Connective Tissue Growth Factor mRNA Levels Research Articles

Galectin-3 promotes fibrosis in ovarian endometriosis.

This study aimed to investigate the potential role of galectin-3 (Gal-3) in the pathogenesis of fibrotic alterations in ovarian endometriosis (OVE). In this study, we collected the ectopic endometrial tissues and eutopic endometrial tissues from 31 OVE patients treated by laparoscopy, and the eutopic endometrial tissues from 23 non-OVE patients with leiomyoma or other benign diseases were used as control. Hematoxylin and eosin (H&E) and Masson's trichrome staining were utilized for histopathological assessment. The primary normal endometrial stromal cells (NESC), ectopic endometrial stromal cells (ECSC), and eutopic endometrial stromal cells (EUSC) were isolated. Gal-3 overexpression plasmids (Gal-OE) and short hairpin RNA targeting Gal-3 (Gal-3-shRNA) were transfected into the immortalized human endometriotic cell line 12Z, respectively. RT-qPCR, Western blot analysis, and immunohistochemistry were used to detect the mRNA and protein expression levels of Gal-3, type I collagen (COL-1), connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA), respectively. H&E and Masson staining showed that ovarian ectopic endometrium exhibited glandular hyperplasia, high columnar glandular epithelium, apical plasma secretion, more subnuclear vacuoles, and obvious fibrosis, compared with normal endometrium. The mRNA and protein levels of Gal-3 , CTGF, α-SMA, and COL-1 were all upregulated in the ectopic endometrial tissues of OVE patients compared to the eutopic endometrial tissues from OVE patients and non-OVE patients. Moreover, ECSC expressed higher levels of Gal-3, CTGF, α-SMA, and COL-1 than EUSC and NESC. Follow-up investigations demonstrated that the Gal-3 overexpression substantially increased fibrosis-related markers including CTGF, α-SMA, and COL-1 within the 12Z cell line. Conversely, Gal-3 knockdown showed the opposite effects. Gal-3 promotes fibrosis in OVE, positioning it as a prospective therapeutic target for mitigating fibrosis in endometriosis.

The effect of ET1-CTGF mediated pathway on the accumulation of extracellular matrix in the trabecular meshwork and its contribution to the increase in IOP.

To investigate the effect of endothelin-1 (ET-1) in excessive accumulation of extracellular matrix (ECM) of the trabecular meshwork (TM) and its role in intraocular pressure (IOP) regulation. Cultured human TM cells (HTMCs) were treated with ET-1, ET-1 + ETA receptor (ETAR) antagonist BQ123, ET-1 + ETB receptor (ETBR) antagonist BQ788. The expressions of fibronectin (FN) and collagen type IV (Col IV) were evaluated by western blotting and immunofluorescence. A time course effect of ET-1 on the transcription level of connective tissue growth factor (CTGF) was investigated by qRT-PCR. Next, the transcription level of CTGF was downregulated by using antisense oligodeoxynucleotide sequence. Then HTMCs were treated with ET-1, and the expression levels of FN and Col IV were evaluated by western blotting. In addition, by using an ex-vivo model of cultured anterior eye segment, we explored the effect of ET-1 on IOP changes and the expressions of FN and Col IV. In cultured HTMCs, the expressions of FN and Col IV were significantly increased after ET-1 treatment, which were blocked by the pretreatment of ETAR antagonist BQ123, rather than ETBR antagonist BQ788. Besides, the CTGF mRNA level increased significantly and reached a peak after 48h of ET-1 treatment. However, the effect of ET-1 on increasing the expressions of FN and Col IV in HTMCs could be inhibited by the downregulation of CTGF. In an ex-vivo model, IOP increased significantly after ET-1 administration, which could be blocked by BQ123 but not by BQ788. Furthermore, elevated expressions of FN and Col IV in TM were observed after ET-1 perfusion, and could be inhibited by BQ123 pretreatment. Excessive ET-1 in aqueous humor could lead to the abnormal accumulation of FN and Col IV in TM via the ETA-CTGF pathway, thereby increasing IOP.

EGF promotes human trophoblast cell invasion by downregulating CTGF expression via PI3K/AKT signaling.

Although the pro-invasive role of epidermal growth factor (EGF) has been reported in human trophoblast cells, the underlying mechanism remains largely unexplored. This work reveals that EGF-induced downregulation of connective tissue growth factor (CTGF) mediates the EGF-stimulated human trophoblast cell invasion. During the development of the placenta, trophoblast cell invasion must be carefully regulated. Although EGF has been shown to promote trophoblast cell invasion, the underlying mechanism remains largely undetermined. Our previous study using RNA-sequencing (RNA-seq) has identified that kisspeptin-1 is a downstream target of EGF in a human trophoblast cell line, HTR-8/SVneo, and mediates EGF-stimulated cell invasion. In the present study, after re-analysis of our previous RNA-seq data, we found that the CTGF was also downregulated in response to the EGF treatment. The inhibitory effects of EGF on CTGF mRNA and protein levels were confirmed in HTR-8/SVneo cells by reverse transcription quantitative real-time PCR and western blot, respectively. Treatment with EGF activated both PI3K/AKT and ERK1/2 signaling pathways. Using pharmacological inhibitors, our results showed that EGFR-mediated activation of PI3K/AKT signaling was required for the EGF-downregulated CTGF mRNA and protein levels. Matrigel-coated transwell invasion assays demonstrated that EGF treatment stimulated cell invasion. In addition, the invasiveness of HTR-8/SVneo cells was suppressed by treatment with recombinant human CTGF. By contrast, siRNA-mediated knockdown of CTGF increased cell invasion. Notably, the EGF-promoted HTR-8/SVneo cell invasion was attenuated by co-treatment with CTGF. This study provides important insights into the molecular mechanisms mediating EGF-stimulated human trophoblast cell invasion and increases the understanding of the biological functions of CTGF in the human placenta.

Serotoninergic receptor ligands improve Tamoxifen effectiveness on breast cancer cells

BackgroundSerotonin (or 5-Hydroxytryptamine, 5-HT) signals in mammary gland becomes dysregulated in cancer, also contributing to proliferation, metastasis, and angiogenesis. Thus, the discovery of novel compounds targeting serotonin signaling may contribute to tailor new therapeutic strategies usable in combination with endocrine therapies. We have previously synthesized serotoninergic receptor ligands (SER) with high affinity and selectivity towards 5-HT2A and 5-HT2C receptors, the main mediators of mitogenic effect of serotonin in breast cancer (BC). Here, we investigated the effect of 10 SER on viability of MCF7, SKBR3 and MDA-MB231 BC cells and focused on their potential ability to affect Tamoxifen responsiveness in ER+ cells.MethodsCell viability has been assessed by sulforhodamine B assay. Cell cycle has been analyzed by flow cytometry. Gene expression of 5-HT receptors and Connective Tissue Growth Factor (CTGF) has been checked by RT-PCR; mRNA levels of CTGF and ABC transporters have been further measured by qPCR. Protein levels of 5-HT2C receptors have been analyzed by Western blot. All data were statistically analyzed using GraphPad Prism 7.ResultsWe found that treatment with SER for 72 h reduced viability of BC cells. SER were more effective on MCF7 ER+ cells (IC50 range 10.2 μM - 99.2 μM) compared to SKBR3 (IC50 range 43.3 μM - 260 μM) and MDA-MB231 BC cells (IC50 range 91.3 μM - 306 μM). This was paralleled by accumulation of cells in G0/G1 phase of cell cycle. Next, we provided evidence that two ligands, SER79 and SER68, improved the effectiveness of Tamoxifen treatment in MCF7 cells and modulated the expression of CTGF, without affecting viability of MCF10A non-cancer breast epithelial cells. In a cell model of Tamoxifen resistance, SER68 also restored drug effect independently of CTGF.ConclusionsThese results identified serotoninergic receptor ligands potentially usable in combination with Tamoxifen to improve its effectiveness on ER+ BC patients.

Influence of Silica Exposure for Lung Silicosis Rat.

Objective To investigate the influence of silica exposure on the expression of connective tissue growth factor (CTGF), transforming growth factor beta-1 (TGF-β1), and platelet-derived growth factor (PDGF) in lung silicosis rat. Methods Wistar rats were divided into an experimental group and a control group. In the experimental group, rats were exposed to silica by intratracheal instillation. In the control group, rats were exposed to physiological saline by intratracheal instillation. After 45 days, we compared the level of fibrosis and CTGF, TGF-β1, and PDGF in the lungs by immunohistochemistry or reverse transcription-polymerase chain reaction between the two groups. Results The results showed that the expression levels of CTGF, TGF-β1, and PDGF mRNA were significantly higher in the experimental group than those in the control group (P < 0.05). The positive staining of CTGF, TGF-β1, and PDGF mRNA was found in the cytoplasm, especially in the silicotic nodules of the hyalinisation section and cell endochylema of the alveolar macrophages, type II pneumonocytes, and lung tracheal epithelium. There were significantly positive correlations between CTGF, TGF-β1, and PDGF expressions (P < 0.05). A protein–protein interaction analysis showed interactions between TGF-β1, CTGF, and PDGF. Conclusions TGF-β/CTGF signaling pathway plays an important role in silicosis. Silicon dioxide exposure can induce the expression of CTGF, TGF-β1, and PDGF.

Serum and glucocorticoid-regulated kinase 1 regulates transforming growth factor β1-connective tissue growth factor pathway in chronic rhinosinusitis

PurposeSerum/glucocorticoid-regulated kinase 1 (SGK1) has been identified as a crucial regulator in fibrotic disorders. Herein, we explored SGK1 role in tissue remodeling of chronic rhinosinusitis (CRS). MethodsLentivirus was employed to generate an SGK1-overexpressing human bronchial epithelial cell (16HBE) line. To screen SGK1 downstream genes, RNA sequencing was performed on SGK1-overexpressing and control cell lines. To determine protein and gene expression levels, immunohistochemistry, western blotting, and quantitative real-time polymerase chain reaction were employed. Correlation analysis was performed using mRNA expression levels of SGK1, transforming growth factor β1 (TGF-β1), and connective tissue growth factor (CTGF) derived from CRS mucosal tissue and GEO database. Gene set enrichment analysis was conducted using gene sets from Molecular Signatures Database. The severity of symptoms in CRS patients was assessed using the 22-Item Sinonasal Outcome Test. ResultsSGK1 overexpression significantly increased the expression of connective tissue growth factor (CTGF) in 16HBE cells (P < 0.01). Consistently, CTGF protein level was considerably greater in mucosal tissue of CRS without nasal polyps (CRSsNP) than in CRS with nasal polyps (CRSwNP) (P < 0.05) or in control subjects (P < 0.01). TGF-β1 protein level was higher in mucosal tissue of CRSsNP patients than in CRSwNP patients (P < 0.001) or in the control group (P < 0.01). mRNA levels of SGK1 and CTGF (P < 0.05, r = 0.668; P = 0.001, r = 0.630), TGF-β1 and CTGF (P < 0.05, r = 0.560; P < 0.05, r = 0.420), as well as SGK1 and TGF-β1(P < 0.05, r = 0.612; P < 0.05, r = 0.524) were significantly correlated in CRS mucosal tissue and GSE36830 dataset, respectively. TGF-β1-induced upregulated genes were significantly enriched in SGK1 overexpression group. In vitro assays, TGF-β1 promoted SGK1 and CTGF expression in a concentration- and time-dependent manner. Administrating an SGK1 inhibitor, GSK650394, significantly inhibited TGF-β1-induced CTGF expression in 16HBE and dispersed primary nasal polyp cells. ConclusionsTGF-β1 stimulation significantly increases SGK1 and CTGF expression. By regulating TGF-β1-CTGF pathway, SGK1 may participate in tissue remodeling in the pathological mechanism of CRS.

Intravitreal connective tissue growth factor neutralizing antibody or bevacizumab alone or in combination for prevention of proliferative vitreoretinopathy in an experimental model

Connective tissue growth factor (CTGF) is released by retinal pigment epithelial (RPE) cells and detectable in proliferative membranes (PrMs). This experimental study was performed to investigate the mRNA and protein levels of both CTGF and vascular endothelial growth factor A (VEGF-A) in a rabbit model of proliferative vitreoretinopathy (PVR). In addition, the effects of a single intravitreal injection of the safe dose of anti-CTGF or bevacizumab as monotherapy and in combination were evaluated. PVR was induced in the right eye of albino rabbits by intravitreal injection of cultured adult human RPE cells. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot analysis of CTGF and VEGF-A were performed on whole eye tissue in the PVR model versus controls at different time points. In the next step, the PVR models were assigned to five groups. The monotherapy groups received a single intravitreal injection of 0.1 ml of anti-CTGF 100 μg/ml (final concentration of 6.6 μg/ml in the vitreous) or 0.03 ml of 25 mg/ml bevacizumab. In the combined group, the abovementioned amounts of anti-CTGF and bevacizumab were injected intravitreally from separate sites in one session. No antibody injection was performed in the control group. Intravitreal injection of 0.1 ml of control IgG (1 mg/ml of isotype matched) antibody was performed in the placebo group. After 2 weeks, histologic evaluation including, trichrome staining for collagen, immunostaining by anti-alpha-smooth muscle actin for myofibroblasts, and anti-collagen type-1 antibody on paraffin embedded anterior-posterior sections was done. In addition, fundus photography was performed for clinically equivalent PVR staging. Twenty-four hours following PVR induction, CTGF mRNA and protein levels increased five- and- three-fold compared to controls, respectively (P < 0.001). VEGF-A mRNA and protein levels decreased significantly after 72 h of PVR induction compared to controls (P < 0.05). Means of PrM thickness and myofibroblast cell counts significantly decreased in the anti-CTGF group (P < 0.001 and P < 0.05, respectively). The mean area of collagen type-1 fibers of PrM in the mono- and combination therapy groups that received intravitreal anti-CTGF was significantly reduced (P < 0.001); in addition, mild PVR (stage-1 and 2) formation occurred in comparison with moderate to severe PVR (stage-4 and higher) in other groups. In conclusion, we found that intravitreal injection of CTGF neutralizing antibody resulted in a reduction in PrM thickness, collagen fibers and myofibroblast density in the PVR model. CTGF inhibition may represent a potential therapeutic target for PVR.

Hepatic connective tissue growth factor expression and regulation differ between non-steatotic and non-alcoholic steatotic livers from brain-dead donor

Accurate evaluation of liver steatosis is required from brain-dead donors (BDDs) with nonalcoholic fatty liver disease (NAFLD). Our purposes were to investigate expression and regulation of connective tissue growth factor (CTGF) expression in livers from human and rat after brain death, and further evaluate its potential application. NAFLD and brain death models were established in rats. LX2 cells were cultured under hypoxia/reoxygenation. CTGF protein and mRNA levels were measured in liver samples from BDDs of human and rat by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. YAP-regulated CTGF expression was investigated in LX2 cells via YAP small interfering RNA and Verteporfin treatment. Blood CTGF level from BDDs was measured by enzyme-linked immunosorbent assay. After brain death, CTGF, transforming growth factor-β and YAP were overexpressed in non-alcoholic steatotic liver, whereas CTGF was downregulated in non-steatotic liver. Time-series analysis revealed that CTGF and YAP expression was comparable, as confirmed by inhibited YAP expression in LX2 cells. CTGF level and NAFLD activity were linearly correlated. CTGF expression and regulation differ between non-steatosis and nonalcoholic steatosis livers from BDDs. CTGF may be an important factor to evaluate graft quality from BDDs with NAFLD.

TGF-β Induced CTGF Expression in Human Lung Epithelial Cells through ERK, ADAM17, RSK1, and C/EBPβ Pathways

Background: Lung epithelial cells play critical roles in idiopathic pulmonary fibrosis. Methods: In the present study, we investigated whether transforming growth factor-β (TGF-β)-induced expression of connective tissue growth factor (CTGF) was regulated by the extracellular signal-regulated kinase (ERK)/a disintegrin and metalloproteinase 17 (ADAM17)/ribosomal S6 kinases 1 (RSK1)/CCAAT/enhancer-binding protein β (C/EBPβ) signaling pathway in human lung epithelial cells (A549). Results: Our results revealed that TGF-β-induced CTGF expression was weakened by ADAM17 small interfering RNA (ADAM17 siRNA), TNF-α processing inhibitor-0 (TAPI-0, an ADAM17 inhibitor), U0126 (an ERK inhibitor), RSK1 siRNA, and C/EBPβ siRNA. TGF-β-induced ERK phosphorylation as well as ADAM17 phosphorylation was attenuated by U0126. The TGF-β-induced increase in RSK1 phosphorylation was inhibited by TAPI-0 and U0126. TGF-β-induced C/EBPβ phosphorylation was weakened by U0126, ADAM17 siRNA, and RSK1 siRNA. In addition, TGF-β increased the recruitment of C/EBPβ to the CTGF promoter. Furthermore, TGF-β enhanced fibronectin (FN), an epithelial–mesenchymal transition (EMT) marker, and CTGF mRNA levels and reduced E-cadherin mRNA levels. Moreover, TGF-β-stimulated FN protein expression was reduced by ADAM17 siRNA and CTGF siRNA. Conclusion: The results suggested that TGF-β induces CTGF expression through the ERK/ADAM17/RSK1/C/EBPβ signaling pathway. Moreover, ADAM17 and CTGF participate in TGF-β-induced FN expression in human lung epithelial cells.

Abnormal expression of connective tissue growth factor and its correlation with fibrogenesis in adenomyosis

Research questionDoes connective tissue growth factor (CTGF) expression relate to adenomyotic fibrosis and determine the correlation between fibrosis with adenomyosis-associated dysmenorrhoea? DesignProtein and mRNA expression of CTGF was detected by Western blots and real-time quantitative polymerase chain reaction in the endometrium of the control group and the eutopic and ectopic endometrium of the adenomyosis group. Collagen fibres and type I collagen in the myometrium were detected by immunohistochemistry and Masson's trichrome staining, and the correlations of CTGF protein and mRNA levels with the degree of fibrosis were analysed. Furthermore, the relationship between the severity of dysmenorrhoea and the degree of fibrosis was determined, and the correlation between uterus size and the degree of fibrosis was also analysed. ResultsLevels of CTGF mRNA and protein were significantly higher in patients with adenomyosis than in controls, and CTGF mRNA and protein expression in adenomyosis was positively correlated with fibrosis severity (r = 0.57, P < 0.001 and r = 0.39, P = 0.012), which correlated positively with dysmenorrhoea and uterus size (r = 0.42 and r = 0.6, P < 0.002). ConclusionsIncreased CTGF may contribute to the occurrence and fibrogenic progression of adenomyosis and may play an important role in dysmenorrhoea. The present study may provide ideas for treating adenomyosis-associated dysmenorrhoea.

RGC-32 regulates astrocyte differentiation during experimental autoimmune encephalomyelitis

Abstract Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of response gene to complement 32 (RGC-32) alters astrocyte morphology in spinal cords during experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In addition, we found that RGC-32 regulates TGF-β-induced extracellular matrix production and growth factors expression. We investigated if the lack of RGC-32 alters the expression of the glial fibrillary acidic protein (GFAP) and of astrocytes progenitor markers vimentin and fatty acid binding protein 7 (FABP7) during EAE. Immunohistochemical analysis of spinal cords during the acute phase of EAE (day 14 post EAE induction) showed that the elongated, radial glial cell-like astrocytes from RGC-32 knock-out (KO) mice expressed higher levels of vimentin and FABP7 as compared to wild type (WT) mice, confirming their immature phenotype. In addition, we found that the density of white matter GFAP+ astrocytes was higher in WT as compared to KO mice (p=0.02). Using double staining immunohistochemistry for GFAP and connective tissue growth factor (CTGF), known to be involved in astrocyte differentiation, we found a lower number of CTGF+ astrocytes during EAE in spinal cords of RGC-32 KO when compared with WT mice (p=0.002). We next purified neonatal astrocytes from WT and RGC-32 KO mice, and we found significantly lower levels of GFAP and CTGF mRNA and protein and higher levels of vimentin in RGC-32 KO astrocytes when compared with WT astrocytes. The expression pattern of astrocytic progenitor markers and that of CTGF suggests that RGC-32 is an important regulator of astrocyte differentiation during EAE.

Adventitial growth and lung connective tissue growth factor expression in pulmonary arterioles due to hypobaric hypoxia in broilers

Forty broilers maintained under natural hypobaric hypoxia (2,638 m above sea level) and 20 maintained under relative normoxia (460 m above sea level) were selected as pulmonary hypertensive (PHB) and nonpulmonary hypertensive (NPHB), to estimate the degree of the adventitial vascular thickness in lung arterioles and connective tissue growth factor (CTGF) expression in lung. In each group, the adventitial thickness (%AT) of 20 arterioles with 100 to 250 μm of external diameter was measured in lung samples of 24 and 42-day-old broilers. Also, mRNA extraction and real-time reverse transcription-PCR analysis were used to measure lung CTGF expression. The %AT was higher in PHB at 42 D as compared to NPHB at both ages and PHB at 24 D; however, the same differences were not evidenced at 24 D. In the 2 ages evaluated, differences were observed in the %AT between broilers under hypobaric hypoxia (PHB and NPHB) and under relative normoxia (P < 0.01). In broilers subjected to relative normoxia, no significant differences were found at any of the 2 ages. The expression levels of CTGF mRNA were higher in PHB compared to NPHB at the 2 ages. The %AT was higher in PHB with high levels of expression of CTGF mRNA than those NPHB with low expression of CTGF mRNA. This study showed that adventitial thickening is part of the pulmonary hypertension (PH) physiopathology in broilers exposed to hypobaric hypoxia, in which CTGF appears to be a fibrosis enhancer. Although present data suggest that adventitial engrossment could be a time-dependent process, individual susceptibility and the variable time-course of PH pathophysiology have to be considered.

YAP/TAZ regulates the expression of proteoglycan 4 and tenascin C in superficial-zone chondrocytes.

The roles of cell division control protein 42 homologue (CDC42) and actin polymerisation in regulating the phenotype of superficial-zone chondrocytes (SZCs) have been demonstrated in vitro; however, the signalling pathway(s) downstream have yet to be fully elucidated. The study hypothesis was that Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) act downstream to regulate proteoglycan 4 (PRG4) and tenascin C (TNC). Bovine SZCs grown in monolayer were treated with ML141 (CDC42 inhibitor) or the actin depolymerising agents, latrunculin B and cytochalasin D, to determine the effect on YAP/TAZ. Verteporfin (YAP/TAZ inhibitor) and YAP/TAZ siRNA-mediated knockdown were used to determine their role in regulating PRG4 and TNC. ML141 treatment reduced total YAP/TAZ protein, nuclear TAZ levels and the YAP/TAZ target gene, connective tissue growth factor (CTGF) mRNA levels. Latrunculin B decreased nuclear TAZ, while cytochalasin D treatment trended towards increased nuclear TAZ (p = 0.06), correlating with decreased and increased CTGF mRNA levels, respectively. Verteporfin treatment decreased PRG4 and TNC expression, with no effect on actin polymerisation. siRNA-mediated knockdown of YAP/TAZ revealed that PRG4 was regulated by YAP/TAZ while TNC was regulated by TAZ only. As cytochalasin D can activate myocardin-related transcription factor-A (MRTF-A), siRNA-mediated knockdown was performed to determine the role of MRTF-A in regulating YAP/TAZ. Although nuclear TAZ decreased, no significant changes in total protein levels were observed. Findings suggested that CDC42 and actin polymerisation regulated SZCs through multiple actin-regulated pathways. Understanding the regulation of these chondroprotective molecules may have important implications for prevention/treatment of osteoarthritis.

CTGF Attenuates Tendon-Derived Stem/Progenitor Cell Aging.

Aged tendon-derived stem/progenitor cells (TSPCs) lead to age-related tendon disorders and impair tendon healing. However, the underlying molecular mechanisms of TSPC aging remain largely unknown. Here, we investigated the role of connective tissue growth factor (CTGF) in TSPC aging. CTGF protein and mRNA levels were markedly decreased in the aged TSPCs. Moreover, recombinant CTGF attenuates TSPC aging and restores the age-associated reduction of self-renewal and differentiation of TSPCs. In addition, cell cycle distribution of aged TSPCs was arrested in the G1/S phase while recombinant CTGF treatment promoted G1/S transition. Recombinant CTGF also rescued decreased levels of cyclin D1 and CDK4 and reduced p27kip1 expression in aged TSPCs. Our results demonstrated that CTGF plays a vital role in TSPC aging and might be a potential target for molecular therapy of age-related tendon disorders.

Effect of microRNA-133b on Myocardial Fibrosis

Objective To investigate the effect of microRNA-133b(miR-133b)on cardiac fibrosis and its mechanism.Methods Human cardiac fibroblasts(CFs)were harvested.The proliferation of CFs was detected by CCK8 during the overexpression and knock-down of miR-133b.The expressions of connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA),collagen Ⅰ,and collagen Ⅲ were detected with qRT-PCR and Western blot analysis after miR-133b overexpression or downexpression.Target genes of miR-133b were predicted by bioinformatics software.Dual-luciferase activity assay were used to verify a target gene of miR-133b.Results qRT-PCR showed that the expression level of miR-133b in the miR-133b mimic group was significantly higher than that in the negative control group(t=26.219,P=0.000).The expression level of miR-133b in the miR-133b inhibitor group was significantly lower than that in the negative control group(t=6.738,P=0.003).After 21,45,69,93,and 117 hours of transfection,the proliferation ability of CFs significantly decreased in the miR-133b mimic group but significantly increased in the miR-133b group(all P<0.05,compared with the negative control group).After overexpression of miR-133b,the mRNA and protein levels of CTGF(t=9.213,P=0.001;t=8.195,P=0.001),α-SMA(t=6.511,P =0.003;t=4.434,P=0.011),collagenⅠ(t=3.172,P=0.034;t=4.053,P=0.015)and collagen Ⅲ(t=6.404,P=0.003;t=5.319,P=0.006)were significantly down-regulated.After the expression of miR-133b was knocked down,the mRNA and protein levels of CTGF(t=9.439,P=0.001;t=14.100,P=0.000),α-SMA(t=4.519,P=0.011;t=4.377,P=0.012),collagen Ⅰ(t=5.966,P=0.004;t=5.514,P=0.005)and collagen Ⅲ(t=4.622,P=0.010;t=4.996,P=0.008)were significantly increased.The relative luciferase activity of the cells co-transfected with miR-133b mimic and WT 3'UTR expression vector was significantly lower than that of the cells co-transfected with mimic control and WT 3'UTR expression vectors(t=5.654,P=0.005);however,there was no significant difference in relative luciferase activity between cells co-transfected with miR-133b mimic and MUT 3'UTR expression vectors and cells co-transfected with mimic control and MUT 3'UTR expression vectors(t=0.380,P=0.724).Conclusion miR-133b may affect the activation and proliferation of CFs by targeting CTGF and thus improve cardiac fibrosis.

Role of miR-133a in regulating TGF-β1 signaling pathway in myocardial fibrosis after acute myocardial infarction in rats.

The aim of this research was to explore the effect of microRNA-133a (miR-133a) on myocardial fibrosis and cardiac function after myocardial infarction in rats, and to investigate the possible regulatory mechanism. Myocardial infarction model was successfully established in rats by ligation of the left anterior descending coronary artery. After miR-133a overexpression in rats myocardium, cardiac function was examined by echocardiography. Meanwhile, the degree of myocardial fibrosis was detected by Masson staining. In addition, the expression of α-smooth muscle actin (α-SMA) in cardiomyocytes was detected by immunohistochemistry. Quantitative Real-time polymerase chain reaction (qRT-PCR) was performed to analyze the expression level of miR-133a in the junction of myocardial infarction. The mRNA expressions of transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), collagen type 1 (col 1), collagen type 3 (col 3) and α-SMA were measured by qRT-PCR as well. Furthermore, the protein levels of the above genes were detected by Western blotting. MiR-133a expression in the infarct border zone of myocardial tissue was significantly decreased on the 28th day after myocardial infarction surgery (p<0.05). In addition, up-regulation of miRNA-133a in myocardial tissue of rats with myocardial infarction could remarkably improve cardiac function and reduce collagen volume fraction. Furthermore, the mRNA and protein expression levels of TGF-β1, CTGF, col1, col3, α-SMA in myocardial tissue were obviously decreased after miRNA-133a up-regulation (p<0.001). Overexpression of miR-133a down-regulates the mRNA and protein levels of TGF-β1 and CTGF after myocardial infarction. Moreover, this may eventually reduce myocardial collagen deposition, inhibit myocardial fibrosis and improve cardiac function.

Effect of herbal cake-partitioned moxibustion on renal function and expression of renal connective tissue growth factor, integrin-linked kinase and bone morphogenetic protein-7 in chronic renal failure rabbits

To observe the effect of herbal cake-partitioned moxibustion on renal function and expression of connective tissue growth factor (CTGF), integrin-linked kinase (ILK) and bone morphogenetic protein-7 (BMP-7) in chronic renal failure (CRF) rabbits, so as to reveal its mechanisms underlying improvement of CRF. Twenty-four male New Zealand rabbits were randomly divided into control, model, medication and herbal cake-partitioned moxibustion (moxibustion) groups (n=6 rabbits in each group). The CRF model was established by gavage of suspension of Adenine (150 mg·kg-1·d-1) for 21 days. Herbal cake-partitioned moxibustion was applied to "Mingmen"(GV4) and bilateral "Shenshu"(BL23), "Pishu"(BL20) and for 5 moxa-cones every time. Rabbits of the medication group was treated by gavage of Losartan Potassium (2.33 mg·kg-1·d-1). All the treatments were conducted once daily,12 times a course for consecutive 3 courses with a two-day rest after each course of treatment. Serum creatinine (Scr), blood urea nitrogen (BUN) and 24 h urine protein contents were detected by using an automatic biochemical analyzer. The expression levels of CTGF, ILK and BMP-7 proteins and mRNA in the kidney tissue were determined by Western blot and quantitative real time-PCR, separately. Following modeling, Scr and BUN and 24 h urine protein contents were significantly increased in the model group in comparison with the control group (P<0.01). After the intervention, Scr and BUN contents were all significantly decreased in both the moxibustion and medication groups relevant to the model group (P<0.01), suggesting an improvement of the renal function. Compared with the control group, the expression levels of ILK and CTGF mRNAs and proteins were obviously up-regulated (P<0.01), and those of BMP-7 mRNA and protein significantly decreased in the model group (P<0.01). In comparison with the model group, the expression levels of ILK and CTGF mRNAs and proteins were significantly down-regulated in the two treatment groups (P<0.01, P<0.05), and those of BMP-7 mRNA and protein markedly increased in the two treatment groups (P<0.01). In comparison with the medication group, the expression level of ILK protein was significantly up-regulated (P<0.01) and BMP-7 protein was significantly down-regulated (P<0.01) in the moxibustion group. No significant differences were found between the medication and moxibustion groups in down-regulating the levels of Scr, BUN and 24 h urine protein and expression of ILK mRNA, CTGF mRNA and protein and BMP-7 mRNA(P>0.05). Herbal cake-partitioned moxibustion can improve renal function in CRF rabbits, which may be related to its effects in suppressing the expression of ILK and CTGF, and in up-regulating the expression of BMP-7 in the kidney tissue.

Farnesyl Pyrophosphate Synthase Blocker Ibandronate Reduces Thoracic Aortic Fibrosis in Diabetic Rats

BackgroundThis study assessed the effect of ibandronate (IBN), a farnesyl pyrophosphate synthase (FPPS) inhibitor, on vascular remodeling in diabetic rats. MethodsA rat model of diabetes was induced by a high-fat and high-sugar diet combined with a small dose of streptozotocin. The diabetic rats received 5 µg/kg of ibandronate solution or normal saline subcutaneously every morning for 16 weeks. The morphology of the thoracic aorta was assessed by hematoxylin and eosin and Masson's trichrome staining techniques. Gene expression levels of connective tissue growth factor (CTGF) and FPPS were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. CTGF and FPPS protein levels were determined by Western blotting analysis. ResultsRats with diabetes mellitus showed moderate hyperglycemia, insulin resistance, hyperlipidemia and thoracic aortic fibrosis. FPPS was significantly upregulated in the thoracic aorta from diabetic animals. Interestingly, IBN treatment for 16 weeks alleviated the diabetes-induced histopathologic changes in the thoracic aortic wall and reduced CTGF protein and mRNA levels. ConclusionsThese findings provided evidence that FPPS is involved in thoracic aortic fibrosis in diabetic rats. Meanwhile, IBN could alleviate vascular remodeling in diabetic animals.

TGFβ1 and HGF regulate CTGF expression in human atrial fibroblasts and are involved in atrial remodelling in patients with rheumatic heart disease.

ObjectiveThis study aimed to investigate the effects of transforming growth factor β1 (TGF β1) and hepatocyte growth factor (HGF) on the expression of connective tissue growth factor (CTGF) in human atrial fibroblasts, and to explore the relationship of these factors in atrial fibrosis and atrial anatomical remodelling (AAR) of patients with atrial fibrillation (AF).MethodsFresh right auricular appendix tissue of 20 patients with rheumatic heart disease undergoing valve replacement surgery was collected during surgeries, 10 patients had sinus rhythm(SR), and 10 patients had chronic atrial fibrillation (CAF). Atrial fibroblasts were then cultured from the tissues with differential attachment technique and treated with either TGFβ1 (10 ng/mL) or HGF (100 ng/mL). CTGF mRNA levels were measured by RT‐PCR, and CTGF protein content was determined using immunofluorescence and Western blotting assays.ResultsCAF group had higher left atrial diameters (LADs) and higher CTGF mRNA expression in atrial fibroblasts compared with SR group. The CTGF protein content in CAF group was higher than that of SR group and positively correlated with LAD and AF duration. After CAF group was treated with TGFβ1, CTGF mRNA and protein expression were significantly down‐regulated, whereas when treated with HGF, expression was up‐regulated compared with SR group.ConclusionsIncreased CTGF expression was associated with enlarged LAD, atrial fibrosis and AAR in patients with AF. TGFβ1 and HGF regulate CTGF expression in human atrial fibroblasts with up‐regulation of mRNA and down‐regulation of protein, therefore, either promote or inhibit atrial fibrosis, which could be related to the incidence and persistence of AF.

Histone deacetylase inhibitor suberoylanilide hydroxamic acid alleviates liver fibrosis by suppressing the transforming growth factor-β1 signal pathway

BackgroundHistone deacetylases (HDACs) inhibitors are new anti-fibrotic drugs that inhibit the activity of hepatic stellate cells. The present study focused on the anti-fibrotic function of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) by suppressing transforming growth factor-β1 (TGF-β1) signaling. MethodsMale Sprague-Dawley rats were used to induce liver fibrosis with carbon tetrachloride (CCl4) and LX2 cell (human hepatic stellate cell line) was stimulated by TGF-β1. Both animals and cells were treated with SAHA. The Smad7 and connective tissue growth factor (CTGF) mRNA levels were detected by real-time polymerase chain reaction (PCR). Western blotting was used to examine the protein levels of CTGF, Histone H3 (H3), Smad7, Smad2/3, Acetyl-Histone H3 (AH3), HDAC2, α-smooth muscle actin (α-SMA), HDAC6, p-Smad2/3 and HDAC8. In addition, the TGF-β1 and liver enzyme levels from rat serum were detected. Histopathological changes were examined by hematoxylin and eosin (HE), Sirius red and Masson trichrome staining. The α-SMA expression was detected by immumohistochemical staining. ResultsCompared with control group, the TGF-β1 and liver enzyme levels from rat serum, together with the mRNA levels of CTGF and protein levels of CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were elevated in fibrotic rats (P < 0.01). But the Smad7 mRNA and AH3 protein levels were notably suppressed in the fibrotic rats (P < 0.01). Pathological examination showed the typical changes of liver fibrosis in the fibrotic rats. After the treatment with SAHA, the levels of liver enzymes, TGF-β1, CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were reduced (P < 0.01) and Smad7 and AH3 protein contents were elevated in liver fibrotic rats (P < 0.01). Moreover, immumohistochemistry showed that SAHA significantly suppressed the α-SMA protein content in fibrotic liver (P < 0.01). ConclusionThe HDAC inhibitor SAHA alleviated liver fibrosis by suppressing the TGF-β1 signaling.