Abstract:Aim To investigate the role of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in cardiomyocyte differentiation. Methods Anti-NOX4 ribozyme clones of mouse embryonic stem (ES) cells were differentiated into embryoid bodies (EBs). To measure reactive oxygenspecies (ROS) generation, H2DCF-DA and quantitative nitro blue tetrazolium (NBT) test was used. The mRNA levels of NOX were assayed by RT-PCR while the protein level of ventricular myosin light chain 2 (MLC2v) was detected by western blotting. And the apo ptosis of ES cells was detected by DNA laddering. Moreover, NOX4 mRNA in the heart of mouse embryo sections was analyzed by in situ hybridization. Results The exposure of embryoid bodies to 1-100 nmol/L H₂O₂ led to an enhanced beating activity, whereas 1 μmol/L H₂O₂ depressed cardiomyocyte differentiation as compared with control conditions (p<0.001). In contrast, ROS scavengers, such as N-acetylcysteine and catalase, and NOX inhibitor diphenyleneiodonium chloride impaired H₂O₂-stimulated cardiogenesis. The results revealed NOX4 as source of ROS responsible for ES cell differentiation into cardiomyocytes. Down-regulation of NOX4 expression in ES cells by ribozyme severely reduced ROS generation (p<0.001) and MLC2v expression, resulting in the suppression of cardiomyocyte differentiation (p<0.001). Moreover, NOX4 overexpression increased the production of ROS (p<0.05) and induced ES cell apo ptosis. Conclusion These results suggested a key role of NOX4 in cardiomyocyte differentiation through the generation of ROS.

Abstract:Aim To investigate the effects of leptin on the expression of interleukin-1β (IL-1β) in macrophages in vitro. Methods RAW264.7 cells were incubated with leptin in different concentrations (1.25, 2.5, 5, 10 nmol/L) for 4 hours, reverse transcription polymerase chain reaction (RT-PCR) was used to measure the expression of IL-1β mRNA level; Meanwhile, RAW264.7 cells were incubated with these concentrations of leptin for 1, 3, 6 and 9 hours, then the expression of IL-1β in the medium was determined by enzyme linked immunosorbent assay (ELISA). Results The expression of IL-1β mRNA was promoted with the concentration of leptin (0.107±0.102, 0.204±0.019, 0.718±0.083, 0.642±0.071), and reached the maximum level at 5 nmol/L of leptin. At the same time, the expression of IL-1β protein was promoted with the concentration and the time of leptin stimulating, and reached the maximum at 6th hour (p<0.05). The expression of IL-1β in both mRNA and protein levels in RAW264.7 cells incubated with leptin were increased with the leptin concentration. Conclusion Leptin can increase the IL-1β expression of RAW264.7 cells directly and that might be one of mechanism of atherosclerosis induced by leptin.

Abstract:Aim Effects of captopril on angiotensin Ⅱ type 1 receptors (AT1R) and angiotensin Ⅱ type 2 receptors (AT2R) were studied in excess load-pressured hypertrophied cardiac muscles of adult rats. Methods The experiment adopts the method of narrowing and contraction the aorta in adult and health rats,hence establishing the model of animals with hypertensions and cardiac hypertrophies. By comprehensive applications of cardiac-tube, immunity tissue chemistry, and the techniques of image disjunction, to observing changes of AT1R and AT2R on hypertrophied cardiac muscles of AC rats in rats are discussed. Results (1) Load-pressured of left-ventricular and hypertrophy aggravates increase remarkably as the pressure loading time lasts. When it comes to 6 weeks after AC, Vwt reaches (1310±55), in group SO6W it is (209±8.8, p<0.01). When captopril is used till 6 week after AC, the hypertrophy in cardiac tissue decrease to the control group. (2)AngⅡ in cardiac tissue of adult rats grows to as high as (1219.5± 30.13 g/ml), which is nearly twice as high as that of control group (613.0±132.3). After using cap, left-ventricular hypertrophied decreases more significantly than control group. (3)expression of AT1Receptors increase remarkably as the pressure loading time lasts. Expression of AT1 in LV in rats (IOD value) reaches (124±32)

Abstract:Aim To determine the genes involved in the mechanisms of LDL oxidation mediated by endothelial cells. Methods Microarray analysis on human signal transduction associated proteins was applied to profile changes in gene expression of human ECV304 endothelial cells induced by low density lipoprotein. Results In 1 990 genes detected,52 genes were identified to be >2-fold difference in expression, among which 23 genes with elevated expression and 29 genes with reduced expression. Some of these 52 genes encoding proteins have been determined to be correlated with atherosclerosis. Conclusion Those genes expressed differently may provide new insights into the possible mechanisms of low density lipoprotein oxidation mediated by endothelial cells.

Abstract:Aim To explore the effect of stromal cell-derived factor-1α (SDF-1α) on rat vascular smooth muscle cells(VSMC)/monocytes adhesion. Methods VSMC was incubated with different concentrations of oxidized low density lipoprotein (ox-LDL), SDF-1α mRNA and protein expression were revealed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. VSMC pretreated with 50 mg/L ox-LDL for 48 h were incubated with monocytes and SDF-1α antibody to observe cells adhesion. Results Compared with control, SDF-1α was upregulated to 5 times, VSMC supported 29 times higher arrest of monocytes, but this effect was obviously inhibited by the antibody to SDF-1α. Conclusion SDF-1α was implicated in VSMC/monocytes adhesion.

Abstract:Aim To observe the effects of G protein inhibitory polypeptide (GCIP)on the proliferation of rat vascular smooth muscle cells (VSMC) promoted by angiotensinⅡ (AngⅡ) in vitro. Methods VSMC were isolated from the media of rat's thoracic aorta obtained by explanted method, and identified by immunocytochemistry assay (α-SM-actin). The experiment was carried out with the AngⅡ to induce cell proliferation, and the GCIP-27 of different density was joined at the same time, then MTT assay, Bradford method and cells count were utilized to test the proliferation and the level of vascular smooth muscle cell protein. Results Compared with the control group, the VSMC proliferation activity, protein level, and cell sum (by number) were obviously increased in AngⅡgroup ( p<0.05 or p<0.01). Meanwhile, GCIP-27 (≥10 μg/L) markedly inhibited the proliferation activity , protein level, and cell sum with a significant difference from AngⅡgroup ( p<0.05 or p<0.01). Conclusions GCIP-27 could inhibit proliferation of VSMC induced by AngⅡ.

Abstract:Aim To explore the effect of auricularia auricula polysaccharide on the proliferation of vascular smooth muscle cells in the formation of atherosclerosis. Methods Thirty New Zealand white rabbits were randomly divided into three groups: normal group, model group and experiment group with ten rabbits in every group. The period of experiment is twelve weeks. Normal group received normal chow.model group received normal chow and cholesterol and, experiment group received normal chow,cholesterol and Polysaccharide. The levels of serum triglyceride (TG), total cholesterol (TC), low density lipoprotein cholester (LDLC), high density lipoprotein cholesterol (HDLC), malondialdehyde (MDA) and supperoxide-dismutase (SOD) were detected at 0 and 12th week. Animals were killed after 12 weeks, and excised artery segments were prepared for histomorphological observation and α-smooth muscle actin was investigated by immunohistochemistry analysis. The images were analyzed with HAIPS1000 software to distinguish the express of α-smooth muscle actin. Results The serum TC, TG, LDLC and MDA were decreased (model group versus experiment group: TC was respectively 18.76±2.32 and 11.68±1.63; TG was respectively 1.52±0.15 and 1.19±0.12; LDLC was respectively 17.62±2.27 and 9.64±1.56; MDA was respectively 4.53±0.21 and 4.14±0.14,p<0.01 or p<0.05), while the levels of serum HDLC and the serum SOD concentration were increased (model group versus experiment group: HDLC was respectively 0.65±0.09 and 0.73±0.76; SOD was respectively 182.29±10.87 and 215.97±7.38, p<0.01 or p<0.05) in experiment group compared with those in model group. The atherosclerotic lesion were alleviated (model group versus experiment group: 0.44±0.10 and 0.30±0.83, p<0.01) and smooth muscle cells also diminished in lesions (model group versus experiment group: 14%±1% and 12%±1%, p<0.05). Conclusion The proliferation of vascular smooth muscle cells can be decreased through Auricularia Auricula Polysaccharide, thus inhibits the formation of atherosclerosis.

Abstract:Aim To explore the structural and functional changes of aortic wall in vessel calcification rats after mesenchymal stem cells (MSC) transplantation and try to find a new way to cure serious vessel damage. Methods 8-weeks old healthy Wistar rats were used to copy calcification model using vitamin D3 plus nicotine and receive MSC transplantation. Pulse wave velocity (PWV), blood pressure, elatic/collagen ratio, type Ⅰ/Ⅲ collagen, calcification degree were measured according to different coloration methods. Results MSC infusion can reduce elastic/collagen ratio (1.5%±0.3% vs 0.9%±0.2%) and PWV (204±20 vs 298±26) in rats compared with the pseudo-operation group (p<0.01). Pathologic analysis showed a obvious decrease of Calcium amounts in cell transplantation group (13.80±1.28 mg/g vs 29.90±1.85 mg/g, p<0.01). Conclusion MSC infusion can slow down or reverse the aortic remodeling progress in calcification rats by changing the ratio of extracellular matrix (ECM). Such structural change came with the reduction of PWV, which was a sign of artery compliance. MSC can regulate calcium content in artery to regress the vessel remodeling during the calcification, which led to function improvement in the end.

Abstract:Aim To study the development of spontaneous atherosclerosis in combined hyperlipidemic mice. Methods Comparison of apolipoprotein (Apo) E/lipoprotein lipase (LPL)-double deficient mice with ApoE-deficient mice on the plasma triglyceride (TG),total cholesterol (TC), high density lipoprotein (HDL) as well as the atherosclerotic lesion area of the aortic root and the whole aorta. Results ApoE/LPL-double deficient mice had an approximately 60% and 380% increase in total cholesterol (7.237±0.448 and 4.435±1.892 g/L)and triglyceride (5.904±0.505 and 1.536±0.860 g/L)respectively, but high density lipoprotein was not significantly different from ApoE deficient mice. The atherosclerotic lesion area of the aortic root and the whole aorta were also not significantly different between the 2 groups of mice. Conclusion ApoE/LPL-double deficient mice display significantly higher plasma TG and Ch levels than ApoE deficient mice but the spontenous atherosclerotic lesions are not different.

Abstract:Aim Discuss the effection of Qingxuankeli on rat's aorta pathology morphology and nuclear factor (NF-κB), plateled derived growth factor (PDGF-B), basic fibroblast growth factor (bFGF) positive expression. Methods Choose 50 Wistar rat,assign to normal group (feed basic fodder), model group (feed high fat fodder), high dosage Qingxuankeli group (feed high fat fodder), low dosage Qingxuankeli group (feed high fat fodder), ginkgo blade group (feed high fat fodder). After the 8 weeks, distilled water, high dosage Qingxuankeli, low dosage Qingxuankeli, liquid of ginkgo blade was irrigated into stomach separately for 4 weeks in succession. Examine rat's aorta pathology morphology and NF-κB,PDGF-B,bFGF positive expression. Results The degree of aorta scleratheroma pathological change of high, low dosage Qingxuankeli group, ginkgo blade group is slighter than model group obviously . Compared with the model group,the ratio of plaquearea to aortic cavity area(respectively 21.7±9.9, 1.96±1.62, 4.25±3.01, 5.11±2.82), the NF-κB (respectively 25.5±4.2, 10.53±7.09, 16.67±5.56, 17.18±6.63,p<0.01), PDGF-B (respectively 25.2±8.8, 8.32±5.17, 12.76±4.38, 13.32±5.16,p<0.01), bFGF (respectively 9.99±1.63, 1.41±1.77, 4.71±3.90, 5.00±2.47,p<0.01) positive cell's percentage of aorta of the high, low dosage Qingxuankeli group, ginkgo blade group is reduced, the percentage of high dosage Qingxuankeli group is lower than ginkgoes blade group (p<0.05). Conclusion The function of preventing and curing scleratheroma of Qingxuankeli is related to the fact that Qingxuankeli suppress NF-κB, PDGF-B, and bFGF to express positively.

Abstract:Aim To investigate the role of epidermal growth factor receptor (EGFR) in proliferation of vascular smooth muscle cells (VSMC) activated by angiotensin Ⅱ (AngⅡ). Methods The cultured VSMC of Sprague-Dawley rat were transfered with antisense sequences of EGFR. EGFR mRNA and protein in VSMC was detected by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, the proliferation of VSMC was determined by 3H-deoxythymidine (3H-TdR) incorporation. Results The expression of EGFR mRNA in VSMC transfected with antisense sequences of EGFR were significantly lower than transfected with sense sequances of EGFR and control group (0.18±0.03, 0.61±0.11, and 0.66±0.09 respectively, p<0.05). The expression of EGFR protein in VSMC transfected with antisense sequences of EGFR were significantly lower than transfected with sense sequances of EGFR and control group (43.1±8.4, 92.6±10.5, and 100.7±11.3 respectively, p<0.05). The 3H-TdR incorporation of VSMC transfered with antisense sequences of EGFR were significantly lower than that transfered with sense sequances of EGFR and control group (1 055.1±95.7, 1882.4±129.7, and 2013.3±121.3 respectively, p<0.05). Conclusion EGFR play an important role in proliferation of VSMC of rat induced by angiotensin Ⅱ.

Abstract:Aim To investigate the role of cardiotrophin-1 (CT-1) in the rat model of pressure overload-induced cardiac hypertrophy and the effect of AG490 on the cardiac hypertrophy and CT-1. Methods The model of pressure overload was established by constriction of abdominal aorta. The left ventricular mass index (LVMI) was observed. The mRNA levels of CT-1 and β-myosin heavy chain (β-MHC) were examined by reverse transcription polymerase chain reaction (RT-PCR) method. The circulating and ventricular angiotensinⅡ (AngⅡ) was measured by radiommunoassay. Results Fourteen days after constriction of abdominal aorta, the LVMI of the hypertrophic group was significantly increased than that of the control group (p<0.01) and it was significantly decreased in AG490 group than in the hypertrophic group, but more than in the control group (p<0.01). The circulating and ventricular AngⅡ of the hypertrophic group was increased compared with the control group, and there was a significant difference between the two groups (p<0.01); the AngⅡlevel of AG490 group was less than that of the hypertrophic group, but still more than the control group (p<0.01). The expression of CT-1 mRNA was similar in the hypertrophic group and the AG490 group (p<0.05), and both groups were more than the control group (p<0.01). The expression of β-MHC mRNA in the hypertrophic group was significantly increased than that in the control group (p<0.01) and decreased in the AG490 group compared with the hypertrophic group (p<0.01), but it was still more than the control group (p<0.01). Conclusions The expression of CT-1 may play an important role in cardiac hypertrophy, AG490 may inhibit the expression of CT-1 and the development of cardiac hypertrophy.

Abstract:Aim To investigate the effect of advanced glycation end product bovine serum albumin (AGE-BSA)on macrophage matrix metalloproteinase-9 (MMP-9) activity and the effects of Simvastatin. Methods Mouse peritoneal macrophages were incubated with AGE-BSA and Simvastain at different levels. MMP-9 activity was determined by Gelatin Zymography. Results AGE-BSA induced morphological changes of macrophage in vitro. After treatment with AGE-BSA (0, 50, 100, 200, 400 mg/L)for 48 hours, macrophage MMP-9 was significantly increased in contrast to the control, showing the dose-dependent effect (n=5, p<0.05). After treatment with 400 mg/L AGE-BSA for different duration, MMP-9 activity showed no significant difference at the time point of 12 hour (n=5, p<0.05), but MMP-9 activity was significantly increased in contrast to the control at the time point of 24, 36 and 48 hour, showing the time dependent effect (n=5, p<0.05). Simvastain decreased MMP-9 activity significantly. Conclusion AGE-BSA can activate macrophages in vitro and enhance MMP-9 activity, indicating its effect of atherogenesis and plaque rupture. Simvastatin can lower MMP-9 activity, showing statins' pleiotropic effects.

Abstract:Aim To assess the outcomes after percutaneous renal artery stent implantation in diabetic patients with renal artery stenosis. Methods Forty-three patients with diabetes mellitus (DM) and severe uni-or bi-lateral renal artery stenosis (luminal diameter narrowing ≥70%) and receiving renal stent implantation were included (DM group), and another 43 non-diabetic patients undergone renal artery stenting were served as controls. Basic characteristics and the changes in blood pressure and renal function including serum creatinine level, urine β₂ micro-globulin (β₂-MG) concentration and assessed glomerular filtration rate were compared between two groups at baseline state and at 6 months' follow-up. Results Before stenting procedure, patients in DM group had higher serum creatinine (135±17 μmol/L vs 107±31 μmol/L, p<0.05) and urine β₂-MG levels (175±72 μg/L vs 139±57 μg/L,p<0.05). And 31 renal arteries with lumen stenosis ≥70% were stented in the DM group, compared with 39 stents in the control group, and the procedural success rate was 100%. At 6 months' follow-up, creatinine level was decreased in both groups, but remains higher in DM group compared with the control (127±31 μmol/L vs 99±22 μmol/L,p<0.05). In DM group the urine β₂-MG was significantly decreased after procedure (134±17 μg/L vs 175±72 μg/L, p<0.05). Despite conventional medical treatment, blood pressure remains poorly controlled in majority of patients (benefit is 44% in DM vs 71% in control,p<0.05). Conclusion Diabetic patients receiving renal artery stenting had a worse prognosis with respect to renal function improvement and blood pressure control than non-diabetic patients.

Abstract:Aim To explore whether there is relationship among the inflammation markers, acute coronary syndrome (ACS) and carotid atherosclerotic plaque. Methods 92 patients with ACS, 97 patients with stable angina pectoris (SAP) who underwent coronary angiography were enrolled in our study, 40 healthy people were selected as control. The bilateral carotid ultrasounding by high sensitive was investigated to measure the intima-media thickness (IMT), the number of the carotid plaques and the characteristics of the plaques, the serum levels of intercellular adhesion molecular-1 (ICAM-1), matrix metalloproteinase-9 (MMP-9) and high sensitive C-reaction protein (hs-CRP) were determined and the Logistic statistic analysis was used to study the relationship of the inflammatory markers and the unstable plaque. Results The levels of MMP-9, ICAM-1 and hs-CRP in the patients with ACS were significantly higher than those of the patients with SAP and the normal people. According to the Logistic analysis, the results indicated that serum MMP-9, ICAM-1 and hs-CRP were related to the unstable plaque (MMP-9>300 μg/L, OR: 3.12, 95%CI: 1.11～8.98, P=0.027; ICAM-1>200 μg/L, OR: 4.78, 95%CI: 1.34～9.89, P=0.010; hs-CRP >4.0 mg/L, OR: 5.37, 95%CI: 1.43～15.21, P=0.003). Conclusion The numbers of the complex carotid plaques in patients with ACS were significantly higher than those of the patients with SAP and normal control people. There was significant relationship between the complex carotid plaques and ACS.

Abstract:Aim To investigate the possible association between serum endostatin, serum vascular endothelial growth factor (VEGF) and the pathogenesis of macroangiopathy in type 2 diabetes mellitus (T2DM). Methods The determination of endostatin,VEGF levels in 100 T2DM patients including 34 cases without macroangiopathy (group N),40 cases with only one kind of macroangiopathy (group A), 26 cases with much more macroangiopathy (group B) and 30 healthy control subjects (group C) was made by ELISA assay. Results The levels of endostatin and VEGF in T2DM patients complicated with macroangiopathy(group A,group B) were significantly higher than those without macroangiopathy (group N,group C),(endostatin:32.4±15.6 μg/L and 35.1±20.2 μg/L vs. 11.2±8.6 μg/L and 9.9±6.7 g/L;VEGF:133.5±36.8 ng/L and 302.1±52.4 ng/L vs. 90.2±42.4 ng/L and 81.3±33.5 ng/L, p<0.01). There was no significant difference between endostatin, VEGF levels in group C and those in group N.It was not endostatin levels but VEGF levels that were significantly elevated between group A and group B in turn 133.5±36.8 ng/L vs.302.1±52.4 ng/L,p<0.01). There was a significantly positive correlation between endostatin and VEGF levels in Group A(r=0.540,p<0.01). Conclusion The study suggests that the variation of serum VEGF levels is closely associated with the initiation and progression of T2DM macroangiopathy and meanwhile VEGF together with endostatin possibly plays an important role in the pathological course of As based on homeostasis regulation.

Abstract:Aim To explore the serum soluble cell differentiation antigen 40 ligand (sCD40L) level and its significance in patients with acute cerebral infarction. Methods Serum sCD40L level was measured by two-layer antibody sandwich enzyme-linked immunosorbent assay (ELISA) in 62 patients with acute cerebral infarction and 30 healthy controls matched in sex and age. Results Serum sCD40L level was significantly higher in patients with acute cerebral infarction than that in control [(2.32±1. 29) μg/L vs (0.68±0.56) μg/L,p<0.01]. Serum sCD40L level in patients with acute cerebral infarction was positively related to apolipoprotein B₁₀₀ (ApoB₁₀₀) (r=0.267, p<0.05) and triglyceride (TG) (r=0.254,p<0.05) and negatively related to high density lipoprotein cholesterol (HDLC) (r=-0.272,p<0.05). Conclusions The sCD40L level was significantly elevated in acute cerebral infarction patients within 72 h of onset, which may be related to the pathogenesis of acute cerebral infarction.