Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.
Abstract-Recent studies suggest the possible therapeutic effect of intramuscular vascular endothelial growth factor (VEGF) gene transfer in individuals with critical limb ischemia. Little information, however, is available regarding (1) the required expression level of VEGF for therapeutic effect, (2) the related expression of endogenous angiogenic factors, including fibroblast growth factor-2 (FGF-2), and (3) the related adverse effects due to overexpression of VEGF.To address these issues, we tested effects of overexpression of VEGF165 using recombinant Sendai virus (SeV), as directly compared with FGF-2 gene transfer. Intramuscular injection of SeV strongly boosted FGF-2, resulting in significant therapeutic effects for limb salvage with increased blood perfusion associated with enhanced endogenous VEGF expression in murine models of critical limb ischemia. In contrast, VEGF165 overexpression, 5-times higher than that of baseline on day 1, also strongly evoked endogenous VEGF in muscles, resulting in an accelerated limb amputation without recovery of blood perfusion. Interestingly, viable skeletal muscles of either VEGF165-or FGF-2-treated ischemic limbs showed similar platelet-endothelial cell adhesion molecule-1-positive vessel densities. Maturation of newly formed vessels suggested by smooth muscle cell actin-positive cell lining, however, was significantly disturbed in muscles with VEGF. Further, therapeutic effects of FGF-2 were completely diminished by anti-VEGF neutralizing antibody in vivo, thus indicating that endogenous VEGF does contribute to the effect of FGF-2. These results suggest that VEGF is necessary, but should be delicately regulated to lower expression to treat ischemic limb. The therapeutic effect of FGF-2, associated with the harmonized angiogenic effects seen with endogenous VEGF, provides important insights into therapeutic angiogenesis.
Abstract-Hepatocyte growth factor (HGF) is a potent angiogenic polypeptide that stimulates angiogenesis. Transcriptional regulation of HGF, however, has not been fully defined, with the exception of the hypoxia-mediated downregulation in cultured cells. In the present study, we report that angiogenic growth factors, including HGF, were upregulated in a murine model of critical limb ischemia in vivo, a finding that was in conflict with previous in vitro data. Mice deficient in basic fibroblast growth factor-2 (FGF-2) showed reduced induction of HGF protein in ischemic muscles, and overexpression of FGF-2 via gene transfer stimulated endogenous HGF, irrespective of the presence of ischemia. In culture, FGF-2 rapidly stimulated HGF mRNA, and a sustained expression was evident in the time course in vascular smooth muscle cells and fibroblasts. FGF-2-mediated induction of HGF was fully dependent on the mitogen-activated protein kinase pathway yet was not affected by either hypoxia or a protein kinase A inhibitor. In the early expression, FGF-2 directly stimulated HGF mRNA without the requirement of new protein synthesis, whereas sustained induction of HGF in the later phase was partly mediated by platelet-derived growth factor-AA. Furthermore, in vivo overexpression of FGF-2 significantly improved the blood perfusion, and the effect was abolished by systemic blockade of HGF in ischemic limbs. This is the first demonstration of a regulational mechanism of HGF expression via FGF-2 that was independent of the presence of hypoxia. The harmonized therapeutic effects of FGF-2, accompanied with the activity of endogenous HGF, may provide a beneficial effect for the treatment of limb ischemia.
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