A novel method of augmenting gene expression and angiogenesis in the normal and ischemic canine myocardium

This study presents a novel method that direct intramyocardial injection of low-dose plasmid DNA and microbubbles combined with insonation could further augment gene expression in normal and ischemic canine myocardium. Plasmids encoding enhanced green fluorescent protein (pEGFP) and hepatocyte growth factor (pHGF) (500 μg) were individually mixed with 0.5 ml of microbubble solution (MB) and injected into the normal or acute ischemic canine myocardium. The dogs in the plasmid + MB/US group underwent insonation (US). Other dogs were randomly divided into three treatment groups: plasmid and insonation, plasmid and MB injection, and plasmid injection only. The EGFP and HGF mRNA expressions were assessed in the myocardium at the injection site and at sites 0.5 and 1 cm remote from the injection site. Compared to plasmid transfer alone, a mean 13.4-fold enhancement of gene expression was achieved in the EGFP + MB/US group at 48 h (p < 0.01). HGF mRNA expression in ischemic zones was markedly elevated after 28 days, with a mean 9.0-fold enhancement in the HGF + MB/US group (p < 0.01). EGFP protein expression was detected in the normal myocardium at 1 cm remote from the injection site in the EGFP + MB/US group. Similarly, HGF protein expression was detected in the ischemic myocardium at 0.5 cm remote from the injection site in the HGF + MB/US group. These findings indicate that the radius of gene expression was partly extended in the two plasmid + MB/US groups. The capillary density increased from 20.9 ± 5.3/mm(2) in control myocardial infarction dogs without treatment to 126.7 ± 38.2/mm(2) in the HGF + MB/US group (p < 0.01). Taken together, the present data demonstrate that direct intramyocardial injection of an angiogenic gene and microbubbles combined with insonation can augment gene expression and angiogenesis. Consequently, this strategy may be a useful tool for gene therapy of ischemic heart disease.