Abstract:Aim Monocyte chemoattractant protein-1 (MCP-1) plays an important role in migration of monocytes into subendothelial space. The purpose of this study is to examine whether thrombin and lipopolysaccharide (LPS) induce expression of MCP-1 mRNA and protein in calf aortic smooth muscle cells (SMCs). Methods Calf aortic SMCs were cultured by a substrate-attached explant method. The SMCs at the third to fifth passage which became confluent were used for the experiment. After a four-hour exposure to 2 ku/L thrombin and 100 μg/L LPS respectively, total RNA of SMCs of different groups were extracted by guanidinium isothiocyanate method. On the other hand, the SMCs of different groups after exposed to the above-mentioned inducers for 24 h, respectively, the conditioned media were collected. The expression of MCP-1 mRNA in SMCs was examined by dot blot analysis using a probe of γ- 32 P-labelled 35 mer oligonucleotide, and the MCP-1 protein in the conditioned media was determined by sandwich ELISA. Results Dot blot analysis showed that cultured SMCs were able to express MCP-1 mRNA at a low level. A 4 h exposure of SMCs to LPS and thrombin, respectively, induced a 1.6-fold and 2.1-fold increase in MCP-1 mRNA expression in the cells. After a 24 h exposure to thrombin, ELISA showed that the MCP-1 protein content in the conditioned media were also markedly increased (1.4-fold,P<0.05), while the MCP-1 protein content was not significantly increased (1.1-fold, P>0.05) after exposed to LPS for 24 h. Conclusions Thrombin is able to induce expression of MCP-1 mRNA and protein in cultured SMCs. It suggests that thrombin may play an important role in atherogenesis through increasing recruitment of monocytes in the arterial intima. The effect of LPS on the expression of MCP-1 in SMCs, hovever, is equivocal.

Abstract:Aim Plasma apolipoprotein AI (apo AI) play an important role in reverse cholesterol transport and have becoming attractive target for prevention or treatment of atherosclerosis. The present study was undertaken to search for the possibility of human apo AI gene expression and secretion to blood by transferred Ad-RSV-apo AI into mouse skeletal muscles and developing mathod of atherosclerotic gene therapy. Methods An E1-/E3- adenoviral vector, with an RSV-driven human apo AI cDNA expression vector (Ad-RSV-apo AI) was constracted. Transfected myogenic cells in vitro or directly injected into mouse skeletal muscle with the vector. The result was examined by ELISA and immuncytochemistry. Results In the primary cultured myoblasts, the expression level of apo AI in the culture medium increased with the increassing MOI of Ad-RSV-apo AI (from 10 5 pfu to 10 8 pfu). The expression of apo AI was also increased with time. It peaked on the 6th day .After direct injection of Ad-RSV-apo AI into muscle, apo AI mass could be detected, which peaked on day 5 both in the muscle and plasma. On day 30, apo AI was disappeared in the plasma, but the expression in the muscle existed till day 40. Conclusions Apo AI gene can be expressed in the skeletal muscle and secreted to the circulation. It might be an approach for the atherosclerotic gene therapy.

Abstract:Aim To investigate the changes of aortic nitric oxide (NO) and NO synthases (cNOS and iNOS) in atherosclerotic rabbits induced by high cholesterol diet and the antiatherosclerotic effect of L-arginine. Methods Male New Zealand white rabbits were divided into three groups in random: the rabbits received either normal rabbit chow (normal group),1% cholesterol diet only (cholesterol group) or 1% cholesterol diet plus L-arginine (22.5% in drinking water, arginine group). Serum total cholesterol (TC), plasma L-arginine, asymmetric dimethlarginine (ADMA) and oxidized low density lipoprotein (ox-LDL) concentrations were detected. After 10 weeks, the aorta were harvested for assessment of NO production, NOS activities and area of intimal lesions. Results Compared with normal group, plasma L-arginine level had no significant alteration in cholesterol group, but serum TC, plasma ADMA and ox-LDL concentrations elevated markedly (P<0.01 orP<0.05). Aortic cNOS activity and NO production decreased about 45% and 30% respectively (P<0.01). Intimal lesions increased to 42.6%±9.2%. Compared with cholesterol group, dietary L-arginine had no effects on serum TC,plasma ADMA and ox-LDLconcentrations, but plasma L-arginine level increased about one time、aortic cNOS activity and NO production increased significantly (P<0.01). Intima lesion formation were inhibited markedly (19.5%±7.4% vs 42.6%±9.2%,P<0.05). Conclusions The activity of aortic NOS-NO system is impaired significantly in atherosclerotic rabbits induced by cholesterol diet. Progressive intimal lesion formation is associated with impaired activity of NOS-NO system. Decrease of cNOS activity results in reduction of NO production. L-arginine inhibits atherosclerotic progression by augmenting cNOS activity and restoring NO elaboration.

Abstract:Aim To establish a RT-PCR method for detecting expression of mRNA for inositol trisphosphate receptor (IP 3R) and to investigate expression of IP 3R subtypes in rat brain, myocardium and cultured vascular smooth muscle cells (VSMC). Methods Total RNA was isolated from different tissue of rat and was reversibly transcripted into cDNA. RT-PCR was performed with β-actin as internal label. Three oligonuclotide primers for each subtype of IP 3R were employed to amplify predicted DNA. Results It was found that all subtypes of IP 3R were expressed in cultured SMC. DNA sequencing indicated that amplified products of IP 3R Ⅱ subtype was identical to reported sequence from gene bank. Conclusion Our results suggested that RT-PCR established by us can be used as a reliable method for investigating expression of mRNA for IP 3R subtypes.

Abstract:Aim To study the relation between apolipoprotein E polymorphism and myocardial infarction. Methods 110 patients with myocardial infarction and 131 normals were enrolled and blood samples were collected. Their apoE genotyping were performed by polymerase chain reaction (PCR). Results Both in cases and controls, the allelic frequency of E3/3 was highest, reaching 70.1 percentage of whole volume, hemozygote of apo E3 was moderate, and homozygote E4/4 was lowest. The frequencies of E3/4 and E4/4 was significantly high in patients with myocardial infarction compared with controls (P<0.01). It was found primarily that the age of first acute myocardial infarction was earlier in the carriers of E4. Conclusions apo E4 is an important genetic mark of the patients with myocardial infaction. The age of first acute myocardial infarction was earlier in the carriers of E4.

Abstract:Aim To determine the allele frequencies of genetic variants 373 Ala→Pro and 451 Arg→Gln of cholesteryl ester transfer protein (CETP) and to explore their potential impacts on serum lipid metabolism. Methods The genotypes in CETP codon 373 and 451 in 91 German healthy students and 409 angiographically confirmed coronary heart disease (CHD) patients were respectively analyzed using the allele specific technique of mutagenically separated polymerase chain reaction (MS-PCR) and their serum lipid parameters were determined.Results It is demonstrated that genetic variants 373 Ala→Pro and 451 Arg→Gln are frequent mutations with allele frequencies around 3% to 5% presented in the healthy individuals as well as in the CHD patients. The high density lipoprotein cholesterol (HDLC) level was significantly decreased in the 373-Pro and 451-Gln alleles heterozygotic carriers in both examined cohorts compared with those in the wild type individuals. The increasing of serum triglyceride (TG) in the mutant alleles carriers was only observed in the healthy volunteers. It was also found that these two genetic variants do not always emerge in the linkage form. Conclusion It is suggested that these two mutations together would possess the phenotype of decreasing HDLC and increasing TG levels.

Abstract:Aim To explore the effects of neuropeptide Y (NPY) on the expression of apoptosis-related genes in cultured vascular smooth muscle cells. Methods The expression of apoptosis-related genes were detected in cultured vascular smooth muscle cells with the immunofluorescent quantitative skill through laser scanning confocal microscope (ACAS 570). Results It was found that at the high concentration of NPY (10 -6 mol/L),bcl-2 and bax were markedly expressed in cultured smooth muscle cells compared with control group, the fluorescent value of bcl-2 and bax were 1834.25±138.55 and 1870.82±162.43, much higher than 1682.87±94.47 and 1688.04±110.91 in control group respectively (P<0.01). With the concentration becoming down, the expression of related genes were also decreased. But the fluorescent value of fas gene was 1987.68±185.39, higher than 1882.38±160.84 in control group only at the 10 -6 mol/L concentration(P<0.05). Conclusion NPY could induced the expression of apoptosis-related genes such as bcl-2, bax and fas in cultured vascular smooth muscle cells.

Abstract:Aim Previous studies performed in vitro indicated that XBP-01, a polypeptide which was refined from Chinese cobra venom (Naja naja atra), could inhibited the cultured vascular smooth muscle cell proliferation. To observe the eliminating atherosclerotic plaques effect of XBP-01 in vivo. Methods After fed with the atherogenic diets for 17 weeks, the atherosclerotic sensitive inbred strain C57BL/6J mice have typical atherosclerotic plaques formed near the aorta sinus, accompanied with the serum total cholesterol and triglyceride significant rise. Then the mice were injected with XBP-01 of 3.2 μg/g weight body per day for 4 weeks. Cryostat frozen sections, 10 μm thick, were cut from heart to aorta, stained with Red oil O. The sections were examined microscopically and scored according to the method of Roberts and Thompson. The serum total cholesterol and triglyceride also were assayed. Results The mice have typical atherosclerotic plaques formed near the aorta sinus, accompanied with the serum total cholesterol and triglyceride significant rise after fed with the atherogenic diets for 17 weeks. The plaques are fewer and less than that of the non-treating with XBP-01 group, the Roberts & Thompson score is 8.0±1.2, 4.1±2.2, respectively, and has a statistical significance. The serum total cholesterol and triglyceride descends to normal level. Conclusions XBP-01 possesses the potential antiatherosclerotic effect in vivo.

Abstract:Aim The effects of melittin (a polypeptide from bee venom) on rat aorta and the relation with Ca 2+ influx were studied in this article. Methods The contractile test of the isolated aorta rings and the assay method of 45 ?Ca influx were used. Results In endothelium-intact aorta rings, melittin did not influence the rapid contractions induced by phenylephrine (PHE), but could inhibit the continuing contractions in the concentration dependent manner. In endothelium-denuded aorta rings,melittin did not have the effect on the contractile responses induced by PHE. Melittin did not cause the contraction in endothelium-intact aorta rings, but induced significantly contractions and increased the 45 ?Ca influx in endothelium-denuded aorta rings, these effects were partially antagonised by cartopril and bepridil respectively. Conclusions Melittin could cause the endothelium dependent relaxations to vascular smooth muscle, and the releasing renin and Na + - Ca 2+ exchange system could play partial role in the contractions of the endothelium-denuded vascular.

Abstract:Aim Effects of nitric oxide (NO),superoxide anion(O . 2 ) and polysaccharide krestin(PSK) on macrophage-mediated oxidation of LDL were investigated. Methods Mouse were given peritoneal injection PSK (control mouse were given injection physiological saline), peritoneal macrophages were collected and stimulated with low-density lipoprotein(LDL), γ-IFN, LPS and PMA. Contents change of nitrite, hydroperoxides and TBARS were measured. Results PSK-treated mouse peritoneal macrophages secreted much NO and produced less LOOH, TBARS than non-PSK-treated. Much NO and less LOOH, TBARS could be found to be produced by macrophage when stimulated with γ-IFN, LPS, macrophages stimulated with PMA appeared opposite effect. Conclusions The results indicated PSK has a protective effect, prevent cell from lipoperoxidative injury. γ-IFN and LPS could induce NO synthase activity and produce substantial NO. Stimulated macrophages were less efficient in oxidizing LDL. PMA can activate the respiratory burst, the stimulated cells were more effective than unstimulated cells in modifying LDL, and that O - 2 was of critical importance.

Abstract:Aim To observe the effects of L-arginine on athersclerosis and serum lipids in rabbits. Methods Forty-five rabbits were divided into three groups accidentally. Normal group received nornal chow, control group received 2.5% cholesterol and normal drink, treatment group received 2.5% cholesterol and 2.25% L-arginine solution for 7 or 14 weeks. The levels of serum total cholesterol (TC) and triglycerides (TG) were investigated, while atherosclerosis was measured by image pattern analysis method after ascending aorta and descending aorta were stained with Oil-Red-O. Results The serum total cholesterol and triglycerides had significantly inhibitted by treatment with L-arginine at 7 weeks.But there was no significant difference between treatment with and without L-arginine at 14 weeks. Similar results of atherosclerosis was seen in ascending aorta. But at 7 or 14 weeks, atherosclerosis was less in descending aorta of arginine fed animals than that of rabbits fed cholesterol only. Conclusion The results suggest that chronic dietary L-arginine suppllemintation does not result in a sustained limitation in atherosclerosis and decrease in serum lipids.

Abstract:Aim To evaluate the preventive effect of vitamin C ,a potent free radical scavenger, on the markers of polymorphonuclear leukocyte (PMN) oxygen free radical (OFR)production, lipid peroxidation and defenses against oxidative stress in patients with acute myocardial infarction (AMI). Methods 30 patients with AMI were randomized to receive either conventional treatment only (control group, n=13) or conventional treatment supplemented with vitamin C infusion (vitamin C group,n=17), 2 g per day, for 7 days. All measurements were performed before treatment, on the 3rd and 7th day after been hospitalized. The PMN OFR production was assayed by a convenient and sensitive method named as polymorphonuclear leukocyte chemiluminescence (PMN-CL). The plasma lipid peroxides concentration measured as malondialdehyde (MDA), activities of superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) were estimated by chemical measurement. Meanwhile, serum CK activity and ECG were also monitored. Results Parameters of PMN-CL decreased significantly in vitamin C group on the 7th day compared with that in controls. Plasma MDA concentration decreased significantly on the 7th day and plasma activity of SOD increased more quickly on the 3rd and 7th day in vitamin C group than that in controls, however, it showed no salutary effects of vitamin C on CK and GSHPx activities in vitamin C group. Arrhythmia occurre less often in vitamin C group than in controls on the 7th day. Conclusions Supplementation with vitamin C may suppress PMN OFR production, decrease the levels of lipid peroxide, increase the plasma activity of antioxidative enzymatic system, and could be beneficial in preventing complications in such patients.

Abstract:Aim To understand the aortic function in the old patients with arteriosclerosis. Methods Aortic elastic property were examined by echocardiography in the 56 cardiovascular old patients and in 60 negative old man. Results There was no significant difference in the diameter of aortic ascending and of abdominal arota between test group and control group (P>0.1), but the clistensibility of aorta in the patients subsides (P<0.001) and the stiffness index rises remarkably. Conclusions There was aclose relationship in the cardiovascular old patients between the aortic elastic property subsidence with the pulse pressure difference (△P), with the diameter difference of aorta(△d)and with other factors. So that the target of aortic elastic property is one of the important aspects in the diagnosis of the old men arteriosclerosis.

Abstract:Aim To explore the changes of leukocyte rheologic characteristics and cellular adhesion molecules (CAMS) expression in aged patients with hypertension. Methods By using the erythrocyte deformability apparatus, thrombus and platelet adhesion dual-purpose apparatus and enzyme linked immunosorbent assay (ELISA) methods, the leukocyte deformability (LD), leukocyte adhesion function (LAF), leukocyte CD18 expression, serum soluble intercellular adhesion molecule-1 (sICAM-1) concentration and serum soluble vascular cellular adhesion molecule-1 (sVCAM-1) concentration were measured in 104 aged patients with hypertension and 56 controls. Results The leukocyte filtration index (LFI), leukocyte adhesion rate (LAR), leukocyte CD18 expression, sICAM-1 concentration and sVCAM-1 concentration were significantly higher in aged patients with hypertension than those in controls (P<0.001). Remarkable differences of these parameters were found in patients with three stages (P<0.001), and the most obvious increase of the changes was found in stage Ⅲ. In aged patients with hypertension, the LFI was positively related to the LAR (r=0.562,P<0.001), LFI and LAR were positively related to leukocyte CD18 expression, sICAM-1 concentration and sVCAM-1 concentration (r=0.579～0.816,P<0.001). Conclusion It showed that the decrease of LD and the increase of LAF, leukocyte CD18 expression, sICAM-1 concentration and sVCAM-1 concentration might participate in the pathogenesis of hypertension.

Abstract:Aim To study the characteristics of coronary lesions and the relation with serum estrogen level in perimenopausal women with coronary artery disease (CAD). Methods The coronary angiograms and the serum estradiol (E 2)and lipid levels were compared between 26 perimenopausal women and 38 postmenopausal women with acute myocardial infarction (AMI). Results There were more cases with mild to moderate coronary artery stenoses and single-vessel diseases in perimenopausal women patients than in postmenopausal ones (P<0.01). In perimenopausal women patients, the serum levels of E 2 and high density lipoprotein cholesterol (HDLC) were significantly lower than in normal control (P<0.01), but higher than in postmenopausal patients (P<0.01). And the serum levels of total cholesterol (TC),triglyceride (TG) and low density lipoprotein-cholesterol (LDLC) were significantly higher than in control (P<0.01), but lower than in postmenopausal patients (P<0.01). Conclusions The coronary lesions in perimenopausal women with CAD were characterized by mild to moderate stenoses and single-vessel diseases. Meanwhile, there were less decrease of serum estrogen level and less abnormality of lipid metabolization in this group of patients.

Abstract:Aim To evaluate the effect of statins on serum triglyceride in patients with hyperlipidemia, and whether the effect is related to baseline triglyceride levels. Methods The data analysed were originated from 2 separate multicenter clinical trials with similar designs. Baseline total cholesterol lecels was more than 5.98 mmol/L, and baseline triglyceride lecels was less than 4.52 mmol/L. The patients were grouped by baseline triglyceride levels. 166 patients had participated in Simvastatin Clinical Trial with simvastatin 10 mg once daily for 8 weeks. 146 patients had participated in Lovastatin Clinical Trial with lovastatin 20 mg once daily for 8 weeks. Results It showed that the higher the baseline triglyceride levels were, the more effective the simvastatin or lovastatin was in reducing serum triglyceride lecels. A positive correlation was found between baseline triglyceride levels and effects of simvastatin (r=0.3155,P<0.01) and lovastatin (r=0.3341,P<0.01) in reducing serum triglyceride lecels . Conclusions statins was effective in decreasing triglyceride levels in patients with hypertriglyceridemia. The change of reduction on triglyceride was closely related to baseline triglyceride lecels.

Abstract:Aim To study the difference between endothelial-monocyte and endothelial-neutrophil adhesion. Methods Cell adhesion was measured by testing leukocyte myeloperoxidase activity, the cell adhesion was blocked by monoclonal antibodies to ICAM-1, E-selectin and VCAM-1, the expression of adhesion molecules was measured by ELISA. Results TNF-α (100～1 000 ku/L)increased the adhesion of EC to neutrophils or monocytes concentration-dependently. The increased expression of ICAM-1, E-selectin and VCAM-1 was contributed to TNF-α induced upregulation of leukocyte-endothelial adhesion. The neutrophil-endothelial adhesion was peaked at 6 h after TNF-α stimulation of endothelial, while monocyte-endothelial adhesion was peaked at 24 h. The monoclonal antibodies to ICAM-1 and E-selectin significantly inhibited TNF-α induced endothelial-neutrophil adhesion, nevertheless, the antibody to VCAM-1 had no effect on endothelial-neutrophil adhesion. On the other hand, the antibodies to ICAM-1 or VCAM-1 but not the antibody to E-selectin inhibited TNF-α induced endothelial-monocyte adhesion. Conclusion Neutrophil and monocyte showed different characteristics in adhesion to TNF-α induced endothelial cells.

Abstract:Aim To study the clinical significance of low serum high density lipoprotein cholesterol (HDLC) levels in patients with coronary heart disease(CHD). Methods 224 patient with CHD were divided into acute myocardial infarction (AMI,n=90), unstable angina pectoris (UAP,n=78), stable angina pectoris (SAP,n=56) groups. Serum total cholesterol, triglyceride, HDLC levels were examined in 224 patients with CHD and in 50 healthy controls. Results There was no significant difference in serum total cholesterol levels between the CHD patient group (4.72±1.05 mmol/L) and healthy control group(4.78±0.72 mmol/L)(P>0.05), while serum HDLC level was significantly lower in CHD patient group (1.14±0.30 mmol/L) than healthy control group (1.45±0.26 mmol/L)(P<0.01). Serum HDLC level was significantly lower in AMI group than in UAP and SAP groups (P<0.01), when serum total cholesterol level was ≤ 5.16 mmol/L, serum HDLC levels were significantly lower not only in AMI group than in UAP and SAP groups (P<0.01), but also in UAP group than in SAP group (P<0.05). In CHD patients ,serum HDLC level was lower in smoking group (n=71) than in non-smoking group(n=57)(P<0.01). Conclusions Decrease of serum HDLC level is the most important lipid risk factor of CHD, especially in CHD patients with average or low average total serum cholesterol levels, and relates to the different CHD patterns.

Abstract:Aim An immunodetection method was developed to determine the contents of serum HDL subclasses. Methods High density lipoprotein (HDL) subclasses in human serum were separated by two-dimensional agarose-gradient polyacrylamide gel electrophoresis, the molecular size and mass of the HDL subclasses were determined by immunoblotting test and density scanning. Results The variation coefficients of the contents of the HDL subclasses are 4.9%～11.1%. The contents of HDL 2b , HDL 2a , HDL 3a , HDL 3b , HDL 3c in normalipidic serum are 30.80%±6.95%, 21.60%±3.88%, 20.28%±3.69%, 13.54%±2.03%, 7.05%±1.75%, respectively; and that of pre β1-HDL and pre β₂-HDL are 6.67%±1.22%, 4.52%±1.12%, respectively. Conclusion The results suggested the method developed could be used to determine the contents of α-HDL and pre-β HDL subclasses in serum sample.

Abstract:Aim To establish a model of vascular wall and to introduce its application . Methods The human umbilical vein endothelial cells (ECs) and aortic smooth muscle cells (SMCs) were cultured sepparately on the two surfaces of a amnion membrane treated with trypsin and collagenase and the effect of lipid peroxidation (LPx) on the monocyte chemotactic protein (MCP-1) expression in the above mentioned cells, was studied immunocytochemically. Monocyte migration through the ECs layer into the SMCs layer was observed under microscope. Results ECs and SMCs could be cultured on two surface of amnion to build a vascular wall model. After incubation with diamide (DM), the ECs and SMCs on the amnion membrane expressed MCP-1, and resulted in significantly increased monocyte migration through ECs and the amnion membrane into the SMCs layer. Conclusion This study established a model of vascular wall which is a very useful tool for the study on blood vessel biology, atherosclerosis and inflammation.