Abstract:Vascular remodeling of ischemic disease rely on not only angiogenesis, but also bone marrow precursor cells and many growth factors， such as vascular endothelial growth factor and platelet-derived growth factor.Ischemia itself can not only increase the number of stem cells, but also enhance their ability for differentiation to endothelial cells.however, ischemia- induced vascular remodeling often cannot completely supply blood decrease of peripheral vascular disease and arterial occlusion.So, to promote blood recovery of ischemic tissues, growth factors are used to mobilize vascular precursor cells and enhance angiogenesis.Synergistic effects of cytokines on neovascularization are reviewed in this paper.

Abstract:AimTo examine the effect of Toll-like receptor 4(TLR4) gene silencing by small interfering RNA (siRNA) on the expression of cytokines produced by peritoneal macrophages in chronic mild stress (CMS) ApoE^-/- mice.MethodsTwo siRNA sequences and one negative control sequence were designed targeting the mouse TLR4 gene.Two complementary single-strand DNA were designed and synthesized based on siRNA sequences.The DNA fragments were annealed and ligated to the GFP expression vector pRNAT-H1.1/Adeno.One siRNA with higher interference efficiency than the other was found after siRNA plasmid transfection into 293A cells mediated by liposome.After adenovirus partical packaging and production, the 293A cells were infected, and the single cell clone was acquired and cultured to establish the stable cell strain.The titer of concentrated virus was detected by hole-by-dilution titer assay.One hundred twenty male ApoE^-/- mice were divided into groups of CMS control, CMS + empty vector and CMS + siRNA (tail vein injection, 10 μL/mouse; n=40 per group).All mice were fed a high-fat (5%, wt/w), high-cholesterol (1%, wt/wt) diet and subjected to daily CMS for 0, 4, 12 weeks, respectively.Peritoneal macrophages were prepared from ApoE^-/- mice and then total proteins from cells were extracted.Western Blotting was used to determine the expressions of TLR4 and nuclear factor-κB (NF-κB) of peritoneal macrophages.The supernatants of cultured peritoneal macrophages were collected and then used for detection of interleukin-1β(IL-1β) and tumor necrosis factor-α（TNF-α） levels by ELISA.ResultsCompared with the blank control group, real-time PCR showed that the expression of TLR4 mRNA in 293A cells was decreased by 56% and 67% after 48 h transfection with siRNA1 and siRNA2, respectively.The hole-by-dilution titer assay showed that viral titer was 3.4×10¹⁴TU/L.After exposure to CMS for 4 and 12 weeks, there was no difference in serum corticosterone levels between siRNA group and the CMS control group(p> 0.05), and siRNA group mice demonstrated markedly decrease in TLR4(p<0.05) and NF-κB(p<0.05) levels of peritoneal macrophages compared with the corresponding control group, respectively.IL-1β (p<0.01) and TNF-α (p<0.01) levels in supernatants of cultured peritoneal macrophages of siRNA group were significantly lower than those of the CMS group.ConclusionThese results showed that siRNA effectively inhibited the expressions of TLR4/NF-κB-IL-1β and TNF-α of peritoneal macrophages in CMS ApoE^-/- mice, which suggested that the activation of TLR4 pathway of macrophages might play an important role in chronic inflammation response induced by CMS.

Abstract:AimTo investigate the effects of angiotensinⅡ (AngⅡ) on apoptosis, and phosphorylation of p38 mitogen-activated protein kinase in endothelial cell, and its possible action mechanism.MethodHuman umbilical vein endothelial cells (HUVEC) were cultured in vitro and intervened by AngⅡ.HUVEC were divided into control group and AngⅡ group (stimulated by AngⅡ10－6 mol/L for 24 h), morphologic changes and percentage of apoptosis were assayed with acridine orange fluorescence staining.The early stage apoptosis was detected by flow cytometery with Annexin V-FITC/PI double staining.The expression of apoptosis-association gene Bcl-2 was detected by RT-PCR and Western blotting at different time points.By means of Western blotting, the activation of p38MAPK was observed at different time points.Results10－6 mol/L AngⅡ stimulated cell apoptosis.The percentage of apoptotic cells in AngⅡ-stimulated cells was significantly increased compared to that in the control cells (32.76%±2.98% vs 2.14%±0.36%, p<0.01) by using acridine orange fluorescence staining.The early-metaphase apoptotic rate was significantly increased in AngⅡ-stimulated cells compared to the control cells (37.4%±1.6% vs 10.2%±1.8%, p<0.01) by Annexin V-FITC/PI double staining flow cytometry.Bcl-2 mRNA and protein expression decreased markedly (p<0.05), the activation of p38MAPK began to increase and reach the peak at 18 h (p<0.01).ConclusionsCell apoptosis is possibly an impor-tant factor for atherosclerosis.One of its molecular mechanisms might be associated with decreasing the expression level of Bcl-2.There is a probability that activated p38MAPK signal pathway is involved in the process of pathologic and physiologic reaction in the apoptosis of endothelial cell induced by AngⅡ.

Abstract:AimTo establish rat model of insulin resistance，then treated by related drugs and to observe the expression of Caspase-3 in renal tissue, which was to investigate the relationship between amount of expression of Caspase-3 and extent of insulin resistance.MethodsThe insulin resistant model of neonatal Wistar rats was induced by a single intraperitoneal injection of streptozotocin(STZ) at a dose of 100 mg/kg.The Wistar rats were divided into four groups, including: normal control(control group),insulin resistance(IR group ), the metformin (metformin group ) and the combinated treatment of tetramethylpyrazine and aminoguanidine(combinated treatment group).The concentrations of fasting blood glucose(FPG), fasting insulin (FIN) and insulin resistance index(IRI)were measured on the eighth and thirty-second week.At the end of thirty-second week， one kidney of each rat was localized for 24 hours using 4% paraformaldehyde, embedded in the paraffin and made into 5um section.Another one was quickly frozen by liquid nitrogen, then stored at -80℃.IHC and western-blot were respectively used to qualitatively and quantitatively evaluate expression of Caspase-3.Results (1) The insulin resistance index in IR group was significantly higher than in control group（p<0.01）. metformin and combinated treatment groups were remarkably lower than IR group (p<0.01); and combinated treatment group was less than metformin group (p<0.05).(2)The alteration of Caspase-3: IR group was obviously higher than control group(p<0.01), metformin and combinated treatment groups were evidently lower than IR group(p<0.01), and combinated treatment group was lower than metformin group (p<0.05).ConclusionThe method of intraperitoneal injection of Streptozotocin can induce insulin resistance in neonatal Wistar rats; In this induced model the concentration of Caspase-3 increased, and there is a positive correlation between up-regulation of Caspase-3 and insulin resistance; metformin and the combined treatment with tetramethylpyrazine and aminoguanidine improved streptozotocin-induced insulin resistance via a mechanism of reducing Caspase-3 expression, furthermore the combination treatment of tetramethylpyrazine and aminoguanidine surpassed single metformin treatment at this aspect.

Abstract:AimTo investigate the association between the lipid-regulating effect of statins with the CETP-69G/A,-629C/A,-971G/A and -1337C/T polymorphisms.MethodsThe target DNA fragments of CETP gene were amplified and analyzed by PCR-RFLP technique in 207 dyslipidemia patients treated with statins. The relationship between each SNP and each blood-fat index was analyzed by SPSS statistic software.ResultsThe A allele was not found in the -69G/A polymorphism; After the treatment with simvastatin, the TC and LDLC decreasing extent were significantly different（P=0.025；P=0.000）between -629C/A genotypes, respectively.The -629AA individuals were 0.495 mmol/L（P=0.020）higher than CC individuals in TC decreasing extent. The LDLC decreasing extent were 0.577 mmol/L and 0.352 mmol/L （P=0.000, P=0.012） higher in -629AA and CA individuals than CC individuals; the -1337C/T polymorphisms was associated with the rising extent of HDLC level(F=3.064, P=0.044).The HDLC rising extent was gradually increased among -1337TT,TA and AA genotypes.There was no significant difference in the change of blood- fat level between the -971G/A genotypes after the treatment with simvastatin. ConclusionThe -629C/A and -1337C/T polymorphisms were associated with the lipid-regulating effect of statins.

Abstract:AimTo explore the effect of PCSK9 siRNA on Bax, Bcl-2 expression in THP-1 derived macrophages apoptosis induced by ox-LDL.MethodsCells were incubated with varied concentrations of ox-LDL for different time.Western blot were conducted to detect the expression of Bax and Bcl-2. The siRNAs for PCSK9 were designed and synthesized, then THP-1 macrophages were transfected with PCSK9 siRNA (30, 50, 80 nmol/L) for 24 h and then a high level of ox-LDL (80 μg/mL) for an additional 48-hr. Bax , Bcl-2 protein levels were measured by Western blot and apoptosis was measured with Hoechst 33258 staining.Resultsox-LDL (20, 40, 60, 80 μg/mL) increased Bax expression in a dose-dependent manner, but inversely decreased Bcl-2 expression. Furthermore, 80 μg/mL ox-LDL increased Bax expression in a time-dependent manner, but inversely decreased Bcl-2 expression. However, PCSK9 siRNA attenuated ox-LDL-induced up-regulation of Bax, down-regulation of Bcl-2, and THP-1 cell apoptosis in a dose-dependent manner.ConclusionThe effect of PCSK9-siRNA aganist ox-LDL-induced THP-1 cell apoptosis is associated with down-regulating of Bax and up-regulating of Bcl-2.

Abstract:AimTo observe the changes of human umbilical vein endothelial cells (HUVEC) in function and membrane surface ultrastructure during the lipid peroxidation.MethodsHUVEC were induced by 100 mg/L oxidized low density lipoprotein (ox-LDL) as experiment group, then incubated for 0 h, 4 h, 8 h and 16 h.HUVEC in the PBS as control group was processed likewise.Cell viability was measured by MTT assay.The functional status of HUVEC was determined by detecting content of nitric oxide (NO) in the cultured cell supernate with nitric acid reduction assay.Atomic force microscope (AFM) was used for observation to membrane surface ultrastructure of HUVEC.ResultsThe research showed that the proliferation ability of HUVEC in the experimental group were inhibited, cell functions were attenuated, and the longer incubation, the more significantly effects.At the same time, the eminentias on cell surface became larger, distribution irregularity, even some caveolaes and holes appeared.However, there was no change in control groups.Cell surface roughness analysis showed that roughness on 0 h, 4 h, 8 h and 16 h experimental groups were 13.666±2.196 nm, 15.904±2.203 nm, 17.688±2.076 nm and 21.609±1.867 nm, respectively.There were significant differences between each two (p<0.05).In the comparison, the control group were 13.627±2.218 nm, 13.659±2.183 nm, 13.665±2.175 nm and 13.974±2.478 nm, respectively, and no significant differences between each two (p>0.05).Compared with the control group, roughness in the experimental groups after 4 hours induction was significantly higher (p<0.05).ConclusionsIt was demonstrated that the cell function of endothelial cells were weakened and membrane surface ultrastructure changed in an early stage of lipid peroxidation, and perform a time-dependent rule.

Abstract:AimTo study the effect of angiotensin(1-7) on inhibiting the inflammation induced by angiotensinⅡ （AngⅡ）.MethodsCultured human umbilical vein endothelial cells(HUVEC) were randomly dividied into different groups ,then incubated in the presence of Ang(1-7), AngⅡ and the specific inhibitor of Ang(1-7), A-779, and so on. The phosphorylation of p38MAPK were determined by Western blot, and the mRNA for the mas receptor were determined by reverse transcriptional PCR.ResultsAng(1-7) dose-dependently inhibited the phosphorylation of p38MAPK induced by AngⅡ in HUVECs. The expression of p38MAPK phosphorylation died down markedly at 1 000 nmol/L of Ang(1-7). Pre-treatment with A-779 for 10 min in HUVEC before Ang(1-7) and AngⅡ used, the expression of p38 MAPK phosphorylation was nonsignificantly changed.ConclusionAng(1-7) effectively represses the phosphorylation of p38MAPK induced by AngⅡ in HUVEC.

Abstract:AimTo observe effect of angiotensin (1-7) (Ang-1-7) on myocardial hypertrophy in endoplasmic reticulum stress ( ERS ) induced cell injury.MethodsExperiment was divided into control group, angiotensin Ⅱ(AngⅡ) group, AngⅡ + Ang-1-7 group.Cultured rat myocardial cells were induced hypertrophy by different concentrations of AngⅡ (100 nmol / L, 1000 nmol / L) for 24, 48 and 72 hours, and intervened by different concentrations of Ang-1-7 (10 nmol/L, 100 nmol/L, 1000 nmol/L).After finishing experiment, the change of myocardial cells was observed in an inverted phase contrast microscope.Coomassie brilliant blue G-250 was used to determine cardiac total protein synthesis.Reverse transcription polymerase chain reaction (RT-PCR) and Western-Blotting were used to detect glucose-regulated protein 78(GRP78) and C/EBP homologous protein (CHOP) expression of endoplasmic reticulum stress(ERS).ResultsCompared with the control group, 100 nmol/L AngⅡ induced cardiac hypertrophy at 24 h, the myocardial cell volume increased, the cell protein content increased（1.59±0.03 g/L,p＜0.05）.mRNA and protein expression levels of endoplasmic reticulum stress proteins GRP78 and CHOP were significantly higher（p＜0.05）; Ang-1-7 can reverse the above to a large extent（p＜0.05）.ConclusionMyocardial hypertrophy induced by AngⅡ exist endoplasmic reticulum stress.Ang-1-7 can reduce the endoplasmic reticulum stress by reducing cardiac hypertrophy, and protect myocardial cells; Protective effect of 1000 nmol/L Ang-1-7 was the strongest.

Abstract:AimTo investigate the association between 7 single nucleotide polymorphisms in intron 2 of hyperplasia suppressor gene (HSG) and essential hypertension.Methods500 normotensive subjects (NT group) and 930 essential hypertensive patients (EH group) were screened and DNA was acquired from white blood cells.Real-time quantitative PCR was used for the detection of 7 SNPs in intron 2 of HSG.ResultsThe results showed that genotypes distribution and allelic frequency of rs873457, rs2336384 and rs4846085 were significantly different (p<0.05) between EH group and NT group, TT∶TC∶CC=21.8%∶46.6%∶31.6%/22.5%∶53.0%∶24.5% (rs873457), CC∶CA∶AA=21.8%∶46.8%∶31.4%/22.8%∶52.6%∶24.6% (rs2336384), TT∶TC∶CC=22.6%∶46.4%∶31.0%/23.4%∶51.8%∶24.7% (rs4846085) for genotypes distribution frequency and T∶C=45.1%∶51.0%/49.0%∶51.0% (rs873457), C∶A=45.2%∶54.8%/49.1%∶50.9% (rs2336384), T∶C=45.8%∶54.2%/49.1%∶50.6% (rs4846085) for allelic frequency.When subgrouped by sex, the genotypes distribution and allelic frequency of all the SNP were significantly different in male (p<0.05 or p<0.01) but not in female groups (p>0.05).Correlation analysis indicated that body mass index (BMI), age and genotype were related with essential hypertension.Logistic regression showed that BMI and rs873457 were closely associated with blood pressure after adjusting for age.The frequency of C-G-A-A-A-C-C haplotype was significantly higher in essential hypertensive patients verse control individuals, either in entire population, in male or female group (p<0.01 for all).As for other haplotypes, most of haplotypes were only significantly different in the entire population and male subjects.ConclusionThe genetic variations of HSG may be associated with essential hypertension in male Chinese.

Abstract:AimTo analyse the association of CYP3A4*1G, CYP3A5*3 and MDR1 C3435T gene polymorphisms with the antihypertensive effect of amlodipine.MethodsA total of 70 patients were screened who were diagnosed as hypertension in the first year after transplantation.The patients were assigned to receive amlodipine (5 mg/d) for 4 weeks.PCR-RFLP were applied to detect CYP3A4*1G， CYP3A5*3 and MDR1 C3435T gene polymorphism, and antihypertensive effects were analyzed according to genotype.ResultsAfter 4-weeks treatment, mean reductions in systolic blood pressure(SBP) and diastolic blood pressure(DBP) were 18.8±6.9 mmHg and 12.7±5.4 mmHg, respectively (p<0.05).Patients with CYP3A5*3*3 genotype (15.0±5.0 mmHg) had higher DBP reduction than those with CYP3A5*1*3 (11.3±5.3 mmHg) and CYP3A5*1*1 genotype (10.0±4.1 mmHg) (p<0.05).No significant relationship was found between CYP3A5*3 gene polymorphism and SBP reduction (p>0.05); Patients with CYP3A4*1G*1G genotype (8.6±4.1 mmHg) had lower DBP reduction than those with CYP3A4*1G*1 genotype（13.2±5.2 mmHg） and CYP3A4*1*1 genotype(13.1±5.5 mmHg)(p<0.05).No significant relationship was found between CYP3A5*3 gene polymorphism and SBP reduction (p>0.05); And no significant relationship was found between MDR1 C3435T and BP reduction (p>0.05).A linkage disequilibrium exists between CYP3A4*1G and CYP3A5*3 allele, GGGG, AAAA and AGAG were the most common combination.Patients with GGGG genotype can reach higher DBP reduction than other ones.ConclusionCYP3A5*3 and CYP3A4*1G gene polymorphisms can affect antihypertensive effect of amlodipine in hypertensive patients after renal transplantation, DBP reduction in patients with CYP3A5*3*3 genotype is higher, and DBP reduction in patients with CYP3A4*1G*1G genotype is lower compared with other genotypes.Patients with GGGG genotype have higher DBP reduction in all the haplotypes composed by CYP3A4*1G and CYP3A5*3.MDR1 3435T gene polymorphism was not found to be related to the effect.

Abstract:AimTo expore the effect of hyperinsulin on the maturation of monocyte-derived dendritic cells in the Acute Coronary Syndrome(ACS) patients.MethodsHuman monocytes of ACS were purifed using successive adherence method, recombinated human granulocyte-macrophage colony stimulating factor （GM-CSF,100 μg/L）and interleukin-4（IL-4,100 μg/L）after 5 days culture in RPMI 1640 medium containing. Immature DC (imDC) were collected, then added RPMI 1640 medium containing with insulin of various concentrations(1nmol/L, 10nmol/L,100 nmol/L)for 48 hours, mature DC (mDC) were derived. Immunophenotypic expression of CD40, CD80 and CD83 were monitored by flow cytometry, and expression of IL-12, IL-10 and TNF-α were measured by ELISA,and the morphological features of dendritic cells were observed with invert optical microscope. ResultsHyperinsulin promoted the expression of CD40, CD80 and CD83 and enhanced the expression of IL-12 and TNF-α significantly and refained the expression of IL-10 in the acute coronary syndrome patients. ConclusionsHyperinsulin contributed to the development of atherosclerosis via stimulating immune maturation of DC，which may be one of its mechanisms in the development of ACS.

Abstract:AimTo investigate the relationship between non-alcoholic fatty liver (NAFL) and peripheral atherosclerosis.Methods300 physical examines had selected to enter the study.All participants were divided into NAFL group and non-NAFL group, NAFL group included 138 cases, non-NAFL group included 162 cases. Age and gender composition had no significant difference in the two groups (p＞0.05). Anthropometry indicators, brachial-ankle pulse wave velocity (baPWV) and ankle-brachial index were measured. Fatty liver was measured via ultrasonography.ResultsWeight, body mass index (BMI), waistline, baPWV，systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure (PP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were significantly higher in the NAFL group than those in the non-NAFL group (p<0.05). The detection rates of obesity, hypertension and peripheral atherosclerosis in the NAFL group were significantly higher than those in the non-NAFL group (p<0.01).Logistic regression analysis showed that BMI, baPWV and ALT were susceptible factors of NAFL.ConclusionbaPWV is closely associated with NAFL. There is a close relationship between NAFL and peripheral atherosclerosis.

Abstract:AimTo analyse the influencing factors of images quality with bolus chasing technology for angiography of lower extremity artery with digital subtraction angiography (DSA), and to discuss how to take some methods for controlling the quality of bolus chasing angiography of lower extremity artery.MethodsWith 154 exposure sequences from 67 cases lower extremity artery angiography with DSA by using bolus chasing technology, the influencing factors of the failed or bad images were analysed. ResultsThe influencing factors of the failed or bad images included motion artifact， saturation artifact and the poor vessels showings， of all the 154 exposure sequences, the motion artifacts were 17 exposure sequences (6 angiogram sequences, 11 mask image sequences), the saturation artifact were 17 exposure sequences (9 angiogram sequence, 8 mask image sequences)and the poor vessel showings were 10 of all the 81 angiogram sequences.ConclusionsThe present analysis demonstrates the important keys of getting best images with bolus chasing technology for angiography of lower extremity artery by DSA as follows： effective fixing the patients lower extremity in ordor to prevent motion artifact， placing rubber cuneus to prevent saturation artifact， suitable speeding and doses of contrast， expertly operating machine and medical staff and patients closely matching with each other.

Abstract:Remnant-like lipoprotein particles have been implicated as potentially atherogenic lipoproteins. A variety of proinflammatory adipocytokines secreted by adipocytes play an important role in the development of chronic inflammation of atherosclerosis. Obesity is an independent risk factor of coronary heart disease and atherosclerosis. Remnant-like lipoprotein particles accelerate atherosclerosis events through stimulating the release of proinflammatory adipocytokines and inducing adipogenic differentiation from preadipocytes to mature adipocytes. In addition, the effects of adipocytes on remnant-like lipoprotein particles metabolism may impact the atherogenic intensity of remnant-like lipoprotein particles in vivo.

Abstract:Platelet glycoprotein Ⅱb/Ⅲa receptor antagonists could block the final common pathway of platelet aggregation and effectively inhibit platelet function, thereby they reduce no-reflow phenomenon in patients treated with percutaneous coronary intervention for acute coronary syndrome. This review summarizes platelet glycoprotein Ⅱb/Ⅲa receptor antagonist therapy in patients with acute coronary syndrome.