Abstract:Aim To study the effects of chloride channels on cultured bovine cerebrovascular smooth muscle cells (CSMC) Ca 2+ store operated Ca 2+ influx. Method Cell culture and single intracellular free Ca 2+ concentration was measured in Fura 2/Am flueorescence probed by MetaFluor Fluorescence Ratio Imaging System. Results ① Chloride channel inhibitors 4,4' diisothiocyanostibene 2,2' disulphhonate (DIDS,0.75 μmol/L) decreased Ca 2+ influx significantly induced by endothelin 1 (ET 1),adenosine triphosphate (ATP),cyclopiazonic acid (CPA);Subsequent addition 5 nitro 2 (3 phenylpropylamino) benzoate (NPPB,10 μmol/L) further produced the decrease effect. ② NPPB (10 μmol/L) decreased Ca 2+ influx significantly induced by ET 1,ATP,CPA;Subsequent addition DIDS (0.75 μmol/L) also further produced the effect of decreasing. Conclusion Chloride channel inhibitors DIDS, NPPB inhibited Ca 2+ store operated Ca 2+ influx induced by ET 1, ATP, CPA. DIDS,NPPB sensitive chloride channels participated in the signal transduction of Ca 2+ store operated Ca 2+ influx in cerebrovascular smooth muscle cells.

Abstract:Aim To explore the changes and significance of adrenomedullin(ADM) mRNA and receptor activity modifying protein 2(RAMP2) mRNA in calcified myocardial cells. Methods Calcified myocardial cells was prepared with β glycerophosphate. Content of ADM in VSMC was measured by radioimmunoassay(RIA). The amount of ADM mRNA and RMAP2 mRNA was determined by competitive RT PCR. Results The content of calcium and ADM in calcified myocardial cells was increased by 138% and 136.9%(all p<0.01), respectively, compared with control. The amount of ADM mRNA and RMAP2 mRNA in calcified myocardial cells was elevated by 24% and 25%( all p<0.05), respectively, compared with control. The elevated level of RAMP2 mRNA was parallel to that of ADM mRNA in calcified myocardial cells. Conclusions Content of ADM was increased, and gene expression of ADM and RAMP2 was up regulated in calcified myocardial cells, which suggested that ADM/RAMP2 may have important regulator role in process of calcification of myocardidal cells.

Abstract:Aim to study the role of caveolin 1 in vascular smooth muscle cells (VSMC) cholesterol homeostasis. Method Western blot and RT PCR was performed to evaluate the effects of native low density lipoprotein (n LDL) or oxidized low density lipoprotein (ox LDL) on expression of caveolin 1 in VSMC cellular cholesterol efflux, cholesterol content and lipid droplets of VSMC were determined by using cholesterol detection, HPLC and nile red stain respectively Results The results showed that n LDL (50 mg/L) increased caveolin 1 expression by 60.2%±3.9%, and promoted cholesterol efflux rate to 81.7%±4.3%. However, ox LDL (50 mg/L) downregulated caveolin 1 abundance by 54.7%±5.8% and deduced cholesterol efflux rate to 26.8%±5.1%. In addition, the cholesterol efflux rate also significantly decreased when VSMC harboring caveolin 1 antisence. Caveolin 1 overexpression by transfected Pcl neo cav 1 in VSMC results a dramatically decrease in total cellular cholesterol content and lipid droplets induced by ox LDL (50 mg/L). Conclusions Caveolin 1 mediates cellular cholesterol efflux in vascular smooth muscle cells, which is an important mechanism of regulating cellular cholesterol balance. Dowmregulation caveolin 1 expression is involved in foam cell formation derived from vascular smooth muscle cell induced by ox LDL.

Abstract:Aim To investigate the nucleolar changes and the effect of heat shock protein 70 (HSP70) during the oxidative stress using an injury model of neonatal rat cardiomyocyte induced by hydrogen peroxide (H 2O 2). Methods The nucleolar impairment induced by 0.5 mmol/L H 2O 2 was observed with toluidine blue staining and electronic microscopy. The protective effect of HSP70 on nucleolar damage was investigated with heat shock pretreatment and antisense oligonucleotides of HSP70. Results H 2O 2 (0.5 mmol/L) significantly increased numbers of stained nucleolar granules, and resulted in obvious segregation of nucleolar components as indicated by staining with toluidine blue and electronic microscopy. Western blot analysis demonstrated that heat shock pretreatment induced expression of HSP70. Heat shock pretreatment significantly relieved the nucleolar impairment induced by H 2O 2. Moreover, it was showed by immunocytochemistry that H 2O 2 mediated HSP70 translocation from cytoplasm to nucleus and nucleolus in cardiomyocyte. Additional investigation showed that HSP70 antisense oligonucleotide can remarkably block the protective effect of HSP70 against the nucleolar injury induced by H 2O 2. Conclusions The nucleolar impairment of primary cultured cardiomyocyte can be induced by H 2O 2 and be protected by HSP70, which is over expressed and translocated to the nucleolus in the process.

Abstract:Aim To separate and purify human recombinant apolipoprotein E (Apo E) from Escherichia coli strain BL21(DE3). Methods PET32a Apo E constructs were transformed into BL21 and protein expression was induced. Apo E thioredoxin fusion protein was separated by Ni 2+ affinity column. After digestion of thioredoxin by thrombin, Apo E was purified by Sephacryl S 300 gel filtration column. Results Apo E₂, Apo E3 and Apo E4 were produced by this method, with high yield and high purity. Conclusions A new system for separation and purification of human recombinant Apo E was successfully established.

Abstract:Aim To study the structural changes of basilar arteries during hypertension and the effects of captopril. Methods The animal model of stroke prone renovascular hypertensive rats might be divided into two groups, hypertension group and captopril treated group; one sham operated group was used asnormotensive control group. Each group took the observation of the structure of the basilar arteries under light microscope and transmission electrical microscope as well as analyses of morphometry at 4, 8, 12 weeks respectively. Results There were no obvious microscopic changes in basilar arteries of the hypertension group postoperation 4 weeks. The media thickness and wall to lumen ratio of the hypertension group were higher than those of captopril and sham operated groups (p<0.05). The intercellular spaces are broadened slightly at 4 weeks after operation; there were moderate ultrastructural damages at the end of 8 weeks, with the organelle oedema and partial lysis, interstitial dropsy; there were obvious ultrastructural damages at the end of 12 weeks, such as smooth muscle cell myofilament degeneration, fragmentation, lysis, and endoplasmic reticulumnecrosis dilataltion, mitochondria vacuolation, smooth muscle cell necrosis. The ultrastructure appearances of the captopril treated group were nearly normal. Conclusions Hypertension can cause hypertrophy and ultrastructural damage of basilar arteries. Captopril can prevent media hypertrophy of basilar arteries in experimental hypertensive rats and protect the ultrastructure against injury factors.

Abstract:Aim To establish a new technique for rapid isolation full length cDNA sequence of human novel gene from cDNA library using hot start PCR. Methods The library vector specific primers and gene specific primers have been designed for performing hot start PCR to attain the full length cDNA of differential expressed sequence tag(FRG4) of U937 foam cell formation induced by ox LDL from human fetal liver cDNA library. The products of PCR are cloned to pGEM T vector and sequenced. Results We have successfully attained the full length cDNA sequence of FRG4. Conclusion This technique is a rapid and efficient method for isolating full length cDNA sequence of human novel gene from cDNA library.

Abstract:Aim To investigate the long term effect of dietary fiber complex (DFC) B at different levels on cholesterol metabolism in hypercholesterolemic rats. Methods 84 healthy SD rats were fed with diet rich in lipids and hypercholesterolemic animal models were established. The rats were randomly divided into 7 groups: hypercholesterolemic control group(HC), 4%DFC group, 8%DFC group, 16%DFC group, 32%DFC group and 64%DFC group. Wheat fiber was used in the hypercholesterolemic control group and rats fed with normal diet were used as control group. Different levels of DFC B were given in the experimental groups. Results The concentrations of plasma total cholesterol (TC),low density lipoprotein cholesterol (LDLC) were all significantly lowered by DFC B at different levels and those of high density lipoprotein cholesterol (HDLC) were significantly elevated compared with hypercholesterolemic control group (p<0.05);Dose response relationship was observed in the effect of DFC on plasma TC. Conclusion DFC composed mainly of Hippophae rhamnoides L bran, oat bran and guar gum can reduce the plasma TC, LDLC and raise HDLC effectively in hypercholesterolemic rats, thus could be used for preventing and therapying atherosclerosis and coronary heart disease.

Abstract:Aim To evaluate the relationship of the erythrocytes immunity and atherosclerosis. Methods Twenty five male rabbits were fed with a high fatty diet for 12 weeks. Serum lipid profile, rosette rate of red blood cell C 3b (RBC C 3bRR), the rosette rate of red blood cell immune complexes (RBC ICR), and circulating immune complexes (CIC) were measured before and after high fatty diet. The intimal medial thickness (IMT) of abdominal aorta were measured by echocardiography at the same time. Results Serum lipid level were elevated,and RBC ICR, CIC, IMT were increased (13.25%± 2.97% vs 20.27%±3.02%, p<0.01; 156.60±40.27 μg vs 537.55±141.95 μg, p<0.01; 0.32±0.02 mm vs 0.89± 0.19 mm , p<0.01), and RBC C 3bRR were decreased (10.36%±2.14% vs 6.44%±1.95%, p<0.01) significantly after 12 weeks high fatty diet compared with base line. Linear correlation analysis demonstrated that CIC has a positive relation with IMT(r=0.63, p<0.01)and RBC ICR(r=0.86, p<0.001). RBC C 3bRR has a negative relation with CIC(r=69, p<0.01). Conclusion The decreasing of erythrocytes immune adhesive ability may be an important risk factor of the atherosclerosis.

Abstract:Aim To investigate the role of angiotensin Ⅱ(AngⅡ) 1 type recepter antagonist and aldosterone (Ald) receptor antagonist in reversing myocardial remodeling in hypertensive rats by blocking renin angiotensin system at receptor level and the mechanism of hypertensive myocordial remodeling. Methods Thirty nine male Wistar rats were devided into the shamo operated group, the hypertension group, the valsartan group [10 μg/(kg·d)] and spironolactone group [40 mg/(kg·d)]. Systolic blood pressure (SBP), left ventricular weight index (LVWI), plasma and myocardial AngⅡ/Ald concentration, myocardial collagen concentration (MCC) and collagen volume fraction (CVF) were measured at the sixteenth week, the twentieth week and the twenty eighth week after operation. The pathologic characterization of myocardial Ⅰ type and Ⅲ type collagen was observed at the same time. Results All measured datas in 2K1C hypertension group were significantly higher than in shamo operated group (p<0.05 or 0.005). Compared with hypertension group, plasma AngⅡ concentration was significantly higher (p<0.05), but SBP, LVWI, MCC mainly Ⅰ type collagen, CVF and plasma Ald concentration in the valsartan group decreased significantly (p<0.05). Treatment with spironolactone lightly decreased SBP, LVWI, MCC and CVF in early, but significantly lowered LVWI, MCC and CVF (p<0.05), especially Ⅲ type collagen. Conclusions It indicated that development of the LVH and myocardial fibrosis is heterochronia. This study suggested AngⅡ and aldsterone play a vital role in hypertensive myocardial remodeling. AT 1 receptor mainly mediats type I collagen deposition while aldosterone receptor mainly mediates type Ⅲ collagen deposition.

Abstract:Aim To investigate the effects of interferon γ(IFN γ) on rat vascular smooth muscle cell(VSMC) proliferation and its expression of proliferating cell nuclear antigen(PCNA). Methods The changes of tritium labelled thymidine ( 3 H TdR) incorporation into rat VSMC and PCNA expression (Western blotting) were observed after 24 h stimulation with IFN γ. Results IFN γ(500 ku/L) markedly suppressed serum stimulated 3 H TdR incorporation into VSMC from 7 865±1 054 cpm/well in the serum group to 4 914±1 078 cpm/well in the co cultured group with serum and IFN γ(p<0.01), and decreased PCNA expression from 167.62±8.03 in serum group to 120.65±10.2(Western blotting, absorbance) in the co cultured group(p<0.01). Conclusions The results suggested that IFN γ inhibit PCNA expression and DNA synthesis of rat VSMC, and suppress VSMC proliferation.

Abstract:Aim To explore the effect of black rice outlayer fraction (BRF) on atherosclerotic plque formation in apolipoprotein E deficient mice and find the possible mechanisms of BRF in anti atherogenesis. Methods After 16 weeks intervention by 5% BRF, the atherosclerotic lesion area in aortic sinus were assessed by the method of image analysis and the serum anti ox LDL antibody levels were determined by ELISA. Results Serum anti ox LDL antibody levels in BRF group were lower than positive group and WRF group. This results correlated with the lesion area in aortic sinus. Conclusion BRF has dramatically reduced serum anti ox LDL antibody levels and this effect may be related to its anti atherogenesis.

Abstract:Aim The purpose of this study was to compare the lipid lowering and anti atherosclerosis efficacy of homemade Simvastatin with Zocor (Simvastatin). Methods 56 rabbits were randomized into four groups. One group was fed with regular chow and did not receive treatments (served as normal control group). The second group was fed with cholesterol enriched diet and served as atherosclerosis group (As group). The other two groups were fed with the cholesterol enriched diet in conjunction with either domestic simvastatin or Zocor in a daily oral dose (domestic Simvastatin group and Zocor group respectively). The hypolipidemic and anti atherosclerosis effect of the above treatments was examined by estimating the levels of serum total cholesterol (TC) and triglycerides (TG). The extent of aortic atherosclerosis was measured by planimetry of the sudanophilic area. Results At the end of 8 and 12 week treatment, levels of TC and TG in As group were increased significantly compared with normal control group at the same phase. The histopathological examination revealed marked alteration in aortic wall with the appearance of large multiple atheromatous plaques in As group, domestic Simvastatin group and Zocor group after treatment, but both the domestic Simvastatin group and the Zocor group were significantly less than that in As group (p<0.01). The total lipids lowering effective rate and anti atherosclerosis efficacy of domestic simvastatin group was equal to Zocor group. Conclusion homemade Simvastatin is an effective medicine for the treatment of hypercholesterolemia and atherosclerosis.

Abstract:Aim To investigate the influence of high density lipoprotein(HDL) and oxidized high density lipoprotein(ox HDL) on secretion of nitric oxide(NO),nitric oxide synthase(NOS),and endothelin 1(ET 1) by ECV 304;To provide evidence for protection effect of HDL on endothelial cells. Methods Different concentrations of HDL and ox HDL were used to treat human umbilical endothelial cell line ECV 304. NO, NOS and ET 1 secreted by ECV 304 were detected with corresponding kits respectively. Results The results indicated that HDL increased NO and NOS secreting,and decreased ET 1 secreting.On the contrary,ox HDL decreased NO and NOS level,and increased ET 1 level in ECV 304. Conclusions HDL might protect endothelial cells and antiatherosclerosis by increasing NO and NOS secreting and decreasing endothelin 1 secreting,but after oxidized such effects were lost.

Abstract:Aim To investigate the features of coronary artery calcification (CAC) determined by electron beam computed tomography (EBCT) in patients with coronary artery ectasia. Methods In 27 patients who were diagnosed as coronary artery ectasia by coronary angiography, EBCTs were performed for calculating calcium score (CS). The calcium scores were compared between patients and angiographically normal subjects matched for sex and age. Results The CAC prevalence, CS median and LN (the mean natural logarithm transformation of calcium score) were all greater in ectasia group than in control group (p<0.01 or 0.001). Coronary artery calcification existed in 17 of the 21 patients with atherosclerotic coronary artery ectasia (81.0%). The mean LN was significantly lower in vessels with diffuse ectasia than in those with discrete ectasia (1.22±1.79 vs 2.86±1.85, p<0.05). Conclusion Coronary artery calcification exists in most of the patients with atherosclerotic coronary artery ectasia, and the extent of calcium is related to the types of ectasia.

Abstract:Aim To explore the relationship between the abnormality of postprandial triglyceride (TG) metabolism and the intimal medial thickness (IMT) in patients with essential hypertension (EH). Methods In 38 EH patients and 30 healthy subjects, TG, high density lipoprotein (HDL), total cholesterol (TC), glucose before and 2,4,6,8 hours after a standardized fat loading and low density lipoprotein (LDL) in fasting serum were measured. Triglyceride peak response (TGPR) and the triglyceride area under curve (TG AUC) over 8 hours were calculated and the index was regarded to show the response level of the subjects to the fat loading. IMT of carotid was detected through two dimensional ultrasound before the fat loading. Results EH patients had higher TGPR (4.68±1.74 vs 1.76±0.64 mmol/L) and TG AUC(23.59±6.48 vs 9.56±3.38 mmol/L,8h)(p<0.05). The net increasing value and net increasing rate of serum TG concentration at 6,8 hours after fat loading in EH group were higher than that of control group. The net increasing value at the two time points of 2,4,6,8 hours were also higher in the patients. Besides,IMT of carotid in EH was higher than that in control group (0.767±0.150 vs 0.603±0.050, P=0.009). From regression analysis, IMT was positively correlated with TG AUC,age,TGPR,BMI (r=0.497,0.448,0.382,0.352,p< 0.05 ), while negatively correlated with fasting HDL level. Conclusions EH patients accompanied with the abnormality of postprandial TG metabolism was correlated with IMT of carotid.

Abstract:Aim To investigate the relationship between angiotensin converting enzyme (ACE) gene polymorphism and microalbuminuria in patients with essential hypertension Methods Polymerase chain reaction (PCR) was used to determine the genotypes for an insertion/deldtion polymorphism of the ACE gene in 50 hypertensive patients with microalbuminuria and 49 hypertensive patients without microalbuminuria and 50 healthy controls. Urine MAlb concentrations were determined by radioimmunoassay. Results There was significant difference in the frequencies of D allele and DD genotype between hypertensive patients with microalbuminuria and healthy controls. The frequencies of D allele and DD genotype was higher in hypertensive patients with microalbuminuria and lower in those without microalbuminuria. However, ACE I/D polymorphism did not differ in hypertensive patients with non microalbuminuria and healthy controls. Conclusion ACE gene polymorphism was closely related with hypertensive microalbuminuria. DD genotype was possibly involved in the renal injury in patients with hypertension.

Abstract:Aim To explore the relationship between carotid artery intima media thickness(IMT) and apolipoprotein E(apoE) genotypes among patients with premature coronary heart disease(CHD). Methods Eight five patients with documented premature CHD were recruited to participate in this study. The carotid artery IMT was checked by ultrasound imaging and apoE genotype was classified with the method of polymerase chain reaction restriction refraction length peptide. The relationship between carotid artery IMT and lipid parameters and apoE genotype were discussed. Results There were five kinds of apo egenotypes. Three main genotype according to the frequency were apoE3/3>apoE3/4>apoE₂/3 and apoE₂/4, and apoE4/4 were rare genotype. The allele of apoE also had different frequencies, ε3>ε4>ε2. Levels of serum TC and LDLC were higher in patients with apoE3/4 genotype than those with apoE₂/3 genotype, in verse to HDLC (p<0.05). There were higher values of IMT in apoE3/4 group than that in apoE₂/3. There was positive correlation between IMT and TC(r=0.401,p<0.01) and LDLC(r=0.376,p<0.01). Conclusions ApoE genotype influence carotid artery IMT at least partly through its lipid effect.

Abstract:Aim To investigate the insulin receptor substrate 1 (IRS 1) gene polymorphism and its relationship with serum lipids and apolipoproteins (Apo) levels in patients with endogenous hypertriglyceridemia (HTG) in Chinese population. Methods The genotype and allele frequency of IRS 1 gene polymorphism was assayed by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). Serum lipids were measured by enzymatic kits and apolipoproteins A_Ⅰ, A_Ⅱ, B₁₀₀, CⅡ, CⅢ and E by RID kits developed by Apolipoprotein Research Unit of WCUMS in 117 HTG patients whose fasting serum TG levels were ≥2.26 mmol/L and 194 healthy subjects whose fasting serum TG levels were <1.82 mmol/L and TC levels <6.2 mmol/L from a population of Chinese Han nationality in Chengdu area. Results Both in HTG group and control group, the GG genotype of IRS 1 gene was the major one, and its frequency was 0.995 and 0.985 respectively. In the HTG group, subjects with the genotype GR had a lower serum ApoCⅢ levels than those with the genotype GG (p>0.05). Conclusion These results suggest that the IRS 1 gene G 972 R polymorphism was not associated with endogenous hypertriglyceridemia in Chinese population.

Abstract:Aim To determine the serum cholesteryl ester transfer protein(CETP) level in patients with coronary heart disease(CHD) in Han nationality and explore its association with serum lipids, lipoproteins and apolipoproteins levels. Methods To undertake a case control study, we detected the serum CETP level in 64 unrelated healthy individuals and 106 patients with evidence of CHD by enzyme linked immunosorbent assay. Results The results showed that there was not statistically different between patients and controls(2.15±0.90 mg/L vs 2.31±1.08 mg/L ,p>0.05). No significant correlation was found between CETP concentration and lipids and lipoproteins concentration(p>0.05). Conclusions There is no significant relation between serum CETP level and CHD in Han nationality from northern China.

Abstract:Aim To study a family of hypertriglyceridemia with the metabolism of the lipids and apolipoproteins. Results The results showed that several mumbers of the family suffer from the hypertriglyceridemia. The serum, from the patients of the hypertriglyceridemia which was laid up icebox over night was turbid observed by naked eye. The level of the total cholesterol (TC) in the serum was normal and the values of triglyceride (TG) increased and the level of the high density lipoprotein cholesterol decreased. Although the concentrations of the apolipoprotien A_Ⅰ, AⅣ and B₁₀₀ had no significantly change, the concentration of the apolipoprotein E (apo E) had raised. The concentration of serum TG is positively correlated with that of serum apo E. Conclusions The family was appointed as a family of type Ⅳ hypertriglyceridemia. Experiments have shown that apo E metabolic disturbance may be one of factors that lead to hypertriglyceridemia. The reason why the family members of this familial hypertriglyceridemia case have high frequency to suffer liver cancer and appendicitis requires further research.