Abstract:AimTo discover the expression of ZNF297B in the differentiation of smooth muscle cells and investigate the related mechanism.MethodsThe mRNA expression level of ZNF297B in human, rat and mouse smooth muscle cells and the change of ZNF297B expression level in smooth muscle cell differentiation and proliferation was tested by real-time PCR.The ZNF297B-Myc/His plasmid was constructed and transfected into rat smooth muscle cells to test the distribution of ZNF297B in cultured cells. The binding and interaction of ZNF297B and Myocardin was tested by luciferase assay and confirmed by co-immunoprecipitation.ResultsZNF297B was specificly highly expressed in smooth muscle cells.The expression level of ZNF297B was upregulated in the process of RA induced smooth muscle cell differentiation from mouse embryonic stem cells. ZNF297B expression was downregulated in PDGFBB stimulated smooth muscle cell proliferation process. In balloon-injured rat carotid arteries, ZNF297B was upregulated after 6 hours and then downregulated. ZNF297B mainly expressed in nucleus and the cytoplasm around nucleus. ZNF297B can bind and interact with Myocardin in protein level.ConclusionZNF297B may play an important role in smooth muscle cell differentiation.This function may be related to the interaction between ZNF297B and Myocardin.

Abstract:AimTo construct the human myofibrillogenesis regulator-1(hMR-1) full length eukaryotic expression plasmid and measure its expression in HEK293T cell lines and Sprague-Dawley neonatal rat cardiomyocytes.MethodsThe whole hMR-1 coding gene was cloned from NCBI GenBank database (AF417001), recombined with plasmid pcDNA3.1/Myc-His(-)B and transformed into Escherichia coli XL1-Blue.Positive clone was selected and sequenced by T7 primers.The recombined plasmid pcDNA3.1/Myc-His(-)B -hMR-1 was transfected by Lipofectamin2000 to cells, reverse transcription polymerase chain reaction (RT-PCR) and Western Blotting was employed for measuring the expression of hMR-1.ResultsPlasmid pcDNA3.1/Myc-His(-)B-hMR-1 was verified by sequencing that the target gene was correct, without any mutation; the expression level at mRNA and protein were both increased significantly after hMR-1 transfection.ConclusionA full-length hMR-1 coding gene eukaryotic vector was successfully constructed, based on which, a convenient and effective transient transfection method in neonatal rat cardiomyocytes was established as well.

Abstract:imTo explore the relationship of asymmetric dimethylarginine （ADMA）and senescence of endothelial progenitor cells (EPC) in type 1 diabetic rats.MethodsThe rat model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) for one time.The concentration of fasting blood glucose, the level of ADMA in plasma, the percentage of senescence of EPC from bone marrow and the mRNA expression of dimethylarginine dimethylaminohydrolase 2 (DDAH2) and SIRT1 were detected.ResultsThe senescence percentage of EPC（39 % ± 8 % vs 11 %±2%） and the level of ADMA （0.67± 0.06 μmol/L vs 0.55± 0.07 μmol/L ）in plasma were increased and the expression of DDAH2 and SIRT1 mRNA were down-regulated （0.56 ± 0.17 vs 1.00 ± 0.22，0.08 ± 0.17 vs 1.00 ± 0.39）.ConclusionThe senescence of EPC was related with the increased level of ADMA in plasma, which may involve the SIRT1/DDAH2 pathway.

Abstract:AimTo explore the potential mechanism of endothelial progenitor cells (EPC) on the regulation of AngⅡ-induced phenotype transformation of vascular smooth muscle cell (VSMC).MethodsEPC were obtained by isolating mononuclear cells using Ficoll density-gradient centrifugation and were identified by morphology, fluorescence double-staining and flow cytometry according to previous methods.ELISA were performed to analyze the secretion of calcitonin gene-related peptide (CGRP) in EPC and HUVEC.Early endothelial progenitor cells conditioned medium (E-EPC-CM) was pre-incubated with functional blocking antibodies against CGRP for 1h or VSMC was preteated with CGRP837(CGRP receptor antagonist) for 1 h before VSMC were pretreated with CM for 30 min, RT-PCR and Western-blot were performed to analyze the effect of E-EPC-CM on AngⅡ-induced the expression of α-SM-actin, calponin and phosphorylation of ERK, NF-κB(p65) in VSMC.ResultsThe level of CGRP was higher than that observed in the L-EPC-CM and HUVEC-CM groups.After stimulation with angiotensin Ⅱfor 48h, the expression of the α-SM-actin mRNA and protein significantly decreased and the osteopontin mRNA and protein markerly increased compared with control group, suggesting that VSMC was changed from contractile to synthesize type by angiotensinⅡ.But treatments with E-EPC-CM could up-regulate the expression of the α-SM-actin and down-regulate the expression of the osteopontin, suggesting that E-EPC-CM could inhibit the phenotype of VSMC from contractile to synthesize type induced by angiotensin Ⅱ.Moreover, we demonstrated that treatments with CGRP837 or anti-CGRP antibody could partly reverse the inbibiton effect of phenotype transformation of E-EPC-CM on the Ang II-stimulated VSMC.Likewise, we also demonstrated that treatment with anti-CGRP antibody or CGRP837 could partly reverse AngⅡ-induced phosphorylation of ERK, NF-κB(p65).ConclusionIt is showed that the inhibitory effect of EPCs on phenotype transformation of VSMC is associated at least partly with release of CGRP, which inactivates ERK and NF-κB signaling pathway.

Abstract:AimTo explore the glycoprotein Ⅱb/Ⅲa receptor antagonist, tirofiban, on the plasma inflammatory factors of rabbits in vivo, and find some theoretical basis for appropriately using the glycoprotein Ⅱb/Ⅲa receptor antagonists in atherosclerosis.MethodsNew Zealand rabbits were fed with hyper-cholesterol forage for 12 weeks to form atherosclerosis.The rabbits were divided to 4 groups (n=8), and had intravenous drip natural saline, 3.125 mg/L, 12.5 mg/L or 50 mg/L tirofiban in different group respectively. After the intravenous drip started for 0 h, 12 h, 24 h and 48 h, the platelet maximal aggregating rates ［PAG(M)］ were detected using turbidity method, the interleukin-6, interleukin-1β and tumor necrosis factor-α (TNF-α) level in the plasma were detected using enzyme linked immunosorbent assay (ELISA).Parameters differences among 4 groups were compared.ResultsAfter intravenous dripping started for 12 h, 24 h and 48 h, PAG(M) was reduced (PANOVA<0.05), P-selectin, interleukin-6, interleukin-1β and TNF-α were reduced (PANOVA<0.05) along with the tirofiban concentration increase.In control group and 3.125mg/L tirofiban group, PAG(M) had no difference after intravenous dripping started for 12 h, 24 h and 48 h (P>0.05). PAG(M), P-selectin, interleukin-6, interleukin-1β and TNF-α were reduced significantly (P<0.05) when ≥12.5 mg/L tirofiban was intravenous dripped.ConclusionThe PAG(M) and inflammatory factors were not affected when tirofiban was intravenous dripped in small dosage.When the tirofiban was intravenous dripped in medium or large dosage, the PAG(M) and inflammatory factors were inhibited significantly.

Abstract:AimTo investigate the expression of angiotensin-coverting enzyme (ACE) , angiotensin-coverting enzyme Ⅱ（ACEⅡ) and the effects of Valsartan on them after vascular balloon injury in rats.MethodsRat models of aortic endothelial denudation were made by an 2F balloon catheter.Rats were randomly allocated into three groups: Group 1 served as controls; Group 2 were given vascular balloon injury; Group 3 were given Valsartan from 1day before injury to 30 days after injury. The expression of ACE mRNA and the level of protein of ACE or ACEⅡ were investigated at day 14 and 30 after injury by reverse transcription polymerse chain reaction (RT-PCR) technique and immunohistochemistry method, respectively. ResultsIntimal thickening was significant at 14 days and 30 days after injury.Valsartan significantly and comparably prevented intimal thickening at 14 days and 30 days after balloon injury.The expression of ACE mRNA and protein was significantly increased at 14 days and 30 days after injury（P<0.05）, while the expression of ACEⅡ protein was significantly increased at 14 days and 30days after injury（P<0.05）.Valsartan can decrease the expression of ACE mRNA and protein and increase the expression of ACEⅡ protein（P<0.05）.ConclusionThe effects of Valsartan on aortic endothelial denudation and regression of vascular remodeling are related with the increased expression of ACEⅡ protein and the decreased expression of ACE.

Abstract:AimTo investigate the contribution of platelet activation and the damage marker of blood vessel endothelium in stroke.Methods300 SD rats were divided into three groups: model group, sham operated group and normal control group, the sham operated group were divided into sham operated group and sham operated+endothelium damage group; the model group were divided into hypertension group, endothelium damage group and hypertension+endothelium damage group.In the 12th week the rats were divided into artificial cold exposure (ACE) and non-ACE, only ACE were endured by artificial climate cabinet, blood samples were collected for measuring CD62p expression and brain tissues were got to analyse intercellular adhesion molecule-1 (ICAM-1) expression.ResultsIn model group, the CD62p and ICAM-1 expression were higher than other groups (P<0.05); the CD62p and ICAM-1 expression in ACE (model group) were higher than non-ACE group (P<0.05); the CD62p and ICAM-1 expression in hypertension+endothelium damage group was the highest in model group (P<0.05).ConclusionPersistent hypertension could damage the endothelium system of rat, artificial cold would worse this damage, and make it close to the state of pro-stroke.

Abstract:AimTo study the effect of angiotensin Ⅱ (AngⅡ) on cytoskeleton rearrangement of endothelial cells and the mechanisms within it.MethodsHuman umbilical vein endothelial cells (HUVEC) were treated with AngⅡ (10－6 mol/L) for different time points.The morphological images of filamentous actin (F-actin) cytoskeleton were observed with confocal laser scanning microscope.The phosphorylated protein expression of mitogen-activated protein kinase (MAPK) was tested by Western blotting.ResultsF-actin was mainly enriched along the cell membrane, and well distributed in normal HUVEC; After treatment with AngⅡ, most of the peripheral fibers disappeared, dense stress fibers were observed in the cytoplasm and paracellular gaps formed in a time-dependent manner.AngⅡ increased the phosphorylation of p38 MAPK, JNK1/2 and HSP27, but not ERK1/2.Furthermore, SB203580 (a p38 MAPK specific inhibitor) completely attenuated AngⅡ-induced formation of actin stress fibers and paracellular gaps.In contrast, SP600125 (a JNK1/2 specific inhibitor) had no effect on these responses.ConclusionAngⅡ induces F-actin rearrangement in HUVEC through the activation of p38 MAPK/HSP27 pathway， which results in cytoskeletal damage.

Abstract:AimTo explore the role of advanced glycation end products (AGE) in pathogenesis and development of diabetic complications.MethodsIndirect ELISA and Western blotting were used to analyse the immunological property of McAb and the epitope which would be combined with McAb.AGE in serum of human and aortas, renal, heart from diabetic rats were also detected by McAb.ResultsMcAb reacted with AGE specially and was combined with non-carboxymethyl lysine (non-CML).AGE in renal and heart of diabetic rats had been detected, but were not obviously detected in normal rats.ConclusionMcAb（non-CML）reacted with AGE specially, and might be of value for AGE measurement.

Abstract:AimTo investigate the effect of melatonin (MT) on the activity of inducible nitric oxide synthase in thoracic aorta of endotoxemia rats.MethodsSprague-Dawley rats were divided into four groups randomly: Vehicle group,Lipopolysaccharide (LPS) group(LPS 4 μg/g), LPS+MT group(MT5 μg/g, i.p.) which was given in twice, one injected 30 min before LPS and the other injected 60 min after LPS(4 μg/g ,i.p), and MT group.Six hours after LPS injection， rats were killed and thoracic aortic rings were prepared.The contraction response to phenylephrine and the endothelium-dependent relaxation response to acetylcholine before and after the incubation of aminoguanidine and Nω-nitro-L- arginine (L-NNA) were observed with the isolated artery rings technique.Inducible nitric oxide synthase activity in the artery tissues were detected.The ultrestructure of endothelium on the artery was observed by scanning electron microscope.ResultsCompared with LPS group, thoracic aortic rings in LPS+MT group exhibited an increased contraction response to phenylephrine.After incubation with aminoguanidine, the contractile response to phenylephrine increased significantly in LPS and LPS+MT group.Inducible nitric oxide synthase activity in LPS+MT group decreased markedly compared with that of LPS group in thoracic aorta.ConclusionThe protective role of MT on vascular activity in endotoxemia may be due to its properties to inhibit inducible nitric oxide synthase in a certain degree.

Abstract:AimTo investigate the effect of diallyl trisulfide (DT)-coated coronary stents on expression of metalloproteinase-2 （MMP-2） and tissue inhibitor of matrix metalloproteinase-2 （TIMP-2） in canine model of restenosis.MethodsDT-coated stents (210 μg/stent) were deployed with mild oversizing in the left circumflex coronary arteries (LCX) or the left anterior descending coronary arteries （LAD）of 14 canines.The implantation of protein-coated stents were used as control.Histopathologic examination was performed 28th days after stents implantation.Total RNA was isolated from the arterial wall at different time (5 h, 1 d, 7 d, 14 d and 28 d) after stents implantation.RT-PCR method was adopted for detection of MMP-2 and TIMP-2 genes expression.ResultsNeointimal hyperplasia and the restenosis rate were less in DT-coated stents than those in control group (P<0.01).MMP-2 was constitutively expressed in uninjured coronary arteries, and its activity did not increase until 7 days and remained elevated up to 28 days after the implantation of protein-coated stents.MMP-2 genes expression in DT group was reduced at 5 h, 1 d, 14 d and 28 d compared with that in control groups (P<0.01).The expression of TIMP-2 genes was not significantly different at 5 h, 1 d and 14 d after the implantation of DT-coated stents and protein-coated stents.At 7 d and 28 d ,TIMP-2 genes expression increased in DT group compared with that in control groups (P<0.05).ConclusionDT-coated coronary stents can decrease the expression of MMP-2 and enhance the expression of TIMP-2 in the canine LAD or LCX after implantation, which could therefore contribute to decreasing the effects of its neointimal hyperplasia.

Abstract:AimTo explore the possibility of homocysteine-induced vascular smooth muscle cell (VSMC) proliferation via N-methy-D-aspartate (NMDA) receptor and its possible terminal target molecules.MethodsVSMC proliferation was induced by homocysteine, and the VSMC was treated with NMDA receptor antagonist MK801.The rate of proliferation of VSMC were detected by the way of MTT.Cell cycle distribution were determined by flow cytometer.The cyclin D1 mRNA expression in cultured VSMC were measured by RT-PCR.ResultsHomocysteine significantly stimulated VSMC proliferation, promoted VSMC convert from the G0/G1 to S phase, and increased cyclin D₁ mRNA expression. MK801, however, inhibited the proliferation of VSMC by homocysteine, the conversion from G0/G1 to S phase and the expression of cyclin D₁ mRNA were synchronously inhibited.All these effects showed significant dose-dependent manner.ConclusionThe proliferation-promoting effect of homocysteine on VSMC might be partly mediated by NMDA receptor and ultimately achieved through the cyclin D pathway.

Abstract:AimTo explore changes in the amount and function of the late endothelial progenitor cells(EPC) colonies derived from peripheral blood in patients with coronary heart disease（CHD）.Methods Fifty four cases were divided into CHD group(n=27) and control group(n=27).Mononuclear cells were isolated from peripheral venous blood with density-gradient centrifugation and were cultured.After 21 days, the late EPC were identified and the late EPC colonies were counted.The late EPC proliferation and migration were assayed by MTT assay and modified Boyden chamber assay, respectively.The late EPC adhesion assay was performed by replating those on fibronectin-coated dishes, then adherent cells were counted.ResultsThe amount of the late EPC colonies （2.5±1.2） were significantly reduced in patients with CHD compared with control subjects（3.8±1.6）(P<0.05) .The function of proliferation， migration and adhesion of the late EPC in CHD group were significantly lower than control group （0.324±0.024 vs 0.433±0.064，9.9±2.5 vs 13.9±4.P>1，2P>1.3±5.1 vs 31.0±7.1）(P<0.05).ConclusionDecreased late colonies amount can be observed in CHD group,and impaired function can be observed.

Abstract:AimCardio-ankle vascular index (CAVI) was newly developed index of arterial wall stiffness.To compare the validity of both diagnostic technique CAVI and carotid intima-media thickness (IMT) which was known to reflect cervical arterial atherosclerosis, both indices were measured in patients with coronary stenosis and cerevral infarction and non-atherosclerosis diseases.MethodsBetween January and June 2006, a total of 183 (55～70 years old ) consecutive subjects were measured with CAVI and IMT.The percentage of difference of left and right sidedness was compared with measured value for CAVI and IMT.In the comparison study for validity of both diagnostic technique, miocardial infarction(n=42), cerebral infarction ( n=47) and non-atherosclerosis (n=30)were included.CAVI and IMT were simultaneously measured.Validities of CAVI and IMT for various cardio-and cerebro-vascular diseases were evaluated by ROC analysis.Instrument for CAVI was applied in Sakura hospital (TOHO university ).In all, 3 autopsied patients had performed concurrently CAVI measurement in life.The study compared respective agreement degree between measurement value and pathological change in main artery.Results The percentage of difference of left and right sideness with measured value for CAVI was remarkably smaller than IMT.The area under the ROC curves of CAVI and IMT for coronary stenosis were 0.894(95%CI 0.810～0.978) and 0.700(95%CI 0.566～0.834) respectvely.And the area under the ROC curves of CAVI and IMT for cerebral infarction were 0.831(95%CI 0.747～0.914) and 0.576 (95%CI 0.458～0.695) respectively.Obviously areas under the ROC curve of CAVI were larger compared with area under ROC curve of IMT whether in coronary artery or cerebro-artery atherosclerosis.Three autopsy cases demonstrated that CAVI and IMT during in life was agreement with severity of aortic atherosclerosis.ConclusionCAVI has a high validity for diagnosis to IMT, it is a good indicator of cardio-and cerebro-vascular event, and is more likely reflecting arterioscleosis in a whole body than IMT.

Abstract:AimTo investigate the association between apolipoprotein B(ApoB) gene polymorphism and coronary heart disease with diabetes in Han and Uygur.MethodsUsing PCR-LDR,we detected the genotypes of EcoRⅠ of ApoB gene in 136 unrelated healthy individuals and 266 patients with evidence of CHD, which included 154 diabetes.The relation of gene polymorphism ApoB and levels of serum lipids, apolipoproteins was also studied.ResultsNeither the frequencies of genotypes nor those of alleles of ApoB gene EcoRⅠ polymorphism was statistically different between patients and controls (P>0.05), both in Uygur and Han in CHD and CHD with diabetes.No relation was found between A allele and lipid level.ConclusionThe EcoRⅠ polymorphism of ApoB gene maybe affect the levels of serum lipids and lipoproteins, but were not major genetic factors of CHD and CHD with dibetes in Han and Uygur.

Abstract:AimTo investigate the change of plasma tissue factor (TF) level in the ischemic smoker and the healthy smoker, and discuss the influence of cigarette smoking on cerebral thrombosis.Methods Plasma TF levels of 45 smoking brain ischemic patients, 43 non-smoking brain ischemic patients, 42 healthy smokers and 45 healthy non-smokers were detected with two-layer antibody sandwich enzyme-linked immunosorbent assay (LWISA). ResultsThe plasma TF level of smoking brain ischemic group was obviously higher than that of non-smoking brain ischemic group (P<0.01) and healthy non-smoking group (P<0.01); TF level of healthy smoking group was obviously higher than that of non-smoking healthy group (P<0.01), and heavy smoking group was obviously higher than middle smoking group (P<0.05), little smoking group (P<0.01)and healthy group (P<0.01).The smoking index was positively correlated with the plasma TF value（r=0.43，P<0.01）.ConclusionThe improvement of plasma TF level induced by smoking may be a pathway through which smoking induce brain atherosclerosis and cerebral thrombosis.

Abstract:AimTo investigate the operative outcomes of patients between on-pump coronary artery bypass grafting (CABG) and off-pump CABG.Methods59 patients were randomly assigned to undergo on-pump CABG and 47 to undergo off-pump CABG.Data on postoperative and operative variables were analysed.ResultsThere was no difference in the parameters including the mean number of grafts, artery grafts and revised grafts, mortality and postoperative complications (P>0.05).But the parameters such as the mean duration of postoperative ventilation support, days in the ICU or in hospital, postoperative pleural fluid drainage and blood transfusion requirement of off-pump CABG group were lower than those in CABG group (P<0.05).ConclusionIn this study, off-pump CABG was effective as on-pump CABG, there are advantages in off-pump CABG, especially in the mean duration of postoperative ventilation support, days in the hospital, postoperative pleural fluid drainage and blood transfusion.

Abstract:AimTo discuss the concentration of sercum total bilirubin in the etiology of atherosclerotic thrombotic cerebral infarction.MethodsThe concentration of fast serum total bilirubin were compared in the group of atherosclerotic thrombotic cerebral infarction and control persons.ResultsIn the normal range of serum total bilirubin,the lower of serum total bilirubin, the higher of incidence of atherosclerotic thrombotic cerebral infarction.ConclusionIn the normal range, serum total bilirubin have a certain preventive effect for incidence of atherosclerotic thrombotic cerebral infarction.

Abstract:AimTo approach polymorphism distribution of angiotensin converting enzyme （ACE） and cytochrome P450 （CYP） 3A5 gene of hypertensive patients in Hunan province.MethodsThere were 583 hypertensive patients.ACE gene polymorphisms of 403 hypertensive patients in Loudi and Xiangtan were investigated.While, CYP3A5 gene polymorphisms of 180 hypertensive patients were investigated.ResultsACE allele frequency was I (55.8%) and D (44.2%), in order with ID as the highest genotype.CYP3A5 allele frequency was *1 (35.0%) and *3 (65.0%), in order with *1*3 as the highest genotype.No sex difference was found in genotypes and allele frequency distributions (all P>0.05).ConclusionPolymorphism distribution of ACE and CYP3A5 gene of hypertensive patients in Hunan province reached Hardy-Weinberg genetic balance, hypertension related drug metabolism enzyme and receptor gene polymorphisms showed the individual differences, which provided the previous genetical evidences for single-drug therapy in hypertension population.

Abstract:Growth differentiation factor-15 (GDF-15) which belongs to the GDF family is a member of TGF-β super family.The previous researches indicated that GDF-15 increased its expression in the condition of rheumatoid arthritis, inflammation, tumor, anemia and so on.During recent years, it was found that increased level of GDF-15 was associated with heart failure (HF), acute coronary syndrome (ACS), idiopathic pulmonary arterial hypertension (IPAH) and other cardiovascular diseases.GDF-15 which provides independent prognostic information in HF and other cardiovascular diseases might be a new promising biomarker.

Abstract:The soluble epoxide hydrolase (sEH) in mammalian systems is a member of the α/β-hydrolase fold family of enzymes and it shows a high degree of selectivity for epoxides of fatty acids.The epoxides of arachidonic acid or epoxyeicosatrienoic acids (EET) are particularly good substrates.These EET appear to be major components of the endothelium-derived hyperpolarizing factors (EDHF).As such, EET cause vasodilation and reduce blood pressure.The EET also have variable biological effects, such as anti-inflammatory, fibrinolysis, stimulating angiogenesis, etc.Whereas inhibitors of the regulatory enzyme—soluble epoxide hydrolase inhibitors (sEHi), can increase the level of EET and mimic those effects.Therefore, sEH inhibitors are being extensively studied as a therapeutic potential for the treatment of hypertension, atherosclerosis, and inflammatory diseases.