Abstract:Aim To investigate the expression pattern of plasma exosome mRNA inAMI(acute myocardial infarction ) patients using NCCP(noncardiac chest pain)patients as control group, and determine the possible effects of differentially expressed mRNAs in AMI patitents. Methods Fifteen NCCP andAMI patients were enrolled from Beijing Chao-yang Hospital and Baotou Central Hospital. Exosomes and exosome RNA were extracted from the plasma of selected patients. High-throughput sequencing technology was used to detect the expression of mRNA(message RNA )profile in exosomes in the plasma of patients with AMI and NCCP. GO (gene ontology) and KEGG (kyoto encyclopedia of genes and genomes) were used to analyze differentially expressed mRNAs. Results The size of exosomes was between 30-200 nm. These exosomes express CD63(cluster of differentiation 63) and HSP70 (heat shock proteins 70), but not GAPDH (glyceraldehyde-3-phosphate dehydrogenase), conforming with the characteristics of exosomes. 64258 mRNAs were detected in the plasma exosomes of AMI patients and 64374 mRNAs were detected in the plasma exosomes of NCCP patients.Among these detected mRNAs, 1800 mRNAs were differentially expressed in AMI group (951 mRNAs were upregulated, and 849 mRNAs were down regulated). GO and KEGG analysis showed that these differentially expressed mRNA participated in the biological regulatory functions and pathways of AMI. Conclusion The mRNA expression pattern of plasma exosomes in patients with AMI was significantly different from that in the NCCP group. These differentially expressed exosome mRNAs may play important roles in the occurrence and development of AMI.

Abstract:Aim To observe the effects of lysophosphatidic acid(LPA) on system generation of adrenomedullin(ADM) with its receptors:calcitonin receptor like receptor(CRLR) and receptor activity modifying protein(RAMP),and the effects of ADM on proliferation of the vascular smooth muscle cells(VSMC) induced by LPA.Methods VSMC of rat aortic were cultured by explant method.DNA synthesis of VSMC were measured by 3H-TdR incorporation.The ADM concentration in VSMC were determined by radioimmunoassay.Results LPA stimulated the production of ADM on vascular smooth muscle cells in rats.LPA up-regulated the expression of mRNA of ADM and its receptors CRLR,RAMP2 and RAMP3 in VSMC.ADM can inhibit 3H-TdR incorporation of rat VSMC stimulated by LPA.ADM inhibited activation of mitogen-activated protein kinase(MAPK) in VSMC induced by LPA.Conclusions ADM and its receptors inhibit VSMC proliferation which involves in its inhibition on MAPK activity.

Abstract:Aim To observe the effect of vascular endothelial growth factor(VEGF) on the apoptosis and the expression of Fas mRNA and Bcl-2 mRNA in human umbilical vein endothelial cell(hUVEC). Methods hUVEC were randomly divided into three groups: control group,hydrogen peroxide group and hydrogen peroxide + VEGF group.After 12 hours,the apoptosis of hUVEC was determined by flow cytometry(FCM) and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL),the expression of Bcl-2 mRNA and Fas mRNA were examined by reverse transcription polymerase chain reaction(RT-PCR). Results Apoptosis number in hydrogen peroxide group was higher than that in control group(27.83±2.14 vs 2.50±1.05,p<0.01).However,in hydrogen peroxide+VEGF group,apoptosis number was lower than that in hydrogen peroxide group(13.00±2.10 vs 27.83±2.14,p<0.01).Apoptosis rate in hydrogen peroxide group was higher than that in control group (14.17%±0.45% vs 1.55%±0.87%,p<0.01) and in hydrogen peroxide+VEGF group(14.17%±0.45% vs 5.69%±0.38%,p<0.01).Compared with control group,hydrogen peroxide markedly increased Fas mRNA expression(94.50%±3.45% vs 21.17%±1.17%,p<0.01) and decreased Bcl-2 mRNA expression(23.17%±1.17% vs 85.17%±1.47%,p<0.01).Compared with hydrogen peroxide group,VEGF significantly decreased Fas mRNA expression((40.67%)±2.16% vs 94.50%±3.45%,p<0.01) and increased Bcl-2 mRNA expression(60.33%±1.75% vs 23.17%±1.17%,p<0.01). Conclusions VEGF can inhibit the apoptosis induced by hydrogen peroxide in HUVEC,which may correlated with upregulation Bcl-2 mRNA expression and inhibiting Fas mRNA expression.

Abstract:Aim To investigate the process of neointimal formation, level of platelet activation and thrombin receptor mRNA by means of a rat aortic balloon injury model in order to study the mechanism of acute and late restenosis after percutaneous transluminal coronary angioplasty(PTCA). Methods Forty eight male Wistar rats were divided into two groups randomly. Group 1 (n=24) served as controls, group 2 (n=24) were given aortic balloon injury by self-made 2F balloon catheters. The process of intimal thickening, number of platelet GMP-140 and level of thrombin receptor mRNA were investigated at day 3, 7, 14 and 28 after balloon injury by histological method, radioimmunological method and reverse transcription polymerase chain reaction (RT-PCR) technique, respectively. Six rats were examined at each group. Results The expression of thrombin receptor mRNA was at a low level in endothelium and smooth muscle cell of normal arteries, but increased significantly at day 3 after balloon injury, reached its peak at day 14 and decreased at day 28. The number of platelet GMP-140 was higher at day 3 and began to decrease at day 7 after injury. The migration and proliferation of vascular smooth muscle cell had existed at day 3 after balloon injury. The intimal thickening began at day 7 after injury, and it was more significant at day 14. The proliferation of VSMC decreased at day 28, but extracellular matrix increased and the intimal thickening continued. Conclusions The expression of TR mRNA and the number of platelet GMP-140 increase in the process of intimal thickening after balloon injury.

Abstract:Aim To evaluate the effect and explore the mechanism of the Compound Salvia Miltiorrhiza Bunge Injection on the vascular endothelium-dependent vasodilatation in patienents with CHD. Methods 84 patienents were randomly divided into three groups(n=28): all patienents were given basic therapy according to the therapeutic guide of CHD, while the two therapeutic groups were respectively added the Compound Salvia Miltiorrhiza Bunge Injection and VitC. We examined the baseline(D 0) and the flow-mediated vasodilation (FMD) of brachial artery by high-resolution ultrasound technique and drew blood to examine the concentration of ET-1,NO and the expression of ET-1 and eNOS mRNA before experiment and after three week’s therapy. The expanding percentage of the flow-mediated vasodilation(FMD) of brachial artery represented the endothelium-dependent vasodilatation(EDD) [EDD=(FMD-D0)/D0×100%]. Results Three groups had no significant difference in D 0, FMD and EDD of brachial artery before experiment. But the EDD of two therapeutic groups improved notably after three weeks’ therapy, and the Compound Salvia Miltiorrhiza Bunge Injection group was better than VitC group. There was no difference of ET-1 and NO among three groups before experiment. After three week’s therapy, the compound salvia miltiorrhiza bunge injection group showed decreasing in the concentration of ET-1 and expression of ET-1 mRNA,increasing in the concentration of NO and expression of eNOS mRNA, all that were significantly different from that of the control group(p<0.05). The VitC group also showed increasing in the concentration of NO and expression of eNOS mRNA, but lower than that of the Compound Salvia Miltiorrhiza Bunge Injection group(p<0.05). Conclusion The Compound Salvia Miltiorrhiza Bunge Injection could improve the brachial endothelial function of CHD patients by regulating the expressions of ET-1 and eNOS mRNA.

Abstract:Aim To observe the change of endothelin content and to explore the effects of vascular calcification on endothelin expression on the model of vascular calcification in rats induced by vitamin D 3 plus nicotine and calcified vascular smooth muscle cells induced by β glycerophosphate. Methods Arterial calcification of Sprague Dawley rats was induced by vitamin D 3 plus nicotine (VDN). Calcification of cultured rat vascular smooth muscle cells (VSMCs) was prepared by incubation with β glycerophosphate. Calcification was confirmed by Von Kossa staining, measurerment of calcium content, 45 Ca 2+ accumulation and alkaline phosphatase (ALP) activity of intracellular and vascular tissue. Endothelin levels in the plasma, vascular tissue and medium were measured by using radioimmunoassary. Endothelin mRNA level was determined by using competitive quantitative reverse transcription polymerase chain reaction. Results The results showed that the content of calcium, 45 Ca 2+ uptake and alkaline phosphatases activity in calcified VSMCs were increased by 118%, 174% and 7 fold respectively (all p<0.01), compared with control VSMCs. Content of endothelin in medium was increased by 35% (p<0.01). It was found that the amount of endothelin mRNA was elevated by 120% (p<0.01) compared with control. The calcium content, 45 Ca 2+ accumulation and ALP activity in calcified arteries were increased by 5.0 fold, 1.4 fold and 1.4 fold respectively (p<0.01), compared with control. Furthermore, it was showed that endothelin 1 levels in plasma and arteries tissues increased 102% and 103% respectively, compared with control (p<0.01). The amount of endothelin mRNA in calcified aorta was elevated by 22% (p<0.01) compared with control. However, the content of calcium, 45 Ca 2+ uptake and ALP activity in VDN plus endothelin receptor inhibitor groups were decreased by 33%, 36.7% and 40.4% respectively (p<0.01), compared with VDN group alone. Conclusions These results showed that in calcified artery and VSMCs the production of endothelin was increased,and the gene expression of endothelin was up regulated. The Bosentan (inhibitor of endothelin receptor) significantly reduced the vascular calcification. These results suggested that endothelin could play a role in the pathogenesis of vascular calcification.

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

Abstract:Aim To 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 determine the expression of insulin like growth factor 1 receptor (IGF 1R)gene in atherosclerotic plaques. Methods White rabbits were fed with either regular feed or high cholesterol diets for 12 weeks. By in situ hybridization we detected the expression of IGF 1R mRNA in rabbit atherosclerotic plaques and normal artery wall. Results Control rabbits, hybridization with antisense RNA probe only localized IGF 1R mRNA to adventitia. In atherosclerotic aortas, the positive signals were widely distributed in the vessel wall, including neointima, media, adventitia and atherosclerotic plaques. Conclusion Smooth muscle cells, endothelial cells and foam cells are the major source of IGF 1 receptor mRNA in atherosclerotic lesions. Over expression of IGF 1R mRNA might be related to the genesis of the atheromatous plaque. 还原

Abstract:Aim To well understand the role of oxidized low density lipoprotein (ox-LDL) in the pathogenesis of thrombotic complications in atherogenesis. Methods LDL was isolated from normal heparinized blood by density gradient ultracentrifugation and oxidized by CuCl 2. Total RNA was extracted from human umbilical vein endothelial cells (hUVECs) exposed to LDL or ox LDL by guanidinium isothiocyanate method. The quantification of tissue factor pathway inhibitor (TFPI) mRNA in hUVECs was carried out by reverse transcriptase PCR (RT PCR). Results hUVECs were able to express TFPI mRNA constitutively. The expression was not affected by LDL but effectively inhibited by ox LDL in a time and dose dependent manner. Conclusions Oxidized LDL may play an important role in inducing coagulation in atherosclerotic lesion by inhibition of expression of TFPI in vascular endothelial cells.