Characterization of the biological activities of lyophilized recombinant adenovirus expressing the triple mutant of hypoxia inducible factor-1α / 南方医科大学学报

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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 (P>0.05). 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 (P>0.05).</p><p><b>CONCLUSION</b>Lyophilized Ad-HIF-1α-564/402/803 can maintain its biological activities for a long time.</p>

Management of subacute myocardial infarction in a patient with left coronary artery originating from the right coronary artery / 南方医科大学学报

Although the majority of coronary artery anomalies are found incidentally and not clinically significant, the interarterial course between the major vessels of the aberrant artery may be responsible for syncope, angina, arrhythmias or sudden death. There are only a few case reports describing the origination of all the coronary arteries from a single ostium. This anomaly occurs in only 0.024%-0.044% of the population. Left coronary artery originating from the right coronary is a rare coronary abnormality. Here we report a case of acute myocardial infarction in a patient with anomalous left coronary artery originating from the right coronary artery, as was confirmed by computerized tomography angiogram, which showed that only one single coronary artery stem originating from the right sinus of Valsalva trifurcated into a right coronary artery, left circumflex artery and a hypoplastic left anterior descending artery. Subsequent percutaneous coronary intervention (PCI) procedures were performed successfully. PCI procedures should be carried out with great caution in such cases, and this condition should be managed as a left main lesion.

Effect of different doses of recombinant adenovirus mediated hypoxia-inducible factor-1alpha on angiogenesis in ischemic lower hind limb of rabbits / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>Real-time PCR showed that Ad-HIF-1alpha significantly increased the expression of HIF-1alpha mRNA (P<0.01) in a dose-dependent manner as compared with that in the saline and Ad-null groups (P<0.01). CEU revealed greater blood perfusion in the hind limb of rabbits in association with increased dose of Ad-HIF-1alpha (P<0.05 or P<0.01); similar changes in the MVD was observed following Ad-HIF-1alpha injections as shown by immunohistochemistry (P<0.05 or P<0.01). No significant differences were found either in the blood perfusion or MVD between saline and Ad-null groups (P>0.05).</p><p><b>CONCLUSION</b>Ad-HIF-1alpha can dose-dependently promote the angiogenesis in the ischemic limb of rabbits.</p>

Effect of erythropoietin on reperfusion arrhythmias in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect of erythropoietin (EPO) on reperfusion arrhythmias in rats and identify the possible mechanism involved.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>The arrhythmia score in EPO group was significantly lower than those in IR, LY294002 and EPO+ LY294002 groups (P<0.05). The levels of serum CK-MB and cTnI were significantly lower in EPO group than in the other 3 IR groups (P<0.001). The EPO group showed also significantly lower MDA content (P<0.001) and higher SOD content than the other 3 IR groups (P<0.001).</p><p><b>CONCLUSION</b>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.</p>

Targeted delivery of bone marrow mesenchymal stem cells by ultrasound-mediated microbubble destruction / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the feasibility of bone marrow mesenchymal stem cell (MSC) transplantation with ultrasound-targeted microbubble destruction.</p><p><b>METHODS</b>Twenty-one Wistar rats were divided into MSCs-iv group (MSCs-iv), ultrasound+MSCs-iv group (US+MSCs-iv), ultrasound+microbubble+MSCs-iv group (US+MB+MSCs-iv) with intravenous MSC transfer, ultrasound and microbubble treatment as indicated. The skeletal muscles were obtained from the rats for microscopic examination with HE staining. The hindlimb gracilis and semimembranosus muscles were sampled 7 days after MSC transplantation, and the transplanted MSCs were detected by immunohistochemistry. The vital organs were collected from rats in US+MB+MSCs-iv group for immunohistochemistry.</p><p><b>RESULTS</b>In US+MB+MSCs-iv group, HE staining demonstrated the presence of red blood cell leakage into the tissue space in the gracilis and semimembranosus muscles, and immunohistochemistry identified large numbers of transplanted MSCs in the the gracilis and semimembranosus muscles and the spleen, whereas no labeled cells were detected in the skeletal muscles in other groups.</p><p><b>CONCLUSION</b>Ultrasound-targeted microbubble destruction provides a useful means for enhancing the efficiency of stem cell transplantation.</p>

Evaluation of left ventricular diastolic function using velocity vector imaging and quantitative tissue velocity imaging / 中华心血管病杂志

<p><b>OBJECTIVE</b>To validate the efficacy of velocity vector imaging (VVI) and quantitative tissue velocity imaging (QTVI) for evaluating left ventricular diastolic function.</p><p><b>METHODS</b>Fifty-one patients underwent left heart catheterization were included in this study. Mean of peak early diastolic velocity (Em), EF and the ratio of early (E) to late (A) mitral valve flow velocity (E/A) were measured by echocardiography and the ratio of E to Em (E/Em) was calculated. Left ventricular end diastolic pressure (LVEDP) was measured during catheterization examination.</p><p><b>RESULTS</b>E/Em derived from VVI or QTVI was significantly correlated with LVEDP (r = 0.808, P < 0.01 and r = 0.692, P < 0.01, respectively) and the correlation coefficient between VVI and LVEDP was significantly higher than that between QTVI and LVEDP (Z = 2.246, P = 0.025). Em derived from VVI and QTVI also negatively correlated with LVEDP (r = -0.740, P < 0.01 and r = -0.567, P < 0.01) and the correlation coefficient between VVI and LVEDP was significantly higher than that between QTVI and LVEDP (Z = 2.595, P = 0.009). However, there was no correlation between E/A and LVEDP (r = 0.117, P = 0.415).</p><p><b>CONCLUSION</b>E/Em and Em derived from VVI and QTVI are valuable parameters for evaluating LV diastolic function.</p>

Plasma matrix metalloproteinases-2 and -9 levels are elevated in patients with acute coronary syndrome and coronary chronic total occlusion / 南方医科大学学报

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>Both the ACS and non-ACS groups showed significantly higher MMP9 and MMP2 levels than the NCA group (P<0.05), and MMP2 and MMP9 levels were significantly higher in ACS group than in non-ACS group (P<0.05). Compared with the NCA group, the UAP, AMI and CTO subgroups showed obvious increases in plasma MMP2 and MMP9 levels (P<0.01). Significantly increased MMP9, but not MMP2 level was noted in AMI subgroup in comparison with SAP (P<0.01) and UAP subgroups (P<0.05); both MMP2 and MMP9 levels were elevated in CTO subgroup in comparison with those in SAP (P<0.001), UAP (P<0.01), and AMI subgroups (P<0.05).</p><p><b>CONCLUSION</b>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.</p>

Mechanism of recombinant adenovirus-mediated mutations of hypoxia inducible factor 1alpha in modulation of cell apoptosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanism by which recombinant adenovirus (Ad)-mediated mutations of hypoxia inducible factor 1alpha (Ad-HIF-1alpha-Ala564-Ala803) regulates cell apoptosis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>HIF-1alpha may induce cell cycle arrest by up-regulating p21WAF1/CIP1 expressions to promote LoVo cell apoptosis.</p>

Assessment of the cardiotoxicity of recombinant human endostatin using myocardial biochemical markers in cancer patients / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the value of the myocardial biochemical markers including creatine kinase MB isoenzyme (CK-MB), cardiac isoform of Tropnin-T (cTnT) and N-termimal pro-brain natriuretic peptide (NT-proBNP) and electrocardiogram (ECG) in monitoring the cardiotoxicity of recombinant human endostatin (rh-endostatin) in cancer patients.</p><p><b>METHODS</b>Forty cancer patients were divided into two groups and received rh-endostatin in addition to chemotherapy (group A, n=24) or chemotherapy only (Group B, n=24). Serum CK-MB, cTnT levels and plasma NT-proBNP levels were measured and the ECG was recorded in all the patients before and after each of the two therapy cycles.</p><p><b>RESULTS</b>In group A, serum CK-MB, cTnT and plasma NT-proBNP levels were significantly increased after the treatment in comparison with the baseline levels (P<0.05), but such increment was not observed in group B (P>0.05). With comparable baseline levels of CK-MB, cTnT and NT-proBNP before the treatment (P>0.05), patients in group A showed significantly higher levels of the indices than those in group B after each therapy cycle (P<0.05). Increased ECG abnormality were observed after rh-endostatin treatment in Group A (P<0.05) at a rate significantly higher than that of Group B after the second treatment cycle (P<0.05).</p><p><b>CONCLUSION</b>Rh-endostatin has definite cardiotoxicity, and detection of the myocardial biochemical markers of CK-MB, cTnT and NT-proBNP may help predict the occurrence of cardiotoxicity.</p>

Construction and identification of adenovirus vector for double-mutant human hypoxia-inducible factor-1alpha gene / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULT AND CONCLUSION</b>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.</p>