Vegf165 Was, And Of Angiopoietin-1 Expression In The Ischemic Myocardium-specific Treatment Of Acute Myocardial Infarction

Background: Vascular endothelial growth factor 165 (VEGF165) and angiopoietin-1 (Ang1) are major angiogenic factors being studied for the treatment of myocardial infarction (MI). The over-expression of VEGF165 and/or Ang1 in heart could improve cardiac founction. However, uncontrolled expression of angiogenic factors in vivo may cause some unwanted side effects, such as hemangioma formation, retinopathy, and arthritis. Hence, it is important to control VEGF165 and Ang1 expression when gene transduction is used.Aims: The present study was designed to detect whether 250bp MLC-2v promoter and nine copies of HRE could mediate the expression of cardiac-specific and hypoxia-induced VEGF165 and Ang1 in ischemic myocardium and evaluate whether the expression could improve myocardial perfusion and cardiac function in pig with acute myocardial infarction(AMI).Methods: Left ventricular anterior transmural AMI model was established by permanent ligation of the left descending coronary artery (LAD) with silk ligature using male Chinese minipigs. Adeno-associated virus 1 (AAV1) was chosen to vector. To limit the expression of VEGF and Ang1 in organs other than the heart, the cardiac myosin light chain 2v (MLC-2v) promoter and the cis-acting hypoxia-responsive element (HRE) were used to mediate the cardiac-specific and hypoxia-induced VEGF165 and Ang1 expression. AAV1-MLCVEGF165 and AAV1-MLCAng1, with 250bp of the MLC-2v promoter and nine copies of the HRE were constructed for the local the co-expression of VEGF165 and Ang1 in infarcted myocardium. AAV1-MLCVEGF165 and AAV1-MLCAng1 were injected together (VEGF/Ang1 group) into six different sites of the porcine myocardium at the peri-infarct zone immediately after ligating the LAD. Identical doses of AAV1-CMVLacZ or saline were injected into control animals. Coronary angiography and Electrocargiogram (ECG) were used for estimating the construction of porcine infarction heart model. AAV1 genomes were detected with PCR. RT-PCR, Western blot, and ELISA analyses were used to determine the expression of cardiac-specific and hypoxia-induced VEGF165 and Ang1. Gated-Single Photon Emission Computed Tomography (G-SPECT) analyse was used for cardiac function and myocardial perfusion two weeks and eight weeks after the treatment.Results: Coronary angiography and ECG proved that acute myocardial infarction model was successfully created by ligation of the porcine LAD. AAV1 genomes were detected in the liver in addition to the heart by PCR. RT-PCR, Western blot, and ELISA analyses demonstrated that VEGF and Ang1 were predominantly expressed in infarct core and border in myocardial infarcted heart. G-SPECT analyse showed that the VEGF/Ang1 group had better cardiac function and myocardial perfusion eight weeks than two weeks after the treatment.Conclusion: Acute myocardial infarction model is created by ligation of the LAD. AAV1-MLCVEGF165 and AAV1-MLCAng1 mediate cardiac-specific and hypoxia-induced the co-expression of VEGF165 and Ang1. The co-expression of VEGF165 and Ang1 improves the perfusion and function of porcine myocardial infarcted heart. Background: VEGF165 and Ang1 is usually used for promoting neovascularization, its role of angiogenesis and anti-apoptosis improves myocardial perfusion and cardiac function. Previous studies have showed that several factors had the effect of myocardial proliferation, such as bFGF, Akt, cyclin A and cyclin D1. However, it is unknown whether VEGF165 and Ang1 may promote myocardial proliferation.Aim: To explain the mechanism that cardiac-specific and hypoxia-induced co-expression of hVEGF165 and hAng1 improves myocardial perfusion and cardiac function in porcine acute infarcted heart, angiogenesis, myocardial apoptosis and proliferation were analysed by flow cytometry (FCM), immunohistochemistry (IHC), Western blot and immunoprecipitation (IP).Methods: The hearts were fixed in 10% formalin, embedded in paraffin and sectioned. Serial sections were made from the apex of the heart to the site of the ligation. Sections were stained with specific antibodies against Lectin,α-smooth muscle actin (α-SMA), Ki67 and cardiac smarcomericα-actinin (α-actinin) (Santa Cruz Biotech) antibodies. Nuclei were stained with 4',6'-diamino-2-phenilindole (DAPI). Lectin-FITC (Santa Cruz Biotech) was used to visualize the blood vessels. The immunohistochemical staining was carried out according to the manufacturer's instructions. For quantification of vessel-densities in myocardium, four sections from each group were randomly selected, and six visual fields from each section were observed. Cardiomyocyte apoptosis was measured by double immunofluorescence staining of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) using In Situ Cell Death Detection Kit, Fluorescein. The cell suspensions were prepared using Medimachine System (Becton Dickinsin, U. S. A.). Cardiomyocyte cells were collected by flow cytometry with the use of an antibody to sarcomericα-actinin. The Cells (1×106/sample) were fixed in 70% ethanol and double staining for FITC–Annexin V binding and propidium iodide (PI), and analyzed by flow cytometry using a FACS-SCAN apparatus (Becton-Dickinson, U. S. A). Cell cycle distribution was analyzed for DNA content using PI in flow cytometry. The protein was isolated from normal, peri-infarct, infarct myocardium in three groups according to the manufacturer's instructions. The total protein concentration was determined by modified Bradford assay. Immunoprecipitation and Western blot analysis were performed for detecting the pathway of antiapoptosis and proliferation. Caspase-3, Akt, phosphorylated Akt (p-Akt), Bad, Bcl-xL, Bcl-2, ERK1/2, p21, p27, cyclin A, cyclin E, cyclin D1, cyclin D2, cdk2 and cdk4 antibodies were used.Results: The VEGF/Ang1 group had significantly more capillaries in the myocardial infarct and peri-infarct zones than those in the saline and LacZ group (P<0.01). Similarly, the density ofα-SMA-positive vessels in the myocardial infarct and peri-infarct zones of VEGF/Ang1 group were significantly higher than those of the saline and LacZ group (P<0.01). The densities of capillaries andα-SMA-positive vessels were not significantly different between the LacZ and saline group (P>0.05). More Ki67-positive endothelial cells wrer significantly detected in the VEGF/Ang1-injected heart than those in the saline- and LacZ-injected hearts (P<0.01).The hearts of the VEGF/Ang1 group have significantly fewer TUNEL-positive nuclei in the myocardium of infarct and peri-infarct zones than those in the LacZ and saline-injected pigs (P<0.01). The ratio of apoptotic cell in the infarct and peri-infarct zones of VEGF/Ang1 group was significantly lower compared to the saline and LacZ groups (P<0.01), the number of apoptotic cells in normal myocardial zone was similar among the three groups by flow cytometry (P>0.05). Consistent with the cytological analysis, caspase-3 expression in the infarct and peri-infarct zones of VEGF/Ang1 group was the lowest among the three groups. To analyze the mechanism of anti-apoptosis in the VEGF/Ang1 group, apoptotic pathway was analyzed by Western blot. There was more p-Akt expression in the VEGF/Ang1-injected hearts compared to those in the hearts injected with LacZ and saline. Bad expression was lower in the myocardial infarct and peri-infarct zones of the VEGF/Ang1 group than that in LacZ and saline group. On the other hand, Bcl-xl was higher in the VEGF/Ang1-injected group compared to the LacZ and saline group. However, the expression of Bcl-2 and ERK1/2 was similar among all three zones of the three groups. To determine whether over-expression of VEGF165 and Ang1 promoted cardiomyocyte proliferation, antibodies against Ki67 and sarcomericα-actinin (a cardiomyocyte-specific marker) were used for double labeling. There were more Ki67 positive cardiomyocytes in the VEGF/Ang1-injected hearts than those in the saline- and LacZ- injected hearts (P<0.01). FCM data were consistent with the immunostaining data. There were more S phase cardiomyocytes in the VEGF/Ang1 group compared to those in the saline and LacZ group (P<0.01). To determine pro-survival pathways, the expression of cell cycle proteins was analyzed. p27, a cell cycle inhibitor, was lower in the infarct and peri-infarct zones of the VEGF/Ang1 group compared to that in the saline and LacZ group. The expression of cyclin D2 in the infarct and peri-infarct zones of the VEGF/Ang1 group significantly increased compared to that in the saline and LacZ group. Similarly, cdk4 level in the infarct and peri-infarct zones of VEGF/Ang1 group increased compared to that in the saline and LacZ group. The infarct and peri-infarct zones of VEGF/Ang1 group expressed higher cyclin A protein than that in the saline and LacZ group. The expression of cdk2 in the infarct and peri-infarct zones of the VEGF/Ang1 group significantly increased compared to that in the saline and LacZ group.Conclusion: Cardiac-specific and hypoxia-induced co-expression of VEGF and Ang1 improves the perfusion and function of porcine MI heart through the induction of angiogenesis and cardiomyocyte proliferation, activation of pro-survival pathways, and reduction of cell apoptosis.