Abstract:Aim To study the relationship of very low density lipoprotein receptor (VLDLR) structure and its function. Methods The present paper reported the cloning of VLDLR gene cDNA from a chinese heart tissue by RT-PCR technology. A recombinant named pCD-VR was constructed in which the VLDLR cDNA was inserted a vector pcDNA3. Results Sequencing result confirmed that the chinese oriented VLDLR gene cDNA was in line with previously one. Then, the pCD-VR was transfected into CHO cells. Lipoprotein combination test and mRNA detection confirmed that this human VLDLR gene could express effectively in the CHO cells. Conclusion It was provided a new idea about the roles of VLDLR in atherosclerosis.

Abstract:Aim To investigate the effects of phosphatidylinositol 3-kinase (PI3K)/protein kinase C-ζ (PKC-ζ)complex on regulating angiotensin Ⅱ(AngⅡ) activation of p70 ribosomal S6 kinase (p70S6K) in vascular smooth muscle cells(VSMC). Methods VSMC isolated from 200 g to 250 g male Sprague-Dawley rats were cultured to 70% to 80% confluence and were growth-arrested by incubation in 0.1% calf serum/DMEM for 48 h before use. Both 3 H-TdR incorporation and cell counting were used to estimate the proliferation of VSMC. Western blot, immunoprecipitation and immunoblot analyses were performed to valuate the AngⅡ-stimulated kinase expression and Ras-PI3K-PKC-ζ association of VSMC. Phosphotransferase assay by using S6 peptide as substrate was employed to measure the p70 S6K activity. Results VSMC expressed p85PI3K, PKC-ζ and p70S6K; AngⅡ and PDGF stimulation did not affect the p85PI3K, PKC-ζ and p70S6K expression. 2) 100 nmol AngⅡ

Abstract:Aim To investigate in vitro calcification of aortic medial cells and the acceleration by 25-hydroxycholesterol. Methods Aortic medial cells were obtained by explantation. Von Kossa staining was performed to show calcification. Insoluble calcium of cellular layer was determined by biochemical method. And osteocalcin in the media was analyzed with radioimmunoassay. Results Primary cells appeared two different types: cells with the features of parallel growth and negative von Kossa staining, and cells forming nodules with positive von Kossa staining. Passaged cells formed no nodules after 28 days. Cells treated with 25-hydroxycholesterol appeared many cell nodules with positive von Kossa staining. Both insoluble calcium and medium osteocalcin increased significantly. Conclusions There are two subgroups of the cultured aortic medial cells: one with the characters of smooth muscle cells; the other like micro vascular pericytes, which can calcify the extracellular matrix. 25-hydroxycholesterol can promote in vitro calcification. Osteocalcin secretion increases during pericyte-like cell calcification, suggesting that osteocalcin may take part in aortic calcification.

Abstract:Aim To understand whether lipid peroxidation in endothelial cells (EC) induces the expression of macrophage inflammatory protein-1α (MIP-1α)and vascular cell adhesion molecule-1 (VCAM-1). Methods The cultured human umbilical vein EC were divided at random into experimental groups (cultured in the media containing 1 μmol/L, 5 μmol/L and 10 μmol/L diamide, respectively) and control group (cultured in standard medium without diamide). The EC of all groups were hybridized, insitu, with the digoxigenin-labeled MIP-1α and VCAM-1 cDNA probes. In addition , the total RNA in EC of all groups extracted by the single-step method, and MIP-1α and VCAM-1 mRNA expression in EC was determined by dot blotting analysis. Results Insitu hybridization showed that both the cytoplasm and nuclei of the normal EC expressed MIP-1α and VCAM-1 mRNA, that were granular blue substances. Diamide induced stronger expression of cytokines in a dose dependent manner. Of which, the expression of MIP-1α and VCAM-1 mRNA in EC in 1 μmol/L diamide group was 1.28- and 1.26-fold, in 5 μmol/L diamide group, 1.87- and 1.54-fold, and in 10 μmol/L diamide group, 2.41- and 2.01-fold as much as that in control group, respectively. The analysis of variance showed that there were significant differences between groups (P<0.05). Dot blotting showed that the expression of MIP-1α and VCAM-1 mRNA in 1 μmol/L diamide group was 1.40- and 1.22-fold, in 5 μmol/L diamide group, 1.90- and 1.56-fold , and in 10 μmol/L diamide group, 2.50- and 2.04-fold as much as that of the control group, and the increased expression of both cytokine mRNA was positively correlated with the diamide concentrations. Conclusions Lipid peroxidation injury might induce adhesion of monocytes to the endothelium and migration into subendothelial space through stimulating EC to produce increased MIP-1α and VCAM-1 and may play an important role in the recruitment of monocytes into the intima in atherogenesis.

Abstract:Aim [WT5”BZ]To establish whether the changes of local blood flow involved in the vessel wall remodeling, we investigated the effects of culture medium conditioned with endothelial cells exposed to hemodynamic shear forces on modulation of smooth muscle cells(SMC) proliferation and collagen synthesis. Methods SMC were isolated and cultured with collagenase digestion. Confluent monolayers of EC were exposed to static or to laminar flow conditions for 24 h using a parallel-plated flow chamber. Endothelial cell-conditioned medium was used to study the growth of PASMC by 3 H-thymidine uptake and collagen synthesis of PASMC using 2,3- 3 H-proline incorporation into 3 H hydroxyproline. Results The proliferative response of SMC to culture medium from endothelial cells under static flow is higher than to fresh medium(1 202±63 cpm/10 3 cells vs. 749±53 cpm/103 cells,P<0.01). SMC synthesized more collagen cultured with medium from endothelial cells under static flow than fresh medium(47±4 cpm/10 3cells vs. 30±6 cpm/10 3 cells,P<0.01). In contrast, medium conditioned with endothelial cells exposed to shear stress of 10 dyn/cm 2 and 25 dyn/cm 2 after 18 h reduced SMC DNA and collagen synthesis by 10.41%±5.66%, 23.97%±6.23% and 45.71%±2.93%, 64.53±2.42% comparison with medium from endothelial cells under static flow. Conclusions Our experimental results showed lower shear stress led to enhance the underlying SMC proliferation and collagen synthesis. It suggests a steady level of shear stress acting on artery plays an important role of preventing it of remodeling.

Abstract:Aim To investigated the polymorphisms of the apolipoprotein AI(apoAI) gene with coronary heart disease (CHD) and atherosclerotic cerebral infarction (ACI) patients in Beijing area. And to evaluate the association of polymorphisms of MspI with variable of lipid. Methods Using the polymerase chain reaction (PCR), We studied the polymorphism of MspI in 226 patients with CHD and 236 patients with ACI and 321 age-matched healthy individuals. Results The polymorphic site was the MspI site in the third intron of apoAI gene, whose presence has previously been shown to be associated with CHD. The frequencies of the MspI minor allele M 2 were 0.387 in CHD patients (P<0.05). There were no difference in the frequencies of minor allele M 2 between ACI patients and controls (P>0.05). There were apparent differences in the genotypes of apoAI gene and several lipid variables among the CHD patients, ACI patients and control group. Conclusions M 2 allele in the third intron of the apoAI gene may associated with CHD and ACI and presumably through its influences on lipid metabolism to increase the susceptibility to atherosclerosis disease.

Abstract:Aim To compare the difference between human cytomegalovirus (hCMV)-induced and hyperlipemia-induced in rabbits. Methods The rabbits were randomly divided into three groups: the control group, the group fed on diet containing high lipid, and the group infected with hCMV AD169 strain. In 60th day the arterial tissue speciments were detected by immunohistochemistry, pathological method and electron microscopy. Results The pathological alterations of the arterial intima of the rabbits inoculated with hCMV, were EC swelling, degeneration, necrosis and dropping from intima with adhering fibrin-like materials. Four thrombus-like materials were found in nineteen of arterial speciments. The hCMV antigens were detected in EC and virion-like materials were found in smooth muscle cells. Fibrin-like materials adhering to the surface of ECs were manifested by electron microscopy;The pathological alterations of the arterial intima of the rabbits which fed on diet containing high lipid were atherosclerosis. A large number of red blood cells adhering to the surface of the EC were found by electron microscopy. Conclusion There are some differences in the pathological changes of the intimal injury between the hCMV-induced and hyperlipemia-induced of in Rabbits.

Abstract:Aim To study the relation between cholesterol ester transfer protein (CETP) gene mutation and size of low density lipoprotein (LDL) particle. Methods Exon 15 missense mutation (442D∶G) in CETP was determined using PCR-RFLP method in 200 patients with coronary artery disease (CHD). LDL particle size was analyzed by non-denaturing polyacrylamide gradient gels. Results 6 heterozygotes and 1 homozygote were found to had the 442D∶G mutation among 200 CHD patients. Frequency of this mutation was 3.5%. The patients with gene mutation (n=7) had significantly larger particle diameters than those without the mutation (n=40) (26.92±0.79 nm vs. 25.71±0.66 nm, respectively;P<0.01). LDL subfraction pattern in all the patients with gene mutation is pattern A. The distribution of LDL subfraction pattern was significantly difference between two groups. The patients with gene mutation had decreased plasma CETP level, while elevated level of HDLC and apoA_Ⅰ compared with control. Conclusion LDL particle size in patients with CETP gene mutation increases.

Abstract:Aim To study the effects of glucagon, dibutyryl cAMP on secretion of apolipoproteins A_Ⅰ,A_Ⅱ,CⅢ,B100 and E by HepG2 cells. Methods The apolipoprotiens contents in culture media were measured by radioimmuodiffusion assay (RID) kits developed by authors' research laboratory. 20-fold lyophilizely condensed culture media were used for the assays. Results When the concentration of glucagon is 4×10 -7 mol/L in culture media, the secretion of apoA_Ⅰ,A_Ⅱ,B100 and E decreased 47.7%, 54.1%, 32.7% and 33.2%(P<0.01) respectively, while the secreation of apoCⅢ increased 89.1%(P<0.01); When the concentration of dibutyryl cAMP is 1×10 -3 mol/L in culture media, the secretion of apoA_Ⅰ,B100 and E decreased 12.3%, 24.9% and 16.1% (P<0.01)respectively, while the secretion of apoA_Ⅱ and CⅢ increased 66.1% and 142.6%(P<0.01). Conclusion [WT5”BZ]Glucagon and dbcAMP had similar effects on the secretion of apolipoproteins by cultured HepG2 cells.

Abstract:Aim To study the mechanisms of the high restenosis rate of the patients with diabetes mellitus after percutaneous transluminal coronary angioplasty (PTCA) and the influence of L-arginine (L-Arg) and vitamin C (Vit C) on intimal hyperplasia. [WT5”HZ]Methods The iliac arteries of 36 diabetic rabbits induced by alloxan and 8 normal rabbits were injured by balloon. All rabbits were divided randomly into control group, DM group, L-Arg group, Vit C group and L-Arg+Vit C group, respectively provided with normal drinking water, normal drinking water, 2.25% L-Arg solution as the drinking water, Vit C (0.25 mg/g·d), or combided L-Arg with Vit C for 5 weeks. 4 weeks after the balloon injury, all the illacs were excised to being analyzed by computer after formaldehyde-intu-perfuse. Results The neointimal area of DM group was significantly larger than that of control group (0.255±0.024 mm 2 vs 0.176±0.029 mm 2,P<0.01), however, the neointimal area of L-Arg+Vit C group was significantly reduced than that of DM group (0.153±0.016 mm 2 vs 0.255±0.024 mm 2,P<0.01), together with a significant decrease in malondialdehyde (MDA) plasma level and significant increase in both superoxide dismutase (SOD) and nitric oxide (NO) plasma levels than those of DM group. Conclusions The therapy of combining L-Arg with Vit C could reduced the neointimal hyperplasia induced by balloon injury in diabetic rabbits. It might be effective by increasing the synthesis of endothelium-derived relaxing factor (EDRF) and protecting EDRF from being quenched by oxygen free radical.

Abstract:Aim To investigate the influence of oxidized low density lipoprotein (ox-LDL) and pravastatin on expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC) in order to elucidate one of the molecular mechanisms of ox-LDL on pro-atherosclerosis and to explore non-lipid mechanism of pravastatin on anti-atherosclerosis. Methods HUVEC was incubated in vitro. Ox-LDL of 50 mg/L, 100 mg/L, 200 mg/L and pravastatin of 10 -6～-4 mol/L were co-incubated with HUVEC for 12 h, 24 h and 36 h respectively. The expression of ICAM-1 in protein level and mRNA level was detected by enzyme linked immunosorbent assay (ELISA), flow cytometric technique and reverse transcription-polymerase chain reaction (RT-PCR). Results Ox-LDL induced ICAM-1 expression in HUVEC in concentration-dependent and time-dependent manner. Pravastatin exerted inhibitory effect on ox-LDL-induced ICAM-1 expression, which was also dosage-dependent and time-dependent. Conclusions Ox-LDL could induce or up-regulate ICAM-1 expression in HUVEC, which probably reflected a new mechanism of ox-LDL to atherogenesis. Pravastatin inhibited ox-LDL-induced ICAM-1 expression, which may crucially contribute to the clinical benefits of HMG-CoA reductase inhibitors on coronary artery disease, beyond the cholesterol-lowering effects.

Abstract:Aim To observe the synergistic effect of basic fibroblast growth factor and interleukin-6 on vascular smooth muscle cells proliferation. Methods BrdU incorporation and cell counting method were adopted to observe the effects of basic fibroblast growth factor and interleukin-6 on the proliferation of rat aortic cultured smooth muscle cells. Results 0～10.0 μg/L basic fibroblast growth factor and 0～100 μg/L interleukin-6 were able to promote smooth muscle cells proliferation separately, and synergistically in combination in dose- and time-dependent manner. The pro-proliferation effect reached climax at 24 hours. Conclusion Basic fibroblast growth factor and interleukin-6 had synergistic effect on vascular smooth muscle cells proliferation.

Abstract:Aim The effects of heparin and low molecular weight heparin (LMWH) on the growth of cultured human aortic smooth muscle cells(hASMCs) was studied in this article. Methods The thirth-passage hASMCs were planted onto 24-well plate. After incubated for 24 h in media supplemented with 10%human serum(HS) and 10%fetal calf serum(FCS), media were changed into low concentration serum(0.5% HS, 0.5%FCS)and continued to incubate for 24 h and cultured by DMEM containing 10%HS either old or fresh, 10%FCS and different concentration of heparin or LMWH (heparin and LMWH were presented as hexuronic acid) together with corresponding control groups (without heparin or LMWH) for 24 h, hASMCs growth was estimated both morphologically and by 3 H-TdR incorporation. Results Both heparin and LMWH inhibited the proliferation of well-growth hASMCs, the inhibition rates were 66%, 69%, 69%, and 60%, 68%, 67%, respectively, with no concentration dependent relationship. hASMCs of control (with old HS stored at 4℃ for 2 years) shrank and showed poor-growth pattern while those in groups of different concentrations of LMWH or heparin became well-spread and well-growth pattern. Both heparin and LMWH promoted the proliferation of poor-growth hASMCs, the stimulation rates were 149%, 140%, 180%, and 207%, 246%, 309%, respectively. Conclusions Heparin and LMWH have a dual regulative role (inhibition and promotion) in hASMCs growth. It indicate that they may play an important role in controlling the proliferation of vascular smooth muscle cells and maintaining the integrity of vascular structure.

Abstract:Aim Study on the accurence and regluar patterns of atrial natriuretic factor (ANF) in the human heart befor birth. Methods Collection of 65 embryo cares from accidental abortionsor miscarriages or induced labour of the pregrant women with no cardiovascular disease in clinical diaynosio, measurement of the amount of ANF in different locations of the embryocal heart means of radioimmunoassay. Results ANF in the atria appeared in the sixth week ,but in a minute amount, increasing with the growth of the embryo, while the amount of ANF in the ventricals reached a rather higher level in the sixth week, decreasing with the growth of the embryo and then nearly disappeared after a full term. In the right auriclua of the embryos at the same age ANF reached the maximam, with the amount in the right atrium over that in the left, and the amount in the left ventricle over that in the right. Conclusions ANF in the human embryocal atria appeared in the sixth week, increasing with the growth of the embryo while ANF was also found in the ventricles and the interventricular septa, decreasing with the growth of the embryo.

Abstract:Aim To investigate the effects of transmyocardial laser revascularization (TMLR) on acute myocardial ischemic injuries. Methods Nd: YAG laser with a guartz optical fiber was treated to irradiate the canine acute myocardial ischemic area and the dynamic changes of epicardial ST-segment electrocardiograms serum creatine kinase level were measured following TMLR. Results The deviant summation of ST segment (∑ST) in single channel group was 42.3±12.8 mm, the total numder of extraordinary elevation point in ST segment(NST) was 6.5±1.3 and ∑ST in the multi-channel group was 20.1±6.72 mm, NST 5.3±0.6. Both were significantly lower than ∑ST 80.4±24.2 mm, NST 9.8±1.1 before being laser-treated respectively (P<0.05 orP<0.01). As being compared to the peak valve of contrast group in the corresponding period, ∑ST and NST in laser single channel group and multi-channel group were significantly lower than those in the contrast group(P<0.05). There was no significant difference(P>0.05) in serum activity CK level in all groups. Conclusion Transmyocardial laser revascularization (TMLR) can reduce the injuries of ischemic myocardium when canine coronary artery is ligated.

Abstract:Aim To study the effects and mechanism aroused from inhalation of nitric oxide (NO) in patients with chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH). Methods The pulmonary and systemic hemodynamics, blood gas exchange, oxygen transportation, guanosine 3', 5'-cyclic monophosphate (cGMP), and methemoglobin (MetHb) level were measured with Swan-Ganz catheter in 18 patients with COPD-PH before and after inhalation of 40 ppm NO. Results The pulmonary vascular resistance (PVR) was decreased by 28.1%, 31.3% and 34.4%, respectively, meanwhile the mean pulmonary arterial pressure (mPAP) was also decreased after continuous inhalation of NO for 2 min, 8 min and 15 min (P<0.01). Both mPAP and PVR were returned to basic line in 2 min after a stop of inhaled NO. Moreover, few effects of inhaled NO on systemic arterial pressure and systemic vascular resistance was observed. After inhaled NO 15 min, PaO 2 was increased from 6.9±1.5 kPa to 7.8±1.9 kPa, Qs/Qt was decreased from 26%±6% to 20%±7% , and mixed venous cGMP level was increased from 14.6±7.8 pmol/L to 28.9±6.7 pmol/L (P<0.05), but VA/Q, arterial oxygen transport, coefficient of oxygen delivery, and MetHb level were not altered during inhalation of NO. Conclusion The data indicate that the pulmonary blood vessel in COPD-PH can be dilated immediately and selectively, gas exchange in lung can be improved during inhalation of NO , the mechanism of which may be related to increase of cGMP.

Abstract:Aim To explore the effects of different ratios of saturated, monounsaturated and polyunsaturated fatty acids of oral fatty loads on the postprandial lipids and lipoproteins metabolism. Methods The oral fatty loaded tests with three different fatty acids ratio. Results (1) Lipid concentrations: Triglyceride responded greatly to the oral fatty loads in three groups. The triglyceride in 2, 4, 6 hour plasma after the loads was significantly higher than fasting triglyceride, respectively (P<0.05). No significant total cholesterol changes occurred but high density lipoprotein cholesterol and low density lipoprotein cholesterol significantly decreased in group Ⅱ and Ⅲ. The increment of triglyceride at 2 hour after the test meal was significantly higher in group Ⅱ than group Ⅲ (P<0.05). Low density lipoprotein cholesterol in 6 hour plasma in group Ⅲ was significantly lower than that in group Ⅱ and Ⅲ (P<0.05). (2)Peak time:Triglyceride in group Ⅰ, Ⅱ and Ⅲ peaked at 2～4, 4 and 6 hours after the fatty load. High density lipoprotein cholesterol and apoprotein A1 in group Ⅱ and Ⅰ were lowest at 4 hours after the loads but at 6 h in group Ⅲ. (3)Area under the curve: Area under the curve of low density lipoprotein cholesterol in group Ⅲ was markedly smaller than in group Ⅱ. Conclusion The study showed that fatty acids composition ratios of oral fatty loads influenced the dynamic metabolism of the postprandial lipids and lipoproteins metabolism in type Ⅱ diabetic patients. We suggested that the fatty acid composition ratio of 1∶1.7∶1.2 could be used in the fatty load.

Abstract:Aim To explore human interleukin-17 (hIL-17)gene in the pathogenesis of coronary disease. Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to amplified hIL-17 gene from activated peripheral blood mononuclear cells (PBMC), hIL-17 gene was confirmed by DNA sequencing. Expression of hIL-17 mRNA in PBMC was determined by dot hybridization techniques. Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) was determined by enzyme linked immunosorbent assay (ELISA) method. Results With reference to the published sequence of the human IL-17 gene, a pair of DNA primer were designed and synthesized. A 473 bp fragment encoding fall length of hIL-17 cDNA gene was successfully amplified by using RT-PCR, and then cloned into pUC18 vector. The recombinant plasmid was sequenced to confirm hIL-17gene. Meanwhile, Using random-prime synthesis of probe for hIL-17 gene. We apply for dot hybridization techniques to determine the over expression of hIL-17 mRNA in peripheral blood monocytes (PBMC) with coronary disease, and their high levels of sICAM-1. Conclusions hIL-17 gene may play an important role in the pathogenesis of coronary Disease. Our experiment also lay a profound foundation for further analysis of hIL-17 biological functions.

Abstract:Aim To study the association between prior infection with chlamydia pneumoniae and chronic coronary heart disease. Methods Chlamydia pneumoniae-specific lgG and lgM antibodies were measured in 45 patients with acute myocardial infarction (AMI), 32 patients with old myocardial infarction (OMI) and 101 control by microimmunofluorescence test. Results The positive rate of specific lgG antibodies was 95.6% (43/45) in patients with AMI, 81.3% (26/32) in patients with OMI and 56.4% (57/101) in controls, the geometric mean titres of spesifc lgG antibodies were 95.6±113.5, 51.7±39.1 and 35.3±33.7 respectively. The positive rate and geometric mean titres of specific lgG antibodies in patients with AMI and OMI were significantly higher than those in controls. Conclusion There may exist an association between chronic chlamydia pneumoniae infection and coronary heart disease.

Abstract:Aim To study the association of cytomegalovirus (CMV) with atherosclerosis. Methods The present of cytomegalovirus (CMV) nucleic acids was demonstrated in carotid artery and coronary artery of patients with and without atherosclerosis by polymerase chain reaction (PCR), and detected CMV by PCR in the blood of cerebral infarction patients with and without atherosclerosis. Results 83.3%-86.7% of the samples obtained from atherosclerotic patients' arterial wall were shown to contain viral nucleic acids (CMV), among 6.6% of patients' arterial wall without atherosclerosis CMV could be found, significant deference can be found between them (P<0.01). In blood CMV could be found in 42.4% of patients with atherosclerosis, in the comparison only 3% of samples had CMV (P<0.01). Conclusion CMV plays an important role in the pathologic process of the atherosclerosis and the atherosclerotic cerebral infarction.