Chen Bangdang

e0228 Transfection of recombinant adeno-associated virus serotype 9 to mouse heart in vivo and the effects on cardiac function

Objective To evaluate the transfection efficiency of recombinant adeno- associated virus serotype 9 carrying enhanced green fluorescent protein (rAAV9- eGFP) to mouse heart in vivo and the effects on cardiac function. Methods 1. 16 C57BL/6 mice were transfected rAAV9-eGFP by tail injection. EGFP expression in the heart, liver, lung, kidney and brain cryosections was observed under inverted fluorescence microscope 7, 14, 21, 28u2005days after the injection of rAAV9-eGFP and eGFP was quantitated by Western Blot. 2. 20 C57BL/6 mice were divided into control group and rAAV9-eGFP group randomly, and were received with saline or rAAV9-eGFP. The echocardiography and haemodynamics were performed 28u2005days after the injection of saline or rAAV9-EGFP. Results 1. EGFP expression in the heart reached the maximum at day 21, at the point of which the transduction efficiency of rAAV9-eGFP in myocardium was 32%. The other tissues had a little or no eGFP expression. 2. The cardiac function did not reveal significant difference between rAAV9-eGFP group and the control group after transfection (p>0.05). Conclusion rAAV9-eGFP gene can be stably and efficiently expressed in mouse heart, and has no toxic effect on cardiac function.

ASSA13-11-7 Distributional Characteristics of Genetic Polymorphisms of Serum Amyloid Protein A1 in Healthy Chinese Han and Uighur Population in Xinjiang

Objective To explore the distributional characteristics of genetic polymorphisms (rs2229338 and rs12218) of serum amyloid protein A1 (SAA1) in healthy Chinese Han and Uighur population of Xinjiang. Methods 316 Uighur and 362 Han healthy persons were detected the genotypes of the SAA1 by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results The genotype distributions of the Uighur group and the Han group were in the Hardy-Weinberg equilibrium (both P > 0.05). The frequencies of AA, AG and GG of rs2229338 were 76.6%, 23.4%, and 0 in Uighur group while ones were 91.7%, 7.7% and 0.6% in Han group. There was significant difference in distribution of genotypes between these two groups (P < 0.001). The frequencies of CC, CT and TT of rs12218 were 10.1%, 47.5%, and 42.4% in Uighur group while ones were 3.3%, 34.3% and 62.4% in Han group. There was also significant difference in distribution of genotypes between these two groups (P < 0.001). The A-C and G-T haplotypes were more frequency in the Uighur but the A-T haplotype was more common in the Han population, respectively (both P < 0.001). Conclusions The mutational frequencies of the tagging SNP (rs2229338 and rs12218) in the SAA1 gene of Uighur population were higher than those of Han.

ASSA13-03-18 Precondition with CyclinA2 Promote Cardiac Regeneration After Myocardium Infarction Via the Recruitment of C-Kit+ Cells

Objective Downregulation of CyclinA2 till disappearance in mammalian heart after birth is associated with the withdrawal of cardiomyocytes from cell cycle. We hypothesis that exogenous CyclinA2 transferred into infracted myocardium might induce cardiac regeneration and reactivate cardiomyocytes cell cycle. Methods Adeno-associated virus type 9 -CyclinA2-CMV recombinant was constructed and 2 × 1011vg constructs in 200ul of saline were deliveried into mice myocardium through tail vein one week before MI created (n = 20). The control group (n = 20) were injected with saline at a same volume and time. Post-MI observation were respectively 1 week and 3weeks. Echocardiography was performed to measure Left Ventricular End Diastole Diameter (LVEDD), Left Ventricular End Systolic Diameter (LVESD) and Ejection Fraction (EF). Western blot and immunohistochemical analysis were used to detect the expression and location of CyclinA2. DNA synthesis and mitosis were respectively analysed by PCNA and phosphohistone-H3. C-kit and connexin 43 were also measured. Masson’s trichrome stain was used to measue collagen volume in two groups. Results CyclinA2 located in cytoplasm but not nuclear after transferred. Western blot showed that expression of CyclinA2 in two groups has a significant statistical difference (p group than control (0.75 + 0.03 VS 0.43 + 0.02, p = 0.036). However mitosis specific protein H3P had no statistical difference in two groups (p > 0.05). C-kit expression showed an increase in CyclinA2 treated group but no change in control (p < 0.05). Immunohistochemical analysis showed that PCNA positive cells in CyclinA2 treated group were 41.3 ± 2.1 VS control 25 ± 1.1 (p = 0.017). C-kit and connexin 43 positive cells and areas exhibit higher level in CyclinA2 treated group verus control. Masson’s trichrome stain showed a decrease level of collagen in CyclinA2 treated group than control (p = 0.029). Echocardiography showed that LVEDD, LVESD and EF in CyclinA2 treated groups and control were respectively 0.31 ± 0.02cm VS 0.44 ± 0.01cm (p < 0.05) and 0.21 ± 0.02cm VS 0.34 ± 0.01cm (p < 0.05) and 55 ± 2.3% VS 40 ± 1.7% (p < 0.05). Conclusions Expression of Cyclin A2 via gene transfer induced cardiomyocyte DNA synthesis but no mitosis. Cyclin A2 might promote cardiac regeneration via the recruitment of c-kit+ cells which had been known as a cardiac stem cell marker. Continuous expression of CyclinA2 improved cardiac function after MI.

ASSA13-11-2 Relationship Between Novel Polymorphisms of the C5L2 Gene and Coronary Artery Disease

Objective C5L2, a G protein-coupled 7-transmembrane domain complement, has been demonstrated to be a functional receptor of acylation-stimulating protein (ASP), which is a stimulator of triglyceride synthesis or glucose transport. In this study, we will investingate the variations in the coding region of the C5L2 gene (C698T and G901A) and their association with coronary artery disease (CAD). Methods We identified novel single nucleotide polymorphisms (SNPs), (C698T and G901A) in exon 2 using a polymerase chain reaction direct-sequencing method. We examined the role of this SNP for CAD using two independent case-control studies: one was in the Han population (492 CAD patients and 577 control subjects) and the other was in the Uygur population (319 CAD patients and 554 control subjects). Results Using a polymerase chain reaction direct-sequencing method. We identified novel SNPs, 698C > T (P233L), and G901A (A300H) in exon 2. Heterozygote carriers of the 698CT genotype were more frequent among CAD patients than among controls not only in the Han population (7.3% versus 1.7%) but also in the Uygur population (4.7% versus 1.6%). The odds ratio (OR) for carriers of the 698CT genotype for CAD was 4.484 (95% confidence interval (CI): 2.197–9.174) in the Han group and 2.989 (95% CI: 1.292–6.909) in the Uygur population. After adjustment of confounding factors such as sex, age, smoking, alcohol consumption, hypertension, diabetes, as well as serum levels of triglyceride, total cholesterol, high-density lipoprotein, the difference remained significant in the Han group (OR = 6.604, 95% CI: 2.776–15.711, P < 0.001) and in the Uygur group (OR = 2.602, 95% CI: 1.015–6.671, P = 0.047). Heterozygote carriers of the 901GA genotype were more frequent among controls than among CAD patients not only in the Han population (8.6% versus 1.8%) but also in the Uygur population (5.2% versus 0.9%). The OR for carriers of the 901GA genotype for CAD in the Han population was 0.205 and 0.172 in the Uygur population. After adjustment of confounding factors, the difference remained significant in the Han group (OR = 0.143, 95% CI: 0.068–0.302, P < 0.001) and in the Uygur group (OR = 0.246, 95% CI: 0.072–0.837, P < 0.001).Two haplotypes in the Han population in the result analysis, which the C-G haplotype frequencies in the CHD group was significantly lower than the control subjects (χ2 = 22.713, P < 0.001), the T-G haplotype distribution in the the CHD group was significantly higher (χ2 = 19.022, P < 0.001). In Uighur population, analysis of the T-G haplotypes frequency in case group and the control group was (0.025 vs 0.008, P = 0.004); C-A haplotypefrequency in CAD group and the control group (0.026 vs 0.005, P = 0.001). Conclusions (1) C698T and the G901A in Han and Uygur population are association with coronary heart disease; (2) The 698CT and 901GA genotype of C5L2 may be a genetic maker of CAD in the Han and Uygur population in western China.

ASSA13-03-45 Effects from Age to Wnt and NF-kB Signal Pathway in Heart of Mice

Background Wnt and NF-κB signal pathway are all related to cardiac remodelling post-infarction and there were age-related differences of outcome post infarction. We study changes of these two signal pathways in heart in old mice to prepare some materials for advanced study. Methods We used 20 mice to study changes of these two signal pathways: 10 in young (3-mo) and old group (18-mo) respectively. They were detected expression of dvl-1, β-catenin, p/t GSK-3β and connexin 43 in the left ventricle (LV) by western-blot. And they were detected expression of p65 and p50 by immunohistochemistry and western-blot. At the same time they were detected expression of ICAM-1 and VCAM-1. Results (1) Expression of dvl-1 increased by 2.41 fold in old group compared with young mice in the LV (P = 0.000). There were no statistically differences of expression of β-catenin between the old and young group (P = 0.647) in LV. Ratio of p/t GSK-3β is much lower in old compared with young (P = 0.000). When talking about connexin 43, there were statistically significant differences in old group compared with young (P = 0.001) in the LV. (2) There was no difference of numbers of p65 and p50 positive cells between old and young group by immunohistochemistry (P < 0.05). But expression of p65 and p50 in nuclear protein in old group is higher than it in young group (P = 0.000). There was no difference of ICAM-1 between old and young group (P = 0.401). Expression of VCAM-1 in old group was higher than it in young group (P = 0.000). Conclusions There were age-related differences of protein associated with Wnt and NF-κB signal pathway in heart. Progressing study about changes in these two signal pathway post myocardial-infarction might find new mechanisms about age-related differences of cardiac remodelling.

CONSTRUCTION OF RECOMBINANT ADENO-ASSOCIATED VIRUS SEROTYPE 9 WITH RIBOZYME GENE TARGETING NF-κB AND ITS SUPPRESSION OF NF-κB ACTIVITY IN HELA CELLS

Objectives To construct the recombinant adeno-associated virus serotype 9 containing ribozyme gene (R65) targeting nuclear factor-κ B (NF-κB), and investigate the inhibitory effect of rAAV9-eGFP-R65 on activation of NF-κB as well as on protein expression of NF-κB P65 in HeLa cells. Methods The synthesised ribozyme gene targeting NF-κB was inserted into the plasmid pFB-CMV-eGFP with definite direction, and packaged into the recombinant adeno-associated virus serotype 9 by three plasmids co-transfection, then recombinant adeno-associated virus was purified by cesium choride density centrifugation. The purity of recombinant virus rAAV9-eGFP-R65 was observed and verified by transmission electron microscopy and SDS-PAGE and viral tite was checked by GFP. Finally, HeLa cells were infected by the recombinant adeno-associated virus, the activation of NF-κB and protein expression of P65 were analysed by electrophoretic mobility shift assay [1] and Western blot. Results The high expression of green fluorescence protein expression in HEK293 and HeLa cell lines were found under fluorescent microscope. Electron microscopy and SDS-PAGE test indicated that recombinant adeno-associated virus rAAV9-eGFP-R65 was successfully constructed, and the titre of the virus reached 4.63×1012 vg/ml. Western blot and EMSA showed that the protein expression of P65 and the activation of NF-κB in HeLa cells were markedly inhibited after transfection with rAAV9-eGFP-R65. Conclusions Activation of NF-κB and expression of NF-κB p65 protein in HeLa cells are effectively inhibited by recombinant adeno-associated virus rAAV9-eGFP-R65, which lays the foundation for further researches into therapy nuclear factor-κ B related ischemic diseases.

e0229 Inhibition of NF-κB attenuates post-infarct left ventricular rupture and remodelling in aged mice by ribozyme gene transfer with adeno-associated virus serotypes 9

Objective Using intravenous injection of adeno-associated virus serotypes 9 carring ribozyme gene (AAV9-R65), we examined whether inhibition NF-κB would prevent post-infarct left ventricular rupture and remodelling in aged mice. Methods and results Old (18-month-old) C57BL/6 male mice were given AAV9-R65 by tail vein injection 16u2005days before operation (MI+R65). Myocardial infarction was induced by ligation of the left coronary artery in MI+R65 group and myocardial infarction (MI) group mice. NF-κB activity was inhibited in MI+R65 mice. Inhibition of NF-κB reduced cardiac rupture in MI+R65 group (15.2% vs 32.8%, p=0.018). Echocardiographic measurements revealed that diameter of LV was significantly decreased, and ventricular wall thickness, fraction shortening were significantly increased in MI+R65 mice compared with MI mice (p<0.05). MMP-9 and TNF-α were decreased in MI+R65 group (p<0.05). And collagen was also decreased in MI+R65 group (p<0.05). But there were no changes of IL-1β in MI+R65 group. Conclusions Cardiac rupture and remodelling were attenuated in aged mice by ribozyme gene transfer with adeno-associated virus serotypes 9. It maybe caused by decreased collagen as the result of decreased MMP-9, TNF-α which proved that NF-κB signal pathway may be associated with cardiac rupture and remodelling in aged mice.

e0081 Association of genetic polymorphisms of SAA1 and SAA2 with coronary artery disease in Chinese Han population

Background Both Low plasma HDL cholesterol (HDL-C) and inflammatory responses are associated with an increased risk of coronary artery disease (CAD). Serum amyloid A protein (SAA) is not only an inflammatory factor but also an apolipoprotein that can replace apolipoprotein A1 (apoA1) as the major apolipoprotein of HDL. However, the relationship between genetic polymorphisms of SAA and CAD remains unclear. Methods 4 Single Nucleotide Polymorphisms (SNPs) (rs12218, rs1059559, rs2229338, and rs2468844) of SAA1 and SAA2 gene were genotyped in 1580 CAD patients and 1914 age- and sex-matched controls by the use of PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Results The CC genotype and C allele of rs12218 and the GG genotype and G allele of rs2468844 were more common in the CAD patients than in the control subjects, respectively (all p<0.001). After adjusted for diabetes mellitus, hypertension, smoking, drinking and lipid disorders by use of logistic regression, the SNPs rs12218 (OR=5.906, 95% CI 2.877 to 12.124, p<0.001) and rs2468844 (OR=4.102, 95% CI 2.018 to 8.129, p<0.001) still differed significantly between the CAD patients and control subjects. Conclusion These data suggest that genetic polymorphisms of SAA1/2 gene significantly increased the risk of CAD in a Chinese Han population.

e0255 Relationship of alcohol consumption and carotid atherosclerosis in Chinese Han Uighur and Hazakh cohort

Aim The relationship between alcohol consumption and carotid atherosclerosis had been reported in some epidemiologic studies. But the results were conflicting in different researches. In the present study, we investigated the association between alcohol intake and carotid atherosclerosis in Chinese Han, Uighur, and Hazakh population. Methods and Results The study population sample comprised 13u2008037 Chinese people (5277 Han, 4572 Uighur, and 3188 Hazakh) aged 35u2005years and over, who participated a Cardiovascular Risk Survey between June 2007 and September 2009. Daily alcohol consumption was determined by the number and frequency of alcoholic beverages consumed. The carotid artery parameters including common carotid artery intima-media thickness (CCA-IMT) and carotid plaques were measured using high-resolution B-mode ultrasonography. In Han or Hazakh, carotid IMT as a function of alcohol consumption was depicted as a J-shaped curve with a nadir for the alcohol intake category of 20 to 29.9u2005g/d; In Uighur, the similar curve with a nadir of 30 to 49.9u2005g/d was observed. For the prevalence of carotid plaques, we also observed the similar curves in Han and Hazakh but not in Uighur. After adjusted for the age, sex, blood pressure, body mass index, smoking, GLU, total cholesterol, HDL, and LDL, the J-shaped curves remain existence. Conclusions Our results indicated that moderate drinking is a protective factor for carotid atherosclerosis. But the definition of moderate drinking should be difference in Han, Uighur and Hazakh population.

e0256 Alcohol consumption and ankle-to-brachial index

Aim A low ankle-to-brachial index (ABI) is a strong correlate of cardiovascular disease and subsequent mortality. The relationship between ABI and alcohol consumption remains unclear. Methods and Results Data are from the Cardiovascular Risk Survey (CRS), a multi-ethnical community-based study of 14u2008593 Chinese people (5749 Han, 4747 Uighur, and 4097 Hazakh) aged 35u2005years and over at baseline in June 2007 to March 2010. The relationship between alcohol intake and ABI were determined by use of analysis of covariance and multivariable regressions. In men, a linear correlation between alcohol consumption and ABI was found by one-factor analysis of variance (p<0.001); After adjusted for the age, sex, ethnicity, blood pressure, body mass index, smoking, GLU, total cholesterol, HDL, and LDL, the difference remains significant (p=0.007). The multivariate-adjusted OR for peripheral artery disease was significantly higher in men who consumed >60.0 g/d (OR=1.997, 95% CI 1.500 to 2.989) and was significantly lower in men who consumed 20.0–40.0u2005g/d compared with never drinking, respectively (p=0.01, p=0.027, respectively, data not shown). Neither ABI nor PAD was correlated with alcohol intake in women. Conclusions Our results indicated that in Chinese men, moderate drinking is a protective factor but heavier drinking is a risk factor for peripheral arteriosclerosis.