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Huang H.,Sun Yat Sen University | Cao D.,Southern Medical University | Qin L.,Sun Yat Sen University | Qin L.,Key Laboratory on Assisted Circulation | And 4 more authors.
Molecular Pharmaceutics | Year: 2014

Numerous preclinical studies have demonstrated that polycation mediated gene delivery systems successfully achieved efficient gene transfer into cells and animal models. However, results of their clinical trials to date have been disappointing. That self-assembled gene and polycation systems should be stable undergoing dilution in the body is one of the prerequisites to ensuring efficiency of gene transfer in clinical trials, but it was neglected in most preclinical studies. In this account, we developed the dilution-stable PAMAM G1-grafted polyrotaxane (PPG1) supermolecules in which PAMAM G1-grafted α-cyclodextrins are threaded onto a PEG chain capped with hydrophobic adamantanamine. The PPG1/pDNA polyplex (approximate 100 nm in diameter) was very stable and kept its initial particle size and a uniform size distribution at ultrahigh dilution, whereas DNA/PEI 25K polyplex was above three times bigger at a 16-fold dilution than the initial size and their particle size distribution indicated multiple peaks mainly due to forming loose and noncompacted aggregates. PPG1 supermolecules showed significantly superior transfection efficiencies compared to either PEI 25K or Lipofectamine 2000 in most cell lines tested including normal cells (HEK293A) and cancer cells (Bel7402, HepG2, and HeLa). Furthermore, we found that the PPG1 supermolecules delivered DNA into HEK293A through a caveolae-dependent pathway but not a clathrin-dependent pathway as PEI 25K did. These findings raised the intriguing possibility that the caveolae-dependent pathway of PPG1 supermolecule/pDNA polyplex avoiding lysosomal degradation was attributed to their high transfection efficiency. The dilution-stable PPG1 supermolecule polyplex facilitating caveolae-dependent internalization has potential applications to surmount the challenges of high dilutions in the body and lysosomal degradation faced by most gene therapy clinical trials. © 2014 American Chemical Society.


Ma Y.,Sun Yat Sen University | Chen B.,Peoples Hospital of Guizhou Province | Liu D.,Sun Yat Sen University | Yang Y.,Sun Yat Sen University | And 4 more authors.
Biochemical Pharmacology | Year: 2011

Although MG132, a proteasome inhibitor, is suggested to impede secondary cardiac remodeling after hypertension, the mechanism and optimal duration of treatment remain unknown. This study was designed to investigate the effects and possible mechanism of MG132 on hypertension-induced cardiac remodeling. Male Sprague-Dawley rats subjected to abdominal aortic constriction (AAC) or sham operation received an intraperitoneal injection of MG132 (0.1 mg kg-1 day-1) or vehicle over a 2- or 8-week period. In the end, left ventricular (LV) function was evaluated with echocardiography and pressure tracing. Collagen deposition within the LV myocardium was assessed with Masson's trichrome staining. Ubiquitin-proteasome system (UPS), NF-κB, I-κB, TGFβ1 and Smad2 within the LV tissue were evaluated. In addition, angiotensin II within both plasma and LV tissue was also examined. Compared with the sham groups, the vehicle-treated AAC group exhibited a higher angiotensin II level, LV/body weight ratio, septal and posterior wall thicknesses, and a markedly reduced cardiac function (P < 0.05). Treatment with MG132 for 8 weeks attenuated these cardiac remodeling parameters and improved cardiac function (P < 0.01). 2- and 8-week hypertension led to activation of UPS, which was followed by activation of NF-κB and increased expression of TGFβ1 and Smad2 (P < 0.01). MG132 significantly inhibited NF-κB activity and down-regulate the levels of TGFβ1 and Smad2 expression by 2 and still at 8 weeks (P < 0.01). Short- and long-term treatment with MG132 significantly attenuated hypertension-induced cardiac remodeling and dysfunction, which may be mediated by the NF-κB/TGFβ1 signaling pathway. © 2011 Elsevier Inc. All rights reserved.


Ma Y.,Sun Yat Sen University | Chen Y.,Sun Yat Sen University | Yang Y.,Sun Yat Sen University | Chen B.,Peoples Hospital of Guizhou Province | And 5 more authors.
Biochemical Pharmacology | Year: 2013

Although the role of the ubiquitin-proteasome system (UPS) in cardiac hypertrophy induced by pressure overload has been consistently studied, the fundamental importance of the UPS in cardiac fibrosis has received much less attention. Our previous study found that proteasome inhibitor (MG132) treatment attenuated cardiac fibrosis and heart failure during the early and middle stages of pressure overload. However, the effects of this inhibitor on late-stage pressure overload hearts remain unclear and controversial. The present study was designed to investigate the effects and possible mechanisms of MG132 on cardiac fibrosis and dysfunction during the late stages of pressure overload. Male Sprague Dawley rats with abdominal aortic constriction (AAC) or a sham operation received an intraperitoneal injection of MG132 (0.1 mg kg-1 day -1) or vehicle for 16 weeks. Left ventricular (LV) function, collagen deposition and Ang II levels were evaluated at study termination. Ang II-stimulated adult rat cardiac fibroblasts were utilized to examine the effects of MG132 on collagen synthesis and the relationship between the renin-angiotensin-aldosterone system (RAAS) and the UPS. MG132 treatment attenuated ventricular dysfunction by suppressing cardiac fibrosis rather than inhibiting cardiac hypertrophy during the late-stages of pressure overload. We also found that Ang II activates UPS in the heart and MG132 attenuates Ang II-induced collagen synthesis via suppression of the NF-κB/TGF-β/ Smad2 signaling pathways. Proteasome inhibition therefore could provide a new promising therapeutic strategy to prevent cardiac fibrosis and progression of heart failure even during the late-stages of pressure overload. © 2012 Elsevier Inc.


Huang H.,Sun Yat Sen University | Ma R.,Jiangsu Jiankang Vocational College | Liu D.,Sun Yat Sen University | Liu C.,Sun Yat Sen University | And 4 more authors.
Disease Markers | Year: 2012

Objective: The purpose of the present study was to investigate the value of ox-LDL and oxidation ratio of LDL (ox-LDL/TC, ox-LDL/HDL-C and ox-LDL/LDL-C) in diagnosis and prognosis evaluation in CAD patients. Also, we aimed to observe the effect of statins on reducing level of ox-LDL and oxidation ratio of LDL, and explore whether statins still have similar effect on ox-LDL in a short period of therapy (within 2 weeks). Methods: Blood ox-LDL, TC, HDL-C, LDL-C, and TG were measured in cases with acute myocardial infarction (AMI, n=177), unstable angina pectoris (UAP, n=195), stable angina pectoris (SAP, n=228), normal control (n=120), and high risk control (n=140). Results: Mean value of ox-LDL and oxidation ratio of LDL was significantly higher in the CAD group than in the two control groups. The AUC of ROC curve of ox-LDL, ox-LDL/TC, ox-LDL/HDL-C, ox-LDL/LDL-C and apoA1/apoB were more than 0.50 (P < 0.001). Multivariate logistic regression analysis showed that age and ox-LDL/LDL-C related with short-term, while ox-LDL/LDL-C and ox-LDL/TC related with long-term prognosis (p < 0.05). Furthermore, after treatment with statins for 2 weeks, TC, LDL-C, ox-LDL, ox-LDL/TC, ox-LDL/HDL-C and ox-LDL/LDL-C decreased by 22%, 28%, 38%, 29%, 23% and 25% respectively. And the reduction of ox-LDL by statins is independent of lowering of LDL-C and TC. Conclusions: Ox-LDL and oxidation ratio of LDL are closely related with AS, and they are better biomarkers for discriminating between patients with coronary artery disease and healthy subjects. In addition, statins can decrease level of ox-LDL significantly, which is independent of lowering of LDL-C and TC. © 2012-IOS Press and the authors. All rights reserved.


Yang D.-Y.,Sun Yat Sen University | Yang D.-Y.,Key Laboratory on Assisted Circulation | Wu G.-F.,Sun Yat Sen University | Wu G.-F.,Key Laboratory on Assisted Circulation
International Journal of Cardiology | Year: 2013

A growing pool of evidence has shown that enhanced external counterpulsation (EECP) is a non-invasive, safe, low-cost, and highly beneficial therapy for patients with coronary artery disease. However, the exact mechanisms of benefit exerted by EECP therapy remain only partially understood. The favorable hemodynamic effects of EECP were previously considered as the primary mechanism of action. Nevertheless, recent advances have shed light on the shear stress-increasing effects of EECP which are vasculoprotective and anti-atherosclerotic. EECP-induced endothelial shear stress increase may lead to improvement in endothelial function and morphology, attenuation of oxidative stress and inflammation, and promotion of angiogenesis and vasculogenesis. This review summarizes evidence of the potential mechanisms contributing to the immediate and long-term benefits of EECP, from the perspective of its shear stress-increasing effects. © 2012 Elsevier Ireland Ltd.


Chen K.,Sun Yat Sen University | Fan W.,Sun Yat Sen University | Wang X.,Sun Yat Sen University | Ke X.,Sun Yat Sen University | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2012

Shear stress associated with blood flow plays an important role in regulating gene expression and cell function in endothelial cells (ECs). MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that negatively regulate the expression of target genes by binding to the mRNA 3'-untranslated region (3'UTR) at the posttranscriptional level involved in diverse cellular processes. This study demonstrates that microRNA-101 in response to laminar shear stress (LSS) is involved in the flow regulation of gene expression in ECs. qRT-PCR analysis showed that miR-101 expression was significantly upregulated in human umbilical vein endothelial cells (HUVECs) exposed to 12dyn/cm2 laminar shear stress for 12h. We found that transfection of miR-101 significantly decreased the luciferase activity of plasmid reporter containing the 3'UTR of mammalian target of rapamycin (mTOR) gene. Western analysis revealed that the protein level of mTOR was significantly reduced in ECs transfected with miR-101. Furthermore, miR-101 overexpression induced cell cycle arrest at the G1/S transition and suppressed endothelial cell proliferation. Finally, transfection of miR-101 inhibitors attenuated the suppressive effects of LSS on mTOR expression, which identified the efficacy of loss-of-function of miR-101 in laminar flow-treated ECs. In conclusion, we have demonstrated that upregulation of miR-101 in response to LSS contributes to the suppressive effects of LSS on mTOR expression and EC proliferation. These studies advance our understanding of the posttranscriptional mechanisms by which shear stress modulates endothelial homeostasis. © 2012 Elsevier Inc.


Cao D.,Southern Medical University | Tian S.,Sun Yat Sen University | Huang H.,Sun Yat Sen University | Huang H.,Key Laboratory on Assisted Circulation | And 2 more authors.
Molecular Pharmaceutics | Year: 2015

The stability and targeting ability of nanocarrier gene delivery systems are necessary conditions to ensure the good therapeutic effect and low nonspecific toxicity of cancer treatment. Poly(ethylene glycol) (PEG) has been widely applied for improving stability and as a spacer for linking ligands and nanocarriers to improve targetability. However, the cellular uptake and endosomal escape capacity of nanocarriers has been seriously harmed due to the introduction of PEG. In the present study, we synthesized a new gene delivery vector by coupling divalent folate-PEG (PEG3.4k-FA2) onto polyamidoamine-polyethylenimine (PME) copolymer (PME-(PEG3.4k-FA2)1.72). Both PEG and monovalent folate-PEG (PEG3.4k-FA1) modified PME were prepared as control polymers, which were named as PME-(PEG3.5k)1.69 and PME-(PEG3.4k-FA1)1.66, respectively. PME-(PEG3.4k-FA2)1.72 exhibited strong DNA condensation capacity like parent polymer PME which was not significantly influenced by PEG. PME-(PEG3.4k-FA2)1.72/DNA complexes at N/P = 10 had a diameter ∼143 nm and zeta potential ∼13 mV and showed the lowest cytotoxicity and hemolysis and the highest transfection efficiency among all tested polymers. In folate receptor positive (FR-positive) cells, the cellular uptake and transfection efficiency were increased with the increase in the number of folates coupled on PEG; the order was PME-(PEG3.4k-FA2)1.72 > PME-(PEG3.4k-FA1)1.66 > PME-(PEG3.5k)1.69. Folate competition assays showed that PME-(PEG3.4k-FA2)1.72 complexes had stronger targeting ability than PME-(PEG3.5k)1.69 and PME-(PEG3.4k-FA1)1.66 complexes due to their higher folate density per PEG molecule. Cellular uptake mechanism study showed that the folate density on PEG could change the endocytosis pathway of PME-(PEG3.5k)1.69 from clathrin-mediated endocytosis to caveolae-mediated endocytosis, leading to less lysosomal degradation. Distribution and uptake in 3D multicellular spheroid assays showed that divalent folate could offer PME-(PEG3.4k-FA2)1.72 complexes stronger penetrating ability and higher cellular uptake. With these advantages, PME-(PEG3.4k-FA2)1.72 may be a promising nonviral vector candidate for efficient gene delivery. This study also indicates that divalent folate modification on PEG can serve as an efficient strategy to improve the cellular uptake and targeting ability of PEGylated cationic polymers for gene delivery. © 2014 American Chemical Society.


Fan W.,Sun Yat Sen University | Fan W.,Key Laboratory on Assisted Circulation | Fang R.,Sun Yat Sen University | Fang R.,Key Laboratory on Assisted Circulation | And 11 more authors.
Journal of Cell Science | Year: 2015

Although many studies have described the roles of microRNAs (miRNAs) in the modulation of the endothelial response to shear stress, the mechanisms remain incompletely understood. Here, we demonstrate that miR-34a expression in endothelial cells was downregulated by atheroprotective physiological high shear stress (HSS), whereas it was upregulated by atheroprone oscillatory shear stress (OSS). Blockade of endogenous miR-34a dramatically decreased basal vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein expression levels. Conversely, miR-34a overexpression increased the protein levels of VCAM-1 and ICAM-1, consequently promoting monocyte adhesion to endothelial cells. Furthermore, miR-34a overexpression attenuated HSS-mediated suppression of VCAM-1 protein expression on endothelial cells, but promoted HSS-induced ICAM- 1 expression. In addition, the OSS induction of endothelial cell VCAM-1 and ICAM-1 was suppressed by using an miR-34a inhibitor, which led to a reduction of monocyte adhesion to endothelial cells. Mechanistically, sirtuin 1 overexpression partially prevented miR-34a-induced VCAM-1 and ICAM-1 expression. Subsequent investigation demonstrated that miR-34a increased nuclear factor kB (NF-κB) p65 subunit (also known as RelA) acetylation (on residue Lys310), and silencing NF-κB signaling reduced miR-34a-induced VCAM-1 and ICAM-1 protein expression. These results demonstrate that miR-34a is involved in the flowdependent regulation of endothelial inflammation. © 2015. Published by The Company of Biologists Ltd.


Lu G.,Sun Yat Sen University | Lu G.,Key Laboratory on Assisted Circulation | Xu S.,Sun Yat Sen University | Xu S.,Key Laboratory on Assisted Circulation | And 4 more authors.
Biochemical and Biophysical Research Communications | Year: 2014

Kv1.5 potassium channel represents a promising target for atrial fibrillation (AF) therapy. During AF, the renin-angiotensin system is markedly activated. Recent evidence indicates that angiotensin II (Ang II) can upregulate Kv1.5 channel, but the mechanism remains unknown. In this study, we report that Ang II-mediated transforming growth factor-beta1 (TGF-β1)/Smad2/3 and extracellular signal-regulated kinase (ERK) 1/2 signalings are involved in atrial Kv1.5 expression. In neonatal rat atrial myocytes, quantitative PCR and Western blotting revealed that Ang II upregulated TGF-β1, synapse-associated protein 97 (SAP97) and Kv1.5 expression in a time- and concentration-dependent manner. The Ang II-induced upregulation of Kv1.5, SAP97 and phosphorylated Smad2/3 (P-Smad2/3) were reversed by the Ang II type 1 (AT1) receptor antagonist losartan, an anti-TGF-β1 antibody and the ERK 1/2 inhibitor PD98059 but not by the AT2 receptor antagonist PD123319. mRNA knockdown of either Smad2 or Smad3 blocked Ang II-induced expression of Kv1.5 and SAP97. These data suggest that AT1 receptor/TGF-β1/P-Smad2/3 and ERK 1/2 signalings are involved in Ang II-induced Kv1.5 and SAP97 expression. Flow cytometry and Western blotting revealed that losartan and the anti-TGF-β1 antibody diminished Ang II-induced reactive oxygen species (ROS) generation and that the antioxidants diphenyleneiodonium and N-acetyl cysteine inhibited Ang II-induced expression of P-Smad2/3, phosphorylated ERK (P-ERK) 1/2, Kv1.5, SAP97, suggesting that ROS participate in Kv1.5 and SAP97 regulation by modulating Ang II-induced P-Smad2/3 and P-ERK 1/2 expression. In conclusion, we demonstrate that ROS-dependent Ang II/AT1 receptor/TGF-β1/P-Smad2/3 and Ang II/ERK 1/2 signalings are involved in atrial Kv1.5 and SAP97 expression. Antioxidants would be beneficial for AF treatment through inhibiting atrial Kv1.5 expression. © 2014 Elsevier Inc. All rights reserved.


Wu X.-Y.,Sun Yat Sen University | Fan W.-D.,Sun Yat Sen University | Fang R.,Key Laboratory on Assisted Circulation | Wu G.-F.,Key Laboratory on Assisted Circulation | Wu G.-F.,Guangdong Medical College
Journal of Cellular Biochemistry | Year: 2014

Increasing evidences have illuminated the fundamental role of inflammation in mediating all stages of atherosclerosis. miR-155, a typical multi-functional miRNA, has recently emerged as a novel component of inflammatory signal transduction in the pathogenesis of atherosclerosis. However, little is known about whether endothelial highly expressed miR-155 can regulate endothelial inflammation-related transcription factors and the predicted role of miR-155 as a negative feedback regulator in endothelial inflammation involved in atherosclerosis. Bioinformatics analysis showed that RELA (nuclear factor-κB p65) is a potential target gene of miR-155 and this was confirmed by a luciferase reporter assay. Our results show that microRNA-155 mediate endothelial inflammation and decrease NFCyrillic small letter kaB p65 and adhesion molecule expression in TNFα-stimulated endothelial cells. Transfection with miR-155 significantly inhibited TNFα-induced monocyte adhesion to endothelium. Inhibition of miR-155 enhanced p65 level and endothelial inflammatory response which was counteracted through the depletion of P65 by Si-P65. On the other hand, knockdown of eNOS, another target of miR-155, while transfecting with miR-155 inhibitor resulted in more significant inflammatory response. miR-155 is highly expressed in TNFα treated HUVECs, deprived of endogenous p65 could reverse TNFα-induced upregulation of miR-155. Thus, TNFα induced miR-155 may serve as a negative feedback regulator in endothelial inflammation involved in atherosclerosis by targeting nuclear transcription factor P65. These results provide a rationale for intervention of intracellular microRNA as possible anti-atherosclerotic targets. J. Cell. Biochem. 115: 1928-1936, 2014. © 2014 Wiley Periodicals, Inc.

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