Abstract:AimTo investigate effects of advanced glycation end products (AGE) modified albumin (AGE-albumin) on functions of late endothelial progenitor cell (EPC) derived from human umbilical cord blood and its mechanisms.MethodsMononuclear cells from human umbilical cord blood were cultured by using EGM-2-MV (Clonetics).Expression of CD45, CD146, and CD105 were detected by fluorescence-activated cell sorter analysis (FACS), vWF was detected by immunocytochemistry.Uptake of acetylated low density lipoprotein (ac-LDL) and binding to Ulex European Agglatinin (UEA-1) were used for fluorescent labeling of EPC.Late EPC were incubated with different concentrations of AGE-albumin which were similar to the diabetic serum for 24 h.MTT was used for EPC proliferation assay.To measure the apoptosis, Annexin V＋/PI－ cells were detected by FACS analysis.Boyden chamber assay was used to detect the migration by vascular endothelial growth factor (VEGF).Tube formation on ECMatrix-gel was performed to assess the capacity for vasculogenesis.The mRNA and protein expression of AGE receptor (RAGE) were evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting, respectively.ResultsThere were two types of cells appeared in the culture system.After 3 to 5 d, attached cells appeared and appeared to be clusters.Their number increased for 2 weeks.Thereafter, they did not replicate in vitro and gradually disappeared.We observed another population of cells with different morphology and growth pattern.These appeared in 10 to 15 d after plating, with

Abstract:AimTo investigate the effect of non-specific matrix metalloproteinases (MMP) inhibitor, doxycycline(DOX), on hypertensive aortic remodeling.Methods80 male stroke-prone spontaneously hypertensive rats（SHR-SP） of 7 weeks age were randomly divided into 2 groups (DOX and control group, respectively, n=40 each).Five male Wistar-Kyoto rats (WKY) with same age were also investigated.SHR-SP were chronically treated from the end of 7 weeks after birth with doxycycline (30 mg/(kg·d)) in drinking water.Follow-up was terminated when the mortality rate of control group reached 50%.Rats were killed, and aorta was dissected and prepared for histological and biochemical assays.For pathological analysis, we used hematoxylin and eosin (HE) staining, Victoria blue (elastic fiber staining) and von Kossa silver (calcium staining). For biochemical analysis, soluble collagen assay and gelatin zymography were carried out.ResultsThere were no difference in intima perimeter, adventitia perimeter and media area in hypertensive rats. MMP-9 activity by gelatin zymography was not detected in all animals.Compared with control group, MMP-2 activity in dox group were significantly reduced ( 2.43±0.14 vs 3.15±0.23, P<0.05).Compared with control group, the ratio of elastic fiber area to intima area was dramatically decreased in DOX group ［(3.46%±1.62% vs 5.72%±3.87%, P<0.05)］. Sircol soluble collagen assay showed that there had no statistical difference between control and DOX groups (26.4±7.0 mg/g vs 28.0±8.7 mg/g, P>0.05).Although calcium deposition as revealed by von Kossa silver staining was no statistical difference between control and DOX groups, there was decreasing trend in DOX group (0.688%±0.718% vs 0.960%±0.999%, P=0.177).ConclusionDespite the fact that doxycycline could inhibit aortic MMP-2 activity in SHR-SP, in the setting of uncontrolled hypertension, doxycycline may aggravate the process of hypertensive aortic stiffening.

Abstract:AimTo investigate the effects of 7-difluoromethyl-5,4’-dimethoxygenistein (dFMGEN) on the adhesion of monocytes to H2O2-induced endothelial cells, and its molecular mechanism.MethodsThe effects of dFMGEN on the adhesion rate of monocytes and endothelial cells were examined by measuring protein content.The expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) in H2O2-induced endothelial cells were measured by enzyme-linked immunosorbant assay (ELISA).The effects of dFMGEN on the expression of nuclear factor-κB (NF-κB) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) were determined using Western Blotting.ResultsWhen endothelial cells were treated with 1.0 mmol/L H2O2 for 24 h, the adhesion rate was added, the release of E-selectin and ICAM-1 were increased, and the expression of NF-κB and p-ERK1/2 were up-regulated. After pretreating with dFMGEN, results showed that dFMGEN can inhibit the adhesion between monocytes and endothelial cells induced by H2O2, reduce the release of E-selectin and ICAM-1, and down-regulate the expression of NF-κB and p-ERK1/2.ConclusiondFMGEN effectually inhibits the adhesion between monocytes and endothelial cells induced by H2O2, which is probably due to the inhibition of the expression of E-selectin and ICAM-1 and downregulation of NF-κB and p-ERK1/2.

Abstract:AimTo investigate the potential of mouse fibroblast cell-derived embryonic-like induced pluripotent stem cells (iPS cells) to differentiate into endothelial cells.MethodsO9 iPS cells were induced to differentiate into endothelial cells by monolayer culture, FACS sorting Flk1+ cells, followed by OP9 stromal cell co-culture, and purified by FACS sorting VE-cadherin+ cells(O9-EC).The endothelial specific protein expression were examined by immunofluorescence.The functions of angiogenesis in vitro, uptaking DiI-Ac-LDL and binding UEA1 were also tested.Gene expression during differentiation process were tested by real-time PCR.ResultsMouse O9 iPS cells derived Flk1+cells could differentiate into endothelial-like cells on OP9 stromal cells.VE-cadherin could be used as a marker to purify O9-EC.O9-EC could express CD31, VE-cadherin and vWF, uptake DiI-Ac-LDL, bind UEA1 and form tube-like structures on matrigel.The endothelial specific genes such as CD31, Tie2 and eNOS were up-regulated in the endothelial differentiation process.Four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that induce the generation of iPS cells were significantly down-regulated in this process.ConclusionThis study supports that iPS cells can differentiate into endothelial cells in vitro.This procedure may contribute to clinic application of patient-specific iPS cells derived endothelial cells in vascular degeneration diseases.

Abstract:AimTo investigate the effects of advanced glycation end products(AGE) and homocystein（Hcy）on the expression of vascular cell adhesion molecule-1(VCAM-1) mRNA via oxidative stress mechanism in human umbilical vein endothelial cell（HUVEC）.MethodsCollagenase was used to isolate the endothelial cell from human umbilical vein.RT-PCR was used to examine the expression of VCAM-1 mRNA.Inversion fluoescence microscope was used to observe the level of oxygen free radical.Results The expression of VCAM-1 mRNA was induced concentration-dependently by AGE(10－4～10－1 g/L) and Hcy(1.35×10－3～1.35 g/L); AGE combined with Hcy group could promote the expression of VCAM-1 mRNA respectively 7.78 times and 6.05 times in comparison with AGE group and Hcy group（1.09±0.18 vs 0.14±0.07；1.09±0.18 vs 0.18±0.06， P<0.01）; By comparison with AGE combined with Hcy group, the VCAM-1 expression of DPI group was obviously reduced(0.20±0.09 vs 1.19±0.23,P<0.01); In comparison with control group, AGE group and Hcy group could enhance oxygen free radical in HUVEC; The level of oxygen free radical in AGE combined with Hcy group was higher than that in AGE group and Hcy group; DPI could significantly inhibit the level of oxygen free radical.ConclusionAGE and Hcy can enhance the level of oxygen free radical in HUVEC and increase the VCAM-1 expression, and they have the cooperative effect; The enhancement of oxygen free radical is an important factor on the expression of VCAM-1 mRNA; The above process is realized possibly through activating the NADPH oxidase.

Abstract:AimTo explore whether SIRT1 interacts with liver X receptor-α, the level of liver X receptor-α acetylation and the relationship between liver X receptor-α acetylation and intracellular cholesterol content during THP-1 macrophages foam-cell formation.MethodsTHP-1 macrophages were used to build foam-cell model, the changes of intracellular cholesterol content was detected by high performance liquid chromatography, the level of liver X receptor-α acetylation, the level of SIRT1 expression, and the interaction between liver X receptor-α and SIRT1 were detected by western blot and co-immunoprecipitation respectively.Results After oxidized low-density lipoprotein treated for 12 h, 24 h, 48 h, the cellular cholesterol content of THP-1 macrophages cell was increased gradually, SIRT1 interact with liver X receptor-α, liver X receptor-α acetylation decreased, whereas SIRT1 expression increased.ConclusionDuring THP-1 macrophages foam-cell formation, decreased level of acetylated liver X receptor-α was related to up-regulated expression of SIRT1.

Abstract:AimTo investigate the influence of benazepril alone and in combination with fluvastatin on adiponectin (APN) in adriamycin (ADR)-induced nephrotic rat models.MethodsMale Sprague-Dawley (SD) rats were randomly seperated into five groups and given different therapies. Normal control group (n=18).Nephrotic model was induced by tail introvenously injection of ADR (6.0 mg/kg).Eighty-four ADR-induced nephrotic male SD rats were randomly seperated into 4 groups: without treatment group (normal saline 3 mL/d; n=21), benazepril treatment group (benazepril 3.5 mg/(kg·d); n=21), fluvastatin treatment group (fluvastatin 10 mg/(kg·d); n=21) and combined treatment group (benazepril 3.5 mg/(kg·d) plus fluvastatin 10 mg/(kg·d), n=21).After the end of therapies for 2, 6 and 10 weeks, the samples of 24 h urine, serum were collected and assayed (six rats were assigned randomly in every group).ResultsCompared with normal control group, 24 h urinary protein excretion, the serium levels of total cholesterol, triglyceride and APN were increased significantly in the other four groups (P<0.01).Treatment with either benazepril or fluvastatin or combined with benazepril and fluvastatin could reduce 24 h urinary protein excretion, reduce the serium levels of total cholesterol, triglyceride and APN (P<0.05 or P<0.01), especially for combined treatment group.Proteinuria, total cholesterol, triglyceride were correlated with APN (P<0.01).ConclusionsBenazepril and fluvastatin can decrease proteinuria, reducse serium triglyceride, total cholesterol and APN in ADR-induced nephrotic rats.Combination of benazepril and fluvastatin has superiority over monotherapies on renal protection.These results suggest that benazepril and fluvastatin may mediated through, at least partly, decreasing serum APN and attenuating renal damage.

Abstract:AimTo explore the effect of stromal cell-derived factor-1α (SDF-1α) on proliferation and vessel-like structure formation in vitro rat bone endothelial progenitor cells ( EPC).MethodsEndothelial progenitor cells derived from rat bone were isolated by method of micropore and characterized by immunofluorescence technique with EPC special markers VEGFR-2/CD133.And the abilities of vessel-like structure formation and cell proliferation were detected by the methods of cell culture, MTT assays after treated with 1, 10, 100 μg/L SDF-1α and CXCR4 antagonist AMD3100.Results The EPC derived from rat bone arrayed into a cobblestone-like structure and formed vessel - like structure， at the same time, EPC differentiated more maturer and expressed endothelial cells special markers vWF. Two groups of SDF-1α （10 and 100 μg/L） significantly promoted EPC proliferation(n=5, P<0.01)， and three groups of SDF-1α （1, 10 and 100 μg/L）significantly promoted EPC vessel-like structure formation with a dose dependent manner (n=5, P<0.01).All these effects were significantly attenuated by AMD3100.ConclusionSDF-1α possibly plays a critical role in regulating proliferation and vasculogenesis of rat bone-devived EPC.

Abstract:AimTo investigate the effects of peroxisome proliferator activated receptor-δ (PPAR-δ) activation by GW0742 on oxidized low density lipoprotein (ox-LDL)induced upregulation of monocyte chemotactic protein-1 (MCP-1),vascular cell adhesion molecule-1 (VCAM-1) and matrix metalloproteinases-9 (MMP-9) in RAW264.7 cells.MethodsCultured RAW264.7 cells were divided into 4 groups: vehicle-treated group,GW0742-treated group, PPAR-δ silencing＋GW0742-treated group and empty vector-transformed group.After stimulated with ox-LDL (50 mg/L) for 24 hours,the mRNA and protein levels of MCP-1, VCAM-1 and MMP-9 were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting, respectively.The monocyte migration activity was tested by micropore filter method using a modified Boyden chamber.ResultsBoth the mRNA and protein levels of MCP-1,VCAM-1 and MMP-9 were significantly decreased in GW0742-treated cells compared with vehicle-treated group (P<0.01 or P<0.05 ).However, PPAR-δ gene silencing by RNA interference markedly attenuated these beneficial effects of GW0742 (P<0.01 or P<0.05).Similarly,the monocyte chemotactic activity was noticeably inhibited in GW0742-treated cells (P<0.05),while this inhibitory effect of GW0742 was completely blocked by PPAR-δ gene silencing.ConclusionsThe present study shows that PPAR-δ activation by GW0742 successfully inhibits the oxidized LDL-induced upregulation of MCP-1,VCAM-1 and MMP-9 in RAW264.7 cells and monocyte migration.The results indicates that activating PPAR-δ might become an effective strategy for the prevention of atherosclerosis.

Abstract:AimTo detect the expression development of proapoptic molecule C/EBP homology protein(CHOP) mRNA and protein, and explore its effect on neuronal apoptosis after cerebral ischemia/reperfusion in rats.MethodsTransient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 2 hours followed by reperfusion in sprague-dawley rats.Then，the expression of CHOP protein and/or mRNA were measured with methods of immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) at 1 h, 3 h, 6 h, 12 h and 24 h after reperfusion in cerebral cortex of rats.The neuronal apoptosis was detected by the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL).ResultsIn cerebral ischemia model group，the expression level of CHOP mRNA reached a peak at 12 h after reperfusion and that of CHOP protein reached a peak at 24 h after reperfusion，which paralleled with the tendency of neuronal apoptosis development.ConclusionCerebral ischemia reperfusion may induce CHOP expression. CHOP may play an important role in neuronal apoptosis induced by cerebral ischemia/reperfusion.

Abstract:AimTo study the recombinant human tumor necrosis factor-α receptorⅡ∶IgG Fc fusion protein （rhTNFR∶Fc）, ischemic postcondition （postcon）, and both combination on the prevention of myocardium ischemia/reperfusion injury.Methods48 SD rats were randomly divided into four groups， all rats were subjected to 1 h of LAD occlusion followed by 6 h of reperfusion.In Control group, no interventions were applied; in rhTNFR∶Fc group, rhTNFR∶Fc solution (2 mL/kg, 1 g/L) was administered intravenouslly at 30 min after ischemia; in postcon group∶ three cycles of 10 s of reperfusion and 10 s of reocclusion was applied immediately at the onset of reperfusion; in combination therapy group∶rhTNFR∶Fc solution (2 mL/kg, 1 g/L) was administered intravenouslly at 30 min after ischemia, and three cycles of 10 s of reperfusion and 10 s of reocclusion was applied immediately at the onset of reperfusion.We measured plasma lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), tumor necrosis factor-α(TNF-α) levels at different time after reperfusion and examined ischemic and infarct area, apoptosis, the activity of Caspase-3, Bcl-2 at risk myocardium at the end of reperfusion.ResultsrhTNFR∶Fc, ischemic postcondition, both combination reduced the plasma LDH, CK-MB, TNF-α levels (at 30 min, 1 h, 3 h, 6 h after reperfusion), infarct area, apoptotic myocytes, Caspase-3 activity, increased Bcl-2 activity when compared with controls(P<0.01). Ischemic postcondition reduced the plasma TNF-α more significantly than rhTNFR∶Fc at 30 min and 1 h after reperfusion (P<0.01).However, at 3 h and 6 h after reperfusion, rhTNFR∶Fc reduced the plasma TNF-α more significantly (P<0.01).ConclusionBoth rhTNFR∶Fc and ischemic postcondition can inhibit TNF-α activity, attenuate myocyte apoptosis, and there is an additive effect when used in combination.

Abstract:AimTo investigate the effects of homocysteine (Hcy) on cholesterol synthesis in THP-1 macrophages and its potential target loci.MethodsThe macrophages were induced by phorbol 12-myristate 13-acetate(PMA)from THP-1 mononuclear cells, and treated by 5 mmol/L Hcy for 0 h,4 h,8 h,18 h and 24 h respectively.The mRNA expression of insulin-induced gene 1 (Insigl) and 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR）were determined by reverse transcription polymerase chain reaction (RT-PCR).Intracellular total cholesterol levels were examined by enzymatic colorimetric assay.ResultsIntracellular total cholesterol level increased significantly compared with the 0 h group at 18 h and 24 h（P<0.01）after Hcy-treatment. The expression of Insigl mRNA demonstrated a time-dependent down-regulation after Hcy-treatment， 8 h, 18 h and 24 h groups had significant lower Insigl mRNA level vs 0h group(P<0.05 and P<0.01).The HMGCR mRNA expression showed obvious upregulation at 18 h and 24 h compared with the 0 h group（P<0.01）.Conclusion A potential mechanism for Hcy-induced lipid accumulation in macrophage and foam cell formation may go through the following way: Hcy down-regulates the expression of Insigl, and sequentially up-regulates the HMGCR expression, which eventually increases the cholesterol synthesis in THP-1 macrophages.

Abstract:AimTo investigate the relationship between dendritic cells and severe atherosclerosis (As) of coronary heart disease patients and explore the possible immune mechanisms of Simvastatin.Methods According to the results of selective coronary angiography, 20 negative control (no obvious atherosclerosis, group A)，20 severe coronary heart disease patients with severe coronary stenosis without any statins (As group, group B) and 20 severe coronary heart disease patients with severe coronary stenosis with simvastatins 40 mg daily for at least 30 days (Simvastatin group, group C).20 mL blood samples were extracted through the angiographic catheter.Density gradient centrifugation was used to separate mononuclear cells (MNC).MNC were cultured in vitro and differentiated to DC in vitro.In B2 group, 100 μmol/L Simvastatin were added on day 5. Flow cytometry was used to detect the immune characteristics of DC.Percent of CD1a+ , CD1a+CD80+, CD1a+CD83+, CD1a+CD86+ cells and the mean fluorescent intensity (MFI) were measured and recorded.ResultsAfter in vitro proliferation, mononuclear cells from coronary artery could differentiate into dendritic cells and the morphology of DC did not differ between groups; Compared with negative controls, the percent of CD1a+, CD1a+CD80+, CD1a+CD83+, CD1a+CD86+ cells, MFI, MNC number, DC number and stimulation index (SI) in group B1 were significantly increased (P<0.05); Compared with group B1, the MNC number in group B2 was

Abstract:AimTo investigate the relationship between －413A/T single nucleotide polymorphism in heme oxygenase-1(HO-1) gene promoter and susceptibility of coronary artery disease.Methods200 patients who were diagnosed as having coronary artery disease by coronary angiography were selected in this study.120 subjects without evidence of coronary artery disease under coronary angiography with their sex and age similar to coronary artery disease patients were selected as controls.Sequencing was carried out on the ABI 3730 DNA analysis platforms to determine the genotypes of －413A/T.ResultsA single nucleotide polymorphism A(－413)T was found in the HO-1 gene promoter. Genotype T/T was protective against coronary artery disease in patients with dyslipimedia (adjusted OR=0.179, 95%CI=0.05～0.642).ConclusionA(－413)T single nucleotide polymorphism may be an important marker to evaluate susceptibility to coronary artery disease in patients with dyslipidemia.

Abstract:AimTo investigate the changes of visfatin and adiponectin(APN) levels in patients with type 2 diabetes mellitus (T2DM), and their relation with intima-media thickness(IMT) of the carotid artery.MethodsAccording to the IMT level, the 73 diabetic patients were divided into two subgroups: IMT thickened group (37 cases) and IMT normal group (36 cases).Plasma visfatin and APN levels were examined.The relationship between visfatin, APN and IMT were investigated.ResultsPlasma visfatin was elevated in T2DM patients as compared with that in normal control group (P<0.01), and more in IMT thickened group than in IMT normal group (P<0.01).Plasma APN was lower in the diabetic group as compared with that in controls(P<0.01). It was also lower in IMT thickened group compared with those in IMT normal group (P<0.01).Multiple stepwise regression analysis revealed that visfatin level was positively related to IMT, visceral fat thickness and fasting insulin.There was positive correlation between adiponectin level and HDLC, but it was negatively correlated with HOMA-IR and waist-hip ratio in these T2DM patients.Visfatin, age and HbAlC were positively correlated with IMT, and APN was negatively correlated with IMT.ConclusionElevated plasma visfatin and descended APN may be one of probable risk factors in the early atherosclerosis in the patients with T2DM.

Abstract:AimTo study the correlation between the cytochrome P450 (CYP) 3A5 gene polymorphism and dyslipidemia.MethodsThe real-time PCR genotyping at CYP3A5*3 (6986 A＞G) position was established using Taqman minor groove binding (MGB) probes.Total 321 dyslipidemia patients and 258 matched controls of Chinese Han population were genotyped at CYP3A5*3 (6986 A＞G) position using this method.Plasma lipid levels were measured by using routine method.ResultsAll three genotypes, AA, GA and GG existed in both dyslipidemia patients and control group.AA, GA, GG genotyped frequencies were 9.0%, 46.1%, 44.9% for patients and 9.7%, 39.5%, 50.8% for control group respectively; A，G allele frequencies were 32.1%, 67.9% for patients and 29.5%, 70.5% for control group respectively, in which the statistic significance was not observed （P>0.05）.But the results demonstrated that the presence of A allele was associated with high density lipoprotein cholesterol (HDLC) in patients group.ConclusionCYP3A5*3 (6986 A＞G) polymorphism was not associated with dyslipidemia in Chinese population, however, this polymorphism has some influence on serum HDLC level.

Abstract:AimTo observe the relationship between vascular lesions in the lower extremities and osteoporosis in elderly male people with type 2 diabetic mellitus.Methods148 elderly male patients with type 2 diabetic mellitus were enrolled.All patients underwent ultrasonography for both sides of artery of lower extremity.They also underwent dual-energy X-ray absorptiometry (DXA) examination to determine the bone mineral density (BMD)of the vertebra and the femoral head.According to a lower extremity vascular disease severity score, the patients were divided into control group, mild-moderate group and severe group.ResultsBMD was significantly different among three groups (P<0.05 or 0.01), the more severe lower extremity vascular disease, the lower the bone mineral density was.Only age, body mass index (BMI) and lower extremity vascular disease severity score correlated to osteoporosis by multiple regression analysis.ConclusionElderly male people with type 2 diabetic mellitus and vascular lesions in the lower extremities tend to have lower BMD, they may have increased prevalence of osteoporosis.

Abstract:AimTo determine the incidence of hypertension and its risk factors among rural men in the countryside of Fuxin, Liaoning province.MethodsA population-based sample of 13 170 men of rural Chinese adults aged ≥35 years and free from hypertension at baseline were followed from 2004 to 2006.In 2008, investigators who had collected contact information on their participants at baseline invited their participants to return to the clinic for follow-up.12 274 individuals attended the follow-up clinic examination.The response rate was 93.2%.Trained and qualified physicians conducted blood pressure measurement on the survey and data collection.Incident hypertension was defined as systolic pressure≥140 mmHg, diastolic pressure ≥90 mmHg, or current use of antihypertensive medication, according to the WHO definition of hypertension.ResultsOver a mean of 28 months of follow-up, 3 639 (29.65%) of men developed hypertension.Among men, independent predictors of incident hypertension were baseline age, Mongolian ethnicity, use of alcohol, high income, prehypertension, overweight and obesity, baseline salt intake, and family history of hypertension.The awareness, treatment and control rates for newly developed hypertension were 29.9%, 19.5% and 1.5% respectively.ConclusionThese data indicate that the incidence of hypertension is high among these rural Chinese adults and it is associated with many risk factors.Most newly developed hypertension cases are not treated.We should actively carry out health promotion, health education, healthy lifestyles, in order to prevent high blood pressure.

Abstract:Vascular endothelial progenitor cell is the precursor cell of endothelial cell.Vascular endothelial progenitor cells can proliferate in the blood circulation，differentiate into endothelial cells，and take part in the formation of human embryonic vasculogenesis and the promotion after birth.It is known that protecting vascular endothelial function is one of the important organism that high density lipoprotein prevent atherosclerosis.It is found that in recent study high density lipoprotein can regulate vascular endothelial progenitor cells,The article reviews the research status in this aspect.