Abstract:Aim To test whether the transgenic mice with expressed human SR AI on endothelial cells are susceptible to atherosclerosis on high fat high cholesterol diet (HFHC). Methods TghSR AI+/+, TghSR AI+/ , TghSR AI / from F3 mice and C57BL/6 mice were fed 1.25% cholesterol, 16% fat and 0.5% sodium cholate for 14 weeks. Tissues of heart and aorta, liver, kidney and others from control and transgenic mice were prepared for morphological analysis. To evaluate the degree of atherosclerosis in the transgenic and control mice, atherosclerotic lesions were quantified in the aortic root area with computer assisted image analysis system in all four groups. Results After animals were fed HFHC diet for 14 weeks, extensive atherosclerotic lesions were found at the junction of the aorta to the heart and the aortic sinus, including valve, valve commissures in the aortic wall of TghSR AI+/+ and TghSR AI+/ transgenic mice. Typical lesions of aorta in transgenic mice was stained with oil red O. In TghSR AI+/+ and TghSR AI+/ mice, the mean lesion area per section was 144 864±17 103 μm 2 and 111 323±10 713 μm 2, respectively, p<0.05 (n=6). In TghSR AI / and C57BL/6 mice, the mean lesion area per section was 79 887±9 228 μm 2 vs 66 305±7 991 μm 2, p>0.05 (n=6). However, the lesion area of TghSR AI+/+ and TghSR AI+/ mice was significantly higher than lesion area of TghSR AI / or C57BL/6 mice. Conclusions The transgenic mice are more susceptible to atherosclerosis than C57BL/6 on HFHC diet. These mice may be of value for use as an in vivo test system for studied of pharmacological or genetic interventions of atherosclerosis.

Abstract:Aim The purpose of the present work was to study the inhibitory effect of a triple helix forming oligonucleotides (TFO) on expression of tissue factor (TF) gene in endothelial cells. Methods Antiparallel oligonucleotides T21GTa sequence targeted to shear stress responese element in promoter of TF gene were designed and synthesized by phosphoramidite method and decorated with all PS alinkage. The affinity of TFO and TFO ps were determined by electrophoretic mobility shift assays. Cellular uptake of γ 32 P labelled TFO ps and the effect of TFO ps on expression of TF gene were observed in ECV304 endothelial cell strain. Results TFO ps (T21GTa ps) formed a triplex binding in antiparallel orientation to the puring rich target strand SSRE, with the Kd 3.6×10 10 mol/L. In endothelial cell strand ECV304 the celluar uptake rate of TFO ps (T21GTa ps) is about 11.65%, mainly localized within the nucleus sediment (77.25%),significantly lower the mRNA expression of and the protein synthesis of TF gene, strikingly decreased the coagulation activity of TF. Conclusions TFO ps(T21GTa ps) have significantly inhibited expression of TF gene in endothelial cell.

Abstract:Aim Identification of genes that differentially expressed during foam cell formation is important for understanding the molecular basis of atherosclerosis. Methods We used suppression subtractive hybridization(SSH) to isolate differentially expressed cDNA species in foam cells induced by incubation of U937 cells in the presence of oxidized LDL. This led to identification of more than twenty differential expressed sequence tags(EST) after further cloned and sequenced and homology searched in Genbank with software advanced BLAST 2.0. Results EST FRG4(a 273 bp cDNA) and FRG14(a 346 bp cDNA) had very low homology with sequence in Genbank, which may be new genes. Moreover, the results from RT PCR confirmed that FRG4 and FRG14 are indeed genes induced by ox LDL. The sequences have been assigned the database accession numbers in Genbank as below: BI50286, BI502587. Also we have found other genes such as human ubiquitin proteasome pathway and human retinoblastoma binding protein 4 differentially expressed in foam cells. Conclusions Suppression subtractive hybridization(SSH) is a kind of high efficient method to clone differentially displayed genes in tester and driver. We have found genes or ESTs may be related genes of atherosclerosis.

Abstract:Aim To explore the relationship between hypertriglyceridemia and blood coagulation. Methods Establishing experimental animal models of endogenous and exogenous hypertriglyceridemia in rats. Determining the levels of plasma triglyceride (TG),total cholesterol (TC), high density lipoprotein cholesterol (HDLC), blood glucose (BG) of the 12～14 h fasting rat, and observing the relationship between plasma lipids and blood platelet (PLT) count, clotting time (CT), fibrinogen (FG), prothrombin time (PT), activated partial thromplastin time (APTT), activity of tissue plasminogen activator (t PA) and plasminogen activator Inhibitor 1 (PAI 1). Results Feeding high carbohydrate diet (HCD)to rats for 3 and 6 days, plasma TG were 2.43 and 2.9 times of the normal dietary rats, respectively (p<0.001). BG were also 80% and 39% higher than that of control group (p<0.01). After taking high fat diet (HFD), plasma TG were 2.3 and 1.6 times of the control group, respectively (p<0.05 or p<0.001). Levels of plasma TC, HDLC in both groups were not obviously different compared with the control group. Feeding HCD and HFD for 3 and 6 days, PT and APTT were obviously shorter than that of control group (p<0.05). After taking 6 days of HCD and 3 days of HFD, FG were 84% and 122% higher than that of control group (p<0.05 or p<0.01). PAI 1 activity of HCD and HFD were higher than control groupm (p<0.01 or p<0.05). CT, PLT and t PA activity were not different compared with the control group. The correlation analysis indicated that TG was positively correlated with FG and PAI 1activity (p<0.01), and negatively correlated with PT and APTT (p<0.05). TC and BG were positively correlated with FG (p<0.01), and HDLC was positively correlated with CT and PT (p<0.01 or p<0.05). Conclusion These results suggest that increasing in triglycerides enhance the activity of clotting system and inhibit the activity of fibrinolytic system.

Abstract:Aim To study effect of homocysteine (Hcy) on taurine uptake in cultured rat vascular smooth muscle cells (VSMC). Methods In cultured VSMC of rats, cellular 3 H taurine uptake were determined by 3 H taurine incubation. Taurine transport velocity was measured by radio ligand method. Results Hcy (10～500 μmol/L) could inhibit taurine transport in a dose dependent manner. As compared to control group, 100 and 500 μmol/L Hcy significantly decreased taurine transport. The maximal transport velocity (Vmax) decreased by 31% (p<0.05) and 51% (p<0.01), respectively. There were no significant changes in the Km value. 5 mmol/L H 2O 2 inhibited taurine transport. Vmax decreased by 48% (p<0.01), but Km value increased by 45% (p<0.01). Taurine could increase taurine transport that Hcy inhibited. Conclusions Hcy could inhibit taurine transport; The exogenous taurine could efficiently inhibit taurine transport dysfunction by Hcy induced.

Abstract:Aim To observe whether protein phosphorylation and dephosporylation in nuclei play roles in the development of myocardial hypertrophy and distribution of protein kinases and phosphatases in cell fractions were determined. Methods The model of hypertensive rat was established by abdominal aortic constriction. Velocity and isopyknic gradient centrifugation was employed to fractionate rat myocardium to membrane, cytosole and nuclei. Enzymatic methods were employed to determine kinases and phosphatases. Results Compared with control group, the activity of mitogen activated protein kinase (MAPK) increased by 82.03% (p<0.05) in nuclear, changed without significance in membranous fraction, whereas declined by 53.69% (p<0.01) in cytosolic fraction; the activity of protein kinase A (PKA) in different fractions were close to those of control; the activity of PPase 2A increased by 44.95% (p<0.05) and 36.75% (p<0.05) respectively in nuclear and membranous fractions, changed without significance in cytosolic fraction; the activity of PPase 2B increase by 43.57% (p<0.05) in nuclear and changed without significance in membranous and cytosolic fractions; the activity of PPase 2C in different fractions were close to those of control. Conclusions Nuclear MAPK, PPase 2A and PPase 2B might be involved in developing overload induced cardiac hypertrophy.

Abstract:Aim To explore the effect of neuropeptide Y on lipid metabolism in human vascular smooth muscle cells. Methods The human vascular smooth muscle cell culture model was used for experiments. The expression of low density lipoprotein receptor in cultured human vascular smooth muscle cells were quantitatively assayed by the immunofluorescent staining under laser scanning confocal micropscope. Results The averaged fluorescent value in control group was 2443±189,while the values in neuropeptide Y groups were 2015±164,1890±145,1841±123 and 1786±136,decreasing by 26.92%, 24.64%, 22.62% and 17.49%(p<0.01),respectively in 10 8 mol/L, 10 7 mol/L, 10 6 mol/L and 10 5 mol/L concentrations of neuropeptide Y, compared with control group. There was significant differences among the different concentrations neuropeptide Y groups in a dose dependent manner. Conclusion Neuropeptide Y may influence the lipid metabolism of human vascular smooth cells by downregulating low density lipoprotein receptor in human vascular smooth muscle cells.

Abstract:Aim To observe the influence of glucose on the polyol pathway in human vascular endothelial cells and to investigate its potential mechanisms. Methods Human umbilical vein endothelial cells were cultured in vitro with glucose at different concentrations or for different cultural time. The level of nitric oxide (NO) and sorbitol, aldose reductase (AR) AR activity and the expression of AR mRNA level were detected with high performance liquid chromatography (HPLC), nitrate reductase method,biochemical assay and reverse transcription polymerase chain reaction (RT PCR). Results The levels of sorbitol in high glucose groups were higher than in control group (p<0.05 or p<0.01), but the levels of NO in high glucose groups were lower than in control group (p<0.05 or p<0.01). Both AR mRNA expression and its activity were dependent on glucose concentrations or cultural times (p<0.05 or p<0.01) AR mRNA expression and its activity with glucose at 22 mmol/L for 48 h or at 44 mmol/L for 24 h did not increase accordingly, but began to decrease(p<0.05). Conclusion Glucose can affect the function of endothelial cells such as the increasing prodction of sorbitol and the decreasing systhesis of NO. The mechanisms may be that glucose can upregulate AR gene expression and enhance its activity, then activates the polyol pathway in endothelial cells.

Abstract:Aim To investigate the effect of captopril on endothelin (ET) and angiotensin Ⅱ (AngⅡ) and their relationship to proto oncogene in atherosclerotic rabbits. Methods 53 male Japan white rabbits were assigned randomly to the control group, cholesterol food (CF) group and high dose (HD ), middle dose (MD ), low dose (LD ) captopril plus cholesterol food group(Cap group), which were fed for 12 weeks. Levels of ET and AngⅡ were determinated by radioimmunoassay and expression of proto oncogene c myc and c fos mRNA were observed by in situ reverse transcription PCR (ISRT PCR) technique. Results (1) Levels of ET and AngⅡ were significantly increased in CF group; There were positive significantly correlation between the change of ET and AngⅡ in different experiment stage. Levels of ET and AngⅡ were significantly decreased in HD Cap group. (2) Positive sign of pro oncogene c myc and c fos mRNA are remarkably increased in proliferated intima, positive sign of pro oncogene c myc and c fos mRNA are remarkably decreased in HD Cap group. (3)There were positive significantly correlation between the change of ET and expression of proto oncogene c myc or c fos mRNA (r=0.9263, p<0.01; r=0.8541, p<0.01). Conclusion The level of ET varied closely with change of AngⅡ and expression of proto oncogene c myc and c fos mRNA. Furthermore, only captopril of high dose may have therapeutic applications to prevent atherosclerotic development effectively.

Abstract:Aim To eclucidate whether Heparin binding epidermal growth factor (HB EGF) and platelet derived growth factor A (PDGF A) were implicated in the pathogenesis of atherosclerosis. Methods 16 healthy chinese rabbits were randomly divided into common feed group and high lipid feed group. After 12 weeks the levels of HB EGF mRNA and PDGF A mRNA in the aortic walls were measured by a reverse transcription polymerase chain reaction (RT PCR)assay. Results The levels of HB EGF mRNA were significantly higher in atherosclerotic rabbit aortic walls in high lipid feed group than in common feed group, while the levels of PDGF A mRNA were significantly lower in atherosclerotic rabbit aortic walls in high lipid feed group than in common feed group. Conclusion HB EGF might play important roles in the formation of atherosclerosis, while PDGF A might have no effects on the progression of atherosclerosis.

Abstract:Aim To explore the protective effect of Vitamin C on the human umbilical vein endothelial cell (hUVECs) injured by lipid peroxidation and its probable mechanism. Methods Confluent hUVECs were incubated with Vitamin C for 24 h and consequently exposed to lipid peroxides induced by high concentration of Copper ions. Lactate dihydrogenase (LDH) activity was measured in cell medium and lysate. Endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production induced by acetylcholine were assessed by endothelial nitric oxide synthase and nitric oxide kits. Results Lipid peroxides enhanced markedly LDH activity and decreased nitric oxide bioavailability via the inhibited eNOS activity (p<0.01). These effects were attenuated by administration of different dose Vitamin C (100 μmol/L, 300 μmol/L); the correlation between eNOS activity and NO production was significant (r=0.9844, p<0.01; r=0.8802, p<0.05, respectively). Conclusions Lipid peroxides could injury hUVECs and inhibit eNOS activity directly. Vitamin C might play a protective role in hUVECs injured by lipid peroxidation.

Abstract:Aim To observe the effect of liver regulating and turbid removing Chinese medicine formular to apolipoprotein content in blood and lipoprotein receptor in membrane of hepatocyte and research the principle of preventing and curing atherosclerosis. Methods The rabbits were fed with heavy dosage of cholesterol in order to obtain the mould of atherosclerosis by assaying the content of apolipoprotein AI and apolipoprotein B in blood. In addition, the hepatocyte of rat was cultured in vitro by adding ox LDL into the culture medium, and the effect of lipoprotein receptor and the regulating function of Chinese medcine formular related with given in different ways by means of hybridization in situ were observed. Results and Conclusions Liver regulating and turbid removing Chinese medicine formular can increase effectively the content of apolipoprotein AI, decrease that of apolipoprotein B and have a good regulating function about the lipoprotein receptor or passway. Consequently, liver regulating and turbid removing Chinese medicine formular can regulate the factors related with lipid metabolism, thus effectively prevent the formation and progress of hyperlipemia.

Abstract:Aim To investigate the mechanisms of hypertensive microalbuminuria and the benefit of early intervention with losartan. Methods The investigation comprised normotensive control (WKY) and hypertensive group (SHR) of rats, of which blood pressure, microalbuminuria, renal function and relevant ultrastructural indexes in vessel and glomerulus with transmission electronic microscope were acquired at three month old and eight month old. Results In three month old SHR, electronic microscopic observation demonstrated early impairment of vessel endothelium and slight lesion of glomerular basement membrane compared with age matched controls. As a result, microalbuminuria that correlated with blood pressure and impairment of endothelium has been shown. At eight month old, creatinine level has not elevated in spite of the aggravation of impairment of vessel endothelium and lesion of glomerulus, which was associated with microalbuminuria. However, microalbuminuria was unrelated to blood pressure at this stage. With the treatment of losartan, not only reduction of microalbuminuria was achieved but also decrease in volume density of vessel endothelium, mesangial cells and mesangial area was shown. Conclusions The mechanism led to microalbuminuria in hypertension may vary according to the severity of the hypertensive condition. Early intervention with losartan can inhibit glomerulosclerosis more than lower blood pressure.

Abstract:Aim To investigate the effects of Doxazosin on the proliferation of rat vascular smooth muscle cells (VSMCs) cultivated in vitro. Methods Various concentrations of Doxazosin were added to the cultured rat VSMCs. 3 H TdR incorporation were used to detect proliferation of VSMCs. Results Doxazosin inhibited the proliferation of VSMCs. The proliferation of NIH3T3 cells,which do not express α 1 receptors, also were inhibited. Pretreatment of cells with phenoxybenzamine, an irreversible α 1 receptor antagonist, did not chang the effect of Doxazosin. Conclusions These results suggest that doxazosin inhibited the proliferation of VSMCs through a mechanism unrelated to α 1 receptor.

Abstract:Aim To explore the molecular mechanisms of the change of whole blood viscoelasticity in the patients with coronary heart disease (CHD) and its clinical significance. Methods Blood lipids, erythrocyte membrane microviscosity (η), sodium pump activity (SPA), relative content of spectrin (SP), erythrocyte deformability parameters (α) and dynamic modulus (G'), dynamic viscosity (η') were measured in 34 CHD with slight coronary atherosclerosis (As), 32 CHD with complicated coronary As and 35 controls. Results Blood lipids, η, G' and η' were significantly higher, but SPA, SP and α were significantly lower in the CHD than those in controls and the changes of these parameters were more obvious along with the development of coronary As. SPA was correlated negatively to η and positively to SP content and α; G' was correlated negatively to α and positively to η'; SP content was positively correlated with α in CHD. Conclusion The increase of whole blood viscoelasticity was closely associated with the increase of blood lipids, η of erythrocyte membrane and the decrease of erythrocyte SPA, SP content and α, and the decreased α and increased viscoelasticity play an important role in the course of coronary As.

Abstract:Aim To study the relationship of polymorphisms of methylenetetrahydrofolate (MTHFR) gene and plasma homcysteine (Hcy) levels with myocardial infarction. Methods 178 myocardial infarction patients and 178 normal subjects were recruited in the study. Their polymorphisms of MTHFR gene were analyzed using PCR RFLP and their plasma total Hcy levels were measured using high performance liquid chromatography with fluorescence detection. Results There were three kinds of genotype: TT (homozygous mutation), TC (heterozygous mutation) and CC (wild type). The frequencies of the three genotypes were as follows: TT, 35.4%; TC, 53.8%; CC, 10.8% in myocardial infarction patients and TT, 20.1%; TC 55.8%; CC, 24.1% in normal subjects, respectively. The frequency of T alleles was significantly higher in myocardial infarction patients than that in normal controls (62.3% and 37.7%, respectively). Mean total plasma homocysteine concentrations were significantly higher in myocardial infarction patients than in the normal subjects. Conclusions These results suggest that polymorphisms of MTHFR gene and hyperhomocysteinemia may be an independent risk factor for myocardial infarction.

Abstract:Aim To investigate the quantitative correlation between coronary calcium score (CS) determined by electron beam computed tomography (EBCT) and angiographically atherosclerotic plaque burden. Methods In 320 patients who were clinically suspected to have coronary artery disease and had undergone coronary angiography, EBCT was performed for calculating calcium score. The relation of calcium score to atherosclerotic plaque burden was quantitatively analyzed. Results The calcium score ranged from 0 to 3 346, with a median of 45. The mean natural logarithm transformation of calcium score ) was 3.64±2.18. LN(CS+1) was poorly related to stenosis severity (r=0.385, p<0.001) and well related to the total score of plaque burden (r=0.768, p<0.001). Conclusion Calcium score determined by electron beam computed tomography correlates intimately with plaque burden but poorly with stenosis severity.

Abstract:Aim To investigate the relationship between glycopropean complex of activated platelets (PAC 1) and cervical atherosclerosis in type 2 diabetic patients. Methods PAC 1 were measured by FCA in 118 type 2 diabetic patients (73 with cervical atherosclerosis, 45 without cervical atherosclerosis). The correlation of PAC 1, age, diabetic courses, blood pressure, blood lipids, blood glucose and renal function with cervical atherosclerosis was evaluated. Results The level of PAC 1 in diabetic group was significantly higher than that in control group. PAC 1 in diabetic group with cervical atherosclerosis was higher than those without cervical atherosclerosis. Moreover PAC 1, age, disease course, plasma glucose, GHbA 1, blood pressure and low density lipoprotein cholesterol (LDLC) was positively correlated with cervical atherosclerosis. Conclusion The high level of PAC 1 is the independent risk factor for diabetic patients suffering from cervical atherosclerosis.

Abstract:Aim To evaluate the clinical effect of delayed thrombolytic therapy with low dose urokinase in acute myocardial infarction. Methods 66 cases of acute myocardial infarction with 12 h to 24 h after onset were divided randomly into two groups, low dose UK group (n=30) and aspirin plus heparin group (n=36). In low dose UK group, low dose UK 5 kIU/kg was given per half hour for 5 consecutive days. Results The number of patients with frequent angina was significant decrease in patients treated with low dose UK as compare with patients in aspirin plus heparin group, the differences were significant (first week: 26.7% vs 36.1%, p<0.05). The rate of heart failure was much lower in low dose UK group than aspirin plus heparin group, the differences were significant (13.3% vs 33.3%, p<0.05). There was no statistically significant difference in four week mortality(3.3% vs 2.8%, p>0.05). Frequency of shock, cardiac arrhythmias, adverse bleeding actions had no statistical significance between the two groups. Conclusion The number of patients with frequent angina and the rate of heart failure might be reduced by using low dose UK in acute myocardial infarction with 12 h to 24 h after onset.

Abstract:Aim To introduce a multilocus genotyping assay for candidate genotypes of cardiovascular disease (CVD) established by Roche Molecular Systems (RMS). Methods By applying the regents, materials and the protocol provided by RMS, genotyping was done for the Chinese from different regions (Beijing and Hong Kong) and a French population. Results By comparing the allelic frequencies between the Chinese from different regions and between the Chinese and the French populations, no significant difference was found for each allelic frequency between the two Chinese groups, but some allelic frequencies significantly differed between the Chinese and the French populations. Conclusions This multilocus genotyping assay established by RMS is a rapid, convenient and efficient method in determining the genotypes possibly related to the development of CVD in a large scale population study for the genetic risk of CVD.