Abstract:Aim To construct short hairpin RNA(shRNA)retroviral vector of PKB/Akt and study the effect on the proliferation of vascular smooth muscle cell(VSMC).Methods The sequence of shRNA of PKB/Akt was constructed by the software and synthesized by chemical method and then was cloned into pGEM-T vector,which was certified by DNA sequencing.The sequence was inserted into a retroviral vector pLXIN,then the vector was packaged in PT67 cells.The efficiency of inhibition was verified by Northern blot and Western blot after transfection to VSMC.The change of p70s6k was also determined at the same time.The proliferation activity was determined by flowcytometry and MTT.Results The PKB/Akt shRNA vector was successfully constructed by means of recombinant DNA technology and cell transfection.Additionally,the vector could efficiently decrease mRNA and protein expression of PKB/Akt,while the expression of p70s6k also decreased significantly.The cell cycle VSMC was also stunned in phase G0/G1.Conclusion The PKB/Akt shRNA retroviral vector has been constructed successfully,which can significantly inhibit the proliferation of VSMC.

Abstract:Aim To explore the role of endothelial gap junctions in intercellular communication and vascular endothelial wound repair.Methods Rat aortic endothelial cells(RAEC)were cultured by explanted rat aortic wall tissue.Cell immunofluorescence staining was applied to detect the expressions of connexin(Cx)37,Cx40 and Cx47 in RAEC.Fluorescence redistribution after photobleaching(FRAP)was used to measure the communications between cells via gap junctions.The monolayer of cultured RAEC was scraped by a mechanical method and the endothelial wound healing rate was quantified by an analysis of the photographs taken every 24 h after endothelial cell layers were wounded.Meanwhile,18α-glycyrrhetinic acid(18α-GA),a specific blocker of gap junction,was administered to observe its effect on endothelial wound repair.Results Cx37,Cx40 and Cx47 were all expressed in RAEC.Fluorescent dye could only be transferred between conjugated cells,and mean fluorescence recovery rate in isolated cells were significantly lower compared with that in conjugated cells(5.70%±0.63% vs 82.26%±1.68%,p<0.01).Compared with control group,mean fluorescence recovery rate in 18α-GA group were significant lower(53.58%±1.73% vs 82.26%±1.68%,p<0.05).Therefore,18α-GA could inhibit dye transfer between conjugated cells.The scrape width of endothelium were similar in two groups at the time of wound.However,at 24 h after endothelial wound,the scrape width in 18α-GA group were significantly bigger than those in control group(237.38±20.40 μm vs 126.29±21.40 μm,p<0.05).The complete recover time of the wound in 18α-GA group were significantly more than those in control group(4.2±0.2 d vs 2.6±0.3 d,p<0.05).Conclusions There are intercellular communications via gap junctions among conjugated RAEC and 18α-GA could inhibit the intercellular communication via gap junctions,slow down endothelial wound repair speed and prolong the complete recover time of endothelial wound.Hence,endothelial gap junctions might play a vital role in the process of endothelial wound repair.

Abstract:Aim To investigate the effect of atorvastatin on oxidized low density lipoprotein(ox-LDL)-induced lipid accumulation and CD36 expression in THP-1 macrophages.Methods THP-1 macrophages were co-incubated with 50 mg/L ox-LDL and different concentrations of atorvastatin(0,0.312,1.25 and 5 μmol/L)for 24 h,and the unmixed THP-1 macrophages culture was used as the control group.3H-labeled Cholesterol influx was determined by FJ-2107P type liquid scintillator.Oil red O staining was used to observe the intracellular lipid droplets.Cellular total cholesterol was determined by high performance liquid chromatography analysis.CD36 mRNA and protein levels were determined by reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting respectively.Results Atorvastatin decreased CD36 in both protein and mRNA levels.Atorvastatin decreased cholesterol influx in THP-1 macrophages.The levels of cholesterol influx were 35.90%±2.36%(control),47.10%±3.18%(0 μmol/L atorvastatin),41.20%±2.88%(0.312 μmol/L atorvastatin),35.10%±2.35%(1.25 μmol/L atorvastatin),28.30%±1.98%(5 μmol/L atorvastatin)respectively.Oil red O staining showed intracellular lipid droplets were minor with the presence of atorvastatin.High performance liquid chromatography analysis demonstrated the amount of cellular total cholesterol were 78.24±11.35 mg/g(control),123.13±15.92 mg/g(0 μmol/L atorvastatin),115.36±13.18 mg/g(0.312 μmol/L atorvastatin),107.52±12.05 mg/g(1.25 μmol/L atorvastatin)and 98.03±10.24 mg/g(5 μmol/L atorvastatin).Conclusion Atorvastatin may downregulate ox-LDL-induced CD36 expression and decrease lipid accumulation in THP-1 macrophages.

Abstract:Aim To investigate the effect of niacin on serum leptin levels in hypercholesterolemic rabbits and the expression of leptin,peroxisome proliferator-activated receptor γ(PPARγ)and CD36 in adipocytes from hypercholesterolemic rabbits.Methods Twelve rabbits fed with high-cholesterol diet for 8 weeks were randomly divided into two groups:high cholesterol group(n=6)maintained on high cholesterol diet for 6 weeks,niacin group(n=6)fed with the same cholesterol diet plus niacin 0.2 g/(kg·d)for 6 weeks.Control group(n=6)was fed with normal diet for 14 weeks.Subcutaneous adipose was collected for RNA analysis.Leptin levels in serum and adipocytes culture supernatant were measured by enzyme linked immunosorbent assay(ELISA).Reverse transcription polymerase chain reaction(RT-PCR)was used to evaluate leptin,PPARγ and CD36 mRNA expressions in adipose and adipocytes.Results Compared with control group,rabbits fed with high cholesterol diets showed higher levels of serum and adipocyte leptin,all of which were significantly reduced by niacin treatment.Leptin mRNA expression of adipose was significantly lower in rabbits treated with niacin than those fed with high cholesterol diet.Leptin mRNA expression of hypercholesterolemic rabbits by niacin was negatively correlated with the mRNA expression of PPARγ and CD36 by niacin.Niacin dose-dependently inhibited leptin secretion and increased CD36 and PPARγ expression in cultured adipocytes.Conclusion Niacin can reduce serum level and adipose mRNA expression of leptin,and upregulate PPARγ and CD36 mRNA expression in hypercholesterolemic rabbits.

Abstract:Aim To investigate the impact by delivery of different number of endothelial progenitor cells(EPC)on repair of injured vessels.Methods Mononuclear cells(MNC)were isolated from rabbit peripheral blood by density-gradient centrifugation.MNC were cultured in endothelial growth medium for 7 days,yielding EPC.Autologous EPC(5×105 cells or 2×105 cells in 100 μL saline)or 100 μL saline alone(control)were administered into a rabbit model of balloon carotid injury.Meanwhile the cells were labeled by CM-DiI for cells tracking.Four weeks after transplantation,rabbits were killed.Fluorescence-labeled EPC,endothelial regeneration rate and interal area/media area(IA/MA)ratio were detected.Results Four weeks after transplantation,fluorescence-labeled EPC were detected within the media,neointima and on the luminal surface of injured vessel.Local transplantation of EPC compared with saline administration markedly accelerated endothelialization of denuded vessel,especially in the group of 5×105 EPC.Meanwhile EPC treatment significantly reduced neointimal formation.Conclusions Delivery of EPC to balloon-injured arteries is associated with accelerated reendothelialization and reduced neointimal formation.More notable effect may turn out when larger number of EPC are deliverd.

Abstract:Aim To investigate the special pharmacological effect of phenytoin on the healing process after ballon injury in rat carotid artery.Methods Study was performed on rat model of balloon-injury in common carotid artery.Rats survived from successful operation were divided into phenytoin group and injury group.On the 28th day after operation,rats were anesthetized,then the left carotids and the corresponding part of the right carotids were separated,embedded with paraffin,sectioned into 5 μm and stained.Results On the 28th day after operation,the intima area(0.154±0.018 mm2 vs 0.204±0.054 mm2,p<0.01),intima/media area(1.70±0.08 vs 2.26±0.46,p<0.01)and the rate of restenosis(59.5%±3.2% vs 75.9%±13.3%,p<0.01)in phenytoin group were all less than those in injury group,while lumen area(0.106±0.024 mm2 vs 0.063±0.034 mm2,p<0.01)was larger than that in injury group,cell density in intima(72.18±20.08/cm2 vs 84.85±10.77/cm2,p<0.05),proliferating cell nuclear antigen(PCNA)positive cell counting(9.89±7.63 per 200 magnification vs 23.03±13.95 per 200 magnification,p<0.01)and α-smooth muscle actin positive cell counting(30.91±20.05 per 200 magnification vs 61.81±16.57 per 200 magnification,p<0.01)were all smaller than those in injury group;under polarized light,collagen could be visualized deposite in intima,but there were no statistic difference in collagen area and density(p>0.05).Conclusion 28 days after vascular injury,phenytoin can decrease the cell number in neointima and promote the synthesis of extracellular matrix which result in diminished neointima thickening.

Abstract:Aim To investigate the effect of long-term folate supplementation on endothelial function of thoracic aortea in rats with type 2 diabetes mellitus and explore its mechanisms.Methods After the rat model of type 2 diabetes mellitus was established by a single injection of low dose streptozotocin(STZ)after 6 weeks of high calorie diet,the animals were divided into model group,low-dose folate group and high-dose folate group.Other 11 rats were employed as normal control group.The levels of nitric oxide(NO),superoxide dismutase(SOD)and maleic dialdehyde(MDA)in the serum were determined.Acetylcholine-induced endothelium-dependent vascular relaxation(EDVR)of aorta rings was tested.Results The levels of NO and SOD in serum were significantly lower and the level of MDA in serum was notably higher in model group than those in normal control group;After 11 weeks of folate supplementation,compared with model group,the levels of NO and SOD in serum significantly increased in low-dose folate group and high-dose folate group,while the level of MDA in serum decreased markedly.Compared with normal control group,EDVR responses to acetylcholine(Ach)of each concentration were obviously weakened in model group.Compared with model group,EDVR responses to Ach of each concentration were significantly improved in low-dose folate group and high-dose folate group,and further increment of folate led to no further improvement of EDVR response.Conclusion Long-term folate supplementation can effectively prevent the EDVR impairment.The effect may be associated with the increase of NO formation and its antioxidant effect.

Abstract:Aim To investigate the effects of asymmetric dimethylarginine(ADMA)on the proliferation of endothelial progenitor cell(EPC)of human umbilical cord blood and observe whether L-arginine can antagonize the effects of ADMA.Methods Mononuclear cells were isolated from fresh cord blood and cultured for 7days,attached cells were incubated with different concentration of ADMA(1,5 and 10 μmol/L)among different times(24,48 and 72 h).MTT assay was used and colony forming units(CFU)were quantified to evaluate the proliferation of EPC after treated with ADMA.Meanwhile,MTT assay was used and DiI-acLDL uptake cells under high-power field of fluorescence microscopy were quantified to assay effects of L-arginine on ADMA-induced EPC proliferation.Nitric oxide(NO)and nitric oxide synthase(NOS)in the supernatant were measured by nitrate reductase assay and chemical colorimetry assay.Results Incubation of EPC with ADMA dose and time-dependently decreased the number and the proliferation of EPC.In addition,the depressant effect of ADMA on EPC were dismissed with the inceasing dosage of L-arginine.Conclusions It is suggested that ADMA can promote endothelial dysfunction by means of depressant EPC proliferation and exogeno L-arginine maybe antagonize the effects of ADMA.

Abstract:Aim To investigate the effect of oxidized low density lipoprotein(ox-LDL)on proliferation,apoptosis,and expression of bcl-2 of endothelial progenitor cells(EPC)from human umbilical cord blood in vitro.Methods Total mononuclear cells were isolated from human umbilical cord blood in vitro by Ficoll density gradient centrifugation and then the cells were plated on fibronectin-coated culture dishes.After 7 d of culture,attached cells were stimulated with different cencentrations of ox-LDL(5,10 and 20 mg/L)for 48 h.EPC were identified by demonstrating the expression of CD34,KDR and CD133 under a laser scanning confocal microscope.MTT assay was used to detect the effect of ox-LDL on the multiplication ability of EPC.Flow cytometry was used to detect the apoptosis caused by ox-LDL.The expression of bcl-2 genes mRNA and protein were detected respectively by RT-PCR and immunohistochemistry technology.Results After exposure to ox-LDL,the proliferation of EPC was lower than that of control group,apoptosis rate was higher than that of control group(p<0.01),and it was dose-dependent in experiment range;expression of bcl-2 mRNA and protein were down-regulated(p<0.05).Conclusion Ox-LDL can inhibit the proliferation of EPC and promote the apoptosis of the cells by down-regulation of bcl-2 expression,which may contribute to vasculogenesis and reparation of blood vessel endothelium.

Abstract:Aim To investigate the relationship between glucose fluctuation and subclinical atherosclerosis(As)in type 2 diabetic patients.Methods A random-controlled study was used,170 type 2 diabetic patients(35～70 year-old,duration≤1 year)without As were randomly divided into 4 groups:group A,B,C and D.Each group was given different treatment.All patients accepted following-up once a month.Fasting blood sugar(FBS)and postprandial blood sugar(PBS)were measured after breakfast each time.Other metabolic variables were measured,including body mass index,waist circumference,glycosylated hemoglobin,plasma lipid,ultrasound measurement of the carotid and femoral artery intimal-medial thickness and plaque.Results Over three-year follow-up,data of four groups show no statistical difference in blood glucose fluctuation.Following blood glucose variation intensify,the morbidity of subclinical As increase.Logistic regression analysis show that the risk factors related to subclinical As are smoking,hyperlipemia history,age,WHR and PFSD.Conclusions The instable margin of PBS and FBS may be one of risk factors to subclinic atherosclerosis development in type 2 diabetic patiants.Smoking and hyperlipemia history,age,waist-to-hip ratio are all risk factors to subclinic atherosclerosis in type 2 diabetic patients.

Abstract:Aim To explore the relationship between brachial-ankle pulse wave(baPWV)and flow-mediated vasodilation(FMD)in patients with coronary heart disease(CHD).Methods 98 patients with CHD and 33 control subjects were recruited in the study.baPWV velocity was measured non-invasively using VP 1000 automated ABI/PWV analyzer.Flow-mediated vasodilation(FMD)in the brachial artery was measured by high-resolution ultrasound.Results FMD was significantly reduced in CHD patients compared with that in control subjects(5.4%±2.5% vs 11.1±4.4%,p<0.01);and baPWV was increased in CHD group as compared to the control group(1745.3±215.2 cm/s vs 1495.3±202.3 cm/s,p<0.01).There was a significant correlation between baPWV and FMD in patients with CHD(r=-0.70,p<0.001).Conclusions There were increased baPWV and impaired endothelial function in patients with CHD.The correlation between flow-mediated vasodilation and brachial-ankle pulse wave velocity was high.

Abstract:Aim Aim To study the relationship between arginine vasopressin(AVP),brain natdurefic peptide(BNP)and left ventricular hypertrophy(LVH)in patients with essential hypertension(EH).Methods There were 71 cases of essential hypertension,36 of whom were complicated with left ventricular hypertrophy(LVH)and 35 of whom were complicated with non left ventricular hypertrophy(NLVH).There were 28 cases in normal control group.Echocardiography examination was performed to determine left ventricular mass index(LVMI)and the ratio of early diastolic E and late diastolic A(E/A).The plasma AVP and BNP levels were measured by radioimmunoassay(RIA).The linear correlation analysis was made among AVP,BNP,LVMI and the ratio of E/A.Results ①The plasma AVP and BNP levels were higher in EH patients with NLVH and LVH than in normal Subjects(p<0.01),the plasma AVP and BNP levels were higher in patients with LVH than those in NLVH(p<0.01).The LVMI were higher in EH patients with NLVH and LVH than in normal subjects(p<0.01),the LVMI were higher in patients with LVH than those in NLVH.The the ratio of were lower in EH patients with NLVH and LVH than in normal subjects(p<0.01).②The plasma BNP level was positively corelated with LVMI(r=0.552,p<0.01)and negatively corelated with the ratio of E/A(r=-0.803,p<0.01).The plasma AVP level was positively corelated with The plasma BNP level(r =0.437,p<0.01)and negatively corelated with the ratio of E/A(r=-0.506,p<0.01).Conclusions ①The increase of plasma AVP level may play some role in the pathogenesis of LVH in EH patients,and result to decrease of the 1eft ventricular diastolic function(LVDF).②The level of plasma BNP maybe taken as a important marker for the judgement of LVH and LVDF.

Abstract:Aim To investigate clinical characteristic of women ≤45 years old with coronary heart disease.Methods Coronary risk factors,angiographic characterization and revascularization of young women ≤45 years old(group A,n=123)were retrospectively analyzed,compared with that of women >45 years old(group B,n=119).Results Among group A,64(52.0%)patients had hypertension,21(17.0%)patients had diabetes,32(26.0%)patients had hypercholesterolemia,each of which was higher than that of group B(p<0.05);43(35.0%)patients had a history of myocardial infarction,which was similar to group B.Group A were more likely to have angiographically zero and single-vessel disease(p<0.05),and less likely to have three-vessel disease(p<0.05)compared with group B.Drug therapy and single-vessel coronary percutaneous intervention were more often performed for group A than group B(p<0.05).Conclusions Women ≤45 years old had relatively less coronary risk factors and less extensive disease,and hypertension was an important risk factor.No lower morbidity of myocardial infarction was revealed.More attention should be paid on prevention and therapeutic management of young women with coronary heart disease.

Abstract:Aim To observe the efect of cilostazol on the serum levels of inflammatory cytokine in acute coronary syndrome.Methods 60 selected patients were randomly divided into two groups:basic treatment group(conventional medication plus atorvastatin)and cilostazol group(basic treatment plus cilostazol).The change of some indicator levels,including blood lipids,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),high sensitivity C-reactive protein(hs-CRP)was measured after three weeks of treatment.Clinical therapeutic effect and adverse reactions was monitored.Results After the treatment,the serum levels of cholesterol,triglyceride and low density lipoprotein cholesterol(LDLC)in both groups decreased slightly(p<0.05),but there was no significance in the decreased degree between both groups.The increase of high density lipoprotein cholesterol(HDLC)was observed in both groups,but the increased degree was significantly higher in cilostazol group than in basic treatment group(p<0.01).The serum levels of TNF-α,IL-1β,hs-CRP declined in both groups(p<0.05),but the degree of reduction of them was greater in cilostazol group than in basic treatment group(p<0.01).The reduction of TNF-α and IL-1β was negatively correlated with the increase of HDLC in cilostazol group(r=-0.38,P=0.04;r=-0.39,P=0.03).Medication group could obviously ease chest pain with no adverse reactions.Conclusion The use of cilostazol joint atorvastatin on the basis of conventional treatment,compared with atorvastatin alone,could furtherly increased HDLC,reduced inflammatory cytokine concentration,promoted the stability of atherosclerotic plaque.

Abstract:Aim To discover the level and the relationship of the homocysteic acid(HCY),blood fat,fasting blood glucose(FBG)and glycosylated hemoglobin(HbAlc)in the type Ⅱ diabetes with eyeground vascular sclerosis.Methods The HCY,blood fat,FBG and HbAlc were detected in the 37 type Ⅱ diabetes with eyeground vascular sclerosis,compared with those of 45 healthy cases.Results The HCY,triglyeride(TG),total cholesterol,apolipoprotein A,FBG and HbAlc were more higher than those of healthy person(p<0.05 or 0.01).Conclusion The levels of the HCY,TG,FBG,HbAlc in the type Ⅱ diabetes with eyeground vascular sclerosis related to degree of eyeground vascular desease.

Abstract:Aim To investigate the coronary artery lesion characteristics and its clinical implications in patients with acute non-ST-segment elevation myocardial infarction(NSTEMI).Methods 83 patients with acute NSTEMI and 109 patients with acute ST-segment elevation myocardial infarction(STEMI)were enrolled in the study with complete clinical and coronary angiographic data.Comparative analysis was done between the two groups,according to the following aspects such as the number of diseased coronary vessels,the coronary artery stenosis degree,the formation of collateral circulation and the part of clinical features.Results Patients of NSTEMI had less single branch lesions than patients of STEMI,there was a significant difference between them(p<0.05);Three branch lesions in NSTEMI group were more than that in STEMI group,significant difference was found(p<0.05);coronary artery lesions were more than that in patients of STEMI when stenosis degree was not less than 90%,but less in NSTEMI group when stenosis degree was less than 90%,they both have significant differences(p<0.05);cases of total coronary occlusion were similar between the two groups(p>0.05);collateral circulations above grade TIMIⅡ were more likely to see in the patients of NSTEMI,significant difference was also found(p<0.05).All the above coronary artery lesion characteristics decided the clinical features of patients with NSTEMI:the case history of previous angina pectoris and old myocardial infarction occurred more commonly(p<0.05);the peak value of CK-MB was obviously lower(p<0.01);serious acute complications such as acute pulmonary edema,cardiogenic shock and fatal arrhythmia were also remarkablely lower(p<0.05).LVEF,the mortality of patients with coronary heart disease being followed up for 3 years and patients among the hospitalization period showed no significant difference between the two groups(p>0.05);but the postinfarction angina pectoris increased obviously in NSTEMI group than that in STEMI group(p<0.05).Conclusions Complex coronary artery lesions,multiple-vessel lesions and severe stenosis,also the existed ischemic preconditioning before attack and collateral circulations were more evident in patients with acute NSTEMI,this is the main reason the non-ST-segment elevation myocardial infarction came into being and that decided the clinical features of patients with acute NSTEMI.

Abstract:Aim To evaluate the relationship between high-density lipoprotein cholesterol and intracoronary in-stent restenosis during angiographic follow-up.Methods 370 patients were successfully underwent coronary stent implantation with a follow-up study of angiography.They were divided into restenosis group and non-restenosis group.In-stent restenosis was defined as luminal diameter narrowing of intracronary stent by 50% or greater.Results In 370 patients,487 coronary stents were implanted.In-stent restenosis occurred in 123 lesions of 94 patients.Serum concentration of high density lipoprotein in in-stent restenosis group was significant higher than that in non in-stent restenosis group(p<0.05).Multivariate logistic regression analysis revealed that low high density lipoprotein was an independent risk factor of in-stent restenosis.Conclusion There is a close relationship between serum high density lipoprotein and in-stent restenosis.The occurrence of in-stent restenosis may be reduced by regulating serum high density lipoprotein.

Abstract:Aim To assess the current epidemic characterization of myocardial infarction and risk factors among rural adult groups in Fuxin county of Liaoning province of China,and to provide scientific basis for preventing and intervening measurement of stroke in the rural area for government.Methods 30 214 resident people aged more than 35 years were collected with a multi-stage,stratified clustering sampling scheme in seven small towns of Fuxin county of Liaoning province,China.The blood pressure measurement and correlative risk factors about myocardial infarction were investigated by trained doctors.Results The prevalence rate of myocardial infarction for middle and old aged group in rural of Liaoning province was 0.29%,and the standardized prevalence rate was 0.28%.The prevalence and standardized rate of myocardial infarction for male were 0.26% and 0.25%,for female were 0.32% and 0.31% respectively.The difference between the male and female was not significant(0.90,P=0.342).With the age and grade of blood pressure increasing,the prevalence rate was obviously up.By Logistic regression,risk factors of age,overweight,obesity,family history of hypertension,hypertension,smoking,like-eating salty food and so on were close to myocardial infarction occurring.The fatalness of suffering from myocardial infarction among persons who had obesity was 6.08 times(95%CI:2.84～13.03)of the same age “healthy” groups.Conclusions Myocardial infarction was highly prevalent in rural areas of Liaoning province.Obesity and hypertension ranked the distinguished risk factors.However most residents were highly short of prevention about myocardial infarction.We should improve the awareness of risk factors and correlation knowledge on myocardial infarction to prevent myocardial infarction in rural population of Liaoning province.