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Small Interfering RNA-mediated Caveolin-1 Knockout on plasminogen activator inhibitor-1 Expression in Insulin-stimulated Human Vascular Endothelial Cells

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Acta Biochimica et Biophysica Sinica 2007年第3期39卷 224-233页

作者： Huiling YANG~(1,3#) Shuya HE~(2,3#) Zhihua QUAN Weixia PENG~1 Bin YAN~1 Jianghua LIU~1 Fang WEN~1 Renxian CAO~1 Yangyan XU~3 Gebo WEN~1 Weixin HU~2 1 Institute of Clinic Medicine,the First Affiliated Hospital of Nanhua University,Hengyang 421001,China 2 Research Institute of Biochemistry and Molecular Biology.Central South University Changsha 421001,China 3 Institute of Pharmacology and Pharmacy,Nanhua University,Hengyang 421001,China Institute of Clinic Medicine the First Affiliated Hospital of Nanhua UniversityHengyang 421001 China Institute of Pharmacology and Pharmacy Nanhua UniversityHengyang 421001 China Research Institute of Biochemistry and Molecular Biology Central South UniversityChangsha 421001 China

Using human vascular endothelial cells（ECV304）as the target,we studied the effect ofcaveolin（CAV）-1 in the course of insulin-stimulated expression of plasminogen activator inhibitor（PAl）-1.The appropriate single-stranded oligonucleotides representing the RNAi CAV- 1 gene were analyzed by Ambionsoftware.After annealing to generate double-stranded oligonucleotides（ds oligo）,it was cloned into thepENTR/U6 entry vector containing RNA polymerase Ⅲ expression element by T4 DNA ligase.The shorthairpin（shRNA）sequences transferred from the pENTR/U6 entry were cloned into the pLenti6/BLOCK-iT-DEST vector with an LR recombination reaction.After identification by sequencing,we successfully con-structed the CAV-1 RNAi lentiviral expression system using Gateway technology.Silencing efficiency wasassayed by real-time reverse transcription-polymerase chain reaction,immunofluorescence staining and Westernblotting.ECV304 cells were cultured in the medium containing different concentrations of insulin(1×10-9to1×10-7M)with the CAV-1 gene silenced or not.The expression level and subcellular localization of PAI-1 andCAV-1 were compared using reverse transcription-polymerase chain reaction,immunofluorescence stainingand Western blot assay.The results showed that the potent inhibition of CAV-1 expression could reach 85%,and it was specific to the CAV-1-derived shRNA,not the S 100A13-derived shRNA.There was no dramaticdifference in PAI-1 expression between the RNAi+and RNAi-ECV304 cells incubated with physiologicalinsulin,but PAI-1 protein did accumulate under the cell membrane.As the concentration of insulin increased,the expression of PAI-1 was up-regulated,whereas the expression of CAV-1 attenuated.Furthermore,PAI-1 clearly augmented after CAV-1 knockdown.These results indicated that hyperinsulinism could promotePAI-1 expression by inhibiting CAV-1,and stabilizing or up-regulating CAV-1 expression in endothelial cellsmight reduce complications of the great vessels and capillary vessels in diabetes.

关键词： caveolin-1 plasminogen activator inhibitor-1 gene silencing insulin

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Effects of simvastatin on cigarette smoke extract induced tissue-type plasminogen activator and plasminogen activator inhibitor-1 expression in human umbilical vein endothelial cells

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Chinese Medical Journal 2009年第19期122卷 2380-2385页

作者： HU Xiao-yun MA Yu-hui WANG Chen YANG Yuan-hua Department of Respiration Medicine First Hospital Shanxi Medical University Taiyuan Shanxi 030001 China Department of Respiratory Medicine Beijing Chaoyang Hospital Beijing Institute of Respiratory Medicine Capital Medical University Beijing 100020 China

Background Cigarette smoking has an influence on both arterial-type and venous-type thrombosis. However, little is known about the direct effect of cigarette smoke extract （CSE） on fibrinolytic activity of human umbilical vein endothelial cells （HUVECs）. Most recently, simvastatin has been marked in its effect on endothelial cells protection and anticoagulation. In this study, the effect of CSE on the expression of tissue-type plasminogen activator （t-PA） and plasminogen activator inhibitor-l（PAl-1） in HUVECs was addressed. The role of simvastatin in CSE-induced fibrinolytic activity changes was investigated as well. Methods The fourth to fifth generation of HUVECs were incubated respectively with 0, 5%, 10% and 20% CSE for 6 hours or exposed to 5% CSE for 0, 4, 6, 8, 12, 24 hours to determine the expression changes of t-PA and PAl-1 protein. Meanwhile, cells were also accordingly exposed either to 5% CSE alone or simvastatin pre-treated and 5% CSE for 24 hours to assess the role of simvastatin in CSE-induced t-PA and PAl-1 protein and mRNA expression in HUVECs. RT-PCR and ELISA techniques were used for detecting the t-PA or PAl-1 mRNA and protein. Results After 6-hour exposure to CSE, the expression levels of t-PA protein in 10% and 20% CSE-treated groups reduced significantly （（0.0365±0.0083） ng/ml, （0.0255±0.0087） ng/ml） when compared with that of control group （（0.0660±0.0120） ng/ml）（P 〈0.05）. In contrast, the levels of PAl-1 protein in 5%, 10% and 20% CSE-treated groups increased remarkably （（13.3225±0.5680） ng/ml, （14.2675±1.5380） ng/ml, （14.4292±1.6230） ng/ml） when compared with that of control group （（8.5193_±0.7537）ng/ml）（P〈0.05）. After stimulation with 5% CSE for 0, 4, 6, 8, 12, 24 hours, the levels of PAl-1 protein increased over time and reached the peak at 24 hours （（14.6400±1.0651） ng/ml）, which was significantly higher than that of control group （（12.0656±0.6148） ng/ml）（P 〈0.05）. Additionally, CSE could up-regulate PAl-1 expression at both the mRNA and the protein levels. The levels of PAl-1 mRN

关键词： cigarette smoke extract simvastatin tissue-type plasminogen activator plasminogen activator inhibitor-1 human umbilical vascular endothelial cells

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Rhein inhibits transforming growth factor β1 induced plasminogen activator inhibitor-1 in endothelial cells

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Chinese Medical Journal 2003年第3期116卷 35-40页

作者：朱加明刘志红黄海东陈朝红黎磊石 Research Institute of Nephrology Jinling Hospital Nanjing University School of Medicine Nanjing 210002 China

Objectives To investigate the effect of rhein on endothelial plasminogen activator inhibitor-1 (PAI-1) mRNA expression and protein production induced by transforming growth factor β1 (TGFβ1), and to explore the mechanism of the protective action of rhein on endothelial cells. Methods A human umbilical endothelium derived cell line (ECV-304) from ATCC was used in this study. The PAI-1 mRNA expression and protein synthesis in the endothelial cells were detected by Northern blot and flow cytometry analysis, respectively. The activity of phospho-p44/p42 MAP kinase induced by TGFβ1 was determined by immunoprecipitation analysis and western blot. Results TGFβ1 rapidly increased PAI-1 mRNA expression in the endothelial cells, and this effect lasted at least 24 hours. The upregulation of PAI-1 mRNA expression induced by TGFβ1 in endothelial cells was inhibited by rhein in a dose-dependent manner. In addition, rhein inhibited endothelial PAI-1 protein production. Further study revealed that rhein had a significant inhibitory effect on the activity of phospho-p44/p42 MAP kinase induced by TGFβ1 in human endothelial cells. Conclusions Our results showed that rhein may have a protective effect on the endothelial dysfunction by inhibiting overexpression of PAI-1, indicating a way for the treatment of vascular diseases.

关键词： rhein endothelial cells plasminogen activator inhibitor-1

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Pioglitazone suppresses advanced glycation end product-induced expression of plasminogen activator inhibitor-1 in vascular smooth muscle cells

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Journal of Genetics and Genomics 2011年第5期38卷 193-200页

作者： Xiaochen Yuan Naifeng Liu Department of Cardiology Institute for Cardiovascular Medicine Zhongda Hospital of Southeast University Nanjing 210009 China Department of Cardiology Institute for Cardiovascular Medicine of Southeast University the First People's Hospital of Yangzhou Yangzhou 225001 China

glycation end products （AGEs） play an important role in vascular complications of diabetes, including fibrinolytic abnormalities. Pioglitazone, a peroxisome proliferator-activated receptor gamma （PPARy） agonist, has recently been shown to reduce circulating plasminogen activator inhibitor-1 （PAI-1 ） levels in diabetes mellitus. In the present study, we investigated the effects of pioglitazone on the expression of local PAI-1 in rat vascular smooth muscle cells （VSMCs） induced by AGEs and the underlying mechanism. The result showed that AGEs could enhance the PAI-1 expression by 5.1-fold in mRNA and 2.7-fold in protein level, as evaluated by real-time RT-PCR and Western blotting, respectively. Pioglitazone was found to down-regulate the AGE-stimulated PAI-1 expression in VSMCs. However, these inhibitory effects were partially attenuated by the PPARy antagonist, GW9662. Furthermore, we found that AGEs induced a rapid increase in phosphorylation and activation of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. The ERK kinase inhibitor, U0126, partially prevented the induction of PAI-1 by AGEs. Moreover, pioglitazone was also found to inhibit the pbosphorylation of ERKI/2. Taken together, it was concluded that pioglitazone could inhibit AGE-induced PAI-1 expression, which was mediated by the ERK1/2 and PPARy pathways. Our findings suggested pioglitazone had a therapeutic potential in improving fibrinolytic activity, and consequently preventing thromboembolic complications of diabetes and cardiovascular disease.

关键词： AGEs PPARγ Pioglitazone plasminogen activator inhibitor-1

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Study on Effect of Different Dosages of Ligustrazine on Level of plasminogen activator inhibitor-1 Activity in Type 2 Diabetes Mellitus Patients

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Chinese Journal of Integrated Traditional and Western Medicine 2003年第3期9卷 199-203页

作者：薛现中张兆华邢小燕 Tengzhou Municipal Central People's Hospital of Shandong Province Shandong 277500 China-Japan Friendship Hospital

Objective: To observe the effect of different dosages of ligustrazine (LG) on the level of plasminogen activator inhibitor-1 (PAI-1) activity in patients with type 2 diabetes mellitus. Methods: Ninety cases of type 2 diabetes mellitus inpatients were selected, and randomly divided into LG small dosage group (SDG), LG large dosage group (LDG) and control group. The 120 mg LG, 400 mg LG and normal saline 250 ml were given through intravenous dripping respectively, once daily, 20 days as one treatment course. Before and after treatment, all the patients had their fasting blood taken for PAI-1 and tissue plasminogen activator (t-PA) assessment test to perform the comparative study. Results:Seventy-three out of the 90 patients completed the observation course, the PAI-1 activity of three groups after treatment all lowered compared with that before treatment, and the difference between groups was also significant (all P<0. 01). After treatment the PAI-1 level of SDG and LDG of LG were all markedly lowered (all P<0. 01), the LDG's lowering was more evident than that of SDG, and comparison between these two groups of patients showed significant difference (P<0. 01). Although in the control group there was some difference between before and after treatment, it was not so significant like the above-mentioned two groups (P = 0. 0140). No adverse reaction occurred in the 3 groups during the observation period. Conclusion:LG could safely and effectively lower type 2 diabetes mellitus patient's plasma PAI-1 activity level, and LDG of LG proved to be particularly effective.

关键词： type 2 diabetes mellitus ligustrazine plasminogen activator inhibitor-1

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Research on the correlation of serum plasminogen activator inhibitor-1 level to vascular complications in type 2 diabetes mellitus patients with overweight or obesity

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Discussion of Clinical Cases 2019年第2期6卷 20-25页

作者： Jun X Yanan Z Zhijie C Zhihui D Danhua S Xiaojing C Ying W Jixiang J Department of Geriatrics the Third Affiliated Hospital of Inner Mongolia Medical UniversityBaotouInner MongoliaChina

Objective:To explore the relationship between serum plasminogen activator inhibitor(PAI-1)level and Type 2 Diabetes Mellitus(T2DM)accompanied by overweight or obesity by observing not only the changes of PAI-1 level in T2DM patients with overweight or obesity,but also glucose and lipid metabolism related indicators,the changes of the inflammatory cytokines secreted by adipocytes,and then making an analysis on the correlation to PAI-1.Methods:36 cases of healthy examinees were selected as normal control group(NC group),and the experimental group can be divided into T2DM group(54 cases),Overweight/Obesity group(35 cases)and T2DM+Overweight/Obesity group(48 cases).Glucose and lipid metabolism related indicators such as fasting blood glucose(FBG),triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),glycated hemoglobin(HbA1c),fasting insulin(FINS),insulin resistance index(IR),body weight index(BMI)and inflammatory cytokines interleukin-6(IL-6),tumor necrosis factor(TNF-α)and PAI-1 were observed and compared between groups,and then made an analysis to explore the correlation of these factors to PAI-1.Results:(1)Compared with NC group,the levels of FBG,HbA1c,FINS and IR were increased in T2DM group,and the difference was of statistical significance.However,there was no statistically significant difference in TG,TC,LDL-C and BMI between NC group and T2DM group;the levels of FINS,IR,TG,LDL-C,TC and BMI were elevated in Overweight/Obesity group,and the difference was of statistical significance.However,there was no statistically significant difference in FBG and HbA1c;the levels of FBG,HbA1c,FINS,IR,TG,LDL-C,TC and BMI were up-regulated in T2DM+Overweight/Obesity group,and the difference was of statistical significance.Compared with T2DM group,the levels of TG,TC,LDL-C and BMI were increased in Overweight/Obesity group,and the difference was of statistical significance,however,the levels of FBG,HbA1c,FINS and IR were decreased,and the difference was statistically significant;The levels of FINS,

关键词： plasminogen activator inhibitor-1 Type 2 diabetes mellitus Overweight/obesity

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Atorvastatin Reduces plasminogen activator inhibitor-1 Expression in Adipose Tissue of Atherosclerotic Rabbits

Atorvastatin Reduces Plasminogen Activator Inhibitor-1 Expre...

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作者： Jie-Qi Li~1 Shui-Ping Zhao~2 Quan-Zhong Li~3 Yun-Chang Cai~4 Li-Rong Wu~1 Ying Fang~1 Ping Li~1 1.Department of Cardiology,the Affiliated Hospital of GuiYang Medical College.Guiyang,Guizhou 550004,PR China. 2.Department of Cardiology,the Second XiangYa Hospital of Central South University. 3.Department of Cardiology,the Affiliated Hospital of GuiLin Medical College. 4.Department of Cardiology,the Province People’s Hospital of GuiZhou.

plasminogen activator inhibitor-1 (PAI-1)expression are elevated in adipose tissues/ adipocytes of obese mice,which is associated with a hypofibrinolytic state that contributes to throm- bosis.We have recently demonstrated that PAI-1 expression increases in adipose tissues/adipo- cytes of cholesterol-fed rabbit.In this study,we evaluated the ability of atorvastatin to modulate PAI-1 expression in cholesterol-fed rabbit and the regulatory mechanism.Male rabbits were ran- domly fed with normal diet and high-cholesterol diet for 8 weeks,following 4 weeks,those fed high -cholesterol diet were randomly assigned to 2.5 mg/kg per day atorvastatin or starch.At the end of 12 weeks,subcutaneous adipose was collected,and culture adipocyte.PAI-1 mRNA was detec- ted by reverse transcription-polymerase chain reaction (RT-PCR).PAI-1 concentrations were determined with ELISA.The in vitro effect of atorvastatin and mevalonate (MVA)on PAI-1 pro- duction in adipocytes was observed.Atorvastatin significantly reduced serum TC and LDL-C levels (P＜0.05),and decreased plasma PAI-1 concentration and PAI-1 expression in adipose tis- sues/adipocytes from cholesterol-fed rabbits.In vitro,atorvastatin dose-dependently suppressed PAI-1 expression and protein secretion in adipocytes.MVA reversed the inhibitory effect of atorv- astatin on PAI-1 expression in concentration-dependent manner.Results indicate that atorvastatin reduces plasma PAI-1 concentration and PAI-1 expression in adipose tissue and adipocyte of ath- erosclerotic rabbit,and inhibits PAI-1 expression and protein secretion in adipocytes in vitro,sug- gesting that atorvastatin may have an antithrombtic effect.It also indicated that mevalonate pathway may play an important role in PAI-1 expression in adipocyte.

关键词： atherosclerosis plasminogen activator inhibitor-1 adipose tissue/adipocyte atorvastatin mevalonate fibrinolysis

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PAI-1 genetic polymorphisms influence septic patients’outcomes by regulating neutrophil activity

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Chinese Medical Journal 2023年第16期136卷 1959-1966页

作者： Shaowei Jiang Yang Wang Liang Chen Honghua Mu Connor Meaney Yiwen Fan Janesh Pillay Hairong Wang Jincheng Zhang Shuming Pan Chengjin Gao Department of Emergency Xinhua HospitalShanghai Jiao Tong University School of MedicineShanghai 200092China Division of Rheumatology Department of Internal MedicineUniversity of Utah School of MedicineSalt Lake CityUTUSA Department of Pathology Medicine Biology University Medical Center GroningenThe Netherlands Department of Critical Care University Medical Center GroningenThe Netherlands Department of Critical Care Medicine Zhongshan HospitalFudan UniversityShanghai 200032China

Background:plasminogen activator inhibitor-1(PAI-1)plays an important role in the pathophysiology of sepsis,but the exact mechanism remains debatable.In this study,we investigated the associations among the serum levels of PAI-1,the incidence of 4G/5G promoter PAI-1 gene polymorphisms,immunological indicators,and clinical outcomes in septic patients.Methods:A total of 181 patients aged 18-80 years with sepsis between November 2016 and August 2018 in the intensive care unit in the Xinhua Hospital were recruited in this retrospective study,with 28-day mortality as the primary outcome.The initial serum level of PAI-1 and the presence of rs1799768 single nucleotide polymorphisms(SNPs)were examined.Univariate logistic regression and multivariate analyses were performed to determine the factors associated with different genotypes of PAI-1,serum level of PAI-1,and 28-day mortality.Results:The logistic analysis suggested that a high serum level of PAI-1 was associated with the rs1799768 SNP of PAI-1(4G/4G and 4G/5G)(Odds ratio[OR]:2.49;95%confidence interval[CI]:1.09,5.68).Furthermore,a high serum level of PAI-1 strongly influenced 28-day mortality(OR 3.36;95%CI 1.51,7.49).The expression and activation of neutrophils(OR 0.96;95%CI 0.93,0.99),as well as the changes in the expression patterns of cytokines and chemokine-associated neutrophils(OR:1.00;95%CI:1.00,1.00),were both regulated by the genotype of PAI-1.Conclusions:Genetic polymorphisms of PAI-1 can influence the serum levels of PAI-1,which might contribute to mortality by affecting neutrophil activity.Thus,patients with severe sepsis might clinically benefit from enhanced neutrophil clearance and the resolution of inflammation via the regulation of PAI-1 expression and activity.

关键词： Gene polymorphism Neutrophil plasminogen activator inhibitor-1 PAI-1 Sepsis Single nucleotide polymorphism

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The plasminogen activating system in the pathogenesis of Alzheimer’s disease

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Neural Regeneration Research 2021年第10期16卷 1973-1977页

作者： Manuel Yepes Department of Neurology Emory University School of MedicineAtlantaGAUSA Department of Neurology Veterans Affairs Medical CenterAtlantaGAUSA Division of Neuropharmacology and Neurologic Diseases Yerkes National Primate Research CenterAtlantaGAUSA

Dementia is a clinical syndrome that affects approximately 47 million people worldwide and is characterized by progressive and irreversible decline of cognitive,behavioral and sesorimotor functions.Alzheimer’s disease(AD)accounts for approximately 60–80%of all cases of dementia,and neuropathologically is characterized by extracellular deposits of insoluble amyloid-β(Aβ)and intracellular aggregates of hyperphosphorylated tau.Significantly,although for a long time it was believed that the extracellular accumulation of Aβwas the culprit of the symptoms observed in these patients,more recent studies have shown that cognitive decline in people suffering this disease is associated with soluble Aβ-induced synaptic dysfunction instead of the formation of insoluble Aβ-containing extracellular plaques.These observations are translationally relevant because soluble Aβ-induced synaptic dysfunction is an early event in AD that precedes neuronal death,and thus is amenable to therapeutic interventions to prevent cognitive decline before the progression to irreversible brain damage.The plasminogen activating(PA)system is an enzymatic cascade that triggers the degradation of fibrin by catalyzing the conversion of plasminogen into plasmin via two serine proteinases:tissue-type plasminogen activator(tPA)and urokinase-type plasminogen activator(uPA).Experimental evidence reported over the last three decades has shown that tPA and uPA play a role in the pathogenesis of AD.However,these studies have focused on the ability of these plasminogen activators to trigger plasmin-induced cleavage of insoluble Aβ-containing extracellular plaques.In contrast,recent evidence indicates that activity-dependent release of uPA from the presynaptic terminal of cerebral cortical neurons protects the synapse from the deleterious effects of soluble Aβvia a mechanism that does not require plasmin generation or the cleavage of Aβfibrils.Below we discuss the role of the PA system in the pathogenesis of AD and the translational relevance of data published t

关键词： Alzheimer’s disease amyloid precursor protein amyloidβ neuroserpin plasmin plasminogen activating system plasminogen activator inhibitor-1 synapse tissue-type plasminogen activator urokinase-type plasminogen activator

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Protein Microarrays for Quantitative Detection of PAI-1 in Serum

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Chinese Journal of Cancer Research 2012年第3期24卷 220-225页

作者： Xu Ma Qing-yun Zhang Key Laboratory of Carcinogenesis and Translational Research(Ministry of Education) Department of Clinical LaboratoryPeking University Cancer Hospital&InstituteBeijing 200142China

Objective： plasminogen activator inhibitor-1 （PAI-1）, one crucial component of the plasminogen activator system, is a major player in the pathogenesis of many vascular diseases as well as in cancer. High levels of PAI-1 in breast cancer tissue are associated with poor prognosis. The aim of this study is to evaluate rigorously the potential of serum PAI-1 concentration functioning as a general screening test in diagnostic or prognostic assays. Methods： A protein-microarray-based sandwich fluorescence immunoassay （FIA） was developed to detect PAI-1 in serum. Several conditions of this microarray-based FIA were optimized to establish an efficacious method. Serum specimens of 84 healthy women and 285 women with breast cancer were analyzed using the optimized FIA microarray. Results： The median serum PAI-1 level of breast cancer patients was higher than that of healthy women （109.7 ng/ml vs. 63.4 ng/ml）. Analysis of covariance revealed that PAI-1 levels of the two groups were significantly different （P0.001） when controlling for an age effect on PAI-1 levels. However, PAI-1 values in TNM stage I？IV patients respectively were not significantly different from each other. Conclusion： This microarray-based sandwich FIA holds potential for quantitative analysis of tumor markers such as PAI-1.

关键词： Breast cancer plasminogen activator inhibitor-1 Protein microarray Serum

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