Abstract:Aim To study the role of nitric oxide (NO) in the preconditioning of the heart with hyperthemia in rats. Methods The isolated rat heart was perfused in a Langendorff model. Hearts for all groups were subjected to 4 h hypothermia (4℃) and 40 min reperfusion. Heat stress was induced by whole-body hyperthermia (rectal 42℃, 15 min) 24 h before the experiment. Heart rate, coronary flow, left ventricular pressure, and its derivative (±dp/dt max ) were recorded, and tumor necrosis factor-alpha (TNF-α) immunoreactivity in myocardial tissues and the activity of creatine kinase (CK) in the coronary effluent were measured. Results Pretreatment with hyperthermia significantly improved the recovery of cardiac protection, reduced the release of CK, and increased plasma concentrations of NO. Pretreatment with L-NAME, an inhibitor of NOS, abolished the protective effects and the increased level of TNF-α and NO elicited by hyperthermia. Conclusion Endogenous NO is involved in the cardioprotection afforded by heat stress, and the beneficial effects of NO are related with stimulation of TNF-α production in the rat heart.

Abstract:Aim To investigate the influence of NO on expression of intercellular adhesion molecule-1(ICAM-1) in vascular smooth muscle cells (VSMC) and adhesion between monocyte and VSMC induced by TNF-α. Methods Cell adhesion, flow cytometry and RT-PCR were applied to study effects of nitroprusside(SNP) and NO, on adhesion between monocyte and VSMC and expression of ICAM-1 gene. Results Adhesion between monocyte and VSMC was significantly inhibited by SNP. Expression of ICAM-1 gene in VSMC induced by TNF-α was depressed after VSMC was treated with SNP, and level of ICAM-1 on VSMC was reduced. Conclusions It is one of the mechanisms of anti-atherosclerosis by NO that expression of ICAM-1 gene in VSMC and adhesion between monocyte and VSMC induced by TNF-α could be inhibited by NO.

Abstract:Aim This study was to investigate p57kip2 gene expression in hyperplastic or hypertrophic vascular smooth muscle cells (VSMC) stimulated by platelet-derived growth factor (PDGF-BB) and angiotensinⅡ(AngⅡ). Methods Rat aorta media smooth muscle cells were cultured. PDGF-BB and AngⅡwere added into serum-free medium at a concentration of 20 μg/L and 1 μmol/L, respectively. VSMC were harvested after stimulated for 24 h. Protein level of p57kip2 wAs investigated with Western blot analysis and p57kip2 gene mRNA wAs determined by microarray. Results Both p57kip2 mRNA and protein level were higher in PDGF-BB stimulated VSMC than those in AngⅡstimulated ones. The p57kip2 protein level of VSMC was similar in AngⅡ stimulated group as compared with serum-free control. Conclusions There is increased expression of p57kip2 protein during VSMC proliferation stimulated by PDGF-BB, which may prevent VSMC from overhyperplasia.

Abstract:Aim To investigate the possibility of ectopic expression of apolipoprotein AI (apo AI) and lecithin cholesterol acyltrasferase(LCAT) by myogenic cells and developing a new approach of gene therapy for atherosclerosis (As). Methods Recombinant replication-deficient viral particles were prepared with polycistronic retrovirus vectors containing apoAI cDNA, LCAT cDNA and the neomycine phosphotransferase gene (NEO), Mice primary cultured myoblasts and myogenic cell line C₂C₁₂ were transfected by these viruses. The efficiency of transfection and the state of integration were detected by PCR, while the expression of apo AI and LCAT were measured by ELISA and immunohistochemical method. Results All transfected mice myoblasts and C₂C₁₂ cells gained the ability of co-expressing human apo AI and LCAT. Stable transfected C₂C₁₂ cell line selected by G418 maintained the ability of co-expressing apo AI and LCAT for 60 days. PCR shown the apo AI cDNA and IRES sequence were integrated into genomes of target cells effectively. Conclusions These finds indicated mouse primary myoblasts and C₂C₁₂ myoblasts transduced with recombinant retroviral vectors could efficiently express and secrete human apo AI and LCAT. It suggested that the use of polycistronic retrovirus vectors containing human apo AI and LCAT cDNA to genetically modify myoblasts in vitro and then implantation back to skeletal muscle to high efficiently, long-term express apo AI and LCAT in vivo, might be a strategy to prevent or treat hypercholesterolemia and As by promoting the "reverse cholesterol transport" pathway.

Abstract:Aim To explore the time course of expression of ICAM-1 gene and protein in macrophages foamy process induced by ox-LDL. Methods Cell cycle of Human myeloid leukemia U937 cells was arrested in G1 by preincubated with thymidine (2.0 mmol/L) for 24 h, then cells released at the same time. U937 cells were incubated with ox-LDL (80 mg/L) for time course(12 h, 24 h, 48 h or 72 h) treatment. Results Flow cytometric analysis results revealed that ICAM-1 protein expression increased from 12 h to 24 h and was highest at 24 h, but ICAM-1 decreased at 48 h. RT-PCR results showed that ICAM-1 mRNA level increased consistently with protein expression. Conclusions These results proved that ox-LDL could increase ICAM-1 gene and protein expression on U937 cells. Accumulation of cholesteryl esters in U937 cells has no negative feedback control on ICAM-1 gene and protein expression.

Abstract:Aim To investigate the effects of fluvastatin, simvastatin and lovastatin on the proliferation of vascular smooth muscle cells derived from spontaneously hypertensive rats. Methods The aorta smooth muscle cells(ASMC) from spontaneously hypertensive rats were cultured, and proliferation of cells were detected by cell number counting and 3 H-thymidine( 3H-TdR) incorporation after incubation with AngⅡ, fluvastatin, simvastatin, lovastatin and mevalonate acid. Results The increased cell number and 3 H-TdR incorporation stimulated with 2%FCS and AngⅡ(10 -6 mol/L) were significantly inhibited by three agents in a concentration-dependent manner (10 -5 ～10 -7 mol/L), but the inhibitory efficacy was fluvastatin>simvastatin>lovastatin. The inhibitory effects were completely reversed by mevalonate acid(10 -3 mol/L). Conclusions The proliferation of SHR ASMC was inhibited by fluvastatin, simvastatin and lovastatin, which suggested that mevalonate acid pathway may play an important role in the proliferation of SHR ASMC.

Abstract:Aim To explore the levels of blood glucose and insulin and the characteristics of tissue structure changes of livers, kidneys and pancreas in New Zealand white rabbits as diabetogenic diet (high fat and high sucrose) induced atherosclerosis in them. Methods Male New Zealand white rabbits were divided into two groups: normal fodder group (n=15) and high-sugar and high-fat fodder group (n=15, with 10% lard, 37% white cane sugar and 53% nomal fodder), and observed for 6 months. Once a month after a overnight fasting the blood of the rabbits are tested for the levels of blood glucose, insulin. At the end of 6 months all the rabbits were killed to get the livers, kidneys and pancreas, which were fixed with 10% formalin sectioned with regular paraflin wrapping. stained with H.E and PAS to livers sections, and then observed with microscopes. Results One month later the rabbits in the experimental group developed high blood glucose, rising with the experimental time course, while those in the contrast group didn't develop obvious rise in blood glucose. The differece between the two groups was significant (P<0.05). Five months later the liver cells in the rabbits of the experimental group developed clear steatosis and swelling with liver glucogen disappearing in the cell plasma. In the kidney the glomeruli became larger, the cell number increased, the walls of capillary vessel became thicker and the epicyte in the urinitubube became larger. The tissue structure in the pancreas develop obvious pathological changes. The pancreas islets are atrophied, there is a decrease of cell's amount in pancreas islets and there are a few lymphocyte infiltration around pancreas islets. Conclusion High-sugar and high-fat fodder can induce sustained and typical diabetes and atherosclerosis in New Zealand White rabbits within a short period of time.

Abstract:Aim To investigate the effect of platelet derived growth factor β-receptor (PDGFR-β)antisense oligonucleotides (AODN) on platelet derived growth factor (PDGF)induced migration of cultured rat vascular smooth muscle cells(VSMC).Methods In vitro cultured rat VSMC model was established. Interactive Laser Cytometer (ILC) was used to investigate fluoresceinisothiocyanate labeled PDGFR-β AODN cellular uptake and localization. The migration of VSMC was assayed by a modification of Boyden's chamber method. Results Fluorescent AODN localized in the cytoplasm of VSMC at 24 hours and in the nucleus at 48 hours. Compared with control group, PDGFR-β AODN at dose of 5 μmol/L or higher can significantly decrease the number of cells which had migrated through the polycarbon filter induced by PDGF. PDGFR-β AODN at dose of 2.5 μmol/L or lower, sense oligonucleotide and scrambled oligonucleotide could not significantly inhibit PDGF induced migration of VSMC. Conclusion PDGFR-β AODN can localize in the nucleus and significantly inhibit PDGF induced migration of cultured VSMC in a dose and time-dependent fashion.

Abstract:Aim To investigate the effect of testosterone on intimal proliferation after rabbit abdominal aorta balloon injury. Methods 24 male New Zealand white rabbits were divided randomly into control group (n=8, sham castration), hypotestosteronemia group (n=8, castration) and testosterone replacement group (n=8, castration +testosterone undecanoate, 14 mg/kg). Abdominal aorta was not injured with 3 mm PTCA balloon until testosterone undecanoate had been injected for three days. After injury two weeks later, blood samples were obtained for detection of plasma testosterone, nitric oxide (NO), superoxide dismutase (SOD) and malondialdehyde (MDA), and all the abdominal aortas were excised to be analyzed by computer. Results The intimal area and PCNA proliferation index of control group and testosterone replacement group were significantly decreased than those of hypotestosteronemia group (P<0.01), together with a significant increase in NO plasma level and significant decrease in MDA plasma level. No difference was observed between control group and testosterone replacement group in all parameters. Conclusions Testosterone, at physiological level, inhibits the intimal proliferation induced by balloon injury in male rabbit abdominal aorta. It might be related to increasing the synthesis of NO and the effect antioxidation.

Abstract:Aim To evaluate the effect of angiotensin Ⅱ type 1 receptor antagonist Irbesartan on the expression of vascular cell adhesion molecule-1 (VCAM-1)in a rabbit model of high cholesterol diet induced atherosclerosis. Methods Twenty-five rabbits were divided into three groups: control group, high cholesterol group, Irbesartan group [50 mg/(kg·d)]. The later two groups received 1% cholesterol diet for 12 weeks to establish the early atherosclerosis model. Blood samples were collected for cholesterol assessment and angiotensin Ⅱ concentrations. The levels of expression of VCAM-1 mRNA were evaluated by RT-PCR and Northern Blot. Results There is no difference between the two high-cholesterol groups, although the cholesterol level of these groups are much higher than control group. Rabbits received Irbesartan showed reduced intimal thickness and the ratio of aortic intimal thickness to medial thickness compared with high cholesterol group (P<0.05). Irbesartan administration for 12 weeks caused a down-regulation in arterial expression of VCAM-1 normalized to GAPDH (P<0.05). Conclusion Chronic Irbesartan treatment ameliorated the severity of the early atherosclerosis lesions, possibly because of the suppression of VCAM-1 mRNA expression.

Abstract:Aim To Prepare anti-hCD154 monoclonal antibody and analysis its immunological properties.Methods The GST-hCD154 fusion protein was used as antigen to immunize BALB/c mouse. B cell was fused with myeloma cell SP2/0 to produce hybridoma cell line that can secrete anti-hCD154 antibody. The hybridoma cells were identified by using ELISA, Western-blot and FACS method. Results (1) ELISA result demonstrated that culture supernatants of three hybridoma cells(6c5, 1B₆, and5c3) can react with GST-hCD154 ,but not react with GST.(2) Western-blot show that three monoclonal antibodies could bound to a protein band with molecular weight of 56 kDa (GST-hCD154) ,but could not bound to a protein band with molecular weight of 26 kDa (GST). (3) FACS show that McAb 6c5 can bound to peripheral blood T cell activated. Conclusion 6c5 cell is a specific McAb against human CD154 molecules expressed on actived human T cells. Preparation of anti-hCD154 monoclonal antibody be used to study the inhibitory function of atherosclerosis progression.

Abstract:Aim To investigate the clinical implications of the expression of CD40 ligand (CD40L) on peripheral blood monocytes and sera soluble CD40L (sCD40L) changes in patients with unstable angina. Methods 16 normal controls and 40 patients including 20 with SA(8 patients by PTCA), 20 with UA entered in this study. The expression of CD40L on monocytes was analyzed by indirect-immonofluorescence flow cytometry and sera sCD40L levels were measured by ELISA. Results (1) The expression of CD40L on monocytes in UA were higher compared with SA and controls (P<0.01), while no significant difference was found between SA and controls(P>0.05). (2) Patients with UA had significantly raised sCD40L sera levels when compared with patients with SA and controls(P<0.01). Values also differed significantly between patients with SA and controls. (3) PTCA induced a marked rise in sCD40L levels in all patients, while the expression of CD40L on monocytes showed no significantly difference between patients with PTCA before and after. (4) A significant correlation was observed between sCD40L sera concentrations and the number of complex lesions(r=0.54,P<0.01). sCD40L, however, was not significantly associated with disease extent and range. Conclusions The enhanced level of serum soluble CD40L may play an important role in coronary plaque instability and disruption, and may represent a marker of coronary disease activity.

Abstract:Aim To investigate the relation between the expression of lipoprotein lipase mRNA and the different clinical class or severity of coronary atherosclerosis. Methods A total of 34 patients with coronary atherosclerosis and 10 healthy subjects underwent coronary angiography. There were 12 patients with a light coronary atherosclerosis (diameter stenosis <40%)(Group 1),11 patients with moderate coronary atherosclerosis (diameter stenosis 40%-80% in only one coronary artery) (Group 2), 11 patients with severe coronary atherosclerosis (diameter stenosis >80% in one or more coronary artery) (Group 3), 10 healthy subjects with normal coronary angiography (Group 5). Group 4 included 11 patients were on heparin. Circulating human lipoprotein lipase mRNA wAs detected by RT-PCR in all patients and healthy subjects. Results There was no statistic difference in any groups. There was no statistic difference between 21 patients with high serum triglyceride level and 13 patients with normal serum triglyceride level, between Group 3 and Group 4 (P>0.05). But the serum triglyceride level in Group 3 was higher than in Group 4 (P<0.05). Conclusion The similar expression of circulating human lipoprotein lipase mRNA is in healthy and patients with coronary atherosclerotic disease, who have different serum lipid level and severity of coronary stenosis. The expression of lipoprotein lipase mRNA is not stimulated by heparin.

Abstract:Aim To investigate the plasma C-type natriurtic peptide (CNP) and atrial natriuretic peptide (ANP) levels of patients with coronary atherosclerosisor coronary heart disease before and after angioplasty in order to study the relationship between C-type natriurtic peptide and coronary atherosclerosis and angioplasty. Methods Seventeen patients with coronary atherosclerosis (coronary stenosis less than 20%) and ten patients with coronary heart disease (coronary stenosis more than 80%～100%) before and after left coronary PTCA and/or stenting were selected. Blood samples were extracted from coronary sinus, femoral artery and vein respectively as well as from elbow vein at 24 h, 72 h after angioplasty. Blood samples were also extracted from elbow vein of seventeen healthy controls. The plasma CNP and ANP levels were measured using radioimmunoassay. Results Venous plasma CNP levels of patients with coronary atherosclerosis and coronary heart disease were higher than those of controls(P<0.001). The plasma ANP level in coronary sinus increased significantly after angioplasty (P<0.05). Plasma CNP levels in coronary sinus, femoral artery, and femoral vein immediately after angioplasty were all obviously elevated (P<0.001). Venous plasma CNP level after 24 h was the highest. After 72 h, the plasma CNP level returned to that before angioplasty. Conclusions CNP is involved in the pathophysiologic process of coronary atherosclerosis and intervention of coronary heart disease. The changes of plasma CNP and ANP levels after angioplasty in coronary heart disease suggest that CNP can reflect the changes of coronary artery better than ANP.

Abstract:Aim To investigate the effect of simvastatin on endothelium dependent relaxing function, intima-media thickness (IMT) of carotid in elderly patients. Methods With high-resolution ultrasound, before and after simvastatin treatment, endothelium dependent relaxing function and IMT of carotid arteries were measured in 60 subjects: 20 with primary hypercholesterolemia, 20 with coronary heart disease and hypercholesterolemia and 20 controls. Results In elderly patients, IMT of carotid arteries was higher and flow-mediated dilatation was much reduced compared with that of control group. After therapy with simvastatin 20 mg/d for 3～4 months, total cholesterol, triglyceride, low density lipoprotein cholesterol(LDLC) and high density lipoprotein cholesterol(HDLC) were much reduced, and flow-mediated vasodilation was improved than before, though it was still lower than control group. There was no significant changes of IMT. Conclusions Endothelium dependent vasodilation was impaired in elderly hypercholesterolemia patients, whose IMT of carotid arteries was increased. Simvastatin improves impaired endotheliumdependent vasodilatation, but with little effect on increased IMT of carotid arteries.

Abstract:Aim To evaluate the efficacy and safety of the national low molecular weight heparin (LWMH)-Livaracine in treating acute coronary syndrome compared with standard heparin (SH). Methods In a randomized clinical trial, 84 patients, with at least one attack of ischemic chest pain by unstable angina or non-Q wave myocardial infarction within 72 h before treatment enrolled, were divided into two groups, group SH: intravenous SH 5 000 IU, twice daily to maintain the APTT or ACT at 1.5～2.0 times of control for 7 days and group LMWH: subcutaneous Livaracine 5 000 IU, twice daily for 7 days. All patients took 150 mg asprins daily at the same time. The primary indexes were the improvement of clinical symptoms and ECG, composite events (acute myocardial infarction,cardiac or noncardiac death and urgent revascularization) and adverse reaction. The patients enrolled should stay in hospital for at least 14 days. Results By 7 days, the incidence of angina pectoris was not significant between the two groups, but more patients needed oral nitroglycerin for pain relief in group SH. By 14 days, composite events were singificantly reduced in group LWMH than in group SH. Serious bleeding was very few in patients receiving LWMH. Conclusions Subcutaneous Livaracine is at least as effectural as continuous intravenous SH for the suppression of heart attacks in the early and late phase of acute coronary syndrome. LMWH is more effective in reducing composite cardiovascular events 14 days after drug initiation. Monitoring is not necessary in the routine use of LMWH, which shows more convenience and ease of use.

Abstract:Aim To evaluate the relationship between carotid atherosclerosis (CAS) and coronary atherosclerosis. Methods Carotid ultrasound was performed in 94 patients who had been examined by coronary angiography. Carotid plaque score was calculated to quantify the extent of CAS using Sutton' methods. Results The results showed that the carotid intima-media thickness (IMT) and plaque index were significantly increases in all of the diseased groups versus controls (P<0.05). Multivariate analysis of all the subjects revealed the age, cigarette smoking, blood pressure, length and severity of hypertension, high density lipoprotein cholesterol (HDLC), ratio of low density lipoprotein cholesterol (LDLC) and HDLC were independently associated with CAS. As coronary atherosclerosis progressed, the extent of CAS became more severe. Significant differences of carotid plaque score were found among different coronary artery disease groups (P<0.01). Conclusion The results suggest that coronary atherosclerosis and CAS could be parelled.

Abstract:Aim To investigate the changes of platelet activation and vascular endothelial function in patients with impaired glucose tolerance (IGT). Methods 40 patients with IGT were chosen and the level of platelet alpha granule membrane protein (GMP-140) was measured in these patients. Meanwhile, flow-mediated endothelium dependent dilation of branchial arteries was observed with high resolution ultrasound by celermajer's method. The diameter of branchial arteries were respectively measured at rest (basal diameter, BD), during reactive hyperemia (increased flow causing endothelium-dependent dilation, FMD) and after sublingual nitroglycerine (nitroglycerine causing endothelium-independent dilation, NCD). Data of 30 healthy persons were taken as the control group. Results There was no significant difference in the level of fasting blood glucose (FBG) between the control group and the IGT group. However, after 75 g oral glucose loading for 2 h, the level of blood glucose (2hBG) in the IGT group was higher than that in the control group. In the fasting state, the level of FMD was significantly reduced and the level of platelet GMP-140 was significantly increased in the IGT group as compared with those in the control group. After 75 g oral glucose loading, the level of FMD (2hFMD) was lower than that in the fasting state and the level of platelet GMP-140 (2hGMP-140) was higher than those in the fasting state. Linear correlation analysis showed that there was no significant relation between FBG and the levels of GMP-140 and FMD. However, before and after oral glucose loading, the changes of blood glucose [△(2hBG-FBG)] were positively correlated with the changes of platelet GMP-140 [△(2hGMP-140-GMP-140)], and the changes of blood glucose [△(2hBG-FBG)] were negatively correlated with the changes of FMD [△(2hFMD-FDM)]. The level of platelet GMP-140 was negatively correlated with the level of FMD. Conclusion Platelet activation was increased and vascular endothelial function was impaired in the patients with IGT, and oral glucose loading aggravate the increasing platelet activation and the endothelial dysfunction.

Abstract:Aim To explore the relationship among serum cholesterol ester transfer protein (CETP) and serum lipid in 600 healthy Chinese subjects which belonged to six deferent Chinese peoples including Chinese Han, Menggu, Hui, Weiwuer, Dai and Dongxiang, the levels of CETP and the frequency of exon 15 missense mutation (D442G) and intron 14 splice defect (I14A) in CETP gene were examined. Methods A simple and practical method for detecting two prevalent mutations in CETP gene using PCR-RFLP was developed to scan 600 healthy Chinese subjects. Using ELISA established by our own laboratory to detect the level of serum CETP, and at same time serum lipids and apolipoprotein determination was carried out. Results Frequency of CETP gene D442G and I14A mutation was similar among Chinese Han, Menggu, Hui, Weiwuer, Dai and Dongxiang. There was no significant deference in the level of CETP among six deferent Chinese peoples. Serum total cholesterol (TC) level of Chinese Han was higher than other five Chinese peoples in the study (P<0.05). Serum triglyceride (TG) level of Chinese Hui was significantly higher than that of Chinese Han (P<0.05). Serum high density lipoprotein cholesterol (HDLC) level in Chinese Menggu, Hui and Dongxiang was lower than that in Chinese Han (P<0.05). Serum low density lipoprotein cholesterol (LDLC) level in Chinese Weiwuer, Hui and Dongxiang was lower than that in Chinese Han (P<0.05). Conclusion There was no significant deference in frequency of CETP gene D442G and I14A mutation in six deferent Chinese peoples and environment factors including zone and meal had little influence on CETP level in six deferent Chinese peoples.

Abstract:Aim To culture rat vascular smooth muscle cells by novel enzymatic dispersion method. Methods Rat aorta was removed from left subclavian origin to diaphragmatic insertion in sterile conditions. The vascular media was digested,then the cells were centrifuged and plated down in prepared flask. Results The primary vascular smooth muscle cells of rat aorta was performed successfully by using enzymatic dispersion method. The cell viability of third generation is 96% by typan blue,and the cell purity is 97% tested by light microscope and immunocytochemical stains. Conclusions The growth period of enzyme-dispersed vascular smooth muscle cells is shorter by 4～5 days than that of explant method and enzymatic dispersion method reported in domesticity.