The Effects Of Gene Transfer Into Myocardium By Recombination Adenovirus-VEGF₁₆₅ Via Pericardial Cavity In Porcine Acute Myocardial Infarction On Angiogenesis And Cardiac Function

Objective To establish a model of acute myocardial infarction and evaluate the expression efficacy and time of adenovirus vector via the pericardial cavity. Methods Replication-deficient recombinant adenoviral vector carrying LacZ reporter genes(Ad-LacZ) was constructed by the calcium phosphate method. Twelve health porcine were randomly divided into two groups, experimental group(n=6) and control group(n=6). AMI mode was constructed by occluding by balloon the D₁ distance ofLAD then the intra-pericardial injections were performed through a small incision of the abdominal wall below the xyphoid appendix using center venous catheter. Whether the experimental group or the control group, the pericardial cavity pre-treated by injecting a mixture of collagenase and hyaluronidase. Then 2.0×10⁹p.f.u Ad-LacZ was injected into pericardial cavity. The histological changes and beta-galactosidase activity of the ischemic myocardium were observed at the 3rd, 7th and 28th day after injection.Results The LAD was occluded completely and infarction and ischemic were detected by histologic assessment. In experimental group, X-gal staining positive cells were detected at the 3rd day after injection, increased markedly at the 7th day and then declined at the 28th day. In control group, no positive cells were observed at the same time.Conclusion Acute myocardial infarction model of pigs can successfully constructed by occluding by balloon. Ad can transfer into ischemic myocardium and succeed in expressing target gene in the model for 4 weeks via pericardium pre-treated by injecting a mixture of collagenase and hyaluronidase.Part IIEffect Of Gene Transfer By Adenovirus-Vascular Endothelial Growth Factor165 Via The Pericardium On Coronary ArteryAngiogensis In PorcineObjective To explore the influence on angiogenesis and the expressing and proper dose of adenovirus-mediated vascular endothelial growth factor 165(Ad-VEGF₁₆₅) gene in ischemic myocardium via the percardium.Methods Twenty health porcine were randomly divided into two groups,experimental group(n=10) and control group(n=10). There are 2 in each group at 3rd, 7th after injection respectively and 6 at 28th. AMI mode was constructed by ballooning occlusion the D₁ distance of LAD at the same time the intra-pericardial injections were performed through a small incision of the abdominal wall below the xyphoid appendix using center venous catheter. Whether the experimental group or the control group, the pericardium pre-treated by injecting a mixture of collagenase(1200U) and hyaluronidase(3000U). Then 2.0×10⁹p.f.u Ad-VEGF₁₆₅ was injected into pericardium in experimental group. While physiological saline was injected into control group. Immunohistochemistry assay and echocardiography were carried out to evaluate the angiogenesis in the ischemic region and cardio function at 3rd, 7th and 28th after injection respectively and enzyme-linked immunoassay(ELISA) was done to investigate the local expression of Ad-VEGF₁₆₅ in plasma, pericardium and myocardium.Results The peak expression of Ad-VEGF₁₆₅ gene in pericardium and myocardium occurred 7th after the administration of Ad-VEGF₁₆₅ and decreased gradually to baseline level at 28th. And microvenous density and cardiac function of the experimental group significantly increase in the course of transfer and have the dvantage of the control group(in 28 days, MVD 517.0±75.7 vs, 226.5±54.1 P=0.009, LVEF% 72.11±5.2 vs 55.14±4.37, P=0.005).Conclusion Gene transfer of Ad-VEGF₁₆₅ via pericardium can induce protein expression and enhance angiogenesis in ischemic region and improve cardio function in AMI after the pericardium pre-treated by injecting a mixture of collagenase and hyaluronidase.Objective To explore the efficacy of gene transfer into myocardium by recombination adenoviais -VEGF₁₆₅ via the pericardium or the coronary artery.Methods Twelve health porcine were randomly divided into two groups: pericardium transfer group and coronary artery transfer group. AMI mode in the pericardium group was constructed by occluding by balloon the D₁ distance of LAD then the intra-pericardial injections were performed through a small incision of the abdominal wall below the xyphoid appendix using center venous catheter. The pericardium pre-treated by injecting a mixture of collagenase and hyaluronidase and then 2.0×10⁹pfu Ad-VEGF₁₆₅ was injected into pericardium. While 2.0×10⁹p.f.u Ad-VEGF₁₆₅ was injected into myocardial cavity via coronary artery in the coronary group at the same time AMI mode was constructed by the same way. Tissue VEGF concentration microvenous density and cardiac function of the two groups were respectively observed at the 3rd, 7th and 28th day before and after injection.Results VEGF₁₆₅ gene was expressed in the heart of the pericardium group and coronary artery group. Tissue VEGF concentration increased markedly at the 7th day after injection and then declined at the 28th day. Tissue VEGF levels in the pericardium group were higher than the coronary artery group[(702±85) pg/ml vs (592±59)pg/ml, P=0.026]. And microvenous density and cardiac function of the two groups all significantly increase in the course of transfer. However, the pericardium group has the advantage of the coronary artery group in parameters above(in 28 days, MVD [(517.0±75.7)/mm² vs (326.4±24.1)/mm², P=0.001; FS (32.9±2.2)% vs (30.6±2.1)%, P=0.049; LVEF (72.11±5.2)% vs (65.87±2.16)%, P=0.034].Conclusion Catheter mediated Ad-VEGF₁₆₅ gene transfer via coronary artery or pericardium is efficacy and feasibility for ischemic myocardium. And the latter isObjective To explore the feasibility and safety of gene transfer via the pericardial cavity by home-made easy device. Methods Replication-deficient recombinant adenoviral vector carrying LacZ reporter genes(Ad-LacZ) was constructed by the calcium phosphate method. Twelve healthly porcine were randomly divided into two groups, experimental group(n=6) and control group(n=6). AMI mode was constructed by balloon occlusion of the distal part of D₁ branch of LAD at the same time the intra-pericardial cavity injections were performed through a small incision of the abdominal wall below the xyphoid appendix using home-made device. After succeeding, gene transfer were performed by center venous catheter. In both the experimental group and the control group, the pericardium was pre-treated by injecting a mixture of collagenase and hyaluronidase. Then 2.0×10⁹p.f.u Ad-LacZ was injected into pericardium. The histological changes and beta-galactosidase activity of the ischemic myocardium were observed at the 3rd, 7th and 28th day after injection. Results The LAD was occluded completely and infarction and ischemic were detected by histologic assessment. In experimental group, X-gal staining positive cells were detected at the 3rd day after injection, increased markedly at the 7th day and then declined at the 28th day. In control group, no positive cells were observed. Conclusion Ad can be transfered into ischemic myocardium and succeed in expressing target gene in the model for 4 weeks via pericardial cavity pre-treated by injecting a mixture of collagenase and hyaluronidase by home-made easy device.