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Lee T.-M.,Tainan Municipal An Nan Hospital China Medical University | Lee T.-M.,China Medical University at Taichung | Lee T.-M.,Taipei Medical University | Lin S.-Z.,China Medical University at Taichung | And 4 more authors.
PLoS ONE | Year: 2013

Cardiac remodeling was shown to be associated with reduced gap junction expression after myocardial infarction. A reduction in gap junctional proteins between myocytes may trigger ventricular arrhythmia. Therefore, we investigated whether N-acetylcysteine exerted antiarrhythmic effect by preserving connexin43 expression in postinfarcted rats, focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). Male Wistar rats after ligating coronary artery were randomized to either vehicle, or N-acetylcysteine for 4 weeks starting 24 hours after operation. Infarct size was similar between two groups. Compared with vehicle, cAMP levels were increased by N-acetylcysteine treatment after infarction. Myocardial connexin43 expression was significantly decreased in vehicle-treated infarcted rats compared with sham operated rats. Attenuated connexin43 expression and function were blunted after administering N-acetylcysteine, assessed by immunofluorescent analysis, dye coupling, Western blotting, and real-time quantitative RT-PCR of connexin43. Arrhythmic scores during programmed stimulation in the N-acetylcysteine-treated rats were significantly lower than those treated with vehicle. In an ex vivo study, enhanced connexin43 levels afforded by N-acetylcysteine were partially blocked by either H-89 (a PKA inhibitor) or brefeldin A (an Epac-signaling inhibitor) and completely blocked when H-89 and brefeldin A were given in combination. Addition of either the PKA specific activator N6Bz or Epac specific activator 8-CPT did not have additional increased connexin43 levels compared with rats treated with lithium chloride alone. These findings suggest that N-acetylcysteine protects ventricular arrhythmias by attenuating reduced connexin43 expression and function via both PKA- and Epac-dependent pathways, which converge through the inactivation of glycogen synthase kinase-3β. © 2013 Lee et al.


Lee T.-M.,Tainan Municipal An Nan Hospital China Medical University | Lee T.-M.,Taipei Medical University | Lin S.-Z.,Tainan Municipal An Nan Hospital China Medical University | Lin S.-Z.,China Medical University at Taichung | And 4 more authors.
Circulation Journal | Year: 2014

Background: The effect of phosphodiesterase-3 (PDE-3) inhibitors on arrhythmia remains controversial, so the purpose of this study was to determine their differential effects on sympathetic hyperinnervation and the involved mechanisms in a rat model of myocardial infarction. Methods and Results: After ligating the coronary artery, male Wistar rats were randomized to cilostazol or milrinone, chemically unrelated inhibitors of PDE-3, or vehicle for 4 weeks. The postinfarction period was associated with increased myocardial norepinephrine levels and oxidant release, as measured by myocardial superoxide level and dihydroethidine fluorescence staining. Infarcted rats in the milrinone- and cilostazol-treated groups had favorable ventricular remodeling with similar potency. Compared with milrinone, cilostazol significantly increased interstitial adenosine levels and reduced the production of myocardial cAMP and superoxide. Cilostazol significantly blunted sympathetic hyperinnervation, as assessed by immunofluorescent analysis of sympathetic innervation, and western blotting and real-time quantitative RT-PCR of nerve growth factor. Furthermore, the inhibitory effect of cilostazol on nerve growth factor was reversed by 8-cyclopentyl-1,3-dipropylxanthine, a selective A1 receptor antagonist, and enhanced by tempol administration. In spite of similar arrhythmic vulnerability during programmed stimulation in both the vehicle- and cilostazol-treated groups, cilostazol did not have proarrhythmic effects compared with milrinone. Conclusions: Unlike milrinone, cilostazol has therapeutic neutrality in arrhythmias because of adenosine uptake inhibition, which antagonizes the PDE-3-induced increase of sympathetic reinnervation via mediation of an adenosine A1 receptor-mediated antioxidation.


Lee T.-M.,China Medical University An Nan Hospital | Lee T.-M.,China Medical University at Taichung | Lee T.-M.,Taipei Medical University | Lin S.-Z.,China Medical University at Taichung | And 4 more authors.
Free Radical Biology and Medicine | Year: 2014

Glycogen synthase kinase-3 (GSK-3) signaling has been shown to play a role in the regulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of antioxidant genes, including heme oxygenase-1 (HO-1). We assessed whether lithium, a GSK-3 inhibitor, attenuates cardiac sympathetic reinnervation after myocardial infarction, a status of high reactive oxygen species (ROS), by attenuating nerve growth factor (NGF) expression and whether Nrf2/HO-1 signaling is involved in the protection. Twenty-four hours after ligation of the left anterior descending artery, male Wistar rats were treated for 4 weeks. The postinfarction period was associated with increased oxidative-nitrosative stress, as measured by myocardial superoxide, nitrotyrosine, and dihydroethidium fluorescent staining. In concert, myocardial norepinephrine levels and immunohistochemical analysis of sympathetic nerve revealed a significant increase in innervation in vehicle-treated rats compared with sham-operated rats. Arrhythmic scores during programmed stimulation in the vehicle-treated rats were significantly higher than those in sham. This was paralleled by a significant upregulation of NGF protein and mRNA in the vehicle-treated rats, which was reduced after administration of LiCl. LiCl stimulated the nuclear translocation of Nrf2 and the transactivation of the Nrf2 target gene HO-1. Inhibition of phosphoinositide 3-kinase by wortmannin reduced the increase in Nrf2 nucleus translocation and HO-1 expression compared with lithium alone. In addition, the lithium-attenuated NGF levels were reversed in the presence of the Nrf2 inhibitor trigonelline, HO-1 inhibitor SnPP, and peroxynitrite generator SIN-1, indicating the role of Nrf2/HO-1/ROS. In conclusion, lithium protects against ventricular arrhythmias by attenuating NGF-induced sympathetic innervation via antioxidant activation of the Nrf2/HO-1 axis. © 2014 Elsevier B.V. All rights reserved.


Lee T.-M.,Tainan Municipal An Nan Hospital China Medical University | Lee T.-M.,China Medical University at Taichung | Chung T.-H.,Fu Jen Catholic University | Lin S.-Z.,China Medical University Beigan Hospital | And 3 more authors.
Journal of Hypertension | Year: 2014

PURPOSE OF REVIEW:: Excessive production of fibrosis is a feature of hypertension-induced renal injury. Activation of RhoA/Rho-kinase (ROCK) axis has been shown in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We assessed whether selective endothelin receptor blockers can attenuate renal fibrosis by inhibiting RhoA/ROCK axis in DOCA-salt rats. METHODS:: At 4 weeks after the start of DOCA-salt treatment and uninephrectomization, male Wistar rats were randomized into three groups for 4 weeks: vehicle, ABT-627 (endothelin-A receptor inhibitor) and A192621 (endothelin-B receptor inhibitor). RESULTS:: DOCA-salt was characterized by increased blood pressure, decreased renal function, increased proteinuria, increased glomerulosclerosis and tubulointerstitial fibrosis with myofibroblast accumulation, increased renal endothelin-1 levels and RhoA activity along with increased expression of connective tissue growth factor at both mRNA and protein levels as compared with uninephrectomized control male Wistar rats. Treatment with a selective mineralocorticoid receptor antagonist, eplerenone, ameliorated proteinuria. Impaired renal function and histological changes were overcome by treatment with ABT-627, but not with A192621. The beneficial effects of bosentan, a nonspecific endothelin receptor blocker, on proteinuria, RhoA activity, and connective tissue growth factor levels were similar to ABT-627. Furthermore, in an isolated perfuse kidney, a RhoA inhibitor, C3 exoenzyme, and two ROCK inhibitors, fasudil and Y-27632, significantly attenuated connective tissue growth factor levels. CONCLUSIONS:: These results indicate that DOCA-salt elevates renal endothelin-1 levels and RhoA activity via activation of mineralocorticoid receptor, resulting in renal fibrosis and proteinuria. Endothelin-A receptor blockade can attenuate DOCA-salt-induced renal fibrosis probably through the inhibition of RhoA/ROCK activity and connective tissue growth factor expression. Copyright © Lippincott Williams & Wilkins.


Lee T.-M.,China Medical University An Nan Hospital | Lee T.-M.,China Medical University at Taichung | Lee T.-M.,Taipei Medical University | Lin S.-Z.,China Medical University at Taichung | And 4 more authors.
Journal of Cellular and Molecular Medicine | Year: 2014

Clinical and experimental studies have established that gender is a factor in the development of ventricular hypertrophy. We investigated whether the attenuated hypertrophic effect of oestradiol was via activation of phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) through non-genomic action. Twenty-four hours after coronary ligation, female Wistar rats were randomized into control, subcutaneous oestradiol treatment or a G-protein coupled oestrogen receptor (GPER) agonist, G-1 and treated for 4 weeks starting from 2 weeks after bilateral ovariectomy. Ventricular hypertrophy assessed by cardiomyocyte size after infarction was similarly attenuated by oestradiol or G-1 in infarcted rats. The phosphorylation of Akt and eNOS was significantly decreased in infarcted rats and restored by oestradiol and G-1, implying the GPER pathway in this process. Oestradiol-induced Akt phosphorylation was not abrogated by G-15 (a GPER blocker). Akt activation was not inhibited by actinomycin D. When a membrane-impermeable oestrogen-albumin construct was applied, similar responses in terms of eNOS activation to those of oestradiol were achieved. Furthermore, PPT, an ERα receptor agonist, activated the phosphorylation of Akt and eNOS. Thus, membrane ERα receptor played a role in mediating the phosphorylation of Akt and eNOS. The specific PI3K inhibitor, LY290042, completely abolished Akt activation and eNOS phosphorylation in infarcted hearts treated with either oestradiol or oestradiol + G-15. These data support the conclusions that oestradiol improves ventricular remodelling by both GPER- and membrane-bound ERα-dependent mechanisms that converge into the PI3K/Akt/eNOS pathway, unveiling a novel mechanism by which oestradiol regulates pathological cardiomyocyte growth after infarction. © 2014 The Authors.


Chen K.-C.,China Medical University at Taichung | Sun M.-F.,China Medical University at Taichung | Yang S.-C.,China Medical University at Taichung | Chang S.-S.,China Medical University at Taichung | And 7 more authors.
Chemical Biology and Drug Design | Year: 2011

Microsomal prostaglandin E synthase-1 (mPGES-1) is the key enzyme for prostaglandin E2 (PGE2) generation during inflammation and is a potential target for designing anti-inflammatory drugs. Potential inhibitors of m-PGES-1 were selected from traditional Chinese medicine (TCM Database@Taiwan) based on the pharmacophore map generated by the top HypoGen hypothesis and validated using structure- and ligand-based analysis. Key features for potential m-PGES-1 inhibitors include pi-interactions and H-bond donors. TCM compounds, shanciol B, shanciol A, castilliferol, and aurantiamide acetate, contoured to the quantitative structure-activity relationship pharmacophore and exhibited high docking scores and binding stability with m-PGES-1. Bioactivity models multiple linear regression (MLR) and support vector machine also supported activity predictions for the candidate compounds. Our results indicate that the investigated TCM compounds could be of use for development into mPGES-1 inhibitors. Traditional Chinese medicine compounds that have activity as mPGES-1 inhibitors were identified and validated using structure-based and ligand-based methods in addition to pharmacophore mapping. © 2011 John Wiley & Sons A/S.


Li Y.-S.,National Dong Hwa University | Harn H.-J.,China Medical University at Taichung | Hsieh D.-K.,Chaoyang University of Technology | Wen T.-C.,National Dong Hwa University | And 5 more authors.
Cell Transplantation | Year: 2013

Liver transplantation is currently the most efficacious treatment for end-stage liver diseases. However, one main problem with liver transplantation is the limited number of donor organs that are available. Therefore, liver tissue engineering based on cell transplantation that combines materials to mimic the liver is under investigation with the goal of restoring normal liver functions. Tissue engineering aims to mimic the interactions among cells with a scaffold. Particular materials or a matrix serve as a scaffold and provide a three-dimensional environment for cell proliferation and interaction. Moreover, the scaffold plays a role in regulating cell maturation and function via these interactions. In cultures of hepatic lineage cells, regulation of cell proliferation and specific function using biocompatible synthetic, biodegradable bioderived matrices, protein-coated materials, surface-modified nanofibers, and decellularized biomatrix has been demonstrated. Furthermore, beneficial effects of addition of growth factor cocktails to a flow bioreactor or coculture system on cell viability and function have been observed. In addition, a system for growing stem cells, liver progenitor cells, and primary hepatocytes for transplantation into animal models was developed, which produces hepatic lineage cells that are functional and that show long-term proliferation following transplantation. The major limitation of cells proliferated with matrix-based transplantation systems is the high initial cell loss and dysfunction, which may be due to the absence of blood flow and the changes in nutrients. Thus, the development of vascular-like scaffold structures, the formation of functional bile ducts, and the maintenance of complex metabolic functions remain as major problems in hepatic tissue engineering and will need to be addressed to enable further advances toward clinical applications. © 2013 Cognizant Comm. Corp.


Liu S.-P.,China Medical University at Taichung | Fu R.-H.,China Medical University at Taichung | Wu D.-C.,China Medical University at Taichung | Hsu C.-Y.,China Medical University at Taichung | And 8 more authors.
Stem Cells and Development | Year: 2014

Induced pluripotent stem (iPS) cells are considered as having the greatest potential for use in cell-based therapies. However, at least two hurdles remain: integrating viral transgenes and introducing the c-Myc and Klf4 oncogenes. In a previous study, fibroblasts were incapable of generating iPS cells in the absence of both oncogenes and viral infection. For the present study, we tested our hypothesis that iPS cells can be generated without oncogenes and viral infection under hypoxic conditions and used for cell therapies. By avoiding oncogenic factors and virus integration, this strategy would decrease the potential for cancer formation. According to our observations, the repeated transfection of two expression plasmids (Oct4 and Sox2) into mouse embryonic fibroblasts (MEFs) and combined hypoxic condition resulted in the generation of a novel iPS cell. At 6 h post-transfection, MEFs were subjected to hypoxic conditions (3% O2) for 24 h; this procedure was repeated four times. The resulting MEFs were seeded on feeder cells on day 9; iPS cell clones were observed 12 days post-seeding and designated as iPS-OSH. Data for cell morphology, stem cell marker staining, gene expression profiles, and embryonic body, teratoma, and chimeric mouse formation indicated iPS-OSH pluripotent capability. Neural precursor cells differentiated from iPS-OSH cells were used to treat an ischemic stroke mouse model; results from a behavior analysis indicate that the therapeutic group surpassed the control group. Further, iPS-OSH-derived neural precursor cells differentiated into neurons and astrocytes in mouse stroke brains. In conclusion, we generated a novel iPS-OSH in the absence of viral infection and oncogenic factors and could use it for ischemic stroke therapy. © Copyright 2014, Mary Ann Liebert, Inc. 2014.


Cho D.-Y.,China Medical University at Taichung | Lin S.-Z.,China Medical University at Taichung | Lin S.-Z.,China Medical University Beigan Hospital | Yang W.-K.,China Medical University at Taichung | And 7 more authors.
Cell Transplantation | Year: 2013

Cancer stem cells (CSCs) in glioblastoma multiforme (GBM) are radioresistant and chemoresistant, which eventually results in tumor recurrence. Targeting CSCs for treatment is the most crucial issue. There are five methods for targeting the CSCs of GBM. One is to develop a new chemotherapeutic agent specific to CSCs. A second is to use a radiosensitizer to enhance the radiotherapy effect on CSCs. A third is to use immune cells to attack the CSCs. In a fourth method, an agent is used to promote CSCs to differentiate into normal cells. Finally, ongoing gene therapy may be helpful. New therapeutic agents for targeting a signal pathway, such as epidermal growth factor (EGF) and vascular epidermal growth factor (VEGF) or protein kinase inhibitors, have been used for GBM but for CSCs the effects still require further evaluation. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as cyclooxygenase-2 (Cox-2) inhibitors have proven to be effective for increasing radiation sensitivity of CSCs in culture. Autologous dendritic cells (DCs) are one of the promising immunotherapeutic agents in clinical trials and may provide another innovative method for eradication of CSCs. Bone-morphogenetic protein 4 (BMP4) is an agent used to induce CSCs to differentiate into normal glial cells. Research on gene therapy by viral vector is also being carried out in clinical trials. Targeting CSCs by eliminating the GBM tumor may provide an innovative way to reduce tumor recurrence by providing a synergistic effect with conventional treatment. The combination of conventional surgery, chemotherapy, and radiotherapy with stem cell-orientated therapy may provide a new promising treatment for reducing GBM recurrence and improving the survival rate. © 2013 Cognizant Comm. Corp.


PubMed | Anhui Medical University, China Medical University at Taichung and China Medical University Beigan Hospital
Type: Journal Article | Journal: The Chinese journal of physiology | Year: 2016

The incidence of osteoporosis has increased among the elderly population. Establishing a model of bone remodeling for screening new drugs is critical to identify safe and effective treatments for osteoporosis. In this study, we established a platform to investigate the therapeutic effects of collagenous peptides extracted from scales of two kinds of fish, namely, sparidae and chanos. These peptides were prepared using seven concentrations of collagenous peptide: 100, 80, 60, 40, 20, 10 and 1 mg/ml. Experimental results indicated that collagenous peptides promoted the proliferation of osteoblasts and inhibited the proliferation of mature osteoclasts; the effective concentration of collagenous peptide-sparidae was 10 mg/ml and that of collagenous peptide-chanos was 40 mg/ml. These findings demonstrate that, to a certain extent, collagenous peptides extracted from fish scales can be used to prevent osteoporosis to assist bone remodeling.

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