Abstract:Aim To examine the role of Cl- channels in fetal calf serum (FCS) induced proliferation of vascular smooth muscle cells (VSMC). Methods Through cell count and 3 H-TdR incorporation, together with i measurement technology of fura-2 fluorescence, we studied the effects of Cl- channel blockers on proliferation of VSMC induced by FCS. Results Cl- channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.01 μmol/L～0.1 mmol/L) inhibited 5%FCS-induced increase in cell count and 3 H-TdR incorporation into VSMC, this inhibition was time and dose dependent. Other Cl- channel blockers such as indanyloxyacetic acid (IAA-94,0.1 μmol/L～1 mmol/L), 5-nitro-2-(3-phenylpropylamino)-benzonic acid (NPPB, 0.1 μmol/L～1 mmol/L),diphenylamine-2,2'-dicarboxylic acid (DPC, 0.1 μmol/L～1 mmol/L),4-acetamido-4'-isocyanatostilbene-2,2'-disulphonate (SITS,0.1 μmol/L～1 mmol/L) and furosemide (10 μmol/L～1mol/L), however, had no obvious effects (P>0.05, n=7 for cell count and n=8 for 3 H-TdR incorporation), and DIDS had no direct effect on voltage-gated calcium channel. Conclusion FCS can open DIDS-sensitive Cl- channel, and this channel may play an important role in modulating the proliferation of VSMC induced by FCS.

Abstract:Aim To understand whether lipid peroxidation injury to endothelial cells ( ECs ) induces the production of monocyte chemotactic factor RANTES. Methods After a four hour exposure of the cultured human umbilical vein ECs to 5 μmol/L diamide, the conditioned medium (CM) was collected. To obtain a conditioned medium containing RANTES (MW 8 kDa ), the above CM was put into the double layer dialysis molecular porous membrane tubings (Spectrum Lab. Inc. ) with the molecular weight cut off at 10 kDa ( inner layer ) and 3.5 kDa ( outer layer ) respectively. Appropriate amount of 0.01 mol/L PBS was injected into the interspace between the two membranes. The whole set of the membrane tubings was suspended in a pyramid flask containing PBS, and then the samples were dialysed against PBS. Finally, a conditioned medium with MW of 3.5～10 kDa was taken out from the interspace between the two membranes. The chemotactic activity of this conditioned medium for monocytes was tested by micropore filter method using a modified Boyden chamber. For antibody inhibition studies, the conditioned medium was incubated with anti-RANTES antibody and assayed again. Results The monocyte migration distance induced by diamide-CM group (89.75±11.33 μm) is markedly longer than that of the nondiamide-CM group(77.30±11.53 μm)and that of the random migration group(76.18±10.50 μm ). Analysis of variance showed that there was a significant difference between groups(F=47.20,P<0.01). After the addition of anti-RANTES antibody, monocyte chemotactic activity was markedly inhibited (F=21.31,P<0.01 ). Conclusion On the basis of functional assay it suggests that lipid peroxidation injury might induce ECs to produce increased RANTES and may play an role in the recruitment of monocytes into the intima in atherogenesis.

Abstract:Aim To investigate whether hypothalamus-pituitary neuroendocrine pathway influenced the development of atherosclerosis. Methods The hypothalamic and pituitary tissue from freshly aborted 5-month human fetuses were taken for cellular culture and the condition media were collected respectively according to hypothalamus, pituitary and hypothalamus+pituitary groups. The normal endothelial cells and those induced by diamide were cultured respectively with the condition media and the control group with ordinary medium. After culturing with the condition media, all endothelial cells of the experimental and control groups were cultured in serum-free medium. Then collected the serum-free media of the EC separately and detected their content of lipid peroxide and nitric oxide. Results The pure hypothalamus or pituitary group had intensive effect of lipid peroxidation on endothelial cells and did not affect the release of nitric oxide from endothelial cells when the pure hypothalamus or pituitary conditioned media were used. The content of lipid peroxide in hypothalamus +pituitary group was significantly lower than that in other experimental groups (hypothalamus group and pituitary group), but the nitric oxide showed an inhibited function. However, the hypothalamus+pituitary group performed an anti-lipid peroxidation function to the endothelial cells induced by diamide and an increased releasing of nitric oxide. Conclusion From the result we deduced that the hypothalamus may play a significant role in keeping normal function of the endothelial cells, and the effect may be through the hypothalamus-pituitary pathway. When meeting with the harmful factors it can play a protective role in maintaining the function of endothelium.

Abstract:Aim In order to study the change and role of apoptosis in hypertensive left ventricular remodeling. Methods Hearts from 16-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY) were investigated. Apoptosis in left ventricle sections was assessed by an in situ end-labeling technique (TUNEL), the feature and type of cells undergoing apoptosis were identified ultrastructurally by transmission electron microscope (ECM). Additionally, localization of Fas protein- a mediator of apoptotic cell death was examined by immunohistochemistry. Results Compared with 16-week-old WKY, 16-week-old SHR exhibited increased hypertrophic index, the apoptotic frequency of myocytes was also increased(19.54±7.27 vs 6.92±4.35/104 nucleus,P<0.05), whereas the apoptotic frequency of fibroblasts was significantly decreased (5.88±2.15 vs 14.34±9.56/104 nucleus,P<0.05). Moreover, apoptotic endothelial cells and leukocytes infiltrated into myocardium were also identified in SHR. Features of apoptotic myocytes and endothelial cells were observed by ECM in SHR. The latter often occurred in shrunken capillaries surrounded by collagen fragments The distribution of Fas antigen protein was mainly confined to myocytes in SHR, but limited to interstitial fibroblasts in WKY, consistently with the type of cells undergoing apoptosis in these two strains. Conclusion This study first suggest that apoptosis of different cell type may work synthetically in hypertensive myocardial remodeling, and Fas antigen may be associated with the mechanisms of these apoptotic process. Our findings also have important significance in searching new avenues for treatment with appropriate site-specific physiological or pharmacological regulators of apoptosis.

Abstract:Aim The purpose of the study is to evaluate the role of AT-1 anti-sense nucleotide (AT-1A) in expression of subtypes of angiotensin Ⅱ(AngⅡ) receptor mRNA, and syntheses of protein and nucleic acid in cultured rat adventitial fibroblasts. Methods AT-1 cDNA sequence (476 bp) was cloned with RT-PCR and inserted into PLXSN reversely to construct an intact plasmid containing AT-1A(PAT-1A). It was transfected into cultured rat adventitial fibroblasts,and identified by RT-PCR and Western blot. Syntheses of protein and nucleic acid by 3 H-Leu and 3 H-TdR incorporation, mRNA expressions of AT-1 and AT-2 by RT-PCR, were compared between transfected and nontransfected cultured rat adventitial fibroblasts after being stimulated for 24 h by AngⅡ 10 -7 mol/L. Results AT-1 mRNA and protein were expressed significantly less in cultured rat adventitial fibroblasts than that in control (P<0.01). AT-1 mRNA expression was markedly decreased,and AT-2 mRNA obviously increased (P<0.01); but no apparent difference was found in 3 H-Leu and 3 H-TdR incorporation between transfected and nontransfected cultured rat adventitial fibroblasts after stimulation for 24h of AngⅡ10 -7 mol/L (P>0.05). Conclusion After being blocked by AT1A, expression of AT-1 mRNA in cultured rat adventitial fibroblasts was markedly suppressed,while AT-2 mRNA up-regulated at the same time. It is shown that both syntheses of protein and nucleic acid in cultured rat adventitial fibroblasts mediated by AngⅡ could not be effectively interrupted simply by AT-1A blocking.

Abstract:Aim To investigate the effect of antioxidant vitamin E (Vit E) on the cytotoxicity and proliferation of aortic smooth muscle cell (SMC) induced by oxidized low density lipoprotein (oxLDL). Methods The injury and proliferation of SMC were determined by lactic deydrogenase (LDH) release kit and the incorporation of 3 H- TdR and 3 H- Leu into cells respectively. Results Vit E could significantly inhibit SMC from releasing LDH induced by high levels of oxLDL and the incorporation of 3 H-TdR,3 H-Leu caused by oxLDL at low concentrations in a dose-dependent manner. Conclusion Antioxidant Vit E has both protective effects on the injury and inhibition effects on the proliferation of aortic SMC induced by oxLDL.

Abstract:Aim To observe whether the hyperplasia of pulmonary arteriole wall during pulmonary hypertension can be interfered by calcium channel-blocking. Methods Performed the anastomosis between left pulmonary artery and descending aorta in New Zealand rabbits, and banded the proximal left pulmonary artery of anastomosis, to establish the model of one-side kinetic pulmonary artery hypertension. After operation rabbits in drug group were fed with nifedipine and aspirin, and the blood concentration of endothelin and TXA2/PGI2 was measured regularly. Pulmonary artery pressure was measured and lung biopsy was performed in 2-3 months observation. Results During rabbits formatting pulmonary hypertension, those giving drugs got minor hyperplasia of pulmonary arteriole wall than those not giving drugs. The blood concentration of endothelin increased at all rabbits but the crest value was lower in drugs group. The ratio of TXA2/PGI2 was lower in drugs group too. Conclusion Calcium channel blocker can inhibit the hyperplasia of pulmonary arteriole wall during the formation of pulmonary hypertension, which could be the results that calcium channel blocking and aspirin inhibit the production of endothelin and TXA2 and promote the produce of PGI2.

Abstract:Aim Atherosclerotic cerebral infarction (ACI) as agradually progressive atherosclerotic disorder has been thought be a hetero-ground and multifactor of environment, There are in different epidemiologic state. The relationship of ministellite region (MSR) polymorphisms at 3'-end of apolipoprotein B gene in Chinese ACI and healthy controls was observed. Methods The 3' hypervariableministellite region polymorphism of apolipoprotein B gene was examined by polymerase chain reaction technique in 84 patients with ACI and 107 age-matched Chinese healthy controls. The targeted ministellite region were amplified rapidly and accurately, and then identified oligonucleotide primer 5'-ATG GAA ACG GAG AAA TTTG-3',5'-CCT TCT CAC TTG GCA AAT AC-3' was used to the amplification of DNA genome which in peripheral leucocyte, number repeat unit (MSR) alleles detached by polyacrylamide gel electrophoresis (PAGE). Results ①The frequency distribution of apolipoprotion B 3'ministellite region alleles was in monomold peaks at 37, 39 repeat uints in both group(from MSR29 to MSR53), and the big number repeat unit (MSR39 is 18.5%, and P<0.02). ②The homozygote genotype of the big number repeat units alleles was related to the high level of apolipoprotein and LDL. The blood lipid parametes in differant zygote genotype of the small number repeat unit (MSRS, ≤MSR37) and big number repeat unit (MSRB, ≥MSR39 ) was cmparised (table 2), the homozygote big copied alleles (MSRB/MSRB) related to the increase of apo B and LDLC, There were same tandency in ACI and controls. Conclusions Our investigate resluts suggested the possibility that apolipoprotein B 3' gene ministellite region alleles polymorphism increase the susceptibility to atherosclerosis by means other than by altering lipoprotein levels deserves further consideration.

Abstract:Aim To elucidate the relationship of apolipoproteins E gene polymorphism to coronary artery disease. Methods Apolipoproteins E genotype was analyzed in 60 patients judged by selective coronary angiography and 50 cases with negative coronary angiography as control. PCR-restriction fragment length polymorphism methods was employed using genomic DNA from anti-coagulantion blood. χ2 test and Logistic regression were used for statistical analysis. Result and Conclusion The apolipoproteins E₂/3, E3/3, E3/4, E₂/4, E4/4 and E₂/2 genotypes and ε2, ε3 and ε4 alleles frequency in patients group were 11.7%, 68.4%, 10.7%, 1.7%, 1.7%, 0 and 7%, 82.2%, 10.8%;while 6%, 76%, 16%, 2%, 0, 0 and 4%, 87%, 9% in control group respectively. χ2 test and Logistic regression analysis showed no differences in gene polymorphism between patients and control group. The apolipoproteins E gene polymorphism were also found not related to the severity of coronary artery disease.

Abstract:Aim To investigate the relationship between triglyceride (TG) concentration and tissue plasminogen activator (t-PA) activity, and analyze comparatively. plasminogen activator inhibitor-1 (PAI-1) activity in patients with different types of coronary heart disease (CHD) and high TG concentration. Methods TG concentrations of 73 patients with different types of CHD and 40 patients with high TG concentration and 40 controls were measured. Meanwhile, t-PA and PAI-1 activity were determined. Results PAI-1 activity in patients with high TG or CHD were higher than those of the control group, but t-PA activity were lower than those of the control group in patients with high TG and acute myocardial infarction (AMI) or unstable angina pectoris (UAP) at same time. TG positively correlated with PAI-1 activity but negatively correlated with t-PA activity. Conclusion TG may play an important role in the pathogenesis of CHD by inducing fibrinolytic dysfunction

Abstract:Aim To investigate the role of thoracic epidural anesthesia (TEA) and the change of concentration of nitric oxide (NO) and circulating endothelium cell (CEC) in patients with refractory angina pectoris (RAP). Methods 61 patients with RAP were treated with TEA as the main therapy after cessation of nitroglycerin infusion. The clinical effect of TEA was recorded and plasma concentration of NO,endothelin-1 (ET-1) and CEC was investigated before and 14 days after TEA. Results All patients had reduced severity of chest-pain after TEA. The number of leads with ST depression and degree of ST depression decreased significantly after TEA (P<0.05). Plasma concentration of NO increased after the therapy, whereas ET, CEC decreased significantly (P<0.01). Conclusion TEA may be an effective therapeutic approach for patients with refractory angina pectoris, associated with a protective effect for endothelium cells.

Abstract:Aim We sought to evaluate the markers of polymorphonuclear (PMN) oxygen free radical (OFR) production, the levels of lipid peroxidation (malonaldialdehyde, MDA) and defenses against oxidative stress in the patients with acute myocardial infarction (AMI). Methods and Results A convenient and sensitive method for PMN OFR production was assayed by polymorphonuclear chemiluminescence (PMN-CL). We have measured the parameters of PMN-CL, the plasma MDA concentration, the plasma activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX) in 60 AMI patients and 62 age-matched healthy subjects. Patients with AMI showed significantly increased PMN-CL parameters as peak value, integration and phagocytic index compared to healthy controls (P<0.001,respectively). The plasma MDA concentration increased in AMI patients compared with that in healthy controls (P<0.001). The PMN-CL peak value correlated best with plasma MDA concentration(r=0.58,P<0.01,n=122) and negatively correlated with the plasma activity of SOD (r=-0.63,P<0.01, n=122). Conclusion The PMN act as a potential contributor to extension of myocardial necrosis induced by OFR, thereby indicating the involvement of free radicals in AMI.

Abstract:Aim To investigate the significance of the changes on plasm brain natriuretic peptide (BNP) in patients with acute myocardial infarction(AMI). Methods 63 patients with AMI were divided into losartan (n=19), enapril(n=23) and control (n=19) group. The plasm levels of BNP was measured in patients with AMI on admission and 24 h as well as on the day of 5 and 14, compared with ten health subjects. Left ventricular end diastolic volume index (LVEDVI) and left ventricular end systolic volume index (LVESVI) were determined by echocardiogram on the day of 5 and 14. Result The plasm levels of BNP was significantly increased on admission in control group compared with that of the health subjects(P<0.01) and increased further on the day of 5 and 14 than that on admission(P<0.05). Enapril and losartan treatment decreased plasm BNP level on the day of 5 and 14. The plasm level of BNP on the day of 5 and 14 in patients with AMI correlated with LVEDVI and LVESVI (r=0.62～0.77,P<0.01). The plasm levels of BNP levels in patients with anterior myocardial infarction (AMI) were higher than those in patients with inferior. MI and were lower in patients with early reperfusion than those with non reperfusion. Plasm levels of BNP on the day of 5 and 14 correlated with plasm peak creatine phosphokinase (r=0.32, 0.67,P<0.05, 0.01). Conclusions The plasm level of BNP increased continuously within 14 days after the onset of AMI, and this would reflect infarction area and infarction expansion post AMI.

Abstract:Aim To evaluate the endothelial function in young hypertensive patients with no evidence of plaque formation and other risk factors for arterial sclerosis. Methods 45 young hypertensive patients and 20 matched healthy subjects were included in this study. Two-dimensional ultrasonography was used to determine the diameter of the brachial artery during reactive hyperemia caused by endothelium-dependent dilatation and the diameter of the same artery after sublingual glyceryl trinitrate induced by endothelium-independent dilatation. Result Despite the similarity of 2 groups in age, blood lipids and blood glucose levels, the increased percentage of the brachial artery diameter was lower in patients group than in normal control (12.32%±4.94% versus 16.63%±5.87%,P<0.01) during reactive hyperemia. There was, however, no significant difference in response to glyceryl trinitrate between two groups (18.94%±6.50% versus 21.02%±6.65%, P>0.05). Conclusion Endothelial dysfunction is present in young patients with essential hypertension before clinical evidence of atherosclerosis.

Abstract:Aim To document the safety and feasibility of intramuscular gene transfer by use of naked plasmid DNA encoding vascular endothelial growth factor and to observe potential therapeutic benefits in patients with critical limb ischemia. Methods Gene transfer was performed in 4 limbs of 3 patients with rest pain and nonhealing ischemic ulcers due to peripheral arterial disease. Four milligrams of naked plasmid DNA encoding the 165-amino-acid isoform of human vascular endothelial growth factor (pUC-CAGGS/h VEGF165) was injected directly into the muscles of the ischemic limb at 4 arbitrarily selected sites, two weeks apart. After 1～3 months after gene transfer, contrast angiography was repeated to document newly visible collateral blood vessels. Clinical status was recorded per day after gene transfer. Results Ischemic ulcers healed or markedly improved in 4 of 4 limbs, rest pain was relieved in all patients. Angiography confirmed newly visible collateral blood vessels in 4 of 4 limbs. Complications were limited to transient lower-extremity edema in all patients, consistent with VEGF enhancement of vascular permeability. Conclusions This findings may indicate that intramuscular injection of plasmid DNA expressing VEGF165 is sufficient to induce therapeutic angiogenesis in patients with critical limb ischemia. It may be a safe and effective strategy for patients with critical leg ischemia.

Abstract:Aim To investigate the frequency of variant at MspI Sites of apoA_Ⅰ gene and its relation to endogenous hypertriglyceridemia (HTG) in Chinese population. Methods The restriction fragment length polymorphisms (RFLPS) at two MspI sites in the 5'end at -78 bp and +83 bp of apolipoprotein A_Ⅰ gene were studied using PCR in 134 endogenous hypertriglyceridemics and 255 healthy subjects from a population of Chinese Han nationality in Chengdu area. The loci included MspI 1 (promoter region, -78 bp) and MspI 2 (intron 1,+83 bp)sites. Results Both in HTG group and control group, G/G genotype were the most frequent one, the frequencies of rare A alleles at -78 bp of apoA_Ⅰ gene in Chengdu were significantly higher than those in American and European Caucasians (0.300 vs 0.120～0.191,P<0.01), and the frequency of rare A allele in HTG group was significantly higher than that in the control group (0.350 vs 0.273,P<0.05). But no differences were found in the frequency of rare T allele for +83 bp. Both in the HTG group and control group, subjects with A/A had a higher serum mean concentration of TG, apoCⅢ and higher serum TG/HDLC ratio compared with the subjects with the genotypes G/A and G/G (P<0.05), and the serum apoCⅡ had a tendency to increase (P>0.05). Conclusions These results suggest that only the MspI 1 RFLP at -78 bp of apoA_Ⅰ gene is associated with endogenous hypertriglyceridemia to some extent in Chinese popuplation.

Abstract:Aim To determine the distribution of apolipoprotein E genotype that can veritably reflect the actual apolipoprotein E genotypic frequency of Chinese population. Methods The blood samples were randomly collected from 121 newborn's umbilical cords. The apolipoprotein E genotypes were examined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques. Results The allelic frequencies of apolipoprotein E are ε2 9.50%, ε3 85.13% and ε4 5.37% respectively. Conclusions Although there were some certain differences between the results and those of other research groups', the Apo E allelic frequencies were quite similar to that of the aged and the adults reported previously by the major laboratories in China. Based on the unremarkable difference of the allelic and genotypic frequencies of Apo E among different generations, although the Apo E allele may affect the lipid level and the morbidity of coronary heart disease, it has no obvious impact on the life expectancy.

Abstract:Aim To reveal the distribution characterization of serum levels of lipids,lipoproteins,and apolipoproteins in different nationality. Methods The authors determined quantitative levels of those traits mentioned above and messured the body hight (H) and weight (W), and calculated both body surface area (s) and body mass index [BMI=W/S2(m2)] in 773 Kazaks (men 360 and women 413) and 911 Han nationality (men 466 and women 445) from Xinjiang of China. Results Kazaks men and women, respectively, had significantly higher serum levels of HDLC and apolipoprotein A1 (allP<0.001) and significantly lower serum levels of TG (menP<0.01, womenP<0.001), Lp(a)(allP<0.005), apolipoprotein B (menP<0.005,womenP<0.001) and apolipoprotein B/A1 (menP<0.01, womenP<0.001) than their Han nationality controls; the threshold points of apolipoprotein A1 (<1.2 g/L) and apolipoprotein B (>1.2 g/L) are higher in Hans than in Kazaks for apolipoprotein A1 (20.7% vs 9.8%) and apolipoprotein B (18.6% vs 14.3%); the serum levels of TG,HDLC, Lp(a),apolipoprotein A1 and apolipoprotein B/A1 are all closely correlated with nationality (respectively,P<0.001, P=0.001, andP<0.05). Conclusion The nationality differences exist in serum levels of lipids, lipoproteins, and apolipoproteins, and this differs maybe come from genetic effects, which affect the serum levels of lipids, lipoproteins,and apolipoprotein by the method of controlling the lipid metabolism pattern. Future study will to dissect to the role of genetic factors affecting on serum lipids.

Abstract:Aim To establish a direct, accurate method for detecting the tetranucleotide repeat genotype of human low density lipoprotein receptor related protein(LRP). Methods LRP was amplified by using polymerase chain reaction (PCR). The PCR products were subjected to electrophoresis on 6% denature polyacrylamide gels. Sequences of LRP were analysed by direct sequencing of PCR products. Results Using this method, we detected the LRP genotypes: 5/6, 6/6, 5/5 and two allelic genes 5, 6. The results were in agreement with sequence analysis. Conclusion LRP genotype can be determined directly and accurately. It is a tool for the investigation of large-scale population and study of cardio-cerebro vascular diseases(CCVD).