Abstract:Aim To acquire oligonucleotide aptamers of foam cells derived from macrophages and laying theoretical basis for targeted therapy of atherosclerosis. Methods THP-1 cell was treated with 80 mg/L oxidized low density lipoprotein(ox-LDL) for 72 hours to establish macrophage derived foam cell model.Aptamers acquired from a ssDNA library by complex system evolution of ligands by exponential enrichment(SELEX).Fluorescence microscopy was used to detect the binding specificity of ssDNA library and foam cells.After cloning and sequencing,the primary sequences and second structure of the aptamers were analyzed. Results THP-1 macrophage derived foam cell model was identified successfully by red oil O staining and HPLC.After 18 rounds selection,the ssDNA library did not bind to THP-1 macrophages and vascular smooth cells but bind to foam cells.All aptamers had no conserved motifs,and could be divided into 12 families.The main secondary structures of these aptamers were stem-loops which could be vital in the interaction between aptamers and foam cells. Conclusions We obtained the aptamers of THP-1 macrophage derived foam cells successfully by complex SELEX.

Abstract:Aim To establish an efficient and stable method for isolation,culture and directional differentiation of endothelial progenitor cells from mouse bone marrow. Methods Mononuclear cells were isolated from mouse bone marrow by density gradient centrifugation and differential adhesion method.The remaining cells were cultured and differentiated to endothelial progenitor cells in EBM-2.The expressions of specific antigens(CD34,CD133,Flk-1 and CD31) on cell surface were analyzed by flow cytometer. Results Cells were obtained from mouse bone marrow by density gradient centrifugation and differential adhesion method formed clusters at day 4.At day 12,positive ratios of CD34+,CD133+,Flk-1+ and CD31+ were 65%±4%,48%±3%,37%±3% and 51%±4%,respectively. Conclusion An efficient,stable and replicable method for isolation and culture of endothelial progenitor cells from mouse bone marrow has been established.

Abstract:Aim To approach the effects of mast cell degranulation on plaque stabilization in apolipoprotein E-knock out(ApoE-/-) mice with perivascular common carotid collar placement. Methods 40 male ApoE-/-mice were fed a western-type diet and operated with perivascular right common carotid collar placement.4 weeks after surgery,mice were divided into 2 groups and treated for 7 days as follows(n=20): experimental mice were intraperitoneally injected with mast cell degranulator-Compound 48/80,0.5 mg/kg;control mice were intraperitoneally injected with an equal volume of dissolvent(D-Hank's).Thirty minutes after the 4th injection,animals were sacrificed to obtain blood and carotid.Serum was collected to quantify the activity of tryptase by colorimetric assay.Sections were routinely stained with hematoxylin and eosin.Corresponding sections on separate slides were stained with toluidine blue to detect mast cell degranulation and immunohistochemically with antibodies against a von Willebrand factor(vWF),interleukin-1β(IL-1β) and VE-cadherin. Results There were more foam cells,pericellular lipids and inflammatory cells in plaque in com mon carotids with collarplacement of experimental group.The percentage of degranulated mast cells(P<0.01),the activity of tryptase in serum(P<0.05),the density of neovessel in plaque(P<0.05),intraplaque hemorrhage(P<0.05),the expressions of IL-1β(P<0.05) and VE-cadherin(P<0.05) in plaque were significantly increased in experimental group than in control group. Conclusions Mast cell degranulation increases foam cells,pericellular lipids and inflammatory cells in plaque,and promotes angiogenesis in plaque,intraplaque hemorrhage,the expressions of IL-1β and VE-cadherin in plaque,accordingly weakens plaque stabilization.

Abstract:Aim To investigate the effect of Uncaria alkaloids on protecting rat vascular endothelial cells and inhibiting cell senescence. Methods Aging model of rat aortic endothelial cells(REAC) was established by D-galactose.Treated with Uncaria alkaloids and Captopril,separately,then,the aging vascular endothelial cell morphology were observated by scanning electron microscopy,vascular endothelial cell senescence rate was determinated by β-galactosidase staining method,and telomerase activity was determinated by PCR-ELISA method. Results REAC cell morphology treated with Uncaria alkaloids or captopril was significantly improved,β-galactosidase expression and telomerase activity were significantly decreased(P<0.05).The effects of each dose group of Uncaria alkaloids were: medium dose> low dose> high dose,suggesting that high dose of Uncaria alkaloids(> 400 mg/L) may have a certain degree of cytotoxicity,and the best dose of Uncaria alkaloids dose was 200 mg/L. Conclusion Uncaria alkaloids can inhibit rat aortic endothelial cell senescence,and protect vascular endothelium.

Abstract:Aim To investigate the protective effect of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside(TSG) on human umbilical vein endothelial cells(HUVEC) apoptosis induced by H_2O_2 and further explore whether this effect is related to the NF-κB signaling pathway and the expression of apoptosis-related protein. Methods HUVEC were divided into three groups: control group,H_2O_2 group and TSG group.The proliferation and apoptosis rates of HUVEC were detected by MTT and flow cytometry respectively.The protein expression of NF-κB p65,IκB and bcl-2 were assessed by Western blot. Results According to the MTT and flow cytometry,the proliferation rate of HUVEC decreased significantly compared with control group(P<0.01),while the apoptosis rate increased obviously(P<0.01) after treating with H_2O_2.The proliferation rate of TSG pretreatment group increased significantly(P<0.01) while the apoptosis rate decreased obviously(P<0.01) compared with H_2O_2 group.Compared with H_2O_2 group,Western blot analysis revealed that the expression of NF-κB p65 was declined and the level of IκB and bcl-2 were increased significantly after pretreated by TSG(P<0.01). Conclusions TSG has a protective effect on the HUVEC apoptosis induced by H_2O_2 and this molecular mechanism may be associated with its inhibiting the activation of NF-κB/IκB signaling pathway and the up-regulating expression of bcl-2 protein.

Abstract:Aim To investigate the expression of platelet derived growth factor induced nuclear receptor Nur77 mechanism and relationship with proliferation of vascular smooth muscle cell. Methods Vascular smooth muscle cells were isolated and cultured. The expression of nuclear receptor Nur77,extracellular regulated protein kinases,proliferating cell nuclear antigen induced by platelet derived growth factor were checked after incubation with PD98059,atorvastatin,Nur77 siRNA respectively. Results Platelet derived growth factor induced the expression of nuclear receptor Nur77 in vascular smooth muscle cell through ERK-MAPK signal pathway. Atorvastatin affected the expression of nuclear receptor Nur77 induced by platelet derived growth factor. Vascular smooth muscle cell proliferation was attenuated in nuclear receptor Nur77-deficient cells. Conclusion Nuclear receptor Nur77 regulated vascular smooth muscle cell proliferation induced by platelet derived growth factor. Atorvastatin decreased vascular smooth muscle cell proliferation by affecting the expression of nuclear receptor Nur77. This may be a new mechanism by which statins affected nuclear factor kappa B activity by regulating the nuclear receptor Nur77 expression. Down-regulation of nuclear receptor Nur77 suggests a novel therapy strategy for atherogenesis based on suppression of vascular smooth muscle cell proliferation.

Abstract:Aim To investigate the effect of advanced glycation end products(AGE) on the activation of nuclear factor-κB(NF-κB) in rat retinal microvascular endothelial cells(RMEC) of diabetic retinopathy and the protection of simvastatin. Methods Culturing RMEC in vitro and preparing advanced glycation end products-AGE-BSA.Cells were incubated with AGE-BSA,then NF-κB activation was detected by fluorescence microscope.Tube formation and the expression of monocyte chemotactic protein-1(MCP-1) in microvascular endothelial cells in the presence of simvastatin were studied. Results NF-κB was mainly in cytoplasm without AGE-BSA.After RMEC stimulated by AGE-BSA,NF-κB translocated into nucleus,and reached to the peak in 30 min.AGE-BSA could induce the expression of MCP-1 and tube formation.But obvious inhibitory effects were obtained after treatment with the use of simvastatin. Conclusions AGE is crucial in diabetic retinopathy in which the activation NF-κB is important.Simvastatin significantly reduced the AGE-BSA-induced MCP-1 expression and tube formation.

Abstract:Aim The proliferation of endothelial cells plays an important role in angiogenesis.In present study,we observed the effect of syndecan-4 on human umbilical vein endothelial cells(HUVECs) proliferation as well as its possible mechanism. Methods HUVECs were isolated and cultured.The cells were infected with Ad-syndecan-4 or Ad-null as control.Immunostaining was performed to investigate syndecan-4 expression and clusterization.Western blotting was used to assess phosphorylation of downstream kinase Akt and endothelial nitric oxide synthase(eNOS).Proliferation ability of HUVECs was assessed by p-H3,bromodeoxyuridine(5-BrdU) and 5-ethynyl-2′-deoxyuridine(EdU). Results Compared with the Ad-null group,increased cell proliferation rate was observed in the Ad-syndecan-4 group.Syndecan-4 overexpression induced the clusterization of syndecan-4 themselves.The levels of Akt and eNOS phosphorylation were also increased in HUVECs with syndecan-4 overexpression. Conclusion Overexpressed syndecan-4 induced the self-clusterization of syndecan-4 and the activation of downstream Akt and eNOS,which may contribute to increased proliferation of HUVECs.

Abstract:Aim To observe the effect of probucol on the atherosclerosis of experimental diabetic rats,and study its related mechanism. Methods Fourty rats were randomly divided into two groups,normal control group and the model group.Streptozotocin(STZ)was injected intraperitoneally to establish experimental diabetic rat models.Twenty-two rats became diabetes successfully and were randomly divided into two groups: diabetes group,probucol treatment group.After 8 weeks,all rats were executed,and the level of blood glucose,serum concentration of HDL,LDL,TC,TG,SOD,MDA,8-iso-PGF2α,platelet endothelial cell adhesion molecule-1(PECAM-1) were tested in each group.The structure of aorta and the thickness of intima was observed by the light microscope,and the expression of PECAM-1in aorta were assayed by immunohistochemistry. Results Compared with the normal control group,the blood glucose,body weight and water consumption of the model group increased significantly(P<0.01).The level of TC,TG and LDL were significantly increased(P<0.01),however HDL decreased.The thickness of intima in the light microscope was in creased(P<0.01),the structure of aorta revealed that intima was added thick and plaque formation mildly.Compared with the diabetes control group,the level of TC and LDL in probucol treatment group decreased significantly(P<0.01),but serum HDL and TG did not change significantly(P>0.05).The thickness of intima in the light microscope decrea sed significantly(P<0.01).Compared with the normal control group,the level of MDA,8-iso-PGF2α and PECAM-1 in diabetes group were significantly increased(P<0.01),but the level of SOD was much lower(P<0.01).Compared with the diabetes group,the level of MDA,8-iso-PGF2α and PECAM-1 in probucol treatment group decreased significantly(P<0.01),and the level of SOD increased significantly(P<0.01).The expression of PECAM-1 in probucol treatment group decreased significantly(P<0.01). Conclusions Probucol treatment can improve the atheromatosis of aorta in experimental diabetic rats.Its protection may be related to anti-oxidant and suppressiont of PECAM-1 expression in diabetic rats.

Abstract:Aim To investigate the effect of angiotensinⅡ(AngⅡ),angiotensin-(1-7)(Ang(1-7)) at different concentrations on the expression of peroxisome proliferator activated receptorγ(PPARγ) in cultured human THP-1 macrophages. Methods Monocytic THP-1 cells were cultured with 100 nmol/L phorbol myristate acetate(PMA) for 48 hours to lead cells into THP-1 macrophage.Handle the cells in different conditions for 24 hours: medium added nothing,different concentration of Ang-(1-7)(0.1,1,10 μmol/L),different concentration of AngⅡ(0.1,1,5,10 μmol/L).mRNA and protein expression of PPARγ in THP-1 macrophages was measured by RT-PCR and Western Blotting respectively. Results Compared with control group,mRNA and protein expression of PPARγ were increased in Ang(1-7) groups,and they both expressed more as concentration of Ang(1-7) increasing(0.1,1,10 μmol/L).mRNA and protein expressions of PPARγ were decreased in AngⅡgroups compared with control group,and the mRNA and protein both expressed less as concentration of AngⅡ increasing(0.1,1,5,10 μmol/L)(P<0.05). Conclusion Ang(1-7) can increase PPARγ expression in THP-1 macrophages in dose-dependent manners.AngⅡcan decrease PPARγ expression in dose-dependent manners.

Abstract:Aim To investigate the effect of telmisartan on rabbit apoptotic cardiomyocytes underwent ischemia/reperfusion(I/R) injury. Methods 48 Healthy male New Zealand white rabbits were randomly divided into six groups(n=8): sham operation group,I/R model group,GW9662 group,telmisartan group,telmisartan+GW9662 group,candesartan group.After intragastric administration for 2 weeks,the left anterior descending(LAD) coronary artery was occluded for 60 minutes followed by 360 minutes reperfusion to induce ischemia/reperfuion injury.The concentration of angiotensinⅡ in myocardium was analyzed by radioimmunoassay and the intracellular free calcium concentration was measured by dual wavelength fluorophotometry.Then,protein expression of peroxisome proliferator activated receptor-γ(PPARγ) was detected by Western blot.In addition,apoptosis was detected by transmission electron microscope and by TUNEL staining. Results Compared with sham operation group,the concentration of angiotensinⅡin myocardium was significantly increased in I/R model group,GW9662 group,telmisartan group,telmisartan+GW9662 group,and candesartan group(P<0.01).And,the expression of PPARγ was higher in telmisartan group than that in other groups(P<0.01).Interestingly,telmisartan,telmisartan+GW9662 and candesartan inhibited the morphological chan ges of apoptotic cardiomyocytes,which was induced by myocardial ischemia/reperfusion,by condensing chromatin clumps against the nuclear envelope and presentation of apoptotic body.When compared with I/R model group,the intracellular free calcium concentration and apoptosis index were significantly reduced in telmisartan group,telmisartan+GW9662 group and candesartan group(P<0.01).Among them,apoptosis index was lowest in telmisartan group(P<0.01). Conclusion Telmisartan could reduce myocardial apoptosis by blocking the angiotensinⅡreceptor and up-regulating the expression of PPARγ in the rabbit I/R model.

Abstract:Aim To evaluate the Changes among inflammatory factor diabetic retinopathy and plasma hyperhomocyssteinemia. Methods 150 type 2 diabetic patients were enrolled,and assigned to three groups: no diabetic retinopathy(NDR),background diabetic retinopathy(BDR) and proliferative diabetic retinopathy(PDR).All patients' inflammatory factors and plasma homocyssteine were measured.All patients'blood rheology analysis were measured. Results The plasma homocyssteine of BDR and PDR were significantly higher than NDR,the plasma homocyssteine of PDR was significantly higher than BDR.Whole blood viscosity and fibrinogen,plasma viscosity,ESR in group BDR and PDR were significantly higher than those in group NDR. Whole blood viscosity and fibrinogen,plasma viscosity,ESR in group PDR were significantly higher than those in group BDR. Conclusions Hyperhomocyssteinemia and hyperviscosity can cause diabetic retinopathy,hyperhomocyssteinemia is related with the severities' order of diabetic retinophthy.

Abstract:Aim To make retrospective study of the clinical value of OPCABG in 696 cases of high-risk patients with coronary heart disease. Methods 696 cases were divided into OPCABG and CABG groups.The preoperative data,mortality,the average EuroSCORE and the complication rate(including perioperative myocardial infarction,stroke,new atrial fibrillation,acute renal failure,respiratory insufficiency,etc.) were compared between two groups. Results According to surgical procedure,696 cases of high risk patients were divided into OPCABG groups and CABG groups.OPCABG group had 504 cases,EuroSCORE score 7.71 ± 1.27;CABG group had 192 patients EuroSCORE score 7.64 ± 1.33. The average distal anastomosis was 3.46±1.42.The complication rate was 27.9% and operative mortality was 4.31%.ICU time was 65.4±6.73 hours,tracheal intubation time 16.4±6.9 hours and average EuroSCORE 7.68±1.30.There was no statistical difference among the preoperative data of age,comorbidity,gender,heart function and EuroSCORE score between the two groups.The number of distal anastomosis,operation time,blood transfusion,drainage,ventilation time,intensive care,hospitalization time,operative mortality and complication rates(bleeding,acute renal Failure and respiratory insufficiency) between the OPCABG group and CABG group had statistically significant difference.Conclusions Avoiding cardiopulmonary bypass,OPCABG reduces the surgical risks in high riskpatients and the effect of treatment is satifactory.

Abstract:Aim To investigate the levels of soluble low density lipoprotein receptor(sLR11),myeloid-related protein-8/14(MRP8/14) and eosinophil cationic protein(ECP) in coronary heart disease and their relationships with coronary artery disease. Methods The levels of serum sLR11,MRP8/14 and ECP were measured by enzyme-linked immunosorbent assay in 71 patients with acute coronary syndrome(ACS),51 patients with stable angina pectoris(SAP),and 53 controls.All subjects were underwent coronary angiography.The numbers of impaired coronary arteries,Gensini scoring of coronary stenosis and the levels of serum sLR11,MRP8/14 and ECP were compared respectively. Results The levels of sLR11,MRP8/14 and ECP in ACS group and SAP group were significantly higher than those in the controls(P<0.01),the levels of sLR11,MRP8/14 in ACS group were significantly higher than those in SAP group(30.50±10.48 ng/L vs 22.13±6.33 ng/L and 40.30±12.59 ng/L vs 29.12±10.27 ng/L,P<0.05).The ECP levels of ACS group were slightly higher than SAP group,but there were no significant difference(29.47±7.16 μg/L vs 23.73±5.67 μg/L,P>0.05).The serum levels of sLR11 and ECP were significantly higher in three impaired coronary arteries than two vessels and one vessel impaired group(P<0.01),but there were no significantly difference between 2-vessels and 1-vessel impaired group(P>0.05).There were obvious positive correlations in the levels of serum sLR11,ECP with Gensini score(P<0.01),however,there were no correlations in the levels of serum MRP8/14 with them(P>0.05). Conclusions There were correlations in the levels of serum sLR11,ECP levels with impaired coronary arteries and coronary stenosis,ECP played a key role in plaque growth,predicting atherosclerosis burden;MRP8/14 may suggest atherosclerosis plaque instability,predictability of acute coronary events.The serum levels of sLR11,MRP8/14 and ECP were significantly increased in coronary heart disease patients,improving the classification performance of cardiovascular risk factors,which were expected to become the new circulating biomarkers of coronary atherosclerosis.

Abstract:Aim To investigate the relationship between the thromboxane A2 receptor(TXA2R) gene polymorphism(rs768963) and platelet aggregation in Han people in Shanghai. Methods TXA2R gene types,tested by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP),as well as platelet aggregation of 59 cerebral infarction patients and 40 health controls were detected. Results Triglyceride(TG) and blood pressure of cerebral infraction group were significantly higher when high density lipoprotein cholesterol(HDLC) was lower than that of health control(P<0.05).Mutant of TXA2R(CT and CC) improves the platelet aggregation(1') in health control group and platelet aggregation(1' and max) in cerebral infarction group,compared with wild type(TT).Studied as a whole group,mutant of TXA2R also had more significant impact on the platelet aggregation(1' and max) than wild type. Conclusion Compared with wild type,mutant of TXA2R(rs768963) improves the platelet aggregation,which may increase the incidence of thrombosis.

Abstract:Aim To investigate the risk factors of contrast-induced Nephropathy(CIN) in the percutaneous coronary intervention(PCI). Methods To collect the clinical data of 172 patients who underwent the PCI from January,2008 to December,2009,compare contrast agents dose,recorded the creatinine(Cre) before and after treatment,and analyse the risk factors for occurrence of CIN. Results There were 32 cases occurred CIN,including 21 cases(13%) with GFR >60 mL/min,and 19 cases(40%) with GFR<60 mL/min(P<0.001).The incidence rate was 18.61 %(32/172).The following variables may be the significant factors correlating with CIN: aged≥75 years,three teams of coronary lesions,LVEF<40%,Cre<133 μmol/L and contrast agents dose>300 mL(P<0.001).The incidence of CIN in those patients who had renal dysfunction combining with diabetes mellitus or hypertension was higher than those without renal dysfunction(P<0.01). Conclusion renal damage and the degree of it are the most significant risk factors of CIN development after PCI.Renal insufficiency,aged≥75 years,multivessel coronary artery disease,LVEF<40%,and contrast agents dose>300 mL,are all independent risk factors of CIN.Patients with diabetes or hypertension and pre-existing renal insufficiency have a greater risk of CIN.

Abstract:Aim To investigate the clinical significance of the serum ferritin,plasma homcystein and oxidized low density lipoprotein(ox-LDL) levels in patients with coronary heart diseases. Methods 124 cases were divided into coronary heart disease group(n=64) and control group(n=60) according to the result of coronary angiographic examination.The concentration of the serum ferritin,plasma homcystein,oxidized low density lipoprotein levels and plasma lipids were measured. Results The levels of the serum ferritin,plasma homcystein,oxidized low density lipoprotein levels were significantly higher in patients with CHD than that in control group(P<0.05).In addition,the levels of the serum ferritin,plasma homcystein,oxidized low density lipoprotein levels were positively correlated to the degree of coronary stenosis(P<0.05). Conclusion The levels of the serum ferritin,plasma homcystein,oxidized low density lipoprotein levels are closely correlated with the coronary atherosclerosis and its severity,synchronous determination of the three blood parameters may have some clinical importance in evaluating the severity of coronary artery stenosis.

Abstract:Aim To observe the change of serum Apelin,PON1 and malondialdehyde(MDA) in arteriosclerosis with type II diabetes patients. Method Carotid artery and lower extremity artery ultrasonic doppler were performed on patients with type II diabetes from Jun 2008 to Jun.2010.40 non-atherosclerosis cases were divided into non-atherosclerosis group,while 160 cases with atherosclerosis were divided into atherosclerosis group.They were further divided into four groups according to their grade of atherosclerosis,42 cases of grade Ⅰ,39 of grade Ⅱ,41 of grade Ⅲ,and 38 of grade Ⅳ.40 health cases during the same period were chosen as control group.The level of serum Apelin,PON1 and MDA in each group were compared. Result Grade I group had no obvious change with control group in serum Apelin,PON1 and MDA(P>0.05);Apelin and PON1 in gade Ⅱ,grade Ⅲ and grade Ⅳ group were significantly lower than those in control group,and MDA was higher than control group(P<0.01);Apelin,PON1 and MDA in gade Ⅱ,grade Ⅲ and grade Ⅳ group had significant difference(P<0.05). Conclusion The decrease of Apelin and PON1 is the warning factor of atherosclerosis in type 2 diabetes patients.

Abstract:Oxidative stress is involved in vascular remodeling and development of pulmonary hypertension(PH) through multiple pathways such as promoting pulmonary arterial vascular smooth muscle cells proliferation,enhancing endothelial dysfunction and accelerating extracellular matrix deposition.Recent studies have indicated that antioxidant therapy can effectively inhibit pulmonary vascular remodeling and the progress of PH,which provides further evidence that oxidative stress plays an important role in pulmonary vascular remodeling as well as in PH.It is the basis of antioxidant therapy and the research direction for PH to clarify the redox signaling pathway under pathological conditions and to seek specific drugs for antioxidant therapy.