KLF15 Is an Essential Negative Regulatory Factor for the Cardiac Remodeling Response to Pressure Overload

Cardiology ◽  
2015 ◽  
Vol 130 (3) ◽  
pp. 143-152 ◽  
Author(s):  
Yang Yu ◽  
Jie Ma ◽  
Yingbin Xiao ◽  
Qingjun Yang ◽  
Huali Kang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cardiac Function ◽  
Cardiac Remodeling ◽  
Heart Function ◽  
Pressure Overload ◽  
Tissue Growth ◽  
Mrna Levels ◽  
Expression Levels

Objective: To investigate the mechanism of Krüppel-like factor 15 (KLF15) in cardiac remodeling and interstitial fibrosis. Methods: A rat model was established by in vivo aortic coarctation followed by a period of pressure unloading and used to measure heart function, myocardial pathological changes, and KLF15, transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), and myocardin-related transcription factor A (MRTF-A) expression levels. In addition, cardiac fibroblasts were cultured in vitro and treated with KLF15-shRNA or KLF15 recombinant adenovirus to establish a TGF-β-mediated cardiac fibroblast hypertrophy model and analyze cell morphology, collagen secretion, and changes in the expression levels of 4 cytokines. Results: In vivo pressure overload impaired cardiac function and resulted in myocardial hypertrophy and fibrosis. These changes were accompanied by the downregulation of KLF15 mRNA levels and increased expression of the other factors. The response to unloading was the opposite. In in vitro cell experiments, by specifically targeting the KLF15 gene, changes in the expression levels of the 4 cytokines and the amounts of collagen I and III were observed. Conclusions: In myocardial remodeling processes induced by mechanical or metabolic factors, KLF15 regulates TGF-β, CTGF, and MRTF-A expression and can ameliorate or even reverse myocardial fibrosis and improve cardiac function.

Abstract 462: Cardiac-specific LMCD1/dyxin-knockout in Mice Blunts Pressure Overload-induced Activation of Calcineurin Signaling

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Lucia S Kilian ◽  
Jakob Voran ◽  
Nesrin Schmiedel ◽  
Katharina Stiebeling ◽  
Julika Richter ◽  
...  
Keyword(s):  
Protein Level ◽  
Heart Function ◽  
Pressure Overload ◽  
Protective Role ◽  
Left Ventricular ◽  
Cardiomyocyte Hypertrophy ◽  
Mrna Levels

We and others have shown that LMCD1 expression levels are upregulated in various in vitro and in vivo models of hypertrophy and that LMCD1 is necessary and sufficient to induce cardiomyocyte hypertrophy in vitro . We successfully generated a new mouse line with a conditional cardiac knockout of LMCD1. We performed echocardiographic, morphometric, and molecular analysis in these LMCD1-deficient and appropriate control-mice under basic conditions as well as 14 days after transverse aortic banding (TAC)-induced left ventricular (LV) pressure overload. Our aim was to investigate the hypothesis of potential beneficial effects of LMCD1-downregulation in vivo . These knockout (KO)-mice revealed under basic conditions a significant reduction of LMCD1 in the heart to <10% on protein level compared to control (WT)-mice (females and males n=5 each, p<0.001), while anatomic and functional parameters of the heart as well as LMCD1 levels in all other tested organs remained unchanged. Sham-operated KO-mice also showed significantly reduced level of LMCD1 in the LV compared to Sham-operated WT-mice (protein level <20%, p<0.001, n=8). No significant increase of LMCD1 in TAC- compared to Sham-operated KO-mice was found. TAC-operated KO-mice showed no significant differences in heart anatomy and function when compared to TAC-operated WT-mice. However, we determined a consistent trend toward improved heart function (ejection fraction and fractional shortening). Furthermore, TAC-operated KO-mice showed reduced activation of the fetal gene program in LV-tissue compared to TAC-operated WT-mice: mRNA levels of the hypertrophic markers NppA, NppB, and Rcan1-4 were all decreased (WT-TAC n=8 vs. KO-TAC n=10: NppA 8.5±2.0 vs. 5.1±1.5, p<0.05; NppB 1.9±0.2 vs. 1.7±0.3, p=0.093; Rcan1-4 6.0±0.2 vs. 3.2 vs. 0.7, p<0.05), suggesting a protective role of LMCD1-knockout. The reduction of calcineurin (CnA)-responsive Rcan1-4 specifically suggests a protective role of LMCD1-knockout in CnA-dependent signaling. Taken together, our preliminary data reveals protective effects of LMCD1-knockout against TAC-induced hypertrophic signaling. Ongoing experiments focus on effects of LMCD1-knockout on apoptosis and fibrosis and its role in Angiotensin-induced hypertrophy.

Connective tissue growth factor/CCN-2 is upregulated in epididymal and subcutaneous fat depots in a dietary-induced obesity model

2013 ◽  
Vol 304 (12) ◽  
pp. E1291-E1302 ◽  
Author(s):  
Joanne T. M. Tan ◽  
Susan V. McLennan ◽  
Paul F. Williams ◽  
Alireza Rezaeizadeh ◽  
Lisa W.-Y. Lo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Connective Tissue ◽  
Connective Tissue Growth Factor ◽  
Adipocyte Differentiation ◽  
Subcutaneous Fat ◽  
Tissue Growth ◽  
Mrna Levels ◽  
Fat Depots

Connective tissue growth factor (CTGF), also known as CCN-2, is a cysteine-rich secreted protein that is involved in a range of biological processes, including regulation of cell growth and differentiation. Our previous in vitro studies have shown that CCN-2 inhibits adipocyte differentiation, although whether CCN-2 is regulated in vivo in adipogenesis is undetermined and was investigated in this study. C57BL/6 male mice were fed either standard laboratory chow (ND) or a diet high in fat (HFD; 45% fat) for 15 or 24 wk. HFD animals that gained >5 g in weight (termed HFD-fat) were insulin resistant and were compared with HFD-fed animals, which failed to gain weight (termed HFD-lean). HFD-fat mice had significantly increased CCN-2 mRNA levels in both the subcutaneous and epididymal fat pads, whereas CCN-2 mRNA was not induced in the epididymal site in HFD-lean mice. Also in HFD-fed animals, epididymal CCN-2 mRNA correlated positively with key genes involved in adipocyte differentiation, adiponectin and PPARγ ( P < 0.001 and P < 0.002, respectively). Additionally, epididymal CCN-2 mRNA correlated positively with two markers of tissue turnover, PAI-1 in HFD-fat mice only and TIMP-1, but only in the HFD-lean mice. Collectively, these findings suggest that CCN-2 plays a role in adipocyte differentiation in vivo and thus in the pathogenesis of obesity linked with insulin resistance.

Combined Effects of Bone Marrow-Derived Mesenchymal Stem Cells and PLGA-PEG-PLGA Scaffolds on Repair of Articular Cartilage Defect

2013 ◽  
Vol 815 ◽  
pp. 345-349 ◽  
Author(s):  
Ching Wen Hsu ◽  
Ping Liu ◽  
Song Song Zhu ◽  
Feng Deng ◽  
Bi Zhang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Articular Cartilage ◽  
Growth Factor ◽  
Cartilage Defect ◽  
Cartilage Regeneration ◽  
Tissue Growth ◽  
Cartilage Defects

Here we reported a combined technique for articular cartilage repair, consisting of bone arrow mesenchymal stem cells (BMMSCs) and poly (dl-lactide-co-glycolide-b-ethylene glycol-b-dl-lactide-co-glycolide) (PLGA-PEG-PLGA) triblock copolymers carried with tissue growth factor (TGF-belat1). In the present study, BMMSCs seeded on PLGA-PEG-PLGA with were incubated in vitro, carried or not TGF-belta1, Then the effects of the composite on repair of cartilage defect were evaluated in rabbit knee joints in vivo. Full-thickness cartilage defects (diameter: 5 mm; depth: 3 mm) in the patellar groove were either left empty (n=18), implanted with BMMSCs/PLGA (n=18), TGF-belta1 modified BMMSCs/PLGA-PEG-PLGA. The defect area was examined grossly, histologically at 6, 24 weeks postoperatively. After implantation, the BMMSCs /PLGA-PEG-PLGA with TGF-belta1 group showed successful hyaline-like cartilage regeneration similar to normal cartilage, which was superior to the other groups using gross examination, qualitative and quantitative histology. These findings suggested that a combination of BMMSCs/PLGA-PEG-PLGA carried with tissue growth factor (TGF-belat1) may be an alternative treatment for large osteochondral defects in high loading sites.

Targeting RNF8 Effectively Reverse Cisplatin and Doxorubicin Resistance in Endometrial Cancer

2020 ◽  
Author(s):  
Ben Yang ◽  
Wang Ke ◽  
Yingchun Wan ◽  
Tao Li
Keyword(s):  
Endometrial Cancer ◽  
Cell Lines ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Levels ◽  
Mice Model ◽  
Expression Levels ◽  
Gynecological Malignancy ◽  
Ec Cells

Abstract Background Endometrial cancer (EC) is one of the most frequent gynecological malignancy worldwide. However, resistance to chemotherapy remains one of the major difficulties in the treatment of EC. Thus, there is an urgent requirement to understand mechanisms of chemoresistance and identify novel regimens for patients with EC. Methods Cisplatin and doxorubicin resistant cell lines were acquired by continuous exposing parental EC cells to cisplatin or doxorubicin for 3 months. Cell viability was determined by using MTT assay. Protein Expression levels of protein were examined by western blotting assay. mRNA levels were measured by quantitative polymerase chain reaction (qPCR) assay. Ring finger protein 8 (RNF8) knockout cell lines were generated by clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing assay. Nonhomologous end joining (NHEJ) efficiency were quantified by plasmid based NHEJ assay. DNA double strand breaks (DSB) were generated using laser micro-irradiation. Protein recruitment to DSB was analyzed by immunofluorescent assay. Tumor growth was examined by AN3CA xenograft mice model. Results We found that protein and mRNA expression levels of RNF8 were significantly increased in both cisplatin and doxorubicin resistant EC cells. Cell survival assay showed that RNF deficiency significantly enhanced the sensitivity of resistant EC cells to cisplatin and doxorubicin (P < 0.01). In addition, chemoresistant EC cells exhibited increased NHEJ efficiency. Knockout of RNF8 in chemoresistant EC cells significantly reduced NHEJ efficiency and prolonged Ku80 retention on DSB. Moreover, cisplatin resistant AN3CA xenograft showed that RNF8 deficiency overcame cisplatin resistance. Conclusions Our in vitro and in vivo assays provide evidence for RNF8, which is a NHEJ factor, serving as a promising, novel target in EC chemotherapy.

Vascularized Cardiac Spheroids as Novel 3D in vitro Models to Study Cardiac Fibrosis

2017 ◽  
Vol 204 (3-4) ◽  
pp. 191-198 ◽  
Author(s):  
Gemma A. Figtree ◽  
Kristen J. Bubb ◽  
Owen Tang ◽  
Eddy Kizana ◽  
Carmine Gentile
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Cardiac Fibrosis ◽  
Transforming Growth Factor ◽  
Three Dimensional ◽  
In Vitro Models ◽  
Tissue Growth ◽  
Cardiovascular Research

Spheroid cultures are among the most explored cellular biomaterials used in cardiovascular research, due to their improved integration of biochemical and physiological features of the heart in a defined architectural three-dimensional microenvironment when compared to monolayer cultures. To further explore the potential use of spheroid cultures for research, we engineered a novel in vitro model of the heart with vascularized cardiac spheroids (VCSs), by coculturing cardiac myocytes, endothelial cells, and fibroblasts isolated from dissociated rat neonatal hearts (aged 1-3 days) in hanging drop cultures. To evaluate the validity of VCSs in recapitulating pathophysiological processes typical of the in vivo heart, such as cardiac fibrosis, we then treated VCSs with transforming growth factor beta 1 (TGFβ1), a known profibrotic agent. Our mRNA analysis demonstrated that TGFβ1-treated VCSs present elevated levels of expression of connective tissue growth factor, fibronectin, and TGFβ1 when compared to control cultures. We demonstrated a dramatic increase in collagen deposition following TGFβ1 treatment in VCSs in the PicroSirius Red-stained sections. Doxorubicin, a renowned cardiotoxic and profibrotic agent, triggered apoptosis and disrupted vascular networks in VCSs. Taken together, our findings demonstrate that VCSs are a valid model for the study of the mechanisms involved in cardiac fibrosis, with the potential to be used to investigate novel mechanisms and therapeutics for treating and preventing cardiac fibrosis in vitro.

scholarly journals MicroRNA-27a targets Sfrp1 to induce renal fibrosis in diabetic nephropathy by activating Wnt/β-Catenin signalling

2020 ◽  
Vol 40 (6) ◽  
Author(s):  
MingJun Shi ◽  
PingPing Tian ◽  
ZhongQiang Liu ◽  
Fan Zhang ◽  
YingYing Zhang ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
High Glucose ◽  
Renal Fibrosis ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Expression Levels ◽  
Stage Renal Disease

Abstract Diabetic nephropathy (DN) commonly causes end-stage renal disease (ESRD). Increasing evidence indicates that abnormal miRNA expression is tightly associated with chronic kidney disease (CKD). This work aimed to investigate whether miR-27a can promote the occurrence of renal fibrosis in DN by suppressing the expression of secreted frizzled-related protein 1 (Sfrp1) to activate Wnt/β-catenin signalling. Therefore, we assessed the expression levels of miR-27a, Sfrp1, Wnt signalling components, and extracellular matrix (ECM)-related molecules in vitro and in vivo. Sfrp1 was significantly down-regulated in a high-glucose environment, while miR-27a levels were markedly increased. A luciferase reporter assay confirmed that miR-27a down-regulated Sfrp1 by binding to the 3′ untranslated region directly. Further, NRK-52E cells under high-glucose conditions underwent transfection with miR-27a mimic or the corresponding negative control, miR-27a inhibitor or the corresponding negative control, si-Sfrp1, or combined miR-27a inhibitor and si-Sfrp1. Immunoblotting and immunofluorescence were performed to assess the relative expression levels of Wnt/β-catenin signalling and ECM components. The mRNA levels of Sfrp1, miR-27a, and ECM-related molecules were also detected by quantitative real-time PCR (qPCR). We found that miR-27a inhibitor inactivated Wnt/β-catenin signalling and reduced ECM deposition. Conversely, Wnt/β-catenin signalling was activated, while ECM deposition was increased after transfection with si-Sfrp1. Interestingly, miR-27a inhibitor attenuated the effects of si-Sfrp1. We concluded that miR-27a down-regulated Sfrp1 and activated Wnt/β-catenin signalling to promote renal fibrosis.

Isolated hemoperfused heart model of slaughterhouse pigs

2001 ◽  
Vol 24 (4) ◽  
pp. 215-221 ◽  
Author(s):  
D. Modersohn ◽  
S. Eddicks ◽  
C. Grosse-Siestrup ◽  
I. Ast ◽  
S. Holinski ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Heart Function ◽  
Animal Experiments ◽  
Laboratory Animals ◽  
Coronary Perfusion ◽  
Balloon Catheters ◽  
Wide Range ◽  
Infusion Of Norepinephrine

A model of hemoperfused slaughterhouse pighearts is described providing a wide range of applications which leads to a reduction in animal experiments. The size of a pigheart, heart rate, coronary perfusion, metabolism, etc. are more comparable to conditions in patients than those in hearts of small laboratory animals. Global heart function can be assessed either by measuring stroke volume, ejection fraction, Emaxetc. in the working model or by measuring intraventricular pressure with balloon catheters in the isovolumetric model. Regional cardiac function can be measured by sonomicrometry and ischemic and non-ischemic areas can be compared. Local metabolic changes are measurable as well with microdialysis. Cardiac function can be kept on any given functional level by infusion of norepinephrine in spite of the fact that functional parameters are lower without adrenergic drive in vitro than in vivo. Stable heart function can be maintained for several hours with only 500 to 1000 ml of blood because the blood is permanently regenerated by a special dialysis system. This model can be applied in many research projects dealing with reperfusion injuries, inotropic, antiarrhythmic or arrhythmogenic effects of certain drugs, immunological rejection, evaluation of imaging systems (NMR, echocardiography etc.) or cardiac assist devices.

Abstract 117: Ponatinib Mediated Cardiotoxicity Is Driven By Pro-inflammatory S100A8/A9-NLRP3-IL-1β Signaling Circuit

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Anand P Singh ◽  
Sultan Tousif ◽  
Prachi Umbarkar ◽  
Cristi L Galindo ◽  
Nicholas Wheeler ◽  
...  
Keyword(s):  
Cardiac Function ◽  
High Fat Diet ◽  
Kinase Inhibitor ◽  
Clinical Investigation ◽  
Pressure Overload ◽  
Immunosuppressive Agent ◽  
Myelogenous Leukemia ◽  
High Fat

Background: Ponatinib is a third-generation tyrosine kinase inhibitor (TKI) for chronic myelogenous leukemia (CML) treatment. Of note, ponatinib is the only treatment option for CML patients with T315I (gatekeeper) mutation. Unexpected clinical cardiotoxicity, including fatal myocardial infarction and congestive heart failure, has hampered its clinical use. Herein, we aimed to investigate the cardiotoxic mechanism of ponatinib and strategies to prevent the cardiotoxic manifestations. Methods: We employed wild-type C57BL/6, cardiovascular (CV) comorbidity models e.g., transverse aortic constriction (TAC)-pressure overload (cardiac comorbidity) and high-fat diet fed ApoE -/- (vascular comorbidity), to investigate the cardiotoxic mechanism of ponatinib. Echocardiography was performed to assess cardiac function. Comprehensive immune profiling was performed to identify ponatinib-induced immune dynamics using flow cytometry analysis. Results: Echocardiographic assessment of ponatinib treated high-fat diet fed ApoE -/- and pressure overload (PO) murine model showed significant decline in cardiac function, suggesting the key role of CV-comorbidities in ponatinib-induced cardiomyopathy. An unbiased RNA-Seq analysis identified the enrichment of dysregulated inflammatory genes, including a multi-fold upregulation of alarmins S100A8/A9 as a top hit in ponatinib-treated hearts. A combination of in vitro and in vivo mechanistic analysis, identified that ponatinib activates the S100A8/9-TLR4-NLRP3-IL-1β signaling pathway in cardiac and systemic myeloid cells (monocytes and neutrophils), thereby leading to excessive myocardial and systemic inflammation. Finally, we demonstrate that ponatinib-induced excessive inflammation is central to the cardiac pathology because a broad immunosuppressive agent dexamethasone abolished the adverse cardiac remodeling and dysfunction of ponatinib treated hearts. Conclusions: These findings uncover a novel mechanism of ponatinib-induced cardiac inflammation leading to cardiac dysfunction. Our results provide critical preclinical data and rationale for clinical investigation into immunosuppressive interventions to mitigate ponatinib-induced cardiotoxicity.

Biological and Molecular Evidences for the Antitumor Potential of EGb 761 in Glioma Cells In Vitro and In Vivo

2021 ◽  
Vol 15 (5) ◽  
pp. 685-692
Author(s):  
Xin Sui ◽  
Jia Zhou ◽  
Yan Xu ◽  
Yuchen Wang ◽  
Guangfu Lv ◽  
...  
Keyword(s):  
Growth Factor ◽  
Caspase 3 ◽  
Glioma Cells ◽  
Signalling Pathway ◽  
Migration And Invasion ◽  
Egb 761 ◽  
Western Blots ◽  
Expression Levels

EGb 761, the standardized extract from the Ginkgo biloba leaves, has therapeutic effect on many diseases. However, its mechanisms on glioma remain to be fully established. This study aims to investigate the possible effects of EGb 761 on glioma cells, to explore its potential mechanism. The glioma cells SHG44 and U251 were used as materials, the proliferation, migration and invasion were assessed by the MTT, the scratch-wound and Transwell assays were performed respectively. Levels of insulin-like growth factor-1, Bcl-2, p53, Smad2/3, Bax, cleaved caspase-3 and p-Smad2/3 were determined by western blots. The development and progression of U251 glioma cell were measured in vivo, and the apoptosis was evaluated. The results showed that EGb 761 could inhibit the proliferation, migration, and invasion of SHG-44 and U251 cells in vitro. Meanwhile, the expression levels of IGF-1 and Bcl-2, and the transforming growth factor-β (TGF-β) signaling were inhibited. In contrast, the expression levels of p53, Bax, and cleaved caspase-3 were increased significantly. In conclusion, this study suggested that EGb 761 could suppress the growth of glioma cells in vitro and in vivo, possibly by inhibiting the TGF-β signalling pathway and activating the p53 signalling pathway.

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