Intra-pericardial Repeated Transfection Of Adenovirus-mediated Hepatocyte Growth Factor To Improve Heart Function In Post-infarctionrat Model

Background:Clinical trials testing the effects of a single injection of the adenovirus-mediated human hepatocyte growth factor gene(Ad-HGF)in patients with chronic ischemic heart failure failed to show consistent improvements in cardiac function.The aim of this study was to evaluate the efficacy of repeated injections of the Ad-HGF gene in a rat model of post-infarct heart failure.Methods:First Part:To screen the effective method of repeated injection of Ad-HGF in the rat model of post-infarction heart failure.We performed a thoracotomy on the animals and a myocardial infraction(MI)model was formed by ligating the left descending coronary artery.Infarctions were confirmed by the increase or decrease of the ST segment through electrocardiography.Two-D echocardiography was carried out to evaluate cardiac function at four weeks after the MI.A left ventricle ejection fraction(LVEF)equal to/less than 40%indicated successful preparation of the post-infarct heart failure model.At four weeks after MI,the first intra-pericardial injection or intra-myocardium injection of Ad-GFP(5×107PFU)was performed in all rats.After that,one rat was sacrificed every seven days in the intra-pericardial group and the intra-myocardial group.The hearts were harvested,frozen and sectioned serially and subjected to laser scanning confocal microscopy.At eight weeks after MI,the second injection of Ad-GFP(5×107PFU)was performed in the two groups.And then repeat the above process,the green fluorescence intensity was quantified using the image analysis software of Imagepro-plus.Light intensity from the section was measured by integrated optical density(IOD).Second Part:Effects of adenovirus-mediated hepatocyte growth factor gene therapy on post-infarct heart function:Comparison of single-and repeated-injectionsA myocardial infraction model was prepared by ligating the left descending coronary artery.Magnetic resonance imaging(MRI)was carried out to evaluate cardiac function at four weeks after the MI.A left ventricle ejection fraction(LVEF)equal to/less than 40%indicated successful preparation of the post-infarct heart failure model.According to the LVEF,rats were divided into four groups(n =8 in each group):(a)Ad-HGF repeat-injection group;(b)Ad-HGF single-injection group;(c)null-Ad group;and(d)saline group.At four weeks after MI,the first intra-pericardial injection was performed in all groups:null-Ad group(null-Ad,100?l),saline group(saline,100?l),Ad-HGF single-injection group(1×109PFU/ml Ad-HGF,100?l)and Ad-HGF repeated-injection group(5×108PFU/ml Ad-HGF,100?l).At eight weeks after the MI,the second intra-pericardial injection was performed:null-Ad group(null-Ad,100?l),saline group(saline,100?l),Ad-HGF single-injection group(saline,100?l)and Ad-HGF repeated-injection group(5×108PFU/ml Ad-HGF,100?l).Heart function was monitored via magnetic resonance imaging(MRI)for eight and twelve weeks following ligation.The expression of HGF or Factor VIII/Ki-67 was evaluated by Western blot assay or immunofluorescence.Results:No rat can survive after third times open-heart surgeries,so the repeated intra-myocardial injection method was eliminated.The intra-pericardial repeated injection method showed better effect and better tolerance in rats compared with intra-myocardial injection method,after the second intra-pericardial injection of Ad-GFP,green fluorescence persisted for 14 days.Equal dosages of Ad-HGF administered in a single injection versus repeated injections yielded greater improvements in heart function and angiogenesis with repeated injections.Conclusions:In the rat post-infarction heart failure model,repeated Ad-HGF injections can prolong the expression of HGF gene in vivo,which shows better therapeutic effect than single injection.But whether or not the results for repeated administration of Ad-HGF in rats can be extrapolated to clinical application,further research in large mammals should be carried out.