CTGF mRNA Research Articles

TRPV4 regulates matrix stiffness‐dependent activation of YAP/VEGFR2 signaling via Rho/Rho kinase/LATS1/2 pathway

TRPV4 is a mechanosensitive ion channel in endothelial cells and shown to regulate physiological and pathological angiogenesis. We have recently shown that TRPV4 deletion and/or down regulation activates vascular endothelial growth factor receptor-2 (VEGFR2) via yes-associated protein (YAP) in human EC. However, the molecular mechanisms of TRPV4-dependant YAP/VEGFR2 activation are unknown. We have employed human microvascular endothelial cells (HMEC-1; EC) on ECM gels of varying stiffness that mimic normal and failing hearts (8 and 50 kPa) and examined nuclear translocation of YAP and disappearance of perinuclear VEGFR2, after siRNA knockdown of TRPV4. Next, we investigated the role of upstream and downstream regulators of YAP translocation by focusing on LATS-1, CYR61 and Rho/Rho Kinase, using phospho-specific antibodies (pYAP and pLATS) and pharmacological inhibitor of Rho kinase (Y-27632). siRNA knockdown of TRPV4 significantly increased nuclear translocation of YAP with concomitant disappearance of perinuclear-VEGFR2 in matrix stiffness-dependent manner. Further, we found increased Rho-GTP and decreased LATS-1 phosphorylation in TRPV4 knockdown EC compared to control siRNA treated EC. Furthermore, pharmacological inhibition of Rho kinase with Y-27632, increased phosphorylation of LATS1/2 kinase, inhibited nuclear translocation of YAP and VEGFR2 redistribution. Consistent with above results, YAP target genes CYR61 and CTGF mRNA expression levels were increased in a matrix stiffness-dependent manner in TRPV4 knockdown EC than the control siRNA treated EC. Our results suggest that TRPV4 regulates YAP/VEGFR2 activation in a matrix stiffness dependent manner during angiogenesis via Rho/Rho kinase/LATS1/2 pathway.

Ventricular Fibrosis and Coronary Remodeling Following Short-Term Exposure of Healthy and Malnourished Mice to Bisphenol A.

Bisphenol-A (BPA) is an endocrine disruptor associated with higher risk of insulin resistance, type 2 diabetes, and cardiovascular diseases especially in susceptible populations. Because malnutrition is a nutritional disorder associated with high cardiovascular risk, we sought to compare the effects of short-term BPA exposure on cardiovascular parameters of healthy and protein-malnourished mice. Postweaned male mice were fed a normo- (control) or low-protein (LP) diet for 8 weeks and then exposed or not to BPA (50 μg kg−1 day−1) for the last 9 days. Systolic blood pressure was higher in BPA or LP groups compared with the control group. However, diastolic blood pressure was enhanced by BPA only in malnourished mice. Left ventricle (LV) end diastolic pressure (EDP), collagen deposition, and CTGF mRNA expression were higher in the control or malnourished mice exposed to BPA than in the respective nonexposed groups. Nevertheless, mice fed LP diet exposed to BPA exhibited higher angiotensinogen and cardiac TGF-β1 mRNA expression than mice treated with LP or BPA alone. Wall:lumen ratio and cross-sectional area of intramyocardial arteries were higher either in the LP or BPA group compared with the control mice. Taken together, our data suggest that short-term BPA exposure results in LV diastolic dysfunction and fibrosis, and intramyocardial arteries inward remodeling, besides potentiate protein malnutrition-induced hypertension and cardiovascular risk.

Endoglin Promotes Myofibroblast Differentiation and Extracellular Matrix Production in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a complication of diabetes mellitus that can lead to proteinuria and a progressive decline in renal function. Endoglin, a co-receptor of TGF-β, is known primarily for regulating endothelial cell function; however, endoglin is also associated with hepatic, cardiac, and intestinal fibrosis. This study investigates whether endoglin contributes to the development of interstitial fibrosis in DN. Kidney autopsy material from 80 diabetic patients was stained for endoglin and Sirius Red and scored semi-quantitatively. Interstitial endoglin expression was increased in samples with DN and was correlated with Sirius Red staining (p < 0.001). Endoglin expression was also correlated with reduced eGFR (p = 0.001), increased creatinine (p < 0.01), increased systolic blood pressure (p < 0.05), hypertension (p < 0.05), and higher IFTA scores (p < 0.001). Biopsy samples from DN patients were also co-immunostained for endoglin together with CD31, CD68, vimentin, or α-SMA Endoglin co-localized with both the endothelial marker CD31 and the myofibroblast marker α-SMA. Finally, we used shRNA to knockdown endoglin expression in a human kidney fibroblast cell line. We found that TGF-β1 stimulation upregulated SERPINE1, CTGF, and ACTA2 mRNA and α-SMA protein, and that these effects were significantly reduced in fibroblasts after endoglin knockdown. Taken together, these data suggest that endoglin plays a role in the pathogenesis of interstitial fibrosis in DN.

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

P168 Modelling macrophage-fibroblast interaction in scleroderma to evaluate new treatments

Abstract Background Alternatively-activated M2-like macrophages are known to express CD206 and are thought to have an important role in pathological fibrosis in scleroderma through stimulation of fibroblasts. RP peptides are 10-12 mer synthetic peptides which have been developed to engage with human macrophages via CD206, but the selectivity of this receptor for macrophages requires clarification. In this study, we investigate the relative expression levels of CD206 in SSc macrophages and fibroblasts, and explore two approaches in modelling macrophage-fibroblast interactions to evaluate the efficacy of the RP peptides. Methods Macrophages were derived from peripheral blood mononuclear cells by culture of buffy layer cells in RPMI supplemented by M-CSF (4ng/ml) for 7 days. Skin fibroblasts were obtained by explant culture of 4 mm punch biospies from the involved forearm skin of SSc patients and healthy controls, maintained in DMEM with 10-% FCS and studied at passage 3-5. Stimulation with TGFβ (4ng/ml) was used to further enhance the activation state of fibroblasts. Macrophages and skin fibroblast lysates were evaluated using qPCR to determine the level of expression of CD206. Macrophages were co-cultured with fibroblasts within respective monolayers on 50kPa gels (models stiff scleroderma skin) for four days with or without the peptide inhibitors. Collagen mRNA expression from the cellular monolayers were quantified by qPCR and Western blot. In the second approach, macrophages and fibroblasts were co-cultured together with or without the peptide inhibitors in a collagen gel contraction assay. The gel was weighed and imaged after 48 hours of incubation and the collagen gel contraction was then quantified as a measure of fibrotic activity. Results Expression of CD206 was specific to macrophages and not seen in fibroblast cultures (relative expression level in macrophages 26.63 versus 0.004 in unstimulated fibroblasts versus 0.04 in TGFβ-stimulated fibroblasts. Fibroblasts co-cultured with macrophages in monolayers show increased CTGF and collagen mRNA by qPCR. There was a dose response reduction in CTGF and collagen mRNA in assays cultured with the RP peptide inhibitor. CTGF mRNA was positively correlated with CD206 expression (r2=0.81). Co-culture of SSc macrophages led to enhanced contraction of collagen gels (0.1g in macrophages, 0.08g in fibroblasts, 0.062 for fibroblasts co-cultured with macrophages) which was fully reversed by the RP peptide treatment (0.1g). Conclusion SSc macrophages express CD206 which has potential as a biomarker of ongoing fibrotic activity whereas fibroblasts express very minimal levels of CD206 even when stimulated by the pro-fibrotic TGFβ. RP peptides that are specific for CD206 on macrophages led to a suppression of the macrophage signature and inhibition of the pro-fibrotic cross talk in macrophages and fibroblasts in both monolayer and 3D collagen gels. Both approaches of evaluating macrophage-fibroblast interactions in scleroderma are viable approaches to evaluate new treatments. Disclosures L. Lei None. B. Abdi None. A. Mujkavnovic None. X. Shi Wen None. H. Lopez Shareholder/stock ownership; Scientific director at Riptide Biosciences. R. Stratton None.

ERK1/2 drives IL-1\u03b2-induced expression of TGF-\u03b21 and BMP-2 in torn tendons

Diseased and injured tendons develop fibrosis, driven by factors including TGF-β, BMPs and CTGF. IL-1β and its signal transducer Erk1/2 are known to regulate TGF-β expression in animal tendons. We utilised tissues and cells isolated from patients with shoulder tendon tears and tendons of healthy volunteers to advance understanding of how inflammation induces fibrosis in diseased human tendons. ERK1/2 expression was reduced in torn (diseased) compared to healthy patient tendon tissues. We next investigated the fibrotic responses of tendon-derived cells isolated from healthy and diseased human tendon tissues in an inflammatory milieu. IL-1β treatment induced profound ERK1/2 signalling, TGFB1 and BMP2 mRNA expression in diseased compared to healthy tendon-derived cells. In the diseased cells, the ERK1/2 inhibitor (PD98059) completely blocked the IL-1β-induced TGFB1 and partially reduced BMP2 mRNA expression. Conversely, the same treatment of healthy cells did not modulate IL-1β-induced TGFB1 or BMP2 mRNA expression. ERK1/2 inhibition did not attenuate IL-1β-induced CTGF mRNA expression in healthy or diseased tendon cells. These findings highlight differences between ERK1/2 signalling pathway activation and expression of TGF-β1 and BMP-2 between healthy and diseased tendon tissues and cells, advancing understanding of inflammation induced fibrosis during the development of human tendon disease and subsequent repair.

Time Course of the Effects of Buxin Yishen Decoction in Promoting Heart Function and Inhibiting the Progression of Renal Fibrosis in Myocardial Infarction Caused Type 2 Cardiorenal Syndrome Rats.

This study aimed to investigate the therapeutic effect of traditional Chinese medicine-Buxin Yishen decoction (BXYS) on type 2 cardiorenal syndrome (CRS) caused by myocardial infarction and explore the possible mechanism BXYS works. A chronic heart failure (CHF) rat model induced by left anterior descending coronary artery ligation was used and five groups were created that included a sham group, a CHF model group, a fosinopril group, a BXYS (0.4 g/kg) group and a BXYS (0.8 g/kg) group. Heart function, renal hemodynamics, neuroendocrine factors, serum, and urine concentration of soluble form connective tissue growth factor (sCTGF), expression of CTGF mRNA, CTGF, α-smooth muscle actin (α-SMA), and low-density lipoprotein receptor-related protein (LRP) in renal tissues were evaluated after 28 days and 60 days of drug administration. Histological analysis of kidney tissues was also performed. In vitro experiments were designed to verify the results of in vivo experiments by detecting factors including CTGF, α-SMA, in NRK-52E cells. Rats with CHF showed obvious pathophysiological changes including: altered renal hemodynamic parameters; dysregulated heart function; changes to serum concentrations of angiotensin II (AngII), cyclic guanosine monophosphate (cGMP), serum creatinine (Scr), blood urea nitrogen (BUN), C-reactive protein (CRP), brain natriuretic peptide (BNP); high serum and urine sCTGF concentration; high CTGF mRNA, CTGF, α-SMA and LRP expression in renal tissues; increased extracellular matrix (ECM) deposition and fibrosis in renal tissues. Treatment of BXYS was correlated with a restoration of heart function and improvement of renal hemodynamics, lower serum and urine sCTGF, lower CTGF mRNA, CTGF, α-SMA and LRP expression in renal tissues and lower ECM deposition. In addition, in vitro experiments showed that treatment with BXYS reduced the α-SMA and LRP concentration in NRK-52E cells, which were similar in vivo experiments. In conclusion, the current study provided evidences that BXYS played a role in improving heart function and delaying the progress of renal fibrosis. Meanwhile, the CTGF-LRP pathway might be one of the therapeutic targets for myocardial infarction caused type 2 CRS which showed a positive change after BXYS treatment and is worthy of further exploration.

Autologous Cell Therapy for Aged Human Skin: A Randomized, Placebo-Controlled, Phase-I Study

Introduction: Skin ageing involves senescent fibroblast accumulation, disturbance in extracellular matrix (ECM) homeostasis, and decreased collagen synthesis. Objective: to assess a cell therapy product for aged skin (RCS-01; verum) consisting of ~25 × 10⁶ cultured, autologous cells derived from anagen hair follicle non-bulbar dermal sheath (NBDS). Methods: For each subject in the verum group, 4 areas of buttock skin were injected intradermally 1 or 3 times at monthly intervals with RCS-01, cryomedium, or needle penetration without injection; in the placebo group RCS-01 was replaced by cryomedium. The primary endpoint was assessment of local adverse event profiles. As secondary endpoints, expression of genes related to ECM homeostasis was assessed in biopsies from randomly selected volunteers in the RCS-01 group taken 4 weeks after the last injection. ­Results: Injections were well tolerated with no severe adverse events reported 1 year after the first injection. When compared with placebo-treated skin, a single treatment with RCS-01 resulted in a significant upregulation of TGFβ1, CTGF, COL1A1, COL1A2, COL3A1, and lumican mRNA expression. Limitations: The cohort size was insufficient for dose ­ranging evaluation and subgroup analyses of efficacy. Conclusions: RCS-01 therapy is well tolerated and associated with a gene expression response consistent with an improvement of ECM homeostasis.

Effects of intravitreal connective tissue growth factor neutralizing antibody on choroidal neovascular membrane-associated subretinal fibrosis

Connective tissue growth factor (CTGF) plays an essential role in the regulation of extracellular matrix proteins and pro-fibrotic and angiogenic factors. This experimental research was conducted to evaluate if CTGF is elevated after induction of a choroidal neovascular membrane (CNVM) and whether intravitreal anti-CTGF without and with intravitreal bevacizumab (IVB) may have any effect on the CNVM associated sub-retinal fibrosis. In adherence to ARRIVE guidelines, CNVM was induced by laser spots in the right eye retinas of ninety-four pigmented rats. Quantitative real-time reverse transcription PCR (qRT-PCR) and western-blot analysis were performed on sclerochoroidal tissues of forty-four rats before and at different time intervals after laser application. The remaining fifty rats were randomly divided into five groups after laser application. Group A received intravitreal injection of 2 μl of the 50 μg/ml anti-CTGF. In group B, intravitreal injection of 2 μl of 25 mg/ml bevacizumab was performed. Group C received 1 μl intravitreal anti-CTGF and 1 μl IVB. Group D did not receive any intravitreal injection as the control group. In group E, intravitreal injection of 2 μl of nonspecific purified mouse IgG antibody was performed as the placebo group. After two weeks, double immunohistochemistry was performed by isolectin B4 and anti-collagen type1 on the sclerochoroidal flat-mounts. Masked measurement of the fluorescent images of the CNVM and CNVM associated sub-retinal fibrosis areas was performed using the image J software. Ctgf mRNA and CTGF protein levels increased to the maximum level in 24 h after laser application and remained higher than the control level up to the 14th day for the Ctgf mRNA and up to the 7th day for the CTGF protein level. Means of CNVM associated sub-retinal fibrosis areas in three treatment groups (A, B and C) were significantly less than the control (D) and placebo (E) groups (P < 0.001, <0.05, <0.001 respectively). For groups A and C, mean CNVM associated sub-retinal fibrosis areas were also significantly less than group B (P < 0.05 and < 0.01, respectively). In conclusion, this study showed significant reduction of the CNVM associated sub-retinal fibrosis via inhibition of the CTGF which mediates the final steps of fibrosis in various inflammatory and angiogenic pathways.

COA-Cl prevented TGF-β1-induced CTGF expression by Akt dephosphorylation in normal human dermal fibroblasts, and it attenuated skin fibrosis in mice models of systemic sclerosis

BackgroundSystemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. Although transforming growth factor (TGF)-β1-induced connective tissue growth factor (CTGF/CCN2) expression has been presented in SSc fibrosis, the therapeutic potential of targeting CTGF in SSc has not been fully explored. COA-Cl is a novel nucleic acid analog, which is reported to have pleiotropic beneficial biologic effects. ObjectiveWe examine the effects of COA-Cl on TGF-β1-induced CTGF expression in normal human dermal fibroblast (NHDF). We also examined the effects of COA-Cl on CTGF expression in a mouse SSc model of angiotensin II (Ang II)-induced skin fibrosis. MethodsNHDF was cultured for in vitro experiments. For in vivo experiments, C57BL/6J mice were treated with Ang II for 14 days by subcutaneous osmotic pump. Quantitative real-time polymerase chain reaction, western blot analysis, immunohistochemical staining and immunofluorescence staining were performed to examine the expression levels of CTGF and phosphorylation levels of Smad2/3, ERK1/2 and Akt. ResultsCOA-Cl attenuated the TGF-β1-induced expression of both CTGF mRNA and protein in NHDF. Although COA-Cl did not alter the TGF-β1-induced phosphorylation of Smad2/3 or ERK1/2, it reduced the TGF-β1-induced phosphorylation levels of Akt in NHDF. Notably, COA-Cl dephosphorylated the Akt of lysates of TGF-β1-treated NHDF. COA-Cl reduced the levels of CTGF mRNA, CTGF protein, dermal thickness, collagen content and Akt phosphorylation in the skin of mice SSc model. ConclusionThese results imply that the inhibition of TGF-β1-induced CTGF expression by COA-Cl may be a therapeutic approach for SSc.

The prevalence of cardio-metabolic risk factors is differentially elevated in obesity-prone Osborne-Mendel and obesity-resistant S5B/Pl rats

AimsIndividual susceptibility to develop obesity may impact the development of cardio-metabolic risk factors that lead to obesity-related comorbid conditions. Obesity-prone Osborne-Mendel (OM) rats expressed higher levels of visceral adipose inflammation than obesity-resistant, S5B/Pl (S5B) rats. However, the consumption of a high fat diet (HFD) differentially affected OM and S5B rats and induced an increase in visceral adipose inflammation in S5B rats. The current study examined the effects of HFD consumption on cardio-metabolic risk factors in OM and S5B rats. Materials & methodsGlucose regulation and circulating levels of lipids, adiponectin and C-reactive protein were assessed following 8 weeks of HFD or low fat diet (LFD) consumption. Left ventricle hypertrophy and mRNA expression of cardiovascular disease biomarkers were also quantified in OM and S5B rats. Key findingsCirculating levels of triglycerides were higher, while HDL cholesterol, adiponectin and glycemic control were lower in OM rats, compared to S5B rats. In the left ventricle, BNP and CTGF mRNA expression were higher in OM rats and IL-6, IL-1β, VEGF, and iNOS mRNA expression were higher in S5B rats. SignificanceThese findings support the hypothesis that cardio-metabolic risk factors are increased in obesity-prone individuals, which may increase the risk for the development of obesity-related comorbidities. In the current models, obesity-resistant S5B rats also exhibited cardiovascular risk factors supporting the importance of monitoring cardiovascular health in individuals characterized as obesity-resistant.

Yangyin Yiqi Mixture Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats through Inhibiting TGF-β1/Smad Pathway and Epithelial to Mesenchymal Transition.

Objective The aim of the current study was to investigate the protective effect of Yangyin Yiqi Mixture (YYYQ) on Bleomycin-induced pulmonary fibrosis in rats based on TGF-β1/Smad signal pathway and epithelial to mesenchymal transition (EMT). Methods 120 Wistar rats were randomly divided into six groups: control group, BLM group, BLM + Pred group, BLM+YYYQ-L group, BLM+YYYQ-M group, and BLM+YYYQ-H group. Rats were given an intratracheal instillation of 3 mg/kg BLM to establish the pulmonary fibrosis model and followed by different dosages of YYYQ (11, 22, 44g/kg, via intragastric gavage) or prednisone soluble (4.2mg/kg, via intragastric gavage) or water. After 14 days and 28 days, tissue sections were stained with hematoxylin-eosin and Masson's trichrome to observe histopathological changes. Protein levels of TGF-β1, CTGF, Interleukin 18, and hydroxyproline were detected by ELISA method, and mRNA expressions of TGF-β1, TβRI, TβRII, Smad3, Smad7, α-SMA, E-cadherin, laminin, and collagen I were detected by RT-PCR. Results TGF-β1, CTGF, Interleukin 18, and hydroxyproline levels and mRNA expression of TGF-β1, TβRI, TβRII, Smad3, α-SMA, laminin, and collagen I were significantly increased (p <0.01), while Smad7 and E-cadherin levels were significantly decreased in BLM group (p <0.01). YYYQ-M and YYYQ-H group had downregulated the TGF-β1, CTGF, hydroxyproline contents, and mRNA expression of TGF-β1, TβRI, TβRII, Smad3, α-SMA, laminin, and collagen I and upregulated mRNA levels of Smad7 and E-cadherin significantly (p <0.01 or p <0.05). The result from the present study, which was also supported by histological evidence, suggested that YYYQ-M group and YYYQ-H group exhibited better treatment effect on Bleomycin-induced pulmonary fibrotic rats when compared to that of BLM + Pred group (p <0.01). Meanwhile, the effect of YYYQ, in three different dosages, on the level of interleukin 18 was not significant. Conclusion These results showed that YYYQ has the potential of ameliorating the progression of pulmonary fibrosis, and the mechanism may be related to suppressing TGF-β1/Smad signal pathway and EMT in BLM-induced pulmonary fibrosis of rats.

Swertiamarin ameliorates carbon tetrachloride-induced hepatic apoptosis via blocking the PI3K/Akt pathway in rats.

Swertiamarin (STM) is an iridoid compound that is present in the Gentianaceae swertia genus. Here we investigated antiapoptotic effects of STM on carbon tetrachloride (CCl4)-induced liver injury and its possible mechanisms. Adult male Sprague Dawley rats were randomly divided into a control group, an STM 200 mg/kg group, a CCl4 group, a CCl4+STM 100 mg/kg group, and a CCl4+STM 200 mg/kg group. Rats in experimental groups were subcutaneously injected with 40% CCl4 twice weekly for 8 weeks. STM (100 and 200 mg/kg per day) was orally given to experimental rats by gavage for 8 consecutive weeks. Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins were evaluated by western blot analysis. The expression of TGF-β1, collagen I, collagen III, CTGF and fibronectin mRNA were estimated by qRT-PCR. The results showed that STM significantly reduced the number of TUNEL-positive cells compared with the CCl4 group. The levels of Bax and cleaved caspase-3 proteins, and TGF-β1, collagen I, collagen III, CTGF, and fibronectin mRNA were significantly reduced by STM compared with the CCl4 group. In addition, STM markedly abrogated the repression of Bcl-2 by CCl4. STM also attenuated the activation of the PI3K/Akt pathway in the liver. These results suggested that STM ameliorated CCl4-induced hepatocyte apoptosis in rats.

Follistatin supplementation during in vitro embryo culture improves developmental competence of bovine embryos produced using sex-sorted semen

Using sex-sorted semen to produce offspring of desired sex is associated with reduced developmental competence in vitro and lower fertility rates in vivo. The objectives of the present study were to investigate the effects of exogenous follistatin supplementation on the developmental competence of bovine embryos produced with sex-sorted semen and possible link between TGF-β regulated pathways and embryotrophic actions of follistatin. Effects of follistatin on expression of cell lineage markers (CDX2 and Nanog) and downstream targets of SMAD signaling (CTGF, ID1, ID2 and ID3) and AKT phosphorylation were investigated. Follistatin was supplemented during the initial 72 h of embryo culture. Exogenous follistatin restored the in vitro developmental competence of embryos produced with sex-sorted semen to the levels of control embryos produced with unsorted semen, and comparable results were obtained using sorted semen from three different bulls. The mRNA abundance for SMAD signaling downstream target genes, CTGF (SMAD 2/3 pathway) and ID2 (SMAD 1/5 pathway), was lower in blastocysts produced using sex-sorted versus unsorted semen, but mRNA levels for CDX2, NANOG, ID1 and ID3 were similar in both groups. Follistatin supplementation restored CTGF and ID2 mRNA in blastocysts produced using sex-sorted semen to levels of control embryos. Moreover, levels of phosphorylated (p)AKT (Ser-473 and Thr-308) were similar in embryos derived from sex-sorted and unsorted semen, but follistatin treatment increased pAKT levels in both groups. Taken together, results demonstrated that follistatin improves in vitro development of embryos produced with sex-sorted semen and such effects are associated with enhanced indices of SMAD signaling.

Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor \u03b21

BackgroundChronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-β1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-β1.ResultsCortical fibroblast cultures were successfully established from the kidney tissue of cats with normal kidney function (FCF) and cats with chronic kidney disease (CKD-FCF). Both cell types expressed the mesenchymal markers vimentin, CD44 and CD29, and were negative for the epithelial marker cytokeratin, mesangial cell marker desmin and endothelial cell marker vWF. Only CKD-FCF expressed VCAM-1, a cell marker associated with inflammation. Incubation with TGF-β1 (0–10 ng/ml) induced a concentration dependent change in cell morphology, and upregulation of myofibroblast marker gene α-SMA expression alongside collagen 1α1, fibronectin, TGF-β1 and CTGF mRNA. These changes were blocked by the TGF-β1 receptor 1 antagonist SB431542 (5 μM).ConclusionsFCF and CKD-FCF can be cultured via a simple method and represent a model for the investigation of the progression of fibrosis in feline CKD. The findings of this study suggest TGF-β1 may be involved in fibroblast-myofibroblast transition in feline CKD, as in other species.

Human Epicardial Adipose Tissue cTGF Expression is an Independent Risk Factor for Atrial Fibrillation and Highly Associated with Atrial Fibrosis

Epicardial adipose tissue (EAT) is associated with the incidence, perpetuation, and recurrence of atrial fibrillation (AF), with elusive underlying mechanisms. We analyzed adipokine expression in samples from 20 patients with sinus rhythm (SR) and 16 with AF. Quantitative real-time PCR showed that connective tissue growth factor (cTGF) expression was significantly higher in EAT than in subcutaneous adipose tissue (SAT) or paracardial adipose tissue (PAT) from patients with AF, and in EAT from patients with SR (P < 0.001). Galectin-3 expression was significantly higher in EAT than in SAT or PAT (P < 0.001), with no significant differences between patients with AF and SR (P > 0.05). Leptin and vaspin expression were lower in EAT than in PAT (P < 0.001). Trichrome staining showed that the fibrosis was much more severe in patients with AF than SR (P < 0.001). We found a linear relationship between cTGF mRNA expression level and collagen volume fraction (y = 1.471x + 27.330, P < 0.001), and logistic regression showed that cTGF level was an independent risk factor for AF (OR 2.369, P = 0.027). In conclusion, highly expressed in EAT, cTGF is associated with atrial fibrosis, and can be an important risk factor for AF.

Buyang Huanwu Decoction Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats via Downregulation of Related Protein and Gene Expression.

Little is known about the effects of Buyang Huanwu decoction on pulmonary fibrosis. Herein, 144 healthy SD rats were randomly divided into six groups: blank control group (B), model control group (M), positive medicine control group (Mp), and high-, moderate-, and low-dose Buyang Huanwu decoction groups (Hd, Md, and Ld). A pulmonary fibrosis model was established by endotracheal injection of bleomycin. On the second day of modeling, the corresponding saline, methylprednisolone suspension, and the three doses of Buyang Huanwu decoction were used to treat the 6 groups of rats by intragastric administration for 7, 14, and 28 consecutive days. After 7, 14, and 28 days of treatment, the mRNA expression of CTGF and AKT, the protein level of CTGF, p-AKT, and collagen types I and III were tested. Finally, we found that the serum collagen type I and III level in Hd, Md, and Ld rats on the 14th and 28th day and the collagen type I and III level in Hd rats on 7th day were significantly lower than in M rats (P < 0.01). The protein level of p-AKT and CTGF in Hd and Md rats on the 7th and 14th days and the protein level of p-AKT in Hd rats on the 28th day were lower than in M rats (P < 0.01, P < 0.05). The level of CTGF mRNA in Hd, Md, and Ld rats and the level of AKT mRNA in Hd and Md rats on the 7th, 14th, and 28th days and the expression level of AKT mRNA in Ld rats on the 14th and 28th days were significantly lower than in M rats (P < 0.01). The study suggests that Buyang Huanwu decoction alleviated pulmonary fibrosis of rats by improvement of lung tissue morphology, low level of serum collagen types I and III, and the reduced expression of CTGF and p-AKT protein, which might be a result of its downregulated expression of CTGF and AKT mRNA levels.

MiR-375 attenuates injury of cerebral ischemia/reperfusion via targetting Ctgf.

Ischemic stroke is the leading cause of disability and deaths worldwide. MiRNAs have been shown to play an important role in development and pathogenesis of the nervous system. However, the precise function and mechanism of miRNAs are not fully understood in the brain injury induced by ischemia/reperfusion (I/R). Herein, our study showed that miR-375 expression was significantly down-regulated in the rat I/R brain. With the in vivo and in vitro I/R stroke models, we found that miR-375 mimic provides significant protection from injury to cerebral I/R, which is reflected by reduced infarct volumes and cell apoptosis, and increased proliferation and migration of PC12 cells. Mechanistically, our findings showed that miR-375 binds to 3′-UTR region of Ctgf mRNA, subsequently leading to the decreased expression of Ctgf in the I/R brain. Furthermore, we showed that miR-375/Ctgf-mediated protective effects are associated with p21/PI3K/Akt signaling pathways. Our findings thus provide a new insight into the mechanism of cerebral I/R injury and pave a potential new way for the therapy of cerebral I/R injury.

CTGF Is Expressed During Cystic Remodeling in the PKD/Mhm (cy/+) Rat Model for Autosomal-Dominant Polycystic Kidney Disease (ADPKD)

Connective tissue growth factor (CTGF, also named CCN2) plays an important role in the development of tubulointerstitial fibrosis, which most critically determines the progression to end-stage renal failure in autosomal-dominant polycystic kidney disease (ADPKD), the most common genetically caused renal disease. We determined CTGF expression in a well-characterized animal model of human ADPKD, the PKD/Mhm (cy/+) rat. Kidneys of 12 weeks old (cy/+) as well as (+/+) non-affected rats were analyzed for CTGF RNA and protein expression by RT-PCR, Northern and Western blot analyses, in situ hybridization, and IHC. Besides the established expression of CTGF in glomerular cells in kidneys of wild-type (+/+) animals, in (cy/+) rats, CTGF mRNA and protein were robustly expressed in interstitial, stellate-shaped cells, located in a scattered pattern underlying the cystic epithelium and in focal areas of advanced tubulointerstitial remodeling. Renal CTGF mRNA and protein expression levels were significantly higher in (cy/+) rats compared with their (+/+) littermates. Detection of CTGF expression in cells adjacent to cystic epithelium and in areas of marked fibrosis suggests a role in the local response to cyst development and indicates that CTGF may be a relevant factor contributing to tubulointerstitial fibrosis in polycystic kidney disease.

Caffeine modulates glucocorticoid-induced expression of CTGF in lung epithelial cells and fibroblasts

BackgroundAlthough caffeine and glucocorticoids are frequently used to treat chronic lung disease in preterm neonates, potential interactions are largely unknown. While anti-inflammatory effects of glucocorticoids are well defined, their impact on airway remodeling is less characterized. Caffeine has been ascribed to positive effects on airway inflammation as well as remodeling. Connective tissue growth factor (CTGF, CCN2) plays a key role in airway remodeling and has been implicated in the pathogenesis of chronic lung diseases such as bronchopulmonary dysplasia (BPD) in preterm infants. The current study addressed the impact of glucocorticoids on the regulation of CTGF in the presence of caffeine using human lung epithelial and fibroblast cells.MethodsThe human airway epithelial cell line H441 and the fetal lung fibroblast strain IMR-90 were exposed to different glucocorticoids (dexamethasone, budesonide, betamethasone, prednisolone, hydrocortisone) and caffeine. mRNA and protein expression of CTGF, TGF-β1-3, and TNF-α were determined by means of quantitative real-time PCR and immunoblotting. H441 cells were additionally treated with cAMP, the adenylyl cyclase activator forskolin, and the selective phosphodiesterase (PDE)-4 inhibitor cilomilast to mimic caffeine-mediated PDE inhibition.ResultsTreatment with different glucocorticoids (1 μM) significantly increased CTGF mRNA levels in H441 (p < 0.0001) and IMR-90 cells (p < 0.01). Upon simultaneous exposure to caffeine (10 mM), both glucocorticoid-induced mRNA and protein expression were significantly reduced in IMR-90 cells (p < 0.0001). Of note, 24 h exposure to caffeine alone significantly suppressed basal expression of CTGF mRNA and protein in IMR-90 cells. Caffeine-induced reduction of CTGF mRNA expression seemed to be independent of cAMP levels, adenylyl cyclase activation, or PDE-4 inhibition. While dexamethasone or caffeine treatment did not affect TGF-β1 mRNA in H441 cells, increased expression of TGF-β2 and TGF-β3 mRNA was detected upon exposure to dexamethasone or dexamethasone and caffeine, respectively. Moreover, caffeine increased TNF-α mRNA in H441 cells (6.5 ± 2.2-fold, p < 0.05) which has been described as potent inhibitor of CTGF expression.ConclusionsIn addition to well-known anti-inflammatory features, glucocorticoids may have adverse effects on long-term remodeling by TGF-β1-independent induction of CTGF in lung cells. Simultaneous treatment with caffeine may attenuate glucocorticoid-induced expression of CTGF, thereby promoting restoration of lung homeostasis.