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To study the changes of cardiac function following treatment with granulocyte colony stimulating factor (G-CSF) in patients with heart failure after myocardial infarction.Thirty-eight patients with heart failure after myocardial infarction were randomized into G-CSF treatment group and control group. All the patients received conventional treatment (medication and interventional therapy), and the patients in treatment group were given additional G-CSF (600 µg/day) for 7 consecutive days. The plasma level of brain-type natriuretic peptide (BNP) and the number of endothelial progenitor cells (EPCs) in the peripheral blood were detected before and at 7 days and 4 months after the treatment. The cardiac functions (LVSD, EDV, and LVEF) were evaluated by ultrasonic imaging before and at 2 weeks and 4 months after the treatment.The number of EPCs was significantly higher in the treatment group than in the control group after the treatment especially at 7 days (P0.01). In both groups, BNP level was lowered significantly after the treatment to recover the normal level (P0.01). The cardiac functions were improved in all the patients at 7 days and 4 months after the treatment, and the improvement was more obvious in the treatment group (P0.05), especially in terms of LVEF at 4 months after the treatment (P0.01).EPC mobilization by G-CSF can effectively improve the cardiac functions and lessen ventricular remodeling in patients with heart failure after myocardial infarction.

To investigate the influence of lyophilization on the biological activity of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1α (Ad-HIF-1α-564/402/803).Ad-HIF-1α-564/402/803 was amplified from HEK293A cells and purified by ultracentrifugation in CsCl gradient solutions. The infection efficiency was observed by X-gal staining. The lyophilized adenovirus was prepared under appropriate conditions. Before and after lyophilization, the effect of Ad-HIF-1α-564/402/803 on hMVEC proliferation was evaluated by MTS assay. The recombinant adenovirus was confirmed by PCR and DNA sequence analysis before and 1 day, 6 months and 12 months after lyophilization, and hMVECs infected with Ad-HIF-1α-564/402/803 at these time points were examined for HIF-1α protein expression using Western blotting.No significant changes were observed in the effect of lyophilized Ad-HIF-1α-564/402/803 on hMVECs proliferation at the optimal multiplicity of infection of 100 pfu/cell (P0.05). At the 4 time points, the recombinant adenovirus HIF-1α showed no structural alterations or significant changes in the expression level of HIF-1α protein in the transfected hMVECs (P0.05).Lyophilized Ad-HIF-1α-564/402/803 can maintain its biological activities for a long time.

To study the relationship between atrial structural remodeling and the expressions of matrix metalloproteinase (MMPs) and their tissue inhibitors (TIMPs) in atrial fibrillation (AF).Biopsy samples of the right atrial appendages were collected from 20 patients with sinus rhythm and 30 with AF undergoing heart valve replacement surgery for rheumatic heart diseases. All the patients received echocardiographic examination preoperatively. MMP-1, -3, -7, -9 and TIMP-1, -2, -3, -4 protein expressions were detected by immunohistochemistry and RT-PCR.Compared with those in patients with sinus rhythm, the AF patients had significantly increased left and right atrial diameters and mRNA levels of MMP-3, -7, -9 and TIMP-1, -2, -3, -4 (P0.01). MMP-1 expression also showed an increase in AF patients, but the difference was no statistically significant from that in patients with sinus rhythm.The expressions of MMP-1, -3, -7, -9 and TIMP-1, -2, -3, -4 increase in fibrillating atrial tissue, which may contribute to atrial structural remodeling and atrial dilatation in AF patients.

To investigate the effect of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1alpha (Ad-HIF-1alpha-564/402/803) in modulating angiogenesis in vitro.The recombinant adenoviruses Ad-lacZ, Ad-Null, Ad-HIF-1alpha-nature, and Ad-HIF-1alpha-564/402/803 were amplified in HEK293A cells and purified by ultracentrifugation in CsCl step gradient solutions, and the adenoviral titer was determined by end-point dilution assay. The recombinant adenovirus was confirmed by PCR and DNA sequence analysis, and the infection efficiency was observed by X-gal staining. Human microvascular endothelial cells (HMVECs) were infected with Ad- HIF-1alpha-564/402/803, Ad- HIF-1alpha-nature, or Ad-Null to compare the number of capillary-like tube structures in vitro. The effect of Ad- HIF-1alpha-564/402/803, Ad-HIF-1alpha-nature, and Ad-Null on angiogenesis was evaluated using a chick embryo chorioallantoic membrane (CAM) model.PCR and gene sequencing suggested the correct construction of the recombinant adenovirus HIF-1alpha, and the adenoviral titer reached 1011-1012 PFU/ml. Infection of the hMVECs with Ad-HIF-1alpha-564/402/803 at the optimal multiplicity of infection of 100 pfu/cell resulted in a significantly greater number of capillary-like tube structures than infection by Ad-HIF-1alpha-nature and Ad-Null (P=0.000). Ad-HIF-1alpha-564/402/803 group showed significantly higher microvessel density than Ad-HIF-1alpha-nature, Ad-Null, and PBS groups, with also higher angiogenesis area to CAM area ratio (P=0.01, 0.000, and 0.000, respectively).The triple mutant Ad-HIF-1alpha-564/402/803 can obviously promote the formation of capillary-like tube structures in vitro and modulate angiogenesis in the CAM model, suggesting the capacity of Ad-HIF-1alpha-564/402/803 in promoting angiogenesis under normoxic condition.

To investigate the changes of endothelial function indices and their relation to platelet activation in patients with idiopathic atrial fibrillation (AF).We studied 61 patients with idiopathic AF with 34 age- and sex-matched healthy persons as control. Platelet counts in the blood were tested, and plasma levels of NOx were analyzed using Griess method. The plasma levels of von Willibrand factor (vWF) and soluble P-selectin (sP-selectin) were determined using enzyme-linked immunosorbent assay.Compared to healthy control, the patients with idiopathic AF had significantly lower levels of plasma NOx (18.2-/+7.3 vs 24.3-/+7.8 micromol/L, P=0.049) and higher levels of plasma sP-selectin (25.6-/+6.2 vs 22.4-/+4.8 ng/ml, P=0.007). No significant differences were found in the platelet counts or plasma vWF levels between the two groups. A significant inverse correlation was found between plasma NOx and sP-selectin (r=-0.405, P=0.025) in patients with idiopathic AF.AF per se may impair the endothelial function and activate platelet function, suggesting the role of endothelial dysfunction in activated platelet function in AF patients.

To investigate the effect of recombinant adenovirus-mediated hypoxia-inducible factor-1alpha (Ad-HIF-1alpha) at different doses on angiogenesis in a rabbit model of hind limb ischemia.Left hind limb ischemia was induced in 45 Zealand white rabbits by ligation of the left femoral artery. The rabbits were randomly divided into 5 groups (n=9) to receive intramuscular injections of 0.5 ml saline, 2x10(10) PFU empty vector (Ad-null), or different doses of Ad-HIF-1alpha (2x10(9), 2x10(10) or 2x10(11) PFU) immediately after the operation. On the 7th day after the operation, real-time PCR was used to detect the expression of HIF-1alpha mRNA in the skeletal muscles. Immediately and on the 14th and 28th days after the operation, contrast enhanced ultrasound (CEU) was used to observe the blood perfusion of the hind limb. On the 28th day postoperatively, immunohistochemistry for CD31 was performed to evaluate the microvascular density (MVD).Real-time PCR showed that Ad-HIF-1alpha significantly increased the expression of HIF-1alpha mRNA (P0.01) in a dose-dependent manner as compared with that in the saline and Ad-null groups (P0.01). CEU revealed greater blood perfusion in the hind limb of rabbits in association with increased dose of Ad-HIF-1alpha (P0.05 or P0.01); similar changes in the MVD was observed following Ad-HIF-1alpha injections as shown by immunohistochemistry (P0.05 or P0.01). No significant differences were found either in the blood perfusion or MVD between saline and Ad-null groups (P0.05).Ad-HIF-1alpha can dose-dependently promote the angiogenesis in the ischemic limb of rabbits.

To explore the effect of erythropoietin (EPO) on reperfusion arrhythmias in rats and identify the possible mechanism involved.Forty-five SD rats were randomized into a sham-operated group and 4 cardiac ischemia/reperfusion (IR) injury groups, which were further divided into IR group, LY294002 group, EPO group, and EPO+LY294002 group. Cardiac IR injury was induced in the 4 IR injury groups by ligating the left anterior descending branch of the coronary artery (LAD) for 30 min followed by reperfusion for 3 h, with subsequent treatments accordingly. The occurrence of arrhythmias was monitored and scored during experiment, and the levels of serum CK-MB and cTnI were detected. The content of MDA in the myocardium was determined by thiobarbituric acid (TBA) method, and the content of SOD by xanthine oxidase method.The arrhythmia score in EPO group was significantly lower than those in IR, LY294002 and EPO+ LY294002 groups (P0.05). The levels of serum CK-MB and cTnI were significantly lower in EPO group than in the other 3 IR groups (P0.001). The EPO group showed also significantly lower MDA content (P0.001) and higher SOD content than the other 3 IR groups (P0.001).EPO at the dose of 1000 mg/kg decreases the incidence of reperfusion arrhythmias in rats, and this effect can be attenuated by LY294002 pretreatment, suggesting that the cardioprotective effect of EPO involves antioxidation mediated by the phosphoinositide 3-kinase (PI3K) pathway.

To study the effects of oxygen and calcium on the expression of eukaryotic vectors harboring wild-type or mutated hypoxia-inducible factor-1alpha (HIF-11alpha) in HEK293 cells.HEK293 cells were transiently transfected with pcDNA3.1+/HIF-11alpha, pcDNA3.1+/HIF-11alpha-564Ala and pcDNA3.1+/HIF-11alpha-564Ala-803Ala via lipofectin. Western blotting were used to detect HIF-11alpha protein after normoxic or hypoxic exposure of the transfected HEK293 cells in the presence or absence of Ca(2+). The levels of vascular endothelial growth factor (VEGF) mRNA in the transfected cells in normoxic condition was detected using RT-PCR.The levels of HIF-11alpha protein and VEGF mRNA increased in HEK293 cells transfected with the vectors harboring mutated HIF-11alpha, but not in the cells transfected with wild-type HIF-11alpha vectors in normoxia. Hypoxia increased the levels of HIF-11alpha protein in the cells transfected with wild-type HIF-11alpha vectors, which was inhibited by the application of Ca(2+). Ca(2+) showed no inhibitory effect on HIF-11alpha levels in HEK293 cells transfected with the vectors containing mutated HIF-11alpha.The protein products of pcDNA3.1+/HIF-11alpha-564Ala and pcDNA3.1+/HIF-11alpha- 564Ala-803Ala in HEK293 cells enhance the cell tolerance to oxygen and protease.

To investigate the changes in plasma matrix metalloproteinases-2 and -9 (MMP2 and MMP9, respectively) levels in patients with different types of coronary heart diseases (CHD), and assess the value of MMP2/MMP9 detection in predicting acute coronary syndrome (ACS).According to the findings by coronary angiography and the clinical manifestations, 118 patients were divided in ACS group including 30 patients with unstable angina pectoris (UAP) and 19 with acute myocardial infarction (AMI) and non-ACS group including 23 patients with stable angina pectoris (SAP) and 21 with chronic total occlusion (CTO) of the coronary artery. Twenty-five individuals with normal coronary artery (NCA) served as the control group. Plasma levels of MMP9 and MMP2 were determined in these subjects using enzyme-linked immunosorbent assay (ELISA).Both the ACS and non-ACS groups showed significantly higher MMP9 and MMP2 levels than the NCA group (P0.05), and MMP2 and MMP9 levels were significantly higher in ACS group than in non-ACS group (P0.05). Compared with the NCA group, the UAP, AMI and CTO subgroups showed obvious increases in plasma MMP2 and MMP9 levels (P0.01). Significantly increased MMP9, but not MMP2 level was noted in AMI subgroup in comparison with SAP (P0.01) and UAP subgroups (P0.05); both MMP2 and MMP9 levels were elevated in CTO subgroup in comparison with those in SAP (P0.001), UAP (P0.01), and AMI subgroups (P0.05).Increased MMP2 and MMP9 levels in patients with CHD suggest the instability of the atherosclerotic plaque in correlation to the severity of ACS, and may serve as good indicators for the prediction of ACS and diagnosis of CTO of the coronary artery.

To evaluate the effect of a phospholipid-coated microbubble contrast agent for myocardium opacification in comparison with a albumin-coated microbubble contrast agent (Quanfuxian).In 10 dogs with single coronary artery stenosis involving the anterior descending branch or circumflex branch randomly received infusion of the two contrast agents through the femoral vein. The myocardial blood flow, heart rate and blood pressure were analyzed qualitatively and quantitatively. The concentration and the particle diameter of the two contrast agents were determined.The concentration of the phospholipid-coated microbubbles was (1.06-/+0.22) x10(9)/ml, with a diameter of 3.04-/+0.34 microm, similar to the concentration and diameter of Quanfuxian ((1.31-/+0.33)x10(9)/ml and 2.88-/+0.58 microm, respectively, P0.05). Both of the agents achieved grade three myocardium opacification, and produced no obvious effect on the heart rate and blood pressure. Quantitative analysis of myocardial opacification in terms of myocardial blood volume (A), blood velocity (beta), and blood flow (A x beta) revealed no significant difference between the two agents (P0.05), and the parameters derived from the two agents showed good correlations (P0.05, rA=0.809, r beta=0.932, rA.beta=0.925).The phospholipid-coated microbubble contrast agent shows good effect for myocardial opacification without significant difference from Quanfuxian. Both of the agents are good ultrasound contrast agents for quantitative analysis of myocardium blood flow.

To investigate the mechanism by which recombinant adenovirus (Ad)-mediated mutations of hypoxia inducible factor 1alpha (Ad-HIF-1alpha-Ala564-Ala803) regulates cell apoptosis.LoVo cells were infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 and control virus Ad-lacZ under normoxia condition. Real-time PCR was used to detect HIF-1alpha and p21WAF1/CIP1 mRNA expressions at different time points. Western blotting was employed to verify HIF-1alpha and p21WAF1/CIP1 protein expression. Hoechst 33342 flourescein staining was performed to observe the ratio of apoptotic LoVo cells.The expression levels of HIF-1alpha mRNA and protein increased after infection with Ad-HIF-1alpha- Ala564-Ala803, accompanied by an increase in p21WAF1/CIP1 mRNA and protein expressions. The apoptotic ratio was significantly higher in LoVo cells infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 (16.2%) than in the control cells (5.5%, P=0.00).HIF-1alpha may induce cell cycle arrest by up-regulating p21WAF1/CIP1 expressions to promote LoVo cell apoptosis.

To construct an adenovirus vector containing the double-mutant hypoxia-inducible factor-1alpha (HIF-1alpha) gene for exploring the therapeutic angiogenesis for coronary heart disease.Human double-mutant HIF-1alpha cDNA was obtained from PCR of pShuttle-2-HIF-1alpha containing the mutant HIF-1alpha gene (564). The recombinant adenoviral plasmid containing mutant HIF-1alpha cDNA was constructed by ligation of recombinant pShuttle2 containing double-mutant HIF-1alpha cDNA and Adeno-X viral DNA, followed by its identification and transfection into adenoviral packaging cells HEK293 via lipofectamine 2000.The recombinant pAdeno-HIF-1alpha was correctly constructed and verified by restriction endonuclease and DNA sequencing analyseis. This recombinant adenovirus containing the double-mutant HIF-1alpha gene may facilitate further investigation of mutant HIF-1alphagene therapy for coronary heart disease.

To observe the effects of ATP-binding cassette A1 (ABCA1) on intercellular cell adhesion molecule type 1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1) and interleukin-1beta (IL-1beta) in THP-1 macrophages stimulated with 8-Br-cAMP to identify a possible new mechanism that ABCA1 contributes to atherosclerogenesis (AS).Monocytic THP-1 cells were cultured in the presence of 100 nmol/L phorbol myristate acetate (PMA) for 72 h to transform the cells into THP-1 macrophages. After the macrophages were stimulated with 8-Br-cAMP (final concentration 0.5 mmol/L) for 3, 6, 12 and 24 h respectively, the amounts of ABCA1, ICAM-1 and MCP-1 mRNA were examined by real-time fluorescent quantitative RT-PCR, and the protein amounts of ABCA1, ICAM-1, MCP-1 and IL-1beta were determined by Western blotting and enzyme-linked immunosorbent assay (ELISA). Phosphorothioate antisense oligonucleotides of ABCA1 were add into the culture media at a final concentration of 100 nmol/L and the experiments were repeated.ABCA1, ICAM-1 and MCP-1 mRNA and protein and IL-1beta protein were increased in the macrophages after stimulation with 8-Br-cAMP for 6 and 12 h. The mRNA expressions of ABCA1, ICAM-1 and MCP-1 were decreased significantly at 3 and 6 h (P0.01), and the protein expressions of ABCA1, ICAM-1, MCP-1 and IL-1beta declined significantly at 12 and 24 h (P0.01) after transfection of the macrophages with antisense oligonucleotides of ABCA1.ABCA1 can increase the expressions of the inflammatory cytokines in THP-1 macrophages stimulated by 8-Br-cAMP and plays a role in the pathogenesis of AS.