Abstract:Lipid disorder and inflammation play important roles in the development of atherosclerosis.Reverse cholesterol transport is a key event in lipid metabolism.Evidence from our research results and literatures indicate that caveolae and caveolin-1 not only play a key role in cholesterol transportation, but also mediate anti- inflammation during the inflammation response.Therefore, caveolae and caveolin-1 may be the basic molecular platform for the interaction between reverse cholesterol transport and inflammation in atherosclerosis.

Abstract:AimTo investigate the effect of hematoporphyrin monomethyl ether (HMME) mediated photodynamic therapy (PDT) on macrophage.MethodsFluorescence of HMME with different incubated time was tested.Confocal scanning laser microscope was used to investigate HMME subcellular distribution.The survival and apoptosis rate of macrophages were determined by MTT assay and flow cytometer.ResultsHMME was identified to accumulate in the cytoplasm of macrophages by confocal scanning laser microscope.The number of apoptotic macrophages increased slightly in the photodynamic therapy for 1.5 min group(5.82%±3.27%) at 12 h post irradiation (p<0.05), whereas increased significantly in the photodynamic therapy for 3 min group (65.45%±5.17%, p<0.001).ConclusionsHMME could accumulate in macrophages and HMME-mediaded PDT induced macrophage apoptosis.

Abstract:AimTo construct a pSUPER RNAi system that targets human heart developmental candidate gene hole and a stable virus-producing cell line.MethodsA pair of 60nt oligonucleotides coding for short hairpin RNA and targeting human heart developmental candidate gene hole were chemically synthesized and annealed and inserted into pSUPER plasmids digested with BglⅡ and XhoⅠto construct the recombinant pSUPER RNAi plasmid (pSUPER-hole).Recombinant pSUPER-hole plasmid was identified by enzyme digestion and sequencing analysis.The packaging cell H9c2 was transfected with the recombinant plasmid.The mRNA expression level of hole was detected by RT-PCR.ResultsThe result of enzyme digestion and sequencing analysis demonstrated that 60 nt had been inserted successfully into the vector.Green fluorescent was detected in virus-producing cells, RT-PCR showed pSUPER-hole can inhibit the hole gene expression of H9c2. ConclusionThe pSUPER RNAi vector targeting human heart developmental candidate gene hole are successfully constructed.

Abstract:AimThe effect of tanshinoneⅡA on cholesterol homeostasis in foam cells was studied in vitro.MethodsThe foam cell induced from RAW264.7 macrophage with oxidized low density lipoprotein (ox-LDL) were treated with different concentration of tanshinoneⅡA.The relative cell viability were detected by MTT methods, and the change of total cholesterol and cholesterol ester in the cells were detected with the enzyme colorimetric quantifies.The lipid accumulation in cell was observed by oil red O dyeing, then the expressions of CD36, ATP binding cassette transporter A1(ABCA1), liver X receptor α (LXRα) and peroxisome proliferator activated receptors γ (PPARγ), PPARα were detected with the methods of fluorescent quantitation PCR and Western Blot.ResultsThe cell viability was not affected when tanshinone ⅡA concentrations were in the range of 0～20 mg/L.The ratio of cholesterol ester and total cholesterol decreased and lipid accumulation decreased when the foam cells were treated with 20 mg/L tanshinone ⅡA.Meanwhile, the expression of ABCA1, LXRα, PPARα were increased，but the expression of CD36 had no obvious variance.ConclusionTanshinoneⅡA can inhibit the formation of foam cells induced from RAW264.7 by ox-LDL，the mechanism is possibly related to inducing the expressions of PPARα, LXRα and ABCA1 and promoting the excretion of cholesterol.

Abstract:AimTo clarify the effects of the cardiac mast cells on cardiac function after myocardial infarction(MI),and to study the relationship between mast cells and ventricular remodeling after MI in SD rats.MethodsMI model was established in male Sprague-Dawley（SD） rats by ligation of left anterior descending coronary artery.The rats were randomly divided into three groups before 5 days of ligation：sham-operated group(n=6)，MI model group(n=8) and mast cell stabilizer cromolyn sodium treatment group(n=7).Before 3 days, after 2 and 4 weeks of ligation, the cardiac function was assessed by Echocardiography.After 4 weeks of treatment, the ratio of left or right ventricular weight and body weight were measured，the change of histopathological were observed, and the density of mast cells were measured.ResultsAs compared with sham-operated group, left ventricular ejection fraction(EF) in MI model group and treatment group was markedly decreased(p<0.01)after 2 weeks of treatment.Although the enlargement of the heart has been going in model group and treatment group, there is no significant difference among these three groups.After 4 weeks of the treatment, the Left ventricular end-diastolic volume(EDV) was significantly increased in model group and treatment group more than in sham-operated group（p<0.05）.Compared with MI model group，cromolyn sodium increased EF but not EDV, and decreased left ventricular index(p<0.05) and mast cells density(p<0.01) in left ventricular subepicardial layers of infarted region.The number of mast cells in subepicardial layers had negative correlation with EF, but was

Abstract:AimTo construct PGL3-ApoM luciferase reporter vector containing ApoM gene regulation area, and to investigate the effect of C→T substitution in ApoM promoter －778 bp region on ApoM gene expression.MethodsHuman chromosome DNA fragments containing ApoM gene were amplified by PCR, and the DNA fragments consisting of ApoM TT and CC genetypes were separately selected, then PGL3 vector including above two different DNA fragments was constructed.Recombinant vector were contransfected into HepG2 cells by using cationic liposome method.Cells were cultured for 48 h, activity of firefly luciferase was measured.ResultsRelative activity of luciferase for ApoM CC genetype was significantly lower than that for TT genetype.Conclusion－778 bp C→T substitution in ApoM gene may inhibit ApoM gene transcription.It may be the important factor of the ApoM gene expression.

Abstract:AimTo study cholesterol efflux of macrophages in patients with coronary heart disease after cholesterol load and its mechanism. MethodsPeripheral blood mononuclear cells were separated and induced to macrophages with phorbol ester in patients (test group) and normal individuals (control group).Cells incubated with ［³H］cholesterol for 24 h to charge cholesterol and then divided into 4 groups: control group＋normal serum, control group＋high-cholesterol serum, test group＋normal serum and experimental group＋high-cholesterol serum.ABCA1, ABCG1 expression, cholesterol efflux rates were detected.ResultsCompared with control group, monocyte/macrophage cholesterol efflux in experimental group was impaired.Oil Red O stain showed that the accumulation of intracellular lipid increased.The efflux of intracellular cholesterol decreased.But the expressions of ABCA1 and ABCG1 were not obvious.Compared with normal serum, high-cholesterol serum in the test group and control group significantly affected cholesterol efflux capacity.The accumulation of intracellular lipid increased.The efflux of intracellular cholesterol decreased.But the expressions of ABCA1 and ABCG1 were not obvious.ConclusionMacrophage cholesterol efflux in patients with coronary heart disease is impaired, which may be related to atherosclerosis-related vascular diseases.

Abstract:AimTo explore whether testosterone plays a role in regulation of lectin-like oxidized low-density lipoprotein receptor 1(LOX-1) in atherosclerotic male rabbits.MethodsThe model of castrated and atherosclerotic male rabbits was prepared.Pathological sections of thoracic aorta were performed hematoxylin-eosin (HE) staining.Total plasma testosterone was measured using ADVIA Centaur Immunoassay (Bayer, Germany) with chemical luminescent method.Serum oxidized low-density lipoprotein (ox-LDL) was assayed using ELISA kit.The expression of LOX-1 of thoracic aorta was measured by methods of RT-PCR, immunohistochemistry.ResultsSerum testosterone significantly decreased in castrated rabbits, whereas testosterone replacement restored the circulating testosterone to normal levels.Intimal thickness and ratio of intima and media obviously increased in castrated rabbits compared with sham-operation rabbits.The serum ox-LDL wasn’t significantly different between each animal.The expression of LOX-1 of thoracic aorta in mRNA and protein was significantly increased in castrated rabbits compared with those in sham-operation rabbits, and decreased in the castrated rabbits following testosterone replacement.ConclusionsTestosterone has no effect on serum ox-LDL in atherosclerotic rabbits, but plays key roles in the mRNA and protein expression of LOX-1 of thoracic aorta.Testosterone possibly regulates the development of athesclerosis via mediating LOX-1 but not ox-LDL.

Abstract:AimTo construct a lentivirus vector expressing microRNA (miRNA) Rno-miR-145 and probe function of the vector in platlet derived growth factor (PDGF) induced vascular smooth muscle cells(VSMC) phenotype transform.MethodsThe miR-145 shDNA double chain template sequence was synthesized artificially and put this template sequence clone LV3 pGLV/H1/GFP+Puro-miRNA Lentivirus plasmid.293T cells were transfected.Lentivirus particles(virosome) were harvested and concentrated, then primary cultured VSMC of the rats were infected.Fluorescence expression infected with VSMC was observed by inverted fluorescence microscope.miR-145 expression condition was detected with real-time PCR.Blank control group,PDGF group, PDGF＋miR-145 group and miR-NC group were divided in this test.Influence of miR-145 on related genes c-Jun,PCNA,SM22α expression level was observed with real-time PCR.ResultsmicroRNA-145 lentivirus plasmid was construted successfully.The viral titer was 1×109TU/mL.microRNA-145 lentivirus expression plasmid was infected successfully.The best transfection efficiency was on the 3th day when multiply infection(MOI) was 50.Real-time PCR results revealed PDGF increased PCNA,c-Jun expression level,but reduced SM22α expression; miR-145 made VSMC related genes PCNA,c-Jun expression reduce,but made SM22α expression increase.ConclusionMicroRNA-145 lentivirus plasmid may infect rat VSMC efficiently.VSMC phenotype transformation may be inhibited by microRNA-145.

Abstract:AimTo study the effect of dalteparin on the renal artery atherosclerotic lesion formation in the apolipoprotein E knockout (Apo E-/-) mice.Methods36 male Apo E-/- mice，6 weeks，fed with high-fat diet,were randomized into three groups: control group,dalteparin low dose group［100　IU/（kg·d)］, dalteparin high dose group［200 IU/（kg·d）］.24 male C57BL/6J mice，6 weeks，were randomized into two control groups:nomal diet group and high-fat diet group.At 12 weeks Apo E-/- mice were received dalteparin treatment for 4 weeks,then six mice of each group were killed.Light microscopy was adopted to assess the degree of the atherosclerotic plaque of renal artery wall and image analysis was performed with computer.The expressions of lectin-like oxidized LDL receptor-1(LOX-1) in aortic wall were studied by immunohistochemistry and Western Blot.The expressions of LOX-1 mRNA and vascular endothelial growth factor（VEGF）mRNA were studied by RT-PCR.The rest mice were fed with the same diet as before for another 4 weeks,then were killed.Light microscopy was adopted to assess the degree of atherosclerotic plaque of renal artery.Results

Abstract:AimTo study the role and principle of taurine in inhibiting atherosclerosis induced by hyperlipemia and hyperhomocysteinemia in rabbit.Methods33 healthy male New Zealand big-eared white rabbits were divided into 3 groups: normal, model and intervention group.Blood was drawn for laboratory test at 0, 2nd, 12th week respectively.The rabbits were executed and abdominal aortas were taken out for pathology dyeing at 12th week. ResultsHomocysteine (Hcy), high density lipoprotein cholesterol (HDLC) of intervention group had no significant difference in comparison with model group (P>0.05).However, triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDLC), oxidized low density lipoprotein (ox-LDL) lowering had significant difference (P<0.05).Atheromatous plaques area, endothelium thickness and vascular cell adhesion molecule-1 (VCAM-1) positive cells percentage of intervention group were obviously reduced in comparison with model group (P<0.05).ConclusionTaurine, as endogenous cell protective substance, may inhibit atherosclerosis through suppressing endothelial hyperplasia, vascular smooth muscle cell (VSMC) proliferation and migration, reducing hyperlipemia and suppressing oxidation stress rather than lowering hyperhomocysteinemia.

Abstract:AimTo explore the effect and mechanism of pioglitazone on vascular calcification (VC) in diabetic rats.MethodsRat model of vascular calcification and diabetes (DM) was established by injection of vitamin D3 plus warfarin and streptozocin(STZ).Thirty six ten-week-old male SD rats were randomely divided into six groups: control group, DM group, VC group, VC+DM group, VC+PIO group, VC+DM+PIO group.Calcification was confirmed by Von Kossa staining, and calcium content and alkaline phosphatase (ALP) activity of the vascular tissue were measured.The expression of osteoprotegerin (OPG) mRNA and protein were examined by qRT-PCR and immunohistochemistry.ResultsMassive black granules were observed in the vascular smooth muscle cells and interstitial tissue by Von Kossa staining in VC group and VC+DM group.Calcium content and ALP activity of VC+DM group were 3.63 times and 1.35 times higher than DM group, and 3.69 times and 1.30 times（p<0.05）higher than VC group, but the expression of OPG mRNA and protein were reduced（p<0.05）.Calcium content and ALP activity of VC+DM+PIO group decreased 13.70% and 18.04% than VC+DM group（p<0.05）, but the expression of OPG mRNA and protein increased（p<0.05）.ConclusionPioglitazone can reduce the degree of vascular calcification and increase the expression of OPG mRNA and protein.OPG is likely to be one of the main factors of inhibiting vascular calcification.

Abstract:AimTo investigate pro-apoptosis effects of xeroderma pigmentosum D(XPD) gene on human umbilical vein endothelia cells (HUVEC).MethodsRecombinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transient transfected into HUVEC by liposome 2000 method，with the same genetic background and algebra HUVEC as blank controls.The experiments were divided into three groups: control group, pEGFP-N2 group and pEGFP-N2/XPD group.The expression of green fluorescent protein was observed through fluorescence microscopy; the cell apoptosis rate was examined by flow cytometry; through RT-PCR and Western Blot, the expression levels of XPD,Bcl-2 and Bax and wt-p53 were detected ；the cell growth was detected by MTT.ResultsGreen fluorescences were observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating that the plasmids were transfected successfully.Flow cytometry results showed that overexpression of XPD increased the apoptosic rate of HUVEC (p<0.05 or p<0.01).RT-PCR results and Western Blot results showed that the transfection of pEGFP-N2/XPD increased the expression of XPD,Bax and wt-p53 (p<0.05), decreased the expression of Bcl-2 (p<0.05)；MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth(p<0.05).ConclusionsXPD gene can promote HUVEC apoptosis.Therefore, down-regulating the expression of XPD gene is likely to be potential molecular target for treatment of atherosclerosis.

Abstract:AimUsing neuronal nitric oxide synthase(nNOS) knockout mice and nNOS inhibitor to investigate the effect of nNOS during myocardial ischemic preconditioning.MethodsMice were divided into wild-type ischemia-reperfusion group (WT IR), wild-type ischemic preconditioning group (WT IP), wild-type L-VNIO treatment group (WT IP+L-VNIO), nNOS-/- mice ischemia-reperfusion group (KO IR), nNOS-/- mice ischemic preconditioning group (KO IP).They were subjected to 30 minutes of ischemia by left descending branch of coronary artery ligation followed 3 hours reperfusion.IP was induced by 3 cycles of 5 minutes ischemia and reperfusion before 30 minutes ischemia.After 3 hours reperfusion, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and activities of caspase-8, caspase-9, caspase-3, Bax, Bcl-2 and Fas expression were measured.ResultsIn WT group, compared with IR group, TUNEL positive cells, caspase-8, caspase-9, caspase-3 activities and Bax and Fas protein expression were significantly decreased, Bcl-2 expression was significantly increased (p<0.05).In KO group, compared with IR group, TUNEL positive cells, caspase-8, caspase-9, caspase-3 activities and Bax and Fas protein expression were significantly increased, Bcl-2 expression was significantly decreased (p<0.05).ConclusionnNOS was involved in myocardial ischemic preconditioning by reducing myocardial apoptosis.

Abstract:AimTo study the distribution of plasma HDL subclasses in stroke patients and the relationship between the levels of the blood pressure and the contents of plasma HDL subclasses in stroke patients.MethodsThe contents of HDL subclasses in 79 healthy controls and 98 stroke subjects were determined by two-dimensional gel electrophoresis associated with immunodetection method.ResultsIn contrast to healthy controls, the contents of preβ₁-HDL and HDL3c increased significantly and that of HDL2a and HDL2b were decreased significantly in the cerebral infarction patients (p<0.05).The levels of preβ₁-HDL and HDL3c were significantly higher and HDL2b level was significantly lower (p<0.001) in cerebral hemorrhage subjects.With the levels of systolic pressure increaing, the contents of preβ₁-HDL, HDL3c and HDL3a increased and that of HDL2b decreased.When the pressure was greater than 160 mmHg, the contents of preβ₁-HDL and HDL3c were significantly higher than healthy controls (p<0.05), and the contents of HDL2b were significantly lower than healthy controls (p<0.05).Systolic pressure was positively correlated with preβ₁-HDL and HDL3c(r=0.235 and 0.250 respectively) and negatively correlated with HDL2b (r=-0.228 respectively).ConclusionsThe particle size of HDL in stroke subjects showed a shift toward smaller size in stroke subjects, and the tendency was related with the increase of systolic pressure levels.

Abstract:AimTo determine whether rosuvastatin lowers blood pressure (BP) in hypertension with hyperlipidemia patients. Methods52 cases of hypertension with hyperlipidemia patients were divided into treatment group (using rosuvastation 10 mg/L ＋ candesartan 8 mg/d) and control group (only using candesartan 8 mg/d).Observed for six months, the efficacy of blood pressure control was compared. ResultsSystolic blood pressure (SBP) fell by an average of 10.2 mmHg in the treatment group, SBP fell by an average of 7.5 mmHg in the control group (p<0.05).Pulse pressure (PP) fell by an average of 6 mmHg in the treatment group, PP fell by an average of 3 mmHg in the control group (p<0.05). Diastolic blood pressure (DBP) fell by an average of 4.9 mmHg in the treatment group, DBP fell by an average of 4.2 mmHg in the control group (p>0.05).ConclusionsRosuvastation＋candesartan had a significant effect on lowering SBP and PP in hypertension with hyperlipidemia patients campared with only using candesartan, but had no effect on DBP.

Abstract:AimTo investigate a potential association of the gene polymorphisms of endodermis nitricoxide synthase(eNOS) and N5, N10 methylene tetrahydrofolate reductase (MTHFR) gene with premature coronary artery disease (PCAD).MethodsThe polymorphisms of gene eNOS and MTHFR were measured by polymerase chain reaction and restriction fragment length polymorphism analysis(PCR-RFLP) in 131 patients with PCAD (PCAD group)and 131 non-coronary heart disease patients (control group), who were matched in age and sex.ResultsThe frequencies of eNOS G894T gene polymorphism (χ²=2.072, p=0.355) and its T allele (χ²=0.727, p=0.394) in PCAD group were not significantly different from those in control group.The frequencies of MTHFR C677T gene polymorphism T allele were significantly higher in PCAD group than those in control group(χ²=16.339, p=0.000).The frequencies of the three genotypes (CC, CT and TT) were significantly different between the two groups(χ²=14.290, p=0.001).Logistic regression analysis indicated that MTHFR 677C/T TT genotype was an independent risk factor in PCAD(p<0.05).ConclusionThe eNOS G894T polymorphism may have no association with the pathogenesis of PCAD in Han patients of Swnan region; the TT genotype of the MTHFR C677T gene may increase the risk of PCAD.The genetic polymorphism of MTHFR gene promoter is associated with the susceptibility to PCAD in Suwan Han nationality.

Abstract:AimTo observe the effect of pioglitazone on serum retinol binding protein 4 (RBP4) and omentin 1 in type 2 diabetes mellitus patients.Methods80 cases with type 2 diabetes mellitus were randomly divided into two groups: placebo group and pioglitazone group, 40 cases in each group, for a period of 3 months.Levels of fasting blood glucose, insulin resistance index (HOMA-IR), serum RBP4 and omentin 1 before and after treatment were compared in the two groups.ResultsCompared with placebo group, fasting blood glucose and HOMA-IR in pioglitazone group were obviously decreased after treatment (p<0.05), the level of serum RBP4 was decreased (p<0.01), but omentin 1 was increased (p<0.01).Pearson correlation analysis showed that RBP4 was positively correlated with FPG, BMI and HOMA-IR, but negatively correlated with LDL (p<0.05).Omentin 1 was positively correlated with LDL, but negatively correlated with FPG, BMI and HOMA-IR (p<0.05).ConclusionPioglitazone can improve insulin resistance through the regulation of serum RBP4 and omentin 1 level.

Abstract:Lipoprotein(a) consists of low density lipoprotein (LDL) and the unique constituent apolipoprotein(a).Elevated plasma lipoprotein(a) level is an independent risk factor of atherosclerotic disorders and cardiovascular disease.Lipoprotein(a) not only can participate in the formation of atheromatous plaque but also affect the anti-inflammatory mechanism and the balance between procoagulant and anticoagulant agents of the blood vessel wall.Plasma lipoprotein(a) levels which are under strong genetic control can be quite different between individuals.Lipoprotein(a) plasma levels are relatively resistant to many pharmacologic and nonpharmacologic agents, effective and safe approaches to lower Lipoprotein(a) plasma level in clinic are still lacking.However, in recent years, researchers have found a few methods to lower lipoprotein(a) level in laboratory, such as antisense oligonucleotides and synthetic peptides, but further researches are needed when use them as new therapic methods.This paper reviewed the recent research advances in relationship between lipoprotein(a) and atherosclerosis.

Abstract:Toll like receptors (TLR),one of pathogen-associated molecular pattern recognition receptors in innate immune system,play an important role not only in the development of atherosclerosis,but also in the formation of vulnerable plaque.This review will discuss advances of the relationship between Toll-like receptors and atherosclerotic vulnerable plaque from four aspects.