Hepatocyte growth factor (HGF) has been shown to induce angiogenesis in vivo and has potential as a candidate gene for 'therapeutic angiogenesis'. In vivo, two isoforms of HGF, HGF 723 and HGF 728 , consisting of 723 and 728 amino acids, are generated through alternative splicing between exons 4 and 5, but the biological effects of their coexpression have not yet been elucidated. In this study, we generated a series of genomic-complementary DNA (cDNA) hybrids of the HGF gene by inserting various truncated intron 4 into the junction of exons 4 and 5 of HGF cDNA and analyzed the biological activities of these hybrid constructs. We showed that: (1) the hybrid called HGF-X7, which contained 1502 base pairs of intron 4, could drive a higher level of HGF expression than other hybrid constructs and cDNAs of each isoform alone; (2) the pCK vector was most efficient for the gene expression of HGF-X7; (3) coexpression of both isoforms of HGF could more efficiently induce the migration of human umbilical vein endothelial cell (HUVEC) and of the mouse myoblast cell line C 2 C 12 myoblasts than a single isoform of HGF and human vascular endothelial growth factor (VEGF) 165 at a given concentration; (4) intramuscular administration of pCK-HGF-X7 resulted in transient and localized HGF expression in the injected muscle without an increase in the HGF protein levels in other tissues including serum; and (5) intramuscular injection of pCK-HGF-X7 could more efficiently increase the number of angiographically recognizable collateral vessels, as well as improve an intra-arterial Doppler wiremeasured blood flow in the rabbit model of hindlimb ischemia when compared with the identical vector encoding VEGF 165 gene. These results showed that transfer of the genomiccDNA hybrid of the HGF gene could be used as a potential therapeutic approach to human vascular diseases.

Objective. To determine the efficacy of local therapy with human angiostatin gene in murine collageninduced arthritis (CIA).Methods. DBA/1 mice were immunized with bovine type II collagen. Before the onset of arthritis, NIH3T3 fibroblasts, transduced with angiostatinexpressing retroviral vectors or control vectors, were transplanted into the knee cavity. The incidence of arthritis in the knee joints was evaluated histologically based on pannus formation and cartilage destruction. Paws were evaluated macroscopically for redness, swelling, and deformities and immunologically for levels of interleukin-1␤. Angiogenesis in paws and knee joints was studied by immunohistochemistry using anti-CD31 antibody and measurement of von Willebrand factor levels.Results. Pannus formation and cartilage erosion were dramatically reduced in knees transplanted with angiostatin-expressing cells. In addition, the onset of CIA in the ipsilateral paws below the knees injected with the angiostatin gene was significantly prevented. Furthermore, angiostatin gene transfer inhibited arthritisassociated angiogenesis.Conclusion. Local production of angiostatin in the knee was able to prevent the onset of CIA not only in the knee injected with genetically engineered cells, but also in the uninjected ipsilateral paw. This suggests that transfer of the angiostatin gene, and potentially also its protein, may provide a new, effective approach to the treatment of rheumatoid arthritis.

Intramyocardial injection of pCK-HGF-X7 induced significant angiogenesis at infarct-border zone, and increased the left ventricular stroke volume probably caused by reverse remodeling process.

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