Abstract:Aim To study the role of caveolae and caveolin 1 on atherosclerosis formation in scavenger receptor A_Ⅰ (SR A_Ⅰ) transgenic mice. Methods Wild type and SR A_Ⅰ transgenic mice were randomly divided into control, transgenic, wild type plus high cholesterol diet, and transgenic plus high cholesterol diet groups, fed with normal or high cholesterol diet respectively. Animals were sacrificed after 24 weeks. The area of plaque was measured by electronic computer for image analysis. Immunohistochemical analysis and electron microscopy were used to investigate the expression of caveolae and caveolin 1. Results The atherosclerotic plaque were observed in wild type plus high cholesterol diet group, transgenic group, transgenic plus high cholesterol diet group. The thickness of intima and media were thinner in normal control group than in other three groups (p<0.05). Immunohistochemical analysis showed the caveolin 1 expression in above three groups were lower than that in control group. Furthermore, electron microscopy showed the expression of caveolae were also decreased. Conclusion Decreased expression of caveolae and caveolin 1 is associated with the atherosclerosis formation in SR A_Ⅰ transgenic mice.

Abstract:Aim To investigate the effects of pituitary adenylate cyclase activating polypeptide (PACAP) on vascular remodeling in atherosclerotic rabbits. Methods 60 male New Zealand rabbits were divided randomly and equally into three groups: control group,atherosclerosis (As) group and PACAP group. 5～8 rabbits of each group were sacrificed at the end of the fourth,eighth and twelfth week and the thoracaortae were collected. The segments about 0.5 cm long from the beginning, middle and end part of the above aortae were obtained to be made into paraffin sections and stained with hematoxylin eosin(HE), then the sections were observed by light microscope and the related parameters were measured by image analysis system. The rest part of the aortae were stained with Sudan Ⅳ and surveyed by naked eyes. Results Plaque area and percentage of plaque formation increased gradually with the proceeding of experiment in As group (p<0.05), and plaque mostly located in the beginning segment, decreased in the middle and end segments in turn. Lumen area varied rarely in early plaque formation, while decreased significantly at the advanced stages (p<0.05). Plaque area and maximal plaque thickness in PACAP group were smaller than those in As group, but lumen area was larger than that in As group(p<0.05). Conclusion PACAP can retard the progression of As by inhibiting negative remodeling and promoting positive remodeling.

Abstract:Aim To explore the levels of neurotransmitter of sensory nerves and constrictor peptides of renin angiotensin system in renal vascular hyertensive rats, and the changes of these peptides after treatment with losartan or peridopril. Methods The 2 kidney, 1 cliped rats hypertensive model were used, and the plasma concentration of active peptides were determined by radioimmunoassay method. Results The plasma concentration of calcitonin gene related peptide (CGRP) was slightly raised in hypertensive rats, compared with sham operation group, and treatment with losartan or peridopril had markedly elevated plasma concentration of CGRP as well as angiotensin Ⅱ and rennin activity. Conclusions In the metaphase of renal vascular hypertension, the elevation of CGRP may be a compensation action due to increasing of blood pressure. Chronic treatment with losartan or peridopril not only affects the releasing of CGRP, but also activates the circular renin angiotensin system in renal vascular hypertension.

Abstract:Aim To investigate whether homocysteine (HCY) can induce cultured human umbilical vein endothelial cells (hUVEC) to express regulated upon activation, normal T expressed and secreted (RANTES) protein. Methods After exposure of the cultured hUVEC to HCY at increasing concentrations for 8 h, the RANTES protein expression was determined by immunocytochemistry and Western blot analysis. Results Cultured hUVEC could express RANTES protein. Immunocytochemistry showed the mean absorbance values of RANTES protein expression in hUVEC exposed to HCY at different concentrations (0.1, 0.5 and 1 mmol/L HCY) for 8 h were 0.0434±0.0063, 0.0788±0.0053 and 0.1061±0.0215, respectively, which were significantly higher than that of the control group (0.0200±0.0032). Analysis of variance proved a significant difference between groups (F=319.03, p<0.01). Western blot analysis displayed that exposure of hUVEC to HCY at gradient concentrations (0.1, 0.5 and 1 mmol/L) for 8 h resulted in a 2.29 fold, a 2.63 fold and a 2.78 fold increase in the expression of RANTES protein in the cells, compared with the control group. Conclusions The cultured hUVEC could express RANTES protein, and HCY was able to induce hUVEC to express RANTES protein at a higher level.

Abstract:Aim To investigate the relationship between vascular endothelial growth factor (VEGF) and the development of atherosclerosis (As). Methods 50 male rabbits were divided into two groups: the rabbits in control group received normal feed, each rabbit in As group received the feed with 1.5 g cholesterol every day. The aortas of rabbits were harvested and observed the pathological changes at the end of week 0, week 4, week 8, and week 12. VEGF expression was studied with immunohistochemical techniques in these samples. Results The results of qualitative observation and quantitative measure are coincident. The average plaque thickness, maximal plaque thickness of aortae in As group gradually increased. The percentage of residual luminal area in As group appears the trend of narrowing gradually. At 12th weekend, the average plaque thickness (5.78±3.75 μm) is significantly larger than that at 4th weekend (1.28±0.86 μm)( p<0.05), the maximal plaque thickness reach 26.16μm. Normal arterial segments showed no VEGF expression, while atheromatous lesions showed distinct VEGF expression. Furthermore, VEGF positivity was intensified gradually as the pathology developed. Conclusion VEGF may have some role in the progression of atherosclerosis.

Abstract:Aim Our previous result showed that at the concentration of 25 mg/(kg.d), Momordicin has a distict therapeutic effect on coxsackievirus B3 (CVB3) viral myocarditis. Because nuclear factor kappa B (NF κB) is Key tactor in immunity and inflammatory reaction, we measure the regulation effect of Momordicin to the level of NF κB. Method To purified Momordicin from Momordica as our previous report, 5 groups of animal are used in this experiment: control of virus group (VC group); control of normal group (NC group); control of Momordicin group (MC group); low dose Momordicin therapeutic group (LM group); high dose monordicin therapeutic group (HM group). Animals were killed at 0 d, 3 d, 7 d, 14 d and 21 d, and purified nucleus protein, protein quantifyed by BCA method, then measuring the activity of EMSA and assys the result. Result The activity of NF κB are still measuerable in the DC group at 21 d, and it's activity is higher than the NC group; there are different degree of inhibition effect in HM and LM groups to NF κB activity, and nerely no NF κB was appeared in the HM and MC groups; there are low level NF κB acitivity were found in the NC group. Conclusion The curing effect of momordicin to viral myocarditis may related to it's inhibitory effect to NF κB activity.

Abstract:Aim To study the change of the gene expression pattern of vascular endothelial cells induced by oxidized low density lipoprotein. Methods After human umbilical vein endothelial cells was exposed to oxidized low density lipoprotein(100 mg/L) for 24 h, genes expression was screened with a complementary DNA microarray representing 4 000 different human genes and 96 control genes. Results There were 1 down regulated gene and 3 up regulated genes. The down regulated gene was ubiquitin activating enzyme E1. The up regulate genes were as follows: serum and glucocorticoid inducible protein kinase (SGK), heat shock protein 70 (HSP70), and KM 102 derived reductase like factor (KDRF). Conclusions Oxidized low density lipoprotein could induce the change of gene expression pattern of endothelial cells. It was the first time to find that oxidized low density lipoprotein could induce the change of the gene expression of SGK, HSP70 and KDRF. All the changed genes were related to the stress stimulation. These changes might be related to the dysfunction of endothelial cells induced by oxidized low density lipoprotein.

Abstract:Aim To investigate the effects of one of the flavone puerarin on the apoptosis of the vascular smooth muscle cell (VSMC) and the molecular mechanism of this effect and observe the influence of puerarin on the expression ofdthe relevant genes of apoptosis. Methods By using the method of differential display RT PCR on the puerarin induced rabbit aorta smooth muscle cell (SMC) apoptosis model, the different expressed fragments were recovered, cloned,sequenced and compared with GeneBank databases. Then labeled the probe by 32 P, and observed the gene expression by northern hybridization with the VSMC mRNA stimulated by puerarin in different concentration. Results 1. Some ESTs showed significant higher level of expression in puerarin group than in control group. 2. Three ESTs were isolated from VSMCs stimulated by puerarin, similar to the gene of glucose regulated protein 94 (GRP94) in the rabbit. 3. Northern hybridization showed the high level expression in puerarin treated VSMC. 4. Gray scale scan proved that GRP94 was positive ratio with puerarin concentration. Conclusion The molecular mechanism of the function of promoting apoptosis of VSMC by puerarin perhaps results from promoting the gene expression of the GRP94 of VSMC.

Abstract:Aim To explore the effects of lysophosphatidylcholine (LPC) on cholesterol efflux from apolipoprotein E (apo E) gene deficient mouse peritoneal macrophage foam cells. Methods Cholesterol efflux induced by LPC and apo A_Ⅰ from macrophage foam cells separated from normal and apo E gene deficient mice (E 0) mouse loaded with acLDL were measured by enzymetic fluorometry assay. Results LPC could promote cholesterol efflux from macrophage foam cells in relation to both dosage and time. When LPC was incubated with E 0 mouse macrophage foam cells, the released free cholesterol mass was significantly lower than that of normal mouse macrophage foam cells. It was also found that cholesterol efflux induced by apo A_Ⅰ normally occurred in E 0 mouse macrophage foam cells. Conclusion LPC could induce cholesterol efflux from macrophage foam cells, which may occur via apo E pathway.

Abstract:Aim To investigate the effect of low density lipoprotein(LDL) subfractions on the expression of cell adhesion molecules, such as intercellular adhesion molecule 1(ICAM 1) and vascular cell adhesion molecule 1 (VCAM 1) in cultured human umbilical vein endothelial cells (hUVEC). Methods LDL subfractions, small dense LDL(sLDL) and large buoyant LDL (bLDL), were obtained by two sequential ultracentrifugation, Native sLDL(n sLDL), oxidized sLDL(ox sLDL), native bLDL (n bLDL) and oxidized bLDL (ox bLDL) were incubated with hUVEC at 37℃ for 12 hours. Cell ELISA was used to detect the expression of ICAM 1 and VCAM 1 proteins. Results The expression of ICAM 1 and VCAM 1 proteins were enhanced in the four groups. N sLDL and ox sLDL enhanced the levels of ICAM 1 and VCAM 1 in a dose dependent manner. Compared with the same dosage, ox sLDL was the most potent stimulus for ICAM 1 and VCAM 1 expression at 25 mg and 50 mg, while n sLDL was the most potent stimulus at 100 mg (Expression of ICAM 1 is 0.830±0.09 and of VCAM 1 is 0.37±0.04). Conclusion The sLDL can induce ICAM 1 and VCAM 1 expression in hUVEC, thus influencing the pathogenesis of athersclerosis.

Abstract:Aim To investigate whether antiphospholipid antibody increases the uptaking of oxidized low density lipoprotein and up regulates the expression of CD36 in U937 cells. Methods The cultured U937 cells were incubated with oxidized low density lipoprotein of 80 mg/L and antiphospholipid antibody of 100 mg/L for 48 hours in vitro. Then the contents of cholesterol and cholesterol ester were detected by enzyme fluorescence. The expression of CD36 on U937 was quantified by reverse transcription polymerase chain reaction (RT PCR) and flow cytometry respectively. Results In the present of oxidized low density lipoprotein, the uptaking of cholesterol and CD36 expression increased significantly after by U937 cells incubated with antiphospholipid antibody. Conclusion Antiphosphlipid antibody may accelerate atherosclerosis by up regulating CD36 expression.

Abstract:Aim To study the impact of hyperlipidemia on endothelial lesion during the early stage of atherosclerosis. Methods The investigation included control, hyperlipidemic and gemfibrozil treated groups. Hyperlipidemic model was set up on the 4 week atherogenic diet, followed by a 16 week treatment in the treated group [gemfibrozil 60 mg/(kg·d)]. Serum TC, TG, NO level were measured. Expressions of VCAM 1 and cell adhesiveness on aortic endothelium were observed and analyzed by computer aided system. Results Compared with the control group, hyperlipidemic rats showed lower level of NO, stronger and more expansive endothelial expression of VCAM 1. Moreover there was an increase in leukocyte accumulation on the endothelial surface. In contrast, in treated group, expression of VCAM 1 as well as leukocyte adhesion were significantly decreased which associated with improvement of endothelial dysfunction. Conclusions NO activity would be inhibited by hyperlipidemia which facilitated the endothelial impairment of VCAM 1. Gemfibrozil could prevent atherogenesis by restoring endothelial function.

Abstract:Aim To investigate the cellular signal transduction pathway of vascular smooth muscle cell (VSMC) proliferation stimulated by oxidized low density lipoprotein (ox LDL). Methods Rabbit aortic VSMC was cultured in 3 groups:control, low density lipoprotein (LDL), and ox LDL. Cell proliferating ability was determined by measuring cell number and mitochondrial dehydrogenase activity (MTT assay). Western blotting was used to detect the protein expression of phosphotase and tensin homology deleted on chromosome ten (PTEN). Phosphate concentration released from PTEN specific substrat diC 16 PIP 3 was assessed using green reagent method. Results ox LDL (50 mg/L) increased cell number and MD activity to 1.78～3.21 fold, but the same concentration of LDL has no such mitogenic effect. Phosphatase activity detection shew that the dephosphate action of PTEN was inhibited by ox LDL (20～50 mg/L) in a concentration dependent manner,and this inhibiting effect was at its maximum within 10 min,lasting at least for 24 h (p<0.01, vs. control group). Conclusion Ox LDL improved VSMC proliferation probably by inhibiting the lipid phosphatase activity of PTEN.

Abstract:Aim To discuss the effect of soy isoflavones (Genistein and Daidzein) on preventing the oxidation of LDL mediated by Cu 2+ and to investigate the effect of Genistein on c myc mRNA expression induced by ox LDL in human vascular endothelial cells (ECV304). Methods 1. LDL were isolated from healthy human plasma by gradient ultra centrifugation and oxidized by Cu 2+ . The formation of TBARS and relative electrophoretic mobility were employed to measure the antioxidant activity of Genistein and Daidzein of different concentrations (50, 100 and 200 μmol/L respectively). 2. LDL isolated from healthy human plasma by gradient ultra centrifugation and oxidized by Cu 2+ was used as a stimulus. ECV304 cells were exposed to ox LDL (200 mg/L) in the presence or absence of Genistein 100 μmol/L for 1, 2 and 4 h in vitro. Northern Blot was employed to measure c myc mRNA levels of the treated cells. Results 1. The formation of TBARS and relative electrophoretic mobility of LDL were inhibited by Genistein in a concentration dependent manner (p<0.05 vs control); However, Daidzein made none senses compared with control on the formation of TBARS and relative electrophoretic mobility of LDL (p>0.05). 2. In response to ox LDL 200 mg/L, c myc mRNA expression of hUVEC increased by about three folds in 1 2 h and decreased below the control level at 4 h; Genistein 100 μmol/L effectively inhibited this over expression both at 1 and 2 h. Conclusions Genistein not only prevents the oxidation of LDL but also inhibits the over expression of c myc mRNA induced by ox LDL in ECV304.

Abstract:Aim To discuss the desirable and risk level of triglyceride (TG) and high density lipoprotein cholesterol (HDLC) in cardiovascular diseases. Methods Serum lipids of 2915 middle aged subjects of Beijing professional population were studied during annual physical surveys in year 2001. The effects of different levels of TG on HDLC and TG/HDLC ratio were analyzed statistically. Results HDLC level was relatively low as compared with that of the American whites, less then 5% in this studied group had HDLC below 0.9 mmol/L. According to our long term prospective study of the elderly, HDLC level above 1.03 mmol/L may be considered as desirable. Gradient increase in TG level was accompanied by progressive decrease in HDLC. While TG at 1.14 mmol/L, HDLC levels of both sexes were at desirable level, but with slight decrease in females when the TG level up to >1.7 mmol/L, marked decrease in both sexes when TG increased to>2.27 mmol/L. The acceptable TG/HDLC ratio was about 2 3 (by mg/mg). Conclusion According to this study and our previous experience, we propose that it may be appropriate to adopt the TG and HDLC level classification of ATPШ guidelines of the National Cholesterol Education Program of United States.

Abstract:Aim To explore the effect of apolipoprotein E (Apo E) polymorphism on lipid and essential hypertension. Methods Polymerase chain reaction restricted fragments length polymorphism was used to determine the Apo E genotypes in 112 cases with essential hypertension and 118 controls without hypertension. Results The ε4 allele frequency and the Apo E ε4 carriers was significantly higher in the hypertensive patient group than that in the group of normotensive control (p<0.05). The subjects carrying Apo E ε4 had significantly higher levels of total cholesterol (TC) and low density lipoprotein cholesterol (LDLC) than those carrying Apo E ε2 (p<0.05);the Apo E ε3/ε3 carriers had significantly higher LDLC levels than the Apo E ε2 carriers (p<0.05). The Apo E ε4 was an independent risk factor for essential hypertension (p<0.05). Conclusions The Apo E gene polymorphism affects plasma levels of lipids and lipoproteins. The Apo E ε4 may be an important risk factor for essential hypertension.

Abstract:Aim In order to study effects of estrogen on endothelium dependent vasodilatory function. Methods 32 healthy postmenopausal women regarded as estrogen replacement therapy (ERT) group were treated with conjugated equine estrogen 0.625 mg per day for two weeks, and 24 healthy premenopausal women were regarded as control group. Flow mediated vasodilatory response(FMD) of the brachial artery in ERT and control group were measured by use of high resolution ultrasonography method. Plasma lipids, nitric oxide(NO) and estradiol(E₂) level were also measured. Results Compared with premenopause, plasma E₂ and NO level were significantly decreased(p<0.001), and FMD of the brachial artery also were significantly reduced in postmenopausal women (FMD: 10.05%±3.18% vs. 3.84%±2.18%, p<0.001). After short term ERT, plasma E₂ and NO level were significantly increased(p<0.001), and FMD of the brachial artery also were significantly improved in postmenopausal women (FMD: 9.16%±3.02%, p<0.001), while plasma lipids had no significantly changes before and after ERT. Multiple linear stepwise regression analysis showed that FMD of brachial artery was positively related to NO and E₂(r: 0.729, 0.564, respectively, p<0.001), and was negative related to TC level (r:-0.369, p<0.05). Conclusions Short term ERT can reverse endothelium dependent vasodilatory dysfunction in postmenopausal women. This improvement is not dependent on plasma lipids level, but may be related to the direct vascular protective effects of estrogen, which may be involved in the anti atherogenic action of estrogen.

Abstract:Aim To investigate the clinical characteristics of coronary artery calcification (CAC) detected by electron beam computed tomography (EBCT) in female patients with syndrome X. Methods In 26 consecutive patients with syndrome X (abnormal exercise test results and angiographically normal coronary arteries) and 22 normal group women (normal exercise test and angiographically normal coronary arteries) with chest pain, EBCT was performed. Meanwhile, the concentration of serum lipid and plasma oxidized low density lipoprotein (ox LDL) was detected; the risk factors of coronary artery disease were evaluated as well. Results The prevalence of coronary calcification in the syndrome X group was 73% (19 in 26 patients) compared with 18% (4 in 22 patients) in the normal group (p<0.05). The mean risk factors of coronary artery disease in the women with syndrome X and positive CAC were greater than those of the normal group (1.8±1.3 vs. 1.1±0.9, p<0.05), while the concentration of plasma ox LDL was higher too (52.38±6.89 vs. 39.92±7.87, p<0.05). However, the mean CAC score of the syndrome X group with positive CAC was greater than the normal ones with positive CAC, and the number of calcification was much greater, despite postmenopausal or not menopausal. Conclusions EBCT may be useful in detection of atherosclerosis in women with syndrome X who demonstrate normal coronary arteries at angiography. Careful evaluation and necessary therapy should be done in women with syndrome X and positive CAC.