Abstract:Aim To find out the effects of the combination of Benazepril and Losartan on improving and reversing left ventricular remodeling of the aged spontaneously hypertensive rats(SHR). Methods 18 WistarKyoto rats(WKY) and 54 SHR of 55 weeks old were chosen as the experiment animals.54 SHR were divided into 4 groups randomly: SHR-control group,SHR-Benazepril group [10mg/(kg·d)],SHR-Losartan group [30 mg/(kg·d)],SHR-Benazepril [10 mg/(kg·d)]+Losartan [15 mg/(kg·d)] group.After 12 weeks of treatment,all rats were drawn for detection,including observing systolic blood pressure(SBP) and left ventricular mass index(LVMI),myocardial pathologic changes and detecting the collagen protein level of left ventriculum by the immunohistochemical methods.TUNEL-method was used to detect the apoptosis rate of myocardial cells. Results Both SBP and LVMI of the SHR-control group were higher than those of drug-interfered groups(p<0.01). And it was the combination that can lower the LVMI most notably.Compared with the SHR-control group,pathological changes were obviously improved in all three drug-interfered groups,with the combination group being the most.The expression levels of type Ⅰ and total collagen fibers of each treatment group were lower than those of SHR-control group(p<0.01),especially for the combination group.The expression levels of type Ⅲ collagen fibers in each drug-interfered group were decreased slightly,and also with the combination group being the most(p<0.01).Compared with the SHR-control group,the apoptosis rate of Benazepril group was significantly higher(p<0.05),while that of Losartan group and the combination group were significantly lower(p<0.01). Conclusions Compared with monotherapy,the combination of Benazepril and Losartan seems more efficient in improving and reversing the aging SHR's left ventricular hypertrophy,myocardial apoptosis and myocardial fibrosis.

Abstract:Aim To investigate whether glycation could inhibit the protection role of mutant CD59s against human complement. Methods Site-directed mutagenesis to replace residue 37 or 38 with histidine(H)was performed by recombinant PCR.Mutant CD59 DNAs were inserted into the mammalian expression vector pALTER-MAX and transfected into CHO together with the selection marker pcDNA3,which confered resistance to G418.Expression of mutant CD59s in the G418-resistant clones were tested by Western blot,immunohistochemistry and FCM. A functional dye release assay was used to measure protection role of CD59s against human complement. Results Recombinant plasmids of pALTER-HM CD59 had been successfully constructed according to sequence and enzyme digestion analysis.Stable transfectants were selected by the addition of G418.Stable populations of CHO cell,which expressed relatively high levels of recombinant protein,were sorted by immunolabled technique.In Western blot assay,the mutant proteins expressed on CHO was about 20 kDa.Dye release assays confirmed both mutants still preserved CD59 activity of anticomplement,and glycation of CD59 in CHO increased their sensitivity to MAC-mediated lysis. Conclusions Residues by Site-directed mutagenesis to replace residue 37 or 38 with histidine still preserved CD59 activity of anticomplement.Mutant CD59s can be glycated in vitro and loses its most MAC-inhibitory function.The presence of this glycation motif in human mutant CD59,may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.

Abstract:Aim To investigate vascular endothelial growth factor(VEGF) gene expression and bioactivity in rat cardiomyocytes transfected by a recombinant adeno-associated viral vector 2. Methods Recombinant adeno-associated viral vector 2 encoding the human VEGF 165 were constructed and confected in intro to the neonate rat ventricular myocytes.RT-PCR,Western blot and ELISA were used to detect the expression of VEGF gene,and MTT to detect the biological activity of the conditioned medium after transfection. Results The presence of VEGF165 mRNA in transfected cells was demonstrated by RT-PCR,the expression of VEGF165 protein was confirmed by western blot.The level of VEGF165 protein expressed by transfected cells was higher than that by control accroding to an enzyme-linked immunosorbent assay.The conditioned medium after transfection could stimulate the proliferation of endothelial cells. Conclusion Rat cardiomyocytes transfected by Recombinant adeno-associated viral vector 2 containing the human vascular endothelial growth factor 165 cDNA(rAAV-2/hVEGF165) could express VEGF165 with highly biologial activity,which might be useful for gene therapy of myocardial ischemia.

Abstract:Aim To observe the transcript and protein expression levels of vascular endothelial growth factor(VEGF) in human umbilical vein endothelial cells(hUVEC) cultured with high glucose in vitro and investigate the relationship between it and protein kinase C(PKC) pathway. Methods hUVEC were cultured in vitro with glucose at different concentrations or for different cultural time and cultured with PKC inhibitor GF109203X or PKC activator PMA respectively.The transcript of VEGF was measured by RT-PCR.The protein expression of VEGF and PKCβBⅡ was detected with immunocytochemistry. Results The transcript and protein expression levels of VEGF in high glucose groups or PKC activator PMA group were higher than control group or PKC inhibitor GF109203X group(p<0.05 or p<0.01).Cultured with glucose at 22 mmol/L,the mRNA and protein expression of VEGF increased in 72 h,the protein expression of VEGF reached the summit at 72 h,and then began to decrease(p<0.01).Cultured with PMA,the transcript of VEGF increased within 6 h,and then decreased.The protein of PKCβⅡ seemed to translocate to cell membrane from nucleus after treated with 22 mmol/L glucose for 2 h.However,PKC inhibitor GF109203X can inhibit the phenomena above. Conclusions The transcript and protein expression of VEGF gene increased in hUVEC,cultured with high glucose.High glucose induced the expression of VEGF gene via PKC pathway in hUVEC.

Abstract:Aim To examine whether the protective effect of epigallocatechin gallate(EGCG) on endothelial cells is associated with reduction of asymmetric dimethylarginine(ADMA) concentration in rats treated with low density lipoprotein(LDL). Methods 40 rats were randomly divided into five groups: control group,LDL group,10 mg/kg EGCG group,50 mg/kg EGCG group and Vitamin E group.Vascular endothelial injury was induced by a single injection of native LDL(4 mg/kg) in rats.Vascular endothelium-dependent relaxation to acetylcholine of isolated thoracic aorta and plasma concentrations of ADMA,nitric oxide(NO),malondialdehyde(MDA) and tumor necrosis factor-α(TNF-α) were analyzed. Results A single injection of LDL markedly inhibited endothelium-dependent relaxation to acetylcholine,increased plasma level of ADMA,MDA and TNF-α,and decreased plasma concentrations of NO.Treatment with EGCG significantly improved the endothelium-dependent vasodilation,decreased the elevated level of ADMA,MDA,TNF-α,and enhanced the decreased level of NO in the rats treatd with LDL. Conclusions The present results suggest that the beneficial effect of EGCG on endothelial cells may be related to reduction of endogenous ADMA concentration.

Abstract:Aim To observe the protective role of unsaturated fatty acid of actinidia chinesis planch seed oil(UFAACPSO) on adriamycin-induced myocardium injury in rat and clarify the mechanism of its action. Methods Forty SD rat were randomly divided into four groups:Control group(physical salts filled into stomach and injected into pleural),ADR group(physical salts filled into stomach and adriamycin injected into pleural),ADR+Lg group (low concentration UFAACPSO filled into stomach and adriamycin injected into pleural),ADR+Hg group(high concentration UFAACPSO filled into stomach and adriamycin injected into pleural).The death rate was calculated.Creatine kinase isoenzyme MB(CK-MB),glutathione peroxidase(GSH-Px),catalase(CAT),copper-zinc-superoxide dismutase(Cu-Zn-SOD) were detected.The expression level of Cu-ZnSOD protain and mRNA were detected by immunhistochemical method and semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR). Results The change rate of heart rate of ADR+Hg group was obviously lower than ADR group(p<0.05),the activity of Cu-Zn-SOD,CAT and GSH-Px of ADR+Hg group were obviously than higher than ADR group(p<0.05),serum CK-MB concentration of ADR+Hg group was obviously lower than ADR group(p<0.01),Cu-Zn-SOD protein,mRNA expression were up-regulated remarkably in ADR+Hg group(p<0.05),and the death rate was reduced in ADR+Hg group.But no significantly difference was not found between ADR group and ADR+Lg group and adriamycin group. Conclusion UFAACPSO could significantly decrease adriamycin-induced myocardium injury in rat,the mechanism of its action may related to it's activity of reducing oxygen radical and inhibiting oxidative stress.

Abstract:Aim To investigate the effects of activated renin angiotensin system in the vascular tissue on production of reactive oxygen species and nitric oxide.Methods Ten male Wuzhishan minipigs were divided into two groups,fed with normocholesterolemic(control;n=5) or hypercholesterolemic(2% cholesterol,hyperlipid,n=5) food for 3 months.Carotid arteries were isolated and the changes of TC,AngⅡ,ROS,NO and T-AOC of the vascular tissue were determined.In addition,hUVEC(human umbilical vein endothelial cell) were incubated with Ang I and chymostatin or with Ang I and losartan respectively for 24 h,the suspernatant was collected to measure the concentration of AngⅡ,ROS and NO. Results In comparison to the normocholesterolemic state,hypercholesterolemia led to a significant increase in TC content(108%±28%,p<0.01),AngⅡ generation (115%±20%,p<0.01) and ROS production(144%±28%,p<0.01),but it led to a significant decrease in NO(51%±5%,p<0.01) and T-AOC(56%±5%,p<0.01).Cultrured hUVEC could convert Ang I to AngⅡ.AngⅡ increased by11 times in the presence of 10 nmol/L Ang I as compared with control group,and the level of ROS also increased while NO decreased significantly.When hUVEC were incubated with Ang I plus 100 or 500 μmol/L chymostatin,AngⅡ generation was reduced by 61%±6% or 65%±7% respectively,and the ROS decreased while NO increased significantly as compared with only Ang I.When hUVEC were incubated with Ang I plus Losartan,the generation of ROS and No was almost the same as compared with control group on condition that AngⅡ generation was not reduced. Conclusion hypercholesterolemia could activate renin angiotensin system of the vascular tissue,which results in an increased vascular tissue production of ROS and a decreased NO.This effect might be mediated by AT1 receptor.

Abstract:Aim To investigate the effect of C.pneumoniae infection on the expression of peroxisome proliferator-activated receptor γ(PPARγ),nuclear factor-κB(NF-κB) and activated protein-1(AP-1) in aortic endothelial cell in hyperlipidemia mice. Methods Forty-eight,8-week-old female C57BL/6J mice were divided into 4 groups: control group,infected group,atherogenic diet group and infected atherogenic diet group(each twelve).14 weeks later,the expression of PPARγ,P50(subunit of NF-κB) and cFos(subunit of AP-1) was determined by indirect immunofluorescence in the aortic endothelial cell.Slides of aortic sinus were prepared by cryosection,and stained with Sudan Ⅳ for examination of atherosclerotic plaque.The score of atherosclerotic plaque was determined by microscopy. Results The score of atherosclerotic plaque in infected group was not increased,while it was significant higher in atherogenic diet group and infected atherogenic diet group(p<0.01),still the score in infected atherogenic diet group was higher than in atherogenic diet group(p<0.01).The expression of PPARγ,NF-κB and AP-1 in endothelial cell in aortic sinus was upregulated in infected group,atherogenic diet group and infected atherogenic diet group,in comparison with that in control group(p<0.05).There was no significant difference among infected group,atherogenic diet group and infected atherogenic diet group. Conclusion The inflammatory process was already initiated in the aortic endothelial cell in C57BL/6J mice at the early stage of Chlamydia pneumoniae infection and hyperlipidemia.Chlamydia pneumoniae infection alone would not accelerate the process of atherosclerosis.But Chlamydia pneumoniae infection could accelerate the process of atherosclerosis.

Abstract:Aim To investigate the effects of Ramipril on expression of lectinlike oxidized low density lipoprotein receptor-1(LOX-1)in a rabbit model of atherosclerosis. Methods Twenty-four male New Zealand white rabbits were randomly divided into three groups: normal diet group,hypercholesterol diet group and Ramipril-treated group.Expression of lectin-like oxidized low density lipoprotein receptor-1 in a rabbit model of atherosclerosis was detected by immunochemistry and reverse transcription polymerase chain reaction(RT-PCR). Results Expression of LOX-1 in hypercholesterol group significantly increased compared with normal diet group.There was a significant dicrease in expression of LOX-1 in the Ramipril-treated group(p<0.05). Conclusion Ramipril inhibits vascular LOX-1 expression,which may play an important role in prevention of atherosclerosis.

Abstract:Aim To investigate the inhibiting effect of 17-β estradiol on insulin resistance induced by the high fructose diet in ovariectomized rats. Methods Thirty six ovariectomized female Sprague-Dawley(S-D)rats were fed with the high fructose diet for eight weeks to induce insulin resistance. Then they were randomly divided into three groups: the model group, the 17-β estradiol replacement group,the vehicle control group.The normal control group twelve rats were fed with the normal diet for eight weeks.Body weight(BW),systolic blood pressure(SBP),glucose tolerance,plasma triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDLC)and low density lipoprotein cholesterol(LDLC),fasting blood sugar(FBS)and fasting serum insulin(FSI)and insulin sensitivity index(ISI)were measured. Results Compared with the normal control group,BW(p<0.05),SBP(p<0.05), the levels of plasma TG,TC,LDLC,FBS(all p<0.05) and FSI(p<0.01) were increased significantly and the plasma HDLC level was decreased significantly (p<0.05) in the model group. Glucose tolerance was decreased significantly(p<0.05)and injury of pancreatic β-cell was observed in the model group.ISI was decreased significantly(p<0.05) and insulin resistance was arisesed in the model group.Compared with the model group,these effects were reversed, ISI was increased significantly(p<0.05) and insulin resistance was inhibited in the 17-β estradiol replacement group. Conclusion 17-β estradiol inhibited insulin resistance,injury of pancreatic β-cell and lipid dysbolism induced by the high fructose diet in ovariectomized rats.These findings suggested that estradiol in female rat might provide protection againt the effect of the high fructose diet,which caused hypertension,insulin resistance,injury of pancreatic β-cell and lipid dysbolism.

Abstract:Aim To investigate inhibiting effect of hepatocyte growth factor(HGF) on endothelial apoptosis induced by advanced glycosylation end products(AGE) and its possible mechanism. Methods Human umbilical vein endothelial cells(hUVEC) were cultured in vitro and intervened by different concentration of AGE and HGF.The cell inhibiting rates of each group in different time course(12,24,48 and 72 h)were measured by methyl thiazolyl tetrazolizm(MMT) assay,the early stage apoptosis was detected by flow cytometry with Annexin V-FITC/PI double staining,morphology of cell apoptosis was observed by acridine orange fluorescence staining,and the expression of apoptosis-associated genes Bax and Bcl2 were detected by western blotting.The activity of caspase-3 was detected by enzyme-linked immunosorbent assay. Results Morphological observation indicated that high AGE induced characteristic apoptotic changes in hUVEC.Within a certain concentration range,hUVEC apoptosis inducing rates by AGE were both in concentration-and time-dependent manner.HGF significantly inhibited the apoptosis of hUVEC induced by AGE(p<0.05).High AGE significantly increased Bax protein,but not Bcl-2,whereas HGF significantly promoted the expression of Bcl-2(p<0.01) and decreased the activity of caspase-3(p<0.05) without affecting Bax level. Conclusions AGE can induce the apoptosis of endothelial cells in vitro.HGF effectively attenuate high AGE-induced apoptosis through upregulating Bcl-2 gene expression and inhibiting caspase-3 activation.

Abstract:Aim To observe the influence of simvastatin on tumor necrosis factor α(TNF-α)-induced expression of metalloproteinase-9(MMP-9)in human umbilical vein endothelial cell(hUVEC)and to investigate the non-lipid mechanisms of 3-hydroxy-3-methyl-glutaryl coenzyme A(HMG-CoA)reductase inhibitors on anti-atherosclerosis. Methods The expression of MMP-9 in protein level and mRNA level was detected with immunohistochemistry and reverse transcription polymerase chain reaction(RT-PCR). Results TNF-α could induce the expression of MMP-9 in hUVEC.Simvastatin could inhibit the expression of MMP-9 and their inhibitory effects were concentration-dependent and time-dependent. Conclusion The inhibition of TNF-α-induced MMP-9 expression in hUVEC by simvastatin may crucially contribute to the clinical benefits of HMG-CoA reductase inhibitors on coronary artery disease,independent of cholesterol-lowing effects.

Abstract:Aim To elucidate how advanced glycation end products(AGE) effected cyclooxygenase-2(COX-2) expression in cultured human umbilical vascular endothelial cells (ECV304). Methods ECV304 were cultured in vitro with AGE-human serum albumin(AGE-HSA).The levels of protein COX-2 were measured by Western blot. Results COX-2 expressed little in ECV304,AGE-HSA could induce COX2 expression,and the expression could be blocked by inhibiting the activation of NF-κB. Conclusion AGE-HSA could induce COX-2 expression by activing NF-κB.This pathobiological effect of AGEs might contribute to vascular lesion.

Abstract:Aim To compare the effects of intermittent and constant high glucose media on the production of vascular relaxing factors in cultured human umbilical vein endothelial cells(hUVEC). Methods hUVEC were grown to confluence and were exposed for 7 days to 5 mmol/L glucose(normal control),20 mmol/L glucose(constant high glucose) and 5 mmol/L alternating with 20 mmol/L glucose(intermittent high glucose) on a daily basis.The concentration of 6-keto-PGF1α(the stable hydrolysed product of prostacyclin) was determined by radioimmunoassay,the nitric oxide(NO) was measured by means of Griess reaction and the malondialdehyde(MDA) content was detected by the method described by Schuh. Results Both the 6-Keto-PGF1α and NO in intermittent high glucose group were significantly lower than that in constant high glucose group(21±6 ng/L vs 36±8 ng/L,p<0.01;13.6±2.0 mmol/L vs 18.2±3.7 mmol/L,p<0.001 respectively).However,the intermittent glucose group had increased MDA(16.5±2.7 mmol/L vs 13.2±2.2 mmol/L,p<0.05) compared with constant glucose group. Conclusion These findings suggest that variability in glycemic control could be more deleterious to endothelial cells.

Abstract:Aim To investigate the correlation between uric acid and circulating endothelial progenitor cell(EPC)in patients with coronary artery disease(CAD)and its clinical significance. Methods 78 cases were divided into 3 groups: stable angina pectoris(SAP)group(n=18),acute coronary syndrome(ACS)group(n=24)and control group(n=36),and CAD patients were divided into 3 groups according to the result of coronary angiography: single vessel disease group(n=18),double vessels disease group(n=14)and triple vessels disease group(n=10).The concentration of serum uric acid was measured with method of uric acid enzyme.Total mononuclear cell were isolated from peripheral blood by Ficoll density gradient centrifugation,and were cultured in M199 medium supplemented with 20% fetal bovine serum,50 μg/L vascular endothelial growth factor(VEGF).After 14 days cultured,the number of colony-forming units of EPC were counted by phase-contrast microscope. Results The uric acid level in the CAD group was significantly higher than those in control group(p<0.01),and there was no statistical difference between the uric acid level of single vessel disease group and that of multiple vessels disease groups(p<0.05).The number of colony-forming units of circulating EPC in CAD group(including SAP and ACS groups)was significantly lower than those in control group(p<0.01),and the circulating EPC level of single,double,triple vessels disease group were significantly lower than that of control group(p<0.01). It was also observed there was a strong negative correlation between the concentration of serum uric acid and the number of colony-forming units of circulating EPC(r=-0.382,P=0.037). Conclusions The serum uric acid concentration in CAD group was higher than that of control group,but the levels of uric acid were independent of the degree of coronary artery disease.The levels of circulating EPC of CAD patients were lower than those of healthy person.Uric acid and EPC were associated with CAD and the severity of coronary artery lesion,and the level of uric acid was negatively correlated with the number of colony-forming units of circulating EPC.These data indicate that uric acid may impair EPC-mediated reendothelialization after endothelial cell injury by inhibiting the number of the EPC,and uric acid and EPC are correlated with the degree of patient's condition and the clinical situation of cardiovascular disease.

Abstract:Aim To investigate the relationship of platelet-derived growth factor(PDGF-A) and the pathogenesis of atherosclerosis(As),the expression of PDGF-A in atherosclerosis(As)plaque of arteriosclerosis obliterans(ASO)patient and normal arterial wall were studied. Methods The protein level and area of PDGF-A in As plaques and normal artery of human were studied by means of immunohistochemistry. Results The arterial wall of ASO patient wasn' t smooth,and there were very few normal endothelial cell but a lot of foam cell.In contrast,the normal arterial wall was very perfect,endothelial cell and smooth muscle cell stood on line. The data of immunohistochemistry showed that the expression of PDGF-A was augmented significantly in As plaque of ASO patient.In contrast,in normal arterial wall the expression of PDGF-A was very low(p<0.01). Conclusions The data suggests that the increased expression of PDGF-A in As plaques of ASO related to the pathogenesis of ASO.PDGF-A play very important roles in the formation and progression of atherosclerosis.

Abstract:Aim To investigate TFPI V264M polymorphism in Chinese and its effect on plasma levels of free tissue factor pathway inhibitor(f-TFPI) and severity of acute coronary syndrome(ACS). Methods The genotypes of V264M were detected by PCR and PCR-RFLP in 136 ACS patients and 106 controls undergoing coronary angiography,then the serum f-TFPI level in 57 cases was inspected by enzyme linked immunosorbent assay. Results The allele and genotype frequencies were consistent with that predicted by Hardy weinberg equilibrium in the present study population(χ~2=0.437,p<0.05).Plasma f-TFPI level was significantly lower among carriers of Aallel as compared with non-carriers(GG genotype)[6.9±5.1) vs(14.3±9.5)].ACS group showed significantly higher plasma level of f-TFPI than control group(p<0.05).No meaningful corelationship was found between ACS group and control group for V264M polmorphism(p<0.05). Conclusion The V264M polymorphism do have an effect on the serume f-TFPI level but have no relationship with the occurrence and the severity of ACS.

Abstract:Aim To study the effect of the mutation D~(442)→G in cholesteryl ester transfer protein gene on the level of serum high density lipoprotein cholesterol. Methods 189 normal subjects were randomly collected,cholesteryl ester transfer protein gene D~(442)→G mutation were examined by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP),and the serum lipids of the subjects were determined. Results The allelic frequencies of the cholesteryl ester transfer protein 15 D~(442)→G in the group(58 subjects) with high density lipoprotein cholesterol≥1.7 mmol/L and the control(131 subjects) were 6.03% and 1.53% respectively(p<0.05),the results showed that there was statistical difference between the two groups.The level of high density lipoprotein cholesterol in the group with the cholesteryl ester transfer protein gene exon 15 D~(442)→G was higher than the control group(p<0.01). Conclusion Exon 15 D~(442)→G mutation in cholesteryl ester transfer protein is related to the elevated level of serum high density lipoprotein cholesterol.