Abstract:Aim To understand the features of lipoprotein lipase(LPL) and its role in the distinctive lipid metabolism in tree shrew,which is considered to be an animal protected from atherosclerosis.Methods cDNA clones of tree shrew lipoprotein lipase were isolated from adipose tissue mRNA,benefiting from SMART-RACE technique.The tissue distribution of lipoprotein lipase in tree shrew was identified by Real-Time PCR.The activities of lipoprotein lipase from tree shrew and human expressed in eukaryocytes were measured by Radioactive Substrate Emulsion method,respectively.Results The nucleotide sequence of tree shrew LPL covered 3 293 bp,including a 1 437 bp coding region,which encoded a 20 residues predicted signal pepetide and a 458 residues mature protein that was ten residues longer than that of human at N-terminus.The homology of amino acid sequences of lipoprotein lipase from tree shrew and other mammalian species was more than 90%,highly conserved in functional domains.LPL was expressed in many tissues in tree shrew,highly in cardiac muscle and adipose tissue.In despite of no expression in skeletal muscle,there was less-medium expression in liver,distinctly different from human LPL which was expressed in the contrary way in these two tissues.The tree shrew LPL activity was remarkably higher(7.67 times) than that of human LPL,consistent with lower triglyceride concentration in tree shrew plasma compared with human.Conclusion The higher LPL activity in tree shrew should greatly attribute to its lower plasma triglyceride levels.

Abstract:Aim To determine the influence of testosterone and flutamide on atherosclerotic plaque stability and inflammatory reaction.Methods Pathological sections of aorta were performed with hematoxylin-eosin staining and Masson's trichrome staining.Total plasma testosterone was measured using Advia Centaur immunoassay system.The contents of serum tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) were assayed using radio-immunoassay kit.The concentrations of serum soluble intercellular adhesion molecule-1(sICAM-1) and matrix metallopeptidase-2(MMP-2) were assayed using enzyme linked immunosorbent assay(ELISA) kit according to the procedure described by the manufacturer.Results Testosterone undecanoate replacement reduced the plaque area and the aortic intimal thickness,and increased the fibrous cap thickness and collagen contents in castrated rabbits.However,presence of flutamide increased the plaque area and the aortic intimal thickness,and decreased the fibrous cap thickness and collagen contents in castrated rabbits again.Levels of serum TNF-α,IL-6,sICAM and MMP-2 increased in cholesterol-rich diet rabbits compared with standard diet rabbits.Castration caused an increase in levels of serum TNF-α,IL-6,sICAM and MMP-2 in castrated rabbits compared with sham-operated rabbits.Treatment of testosterone undecanoate reduced the levels of serum TNF-α,IL-6,sICAM and MMP-2 in castrated rabbits.However,presence of flutamide increased the levels of serum TNF-α,IL-6,sICAM and MMP-2 again.Conclusion Testosterone at physiological dose and flutamide can influence atherosclerotic plaque growth and stability and the levels of inflammatory factors.

Abstract:Aim To investigate the effect of protein kinase C inhibitor on expression of intercellular adhesion molecule-1 protein and mRNA in smoking rats.Methods The expression of intercellular adhesion molecule-1 protein and mRNA were abserved by immunohistochemistry and in situ hybrizition.18 non-infarcted rats were randomly divided into three groups including 6 non-smoking rats,6 no medicine treated smoking rats and 6 protein kinase C inhibitor treated smoking rats.48 infarcted rats were randomly divided into two groups including 24 no medicine treated rats and 24 protein kinase C inhibitor treated rats.2 hours,6 hours,12 hours or 24 hours after infarction the medicine were given,6 rats in each time points.Results Compared with no medicine groups,the expression of intercellular adhesion molecule-1 and intercellular adhesion molecule-1 mRNA were decreased with corresponding indentical time points in protein kinase C inhibitor groups,which showed statistical difference(p<0.05).Conclusions Protein kinase C inhibitor could reduce the expression of intercellular adhesion molecule-1 and intercellular adhesion molecule-1 mRNA in smoking rats cerebrovascularendothelial cells,the medicine were given more early,more effect will be produced.

Abstract:Aim To study the effect of rat transient myocardial ischemia/reperfusion and the oxidative stress of C₂C₁₂ cells on the expression of Krüppel-like factor 4(KLF 4) gene.Methods Rat model of transient myocardial ischemia/reperfusion was established by occluding and relaxing of left coronary artery.C₂C₁₂ cells were treated with H₂O₂(1 mmol/L) to build the oxidative stress model.RT-PCR and Western blotting were performed to determine the changes of KLF 4 expression.Results Transient myocardial ischemia/reperfusion induced obvious accumulation of KLF 4 mRNA in rat.Oxidative stress distinctly raised the KLF 4 mRNA and protein levels in C₂C₁₂ cells.Conclusion High expression of KLF 4 gene was induced by rat transient myocardial ischemia/reperfusion and the oxidative stress of C₂C₁₂ cells.

Abstract:Aim To approach the effect of nourishing qi and promoting blood herbs on the apoptosis of vascular smooth muscle cell(VSMC) and expression of Bcl-2 in rabbit model with arteriosclerosis and diabetes.Methods By injecting tetraoxypyrimidine intravenous and breeding high glucose and grease animal feeds,the rabbit animal with arteriosclerosis and diabetes can be obtained.The models were divided into three groups randomly:model group,large dose and low dose treatment group,controlled with the normal group.After 12 weeks,all the animals were killed.The pathomorphology of intima of aorta were observed,and the apoptotic rate and expression of Bcl-2 were detected by flow cytometry.Results In the model group the apoptotic rate of smooth muscle cell was higher than that in the normal group(p<0.01),but the expression of Bcl-2 was lower(p<0.01).The visible atheromatous plaque which caused the serious stenosis were observed in model group.In the treatment group the apoptotic rate of smooth muscle cell was lower than that in the model group(p<0.01),but the expression of Bcl-2 was higher(p<0.01),the atheromatous plaque were decreased,and the stenosis were reduced,which were more obvious in the large dose treatment group than in the low dose treatment group.Conclusion The nourishing qi and promoting blood herbs can influence the apoptosis of VSMC and expression of Bcl-2 in rabbit model with arteriosclerosis and diabetes.The herbs have the better treatment on the diabetic arteriosclerosis obliterans.

Abstract:Aim To investigate the effect of cryopreservation on the proliferative abilitiy and vasculogenesis activity of endothelial outgrowth cells(EOC),and to explore the possibility of its resuscitation.Methods The mononuclear cells were harvested from umbilical cord blood by density gradient centrifugation,induced into EOC and expanded in vitro.The endothelial characteristics of EOC were identified by immunostaining and fluorescent staining.Then EOC were cryopreserved at-20℃ for 4 h,-75℃ overnight and then cryopreserved into liquid nitrogen using culture medium containing 50 mL/L and 80 mL/L dimethyl sulfoxide(DMSO).After 24 h cryopreserved EOC were resuscitated,and the resuscitative rate,proliferative ability and vasculogenesis activity were measured.Results EOC possessed many endothelial characteristics.Immunostaining showed that surface antigens Factor Ⅷ,CD34 and Flk-1 were postive;and fluorescent staining experiment revealed that EOC both bound to FITC-UEA-1 and uptook DiI-ac-LDL.There were no statistical differences between 50 mL/L and 80 mL/L DMSO group in resuscitative rate(79%±6% vs 88%±4%,p>0.05).During early period after resuscitation(within 36 h),two cryopreservated groups(50 mL/L and 80 mL/L DMSO group) both showed decrease in proliferative abilitiy compared with non-cryopreservated group(p<0.05).72 h later there were no significant differences between 50 mL/L,80 mL/L DMSO group and non-cryopreservated group in the proliferative ability(p>0.05).In contrast,vasculogenesis activity analysis revealed attenuation in vasculogenesis activity within 6 h(p<0.05) while 30 h later such attenuation became insignificant(p>0.05).Conclusions EOC can be cryopreservated and resuscitated.The cryopreservated cell shows no significant decrease in proliferative abilitiy and vasculogenesis activity after resuscitation.

Abstract:Aim To investigate the relationship between oxidative stress and Fas/FasL system during lung ischemia reperfusion in rabbits.Methods Rabbit lung model of ischemia reperfusion injury was established in vivo.Forty rabbits were randomly divided into control group and ischemia reperfusion 1 h,3 h,and 5 h group,10 rabbits in each group.The activity of plasma superoxide dismutase,the content of plasma nitric oxide and the concentration of malondialdehyde,pneumocyte apoptosis index and express of Fas/FasL mRNA were measured respectively in different groups.Results Compared with control group,apoptosis index was significantly high(p<0.01),Fas/FasL mRNA heavily expressed in lung vascular endothelial cells,alveolar and bronchial epithelial cells in ischemia reperfusion groups(p<0.01).The activity of superoxide dismutase and the content of nitric oxide showed a time-dependent decrease in ischemia reperfusion groups(p<0.01),while the content of malondialdehyde was gradually increased with the time of reperfusion prolonging(p<0.01).Conclusion Oxidative stress was associated with activation of Fas/FasL during lung ischemia reperfusion.

Abstract:Aim To construct the short hairpin small interfering RNA(shRNA)eukaryotic expression vector specific to human lectin-like oxidized low density lipoprotein receptor-1(LOX-1)gene and to observe its silencing effect on LOX-1 in human umbilical vascular endothelial cells(hUVEC).Methods The pGenesil-1-LOX-1-shRNA expression vector was constructed by gene recombination,then transfected into the cultured hUVEC.At 48 h after transfection,the stable transfection and expression of LOX-1 mRNA in hUVEC were determined by semi-quantitative RT-PCR.Results pGenesil-1-LOX-1-shRNA expression vector was successfully constructed.DOTAP-mediated gene transfection of pGenesil-1-LOX-1-shRNA expression vector into hUVEC down-regulated the mRNA expression level of LOX-1 gene,as compared with the control group.Conclusion The pGenesil-1-LOX-1-shRNA expression vector can inhibit the expression of LOX-1 mRNA in hUVEC,which may be beneficial in searching new gene therapy of atherosclerosis.

Abstract:Aim Hyperglycemia-induced apoptosis in ascular endothelial cells may contribute to the acceleration of atherosclerosis associated with diabetes.This study confirmed that acylated ghrelin attenuates hyperglycemia-induced apoptosis in cultured human umbilical vein endothelial cells(ECV-304).Methods Morphology of cell apoptosis was observed by Acridine Orange fluorescence staining.Cell apoptosis was detected by TUNEL and flow cytometry with PI staining.Caspase-3 activity was detected by colorimetric assay.Results Exposure of ECV-304 to hyperglycemia level for 72 h significantly increased the number of apoptotic cells compared with normal glucose level,but pretreatment of ghrelin significantly attenuated hyperglycemia-induced apoptosis.Exposure to hyperglycemia-induced caspase-3 activation and ghrelin also prevented it.Conclusion Results of our study indicate that acylated ghrelin effectively inhibits apoptosis in cultured vascular endothelial cells and provide a basis for future therapeutic interventions in diabetic vascular complications.

Abstract:Aim To investigate the location and expression of tissue factor pathway inhibitor-2(TFPI-2) in U937 cell-derived foam cells.Methods Foam cell model was established by incubating the human monoblastic leukemia(U937) cells with oxidized low density lipoprotein(ox-LDL).Double immuofluorescence was used to detect the location of TFPI-2 protein.Human umbilical vein endothelial cells(hUVEC) were incubated with U937,the effects of the expression of TFPI-2 on U937 cell-derived foam cells were observed.The expression of TFPI-2 protein was examined by time-resolved fluoroimmunoassay(TRFIA).The expression of TFPI-2 mRNA was examined by reverse transcription-polymerase chain reaction(RT-PCR) and real-time PCR.Results TFPI-2 protein distributed mainly in the cytoplasm of U937 cells.U937 cells were incubated with ox-LDL,the mRNA and protein of TFPI-2 peaked at 6 hours,then the levels of TFPI-2 decreased in a time dependent manner.After being added hUVEC,the expression of TFPI-2 in U937 increased.Conclusion Ox-LDL reduced the expression of TFPI-2 in U937 cells,incubated with hUVEC can modify the inhibitive effect.

Abstract:Aim To discuss optimal reconstruction phase of 16-slice CT coronary angiography.Methods 136 patients with suspected or diagnosed coronary artery disease were studied with 16-slice spiral CT.Choosing left and right coronary artery in the axial enhancement scanning images,previewing 10 images at 0%-100% of the cardiocycle in increments of 10%,We evaluated grade of the image quality and number of the ratable coronary artery in different reconstruction phases.We selected the most clear image of left and right coronary artery,and recorded the phase.Raw data were reconstructed with thin images at the best phase.All images were reconstructed in the mode of maximum intensity projection(MIP),multi-plane reformation(MPR),curved planar reformation(CPR) and volume rendering(VR).Results Three coronary,the left anterior descending(LAD),the left circumflex(LCX) and the right coronary artery(RCA),segments were analyzed in each patient with regard to image quality.There was significance of difference in the number of the ratable coronary artery in different reconstruction phases(χ2=34.14,p<0.05).The number of good image in coronary arteries displayed by 40%,50%,70%,80% and the rest reconstruction phases had significant difference(p<0.05).The optimal reconstruction phase of RCA,LAD and LCX were 80% and 70% of the cardiac cycle.The second phase was 40% and 50% R-R phases.Conclusions Image reconstruction in the optimal phase can decrease the artifacts of cardiac motion as possible and improve the quality of images and raise work efficiency obviously.

Abstract:Aim To explore the relationship between the levels of tumor necrosis factor related apoptosis inducing ligand(TRAIL) and death receptor 5(DR5) and acute coronary syndrome(ACS).Methods The 62 patients with coronary artery disease were divided into two groups:40 in the ACS group and 21 in the stable angina pectoris(SAP) group.Twenty-two healthy volunteers served as normal coronary artery(NCA) group.Plasma TRAIL and DR5 levels from these people were assayed by enzyme-linked immunosorbent assay.Reverse transcription polymerase chain reaction was used for the detection of TRAIL and DR5 mRNA of ascending aortas from 22 subjects,of which 8 were from the ACS group,7 from the SAP group and 7 from the NCA group.Results Plasma TRAIL levels were significantly increased in the ACS group(p<0.001).Patients with ACS had a markedly greater plasma DR5 level than those with NCA(p<0.05).Plasma levels of both TRAIL and DR5 in patients with 3-vessel involved ACS were significantly higher than those in patients with 1-vessel(p<0.05).The patients with 3-vessel involved ACS had markedly raised plasma TRAIL levels when compared with the patients with 2-vessel(p<0.05).In the 2-vessel group,only plasma DR5 levels had a statistical significance greater than those in the 1-vessel group(p<0.05).Logistic stepwise regression analysis revealed that odds ratio of TRAIL is 1.012(P=0.002).Ascending aortas from the ACS group exhibited markedly higher TRAIL expression as compared with those from the SAP and NCA group(p<0.05).Conclusions Increased plasma TRAIL and DR5 levels are closely associated with the presence and severity of ACS.

Abstract:Aim To assess the significance of ankle brachial index(ABI) in the type 2 diabetes(T2DM) with peripheral arterial disease(PAD) in lower extremities.Methods 486 T2DM patients were divided into PAD group and Non-PAD group according to the value of ABI(the cut point is 0.9).Two groups were compared and the factors related to ABI were also analyzed.Finally the risk factors of PAD in T2DM were identified.Results Compared with Non-PAD group,the age(62.09±10.53 years vs 56.77±9.83 years,p<0.01),diabetes duration(86.90±51.94 month vs 57.91±57.64 month,p<0.01) and low density lipoprotein cholesterol(LDLC)(3.07±1.01 mmol/L vs 2.71±0.98 mmol/L,p<0.05) were significantly higher,and ABI(0.77±0.15 vs 1.08±0.15,p<0.01) was significantly lower in PAD group.Pearson correlation analysis showed that age and total cholesterol were negatively correlated with ABI.Logistic regression analysis showed that age,diabetes duration,systolic blood pressure(SBP) and LDLC were major risk factors of PAD in T2DM,however,waist hip ratio was a protective factor of PAD.Conclusions ABI is a feasible and reliable index for screening PAD in T2DM patients.Age,diabetes duration,systolic blood pressure and LDLC are major risk factors of low ankle-brachial index in T2DM patients.

Abstract:Aim To investigate the effects of platelet derived growth factor(PDGF) on the expression of vascular cell adhesion molecular-1(VCAM-1) and the adhesion of monocyte-VSMC.Methods GEArray Gene Array and RT-PCR technique were used to analyse the mRNA expression of VCAM-1 in VSMC after incubated with PDGF for 0 min、20 min、6 h.Meanwhile,the adhesion of monocyte-VSMC was detected by adhesion assay.Results The mRNA expression of VCAM-1 was significantly induced by PDGF,as compared with control group.PDGF-induced VCAM-1 accumulation was appeared at 20 min,and significantly increased at 6 h.PDGF also enhanced the adhesion rate of monocyte and VSMC.Conclusions The results demonstrated that PDGF significantly induced the expression of VCAM-1 in VSMC and enhanced monocyte bind to VSMC.This suggested that PEGF played an important role in early injured lesion and contribution to monocytes in circulation adheresion and migration across the endothelium.

Abstract:Aim To evaluate effects of the G994T,sex,and age on the lipoprotein-Associated phospholipase A2(Lp-PLA2) activity.Methods Genotyping of the G994T was done by the allele-specific PCR.The Lp-PLA2 activity was measured using a commercial kit by the spectrophotometric method.Results The Lp-PLA2 levels were significantly higher in GG subpopulation,as compared to GT population and TT population.The Lp-PLA2 activity was correlated with sex.We found that,in men with the GG genotype,enzyme activity increased significantly with age.In contrast,plasma Lp-PLA2 activity in men with the GT genotype did not significantly increase with age.Conclusions The G994T was strongly correlated with the enzyme activity.The Lp-PLA2 activity in men was higher than that in women.Plasma PAF-AH activity may increase in response to stresses induced by PAF and:or oxidized phospholipids.This compensatory response may be attenuated or not evident in individuals with the m allele.

Abstract:Aim To find the distribution of ankle-brachial index(ABI) and prevalence of peripheral arterial disease(PAD) among Chinese inpatients with equal-risk to coronary heart disease.Methods 2 509 inpatients with equal-risk to coronary heart disease were recruited from Shanghai and Beijing(age≥45 years).Associated factors and medical examinations were collected by a retrospective epidemiological survey.At one time,ABI was measured.PAD was diagnosed when the ABI was <0.9,inpatients with an ABI of >1.4 were excluded because of false negative rate.Results Mean level of ABI was 0.96±0.24 and prevalence of PAD was 31.1%(95%CI:29.3%～33.0%)in this study subjects.Prevalence of PAD in male and female were 33.9% and 28.8%(p<0.05),respectively.Prevalence of PAD was increased with advance age among men and women(p<0.0001),whereas mean level of ABI(p<0.0001).Inpatients with high level of total cholesterol,hypertension,ischemic stroke,and diabetes had higher prevalence of PAD in univariate analysis and lower level of ABI.Prevalence of PAD was up to 60.6% among inpatients combined with coronary heart disease,ischemic stroke,and diabetes.Conclusions PAD had universal occurrence in Chinese inpatients with equal-risk to coronary heart disease.Clinicians should attach importance to peripheral arterial disease and strengthen the management of risk factors of cardiovascular disease.

Abstract:Aim To investigate the current epidemiological characteristics of blood lipid levels among rural hypertensive patients in Liaoning province and to provide scientific basis for the prevention and treatment of dyslipoproteinemia.Methods A cross-sectional survey was conducted during 2004～2006,through cluster multistage and random sampling to a resident(≥5 years) groups of 6 412 individuals(2,805 men,3,607 women) aged ≥35 years old with hypertension in the countryside of Liaoning province and blood chemical analyses were performed at a central,certified laboratory so as to understand the prevalence of dyslipoproteinemia based on criteria of the prevention and treatment of dyslipoproteinemia in 2006 in China,SPSS11.5 of statistical software was used for data analysis.Results(1)The average level of blood lipids(TC,HDLC,LDLC and TG)had higher levels in hypertensive women than that in hypertensive men(p<0.001).(2)The prevalence of isolated hypercholesterolemia(TC≥6.21 mmol/L),low HDLC concentration(HDLC<1.03 mmol/L),increased LDLC concentration(LDLC≥4.16 mmol/L),isolated hypertriglyceridemia(TG≥2.26 mmol/L) was 16.9%,8.8%,4.4%,and 19.8%.(3)The prevalence of TC increased with the increased stage of blood pressure and it showed a significant difference among 3 stages.In contrast,the prevalence of HDLC decreased with the increased stage of blood pressure and it was also significant(p<0.05);however,we didn't see a trend between LDLC and the stage of blood pressure and TG and the stage of blood pressure.Conclusion The prevalence of dyslipidemia was relatively high among rural hypertensive patients in Liaoning province,it is very important to prevent and control dyslipidemia for us.

Abstract:Aim To develop a reliable method for the measurement of serum lipoprotein(a) cholesterol.Methods Serum aliquots were centrifuged in the presence of proline at a density of 1.044 g/mL,the bottom fraction,which contains 95% of lipoprotein(a),was readjusted to density of 1.040 in the presence of mercaptoethanol and recentrifuged.Cholesterol levels in the ultracentrifugation top fractions [lipoprotein(a) cholesterol] were analyzed by high-performance liquid chromatography.Results Mercaptoethanol effectively dissociated lipoprotein(a) into apolipoprotein(a) and LDL-like lipoproteins [Lp(a-)].The dissociated Lp(a) showed a distinctive density distribution from that of the native lipoprotein(a).This finding enabled direct and reliable separation of lipoprotein(a) by ultracentrifugation.The with-in run and total CVs for the measurement of lipoprotein(a) cholesterol were 1.84%～2.17% and 2.85%～4.29%,respectively.Conclusion A new method for the measurement of lipoprotein(a) cholesterol by ultracentrifugation and high-performance liquid chromatography has been established.It is reliable and precise and may play an important role in standardization of lipoprotein(a) measurement.