Abstract:Aim To determine the expression of human tissue-type plasminogen activator (tPA) cDNA in mamalian cells. Methods A recombinant retroviral vector containing tPA gene was constructed and transtected into bovine endothelial cells (EC) by retroviral vector-mediated gene transfer. The tPA activity of transduced cells was measared with casein-plastninogen-agarose plate and a synthetic-peptide substract S-2390.Results The viral titer was 4 ×108 cfu/L. Trans-fection effciency was 6×108 colonies/g DNA/106 cells. The tPA activity of transduced cells was obvi-ously increased. Immunohistochemistry analyses confirmed the expression of tPA gene in the genetically engineered cells.Conclusions Retroviral vector mediated gene trans-fer can be used to enhance the fibrinolytic activity of EC. It might be an approach for tPA gene therapy.

Abstract:Aim The present pater is to study the effect of different 3'end of variable number of tandemly repeat (VNTR) alleles from human apoliopoprotein B gene on eukarytic expression and regulation. Methods HVE36 allele distributed widely in nor- mal Population and HVE44 allele distributed mainly in patients with atherosclerosis were cloned into plasmids of luciferase reporter vecters pGL2-promoter and PGL2-control. Then the recombinants were transfect- ed iuto Hela and HepG2 cell lines.Results The level of expression of pGL2-control plasmid inserted with WE44 allele was 4 times than those of the recombinant with HVE36. The level of expression of pGL2-promoter plasmid inserted by HVE44 is 1. 7 times than those in plasmid inserted by HVE36 allele in Hela cell line, In HepG2 cell line, the level of expression of pGL2-control recombinant with HVE44 is 3. 05 times of those in HVE36 recombi- mant. But in PGL2-promoter system, the level of expression of recombinant with HVE44 alleles is 2.1 time than those in HVE36 recombinant. In differ- ent cell lines, the expression level of recombinant with HVE44 was much higher than those of HVE36 recom- binant. Conclusioas Different 3'VNTR alleles have regula-tory role in gene expression and HVE44 allele can pos-itively regulate the eukarytic expression, which may be one of causes of higher apolipoprotein B level in atherosclerosis patients with VNTR-big alleles like HVE44. More investigations are carried out for regu- latory mechanism of the 3'-end nontranslation region.

Abstract:Aim The search for the relation between macrphage colony-stimulating factor (MCSF), scav- enger receptor (SR), oxidative modified low density lipoprotein (OLDL) and atherosclerosis (As). We studied the effect of recombinant human MCSF (rhM- CSF) on the SR passway of mouse peritoneal macrophage (MPM) and the effect of rhMCSF on cel-lular cholesteryl ester (CE) accumulation caused by OLDL.Methods Mouse peritoneal macrophages (MPM) were collected by routine method. LDL was isolated by density gradient ultracentrifugation, oxidized by CuCl2 and labeled either with 3, 3-dioctadecyl indocar- bocyamine (Dil) or with 125 I. The binding of DiI- OLDL and degradation of 125 I-OLDL by MPM and the intracellular content of CE were measured according to the methods of Stephen, Gold stein and Heider respec- tively.Results Recombinant human MSCF could increase the number of SR on cultured MPMs and enhance the binding and degradation of OLDL with a dose-and time-dependent mode. The cellular CE accumulation caused by OLDL was also enhanced by addition of rhMCSF.Conclusions MCSF may enhance the cellular binding and degradation of OLDL by increasing the number of SR on MPMs, hence increase the cellular CE content and promote the formation of foam cells in the vascular wall.

Abstract:Aim The elevation of the plasma lipoprotein (a) level is an independent genetic risk factor for atherosclerosis and its major manifestations. The wide variation in lipoprotein (a) plasma levels is ex- plained to a large extent by a remarkable size polymor- phism of the apolipoprotein (a ) isoforms and by u- nidentified sequence variation in the apo (a) gene. A search was made for sequence variants in the 5' flank- ing region of the apo (a) gene and its relation with lipoprotein(a) levels in 85 unrelated healthy subjects ' of Chinese Han Nationality.Methods The plasma concentrations of lipoprotein (a) were measured within one week using a sandwich enzyme linked immunosorbent assay. Pairs of 25- base oligonucleotides were used to amplify sequences -59 to +325 of the apo(a) gene from genomic DNA using polymerase chain reaction (PCR). The PCR- omplified DNA fragments were screened for single rand DNA conformation polymorphism (SSCP) using a modification of the method of Orita. Results one single-strand DNA conformation polymorphism in the apo(a) 5' flanking 384 bp frag-ment was reported. The genotype distribution in 85 healthy unrelated subjects were 0. 812 for genotype 1, 0. 188 for genotype Ⅱ. The mean lipoprotein(a) level in the subjects with genotype Ⅱ was higher than that with genotype I, but the difference did not reach statistical significance. The sequence Polymorphism was not associated with the differences in the plasma concentrations of lipoprotein(a).'Conclusions Sequence variants exist in the apolipoprotein (a) 5' flanking fragment in individuals of Chinese Han Nationality. Though no association was found between the SSCP genotyp and the plas- ma concentrations of lipoprotein (a ), the sequence variants reported here may significantly alter gene ex-pression in vitro.

Abstract:Aim Previous studies have demonstrated that ni- tric oxide (No) prostacyclin (PGI2) and ATP-sensi- tive K+ channel all participate in the regulation of hy- poxic cerebrovascular dilatation. but the interrelations of them still keep unclear. The aim of the study is to investigate the interrelations of these three vasodila- tive factors under hypoxia. Methods The experiments were Performed in new-born calf basilar arteral strips to observe the interrela-tions between NO and PGI:, Pt7[, and ATP-sensitive K+ channel, ATP-sensitive K channel and NO, re- spetively, in hypoxic cerebrovascular dilatation (the bypoxic Perfusion solation was prepared with PO2 6. 0 kPa, PCO2, 4. 7 kPa, and pH 7. 40±0. 02 ).Results The NO bad no effect of PGI2, but dilata-tion induced by NO appears to depend on production of PGI2, PGI2 had no obvious effect on ATP-sensitive K+ channel, but the effects of PGI2 may be dependent on openning of ATP-sensitive K - channel ; the effects of No may be not directely mediated by openning of ATP-sensitive K+ channel. and the activities of ATP- sensitive K+ channel seem isolated from effect of NO.Conclusions The response of hypoxic cerebrovas-cular dilatation are influenced obviously by No and PGI2 except reaction induced by ATP-sensitive K+ channel. There are some interrelative effects be-tween NO and PGI2, but the openning of ATP-sensi-tive K+ channel in not innuenced by them.

Abstract:Aim To determine the effects of Liensinine on endothelin-stimulated proliferation of vascular smooth muscle cells (VSMC) and expression of heat stress protein (HSP70) and antioncogene P53. Metbods The experimental models of proliferation of cultured porcine aortic smooth muscle cells induced by endothelin were established, and H-thymidine(~3 H-TdR ) incorporation, flow cytometry, Western blot and Northern blot analysis were used.Rerults Liensinine may drop H-TdR incorporation induced by endotbelin and hold-back VSMC from G0/ G, phase to DNA synthetic phase (S) and mitotic phase (G2/M ). Furthermore, Liensinine could reverse the enkanced expression of HSP70 and HSP70 mRNA and reduced expression of antioncogene P53 induced by endothelin.Conclusions Liensinine may inhibit DNA synthesis and proliferation of VSMC, related to the mechanism of molecular biology of controlling HSP70 and P53.

Abstract:Aim To investigate whether or not the time course of the cellular proliferation and aPoptotic cell deatk is re1ated with expresion of some oncoproteins, c-Myc, c-Fos, Bcl-2 and P53, in miniswine atherosclerotic ili- ac arteries following balloon angioplasty.Methods Atherosclerotic lesions developed by endothelial denudation combined with atherogenic di-et. Balloon angioplasty was performed in the right il-iac artery of atherosclerotic miniswines. After 1, 3,8, 14, 30, and 90 days ; respectively, the experimen-tal animals were killed and iliac arteries were re-moved. Immunohistochemical assays were applied by using primary antibodies against P53, c-Myc. c-Fos,and Bcl-2. With 3-3'-diaminobenzidine (DAB) the sec-tions were stained. Cell proliferation was determined with an antibody to the proliferating cell nuclear antigen (PCNA). comtbined with SMC-specific anti-body. The presence of apoptotic cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL).ResultS Percentages of PCNA-positive cells in the intirna vs mfdia were 22. 4% vs 21. 6%, 27. 8% vs 27. 5%, 30. 6% vs 21. 2% at 1, 3, and 8 days, re- spectively, after balloou angioplasty- Percentages of TUNEL-positive cells in the intima vs media were 3.oN vs 0. 9%, 4. 3% vs 1.4%, 16.0% vs 1. 9% at 1,3, and 8 days, rspectively. Coefficients of linear corre-lation of PCNA witb c -MyC and c-Fos were o. 6472 and 0. 6362 (p<0. 05), and TUNEL with Bcl-2 and P53, -0. 7538 and 0. 8447 (p<0. 05). respectively.Angioplastied and control vessels 8howed different lev-els of peNA and TUNEL, and also different expres-sion levels of c-Myc, c-Fos, Bcl-2, and P53 in cells.Concluslons Arterial injury may result in the in-crease of cell prc1iferation and apoptotic cell death in botb intima and media. Cell proliferation and apoptosis in tke injured vessels associated with the expression of the immediate-early gene (c-myc, c-fos), proto-onco-gene (bcl-2) and relative gene (p53).

Abstract:Aim To investigate the protective effect of apolipoprotein(apo) C Ⅱon the morphology and func- tion of human umbilical vein endcthelial cells,injured by low density lipoprotein (LDL) in vilro. Methods Cultured huynan endothelial cells derived from umbilical vein were divided into four groups:control, high density lipoprotein (HDL)-apo + LDL,apo C Ⅱ+LDL and LDL group. which were observed the morphological changes with phase-contrast and transmission electron microscope and measurcd the re- lease of lacate dehydrogenase (LDH) and level of 6-ke-to-prostaglandin Fla (PGFla).Results Endothelial cells after being injured by LDL showed cell contraction. Increased release of LDH ayld decreased jevel of 6-keto-PGFla. Howev- er, normal morphology and LDH rtlease as well as prostacyclin (PGI2) syntllesis in endothelial cells were found when HDL-apo or apo C Ⅱ was added to culture media before LDL inJury.Conclusious Both HDL-apo and apo C Ⅱ could re-sist the injurious effect of LDL on cultured endothelial cells.

Abstract:Aim observing the anti-atherosclerotic effect of API0134 in vivo.Methods An atheroscletotic rabbit model fed by high cholesterol supplemented with bovine serum albu-min injection was established.Results In model group , the percentage of plaque covering aortic intima (PA ) was 51. 2 % ±20. 0%, the incidence of atherosclernois of small arteries (LA ) and the degree of stenosis of coronarye arteries (DS ) were 62. 4% ± 17. 4 % and 57.6% ±17.4% respectively.In AP10134 treated group, compared with model group. the PA was 7. 1% ± 4. 5 %, LA was 12. 5% ± 4. 8%, DS was 10. 6% ±3.4%. While in nifedipine treated group, the image analvsis gave a significantly bigher values than APIC134 treated group, but lower then those of model group.Conclusion API0134 exeits strong anti-atheroscle- rotic effect in experimental rabbit. Althouth nifedip-ine has the similar effeet but with a slightly weaker ac-tion.

Abstract:Aim To investigate the effects of L-arginine on serum lipid, lipoprotein, lipid peroxide, and hemorhe-ology in petients with coronary heart diseasc and hy-perlipidema.Methods 4 tables (0. 25 g/tab )were given orally trice daily for 30 days in 30 patients with coronary heart disease and hyperlipemia. The serum lipid,lipoprotein apolipoprotein, lipid peroxide and hemorheology were meassured be fore and after treat-ment.Results L-arginine could significantly reduce serum total cholesterol, triglyceride, low density lipoprotein cholesterol and lipoprotein(a) levels (p<0. 01), but increase high density lipoprotein choles-terol level (p< 0.05). L-arginine could greatly lower lipid Peroxide level (p<0. 01 ),but increase su-Perokide dismutase activity(p<0. 01 ). L-arg-inine could decrease low shear of blood speific viscosi-ty, erythrocyte sedimentation rate (ESR),equation K value of ESR,aggregative index of red blood cell and plasma specific viscosity (p<0.01 ). An increase of plasma arginine level was companied with the above changes.Conclusions L-arginine releasing nitric oxide in vi-vo can suppress hyperlipemia and lipid Peroxide and improve helnorhfology in patitnts with coronary heart disease. Thus L-arginine may be used to treat coro-nary heart disease an byperliPemia.

Abstract:Aim To well understand the molecular events governing smooth muscle cell (SMC) prolileratlon In develt,pment of atherosclerosis (As), We investigated SMC ultrastructure and levels of c-ny proto-oncogene transcription in plaque.Matt.for 25 bealthy male Japan bigrear white rab bits were divthed control group (n = b, normal diet ) and experimental group (n = 2O, high cholesterol diet plus belloon deendothelialication) at random. The experimental group animals were carried balloon-in-jUry to aorta with 3F Fogarty catllt.r after being fed atherogenic diet for one week- We observed morpl:o-logical changes of rabbit dorta through microscoPe and ultrostructure of SMC with transmissive electrcn-mi-mpe and asse8sed the levels of c-op mRNA witb Northern blot at 1' 2. 4 and 6 weeks Postangloplasty.ReSultS bondothelialization of rabbit aorta in-dutal a large of SMC to migrate from medium to reointimal and proliferate. The changes of SMC pheotyPe were observed one week after injury. Dering 2~4 weeks, there were a nulllber of prolifcrative SMC in thickening neointima. These SMC displayed 'syn- tkftic' pheotype which cotained large amtlllnts of roue,doplasndc reticulum, free rlbosonlts, mito-ckondrla and littie contractile appartus. Some lipid-droplet in some synthetic SMC were observed. Then the typic atheroma plaque in rabbit aorta formed at 6 weeks. The predominant cell typpe in these plaqes were the SMCderived fem cell which filled witb lipiddroplet and contained a few cellular organel-lae. The levels of c-mM mRNA increased signifi-cantly at one weeks after injury Prior to SMC prolifer-ation. Then c-myc expression gradually decreased at 2,4 and 6 weeks, but the levels of c-mp mRNA at 6 weeks was still higher than that of control group.CoecIasloas Deendothelialization promotfd forma-tion of As, c-myc gene expression were closely related to SMC proliferation in development of As.

Abstract:Aim This study was searched for active mechanism of nitrendipine that prevents and treats atheroscle-rosls.Method The experiments were performed in coro-nary arterial rings of Porcine. Results Nitrendipine inhibited proliferation of cul-tured vascular smooth muscle cell witb M IT colori-nelrc assay in porcine.Chadusio.s Nitrendipine may play an important role on antiatherosclerosis

Abstract:Aim Endothelial cells injured by free radicals is a crucial step in tbe development of many diseases. We investigated the protective effect of probucol. an an-tiatherosclerosis drug,to the cultured hurnan endothe-lial cells.Methods The oxidized low density lipoprotein (OLDL) were used as free radial resources and 'H-TdR incorporation of the cells was estimated as DNA sgnthesis. The microstructure change was studied by using the scanning electron misroscope.Results At 24 hours interval, the k-TdR incorPo-rated into the cells in OLDL group was 48K as that of nLDL group or 56K as that of probucol + OLDL group (p<0.001 ). The scanning electron micro-scopic study showed that the intercellular junctions di-ndnished and the cellular outline changed in the OLDL treated cells but not the probucol treated cells.CoarIasloas Probucol is an useful antioxidant in preventing the cultured cells from injury by free radi-cals and perhaps tbus contributes to the antiathero-genesis effeCt.

Abstract:Aim To evaluate the clinical application of coro-uary artetiography (CAG) in diagnosis of coronary heart disease.Methods Selective coronary arteriography by means of multiple angled projecting cine-nuoroscopy was performed in 245 cases. The patients were divided into four groups, A: 101 mycardial infarction, B: 76 angina pectoris, C: 25 atypical angina pectoris, D: 24 non-coronary heart disease. 224 left cardioventricu lography can be reviewerl satisfactorilv in them. Results 164 CAG cases were abnormal. In group A the positive rate was 96. 1 %, and B 71. 1%,C 28. 9% respetively. In group D. no coronary le-sions can be found. 290 coronary artery branches were steootic. Among them 130 are in LAD. 80 LCX, 67 RCA, 13 LMCA successively. Pathologic manifesta-tions in left ventriculography are segrmental functional disorder or aneurysm of the left ventricular wall. The Positive rate in group A is 86. 5K, B 55. 2%, C 34-1 %, D 5% respectively.Conclusion CAG is still direct, more effcient ma-neuver to probe coronary heart disease.

Abstract:Aim An enzyme linked immunosorbent assay (ELISA ) using HRP-conjugated rabbit antihurnan plasma apo B₁₀₀ IgG was develoPed for measurement of low density liPoprotein(LDL) receptors on purified rabbit liver plasma membranes.Methods Membrane protein adhered to tbe solid phase by an overnight incubation, and was quantified by measuring the protein concentrations te fore and af-ter coating. A curve of anti-apo Bloo bo binding to known amounts of LDL was constructed to quantify the LDL bound. Parallel samples with 25-fold excess rabbit plasma LDL were assay6d to detect nonsPeCific binding.Results Kd and Bmax, worked out by scatchard plot, were l3. 59 mg/L and l24 ng/g membrane pro-tein(n= 5),re8PeCtively.C.ncluslous The results support the use of ELISA to measure LDL receptors, particularly for physiologic studies.