Abstract:Aim To evaluate the efficiency of endovascular stent-based gene delivery system using antibody tethered plasmid DNA. Methods Endovascular stents were formulated with a collagen coating.Anti-DNA antibodies were covalently bound to the collagen surface by a cross linking reagent and then pEGFP-C1(enhanced green fluorescent protein) was immunobound to endovascular stent.Gene transduction efficiency was evaluated in cell culture.Furthermore,inducible nitric oxide synthase(iNOS) was chosen as therapeutic gene and bound to endovascular stent.Stents with antibody-tethered iNOS were implanted in pig coronary artery to evaluate the transduction efficiency and the effect of restenosis prevention. Results Gene delivery from stents carrying antibody-tethered pEGFP-C1 demonstrated efficient and site-specific pEGFP-C1 transduction in cell culture.GFP-positive cells were only observed in the site that directly contact with the collagen matrices and the transduction efficiecny was 21.8% vs less than 5% in control group.In pig coronary artery stent deployment studies,reverse transcription polyerase chain reaction(RT-PCR) analyses showed that iNOS was only observed in the blood vessel that contact with the stent,iNOS was undetectable in distal tissues such as lung,liver and spleen,etc. Conclusion Gene delivery system based on antibody immobilized coronary stents provided localized and highly efficient gene delivery for intravascular site-specific gene therapy.

Abstract:Aim To investigate the effects of in vivo local transfection of zinc finger protein A20 gene on restenosis of balloon injured rat carotid artery and its possible mechanism. Methods Balloon catheter denudation of the endothelium of rat common carotid artery was routinely used as a model of restenosis.104 male Sprague-Dawley rats were randomly divided into 4 groups: the sham group(no injury),the model group(the simple injury),the control group(vacant transfection regent group) and the therapeutic group(A20 gene and transfection regent group).pCAGGS-GFP/A20(20 μg) with 40 μL Lipofectamine 2000 or TE buffered solution(20 μL) with 40 μL Lipofectamine 2000 was instilled into the lumen of the injured segment for 30 min after injury.The transfection efficiency of plasmid in injured vascular wall was evaluated 24 hours after transfection by using fluorescence microscope.Quantification of intimal hyperplasia was determined by pathologic examination.Proliferation index of VSMC in vivo was assessed by thymidine analogue bromodeoxyuridine(BrdU) labeling technique.The expression of nuclear factor-kappaB p65(NF-κBp65) of rat carotid arteries in different groups were confirmed by immunohistochemical staining and Western blot analysis. Results At day 14 significant intimal hyperplasia was detected after arterial injury in the model and control group.A20 gene transfection markedly reduced the neointimal area(47.8% reduction;P<0.05) and intimal to media area ratio(42.9% reduction;P<0.05) in the therapeutic group.Proliferation index of VSMC(BrdU index) at day 10 after operation was decreased significantly in the therapeutic group(9.6%±2.3%) than in the control group(26.7%±5.1%,P<0.05).A significantly lower level of NF-κBp65 positive cells ratio was observed in the therapeutic group than in the control group at 10d after operation(P<0.05).A significantly lower level of NF-κBp65 protein expression was observed in the therapeutic group than in the control group at day 7 d,14 d,28 d after operation(P<0.05).Conclusion Local transfection of A20 gene inhibits intimal hyperplasiaand VSMC proliferation after arterial injury.Its possible molecular mechanism is that A20 negative feedback inhibits NFκ-B sig-naling pathway.This study provides evidence for the inflammatory mechanism of restenosis and suggests that A20 gene therapymay serve as a novel gene therapeutic approach to inhibit restenosis.

Abstract:Aim To investigate the effect of the functional state of nAChRα7 on chemerin and chemerinR gene expression and to initially explore the molecular influencing mechanism of non-cholinergic system to lipolytic hydrolysis in preadipocytes and mature adipocytes. Method 3T3-L1 preadipocytes were differentiated with 1-methyl-3-isobuthylxanthine+Insulin+Dexamethasone+fetal bovine serum+DMEM/F12.The relatively quantitative RT-PCR was used to detect the expression of chemerin and chemerinR at indicated time point including 0 h,6 h,12 h,1 d,3 d,6 d and 9 d during 3T3-LI cells differentiation,and the gene expression change of chemerin and chemerinR in preadipocytes and fully differentiated adipocytes were observed when cells treated by various concentration(10-8 mol/L,10-6 mol/L and 10-4 mol/L) of selective nAChRα7 agonist choline chloride or selective nAChRα7 antagonist methyllycaconitine. Results ①The expression of chemerin and chemerinR mRNA was low but detectable in confluent 3T3-L1 preadipocytes.When hormonally stimulated by IBMX+DEX+Insulin,their expression both increased markedly until day 9.②The results showed that the mRNA level of chemerin in both preadipocytes and adipocytes and that of chemerinR in preadipocytes were significantly increased by methyllycaconitine and down-regulated by choline.However,the above nAChRα7 ligands had no obvious effect on chemerinR expression in mature adipocytes.③The above nAChRα7 ligands have stronger effect on preadipocytes than adipocytes. Conclusion nAChRα7 is probably the important factor that allows non-neuronal cholinergic system play biological effect.nAChRα7 mediated lipolysis,in part by directly activating a nicotinic cholinergic receptor located in adipocyte,in part by regulating other lipolysis-associated adipokines.

Abstract:Aim To observe the effect of probucol on the expression of heme oxygenase-1(HO-1)protein in atherosclerotic lesions and livers in rabbits. Methods The hypercholestrolemic model was built up by 8 weeks high cholesterol diet.Then 16 New Zealand rabbits were randomly fed with starch(n=8,starch group) or probucol(n=8,probucol group) for 6 weeks.Those two groups and another 8 New Zealand rabbits(n=8,control group) were tested for serum lipid,body weight and serum malon dialdehyde(MDA).Expression of HO-1 was detected by immunohistochemistry. Results Probucol treatment increased HO-1 expression in atherosclerotic plaque,aorta smooth muscle cell and liver,decreased atherosclerotic areas,initma thicknesses,and ratio of intimae to media,and down regulated serum total cholesterol(TC),low density lipoprotein cholesterol(LDLC),high density lipoprotein cholesterol(HDLC) and MDA.Expression of HO-1 protein in atherosclerotic plaque,smooth muscle cell and liver in hypercholesterolemic rabbits with and without probucol treatment was negatively correlated with serum levels of MDA,atherosclerotic areas,intimae thickness,and the ratio of intimae to media(P<0.05). Conclusions While lowering serum cholesterol,probucol may also exert anti-oxidative actions through upregulation of HO-1 expressions,which might be an important part of its anti-atherogenic properties.

Abstract:Aim To study the effects of AMD3100 on atherosclerosis and the possible mechanism in apolipoprotein E-/-mice. Methods 12 male apolipoprotein E-/-mice,8 weeks old,were randomly divided into two groups: AMD3100 group(2.5 mg/kg,the next day intraperitoneal injection) and control group(PBS 0.1 mL,the next day intraperitoneal injection).After fed with high fat and cholesterol western food for 12 weeks,all mice tissue specimen and bone marrow cells were harvested.The Hematoxylin/Eosin staining of paraffin section was performed to detect atherosclerotic plaque of aortic root.By counting the typical endothelial progenitor cells-colony forming units(EPC-CFU) and observing the size and cell density of secondary EPC-CFU,the clonality of endothelial progenitor cells was measured.The expression of CXCR4 mRNA and protein were examined by RT-PCR and Western blotting. Results Compared with control group,the area percentage of atherosclerotic lesion and vessel lumina increased 38.8% in AMD3100 treated apolipoprotein E-/-mice(37.2%±3.6% vs 26.8%±2.5%,P<0.05).The clonality of endothelial progenitor cells derived from AMD3100 group decreased in comparison to control group(primary EPC-CFU: 9.67±2.16 vs 21.83±2.64,secondary EPC-CFUs: 1.67±0.31 vs 4.11±0.65;P<0.01).The expression of CXCR4 mRNA and protein of AMD3100 group were also reduced(P<0.01). Conclusions The atherosclerotic lesion was aggravated by administration of AMD3100 in apolipoprotein E-/-mice.Possible mechanism of this action for AMD3100 are associated with the inhibition of the clonality of bone marrow endothelial progenitor cells and down-regulatipon of CXCR4 expression on endothelial progenitor cells.

Abstract:Aim To study the impact of chronic stress on the coronary atherosclerosis and related factors. Method Totally 36 male Wister rats were randomly and averagely divided into three groups,and all groups were fed for 6 weeks: control group were fed with basic food;the rats in hyperlipidemia group were fed with hyperlipidemia diet;the rats in hyperlipidemia stress group were given hyperlipidemia diet,and were exposed under the different stressors at the same times,including limit,circumvolve and huddle.The rats in hyperlipidemia group and hyperlipidemia stress group were injected with a single dose of vitamin D(600,000 IU/kg) at the beginning of the test,and the control group were injected saline with the equal capacity.The serum level of TC,TG,HDL and LDL were determined after 6 weeks,the serum level of TXB2,IL-6,and TNF-α were tested synchronously with radioimmunoassay,and the change of pathology of the coronary artery were observed with HE dyeing method. Results(1)Level of TC,LDL in hyperlipidemia stress group were obviously higher than those in control group(P<0.01);Compared with control group,level of TC、LDL in hyperlipidemia group were higher(P<0.01).(2) Level of TXB2,IL-6 and TNF-α in hyperlipidemia stress group were remarkably higher than those in control group(P<0.01)and those in hyperlipidemia group(P<0.05).(3)TXB2,TNF-α and TC were significantly correlated with chronic mental stress(r were 0.543,0.354 and 0.392,P<0.05).(4) Forepart pathological changes of coronary atherosclerosis were formed in both hyperlipidemia stress group and hyperlipidemia group,but hyperlipidemia stress group group were more obvious. Conclusion Chronic mental stress could promote the development of coronary atherosclerosis,which was involved in the formation and development of coronary atherosclerosis by disturbing lipide metabolize,increasing inflammatory reaction guided by cytokines and elevating platelet activation.

Abstract:Aim To explore the in vitro and in vivo angiogenic capacity and phenotypic properties about different clonal endothelial progenitor cell populations derived from human peripheral circulating blood. Methods Mononuclear cells were isolated by density-gradient centrifugation and incubated onto fibronectin-coated dishes in endothelial medium in the presence of vascular endothelial growth factor.The number of early clones was counted at 7 days and cells from another aliquot were cultivated continually until the late clones generated.Flow cytometry analysis was used to evaluate cells surface antigen expression and von willebrand factor(vWF),the endothelial cells marker was detected by indirect fluorescence staining.The capacities of in vitro and in vivo vascular genesis were assessed by plating cells onto collagen gels and implanting the cellularized gel into nude mice. Results Early clones failed to form second clone and were devoid of the capacity of in vitro and in vivo vascular genesis.Furthermore,cells derived from the early clones expressed mainly CD14 and CD45.In contrast to early clones,the late clones emerged until 21 to 28 days after cultivation and exhibited typically endothelial cells properties.Remarkably,cells originated from late clones had the ability to form second endothelial clones after replanted and generated tube-like structures when seeded onto collagen gels or transplanted into nude mice.In addition to the clearly morphological and functional differences,cells derived from late clones expressed obviously increased CD146(P<0.01) and reduced CD45 and CD14(P<0.001). Conclusions Under endothelial cultivating conditions,human peripheral blood mononuclear cells can generate early and late clones.Only cells originated from late clones exhibit double phenotypes of stem/progenitor and endothelial cells with the capacity of in vitro and in vivo vasculargenesis.

Abstract:Aim To investigate whether five lipoxygenase activating protein(FLAP) is involved in the mechanism of human umbilical vein endothelial cells(hUVEC) oxidation injury induced by oxidized low density lipoprotein(ox-LDL). Methods Isolated and cultured hUVEC were used as experimental model.hUVEC were induced by ox-LDL at 10,50,100 and 200 mg/L for 24 hours.Leukotriene C4(LTC4) levels in the cell supernatant fluid were detected with enzyme-linked immunosorbent assay(ELISA),cell viability was detected by MTT assay,and FLAP mRNA and FLAP protein were detected with fluorescent quantitation PCR and Western blot. Results Ox-LDL at 100 and 200 mg/L significantly induced the release of LTC4(P<0.01),the decrease of cell viability in hUVEC(P<0.01) and upregulation of FLAP mRNA expression and FLAP protein expression.The regressive analysis showed that the amounts of released LTC4 had significantly positive correlation with the concentration of ox-LDL(r=0.953,P<0.01). Conclusion FLAP could be involved in the LTC4 secretion and oxidative injury induced by ox-LDL in hUVEC.

Abstract:Aim To investigate the potential candidate genes related to extracellular matrix(ECM) which might play roles in atherosclerosis by microarray technology. Methods Sixteen male SD rats were randomly subjected to the following treatments: standard diet with saline injection i.p.(control group,n=8) or fat diet with vitamin D3 injection i.p.(containing 3% cholesterol,0.5% sodium cholate,5% refined sugar,10% lard,and 81.5% base feed)(model group,n=8).After 14 w,serum total cholesterol(TC) and triglyeride(TG) were detected.Thoracic aorta was stained with HE and a solution of oil red O to visualize the lesion area.Total RNA was isolated from the aorta for microarray to explore the differential gene expression profiling. Cathepsin D expression was measured by immunofluorescence and western blot. Results Compared with the control group,model group showed elevated serum TC and TG(both P<0.05) and aortic fibrous plaque formation.510 genes were up-regulated and 1 218 ones were down-regulated in aorta tissue of model group by DNA microarray analysis when compared with control group.Further immunohistochemistry and western blot analysis revealed cathepsin D protein expression was significantly increased in aorta of model group whereas normal group showed little or no immuno-detectable cathepsin D expression. Conclusion Cathepsin D might influence vascular remodeling,which plays an important role in As.

Abstract:Aim To investigate the effect of ARL-1 expression on acrolein induced oxidative stress in 293T cells. Methods EGFP/ARL-1 fusion protein was expressed in 293T cells by introducing EGFP/ARL-1 plasmid;the reactive oxygen species(ROS) were probed by CM-H2DCFDA and determined by FACS. Results The 293T show a fluorescence density of 530%±36% when treated with 10μmol/l of acrolein,whereas a fluorescence density of 220%±20% was detected in 293T cells with the expression of EGFP/ARL-1 under the same concentration of acrolein. Conclusion ARL-1 expression can decrease cellular ROS level and reduce acrolein induced oxidative stress.

Abstract:Aim To investigate the effect of cigarette smoking on vascular endothelium and endothelin-1(ET-1) expression in rat brains. Methods The expression of endothelin-1 and endothelin recepter A(ETR-A) were examined in site by means of immunohistochemistry and in quantitative by Westen blotting method in rat brains following smoking.Electronic microscopy was used to observe the ultrastructural changes of endothelial cells. Results It was found that there were prominent ET-1 and ETR-A expressions on the endothelium after smoking;ET-1 and ETR-A expressions in long term-large amount group were significantly increased compared with the other terms and in abstained group was significantly decreased compared with short term group.Meanwhile the damage of ultrastructure of vascular endothelium was most prominent.The apoptosis of neuron could be found most frequently. Conclusions Smoking fume can cause vascular endothelium damage and expression of ET-1 incenment.Abstain from smoking can make above change obviously restore.The damage of vascular endothelium and increment expression of ET-1 play an important role in the course of damage and is one way to cerebral thrombosis.

Abstract:Aim To investigate the relationship between vascular function destroy and microinflammation in the patients with uremia before dialysis. Methods 95 patients were selected randomizedly as test group,and 40 normal individuals as control.Carotid artery intima-media thickness(IMT),cross-sectional calculated intima-media area(IMA),plaques,endothelium-dependent dilation(EDD) and endothelium-independent dilation(EID) of brachial artery were determined with non-invasive high-resolution B-mode ultrasonography.Serum levels of C-reactive protein(CRP) and tumor necrosis factor-α(TNF-α) were also determined. Results Compared with normal control,levels of serum CRP(5.27±1.18 mg/L vs 1.54±0.82 mg/L,P<0.05),TNF-α(2.11±0.34 μg/L vs 0.15±0.03 μg/L,P<0.05)were significantly higher in patients with uremia.Lower levels of serum proalbumin and hemoglobin,and higher systolic and diastolic pressure were found in the patients.However,it has no difference about serum albumin,glucose and lipids between the two groups.There was no difference in IMT,IMA,percentage of plaques,basal inner diameter and blood flow of brachial artery between the two groups.But,endothelium-dependent dilation(EDD),endothelium-independent dilation(EID),blood flow of response hyperemia,blood flow after buccal nitroglycerin of brachial artery were decreased significantly in the patients group.Serum levels of CRP and TNF-α were correlated negatively with EDD and EID(P<0.05). Conclusion These data suggest that the microinflammation plays an important role in the pathogenesis of vascular dysfunction in the pre-dialysis patients with uremia.

Abstract:Aim To investigate the effect of angiotensin system blockers on the levels of serum angiotensin Ⅱ(Ang Ⅱ),interleukin-1(IL-1),C-reaction protein(CRP) and lipid metabolism in patient with chronic glomerular disease. Methods 89 cases of chronic glomerular disease were randomly divided into 3 groups: benazepril group(n=30),valsartan group(n=30) and combination of benazepril and valsartan group(n=29).Serum Ang Ⅱ,IL-1,CRP,quantity of 24 hour urinary protein and lipid were measured before and after 8～12 weeks therapy. Results Serum total cholesterol were effectively decreased in 3 groups after therapy.Combination of benazepril and valsartan therapy reduced triglyceride effectively.Benazepril group and combination of benazepril and valsartan group have more influence on the low density lipoprotein(LDL),apolipoprotein B than valsartan group(P<0.01).Meanwhile the levels of serum Ang Ⅱwas effectively decreased in benazepril group and combination of benazepril and valsartan group(P<0.05),in contrast to valsartan group,the level of serum Ang Ⅱ was increased(P<0.05).The levels of serum IL-1 and CRP were significantly reduced after therapy(P<0.05).Therapeutic alliance group decreased proteinuria more effectively than that of benazepril alone or valsartan alone(P<0.05). Conclusions The combination of benazepril and valsartan may reduce the level of serum Ang Ⅱ,IL-1 and CRP,decrease urine protein excretion and ameliorate lipid metabolism in patients with chronic glomerular disease.

Abstract:Aim To investigate whether volume overload poses its detrimental effect on cardiovascular mortality through endothelial dysfunction. Methods In this cross-sectional study,81 stable patients on continuous ambulatory peritoneal dialysis(CAPD) in a single center were recruited.Volume status was evaluated with extracellular water normalized by individual height(NECW),which was assessed by bioimpedance analysis.Endothelial function was estimated by endothelial-dependent flow-mediated dilatation(FMD) of the brachial artery,which was expressed as the percentage change relative to the baseline diameter. Results There were 37 male and 44 female patients.Their mean age and dialysis duration were 61±12 years and 20±23 months,respectively.The FMD in female patients were significantly higher than that in male patients(9.17%±6.23% vs 6.31%±5.01%,P<0.05).FMD was negatively correlated with weight(r=-0.308,P<0.01),body mass index(r=-0.242,P<0.05),systolic blood pressure(r=-0.228,P<0.05),ECW(r=-0.404,P<0.001) and NECW(r=-0.418,P<0.001).No correlation was found between FMD and other variables.In multiple stepwise regression analysis,calcium x phosphate(Ca x P) product(β=0.422,P<0.001),NECW(β=-0.343,P<0.01) and dialysis duration(β=-0.237,P<0.05) were independent determinants of FMD(adjusted R2=0.327 for this model). Conclusion Higher NECW was related to worse endothelial function.The results in this study may help us to understand the underlying mechanism of volume overload leading to increased cardiovascular morbidity and mortality in dialysis patients.

Abstract:Aim To investigate the surgical therapy for chronic total occlusion of coronary artery. Methods 753 patients with 928 totally occluded coronary arteries(148 coronary arteries lack of opacification while the other 780 arteries with reverse flow) underwent coronary artery bypass grafting(CABG).A total of 2501 grafts were constructed including 155 placed to coronary endarterectomy(CE) targets and 37 arterialized middle cardiac veins.Blood flow was detected during operation in 31 coronary arteries with no opacification in preoperative angiography,while no blood flow was detected in 75 coronary arteries with opacification in preoperative angiography.Cardiopulmonary bypass was applied in 17 cases because of a poor hemodynamics and 8 of which were assisted with IABP.The other 736 cases underwent OPCAB.CVVHDF was performed for 8 cases. Results All patients survived the operation.In the 152 cases of coronary endarterectomy,2 died after operation because of low cardiac output(1 case) and renal failure(1 case).The other 150 cases were followed up for 1 month to 9 years.One died suddenly 2 years after operation.Angina disappeared completely in 132 cases(88.6%) and 14 patients relieved from angina with NYHA function of stage Ⅰ～Ⅱ.For the other 601 patients,5 died in hospital because of low cardiac output(1 case),renal failure(1 case),perioperative cardiac infarction(1 case) and cerebrovascular accident(1 case).Recovery of cardiac function and freedom from cardiac angina was 99%. Conclusions CABG can be well preferred in patients with total occlusion of coronary arteries.The application of IABP and CVVHDF can improve the prognosis of severe case.It is limited to evaluate totally occluded coronary artery only using coronary angiography,and endoscope or intravascular ultrasound techniques may be helpful.

Abstract:Aim To compare the risk factors of small vessel disease(SVD) or its subtypes(lacunar infarction and leukoaraiosis) and carotid atherosclerosis(CAS),deduce the effect of CAS on SVD and provide data for early prevention and cure. Methods Data of age,hypertension,diabetes mellitus,smoking,blood fat,alcohol,fibrinogen(FIB),abdomen circumference and body mass index(BMI) were analysed in the patients with SVD(n=366),CAS(n=266) and controls.Patients with SVD were subdivided into lacunar infarction(LI) and leukoaraiosis(LA). Results A total of 506 patients met the inclusion criteria,among which,174 were lacunar cerebral infarction,192 were leukoaraiosis and 140 were normal.Among the 391 patients who were examined by color Duplex ultrasound,266 were found carotid atherosclerosis.①Multiple logistic regression analysis revealed that age,hypertension,diabetes mellitus,hypercholesterinemia,high low density lipoprotein(LDL) and smoking were more frequent in CAS group compared with the controls(P<0.05).In SVD group,hypertension,diabetes mellitus,hypercholesterinemia were more important.②Age(OR=1.07,95%CI 0.56～1.96,P<0.001),Hypertension(OR=2.28,95% CI 1.88～3.72,P<0.001) were more common in SVD than CAS;but hypercholesterinemia(OR=0.53,95% CI 0.23 ～ 1.22,P<0.001) was more important in CAS,high LDL and smoking were the risk factors of CAS but not SVD.③Age,hypertension were more important in LA than LI;on the contrary,hypercholesterinemia was more common in LI.④The pearson coefficient between SVD and CAS was 0.360,the incidence rate of CAS in LI was more frequent than LA. Conclusion ①There are some differences in risk factor profile between SVD and CAS,the same to LI and LA.②The incidence of CAS is related to SVD to some extent,the relationship of CAS with LI is closer than LA.

Abstract:Aim To investigate the effect of telmisartan on adiponectin in hypertensive patients with type 2 diabetes mellitus. Methods In this prospective study,50 hypertensive patients with type 2 diabetes mellitus were treated either with telmisartan or with amlodipine for 10 weeks.The serum concentration of fasting adiponectin,glucose,insulin and blood pressure(BP) were measured at baseline and after treatment respectively. Results The systolic and diastolic BP decreased significantly after the treatment both in telmisartan group and in amlodipine group.In telmisartan group,serum adiponectin was significantly increased,but blood glucose,insulin and insulin resistance(IR) were significantly decreased(P<0.01);But no notable difference was detected in adiponectin,blood glucose,insulin in amlodipine group(P>0.05). Conclusion These data indicated the potential effect of telmisartan on the improvement of adiponectin and IR,which demonstrate the metabolic effect of telmisartan beyond its BP reduction.