; De Falco, Marianna; Di Lieto, Andrea

2017-01-01

Wound healing is the process by which a complex cascade of biochemical events is responsible of the repair the damage. In vivo, studies in humans and mice suggest that healing and post-healing heterogeneous behavior of the surgically wounded myometrium is both phenotype and genotype dependent. Uterine wound healing process involves many cells: endothelial cells, neutrophils, monocytes/macrophages, lymphocytes, fibroblasts, myometrial cells as well a stem cell population found in the myometrium, myoSP (side population of myometrial cells). Transforming growth factor beta (TGF-β) isoforms, connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha (TNF-β) are involved in the wound healing mechanisms. The increased TGF- β1/β3 ratio reduces scarring and fibrosis. The CTGF altered expression may be a factor involved in the abnormal scars formation of low uterine segment after cesarean section and of the formation of uterine dehiscence. The lack of bFGF is involved in the reduction of collagen deposition in the wound site and thicker scabs. The altered expression of TNF-β, VEGF, and PDGF in human myometrial smooth muscle cells in case of uterine dehiscence, it is implicated in the uterine healing process. The over-and under-expressions of growth factors genes involved in uterine scarring process could represent patient's specific features, increasing the risk of cesarean scar complications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Identification of the key regulating genes of diminished ovarian reserve (DOR) by network and gene ontology analysis.

PubMed

Pashaiasl, Maryam; Ebrahimi, Mansour; Ebrahimie, Esmaeil

2016-09-01

Diminished ovarian reserve (DOR) is one of the reasons for infertility that not only affects both older and young women. Ovarian reserve assessment can be used as a new prognostic tool for infertility treatment decision making. Here, up- and down-regulated gene expression profiles of granulosa cells were analysed to generate a putative interaction map of the involved genes. In addition, gene ontology (GO) analysis was used to get insight intol the biological processes and molecular functions of involved proteins in DOR. Eleven up-regulated genes and nine down-regulated genes were identified and assessed by constructing interaction networks based on their biological processes. PTGS2, CTGF, LHCGR, CITED, SOCS2, STAR and FSTL3 were the key nodes in the up-regulated networks, while the IGF2, AMH, GREM, and FOXC1 proteins were key in the down-regulated networks. MIRN101-1, MIRN153-1 and MIRN194-1 inhibited the expression of SOCS2, while CSH1 and BMP2 positively regulated IGF1 and IGF2. Ossification, ovarian follicle development, vasculogenesis, sequence-specific DNA binding transcription factor activity, and golgi apparatus are the major differential groups between up-regulated and down-regulated genes in DOR. Meta-analysis of publicly available transcriptomic data highlighted the high coexpression of CTGF, connective tissue growth factor, with the other key regulators of DOR. CTGF is involved in organ senescence and focal adhesion pathway according to GO analysis. These findings provide a comprehensive system biology based insight into the aetiology of DOR through network and gene ontology analyses.

Osteopontin and Other Regulators of Angiogenesis and Fibrogenesis in the Vitreous from Patients with Proliferative Vitreoretinal Disorders

PubMed Central

Abu El-Asrar, Ahmed M.; Imtiaz Nawaz, Mohd; Kangave, Dustan; Siddiquei, Mohammed Mairaj; Geboes, Karel

2012-01-01

The aim of this study was to determine the levels of the angiogenic and fibrogenic factors osteopontin (OPN), high-mobility group box-1 (HMGB1), and connective tissue growth factor (CTGF) and the antiangiogenic and antifibrogenic pigment epithelium-derived factor (PEDF) in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and rhegmatogenous retinal detachment with no PVR (RD). Vitreous samples from 48 PDR, 17 PVR and 30 RD patients were studied by enzyme-linked immunosorbent assay. OPN, HMGB1, CTGF, and PEDF levels were significantly higher in PDR patients than in RD patients (P < 0.001; 0.002; <0.001; <0.001, resp.). CTGF and PEDF levels were significantly higher in PVR patients than in RD patients (P < 0.001; 0.004, resp.). Exploratory logistic regression analysis identified significant associations between PDR and high levels of HMGB1, CTGF and PEDF, between PDR with active neovascularization and high levels of CTGF and PEDF, and between PDR with traction retinal detachment and high levels of HMGB1. In patients with PDR, there were significant correlations between the levels of PEDF and the levels of OPN (r = 0.544, P = 0.001), HMGB1 (r = 0.719, P < 0.001), and CTGF (r = 0.715, P < 0.001). In patients with PVR, there were significant correlations between the levels of OPN and the levels of HMGB1 (r = 0.484, P = 0.049) and PEDF (r = 0.559, P = 0.02). Our findings suggest that OPN, HMGB1, and CTGF contribute to the pathogenesis of proliferative vitreoretinal disorders and that increased levels of PEDF may be a response to counterbalance the activity of angiogenic and fibrogenic factors in PDR and PVR. PMID:23055574

Gene expression profile of the fibrotic response in the peritoneal cavity.

PubMed

Le, S J; Gongora, M; Zhang, B; Grimmond, S; Campbell, G R; Campbell, J H; Rolfe, B E

2010-01-01

The cellular response to materials implanted in the peritoneal cavity has been utilised to produce tissue for grafting to hollow smooth muscle organs (blood vessels, bladder, uterus and vas deferens). To gain insight into the regulatory mechanisms involved in encapsulation of a foreign object, and subsequent differentiation of encapsulating cells, the present study used microarray technology and real-time RT-PCR to identify the temporal changes in gene expression associated with tissue development. Immunohistochemical analysis showed that 3-7 days post-implantation of foreign objects (cubes of boiled egg white) into rats, they were encapsulated by tissue comprised primarily of haemopoietic (CD45(+)) cells, mainly macrophages (CD68(+), CCR1(+)). By day 14, tissue capsule cells no longer expressed CD68, but were positive for myofibroblast markers alpha-smooth muscle (SM) actin and SM22. In accordance with these results, gene expression data showed that early capsule (days 3-7) development was dominated by the expression of monocyte/macrophage-specific genes (CD14, CSF-1, CSF-1R, MCP-1) and pro-inflammatory mediators such as transforming growth factor (TGF-beta). As tissue capsule development progressed (days 14-21), myofibroblast-associated and pro-fibrotic genes (associated with TGF-beta and Wnt/beta-catenin signalling pathways, including Wnt 4, TGFbetaRII, connective tissue growth factor (CTGF), SMADs-1, -2, -4 and collagen-1 subunits) were significantly up-regulated. The up-regulation of genes associated with Cardiovascular and Skeletal and Muscular System Development at later time-points suggests the capacity of cells within the tissue capsule for further differentiation to smooth muscle, and possibly other cell types. The identification of key regulatory pathways and molecules associated with the fibrotic response to implanted materials has important applications not only for optimising tissue engineering strategies, but also to control deleterious fibrotic

Nrf2 and Snail-1 in the prevention of experimental liver fibrosis by caffeine

PubMed Central

Gordillo-Bastidas, Daniela; Oceguera-Contreras, Edén; Salazar-Montes, Adriana; González-Cuevas, Jaime; Hernández-Ortega, Luis Daniel; Armendáriz-Borunda, Juan

2013-01-01

AIM: To determine the molecular mechanisms involved in experimental hepatic fibrosis prevention by caffeine (CFA). METHODS: Liver fibrosis was induced in Wistar rats by intraperitoneal thioacetamide or bile duct ligation and they were concomitantly treated with CFA (15 mg/kg per day). Fibrosis and inflammatory cell infiltrate were evaluated and classified by Knodell index. Inflammatory infiltrate was quantified by immunohistochemistry (anti-CD11b). Gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction for collagen I (Col-1), connective tissue growth factor (CTGF), transforming growth factor β1 (TGF-β1), tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, superoxide dismutase (SOD) and catalase (CAT). Activation of Nrf2 and Snail-1 was analyzed by Western-blot. TNF-α expression was proved by enzyme-linked immunosorbant assay, CAT activity was performed by zymography. RESULTS: CFA treatment diminished fibrosis index in treated animals. The Knodell index showed both lower fibrosis and necroinflammation. Expression of profibrogenic genes CTGF, Col-1 and TGF-β1 and proinflammatory genes TNF-α, IL-6 and IL-1 was substantially diminished with CFA treatment with less CD11b positive areas. Significantly lower values of transcriptional factor Snail-1 were detected in CFA treated rats compared with cirrhotic rats without treatment; in contrast Nrf2 was increased in the presence of CFA. Expression of SOD and CAT was greater in animals treated with CFA showing a strong correlation between mRNA expression and enzyme activity. CONCLUSION: Our results suggest that CFA inhibits the transcriptional factor Snail-1, down-regulating profibrogenic genes, and activates Nrf2 inducing antioxidant enzymes system, preventing inflammation and fibrosis. PMID:24379627

MiR-133 modulates TGF-β1-induced bladder smooth muscle cell hypertrophic and fibrotic response: implication for a role of microRNA in bladder wall remodeling caused by bladder outlet obstruction.

PubMed

Duan, Liu Jian; Qi, Jun; Kong, Xiang Jie; Huang, Tao; Qian, Xiao Qiang; Xu, Ding; Liang, Jun Hao; Kang, Jian

2015-02-01

Bladder outlet obstruction (BOO) evokes urinary bladder wall remodeling significantly, including the phenotype shift of bladder smooth muscle cells (BSMCs) where transforming growth factor-beta1 (TGF-β1) plays a pivotal role given the emerging function of modulating cellular phenotype. miR-133 plays a role in cardiac and muscle remodeling, however, little is known about its roles in TGF-β1-induced BSMC hypertrophic and fibrotic response. Here, we verified BOO induced bladder wall remodeling and TGF-β1 expression mainly located in bladder endothelium. Furthermore, we uncovered miR-133a/b expression profile in BOO rats, and then explored its regulated effects on BSMCs' phenotypic shift. Our study found that miR-133 became down-regulated during rat bladder remodeling. Next, we sought to examine whether the expression of miR-133 was down-regulated in primary BSMCs in response to TGF-β1 stimulation and whether forced overexpression of miR-133 could regulate profibrotic TGF-β signaling. We found that stimulation of BSMCs with exogenous TGF-β1 of increasing concentrations resulted in a dose-dependent decrease of miR-133a/b levels and transfection with miR-133 mimics attenuated TGF-β1-induced α-smooth muscle actin, extracellular matrix subtypes and fibrotic growth factor expression, whereas it upregulated high molecular weight caldesmon expression compared with the negative control. Also, downregulation of p-Smad3, not p-Smad2 by miR-133 was detected. Additionally, miR-133 overexpression suppressed TGF-β1-induced BSMC hypertrophy and proliferation through influencing cell cycle distribution. Bioinformatics analyses predicted that connective tissue growth factor (CTGF) was the potential target of miR-133, and then binding to the 3'-untranslated region of CTGF was validated by luciferase reporter assay. These results reveal a novel regulator for miR-133 to modulate TGF-β1-induced BSMC phenotypic changes by targeting CTGF through the TGF-β-Smad3 signaling pathway

RNA Expression Profiling Reveals Differentially Regulated Growth Factor and Receptor Expression in Redirected Cancer Cells.

PubMed

Schmucker, Hannah S; Park, Jang Pyo; Coissieux, Marie-May; Bentires-Alj, Mohamed; Feltus, F Alex; Booth, Brian W

2017-05-01

Tumorigenic cells can be redirected to adopt a normal phenotype when transplanted into cleared mammary fat pads of juvenile female mice in specific ratios with normal epithelial cells. The redirected tumorigenic cells enter stem cell niches and provide progeny that differentiate into all mammary epithelial subtypes. We have developed an in vitro model that mimics the in vivo phenomenon. The shift in phenotype to redirection should be accomplished through a return to a normal gene expression state. To measure this shift, we interrogated the transcriptome of various in vitro model states in search for casual genes. For this study, expression of growth factors, cytokines, and their associated receptors was examined. In all, we queried 251 growth factor and cytokine-related genes. We found numerous growth factor and cytokine genes whose expression levels switched from expression levels seen in cancer cells to expression levels observed in normal cells. The comparisons of gene expression between normal mammary epithelial cells, tumor-derived cells, and redirected cancer cells have revealed insight into active and inactive growth factors and cytokines in cancer cell redirection.

Urinary CCN2 (CTGF) as a possible predictor of diabetic nephropathy: preliminary report.

PubMed

Riser, Bruce L; Cortes, Pedro; DeNichilo, Mark; Deshmukh, Poornima V; Chahal, Parminder S; Mohammed, Ali K; Yee, Jerry; Kahkonen, Dorothy

2003-08-01

It is currently impossible to reliably predict which diabetic patients will develop nephropathy and progress to kidney failure. Microalbuminuria, often regarded as a predictor of overt diabetic renal disease is, in fact, an indicator of established glomerular damage. We have shown that glomerular expression of the prosclerotic cytokine CCN2 (CTGF) is greatly up-regulated early in experimental and in human diabetes and mesangial cell exposure to CCN2 increases its production of extracellular matrix (ECM) molecules responsible for glomerulosclerosis. As an early marker, we therefore investigated the presence of CCN2 in urine and the relationship to diabetes and/or renal disease in an experimental model of diabetes and in a limited patient population. Urine samples from (1) healthy rats, (2) rats made diabetic by streptozotocin (STZ), (3) healthy human volunteers, (4) diabetic patients with renal disease, and (5) diabetic patients without renal disease were examined by Western blotting and/or enzyme-linked immunosorbent assay (ELISA) for qualitative and quantitative analysis of the of CCN2. Low levels of urinary CCN2 were present in healthy, control rats, but were increased approximately sevenfold overall in STZ-diabetic animals. CCN2 levels were the highest at week 3 of diabetes, then decreased with time, but remained significantly elevated over controls even after 32 weeks. Consistently low levels of urinary CCN2 were also detected in healthy volunteers (mean value, 7.1 CCN2/mg creatinine). However, levels were elevated approximately sixfold in the majority of diabetic patients with nephropathy. A small number of the diabetic patients not yet exhibiting evidence of renal involvement demonstrated CCN2 urinary levels that were ninefold greater than controls. The remaining normoalbuminuric diabetic patients demonstrated CCN2 levels indistinguishable from those of healthy volunteers. Analysis by Western blotting confirmed the identity of the urinary CCN2. A molecular

Anterior-posterior regionalized gene expression in the Ciona notochord.

PubMed

Reeves, Wendy; Thayer, Rachel; Veeman, Michael

2014-04-01

In the simple ascidian chordate Ciona, the signaling pathways and gene regulatory networks giving rise to initial notochord induction are largely understood and the mechanisms of notochord morphogenesis are being systematically elucidated. The notochord has generally been thought of as a non-compartmentalized or regionalized organ that is not finely patterned at the level of gene expression. Quantitative imaging methods have recently shown, however, that notochord cell size, shape, and behavior vary consistently along the anterior-posterior (AP) axis. Here we screen candidate genes by whole mount in situ hybridization for potential AP asymmetry. We identify 4 genes that show non-uniform expression in the notochord. Ezrin/radixin/moesin (ERM) is expressed more strongly in the secondary notochord lineage than the primary. CTGF is expressed stochastically in a subset of notochord cells. A novel calmodulin-like gene (BCamL) is expressed more strongly at both the anterior and posterior tips of the notochord. A TGF-β ortholog is expressed in a gradient from posterior to anterior. The asymmetries in ERM, BCamL, and TGF-β expression are evident even before the notochord cells have intercalated into a single-file column. We conclude that the Ciona notochord is not a homogeneous tissue but instead shows distinct patterns of regionalized gene expression. Copyright © 2013 Wiley Periodicals, Inc.

NNMT activation can contribute to the development of fatty liver disease by modulating the NAD + metabolism.

PubMed

Komatsu, Motoaki; Kanda, Takeshi; Urai, Hidenori; Kurokochi, Arata; Kitahama, Rina; Shigaki, Shuhei; Ono, Takashi; Yukioka, Hideo; Hasegawa, Kazuhiro; Tokuyama, Hirobumi; Kawabe, Hiroshi; Wakino, Shu; Itoh, Hiroshi

2018-06-05

Nicotinamide N-methyltransferase (NNMT) catalyses the reaction between nicotinamide (NAM) and S-adenosylmethionine to produce 1-methylnicotinamide and S-adenosylhomocysteine. Recently, this enzyme has also been reported to modulate hepatic nutrient metabolism, but its role in the liver has not been fully elucidated. We developed transgenic mice overexpressing NNMT to elucidate its role in hepatic nutrient metabolism. When fed a high fat diet containing NAM, a precursor for nicotinamide adenine dinucleotide (NAD) + , these NNMT-overexpressing mice exhibit fatty liver deterioration following increased expression of the genes mediating fatty acid uptake and decreased very low-density lipoprotein secretion. NNMT overactivation decreased the NAD + content in the liver and also decreased gene activity related to fatty acid oxidation by inhibiting NAD + -dependent deacetylase Sirt3 function. Moreover, the transgenic mice showed liver fibrosis, with the induction of inflammatory and fibrosis genes. Induced NNMT expression decreased the tissue methylation capacity, thereby reducing methylation of the connective tissue growth factor (CTGF) gene promoter, resulting in increased CTGF expression. These data indicate that NNMT links the NAD + and methionine metabolic pathways and promotes liver steatosis and fibrosis. Therefore, targeting NNMT may serve as a therapeutic strategy for treating fatty liver and fibrosis.

Rac1 GTPase regulates 11β hydroxysteroid dehydrogenase type 2 and fibrotic remodeling.

PubMed

Lavall, Daniel; Schuster, Pia; Jacobs, Nadine; Kazakov, Andrey; Böhm, Michael; Laufs, Ulrich

2017-05-05

The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11β-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11β-HSD2 expression ( r = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11β-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11β-HSD2 expression ( r = 0.788 and r = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11β-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11β-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11β-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11β-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling. © 2017 by The American Society for

[Effect of cryotherapy over the expression of vascular endothelial growth factor and pigment epithelium-derived factor].

PubMed

Toscano-Garibay, Julia Dolores; Quiroz-Mercado, Hugo; Espitia-Pinzón, Clara; Gil-Carrasco, Félix; Flores-Estrada, José Javier

2014-01-01

Cryotherapy is a no invasive technique that uses intense cold to freeze and destroy cancer tissues. There are no descriptions of its effects over the expression of vascular endothelial growth factor and pigment epithelium-derived factor. Experimental study in cryogenic spot were applied in the right sclera of twelve pigs for ten minutes. Other 3 pigs were used as normal controls. Animals were sacrificed at 7, 14 and 21 and the tissues of choriodes and retina were dissected in areas of approximately 1 cm2 surrounding cryogenic spots. Expression levels of vascular endothelial growth factor and pigment epithelium-derived factor were determined analyzed using polymerase chain reaction coupled to reverse-transcription. Vascular endothelial growth factor was significantly downregulated (24%, p< 0.05) seven days post-treatment meanwhile pigment epithelium-derived factor levels increased 44.8% (p< 0.05) as compared to normal controls (untreated). Both vascular endothelial growth factor and pigment epithelium-derived factor levels remain the same until day 14 but returned to basal expression at day 21. This work expose the relation of cryotherapy with the expression of two factors related to angiogenesis. RESULTS showed significant changes on the expression of vascular endothelial growth factor and pigment epithelium-derived factor illustrating that both proteins are regulated in response to cryogenic treatment in relatively short periods (21 days).

Neuronal expression of fibroblast growth factor receptors in zebrafish.

PubMed

Rohs, Patricia; Ebert, Alicia M; Zuba, Ania; McFarlane, Sarah

2013-12-01

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Copyright © 2013 Elsevier B.V. All rights reserved.

Berberine attenuates CCN2-induced IL-1β expression and prevents cartilage degradation in a rat model of osteoarthritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Shan-Chi; Lee, Hsiang-Ping; Department of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan

Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator that is abundantly expressed in osteoarthritis (OA). Interleukin-1β (IL-1β) plays a pivotal role in OA pathogenesis. Berberine exhibits an anti-inflammatory effect, but the mechanisms by which it modulates CCN2-induced IL-1β expression in OA synovial fibroblasts (OASFs) remain unknown. We showed that CCN2-induced IL-1β expression is mediated by the activation of α{sub v}β{sub 3}/α{sub v}β{sub 5} integrin-dependent reactive oxygen species (ROS) generation, and subsequent activation of apoptosis signal-regulating kinase 1 (ASK1), p38/JNK, and nuclear factor-κB (NF-κB) signaling pathways. This IL-1β expression in OASFs is attenuated by N-acetylcysteine (NAC),more » inhibitors of ASK1, p38, or JNK, or treatment with berberine. Furthermore, berberine also reverses cartilage damage in an experimental model of collagenase-induced OA (CIOA). We observed that CCN2 increased IL-1β expression via α{sub v}β{sub 3}/α{sub v}β{sub 5} integrins, ROS, and ASK1, p38/JNK, and NF-κB signaling pathways. Berberine was found to inhibit these signaling components in OASFs in vitro and prevent cartilage degradation in vivo. We suggest a novel therapeutic strategy of using berberine for managing OA. - Highlights: • CCN2 induce IL-1β production via αvβ3/αvβ5 integrin, ROS, ASK1, p38/JNK, and NF-κB. • Berberine attenuates CCN2-induced IL-1β expression in vitro and in OA rat model. • Berberine as natural drug of choice for anti-inflammatory effect to ameliorates OA.« less

Cordyceps cicadae extracts ameliorate renal malfunction in a remnant kidney model*

PubMed Central

Zhu, Rong; Chen, Yi-ping; Deng, Yue-yi; Zheng, Rong; Zhong, Yi-fei; Wang, Lin; Du, Lan-ping

2011-01-01

Background and Objectives: Chronic kidney disease (CKD) is a growing public health problem with an urgent need for new pharmacological agents. Cordyceps cicadae is widely used in traditional Chinese medicine (TCM) and has potential renoprotective benefits. The current study aimed to determine any scientific evidence to support its clinical use. Methods: We analyzed the potential of two kinds of C. cicadae extract, total extract (TE) and acetic ether extract (AE), in treating kidney disease simulated by a subtotal nephrectomy (SNx) model. Sprague-Dawley rats were divided randomly into seven groups: sham-operated group, vehicle-treated SNx, Cozaar, 2 g/(kg∙d) TE SNx, 1 g/(kg∙d) TE SNx, 92 mg/(kg∙d) AE SNx, and 46 mg/(kg∙d) AE SNx. Renal injury was monitored using urine and serum analyses, and hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) stainings were used to analyze the level of fibrosis. The expression of type IV collagen (Col IV), fibronectin (FN), transforming growth factor-β1 (TGF-β1), and connective tissue growth factor (CTGF) was detected by immunohistochemistry. Results: Renal injury, reflected in urine and serum analyses, and pathological changes induced by SNx were attenuated by TE and AE intervention. The depositions of Col IV and FN were also decreased by the treatments and were accompanied by reduced expression of TGF-β1 and CTGF. In some respects, 2 g/(kg∙d) of TE produced better effects than Cozaar. Conclusions: For the first time, we have shown that C. cicadae may inhibit renal fibrosis in vivo through the TGF-β1/CTGF pathway. Therefore, we conclude that the use of C. cicadae could provide a rational strategy for combating renal fibrosis. PMID:22135152

[Effects and mechanisms of ursodeoxycholic acid on isoprenaline-Induced myocardial fibrosis in mice].

PubMed

Li, X; Han, K Q; Shi, Y N; Men, S Z; Li, S; Sun, M H; Dong, H; Lu, J J; Ma, L J; Zhao, M; Li, D; Liu, W

2017-02-07

Objective: To investigate the effects and possible mechanisms of ursodeoxycholic acid (UDCA) on myocardial fibrosis in mice. Method: To observe the expression of transforming growth factor(TGF) -β1, CTGF, MMPs and the degree of myocardial fibrosis, 61 male Kunming mice were randomly divided into normal group, low dose UDCA group, high dose of UDCA group, spironolactone group, and the control group.Isoproterenol (ISO) injection was given subcutaneously (30 d) to make the model of myocardial fibrosis.Corresponding anti-fibrosis drugs (UDCA or spironolactone) were given by gavage.HE staining and Masson staining were performed to explore the inflammation and fibrosis in the myocardium.The expression of collagen Ⅰ and collagen Ⅲ protein was detected by immunohistochemistry to evaluate the degree of fibrosis among the groups.Western blot was used to detect the expression of transforming growth factor, (TGF)-β1, connective tissue growth factor (CTGF), matrix metalloproteinase (MMP)-2, -9, tissue inhibitor of metalloproteinase (TIMP)-4, -1 and anti-phospho-NFKBIA (p-IκB-α) inhibitor of NF-κB (IκB) protein in myocardium. Results: HE and Masson staining results showed that in the normal group, myocardial fibrosis is less, while the control group showed a large amount of fibrotic tissue ( P <0.05). Tissue fibrosis in the low/high dose UDCA group and spironolactone group was significantly reduced compared with the control group ( P <0.05), in which high dose of UDCA reduces fibrosis more significantly.Immunohistochemistry results showed that collagen Ⅰ and collagen Ⅲ protein expression was significantly increased ( P <0.05). Whereas in the low/high UDCA dose group and spironolactone group, collagen Ⅰ and collagen Ⅲ expression were significantly decreased ( P <0.05), the high UDCA dose group decreased more significantly.Western blot results suggest that TGFβ-1 expression in the myocardial tissue was significantly increased compared to the normal group ( P <0

Temporal expression of growth factors triggered by epiregulin regulates inflammation development.

PubMed

Harada, Masaya; Kamimura, Daisuke; Arima, Yasunobu; Kohsaka, Hitoshi; Nakatsuji, Yuji; Nishida, Makoto; Atsumi, Toru; Meng, Jie; Bando, Hidenori; Singh, Rajeev; Sabharwal, Lavannya; Jiang, Jing-Jing; Kumai, Noriko; Miyasaka, Nobuyuki; Sakoda, Saburo; Yamauchi-Takihara, Keiko; Ogura, Hideki; Hirano, Toshio; Murakami, Masaaki

2015-02-01

In this study, we investigated the relationship between several growth factors and inflammation development. Serum concentrations of epiregulin, amphiregulin, betacellulin, TGF-α, fibroblast growth factor 2, placental growth factor (PLGF), and tenascin C were increased in rheumatoid arthritis patients. Furthermore, local blockades of these growth factors suppressed the development of cytokine-induced arthritis in mice by inhibiting chemokine and IL-6 expressions. We found that epiregulin expression was early and followed by the induction of other growth factors at different sites of the joints. The same growth factors then regulated the expression of epiregulin at later time points of the arthritis. These growth factors were increased in patients suffering from multiple sclerosis (MS) and also played a role in the development of an MS model, experimental autoimmune encephalomyelitis. The results suggest that the temporal expression of growth factors is involved in the inflammation development seen in several diseases, including rheumatoid arthritis and MS. Therefore, various growth factor pathways might be good therapeutic targets for various inflammatory diseases. Copyright © 2015 by The American Association of Immunologists, Inc.

Hemoglobin enhances tissue factor expression on human malignant cells.

PubMed

Siddiqui, F A; Amirkhosravi, A; Amaya, M; Meyer, T; Biggerstaff, J; Desai, H; Francis, J L

2001-04-01

Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.

PubMed

Dean, Richard A; Butler, Georgina S; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R; Courty, José; Overall, Christopher M

2007-12-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.

Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis▿ †

PubMed Central

Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R.; Courty, José; Overall, Christopher M.

2007-01-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

Dynamic Analysis of the Expression of the TGFβ/SMAD2 Pathway and CCN2/CTGF during Early Steps of Tooth Development

PubMed Central

Pacheco, Marcos S.; Reis, Alice H.; Aguiar, Diego P.; Lyons, Karen M.; Abreu, José G.

2009-01-01

Background/Aims CCN2 is present during tooth development. However, the relationship between CCN2 and the transforming growth factor β (TGFβ)/SMAD2/3 signaling cascade during early stages of tooth development is unclear. Here, we compare the expression of CCN2 and TGFβ/SMAD2/3 components during tooth development, and analyze the functioning of TGFβ/SMAD2/3 in wild-type (WT) and Ccn2 null (Ccn2−/−) mice. Methods Coronal sections of mice on embryonic day (E)11.5, E12.5, E13.5, E14.5 and E18.5 from WT and Ccn2−/− were immunoreacted to detect CCN2 and components of the TGFβ signaling pathway and assayed for 5′-bromo-2′-deoxyuridine immunolabeling and proliferating cell nuclear antigen immunostaining. Results CCN2 and TGFβ signaling components such as TGFβ1, TGFβ receptor II, SMADs2/3 and SMAD4 were expressed in inducer tissues during early stages of tooth development. Proliferation analysis in these areas showed that epithelial cells proliferate less than mesenchymal cells from E11.5 to E13.5, while at E14.5 they proliferate more than mesenchymal cells. We did not find a correlation between functioning of the TGFβ1 cascade and CCN2 expression because Ccn2−/− mice showed neither a reduction in SMAD2 phosphorylation nor a difference in cell proliferation. Conclusion CCN2 and the TGFβ/SMAD2/3 signaling pathway are active in signaling centers of tooth development where proliferation is dynamic, but these mechanisms may act independently. PMID:18089935

Defining the molecular signatures of human right heart failure.

PubMed

Williams, Jordan L; Cavus, Omer; Loccoh, Emefah C; Adelman, Sara; Daugherty, John C; Smith, Sakima A; Canan, Benjamin; Janssen, Paul M L; Koenig, Sara; Kline, Crystal F; Mohler, Peter J; Bradley, Elisa A

2018-03-01

Right ventricular failure (RVF) varies significantly from the more common left ventricular failure (LVF). This study was undertaken to determine potential molecular pathways that are important in human right ventricular (RV) function and may mediate RVF. We analyzed mRNA of human non-failing LV and RV samples and RVF samples from patients with pulmonary arterial hypertension (PAH), and post-LVAD implantation. We then performed transcript analysis to determine differential expression of genes in the human heart samples. Immunoblot quantification was performed followed by analysis of non-failing and failing phenotypes. Inflammatory pathways were more commonly dysregulated in RV tissue (both non-failing and failing phenotypes). In non-failing human RV tissue we found important differences in expression of FIGF, TRAPPAC, and CTGF suggesting that regulation of normal RV and LV function are not the same. In failing RV tissue, FBN2, CTGF, SMOC2, and TRAPP6AC were differentially expressed, and are potential targets for further study. This work provides some of the first analyses of the molecular heterogeneity between human RV and LV tissue, as well as key differences in human disease (RVF secondary to pulmonary hypertension and LVAD mediated RVF). Our transcriptional data indicated that inflammatory pathways may be more important in RV tissue, and changes in FIGF and CTGF supported this hypothesis. In PAH RV failure samples, upregulation of FBN2 and CTGF further reinforced the potential significance that altered remodeling and inflammation play in normal RV function and failure. Copyright © 2018 Elsevier Inc. All rights reserved.

Nerve Growth Factor Expression Is Not Associated with Perineural Invasion in Extrahepatic Cholangiocarcinoma.

PubMed

Urabe, Kazuhide; Murakami, Yoshiaki; Kondo, Naru; Uemura, Kenichiro; Hashimoto, Yasushi; Nakagawa, Naoya; Sasaki, Hayato; Hiyama, Eiso; Takahashi, Shinya; Sueda, Taijiro

2016-03-01

Although the presence of perineural invasion has been recognized as a poor prognostic factor in extrahepatic cholangiocarcinoma, the molecular mechanisms of perineural invasion in extrahepatic cholangiocarcinoma remain unclear. Nerve growth factor has been reported to be a candidate predictive biomarker of perineural invasion in some cancers. To investigate the impact of intratumoral nerve growth factor expression in resected extrahepatic cholangiocarcinoma on survival. Intratumoral nerve growth factor expression was investigated immunohistochemically in 112 patients with resected extrahepatic cholangiocarcinoma. Associations between nerve growth factor expression and clinicopathological factors were statistically evaluated, and risk factors for poor survival were analyzed using univariate and multivariate analyses. High and low nerve growth factor expression was observed in 62 (55%) and 50 (45%) patients, respectively. For all 112 patients, no significant correlation was found between nerve growth factor expression and presence of perineural invasion (P = 0.942). Moreover, nerve growth factor expression was not associated with recurrence-free survival (P = 0.861) and overall survival (P = 0.973). In multivariate analysis, lymph node metastasis (P = 0.004) was identified as an independent risk factor for early recurrence and the presence of perineural invasion (P = 0.002) and lymph node metastasis (P < 0.001) was identified as independent risk factors for poor survival. Intratumoral nerve growth factor expression is not associated with perineural invasion or recurrence-free and overall survival in patients with resected extrahepatic cholangiocarcinoma.

Developmental expression patterns of candidate co-factors for vertebrate Six family transcription factors

PubMed Central

Neilson, Karen M.; Pignoni, Francesca; Yan, Bo; Moody, Sally A.

2010-01-01

Six family transcription factors play important roles in craniofacial development. Their transcriptional activity can be modified by co-factor proteins. Two Six genes and one co-factor gene (Eya1) are involved in the human Branchio-otic (BO) and Branchio-otic-renal (BOR) syndromes. However, mutations in Six and Eya genes only account for about half of these patients. To discover potential new causative genes, we searched the Xenopus genome for orthologues of Drosophila co-factor proteins that interact with the fly Six-related factor, SO. We identified 33 Xenopus genes with high sequence identity to 20 of the 25 fly SO-interacting proteins. We provide the developmental expression patterns of the Xenopus orthologues for 11 of the fly genes, and demonstrate that all are expressed in developing craniofacial tissues with at least partial overlap with Six1/Six2. We speculate that these genes may function as Six-interacting partners with important roles in vertebrate craniofacial development and perhaps congenital syndromes. PMID:21089078

Tissue factor expression by endothelial cells in sickle cell anemia.

PubMed

Solovey, A; Gui, L; Key, N S; Hebbel, R P

1998-05-01

The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that sickle CEC abnormally express TF antigen (expressed as percent CEC that are TF-positive), with 66+/-13% positive in sickle patients in steady-state, 83+/-19% positive in sickle patients presenting with acute vasoocclusive episodes, and only 10+/-13% positive in normal controls. Repeated samplings confirmed this impression that TF expression is greater when sickle patients develop acute vasoocclusive episodes. Sickle CEC are also positive for TF mRNA, with excellent concurrence between antigen and mRNA expression. The TF expressed on the antigen-positive CEC is functional, as demonstrated by a binding assay for Factor VIIa and a chromogenic assay sensitive to generation of Factor Xa. By establishing that endothelial cells in vivo can express TF, these data imply that the vast endothelial surface area does provide an important pathophysiologic trigger for coagulation activation.

Evaluation of procoagulant tissue factor expression in canine hemangiosarcoma cell lines.

PubMed

Witter, Lauren E; Gruber, Erika J; Lean, Fabian Z X; Stokol, Tracy

2017-01-01

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor-replete or specific coagulation factor-deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII-deficient plasma than in factor-replete plasma and was abolished in factor X-deficient plasma; residual thrombin generation in factor VII-deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

Alternative Sigma Factor Over-Expression Enables Heterologous Expression of a Type II Polyketide Biosynthetic Pathway in Escherichia coli

PubMed Central

Stevens, David Cole; Conway, Kyle R.; Pearce, Nelson; Villegas-Peñaranda, Luis Roberto; Garza, Anthony G.; Boddy, Christopher N.

2013-01-01

Background Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. Methodology We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. Conclusions We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products. PMID:23724102

Regulation of extracellular matrix elements and sarcomerogenesis in response to different periods of passive stretching in the soleus muscle of rats.

PubMed

Peviani, Sabrina M; Guzzoni, Vinicius; Pinheiro-Dardis, Clara M; Silva, Yara P da; Fioravante, Alisson C R; Sagawa, Adriana H; Delfino, Gabriel B; Durigan, João L Q; Salvini, Tania F

2018-06-13

Stretching is a common method used to prevent muscle shortening and improve limited mobility. However, the effect of different time periods on stretching-induced adaptation of the extracellular matrix and its regulatory elements have yet to be investigated. We aimed to evaluate the expression of fibrillar collagens, sarcomerogenesis, metalloproteinase (MMP) activity and gene expression of the extracellular matrix (ECM) regulators in the soleus (SOL) muscle of rats submitted to different stretching periods. The soleus muscles were submitted to 10 sets of passive stretching over 10 (St 10d) or 15 days (St 15d) (1 min per set, with 30 seconds' rest between sets). Sarcomerogenesis, muscle cross-sectional area (CSA), and MMP activity and mRNA levels in collagen (type I, III and IV), connective tissue growth factor (CTGF), growth factor-beta (TGF-β), and lysyl oxidase (LOX) were analyzed. Passive stretching over both time periods mitigated COL-I deposition in the SOL muscle of rats. Paradoxically, 10 days of passive stretching induced COL-I and COL-III synthesis, with concomitant upregulation of TGF-β1 and CTGF at a transcriptional level. These responses may be associated with lower LOX mRNA levels in SOL muscles submitted to 10 passive stretching sessions. Moreover, sarcomerogenesis was observed after 15 days of stretching, suggesting that stretching-induced muscle adaptations are time-dependent responses.

Kruppel-like factor 2 inhibits hypoxia-inducible factor 1alpha expression and function in the endothelium.

PubMed

Kawanami, Daiji; Mahabeleshwar, Ganapati H; Lin, Zhiyong; Atkins, G Brandon; Hamik, Anne; Haldar, Saptarsi M; Maemura, Koji; Lamanna, Joseph C; Jain, Mukesh K

2009-07-31

Hypoxia-inducible factor 1 (HIF-1) is a central regulator of the hypoxic response in many cell types. In endothelial cells, HIF-1 induces the expression of key proangiogenic factors to promote angiogenesis. Recent studies have identified Kruppel-like factor 2 (KLF2) as a potent inhibitor of angiogenesis. However, the role of KLF2 in regulating HIF-1 expression and function has not been evaluated. KLF2 expression was induced acutely by hypoxia in endothelial cells. Adenoviral overexpression of KLF2 inhibited hypoxia-induced expression of HIF-1alpha and its target genes such as interleukin 8, angiopoietin-2, and vascular endothelial growth factor in endothelial cells. Conversely, knockdown of KLF2 increased expression of HIF-1alpha and its targets. Furthermore, KLF2 inhibited hypoxia-induced endothelial tube formation, whereas endothelial cells from mice with haploinsufficiency of KLF2 showed increased tube formation in response to hypoxia. Consistent with this ex vivo observation, KLF2 heterozygous mice showed increased microvessel density in the brain. Mechanistically, KLF2 promoted HIF-1alpha degradation in a von Hippel-Lindau protein-independent but proteasome-dependent manner. Finally, KLF2 disrupted the interaction between HIF-1alpha and its chaperone Hsp90, suggesting that KLF2 promotes degradation of HIF-1alpha by affecting its folding and maturation. These observations identify KLF2 as a novel inhibitor of HIF-1alpha expression and function. Therefore, KLF2 may be a target for modulating the angiogenic response in disease states.

Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems.

PubMed

Vettath, Sunitha Kodengil; Shivashankar, Gaganashree; Menon, Krishnakumar N; Vijayachandran, Lakshmi S

2018-04-15

Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved. Copyright © 2018 Elsevier B.V. All rights reserved.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) expression in colorectal cancer.

PubMed

Nagano, Hideki; Goi, Takanori; Koneri, Kenji; Hirono, Yasuo; Katayama, Kanji; Yamaguchi, Akio

2007-12-01

Vascular endothelial growth factor (VEGF) is known as an important factor in the growth and metastasis of cancer cells. In 2001, a novel angiogenesis factor, endocrine gland-derived vascular endothelial growth factor (EG-VEGF), was cloned. In this study, we investigated the expression of EG-VEGF in colorectal cancer, the relationship between its expression and clinicopathological factors, and the in vitro activity of EG-VEGF transfectants. We determined expression levels of EG-VEGF in 113 advanced colorectal cancers resected in our hospital by quantitative PCR, and compared the expression levels and clinicopathological findings by multivariate analyses. The expression of EG-VEGF mRNA was positive in 31 cancers and negative in 82 cancers. We found that compared with the negative expression of the EG-VEGF gene, its positive expression was more frequently associated with hematogenous metastasis, and was associated with a poorer survival rate. In addition, EG-VEGF transfectants showed a higher degree of in vitro tubular formation than control cells. We speculate that, in colorectal cancers, the EG-VEGF gene functions as an important factor in angiogenesis in primary and metastatic lesions, and consider that it is useful as a novel prognostic factor. EG-VEGF molecule-targeted therapy has the potential for improving survival rates.

Anti-atherogenic activity of wild grape (Vitis thunbergii) extract antagonizing smooth muscle cell proliferation and migration promoted by neighboring macrophages.

PubMed

Kang, Sang-Wook; Kim, Min Soo; Kim, Hyun-Sung; Lee, Yong-Jin; Kang, Young-Hee

2012-06-01

The proliferation and migration of vascular smooth muscle cells (SMCs) play critical roles in intimal thickening and neointimal hyperplasia in early-phase atherosclerosis. This study tested whether wild grape extract (WGE) suppressed the proliferation and migration of human aortic SMCs induced by neighboring macrophages. Cellular expression of fibrogenic connective tissue growth factor (CTGF) and secretion of collagen IV and matrix metalloproteinase (MMP)-2 were determined in SMCs exposed to THP-1-differentiated macrophage-conditioned media. Proliferation was enhanced in SMCs exposed to macrophage-conditioned media collected during the early stage of differentiation, which was attenuated by treatment with ≥ 10 µg/ml WGE. Increased secretion of CTGF and collagen IV macrophage-conditioned media was suppressed in WGE-supplemented SMCs. TGF-β1-promoted production of CTGF and collagen IV was suppressed by blocking TGF-β receptors of R1 and R2 in SMCs. WGE repressed macrophage-conditioned media-upregulated MMP-2 secretion, indicating that WGE had an ability to encumber plaque rupture within atherosclerotic lesions. In addition, ≥ 1 µg/ml WGE ameliorated the migration of SMCs promoted by neighboring macrophages. These results demonstrate that WGE retarded neointimal hyperplasia and thickening within atherosclerotic plaques largely comprising of macrophages and SMCs. Therefore, WGE may be developed as an anti-proliferative and anti-migratory agent targeting SMCs in the proximity of newly differentiated and resident macrophages.

Clinical validation of nuclear factor kappa B expression in invasive breast cancer.

PubMed

Agrawal, Anil Kumar; Pielka, Ewa; Lipinski, Artur; Jelen, Michal; Kielan, Wojciech; Agrawal, Siddarth

2018-01-01

Breast cancer is the most commonly diagnosed cancer in Polish women. The expression of transcription nuclear factor kappa B, a key inducer of inflammatory response promoting carcinogenesis and cancer progression in breast cancer, is not well-established. We assessed the nuclear factor kappa B expression in a total of 119 invasive breast carcinomas and 25 healthy control samples and correlated this expression pattern with several clinical and pathologic parameters including histologic type and grade, tumor size, lymph node status, estrogen receptor status, and progesterone receptor status. The data used for the analysis were derived from medical records. An immunohistochemical analysis of nuclear factor kappa B, estrogen receptor, and progesterone receptor was carried out and evaluation of stainings was performed. The expression of nuclear factor kappa B was significantly higher than that in the corresponding healthy control samples. No statistical difference was demonstrated in nuclear factor kappa B expression in relation to age, menopausal status, lymph node status, tumor size and location, grade and histologic type of tumor, and hormonal status (estrogen receptor and progesterone receptor). Nuclear factor kappa B is significantly overexpressed in invasive breast cancer tissues. Although nuclear factor kappa B status does not correlate with clinicopathological findings, it might provide important additional information on prognosis and become a promising object for targeted therapy.

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

PubMed

Schrevel, Marlies; Osse, E Michelle; Prins, Frans A; Trimbos, J Baptist M Z; Fleuren, Gert Jan; Gorter, Arko; Jordanova, Ekaterina S

2017-06-01

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70-90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, β-cellulin, epidermal growth factor (EGF), epiregulin, heparin-binding EGF-like growth factor (HB‑EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB‑EGF had the highest mRNA expression and HB‑EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB‑EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB‑EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB‑EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB‑EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB‑EGF. Therefore, we propose an autocrine

Factor XIIIa is expressed by fibroblasts in fibrovascular tumors.

PubMed

Nemeth, A J; Penneys, N S

1989-10-01

Factor XIIIa (FXIIIa), a blood and intracellularly produced coagulation factor, has been found in a variety of cell types including fibroblast-like mesenchymal cells, and has been shown to stimulate the proliferation of fibroblasts and some neoplastic cells in vitro. We have already shown that the dendritic fibroblasts composing the fibrous papule contain this factor. We hypothesized that histopathologically similar fibrovascular tumors may also express FXIIIa and, in this report, show that the large stellate fibroblasts found in acquired digital fibrokeratomas, angiofibromas (adenoma sebaceum of Pringle), and oral fibroma (oral fibrous hyperplasia) also express FXIIIa. We postulate that FXIIIa, possibly acting as a growth factor, may be a common denominator in the pathogenesis of these tumors. Another possibility is that these tumors may be the consequence of a local overproduction of FXIIIa in response to an, as yet, unidentified stimulus.

E74-like factor 2 regulates valosin-containing protein expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Binglin; Tomita, Yasuhiko; Qiu, Ying

2007-05-11

Enhanced expression of valosin-containing protein (VCP) correlates with invasion and metastasis of cancers. To clarify the transcription mechanism of VCP, human and mouse genomic sequence was compared, revealing a 260 bp DNA sequence in the 5'-flanking region of VCP gene to be highly conserved between the two, in which binding motif of E74-like factor 2/new Ets-related factor (ELF2/NERF) was identified. Chromatin immunoprecipitation assay showed binding of ELF2/NERF to the 5'-flanking region of VCP gene. Knock-down of ELF2/NERF by siRNA decreased expression level of VCP. Viability of cells under tumor necrosis factor-alpha treatment significantly reduced in ELF2/NERF-knock-down breast cancer cell line.more » Immunohistochemical analysis on clinical breast cancer specimens showed a correlation of nuclear ELF2/NERF expression with VCP expression and proliferative activity of cells shown by Ki-67 immunohistochemistry. These findings indicate that ELF2/NERF promotes VCP transcription and that ELF2/NERF-VCP pathway might be important for cell survival and proliferation under cytokine stress.« less

Periodontal ligament stem/progenitor cells with protein-releasing scaffolds for cementum formation and integration on dentin surface.

PubMed

Cho, Hankyu; Tarafder, Solaiman; Fogge, Michael; Kao, Kristy; Lee, Chang H

2016-11-01

Purpose/Aim: Cementogenesis is a critical step in periodontal tissue regeneration given the essential role of cementum in anchoring teeth to the alveolar bone. This study is designed to achieve integrated cementum formation on the root surfaces of human teeth using growth factor-releasing scaffolds with periodontal ligament stem/progenitor cells (PDLSCs). Human PDLSCs were sorted by CD146 expression, and characterized using CFU-F assay and induced multi-lineage differentiation. Polycaprolactone scaffolds were fabricated using 3D printing, embedded with poly(lactic-co-glycolic acids) (PLGA) microspheres encapsulating connective tissue growth factor (CTGF), bone morphogenetic protein-2 (BMP-2), or bone morphogenetic protein-7 (BMP-7). After removing cementum on human tooth roots, PDLSC-seeded scaffolds were placed on the exposed dentin surface. After 6-week culture with cementogenic/osteogenic medium, cementum formation and integration were evaluated by histomorphometric analysis, immunofluorescence, and qRT-PCR. Periodontal ligament (PDL) cells sorted by CD146 and single-cell clones show a superior clonogenecity and multipotency as compared with heterogeneous populations. After 6 weeks, all the growth factor-delivered groups showed resurfacing of dentin with a newly formed cementum-like layer as compared with control. BMP-2 and BMP-7 showed de novo formation of tissue layers significantly thicker than all the other groups, whereas CTGF and BMP-7 resulted in significantly improved integration on the dentin surface. The de novo mineralized tissue layer seen in BMP-7-treated samples expressed cementum matrix protein 1 (CEMP1). Consistently, BMP-7 showed a significant increase in CEMP1 mRNA expression. Our findings represent important progress in stem cell-based cementum regeneration as an essential part of periodontium regeneration.

Synergistic Effect of Simvastatin Plus Radiation in Gastric Cancer and Colorectal Cancer: Implications of BIRC5 and Connective Tissue Growth Factor.

PubMed

Lim, Taekyu; Lee, Inkyoung; Kim, Jungmin; Kang, Won Ki

2015-10-01

We investigated the synergistic effect of simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor plus radiation therapy, on the proliferation and survival of gastric cancer (GC) and colorectal cancer (CRC) cells. We also studied several genes involved in the simvastatin/radiation-induced effects. Gastric cancer (AGS, SNU601, MKN1, and MKN28) and CRC (CoLo320, SW48, HT29, and HCT8) cell lines were treated with 0.2 μM simvastatin alone, or in combination with 0 to 4 Gy of radiation, and subjected to clonogenic survival and proliferation assays in vitro. To assess the molecular mechanism of the combination treatment, we performed microarray analysis, immunoblot assays, small interfering RNA knockdown experiments, and plasmid rescue assays. The antitumoral effects of simvastatin and radiation were evaluated in vivo using xenograft models. The combination therapy of simvastatin plus radiation inhibited basal clonogenic survival and proliferation of GC and CRC cells in vitro. Simvastatin suppressed the expression of BIRC5 and CTGF genes in these cancer cells. In vivo, the combined treatment with simvastatin and radiation significantly reduced the growth of xenograft tumors compared with treatment with radiation alone. We suggest that simvastatin has a synergistic effect with radiation on GC and CRC through the induction of apoptosis, which may be mediated by a simultaneous inhibition of BIRC5 and CTGF expression. A clinical trial of simvastatin in combination with radiation in patients with GC or CRC is warranted. Copyright © 2015 Elsevier Inc. All rights reserved.

Synergistic Effect of Simvastatin Plus Radiation in Gastric Cancer and Colorectal Cancer: Implications of BIRC5 and Connective Tissue Growth Factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Taekyu; Lee, Inkyoung; Kim, Jungmin

Purpose: We investigated the synergistic effect of simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor plus radiation therapy, on the proliferation and survival of gastric cancer (GC) and colorectal cancer (CRC) cells. We also studied several genes involved in the simvastatin/radiation-induced effects. Methods and Materials: Gastric cancer (AGS, SNU601, MKN1, and MKN28) and CRC (CoLo320, SW48, HT29, and HCT8) cell lines were treated with 0.2 μM simvastatin alone, or in combination with 0 to 4 Gy of radiation, and subjected to clonogenic survival and proliferation assays in vitro. To assess the molecular mechanism of the combination treatment, we performed microarray analysis, immunoblot assays, small interferingmore » RNA knockdown experiments, and plasmid rescue assays. The antitumoral effects of simvastatin and radiation were evaluated in vivo using xenograft models. Results: The combination therapy of simvastatin plus radiation inhibited basal clonogenic survival and proliferation of GC and CRC cells in vitro. Simvastatin suppressed the expression of BIRC5 and CTGF genes in these cancer cells. In vivo, the combined treatment with simvastatin and radiation significantly reduced the growth of xenograft tumors compared with treatment with radiation alone. Conclusion: We suggest that simvastatin has a synergistic effect with radiation on GC and CRC through the induction of apoptosis, which may be mediated by a simultaneous inhibition of BIRC5 and CTGF expression. A clinical trial of simvastatin in combination with radiation in patients with GC or CRC is warranted.« less

Krüppel Like Factors Family Expression in Cervical Cancer Cells.

PubMed

Marrero-Rodríguez, Daniel; la Cruz, Hugo Arreola-De; Taniguchi-Ponciano, Keiko; Gomez-Virgilio, Laura; Huerta-Padilla, Victor; Ponce-Navarrete, Gustavo; Andonegui-Elguera, Sergio; Jimenez-Vega, Florinda; Romero-Morelos, Pablo; Rodriguez-Esquivel, Miriam; Meraz-Rios, Marco; Figueroa-Corona, Ma Del Pilar; Monroy, Alberto; Pérez-González, Oscar; Salcedo, Mauricio

2017-05-01

Krüppel Like Factors (KLF) refers to a family of seventeen members of transcription factors. Involved in several cellular processes. As other cancer types, Cervical Cancer (CC) presents molecular deregulations in transcription factors, but especially Human Papilloma Virus (HPV) sequences. Here in this work we analyzed the mRNA expression of all KLF family members in CC-derived cell lines and CC tissues. The cell lines used were HeLa, INBL, RoVa, C4-I, Ms751, ViPa, CaLo, SiHa, CaSki, C33a and ViBo and the non-tumorigenic HaCaT. mRNA expression was analyzed by means of expression microarray and RT-PCR, and KLF5 protein by immunofluorescence. The cell lines were grouped according to HPV genotype as HPV16, HPV18 positive or HPV negative cells. Heterogeneous expression was observed among the cell lines. Despite the heterogeneous expression profile, KLF3, -5, -12, -15 and -16 transcripts were present in all cell lines, KLF4 and -10 which were not expressed in CaSki; KLF11 and 13 were not expressed by Vipa and C4-I, and KLF7 was not expressed by C4-I and Rova. The CC tissue analysis shows expression of most of the KLF members, such as KLF5. KLF5 immunosignal was positive in the three cell lines analyzed. We suggest that KLF expression could not be related to HPV presence/genotype, at least at transcriptional level, and the expression of KLF family members may be necessary in the biology of the CC cells. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

Mechanical Tension Increases CCN2/CTGF Expression and Proliferation in Gingival Fibroblasts via a TGFβ-Dependent Mechanism

PubMed Central

Guo, Fen; Carter, David E.; Leask, Andrew

2011-01-01

Unlike skin, oral gingival do not scar in response to tissue injury. Fibroblasts, the cell type responsible for connective tissue repair and scarring, are exposed to mechanical tension during normal and pathological conditions including wound healing and fibrogenesis. Understanding how human gingival fibroblasts respond to mechanical tension is likely to yield valuable insights not only into gingival function but also into the molecular basis of scarless repair. CCN2/connective tissue growth factor is potently induced in fibroblasts during tissue repair and fibrogenesis. We subjected gingival fibroblasts to cyclical strain (up to 72 hours) using the Flexercell system and showed that CCN2 mRNA and protein was induced by strain. Strain caused the rapid activation of latent TGFβ, in a fashion that was reduced by blebbistatin and FAK/src inhibition, and the induction of endothelin (ET-1) mRNA and protein expression. Strain did not cause induction of α-smooth muscle actin or collagen type I mRNAs (proteins promoting scarring); but induced a cohort of pro-proliferative mRNAs and cell proliferation. Compared to dermal fibroblasts, gingival fibroblasts showed reduced ability to respond to TGFβ by inducing fibrogenic mRNAs; addition of ET-1 rescued this phenotype. Pharmacological inhibition of the TGFβ type I (ALK5) receptor, the endothelin A/B receptors and FAK/src significantly reduced the induction of CCN2 and pro-proliferative mRNAs and cell proliferation. Controlling TGFβ, ET-1 and FAK/src activity may be useful in controlling responses to mechanical strain in the gingiva and may be of value in controlling fibroproliferative conditions such as gingival hyperplasia; controlling ET-1 may be of benefit in controlling scarring in response to injury in the skin. PMID:21611193

Evaluation of procoagulant tissue factor expression in canine hemangiosarcoma cell lines

PubMed Central

Witter, Lauren E.; Gruber, Erika J.; Lean, Fabian Z. X.; Stokol, Tracy

2017-01-01

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor–replete or specific coagulation factor–deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII-deficient plasma than in factor-replete plasma and was abolished in factor X–deficient plasma; residual thrombin generation in FVII-deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma. PMID:28029283

Accurate expressions for solar cell fill factors including series and shunt resistances

NASA Astrophysics Data System (ADS)

Green, Martin A.

2016-02-01

Together with open-circuit voltage and short-circuit current, fill factor is a key solar cell parameter. In their classic paper on limiting efficiency, Shockley and Queisser first investigated this factor's analytical properties showing, for ideal cells, it could be expressed implicitly in terms of the maximum power point voltage. Subsequently, fill factors usually have been calculated iteratively from such implicit expressions or from analytical approximations. In the absence of detrimental series and shunt resistances, analytical fill factor expressions have recently been published in terms of the Lambert W function available in most mathematical computing software. Using a recently identified perturbative relationship, exact expressions in terms of this function are derived in technically interesting cases when both series and shunt resistances are present but have limited impact, allowing a better understanding of their effect individually and in combination. Approximate expressions for arbitrary shunt and series resistances are then deduced, which are significantly more accurate than any previously published. A method based on the insights developed is also reported for deducing one-diode fits to experimental data.

Factors Expressed in an Animal Model of Anteroinferior Glenohumeral Instability

PubMed Central

Mulcahey, Mary K.; Marshall, Mindy; Gallacher, Stacey E.; Kaback, Lee A.; Blaine, Theodore A.

2015-01-01

Background: There is little information on the molecular factors important in healing and changes that occur in the glenoid labrum in response to injury. Using a novel animal model of acute anterior shoulder dislocation, this study characterizes the factors expressed in the glenoid labrum in response to injury and correlates their expression to glenohumeral stability. Purpose: To study the response of the glenoid labrum to injury both biomechanically and with immunohistochemical testing. Methods: An injury to the anteroinferior labrum was surgically induced in 50 male Lewis rats. Rats were sacrificed at 3, 7, 14, 28, or 42 days. Immunolocalization experiments were performed to localize the expression of growth factors and cytokines. For biomechanical testing, dynamic stiffness for anterior and posterior laxity, load to failure, stiffness, and maximum load were recorded. Statistical differences were determined at P < .05. Study Design: Descriptive laboratory study. Results: Expression of interleukin–1 beta (IL-1β), transforming growth factor–beta 1 (TGF-β1), matrix metalloproteinase 3 (MMP3), and matrix metalloproteinase 13 (MMP13) were increased in injured compared with uninjured specimens. Collagen III expression was increased early and decreased with time. Biomechanical testing verified instability by demonstrating increased anterior displacement and decreased stiffness in injured shoulders at all time points. Conclusion: This novel animal model of acute anterior shoulder dislocation showed increased expression of IL-1β, TGF-β1, MMP3, MMP13, and collagen III in the injured labral tissue at early time points. Increased anterior laxity and decreased stiffness and maximum load to failure were seen after anterior labral injury, supporting the model’s ability to re-create anterior glenohumeral instability. These data provide important information on the temporal changes occurring in a rat model of anterior glenohumeral dislocation. Clinical Relevance

Insulin-like growth factor and fibroblast growth factor expression profiles in growth-restricted fetal sheep pancreas.

PubMed

Chen, Xiaochuan; Rozance, Paul J; Hay, William W; Limesand, Sean W

2012-05-01

Placental insufficiency results in intrauterine growth restriction (IUGR), impaired fetal insulin secretion and less fetal pancreatic β-cell mass, partly due to lower β-cell proliferation rates. Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) regulate fetal β-cell proliferation and pancreas development, along with transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1). We determined expression levels for these growth factors, their receptors and IGF binding proteins in ovine fetal pancreas and isolated islets. In the IUGR pancreas, relative mRNA expression levels of IGF-I, PDX-1, FGF7 and FGFR2IIIb were 64% (P < 0.01), 76% (P < 0.05), 76% (P < 0.05) and 52% (P < 0.01) lower, respectively, compared with control fetuses. Conversely, insulin-like growth factor binding protein 2 (IGFBP-2) mRNA and protein concentrations were 2.25- and 1.2-fold greater (P < 0.05) in the IUGR pancreas compared with controls. In isolated islets from IUGR fetuses, IGF-II and IGFBP-2 mRNA concentrations were 1.5- and 3.7-fold greater (P < 0.05), and insulin mRNA was 56% less (P < 0.05) than control islets. The growth factor expression profiles for IGF and FGF signaling pathways indicate that declines in β-cell mass are due to decreased growth factor signals for both pancreatic progenitor epithelial cell and mature β-cell replication.

Epidermal Growth Factor Increases LRF/Pokemon Expression in Human Prostate Cancer Cells

PubMed Central

Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K.

2011-01-01

Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis. PMID:21640721

Renal sympathetic denervation alleviates myocardial fibrosis following isoproterenol-induced heart failure

PubMed Central

Wang, Neng; Zheng, Xiaoxin; Qian, Jin; Yao, Wei; Bai, Lu; Hou, Guo; Qiu, Xuan; Li, Xiaoyan; Jiang, Xuejun

2017-01-01

The aim of the present study was to determine if renal sympathetic denervation (RSD) may alleviate isoproterenol-induced left ventricle remodeling, and to identify the underlying mechanism. A total of 70 rats were randomly divided into control (n=15), sham operation (n=15), heart failure (HF) with sham operation (HF + sham; n=20) and HF with treatment (HF + RSD; n=20) groups. The HF model was established by subcutaneous injection of isoproterenol; six weeks later, 1eft ventricular internal diameter at end-systole (LVIDs), left ventricular systolic posterior wall thickness (LVPWs), 1eft ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured. Plasma norepinephrine (NE), angiotensin II (Ang II) and aldosterone (ALD) levels were measured by ELISA. Myocardial collagen volume fraction (CVF) was determined by Masson's staining. Reverse transcription-quantitative polymerase chain reaction was used to determine the mRNA expression levels of ventricular transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF) and microRNAs (miRs), including miR-29b, miR-30c and miR-133a. The results demonstrated that LVIDs and LVPWs in the HF + RSD group were significantly decreased compared with the HF + sham group. By contrast, LVFS and LVEF in the HF + RSD group were significantly increased compared with the HF + sham group. RSD significantly reduced the levels of plasma NE, Ang II and ALD. CVF in the HF + RSD group was reduced by 38.1% compared with the HF + sham group. Expression levels of TGF-β and CTGF were decreased, whereas those of miR-29b, miR-30c and miR-133a were increased, in the HF + RSD group compared with the HF + sham group. These results indicated that RSD alleviates isoproterenol-induced left ventricle remodeling potentially via downregulation of TGF-β/CTGF and upregulation of miR-29b, miR-30c and miR-133a. RSD may therefore be an effective non-drug therapy for the treatment of heart failure. PMID

Tissue factor expression as a possible determinant of thromboembolism in ovarian cancer

PubMed Central

Uno, K; Homma, S; Satoh, T; Nakanishi, K; Abe, D; Matsumoto, K; Oki, A; Tsunoda, H; Yamaguchi, I; Nagasawa, T; Yoshikawa, H; Aonuma, K

2007-01-01

Ovarian cancer, and clear cell carcinoma in particular, reportedly increases the risk of venous thromboembolism (VTE). However, the mechanisms remain unclear. Tissue factor (TF) supposedly represents a major factor in the procoagulant activities of cancer cells. The present study examined the involvement of TF expression in VTE for patients with ovarian cancer. Subjects comprised 32 consecutive patients (mean age 49.8 years) with histologically confirmed ovarian cancer. Presence of VTE was examined using a combination of clinical features, D-dimer levels and venous ultrasonography. Immunohistochemical analysis was used to evaluate TF expression into 4 degrees. Venous thromboembolism was identified in 10 of the 32 patients (31%), including five of the 11 patients with clear cell carcinoma. Tissue factor expression was detected in cancer tissues from 24 patients and displayed significant correlations with VTE development (P=0.0003), D-dimer concentration (P=0.003) and clear cell carcinoma (P<0.05). Multivariate analysis identified TF expression as an independent predictive factor of VTE development (P<0.05). Tissue factor (TF) expression is a possible determinant of VTE development in ovarian cancer. In particular, clear cell carcinoma may produce excessive levels of TF and is more likely to develop VTE. PMID:17211468

Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors.

PubMed

Perdigão, Pedro; Gaj, Thomas; Santa-Marta, Mariana; Barbas, Carlos F; Goncalves, Joao

2016-01-01

The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.

Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors

PubMed Central

Perdigão, Pedro; Gaj, Thomas; Santa-Marta, Mariana; Goncalves, Joao

2016-01-01

The presence of replication-competent HIV-1 –which resides mainly in resting CD4+ T cells–is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection. PMID:26933881

Keratinocyte growth factor expression in human gingival fibroblasts and stimulation of in vitro gene expression by retinoic acid.

PubMed

Mackenzie, I C; Gao, Z

2001-04-01

Keratinocyte growth factor (KGF) is a stromally derived growth factor of the fibroblast growth factor (FGF) family with paracrine effects targeted to influence the growth and differentiation of epithelia. Regional and temporal changes in KGF expression play important roles in the development and maintenance of epithelial structures and in epithelial wound healing. Differing patterns of expression of KGF by fibroblasts in the gingival region could therefore be related to the observed regional variation in the differentiation and behavior of gingival epithelia. The in vitro and in vivo patterns of expression of KGF mRNA in human gingival and periodontal fibroblasts were examined using reverse transcription polymerase chain reactions (RT-PCR) and in situ hybridization with digoxigenin-labeled riboprobes. The patterns observed for human gingiva were compared with those for human skin and for murine tissues. Gingival and periodontal fibroblasts showed expression of KGF transcripts in vitro, and the degree of expression was markedly influenced by the presence of retinoic acid, an agent known to influence patterns of epithelial differentiation. Sections of human and murine gingiva and skin showed regionally variable expression of transcripts with the cells expressing KGF in the subepithelial, rather than the deeper, connective tissues and periodontium. The results point to a role of KGF in the maintenance of normal growth and differentiation of gingival epithelia. A lack of KGF expression by periodontal fibroblasts in vivo is expected to hinder apical epithelial migration and thus stabilize the epithelial attachment. The effects of retinoic acid (RA) on KGF expression in vitro provide an indirect mechanism by which RA may regulate the growth and differentiation of gingival epithelia.

Epidermal growth factor increases LRF/Pokemon expression in human prostate cancer cells.

PubMed

Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K

2011-10-01

Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.

Environmental signals modulate ToxT-dependent virulence factor expression in Vibrio cholerae.

PubMed

Schuhmacher, D A; Klose, K E

1999-03-01

The regulatory protein ToxT directly activates the transcription of virulence factors in Vibrio cholerae, including cholera toxin (CT) and the toxin-coregulated pilus (TCP). Specific environmental signals stimulate virulence factor expression by inducing the transcription of toxT. We demonstrate that transcriptional activation by the ToxT protein is also modulated by environmental signals. ToxT expressed from an inducible promoter activated high-level expression of CT and TCP in V. cholerae at 30 degrees C, but expression of CT and TCP was significantly decreased or abolished by the addition of 0.4% bile to the medium and/or an increase of the temperature to 37 degrees C. Also, expression of six ToxT-dependent TnphoA fusions was modulated by temperature and bile. Measurement of ToxT-dependent transcription of genes encoding CT and TCP by ctxAp- and tcpAp-luciferase fusions confirmed that negative regulation by 37 degrees C or bile occurs at the transcriptional level in V. cholerae. Interestingly, ToxT-dependent transcription of these same promoters in Salmonella typhimurium was relatively insensitive to regulation by temperature or bile. These data are consistent with ToxT transcriptional activity being modulated by environmental signals in V. cholerae and demonstrate an additional level of complexity governing the expression of virulence factors in this pathogen. We propose that negative regulation of ToxT-dependent transcription by environmental signals prevents the incorrect temporal and spatial expression of virulence factors during cholera pathogenesis.

Baicalein suppresses 17-β-estradiol-induced migration, adhesion and invasion of breast cancer cells via the G protein-coupled receptor 30 signaling pathway.

PubMed

Shang, Dandan; Li, Zheng; Zhu, Zhuxia; Chen, Huamei; Zhao, Lujun; Wang, Xudong; Chen, Yan

2015-04-01

Flavonoids are structurally similar to steroid hormones, particularly estrogens, and therefore have been studied for their potential effects on hormone-dependent cancers. Baicalein is the primary flavonoid derived from the root of Scutellaria baicalensis Georgi. In the present study, we investigated the effects of baicalein on 17β-estradiol (E2)-induced migration, adhesion and invasion of MCF-7 and SK-BR-3 breast cancer cells. The results demonstrated that baicalein suppressed E2-stimulated wound-healing migration and cell‑Matrigel adhesion, and ameliorated E2-promoted invasion across a Matrigel-coated Transwell membrane. Furthermore, baicalein interfered with E2-induced novel G protein-coupled estrogen receptor (GPR30)-related signaling, including a decrease in tyrosine phosphorylation of epidermal growth factor receptor (EGFR) as well as phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine kinase Akt, without affecting GPR30 expression. The results also showed that baicalein suppressed the expression of GPR30 target genes, cysteine-rich 61 (CYR61) and connective tissue growth factor (CTGF) induced by E2. Furthermore, baicalein prevented GPR30-related signaling activation and upregulation of CYR61 and CTGF mRNA levels induced by G1, a specific GPR 30 agonist. The results suggest that baicalein inhibits E2-induced migration, adhesion and invasion through interfering with GPR30 signaling pathway activation, which indicates that it may act as a therapeutic candidate for the treatment of GPR30-positive breast cancer metastasis.

Expression of vascular endothelial growth factor in Juvenile Angiofibroma.

PubMed

Hota, Ashutosh; Sarkar, Chitra; Gupta, Siddhartha Datta; Kumar, Rakesh; Bhalla, Ashu Seith; Thakar, Alok

2015-06-01

To examine Juvenile Angiofibroma (JA) tissue for expression of vascular endothelial growth factor (VEGF), and to explore its relationship with puberty status, stage, recurrence and the intraoperative blood loss. Retrospective cohort study of 36 histologically proven cases of JA. Minimum follow up period was 3 years. VEGF expression on tumor cells assessed by immunohistochemistry and graded on two criteria--percentage of cells expressing positivity and the intensity of positivity. These two parameters assessed for impact on puberty status, stage, recurrence, and blood loss. VEGF expression noted on the tumor endothelial cells in 36/36, and on the tumor stromal cells in 34/36. The percentage of cells expressing VEGF and the intensity of expression were not significantly related to puberty status, tumor stage, recurrence, or intra-operative blood loss (p values 0.3-1.0). VEGF expression is near universal in JA. Such expression is independent of puberty status and stage, and does not impact on intra operative blood loss and recurrence. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Accuracy of expressions for the fill factor of a solar cell in terms of open-circuit voltage and ideality factor

NASA Astrophysics Data System (ADS)

Leilaeioun, Mehdi; Holman, Zachary C.

2016-09-01

An approximate expression proposed by Green predicts the maximum obtainable fill factor (FF) of a solar cell from its open-circuit voltage (Voc). The expression was originally suggested for silicon solar cells that behave according to a single-diode model and, in addition to Voc, it requires an ideality factor as input. It is now commonly applied to silicon cells by assuming a unity ideality factor—even when the cells are not in low injection—as well as to non-silicon cells. Here, we evaluate the accuracy of the expression in several cases. In particular, we calculate the recombination-limited FF and Voc of hypothetical silicon solar cells from simulated lifetime curves, and compare the exact FF to that obtained with the approximate expression using assumed ideality factors. Considering cells with a variety of recombination mechanisms, wafer doping densities, and photogenerated current densities reveals the range of conditions under which the approximate expression can safely be used. We find that the expression is unable to predict FF generally: For a typical silicon solar cell under one-sun illumination, the error is approximately 6% absolute with an assumed ideality factor of 1. Use of the expression should thus be restricted to cells under very low or very high injection.

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed Central

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-01-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver. PMID:9343392

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-11-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver.

Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

PubMed

Nakagawa, Tateo; Shimada, Mitsuo; Kurita, Nobuhiro; Iwata, Takashi; Nishioka, Masanori; Yoshikawa, Kozo; Higashijima, Jun; Utsunomiya, Tohru

2012-06-01

The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

Expression dynamics of self-renewal factors for spermatogonial stem cells in the mouse testis.

PubMed

Sakai, Mizuki; Masaki, Kaito; Aiba, Shota; Tone, Masaaki; Takashima, Seiji

2018-04-16

Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells (SSCs). Although GDNF is indispensable for the maintenance of SSCs, the role of FGF2 in the testis remains to be elucidated. To clarify this, the expression dynamics and regulatory mechanisms of Fgf2 and Gdnf in the mouse testes were analyzed. It is well known that Sertoli cells express Gdnf, and its receptor is expressed in a subset of undifferentiated spermatogonia, including SSCs. However, we found that Fgf2 was mainly expressed in the germ cells and its receptors were expressed not only in the cultured spermatogonial cell line, but also in testicular somatic cells. Aging, hypophysectomy, retinoic acid treatment, and testicular injury induced distinct Fgf2 and Gdnf expression dynamics, suggesting a difference in the expression mechanism of Fgf2 and Gdnf in the testis. Such differences might cause a dynamic fluctuation of Gdnf/Fgf2 ratio depending on the intrinsic/extrinsic cues. Considering that FGF2-cultured spermatogonia exhibit more differentiated phenotype than those cultured with GDNF, FGF2 might play a role distinct from that of GDNF in the testis, despite the fact that both factors are self-renewal factor for SSC in vitro.

Combined blockade of angiotensin II and prorenin receptors ameliorates podocytic apoptosis induced by IgA-activated mesangial cells.

PubMed

Leung, Joseph C K; Chan, Loretta Y Y; Saleem, M A; Mathieson, P W; Tang, Sydney C W; Lai, Kar Neng

2015-07-01

Glomerulo-podocytic communication plays an important role in the podocytic injury in IgA nephropathy (IgAN). In this study, we examine the role of podocytic angiotensin II receptor subtype 1 (AT1R) and prorenin receptor (PRR) in podocytic apoptosis in IgAN. Polymeric IgA (pIgA) was isolated from patients with IgAN and healthy controls. Conditioned media were prepared from growth arrested human mesangial cells (HMC) incubated with pIgA from patients with IgAN (IgA-HMC media) or healthy controls (Ctl-HMC media). A human podocyte cell line was used as a model to examine the regulation of the expression of AT1R, PRR, TNF-α and CTGF by IgA-HMC media. Podocytic nephrin expression, annexin V binding and caspase 3 activity were used as the functional readout of podocytic apoptosis. IgA-HMC media had no effect on AngII release by podocytes. IgA-HMC media significantly up-regulated the expression of AT1R and PRR, down-regulated nephrin expression and induced apoptosis in podocytes. Mono-blockade of AT1R, PRR, TNF-α or CTGF partially reduced podocytic apoptosis. IgA-HMC media activated NFκB, notch1 and HEY1 expression by podocytes and dual blockade of AT1R with PRR, or anti-TNF-α with anti-CTGF, effectively rescued the podocytic apoptosis induced by IgA-HMC media. Our data suggests that pIgA-activated HMC up-regulates the expression of AT1R and PRR expression by podocytes and the associated activation of NFκB and notch signalling pathways play an essential role in the podocytic apoptosis induced by glomerulo-podocytic communication in IgAN. Simultaneously targeting the AT1R and PRR could be a potential therapeutic option to reduce the podocytic injury in IgAN.

Endometrial Expression of Steroidogenic Factor 1 Promotes Cystic Glandular Morphogenesis

PubMed Central

Vasquez, Yasmin M.; Wu, San-Pin; Anderson, Matthew L.; Hawkins, Shannon M.; Creighton, Chad J.; Ray, Madhumita; Tsai, Sophia Y.; Tsai, Ming-Jer; Lydon, John P.

2016-01-01

Epigenetic silencing of steroidogenic factor 1 (SF1) is lost in endometriosis, potentially contributing to de novo local steroidogenesis favoring inflammation and growth of ectopic endometrial tissue. In this study, we examine the impact of SF1 expression in the eutopic uterus by a novel mouse model that conditionally expresses SF1 in endometrium. In vivo SF1 expression promoted the development of enlarged endometrial glands and attenuated estrogen and progesterone responsiveness. Endometriosis induction by autotransplantation of uterine tissue to the mesenteric membrane resulted in the increase in size of ectopic lesions from SF1-expressing mice. By integrating the SF1-dependent transcriptome with the whole genome binding profile of SF1, we identified uterine-specific SF1-regulated genes involved in Wingless and Progesterone receptor-Hedgehog-Chicken ovalbumin upstream promoter transcription factor II signaling for gland development and epithelium-stroma interaction, respectively. The present results indicate that SF1 directly contributes to the abnormal uterine gland morphogenesis, an inhibition of steroid hormone signaling and activation of an immune response, in addition to previously postulated estrogen production. PMID:27018534

Molecular analysis of arterial remodeling: a novel application of infrared imaging

NASA Astrophysics Data System (ADS)

Herman, Brad C.; Kundi, Rishi; Yamanouchi, Dai; Kent, K. Craig; Liu, Bo; Pleshko, Nancy

2009-02-01

Arterial remodeling, i.e. changes in size and/or structure of arteries, plays an important role in vascular disease. Conflicting findings have been reported as to whether an abundance of collagen causes inward or outward remodeling, phenomena that result in either a smaller or larger lumen, respectively. We hypothesize that the amount, type and quality of collagen influence the remodeling response. Here, we create mechanical injury to the rat carotid artery using a balloon catheter, and this leads to inward remodeling. Treatment of the artery with Connective Tissue Growth Factor (CTGF) causes outward remodeling. We investigated the arterial composition in injured CTGF-treated and non-CTGF-treated and sham CTGF-treated and non-CTGF treated arteries 14 days post-injury (n = 7-8 per group) using infrared imaging. A Perkin Elmer Spotlight Spectrum 300 FT-IR microscope was used for data collection. Cross-sections of paraffinembedded arteries were scanned at 2 cm-1 spectral resolution with spatial resolution of 6.25 μm/pixel, and data analyzed using Malvern Instruments ISys 5.0. Post-injury, we found a nearly 50% reduction in the average 1338/AM2 area ratio (correlated to collagen helical integrity). The most dramatic change was a 600% increase in the 1660/1690 peak height ratio, which has previously been related to collagen crosslink maturity. In all cases, CTGF treatment resulted in the observed changes in peak parameters normalized back to control values. Overall, these preliminary studies demonstrate that infrared imaging can provide insight into the underlying molecular changes that contribute to arterial disease.

p27{sup Kip1} inhibits tissue factor expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Breitenstein, Alexander, E-mail: alexander.breitenstein@usz.ch; Cardiovascular Research, Physiology Institute, University of Zurich; Center for Integrative Human Physiology

2013-10-04

Highlights: •p27{sup Kip1}regulates the expression of tissue factor at the transcriptional level. •This inhibitory effect of p27{sup Kip1} is independently of its cell regulatory action. •The current study provides new insights into a pleiotrophic function of p27{sup Kip1}. -- Abstract: Background: The cyclin-dependent kinase inhibitor (CDKI) p27{sup Kip1} regulates cell proliferation and thus inhibits atherosclerosis and vascular remodeling. Expression of tissue factor (TF), the key initator of the coagulation cascade, is associated with atherosclerosis. Yet, it has not been studied whether p27{sup Kip1} influences the expression of TF. Methods and results: p27{sup Kip1} overexpression in human aortic endothelial cells wasmore » achieved by adenoviral transfection. Cells were rendered quiescent for 24 h in 0.5% fetal-calf serum. After stimulation with TNF-α (5 ng/ml), TF protein expression and activity was significantly reduced (n = 4; P < 0.001) in cells transfected with p27{sup Kip1}. In line with this, p27{sup Kip1} overexpression reduced cytokine-induced TF mRNA expression (n = 4; P < 0.01) and TF promotor activity (n = 4; P < 0.05). In contrast, activation of the MAP kinases p38, ERK and JNK was not affected by p27{sup Kip1} overexpression. Conclusion: This in vitro study suggests that p27{sup Kip1} inhibits TF expression at the transcriptional level. These data indicate an interaction between p27{sup Kip1} and TF in important pathological alterations such as atherosclerosis and vascular remodeling.« less

Tumour cells down-regulate CCN2 gene expression in co-cultured fibroblasts in a Smad7- and ERK-dependent manner.

PubMed

van Rooyen, Beverley A; Schäfer, Georgia; Leaner, Virna D; Parker, M Iqbal

2013-10-03

Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix components found in the stroma. The aim of this study was to investigate mechanisms involved in tumour cell-mediated regulation of extracellular matrix and adhesion molecules in co-cultured fibroblasts. To this end, microarray analysis was performed on CCD-1068SK human fibroblast cells after direct co-culture with MDA-MB-231 human breast tumour cells. We found that the expression of both connective tissue growth factor (CTGF/CCN2) and type I collagen was negatively regulated in CCD-1068SK fibroblast cells under direct co-culture conditions. Further analysis revealed that Smad7, a known negative regulator of the Smad signalling pathway involved in CCN2 promoter regulation, was increased in directly co-cultured fibroblasts. Inhibition of Smad7 expression in CCD-1068SK fibroblasts resulted in increased CCN2 expression, while Smad7 overexpression had the opposite effect. Silencing CCN2 gene expression in fibroblasts led, in turn, to a decrease in type I collagen mRNA and protein levels. ERK signalling was also shown to be impaired in CCD-1068SK fibroblasts after direct co-culture with MDA-MB-231 tumour cells, with Smad7 overexpression in fibroblasts leading to a similar decrease in ERK activity. These effects were not, however, seen in fibroblasts that were indirectly co-cultured with tumour cells. We therefore conclude that breast cancer cells require close contact with fibroblasts in order to upregulate Smad7 which, in turn, leads to decreased ERK signalling resulting in diminished expression of the stromal proteins CCN2 and type I collagen.

Methionine-supplemented diet affects the expression of cardiovascular disease-related genes and increases inflammatory cytokines in mice heart and liver.

PubMed

Aissa, Alexandre Ferro; Amaral, Catia Lira do; Venancio, Vinicius Paula; Machado, Carla da Silva; Hernandes, Lívia Cristina; Santos, Patrick Wellington da Silva; Curi, Rui; Bianchi, Maria de Lourdes Pires; Antunes, Lusânia Maria Greggi

2017-01-01

Some important environmental factors that influence the development of cardiovascular diseases (CVD) include tobacco, excess alcohol, and unhealthy diet. Methionine obtained from the diet participates in the synthesis of DNA, proteins, lipids and affects homocysteine levels, which is associated with the elevated risk for CVD development. Therefore, the aim of this study was to investigate the manner in which dietary methionine might affect cellular mechanisms underlying CVD occurrence. Swiss albino mice were fed either control (0.3% DL-methionine), methionine-supplemented (2% DL-methionine), or a methionine-deprived diet (0% DL-methionine) over a 10-week period. The parameters measured included plasma homocysteine concentrations, oxidative stress by reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, levels of inflammatory cytokines IL-1ß, TNF-α, and IL-6, as well as expression of genes associated with CVD. The levels of apolipoprotein A5 (APOA5), a regulator of plasma triglycerides, were measured. The methionine-supplemented diet increased oxidative stress by lowering the GSH/GSSG ratio in heart tissues and decreased expression of the genes Apob, Ctgf, Serpinb2, Spp1, Il1b, and Sell, but elevated expression of Thbs4, Tgfb2, Ccr1, and Vegfa. Methionine-deprived diet reduced expression of Col3a1, Cdh5, Fabp3, Bax, and Hbegf and increased expression of Sell, Ccl5, Itga2, Birc3, Msr1, Bcl2a1a, Il1r2, and Selp. Methionine-deprived diet exerted pro-inflammatory consequences as evidenced by elevated levels of cytokines IL-1ß, TNF-α, and IL-6 noted in liver. Methionine-supplemented diet increased hepatic IL-6 and cardiac TNF-α. Both methionine supplementation and deprivation lowered hepatic levels of APOA5. In conclusion, data demonstrated that a methionine-supplemented diet modulated important biological processes associated with high risk of CVD development.

Fibroblast growth factor 8 is expressed at higher levels in lactating human breast and in breast cancer.

PubMed

Zammit, C; Coope, R; Gomm, J J; Shousha, S; Johnston, C L; Coombes, R C

2002-04-08

Fibroblast growth factor 8 can transform NIH3T3 cells and its expression has been found to be associated with breast and prostate cancer. Following our finding that fibroblast growth factor 8 mRNA expression is increased in breast cancer, we have undertaken an immunohistochemistry study of fibroblast growth factor 8 expression in a series of human breast tissues and other normal tissues. Our findings confirm increased expression of fibroblast growth factor 8 in malignant breast tissue but also show significant fibroblast growth factor 8 expression in non-malignant breast epithelial cells. No significant difference in fibroblast growth factor 8 expression was found between different grades of ductal carcinoma, lobular carcinoma and ductal carcinoma in-situ or cancer of different oestrogen receptor, progesterone receptor or nodal status. The highest levels of fibroblast growth factor 8 expression were found in lactating breast tissues and fibroblast growth factor 8 was also detected in human milk. A survey of other normal tissues showed that fibroblast growth factor 8 is expressed in the proliferative cells of the dermis and epithelial cells in colon, ovary fallopian tube and uterus. Fibroblast growth factor 8 appears to be expressed in several organs in man and appears to have an importance in lactation.

The strategy of fusion genes construction determines efficient expression of introduced transcription factors.

PubMed

Adamus, Tomasz; Konieczny, Paweł; Sekuła, Małgorzata; Sułkowski, Maciej; Majka, Marcin

2014-01-01

The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

PubMed

Sun, Wan-Wei; Zhang, Xin-Xu; Wan, Wei-Song; Wang, Shu-Qi; Wen, Xiao-Bo; Zheng, Huai-Ping; Zhang, Yue-Ling; Li, Sheng-Kang

2017-02-01

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain. Copyright © 2016. Published by Elsevier Ltd.

Expression of a transmembrane phosphotyrosine phosphatase inhibits cellular response to platelet-derived growth factor and insulin-like growth factor-1.

PubMed

Mooney, R A; Freund, G G; Way, B A; Bordwell, K L

1992-11-25

Tyrosine phosphorylation is a mechanism of signal transduction shared by many growth factor receptors and oncogene products. Phosphotyrosine phosphatases (PTPases) potentially modulate or counter-regulate these signaling pathways. To test this hypothesis, the transmembrane PTPase CD45 (leukocyte common antigen) was expressed in the murine cell line C127. Hormone-dependent autophosphorylation of the platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) receptors was markedly reduced in cells expressing the transmembrane PTPase. Tyrosine phosphorylation of other PDGF-dependent phosphoproteins (160, 140, and 55 kDa) and IGF-1-dependent phosphoproteins (145 kDa) was similarly decreased. Interestingly, the pattern of growth factor-independent tyrosine phosphorylations was comparable in cells expressing the PTPase and control cells. This suggests a selectivity or accessibility of the PTPase limited to a subset of cellular phosphotyrosyl proteins. The maximum mitogenic response to PDGF and IGF-1 in cells expressing the PTPase was decreased by 67 and 71%, respectively. These results demonstrate that a transmembrane PTPase can both affect the tyrosine phosphorylation state of growth factor receptors and modulate proximal and distal cellular responses to the growth factors.

Baicalin Ameliorates Experimental Liver Cholestasis in Mice by Modulation of Oxidative Stress, Inflammation, and NRF2 Transcription Factor

PubMed Central

Feng, Xiaowen; Zhang, Feng; Xie, Haiyang

2017-01-01

Experimental cholestatic liver fibrosis was performed by bile duct ligation (BDL) in mice, and significant liver injury was observed in 15 days. Administration of baicalin in mice significantly ameliorates liver fibrosis. Experimental cholestatic liver fibrosis was associated with induced gene expression of fibrotic markers such as collagen I, fibronectin, alpha smooth muscle actin (SMA), and connective tissue growth factor (CTGF); increased inflammatory cytokines (TNFα, MIP1α, IL1β, and MIP2); increased oxidative stress and reactive oxygen species- (ROS-) inducing enzymes (NOX2 and iNOS); dysfunctional mitochondrial electron chain complexes; and apoptotic/necrotic cell death markers (DNA fragmentation, caspase 3 activity, and PARP activity). Baicalin administration on alternate day reduced fibrosis along with profibrotic gene expression, proinflammatory cytokines, oxidative stress, and cell death whereas improving the function of mitochondrial electron transport chain. We observed baicalin enhanced NRF2 activation by nuclear translocation and induced its target genes HO-1 and GCLM, thus enhancing antioxidant defense. Interplay of oxidative stress/inflammation and NRF2 were key players for baicalin-mediated protection. Stellate cell activation is crucial for initiation of fibrosis. Baicalin alleviated stellate cell activation and modulated TIMP1, SMA, collagen 1, and fibronectin in vitro. This study indicates that baicalin might be beneficial for reducing inflammation and fibrosis in liver injury models. PMID:28757911

Protein-releasing polymeric scaffolds induce fibrochondrocytic differentiation of endogenous cells for knee meniscus regeneration in sheep.

PubMed

Lee, Chang H; Rodeo, Scott A; Fortier, Lisa Ann; Lu, Chuanyong; Erisken, Cevat; Mao, Jeremy J

2014-12-10

Regeneration of complex tissues, such as kidney, liver, and cartilage, continues to be a scientific and translational challenge. Survival of ex vivo cultured, transplanted cells in tissue grafts is among one of the key barriers. Meniscus is a complex tissue consisting of collagen fibers and proteoglycans with gradient phenotypes of fibrocartilage and functions to provide congruence of the knee joint, without which the patient is likely to develop arthritis. Endogenous stem/progenitor cells regenerated the knee meniscus upon spatially released human connective tissue growth factor (CTGF) and transforming growth factor-β3 (TGFβ3) from a three-dimensional (3D)-printed biomaterial, enabling functional knee recovery. Sequentially applied CTGF and TGFβ3 were necessary and sufficient to propel mesenchymal stem/progenitor cells, as a heterogeneous population or as single-cell progenies, into fibrochondrocytes that concurrently synthesized procollagens I and IIα. When released from microchannels of 3D-printed, human meniscus scaffolds, CTGF and TGFβ3 induced endogenous stem/progenitor cells to differentiate and synthesize zone-specific type I and II collagens. We then replaced sheep meniscus with anatomically correct, 3D-printed scaffolds that incorporated spatially delivered CTGF and TGFβ3. Endogenous cells regenerated the meniscus with zone-specific matrix phenotypes: primarily type I collagen in the outer zone, and type II collagen in the inner zone, reminiscent of the native meniscus. Spatiotemporally delivered CTGF and TGFβ3 also restored inhomogeneous mechanical properties in the regenerated sheep meniscus. Survival and directed differentiation of endogenous cells in a tissue defect may have implications in the regeneration of complex (heterogeneous) tissues and organs. Copyright © 2014, American Association for the Advancement of Science.

Pirfenidone inhibits migration, differentiation, and proliferation of human retinal pigment epithelial cells in vitro

PubMed Central

Wang, Jing; Yang, Yangfan; Xu, Jiangang; Lin, Xianchai; Wu, Kaili

2013-01-01

Purpose To investigate the effects of pirfenidone (PFD) on the migration, differentiation, and proliferation of retinal pigment epithelial (RPE) cells and demonstrate whether the drug induces cytotoxicity. Methods Human RPE cells (line D407) were treated with various concentrations of PFD. Cell migration was measured with scratch assay. The protein levels of fibronectin (FN), connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), transforming growth factor beta (TGFβS), and Smads were assessed with western blot analyses. Levels of mRNA of TGFβS, FN, and Snail1 were analyzed using reverse transcriptase–polymerase chain reaction. Cell apoptosis was detected with flow cytometry using the Annexin V/PI apoptosis kit, and the percentages of cells labeled in different apoptotic stage were compared. A Trypan Blue assay was used to assess cell viability. Results PFD inhibited RPE cell migration. Western blot analyses showed that PFD inhibited the expression of FN, α-SMA, CTGF, TGFβ1, TGFβ2, Smad2/3, and Smad4. Similarly, PFD also downregulated mRNA levels of Snail1, FN, TGFβ1, and TGFβ2. No significant differences in cell apoptosis or viability were observed between the control and PFD-treated groups. Conclusions PFD inhibited RPE cell migration, differentiation, and proliferation in vitro and caused no significant cytotoxicity. PMID:24415895

Activated Hippo/Yes-Associated Protein Pathway Promotes Cell Proliferation and Anti-apoptosis in Endometrial Stromal Cells of Endometriosis.

PubMed

Song, Yong; Fu, Jing; Zhou, Min; Xiao, Li; Feng, Xue; Chen, Hengxi; Huang, Wei

2016-04-01

The imbalance in cell proliferation and apoptosis is considered an important role in the pathogenesis of endometriosis, but the exact mechanisms remains unclear. A newly established signaling pathway–Hippo/Yes-associated protein (YAP) pathway plays a critical role in the proliferation and apoptosis processes. However, studies focusing on Hippo/YAP pathway and endometriosis are lacking. The objective was to explore the function of the Hippo/YAP pathway in endometriosis. The expression of YAP was first investigated in endometrium of women with or without endometriosis. The role of YAP in cell proliferation and apoptosis is identified by transfection of endometrial stromal cells (ESCs) in vitro, subsequent Verteporfin treatments in eutopic ESCs in vitro, and endometriosis animal model of nude mice in vivo. Our results revealed that increased expression of YAP and decreased expression of p-YAP in ectopic and eutopic endometrium compared with normal endometrium. YAP knockdown in eutopic ESCs decreased cell proliferation and enhanced cell apoptosis companied with decreased expression of TEAD1, CTGF, and B-cell lymphoma/leukemia (BCL)-2; whereas overexpression of YAP resulted in increased proliferation and decreased apoptosis of normal ESCs with increased expression of TEAD1, CTGF, and BCL-2. By chromatin immunoprecipitation qPCR CTGF and BCL-2 were identified as directly downstream target genes of YAP-TEAD1 active complex. Eutopic ESCs treated with Verteporfin revealed decreased proliferation and enhanced apoptosis whereas in endometriosis animal models of nude mice treated with Verteporfin, the size of endometriotic lesions was significantly reduced. Our study suggests that the Hippo/YAP-signaling pathway plays a critical role in the pathogenesis of endometriosis and should present a novel therapeutic method against endometriosis.

The G protein-coupled receptor 30 is up-regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) in breast cancer cells and cardiomyocytes.

PubMed

Recchia, Anna Grazia; De Francesco, Ernestina Marianna; Vivacqua, Adele; Sisci, Diego; Panno, Maria Luisa; Andò, Sebastiano; Maggiolini, Marcello

2011-03-25

GPR30, also known as GPER, has been suggested to mediate rapid effects induced by estrogens in diverse normal and cancer tissues. Hypoxia is a common feature of solid tumors involved in apoptosis, cell survival, and proliferation. The response to low oxygen environment is mainly mediated by the hypoxia-inducible factor named HIF-1α, which activates signaling pathways leading to adaptive mechanisms in tumor cells. Here, we demonstrate that the hypoxia induces HIF-1α expression, which in turn mediates the up-regulation of GPER and its downstream target CTGF in estrogen receptor-negative SkBr3 breast cancer cells and in HL-1 cardiomyocytes. Moreover, we show that HIF-1α-responsive elements located within the promoter region of GPER are involved in hypoxia-dependent transcription of GPER, which requires the ROS-induced activation of EGFR/ERK signaling in both SkBr3 and HL-1 and cells. Interestingly, the apoptotic response to hypoxia was prevented by estrogens through GPER in SkBr3 cells. Taken together, our data suggest that the hypoxia-induced expression of GPER may be included among the mechanisms involved in the anti-apoptotic effects elicited by estrogens, particularly in a low oxygen microenvironment.

Effects and Mechanism of SO2 Inhalation on Rat Myocardial Collagen Fibers.

PubMed

Chen, Ping; Qiao, Decai; Liu, Xiaoli

2018-03-21

BACKGROUND This study investigates the effects and mechanism of sulfur dioxide (SO2) inhalation and exercise on rat myocardial collagen fiber. MATERIAL AND METHODS The rats were randomly divided into 4 groups: a control group (RG), an exercise group (EG), an SO2 pollution group (SRG), and an SO2 pollution and exercise group (SEG). Body weight, cardiac index, and left ventricular index in each group were compared. The myocardial hydroxyproline (Hyp) concentration was determined by pepsin acid hydrolysis. The interstitial myocardial collagen expression was measured by Sirius Red F3B in saturated carbazotic acid. The local myocardial angiotensin II type 1 receptor (AT1R) and connective tissue growth factor (CTGF) expression was tested by immunohistochemistry SABC method. RESULTS Compared with RG, the weight growth rate of EG, SRG, and SEG decreased significantly (P<0.01). Compared with EG, the body weight growth rate of SEG significantly decreased (P<0.01) and cardiac index and left ventricular index decreased but without a significant difference. Compared with EG, myocardial Hyp and collagen concentration, myocardial collagen volume fraction (CVF), perivascular collagen area (PVCA), and the expression of AT1R and CTGF in myocardium of SEG increased significantly (P<0.01). CONCLUSIONS SO2 inhalation and exercise will not only offset beneficial health effects of movement on the cardiovascular system, but also produce more unfavorable influences. People should pay attention to their environment when exercising, and try to avoid exercising in environments with SO2 pollution.

CCN2 plays a key role in extracellular matrix gene expression in severe hypertrophic cardiomyopathy and heart failure.

PubMed

Tsoutsman, Tatiana; Wang, Xiaoyu; Garchow, Kendra; Riser, Bruce; Twigg, Stephen; Semsarian, Christopher

2013-09-01

Hypertrophic cardiomyopathy (HCM) is the most common inherited primary myocardial disorder. HCM is characterized by interstitial fibrosis and excessive accumulation of extracellular matrix (ECM) proteins. Fibrosis in HCM has been associated with impaired cardiac function and heart failure, and has been considered a key substrate for ventricular arrhythmias and sudden death. The molecular triggers underpinning ECM production are not well established. We have previously developed a double-mutant mouse model of HCM that recapitulates the phenotype seen in humans with multiple mutations, including earlier onset of the disease, progression to a dilated phenotype, severe heart failure and premature mortality. The present study investigated the expression of ECM-encoding genes in severe HCM and heart failure. Significant upregulation of structural Fn1, regulatory Mmp14, Timp1, Serpin3A, SerpinE1, SerpineE2, Tgfβ1, and Tgfβ2; and matricellular Ccn2, Postn, Spp1, Thbs1, Thbs4, and Tnc was evident from the early, pre-phenotype stage. Non-myocytes expressed ECM genes at higher levels than cardiomyocytes in normal and diseased hearts. Synchronous increase of secreted CCN2 and TIMP1 plasma levels and decrease of MMP3 levels were observed in end-stage disease. CCN2 protein expression was increased from early disease in double-mutant hearts and played an important role in ECM responses. It was a powerful modulator of ECM regulatory (Timp1 and SerpinE1) and matricellular protein-encoding (Spp1, Thbs1, Thbs4 and Tnc) gene expression in cardiomyocytes when added exogenously in vitro. Modulation of CCN2 (CTGF, connective tissue growth factor) and associated early ECM changes may represent a new therapeutic target in the treatment and prevention of heart failure in HCM. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quantitative gene expression deregulation in mantle-cell lymphoma: correlation with clinical and biologic factors.

PubMed

Kienle, Dirk; Katzenberger, Tiemo; Ott, German; Saupe, Doreen; Benner, Axel; Kohlhammer, Holger; Barth, Thomas F E; Höller, Sylvia; Kalla, Jörg; Rosenwald, Andreas; Müller-Hermelink, Hans Konrad; Möller, Peter; Lichter, Peter; Döhner, Hartmut; Stilgenbauer, Stephan

2007-07-01

There is evidence for a direct role of quantitative gene expression deregulation in mantle-cell lymphoma (MCL) pathogenesis. Our aim was to investigate gene expression associations with other pathogenic factors and the significance of gene expression in a multivariate survival analysis. Quantitative expression of 20 genes of potential relevance for MCL prognosis and pathogenesis were analyzed using real-time reverse transcriptase polymerase chain reaction and correlated with clinical and genetic factors, tumor morphology, and Ki-67 index in 65 MCL samples. Genomic losses at the loci of TP53, RB1, and P16 were associated with reduced transcript levels of the respective genes, indicating a gene-dosage effect as the pathomechanism. Analysis of gene expression correlations between the candidate genes revealed a separation into two clusters, one dominated by proliferation activators, another by proliferation inhibitors and regulators of apoptosis. Whereas only weak associations were identified between gene expression and clinical parameters or blastoid morphology, several genes were correlated closely with the Ki-67 index, including the short CCND1 variant (positive correlation) and RB1, ATM, P27, and BMI (negative correlation). In multivariate survival analysis, expression levels of MYC, MDM2, EZH2, and CCND1 were the strongest prognostic factors independently of tumor proliferation and clinical factors. These results indicate a pathogenic contribution of several gene transcript levels to the biology and clinical course of MCL. Genes can be differentiated into factors contributing to proliferation deregulation, either by enhancement or loss of inhibition, and proliferation-independent factors potentially contributing to MCL pathogenesis by apoptosis impairment.

Expression of connective tissue growth factor is a prognostic marker for patients with intrahepatic cholangiocarcinoma.

PubMed

Gardini, A; Corti, B; Fiorentino, M; Altimari, A; Ercolani, G; Grazi, G L; Pinna, A D; Grigioni, W F; D'Errico Grigioni, A

2005-04-01

Connective tissue growth factor is a member of the 'CCN' protein family. Consistent with its profibrotic properties, it is over-expressed in several human epithelial malignancies. We have retrospectively evaluated by immunohistochemistry the presence of connective tissue growth factor in archival tissues from 55 resected intrahepatic cholangiocarcinomas and compared its expression to the main pathological parameters, disease free and overall survival. Tumours were scored as high and low/absent expressers (> or =50%, 0-50% cells, respectively). Thirty-three of 55 cholangiocarcinomas (60%) were high and 22 (40%) low expressers. No significant correlation was found between connective tissue growth factor and tumour grade, tumour location, vascular and perineural invasion. Eighteen of 22 (82%) low/absent expressers and 12/33 (36%) high expressers had recurrence of disease (P=0.001). Low/absent expressers showed a poor disease free and overall survival compared with the higher expressers (P<0.001). Vascular invasion was related to tumour recurrence (P=0.025) and to decreased disease free survival (P<0.05). During proportional hazard regression analysis, only connective tissue growth factor was found to influence disease free survival (P=0.01). Expression of connective tissue growth factor is an independent prognostic indicator of both tumour recurrence and overall survival for intrahepatic cholangiocarcinoma patients regardless of tumour location, tumour grade, vascular and perineural invasion.

Several fibroblast growth factors are expressed during pre-attachment bovine conceptus development and regulate interferon-tau expression from trophectoderm.

PubMed

Cooke, Flavia N T; Pennington, Kathleen A; Yang, Qien; Ealy, Alan D

2009-02-01

The trophectoderm-derived factor interferon tau (IFNT) maintains the uterus in a pregnancy-receptive state in cattle and sheep. Fibroblast growth factors (FGFs) are implicated in regulating IFNT expression and potentially other critical events associated with early conceptus development in cattle. The overall objectives of this work were to identify the various FGFs and FGF receptors (FGFRs) expressed in elongating pre-attachment bovine conceptuses and determine if these FGFs regulate conceptus development and/or mediate IFNT production. In vitro-derived bovine blastocysts and in vivo-derived elongated conceptuses collected at day 17 of pregnancy express at least four FGFR subtypes (R1c, R2b, R3c, R4). In addition, transcripts for FGF1, 2, and 10 but not FGF7 are present in elongated bovine conceptuses. The expression pattern of FGF10 most closely resembled that of IFNT, with both transcripts remaining low in day 8 and day 11 conceptuses and increasing substantially in day 14 and day 17 conceptuses. Supplementation with recombinant FGF1, 2 or 10 increased IFNT mRNA levels in bovine trophectoderm cells and bovine blastocysts and increased IFNT protein concentrations in trophectoderm-conditioned medium. Blastocyst development was not affected by any of the FGFs. In summary, at least four FGFRs reside in pre- and peri-attachment bovine conceptuses. Moreover, conceptuses express at least three candidate FGFs during elongation, the time of peak IFNT expression. These findings provide new insight for how conceptus-derived factors such as FGF1, 2, and 10 may control IFNT expression during early pregnancy in cattle.

Hypoxia-inducible factor regulates alphavbeta3 integrin cell surface expression.

PubMed

Cowden Dahl, Karen D; Robertson, Sarah E; Weaver, Valerie M; Simon, M Celeste

2005-04-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of alpha and beta aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt(-/-) and Hifalpha(-/-) TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin alphavbeta3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O(2)). Culturing B16F0 melanoma cells at 1.5% O(2) resulted in increased alphavbeta3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O(2) tension influence placental invasion and tumor migration by increasing cell surface expression of alphavbeta3 integrin.

Transcription Factor Binding Site Enrichment Analysis in Co-Expression Modules in Celiac Disease

PubMed Central

Romero-Garmendia, Irati; Jauregi-Miguel, Amaia; Plaza-Izurieta, Leticia; Cros, Marie-Pierre; Legarda, Maria; Irastorza, Iñaki; Herceg, Zdenko; Fernandez-Jimenez, Nora

2018-01-01

The aim of this study was to construct celiac co-expression patterns at a whole genome level and to identify transcription factors (TFs) that could drive the gliadin-related changes in coordination of gene expression observed in celiac disease (CD). Differential co-expression modules were identified in the acute and chronic responses to gliadin using expression data from a previous microarray study in duodenal biopsies. Transcription factor binding site (TFBS) and Gene Ontology (GO) annotation enrichment analyses were performed in differentially co-expressed genes (DCGs) and selection of candidate regulators was performed. Expression of candidates was measured in clinical samples and the activation of the TFs was further characterized in C2BBe1 cells upon gliadin challenge. Enrichment analyses of the DCGs identified 10 TFs and five were selected for further investigation. Expression changes related to active CD were detected in four TFs, as well as in several of their in silico predicted targets. The activation of TFs was further characterized in C2BBe1 cells upon gliadin challenge, and an increase in nuclear translocation of CAMP Responsive Element Binding Protein 1 (CREB1) and IFN regulatory factor-1 (IRF1) in response to gliadin was observed. Using transcriptome-wide co-expression analyses we are able to propose novel genes involved in CD pathogenesis that respond upon gliadin stimulation, also in non-celiac models. PMID:29748492

Transcription Factor Binding Site Enrichment Analysis in Co-Expression Modules in Celiac Disease.

PubMed

Romero-Garmendia, Irati; Garcia-Etxebarria, Koldo; Hernandez-Vargas, Hector; Santin, Izortze; Jauregi-Miguel, Amaia; Plaza-Izurieta, Leticia; Cros, Marie-Pierre; Legarda, Maria; Irastorza, Iñaki; Herceg, Zdenko; Fernandez-Jimenez, Nora; Bilbao, Jose Ramon

2018-05-10

The aim of this study was to construct celiac co-expression patterns at a whole genome level and to identify transcription factors (TFs) that could drive the gliadin-related changes in coordination of gene expression observed in celiac disease (CD). Differential co-expression modules were identified in the acute and chronic responses to gliadin using expression data from a previous microarray study in duodenal biopsies. Transcription factor binding site (TFBS) and Gene Ontology (GO) annotation enrichment analyses were performed in differentially co-expressed genes (DCGs) and selection of candidate regulators was performed. Expression of candidates was measured in clinical samples and the activation of the TFs was further characterized in C2BBe1 cells upon gliadin challenge. Enrichment analyses of the DCGs identified 10 TFs and five were selected for further investigation. Expression changes related to active CD were detected in four TFs, as well as in several of their in silico predicted targets. The activation of TFs was further characterized in C2BBe1 cells upon gliadin challenge, and an increase in nuclear translocation of CAMP Responsive Element Binding Protein 1 (CREB1) and IFN regulatory factor-1 (IRF1) in response to gliadin was observed. Using transcriptome-wide co-expression analyses we are able to propose novel genes involved in CD pathogenesis that respond upon gliadin stimulation, also in non-celiac models.

Growth Factors and COX2 Expression in Canine Perivascular Wall Tumors.

PubMed

Avallone, G; Stefanello, D; Boracchi, P; Ferrari, R; Gelain, M E; Turin, L; Tresoldi, E; Roccabianca, P

2015-11-01

Canine perivascular wall tumors (PWTs) are a group of subcutaneous soft tissue sarcomas developing from vascular mural cells. Mural cells are involved in angiogenesis through a complex crosstalk with endothelial cells mediated by several growth factors and their receptors. The evaluation of their expression may have relevance since they may represent a therapeutic target in the control of canine PWTs. The expression of vascular endothelial growth factor (VEGF) and receptors VEGFR-I/II, basic fibroblast growth factor (bFGF) and receptor Flg, platelet-derived growth factor B (PDGFB) and receptor PDGFRβ, transforming growth factor β1 (TGFβ1) and receptors TGFβR-I/II, and cyclooxygenase 2 (COX2) was evaluated on frozen sections of 40 PWTs by immunohistochemistry and semiquantitatively scored to identify their potential role in PWT development. Statistical analysis was performed to analyze possible correlations between Ki67 labeling index and the expression of each molecule. Proteins of the VEGF-, PDGFB-, and bFGF-mediated pathways were highly expressed in 27 (67.5%), 30 (75%), and 19 (47.5%) of 40 PWTs, respectively. Proteins of the TGFβ1- and COX2-mediated pathways were highly expressed in 4 (10%) and 14 (35%) of 40 cases. Statistical analysis identified an association between VEGF and VEGFR-I/II (P = .015 and .003, respectively), bFGF and Flg (P = .038), bFGF and PDGFRβ (P = .003), and between TGFβ1 and COX2 (P = .006). These findings were consistent with the mechanisms that have been reported to play a role in angiogenesis and in tumor development. No association with Ki67 labeling index was found. VEGF-, PDGFB-, and bFGF-mediated pathways seem to have a key role in PWT development and growth. Blockade of tyrosine kinase receptors after surgery could represent a promising therapy with the aim to reduce the PWT relapse rate and prolong the time to relapse. © The Author(s) 2015.

Differential patterns of replacement and reactive fibrosis in pressure and volume overload are related to the propensity for ischaemia and involve resistin

PubMed Central

Chemaly, Elie R; Kang, Soojeong; Zhang, Shihong; McCollum, LaTronya; Chen, Jiqiu; Bénard, Ludovic; Purushothaman, K-Raman; Hajjar, Roger J; Lebeche, Djamel

2013-01-01

Pathological left ventricle (LV) hypertrophy (LVH) results in reactive and replacement fibrosis. Volume overload LVH (VOH) is less profibrotic than pressure overload LVH (POH). Studies attribute subendocardial fibrosis in POH to ischaemia, and reduced fibrosis in VOH to collagen degradation favouring dilatation. However, the mechanical origin of the relative lack of fibrosis in VOH is incompletely understood. We hypothesized that reduced ischaemia propensity in VOH compared to POH accounted for the reduced replacement fibrosis, along with reduced reactive fibrosis. Rats with POH (ascending aortic banding) evolved into either compensated-concentric POH (POH-CLVH) or dilated cardiomyopathy (POH-DCM); they were compared to VOH (aorta–caval fistula). We quantified LV fibrosis, structural and haemodynamic factors of ischaemia propensity, and the activation of profibrotic pathways. Fibrosis in POH-DCM was severe, subendocardial and subepicardial, in contrast with subendocardial fibrosis in POH-CLVH and nearly no fibrosis in VOH. The propensity for ischaemia was more important in POH versus VOH, explaining different patterns of replacement fibrosis. LV collagen synthesis and maturation, and matrix metalloproteinase-2 expression, were more important in POH. The angiotensin II-transforming growth-factor β axis was enhanced in POH, and connective tissue growth factor (CTGF) was overexpressed in all types of LVH. LV resistin expression was markedly elevated in POH, mildly elevated in VOH and independently reflected chronic ischaemic injury after myocardial infarction. In vitro, resistin is induced by angiotensin II and induces CTGF in cardiomyocytes. Based on these findings, we conclude that a reduced ischaemia propensity and attenuated upstream reactive fibrotic pathways account for the attenuated fibrosis in VOH versus POH. PMID:24018949

Expression of the Maize Dof1 Transcription Factor in Wheat and Sorghum

PubMed Central

Peña, Pamela A.; Quach, Truyen; Sato, Shirley; Ge, Zhengxiang; Nersesian, Natalya; Changa, Taity; Dweikat, Ismail; Soundararajan, Madhavan; Clemente, Tom E.

2017-01-01

Nitrogen is essential for plant growth and development. Improving the ability of plants to acquire and assimilate nitrogen more efficiently is a key agronomic parameter that will augment sustainability in agriculture. A transcription factor approach was pursued to address improvement of nitrogen use efficiency in two major commodity crops. To this end, the Zea mays Dof1 (ZmDof1) transcription factor was expressed in both wheat (Triticum aestivum) and sorghum (Sorghum bicolor) either constitutively, UBI4 promoter from sugarcane, or in a tissue specific fashion via the maize rbcS1 promoter. The primary transcription activation target of ZmDof1, phosphoenolpyruvate carboxylase (PEPC), is observed in transgenic wheat events. Expression ZmDof1 under control of the rbcs1 promoter translates to increase in biomass and yield components in wheat. However, constitutive expression of ZmDof1 led to the down-regulation of genes involved in photosynthesis and the functional apparatus of chloroplasts, and an outcome that negatively impacts photosynthesis, height, and biomass in wheat. Similar patterns were also observed in sorghum transgenic events harboring the constitutive expression cassette of ZmDof1. These results indicate that transcription factor strategies to boost agronomic phenotypic outcomes in crops need to consider expression patterns of the genetic elements to be introduced. PMID:28424717

Hepatocyte growth factor/scatter factor enhances the invasion of mesothelioma cell lines and the expression of matrix metalloproteinases

PubMed Central

Harvey, P; Clark, I M; Jaurand, M-C; Warn, R M; Edwards, D R

2000-01-01

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional factor involved both in development and tissue repair, as well as pathological processes such as cancer and metastasis. It has been identified in vivo in many types of tumours together with its tyrosine kinase receptor, Met. We show here that exogenous HGF/SF acts as a strong chemoattractant for human mesothelioma cell lines. The factor also enhanced cell adhesion to and invasion into Matrigel. The mesothelioma cell lines synthesized a panel of matrix metalloproteinases critical for tumour progression such as MMP-1, 2, 3, 9 and membrane-bound MT1-MMP. HGF/SF stimulated the expression of MMP-1, 9 and MT1-MMP and had a slight effect on expression of the MMP inhibitor TIMP-1 but not TIMP-2. However, there was no simple correlation between the levels of MMPs and TIMPs of the cell lines and their different invasion properties or between HGF/SF stimulatory effects on MMP expression and invasion. In addition, effects of protease inhibitors on invasion suggested that serine proteases were also expressed in human mesothelioma cell lines and were involved in HGF/SF-induced invasion. The results show a predominant role for HGF/SF in mesothelioma cell invasion, stimulating simultaneously adhesion, motility, invasion and regulation of MMP and TIMP levels. © 2000 Cancer Research Campaign PMID:11027427

Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa.

PubMed

Shoji, Mamoru; Sun, Aiming; Kisiel, Walter; Lu, Yang J; Shim, Hyunsuk; McCarey, Bernard E; Nichols, Christopher; Parker, Ernest T; Pohl, Jan; Mosley, Cara A; Alizadeh, Aaron R; Liotta, Dennis C; Snyder, James P

2008-04-01

Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.

Kinase inhibitors of the IGF-1R as a potential therapeutic agent for rheumatoid arthritis.

PubMed

Tsushima, Hiroshi; Morimoto, Shinji; Fujishiro, Maki; Yoshida, Yuko; Hayakawa, Kunihiro; Hirai, Takuya; Miyashita, Tomoko; Ikeda, Keigo; Yamaji, Ken; Takamori, Kenji; Takasaki, Yoshinari; Sekigawa, Iwao; Tamura, Naoto

2017-08-01

We have previously shown that the inhibition of connective tissue growth factor (CTGF) is a potential therapeutic strategy against rheumatoid arthritis (RA). CTGF consists of four distinct modules, including the insulin-like growth factor binding protein (IGFBP). In serum, insulin-like growth factors (IGFs) bind IGFBPs, interact with the IGF-1 receptor (IGF-1 R), and regulate anabolic effects and bone metabolism. We investigated the correlation between IGF-1 and the pathogenesis of RA, and the inhibitory effect on osteoclastogenesis and angiogenesis of the small molecular weight kinase inhibitor of the IGF-1 R, NVP-AEW541, against pathogenesis of RA in vitro. Cell proliferation was evaluated by cell count and immunoblotting. The expression of IGF-1 and IGF-1 R was evaluated by RT-PCR. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase staining, a bone resorption assay, and osteoclast-specific enzyme production. Angiogenesis was evaluated by a tube formation assay using human umbilical vein endothelial cells (HUVECs). The proliferation of MH7A cells was found to be inhibited in the presence of NVP-AEW541, and the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was downregulated in MH7A cells. IGF-1 and IGF-1 R mRNA expression levels were upregulated during formation of M-colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL)-mediated osteoclast formation. Moreover, osteoclastogenesis was suppressed in the presence of NVP-AEW541. The formation of the tubular network was enhanced by IGF-1, and this effect was neutralized by NVP-ARE541. Our findings suggest that NVP-AEW541 may be utilized as a potential therapeutic agent in the treatment of RA.

Understanding Transcription Factor Regulation by Integrating Gene Expression and DNase I Hypersensitive Sites.

PubMed

Wang, Guohua; Wang, Fang; Huang, Qian; Li, Yu; Liu, Yunlong; Wang, Yadong

2015-01-01

Transcription factors are proteins that bind to DNA sequences to regulate gene transcription. The transcription factor binding sites are short DNA sequences (5-20 bp long) specifically bound by one or more transcription factors. The identification of transcription factor binding sites and prediction of their function continue to be challenging problems in computational biology. In this study, by integrating the DNase I hypersensitive sites with known position weight matrices in the TRANSFAC database, the transcription factor binding sites in gene regulatory region are identified. Based on the global gene expression patterns in cervical cancer HeLaS3 cell and HelaS3-ifnα4h cell (interferon treatment on HeLaS3 cell for 4 hours), we present a model-based computational approach to predict a set of transcription factors that potentially cause such differential gene expression. Significantly, 6 out 10 predicted functional factors, including IRF, IRF-2, IRF-9, IRF-1 and IRF-3, ICSBP, belong to interferon regulatory factor family and upregulate the gene expression levels responding to the interferon treatment. Another factor, ISGF-3, is also a transcriptional activator induced by interferon alpha. Using the different transcription factor binding sites selected criteria, the prediction result of our model is consistent. Our model demonstrated the potential to computationally identify the functional transcription factors in gene regulation.

A sigma factor toolbox for orthogonal gene expression in Escherichia coli

PubMed Central

Van Brempt, Maarten; Van Nerom, Katleen; Van Hove, Bob; Maertens, Jo; De Mey, Marjan; Charlier, Daniel

2018-01-01

Abstract Synthetic genetic sensors and circuits enable programmable control over timing and conditions of gene expression and, as a result, are increasingly incorporated into the control of complex and multi-gene pathways. Size and complexity of genetic circuits are growing, but stay limited by a shortage of regulatory parts that can be used without interference. Therefore, orthogonal expression and regulation systems are needed to minimize undesired crosstalk and allow for dynamic control of separate modules. This work presents a set of orthogonal expression systems for use in Escherichia coli based on heterologous sigma factors from Bacillus subtilis that recognize specific promoter sequences. Up to four of the analyzed sigma factors can be combined to function orthogonally between each other and toward the host. Additionally, the toolbox is expanded by creating promoter libraries for three sigma factors without loss of their orthogonal nature. As this set covers a wide range of transcription initiation frequencies, it enables tuning of multiple outputs of the circuit in response to different sensory signals in an orthogonal manner. This sigma factor toolbox constitutes an interesting expansion of the synthetic biology toolbox and may contribute to the assembly of more complex synthetic genetic systems in the future. PMID:29361130

[Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

PubMed

Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

2007-09-01

To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

Characterization of renal biomarkers for use in clinical trials: effect of preanalytical processing and qualification using samples from subjects with diabetes.

PubMed

Brott, David A; Furlong, Stephen T; Adler, Scott H; Hainer, James W; Arani, Ramin B; Pinches, Mark; Rossing, Peter; Chaturvedi, Nish

2015-01-01

Identifying the potential for drug-induced kidney injury is essential for the successful research and development of new drugs. Newer and more sensitive preclinical drug-induced kidney injury biomarkers are now qualified for use in rat toxicology studies, but biomarkers for clinical studies are still undergoing qualification. The current studies investigated biomarkers in healthy volunteer (HV) urine samples with and without the addition of stabilizer as well as in urine from patients with normoalbuminuric diabetes mellitus (P-DM). Urine samples from 20 male HV with stabilizer, 69 male HV without stabilizer, and 95 male DM without stabilizer (39 type 1 and 56 type 2) were analyzed for the following bio-markers using multiplex assays: α-1-microglobulin (A1M), β-2-microglobulin, calbindin, clusterin, connective tissue growth factor (CTGF), creatinine, cystatin-C, glutathione S-transferase α (GSTα), kidney injury marker-1 (KIM-1), microalbumin, neutrophil gelatinase-associated lipocalin, osteopontin, Tamm-Horsfall urinary glycoprotein (THP), tissue inhibitor of metalloproteinase 1, trefoil factor 3 (TFF3), and vascular endothelial growth factor. CTGF and GSTα assays on nonstabilized urine were deemed nonoptimal (>50% of values below assay lower limits of quantification). "Expected values" were determined for HV with stabilizer, HV without stabilizer, and P-DM without stabilizer. There was a statistically significant difference between HV with stabilizer compared to HV without stabilizer for A1M, CTGF, GSTα, and THP. DM urine samples differed from HV (without stabilizer) for A1M CTGF, GSTα, KIM-1, microalbumin, osteopontin, and TFF3. A1M also correctly identified HV and DM with an accuracy of 89.0%. These studies: 1) determined that nonstabilized urine can be used for assays under qualification; and 2) documented that A1M, CTGF, GSTα, KIM-1, microalbumin, osteopontin, and TFF3 were significantly increased in urine from P-DM. In addition, the 89.0% accuracy of A

Diuretics prevent Rho-kinase activation and expression of profibrotic/oxidative genes in the hypertensive aortic wall.

PubMed

Araos, Patricio; Mondaca, David; Jalil, Jorge E; Yañez, Cristián; Novoa, Ulises; Mora, Italo; Ocaranza, María Paz

2016-12-01

Diuretics are current antihypertensive drugs since they reduce blood pressure and cardiovascular risk. Increased vascular tone is modulated in a relevant way by the RhoA/Rho-kinase (ROCK) pathway, by acting on vascular smooth muscle cell contraction. This pathway has also proremodeling vascular effects. There are few data on the role of diuretics on both vascular ROCK activation and on proremodeling effects. We assessed the effects of hydrochlorothiazide (HCTZ) and spironolactone (spiro) alone and in combination with the ROCK inhibitor fasudil (FAS) on ROCK activation, gene expression of proremodeling markers and on hypertrophy in the aortic wall of hypertensive rats. Deoxycorticosterone acetate (DOCA)-salt hypertensive rats (male, Sprague-Dawley) were randomized to the specific ROCK inhibitor FAS, HCTZ, spiro or the combinations of FAS/HCTZ or FAS/spiro for 3 weeks. At the end of the study, ROCK activation (by western blot), gene expression of proremodeling markers (by reverse transcription polymerase chain reaction, RT-PCR) and vascular hypertrophy (by morphometry) were determined in the aortic wall. All treatments significantly reduced blood pressure. In the DOCA rats the p-myosin phosphatase target protein-1 (MYPT1)/t-MYPT1 ratio, index of ROCK activation was higher by 2.8 fold (p < 0.05) compared with control rats. All treatments reduced ROCK activation in the aortic wall to control levels (p < 0.05). Besides, significantly increased protein levels of transforming growth factor β1 (TGF-β 1 ), gene expression of TGF-β 1 , connective tissue growth factor (CTGF), p22 phox and gp91 phox subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, as well as increased media thickness and aortic media area/lumen area (AM/LA) in the untreated hypertensive rats were significantly reduced (p < 0.05) to control levels by all treatments. Similar effects were observed using both diuretics alone or in combination with FAS. In the aortic wall, both HCTZ and

Diuretics prevent Rho-kinase activation and expression of profibrotic/oxidative genes in the hypertensive aortic wall

PubMed Central

Araos, Patricio; Mondaca, David; Jalil, Jorge E.; Yañez, Cristián; Novoa, Ulises; Mora, Italo; Ocaranza, María Paz

2016-01-01

Background: Diuretics are current antihypertensive drugs since they reduce blood pressure and cardiovascular risk. Increased vascular tone is modulated in a relevant way by the RhoA/Rho-kinase (ROCK) pathway, by acting on vascular smooth muscle cell contraction. This pathway has also proremodeling vascular effects. There are few data on the role of diuretics on both vascular ROCK activation and on proremodeling effects. We assessed the effects of hydrochlorothiazide (HCTZ) and spironolactone (spiro) alone and in combination with the ROCK inhibitor fasudil (FAS) on ROCK activation, gene expression of proremodeling markers and on hypertrophy in the aortic wall of hypertensive rats. Methods: Deoxycorticosterone acetate (DOCA)-salt hypertensive rats (male, Sprague–Dawley) were randomized to the specific ROCK inhibitor FAS, HCTZ, spiro or the combinations of FAS/HCTZ or FAS/spiro for 3 weeks. At the end of the study, ROCK activation (by western blot), gene expression of proremodeling markers (by reverse transcription polymerase chain reaction, RT-PCR) and vascular hypertrophy (by morphometry) were determined in the aortic wall. Results: All treatments significantly reduced blood pressure. In the DOCA rats the p-myosin phosphatase target protein-1 (MYPT1)/t-MYPT1 ratio, index of ROCK activation was higher by 2.8 fold (p < 0.05) compared with control rats. All treatments reduced ROCK activation in the aortic wall to control levels (p < 0.05). Besides, significantly increased protein levels of transforming growth factor β1 (TGF-β1), gene expression of TGF-β1, connective tissue growth factor (CTGF), p22 phox and gp91 phox subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, as well as increased media thickness and aortic media area/lumen area (AM/LA) in the untreated hypertensive rats were significantly reduced (p < 0.05) to control levels by all treatments. Similar effects were observed using both diuretics alone or in combination with FAS

Advanced Glycation End-Products affect transcription factors regulating insulin gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Puddu, A., E-mail: alep100@hotmail.com; Storace, D.; Odetti, P.

2010-04-23

Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic {beta}-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation preventsmore » FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.« less

Placental growth factor expression is reversed by antivascular endothelial growth factor therapy under hypoxic conditions.

PubMed

Zhou, Ai-Yi; Bai, Yu-Jing; Zhao, Min; Yu, Wen-Zhen; Huang, Lv-Zhen; Li, Xiao-Xin

2014-08-01

Clinical trials have revealed that the antivascular endothelial growth factor (VEGF) therapies are effective in retinopathy of prematurity (ROP). But the low level of VEGF was necessary as a survival signal in healthy conditions, and endogenous placental growth factor (PIGF) is redundant for development. The purpose of this study was to elucidate the PIGF expression under hypoxia as well as the influence of anti-VEGF therapy on PIGF. CoCl2-induced hypoxic human umbilical vein endothelial cells (HUVECs) were used for an in vitro study, and oxygen-induced retinopathy (OIR) mice models were used for an in vivo study. The expression patterns of PIGF under hypoxic conditions and the influence of anti-VEGF therapy on PIGF were evaluated by quantitative reverse transcription-polymerase chain reaction (RTPCR). The retinal avascular areas and neovascularization (NV) areas of anti-VEGF, anti-PIGF and combination treatments were calculated. Retina PIGF concentration was evaluated by ELISA after treatment. The vasoactive effects of exogenous PIGF on HUVECs were investigated by proliferation and migration studies. PIGF mRNA expression was reduced by hypoxia in OIR mice, in HUVECs under hypoxia and anti-VEGF treatment. However, PIGF expression was reversed by anti-VEGF therapy in the OIR model and in HUVECs under hypoxia. Exogenous PIGF significantly inhibited HUVECs proliferation and migration under normal conditions, but it stimulated cell proliferation and migration under hypoxia. Anti-PIGF treatment was effective for neovascular tufts in OIR mice (P<0.05). The finding that PIGF expression is iatrogenically up-regulated by anti-VEGF therapy provides a consideration to combine it with anti-PIGF therapy.

Nuclear factor I-A represses expression of the cell adhesion molecule L1

PubMed Central

2009-01-01

Background The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression. Results We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold. Conclusion Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF). PMID:20003413

Expression of transcription factors during sodium phenylacetate induced erythroid differentiation in K562 cells.

PubMed

Rath, A V; Schmahl, G E; Niemeyer, C M

1997-01-01

During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.

Extracellular matrix metalloproteinase inducer (EMMPRIN) expression correlates positively with active angiogenesis and negatively with basic fibroblast growth factor expression in epithelial ovarian cancer.

PubMed

Szubert, Sebastian; Szpurek, Dariusz; Moszynski, Rafal; Nowicki, Michal; Frankowski, Andrzej; Sajdak, Stefan; Michalak, Slawomir

2014-03-01

The primary aim of this paper was to evaluate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its relationship with proangiogenic factors and microvessel density (MVD) in ovarian cancer. The study group included 58 epithelial ovarian cancers (EOCs), 35 benign ovarian tumors, and 21 normal ovaries. The expression of EMMPRIN, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) was assessed by ELISA of tissue homogenates. Antibodies against CD105, CD31, and CD34 were used to immunohistochemically assess MVD. We have found significantly higher EMMPRIN expression in EOC than in benign ovarian tumors and normal ovaries. Similarly, the VEGF expression was higher in EOC than in benign ovarian tumors and normal ovaries. By contrast, bFGF expression was lower in EOC than in benign ovarian tumors and ovary samples. EMMPRIN expression in EOC was directly correlated with VEGF expression and CD105-MVD, but inversely correlated with bFGF expression. Grade 2/3 ovarian cancers had increased expression of EMMPRIN and VEGF, increased CD105-MVD, and lowered expression of bFGF compared to grade 1 ovarian cancers. Moreover, EMMPRIN expression was higher in advanced (FIGO III and IV) ovarian cancer. The upregulation of EMMPRIN and VEGF expression is correlated with increased CD105-MVD and silenced bFGF, which suggests early and/or reactivated angiogenesis in ovarian cancer. Aggressive EOC is characterized by the following: high expression of EMMPRIN and VEGF, high CD105-MVD, and low expression of bFGF.

Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro

NASA Technical Reports Server (NTRS)

Long, C. J.; Roth, M. R.; Tasheva, E. S.; Funderburgh, M.; Smit, R.; Conrad, G. W.; Funderburgh, J. L.

2000-01-01

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.

HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs)

PubMed Central

2013-01-01

Introduction Carcinoma-associated fibroblasts (CAFs) play a pivotal role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors possess a unique microenvironment characterized by local hypoxia, which induces gene expression changes and biological features leading to poor outcomes. Hypoxia Inducible Factor 1 (HIF-1) is the main transcription factor that mediates the cell response to hypoxia through different mechanisms that include the regulation of genes strongly associated with cancer aggressiveness. Among the HIF-1 target genes, the G-protein estrogen receptor (GPER) exerts a stimulatory role in diverse types of cancer cells and in CAFs. Methods We evaluated the regulation and function of the key angiogenic mediator vascular endothelial growth factor (VEGF) in CAFs exposed to hypoxia. Gene expression studies, Western blotting analysis and immunofluorescence experiments were performed in CAFs and breast cancer cells in the presence of cobalt chloride (CoCl2) or cultured under low oxygen tension (2% O2), in order to analyze the involvement of the HIF-1α/GPER signaling in the biological responses to hypoxia. We also explored the role of the HIF-1α/GPER transduction pathway in functional assays like tube formation in human umbilical vein endothelial cells (HUVECs) and cell migration in CAFs. Results We first determined that hypoxia induces the expression of HIF-1α and GPER in CAFs, then we ascertained that the HIF-1α/GPER signaling is involved in the regulation of VEGF expression in breast cancer cells and in CAFs exposed to hypoxia. We also assessed by ChIP assay that HIF-1α and GPER are both recruited to the VEGF promoter sequence and required for VEGF promoter stimulation upon hypoxic condition. As a biological counterpart of these findings, conditioned medium from hypoxic CAFs promoted tube formation in HUVECs in a HIF-1α/GPER dependent manner. The functional cooperation between HIF-1α and GPER in CAFs was also

Transforming growth factor-beta inhibits the expression of clock genes.

PubMed

Gast, Heidemarie; Gordic, Sonja; Petrzilka, Saskia; Lopez, Martin; Müller, Andreas; Gietl, Anton; Hock, Christoph; Birchler, Thomas; Fontana, Adriano

2012-07-01

Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-β) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-β2 inhibits the expression of the clock genes Period (Per)1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein (Dbp) and thyrotroph embryonic factor (Tef). However, our results showed that TGF-β2 did not alter the expression of brain and muscle Arnt-like protein-1 (Bmal1). The concentrations of TGF-β2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-β2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients. © 2012 New York Academy of Sciences.

Obesity-induced endoplasmic reticulum stress suppresses nuclear factor-Y expression.

PubMed

Liu, Yulan; Zhang, Yuwei; Zhang, Yanjie; Zhang, Jinlong; Liu, Yin; Feng, Peiqun; Su, Zhiguang

2017-02-01

Nuclear transcription factor Y (NF-Y) is an evolutionarily conserved transcription factor composed of three subunits, NF-YA, NF-YB, and NF-YC. NF-Y plays crucial roles in pre-adipocyte maintenance and/or commitment to adipogenesis. NF-YA dysfunction in adipocyte resulted in an age-dependent progressive loss of adipose tissue associated with metabolic complications. Endoplasmic reticulum (ER) stress has emerged as an important mediator in the pathogenesis of obesity. However, it is not known if NF-YA is involved in the ER stress-mediated pathogenesis of obesity. We first examined the effects of ER stress on the NF-YA expression in cultured 3T3-L1 adipocytes; then in ob/ob genetic obesity mice, we tested the effect of chemical chaperones alleviating ER stress on the expression levels of NF-YA. Subsequently, we inhibited the new mRNA synthesis using actinomycin D in 3T3-L1 cells to explore the mechanism modulating NF-YA expression. Finally, we evaluated the involvement of PPARg in the regulation of NF-YA expression by ER stress. We demonstrated that both obesity- and chemical chaperone -induced ER stress suppressed NF-YA expression and alleviation of ER stress by chemical chaperone could recover NF-YA expression in ob/ob mice. Moreover, we showed that ER stress suppressed NF-YA mRNA transcription through the involvement of peroxisome proliferator-activated receptor gamma (PPARg). Activation of PPARg ameliorates the ER stress-induced NF-YA suppression. Our findings may point to a possible role of NF-YA in stress conditions that occur in chronic obesity, ER stress might be involved in the pathogenesis of obesity through NF-YA depletion.

High-Dimensional Sparse Factor Modeling: Applications in Gene Expression Genomics

PubMed Central

Carvalho, Carlos M.; Chang, Jeffrey; Lucas, Joseph E.; Nevins, Joseph R.; Wang, Quanli; West, Mike

2010-01-01

We describe studies in molecular profiling and biological pathway analysis that use sparse latent factor and regression models for microarray gene expression data. We discuss breast cancer applications and key aspects of the modeling and computational methodology. Our case studies aim to investigate and characterize heterogeneity of structure related to specific oncogenic pathways, as well as links between aggregate patterns in gene expression profiles and clinical biomarkers. Based on the metaphor of statistically derived “factors” as representing biological “subpathway” structure, we explore the decomposition of fitted sparse factor models into pathway subcomponents and investigate how these components overlay multiple aspects of known biological activity. Our methodology is based on sparsity modeling of multivariate regression, ANOVA, and latent factor models, as well as a class of models that combines all components. Hierarchical sparsity priors address questions of dimension reduction and multiple comparisons, as well as scalability of the methodology. The models include practically relevant non-Gaussian/nonparametric components for latent structure, underlying often quite complex non-Gaussianity in multivariate expression patterns. Model search and fitting are addressed through stochastic simulation and evolutionary stochastic search methods that are exemplified in the oncogenic pathway studies. Supplementary supporting material provides more details of the applications, as well as examples of the use of freely available software tools for implementing the methodology. PMID:21218139

Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue.

PubMed

Mydlo, J H; Michaeli, J; Cordon-Cardo, C; Goldenberg, A S; Heston, W D; Fair, W R

1989-06-15

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

IRE1 inhibition affects the expression of insulin-like growth factor binding protein genes and modifies its sensitivity to glucose deprivation in U87 glioma cells.

PubMed

Minchenko, D O; Kharkova, A P; Tsymbal, D O; Karbovskyi, L L; Minchenko, O H

2015-10-01

The aim of the present study was to investigate the effect of inhibition of endoplasmic reticulum stress signaling mediated by IRE1/ERN1 (inositol-requiring enzyme 1/endoplasmic reticulum to nucleus signaling 1) on the expression of genes encoding different groups of insulin-like growth binding proteins (IGFBP6 and IGFBP7) and CCN family (IGFBP8/CTGF/CCN2, IGFBP9/NOV/CCN3, IGFBP10/CYR61/CCN1, WISP1/CCN4, and WISP2/CCN5) and its sensitivity to glucose deprivation in U87 glioma cells. The expression of IGFBP6, IGFBP7, IGFBP8, IGFBP9, IGFBP10, WISP1, and WISP2 genes was studied by qPCR in control U87 glioma cells (wild-type) and its subline with IRE1 signaling enzyme loss of function upon glucose deprivation. The expression of IGFBP8, IGFBP9, and WISP2 genes was up-regulated in control glioma cells upon glucose deprivation with most significant changes for IGFBP9 gene. At the same time, the expression of IGFBP6, IGFBP10, and WISP1 genes was resistant to glucose deprivation in these glioma cells, but the IGFBP7 gene expression was down-regulated. The inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells modified the sensitivity of most studied gene expressions to glucose deprivation condition: introduced sensitivity of IGFBP10 and WISP1 genes to glucose deprivation, enhanced the effect of this deprivation on IGFBP7 and IGFBP9 gene expressions, and reduced this effect on WISP2 gene and induced suppressive effect of glucose deprivation on the expression of IGFBP8 gene. Furthermore, the inhibition of IRE1 strongly affected the expression of all studied genes in glioma cells upon regular growing condition in gene specific manner: up-regulated the expression levels of IGFBP7, IGFBP8, IGFBP10, WISP1, and WISP2 genes and down-regulated the IGFBP6 and IGFBP9 genes. The data of this investigation demonstrate that the expression of IGFBP7, IGFBP8, IGFBP9, and WISP2 genes are sensitive to glucose deprivation in U87 glioma cells and that

Neural progenitor cell implants modulate vascular endothelial growth factor and brain-derived neurotrophic factor expression in rat axotomized neurons.

PubMed

Talaverón, Rocío; Matarredona, Esperanza R; de la Cruz, Rosa R; Pastor, Angel M

2013-01-01

Axotomy of central neurons leads to functional and structural alterations which largely revert when neural progenitor cells (NPCs) are implanted in the lesion site. The new microenvironment created by NPCs in the host tissue might modulate in the damaged neurons the expression of a high variety of molecules with relevant roles in the repair mechanisms, including neurotrophic factors. In the present work, we aimed to analyze changes in neurotrophic factor expression in axotomized neurons induced by NPC implants. For this purpose, we performed immunofluorescence followed by confocal microscopy analysis for the detection of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) on brainstem sections from rats with axotomy of abducens internuclear neurons that received NPC implants (implanted group) or vehicle injections (axotomized group) in the lesion site. Control abducens internuclear neurons were strongly immunoreactive to VEGF and BDNF but showed a weak staining for NT-3 and NGF. Comparisons between groups revealed that lesioned neurons from animals that received NPC implants showed a significant increase in VEGF content with respect to animals receiving vehicle injections. However, the immunoreactivity for BDNF, which was increased in the axotomized group as compared to control, was not modified in the implanted group. The modifications induced by NPC implants on VEGF and BDNF content were specific for the population of axotomized abducens internuclear neurons since the neighboring abducens motoneurons were not affected. Similar levels of NT-3 and NGF immunolabeling were obtained in injured neurons from axotomized and implanted animals. Among all the analyzed neurotrophic factors, only VEGF was expressed by the implanted cells in the lesion site. Our results point to a role of NPC implants in the modulation of neurotrophic factor expression by lesioned central neurons, which might

Expression of LIM-homeodomain transcription factors in the developing and mature mouse retina

PubMed Central

Balasubramanian, Revathi; Bui, Andrew; Ding, Qian; Gan, Lin

2014-01-01

LIM-homeodomain (LIM-HD) transcription factors have been extensively studied for their role in the development of the central nervous system. Their function is key to several developmental events like cell proliferation, differentiation and subtype specification. However, their roles in retinal neurogenesis remain largely unknown. Here we report a detailed expression study of LIM-HD transcription factors LHX9 and LHX2, LHX3 and LHX4, and LHX6 in the developing and mature mouse retina using immunohistochemistry and in situ hybridization techniques. We show that LHX9 is expressed during the early stages of development in the retinal ganglion cell layer and the inner nuclear layer. We also show that LHX9 is expressed in a subset of amacrine cells in the adult retina. LHX2 is known to be expressed in retinal progenitor cells during development and in Müller glial cells and a subset of amacrine cells in the adult retina. We found that the LHX2 subset of amacrine cells is not cholinergic and that a very few of LHX2 amacrine cells express calretinin. LHX3 and LHX4 are expressed in a subset of bipolar cells in the adult retina. LHX6 is expressed in cells in the ganglion cell layer and the neuroblast layer starting at embryonic stage 13.5 (E13.5) and continues to be expressed in cells in the ganglion cell layer and inner nuclear layer, postnatally, suggesting its likely expression in amacrine cells or a subset thereof. Taken together, our comprehensive assay of expression patterns of LIM-HD transcription factors during mouse retinal development will help further studies elucidating their biological functions in the differentiation of retinal cell subtypes. PMID:24333658

Clinically significant association of elevated expression of nuclear factor E2-related factor 2 expression with higher glucose uptake and progression of upper urinary tract cancer.

PubMed

Nukui, Akinori; Narimatsu, Takahiro; Kambara, Tsunehito; Abe, Hideyuki; Sakamoto, Setsu; Yoshida, Ken-Ichiro; Kamai, Takao

2018-05-02

There is growing evidence that the transcription factor nuclear factor E2-related factor 2 (Nrf2) is the major participant in regulating antioxidants and pathways for detoxifying reactive oxygen species (ROS), as well as having a vital role in tumor proliferation, invasion, and chemoresistance. It was also recently reported that Nrf2 supports cell proliferation by promoting metabolic activity. Thus, Nrf2 is involved in progression of cancer. Upper urinary tract urothelial carcinoma (UTUC) is a biologically aggressive tumor with high rates of recurrence and progression, resulting in a poor prognosis. However, the role of Nrf2 in UTUC is largely unknown. In order to study the role of Nrf2 in UTUC from the metabolic perspective, we retrospectively assessed Nrf2 expression in the surgical specimen and the preoperative maximum standard glucose uptake (SUVmax) on [ 18 F]fluorodeoxy-glucose positron emission tomography ( 18 F-FDG-PET) of 107 patients with UTUC who underwent radical nephroureterectomy. Increased expression of Nrf2 in the primary lesion was correlated with less differentiated histology, local invasion, and lymph node metastasis, and was also an independent indicator of shorter overall survival according to multivariate analysis. Furthermore, increased expression of Nrf2 was associated with higher preoperative SUVmax by the primary tumor on 18 F-FDG-PET, while Nrf2 expression and SUVmax were also significantly correlated in the metastatic lymph nodes. Among the 18 patients with lymph node metastasis at nephroureterectomy who underwent retroperitoneal lymph node dissection and received adjuvant chemotherapy, the patients with higher Nrf2 expression in the primary tumor had worse recurrence-free survival. These results suggest that constitutive activation of Nrf2 might be linked with tumor aerobic glycolysis and progression of UTUC, indicating that Nrf2 signaling in the tumor microenvironment promotes progression of UTUC.

FOXO1 expression in keratinocytes promotes connective tissue healing

PubMed Central

Zhang, Chenying; Lim, Jason; Liu, Jian; Ponugoti, Bhaskar; Alsadun, Sarah; Tian, Chen; Vafa, Rameen; Graves, Dana T.

2017-01-01

Wound healing is complex and highly orchestrated. It is well appreciated that leukocytes, particularly macrophages, are essential for inducing the formation of new connective tissue, which requires the generation of signals that stimulate mesenchymal stem cells (MSC), myofibroblasts and fibroblasts. A key role for keratinocytes in this complex process has yet to be established. To this end, we investigated possible involvement of keratinocytes in connective tissue healing. By lineage-specific deletion of the forkhead box-O 1 (FOXO1) transcription factor, we demonstrate for the first time that keratinocytes regulate proliferation of fibroblasts and MSCs, formation of myofibroblasts and production of collagen matrix in wound healing. This stimulation is mediated by a FOXO1 induced TGFβ1/CTGF axis. The results provide direct evidence that epithelial cells play a key role in stimulating connective tissue healing through a FOXO1-dependent mechanism. Thus, FOXO1 and keratinocytes may be an important therapeutic target where healing is deficient or compromised by a fibrotic outcome. PMID:28220813

Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

PubMed

Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

2014-10-17

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

Identification of trans-acting factors regulating SamDC expression in Oryza sativa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basu, Supratim, E-mail: supratim_genetics@yahoo.co.in; Division of Plant Biology, Bose Institute, Kolkata; Roychoudhury, Aryadeep

2014-03-07

Highlights: • Identification of cis elements responsible for SamDC expression by in silico analysis. • qPCR analysis of SamDC expression to abiotic and biotic stress treatments. • Detection of SamDC regulators using identified cis-elements as probe by EMSA. • Southwestern Blot analysis to predict the size of the trans-acting factors. - Abstract: Abiotic stress affects the growth and productivity of crop plants; to cope with the adverse environmental conditions, plants have developed efficient defense machinery comprising of antioxidants like phenolics and flavonoids, and osmolytes like polyamines. SamDC is a key enzyme in the polyamine biosynthesis pathway in plants. In ourmore » present communication we have done in silico analysis of the promoter region of SamDC to look for the presence of different cis-regulatory elements contributing to its expression. Based on the presence of different cis-regulatory elements we completed comparative analysis of SamDC gene expression in rice lamina of IR-29 and Nonabokra by qPCR in response to the abiotic stress treatments of salinity, drought, cold and the biotic stress treatments of ABA and light. Additionally, to explore the role of the cis-regulatory elements in regulating the expression of SamDC gene in plants we comparatively analyzed the binding of rice nuclear proteins prepared from IR-29 and Nonabokra undergoing various stress treatments. The intensity of the complex formed was low and inducible in IR-29 in contrast to Nonabokra. Southwestern blot analysis helped in predicting the size of the trans-acting factors binding to these cis-elements. To our knowledge this is the first report on the comprehensive analysis of SamDC gene expression in rice and identification of the trans-acting factors regulating its expression.« less

Cancer Associated Fibroblasts express pro-inflammatory factors in human breast and ovarian tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erez, Neta, E-mail: netaerez@post.tau.ac.il; Glanz, Sarah; Raz, Yael

Highlights: •CAFs in human breast and ovarian tumors express pro-inflammatory factors. •Expression of pro-inflammatory factors correlates with tumor invasiveness. •Expression of pro-inflammatory factors is associated with NF-κb activation in CAFs. -- Abstract: Inflammation has been established in recent years as a hallmark of cancer. Cancer Associated Fibroblasts (CAFs) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation and invasion. We previously demonstrated that CAFs also mediate tumor-enhancing inflammation in a mouse model of skin carcinoma. Breast and ovarian carcinomas are amongst the leading causes of cancer-related mortality in women and cancer-related inflammation is linked with both these tumor types. However, themore » role of CAFs in mediating inflammation in these malignancies remains obscure. Here we show that CAFs in human breast and ovarian tumors express high levels of the pro-inflammatory factors IL-6, COX-2 and CXCL1, previously identified to be part of a CAF pro-inflammatory gene signature. Moreover, we show that both pro-inflammatory signaling by CAFs and leukocyte infiltration of tumors are enhanced in invasive ductal carcinoma as compared with ductal carcinoma in situ. The pro-inflammatory genes expressed by CAFs are known NF-κB targets and we show that NF-κB is up-regulated in breast and ovarian CAFs. Our data imply that CAFs mediate tumor-promoting inflammation in human breast and ovarian tumors and thus may be an attractive target for stromal-directed therapeutics.« less

Beetroot and Sodium Nitrate Ameliorate Cardiometabolic Changes in Diet-Induced Obese Hypertensive Rats.

PubMed

Bhaswant, Maharshi; Brown, Lindsay; McAinch, Andrew J; Mathai, Michael L

2017-12-01

Dietary intake of beetroot by humans reduces blood pressure but whether this is caused by nitrate or betanin is not well-defined; neither are effects on other signs of metabolic syndrome. Rats fed a high-carbohydrate, high-fat diet (H) for 16 weeks developed abdominal obesity, hypertension, altered cardiovascular and liver structure and function, and impaired glucose tolerance compared to rats fed a corn starch diet (C). H rats treated with ∼16 mg/kg/day of nitrate either from beetroot juice (H+B) or sodium nitrate (H+N) for the last 8 weeks reduced systolic blood pressure by ∼25 mmHg, improved cardiac structure and function, plasma lipid profile and plasma markers of liver function, reduced inflammatory cell infiltration in heart and liver and decreased left ventricular fibrosis. In the left ventricle, H rats increased mRNA expression of connective tissue growth factor (CTGF), monocyte chemoattractant protein 1 (MCP-1), matrix metalloproteinase-2 (MMP-2), and adenosine monophosphate-activated protein kinase-alpha (AMPK-α) and decreased mRNA expression of peroxisome proliferator-activated receptor-alpha (PPAR-α); both beetroot and sodium nitrate diet-fed rats decreased CTGF threefold, MCP-1, and MMP-2 twofold, and doubled PPAR-α mRNA expression in left ventricular tissue. The similar functional and molecular responses to beetroot and sodium nitrate indicate that the nitrate content of beetroot reduced inflammation and improved cardiovascular, liver, and metabolic function in rats with metabolic syndrome, rather than betanin. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immunohistochemical expression of vascular endothelial growth factor in canine oral squamous cell carcinomas.

PubMed

Martano, Manuela; Restucci, Brunella; Ceccarelli, Dora Maria; Lo Muzio, Lorenzo; Maiolino, Paola

2016-01-01

Angiogenesis is crucial for the growth and metastasis of malignant tumours, and various proangiogenic factors promote this process. One of these factors is vascular endothelial growth factor (VEGF), which appears to play a key role in tumour angiogenesis. The aim of the present study was to assess whether VEGF expression is associated with angiogenesis, disease progression and neoplastic proliferation in canine oral squamous cell carcinoma (OSCC) tissue. VEGF immunoreactivity was quantified by immunohistochemistry in 30 specimens, including normal oral mucosa and OSCC tissues graded as well, moderately or poorly differentiated. VEGF expression was correlated with tumour cell proliferation, as assessed using the proliferating cell nuclear antigen (PCNA) marker and microvessel density (data already published). The present results revealed that VEGF and PCNA expression increased significantly between normal oral tissue and neoplastic tissue, and between well and moderately/poorly differentiated tumours. In addition, VEGF expression was strongly correlated with PCNA expression and microvessel density. It was concluded that VEGF may promote angiogenesis through a paracrine pathway, stimulating endothelial cell proliferation and, similarly, may induce tumour cell proliferation through an autocrine pathway. The present results suggest that the evaluation of VEGF may be a useful additional criterion for estimating malignancy and growth potential in canine OSCCs.

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression.

PubMed

Shannon, Diane B; McKeown, Scott T W; Lundy, Fionnuala T; Irwin, Chris R

2006-01-01

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.

Dynamic expression of transcription factor Brn3b during mouse cranial nerve development

PubMed Central

Sajgo, Szilard; Ali, Seid; Popescu, Octavian; Badea, Tudor Constantin

2015-01-01

During development transcription factor combinatorial codes define a large variety of morphologically and physiologically distinct neurons. Such a combinatorial code has been proposed for the differentiation of projection neurons of the somatic and visceral components of cranial nerves. It is possible that individual neuronal cell types are not specified by unique transcription factors, but rather emerge through the intersection of their expression domains. Brn3a, Brn3b and Brn3c, in combination with each other and/or transcription factors of other families, can define subgroups of Retinal Ganglion Cells (RGC), Spiral and Vestibular Ganglia, inner ear and vestibular hair cell neurons in the vestibuloacoustic system, and groups of somatosensory neurons in the Dorsal Root Ganglia (DRG). In the present study we investigated the expression and potential role of the Brn3b transcription factor in cranial nerves and associated nuclei of the brainstem. We report the dynamic expression of Brn3b in the somatosensory component of cranial nerves II, V, VII and VIII and visceromotor nuclei of nerves VII, IX, X, as well as other brainstem nuclei during different stages of development into adult stage. We find that genetically identified Brn3bKO RGC axons show correct but delayed pathfinding during the early stages of embryonic development. However loss of Brn3b does not affect the anatomy of the other cranial nerves normally expressing this transcription factor. PMID:26356988

Increased expression of placental growth factor in high-grade endometrial carcinoma.

PubMed

Coenegrachts, Lieve; Schrauwen, Stefanie; Van Bree, Rita; Despierre, Evelyn; Luyten, Catherine; Jonckx, Bart; Stassen, Jean Marie; Vergote, Ignace; Amant, Frédéric

2013-02-01

Placental growth factor (PlGF), a homolog of vascular endothelial growth factor (VEGF), exerts pleiotropic functions in cancer by affecting tumor cells as well as endothelial and inflammatory cells. Moreover, PlGF expression correlates with tumor stage, recurrence, metastasis and patient outcome in different types of cancer. Recently, administration of anti-PlGF therapy reduced tumor growth and metastasis in preclinical tumor models. In the present study, we evaluated the diagnostic and prognostic value of systemic and local expression of PlGF in primary endometrial carcinomas. PlGF levels in tumor lysates (n=128) and serum (n=88) of patients with primary endometrial cancer were determined using ELISA. PlGF mRNA expression in endometrial carcinoma tissues was quantified by quantitative qRT-PCR. Results were compared to endometrial cancer stage and grade. Systemic PlGF levels were not altered in patients with endometrial cancer (FIGO stage I-II-III) as compared to healthy controls. Only in FIGO stage IV patients, serum PlGF levels were slightly increased. Local PlGF mRNA and protein expression in endometrial tumors progressively increased with tumor grade. In endometrioid carcinomas, PlGF mRNA expression was significantly increased in endometrioid grade 3 tumors as compared to normal endometrial tissue. PlGF protein expression was significantly increased in endometrioid grade 2 and 3 carcinomas and in serous carcinomas as compared to normal endometrial tissue. Our study showed that systemic/serum PlGF levels cannot be used as a diagnostic or prognostic marker in endometrial cancer. However, the increased local expression of PlGF, primarily in high-grade carcinomas, underscores the possibility for preclinical assessment of anti-PlGF therapy in endometrial cancer.

Correlation between spontaneous apoptosis and the expression of angiogenic factors in advanced gastric adenocarcinoma.

PubMed

Ikeguchi, M; Cai, J; Fukuda, K; Oka, S; Katano, K; Tsujitani, S; Maeta, M; Kaibara, N

2001-06-01

The aim of this study was to investigate whether angiogenic factors influence the occurrence of spontaneous apoptosis in advanced gastric cancer. The apoptotic indices (AIs) of 97 tumors from 97 patients with advanced gastric cancer (pT3, pN0, pM0, Stage II) were analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method. Intratumoral microvessel densities (IMVDs) of tumors stained with anti-CD34 monoclonal antibody were quantified under x 200 magnification using computer-assisted image analysis. The expressions of angiogenic factors, such as vascular endothelial growth factor (VEGF), thymidine phosphorylase (dThdPase), transforming growth factor-alpha (TGF-alpha), and p53 were analyzed immunohistochemically and compared with IMVDs and AIs. The mean IMVD of the 97 tumors was 365/mm2 (range 147-990/mm2). The mean AI of tumors was 2.1% (range 0-11.3%). A significant inverse correlation between the AIs and the IMVDs was shown (p = -0.278, P = 0.0064). The mean IMVDs of tumors with high expressions of dThdPase, TGF-alpha, or p53 were significantly higher than those of tumors with low expressions of these factors. The mean AI of tumors with high expressions of dThdPase was significantly lower than that of tumors with low expressions of dThdPase (P = 0.023). However, no significant correlations were detected between AIs and the expression levels of VEGF, TGF-alpha, or p53. In gastric cancer, dThdPase may play an important role in tumor progression by increasing microvessels and by suppressing apoptosis of cancer cells.