More recent investigations have established the feasibility of using recombinant formulations of 
such angiogenic growth factors to expedite and/or augment collateral artery development in animal models 
of myocardial and hindlimb ischemia. This novel strategy for the treatment of vascular insufficiency has 
been termed "therapeutic angiogenesis" 19. The angiogenic growth factors first employed for this purpose 
comprised members of the FGF family. Baffour et al administered bFGF in daily intramuscular (IM) 
doses of 1 or 3 pig to rabbits with acute hindlimb ischemia; at the completion of 14 days of treatment, 
angiography and necropsy measurement of capillary density showed evidence of augmented collateral 
vessels in the lower limb, compared to controls 20. p u e t al used acidic fibroblast growth factor (aFGF) to 
treat rabbits in which the acute effects of surgically-induced hindlimb ischemia were allowed to subside for 
10 days before beginning a 10-day course of daily 4-mg IM injections; at the completion of 30 days 
follow-up, both angiographic and hemodynamic evidence of collateral development was superior to 
ischemic controls treated with IM saline 21. Yanagisawa-Miwa et al likewise demonstrated the feasibility 
of bFGF for salvage of infarcted myocardium, but in this case growth factor was administered intra- 
arterially at the time of coronary occlusion, followed 6 hrs later by a second intra-arterial bolus 22. 
We have used the same animal model developed by Pu et al 21 to investigate the therapeutic 
potential of a 45 kDa dimeric glycoprotein, vascular endothelial growth factor (VEGF), isolated initially as 
a heparin-binding factor secreted from bovine pituitary folliculo-stellate cells 23. VEGF was also purified 
independently as a tumor-secreted factor that induced vascular permeability by the Miles assay 24,25 ^ and 
thus its alternate designation, vascular permeability factor (VPF). Two features distinguish VEGF from 
other heparin-binding, angiogenic growth factors. First, the NH2 terminus of VEGF is preceded by a 
typical signal sequence; therefore, unlike bFGF, VEGF can be secreted by intact cells 26. Second, its 
high-affinity binding sites, shown to include the tyrosine kinase receptors Flt-1 27 and Flk-1 /KDR 28,29 
are present on endothelial cells, but not other cell types; consequently, the mitogenic effects of VEGF - in 
contrast to acidic and basic FGF, both of which are known to be mitogenic for smooth muscle cells 20,3 1 
and fibroblasts as well as endothelial cells - are limited to endothelial cells 23,32 (interaction of VEGF 
with lower affinity binding sites has been shown to induce mononuclear phagocyte chemotaxis) 33,34. 
Evidence that VEGF stimulates angiogenesis in vivo had been developed in experiments 
performed on rat and rabbit cornea 35,36 ? the chorioallantoic membrane 23 ? and the rabbit bone graft 
model 36 We investigated the hypothesis that the angiogenic potential of VEGF was sufficient to 
constitute a therapeutic effect 2 (see Appendix for manuscript). The soluble 165-amino acid isoform of 
VEGF (VEGF165) was administered as a single intra-arterial bolus to the internal iliac artery of rabbits in 
which the ipsilateral femoral artery was excised to induce severe, unilateral hindlimb ischemia. Doses of 
500-1,000 [ig of VEGF produced statistically significant augmentation of angiographically visible 
collateral vessels, and histologically identifiable capillaries; consequent amelioration of the hemodynamic 
deficit in the ischemic limb was significantly greater in animals receiving VEGF than in non-treated 
controls (calf blood pressure ratio=0.75±0.14 vs 0.48±0.19, p<0.05). Serial (baseline, as well as 10 and 
30 days post-VEGF) angiograms disclosed progressive linear extension of the collateral artery of origin 
(stem artery) to the distal point of parent-vessel (reentry artery) reconstitution in 7 of 9 VEGF-treated 
animals. Similar results were achieved in a separate series of experiments in which VEGF was 
administered by an IM route daily for 10 days 37. These findings thus established proof of principle for 
the concept that the angiogenic activity of VEGF is sufficiently potent to achieve therapeutic benefit. 
While each of these studies documented an increase in the number of angiographically visible 
collaterals, and increased capillary density in the muscles studied at necropsy, evidence regarding the 
physiological consequences of such anatomical improvement was limited to blood pressure measurements 
recorded in the ischemic versus the normal limb. Accordingly, we performed a series of studies in the 
ischemic hindlimb model in which an intra-arterial Doppler wire 38 ? sufficiently diminutive (0.018 in.) to 
measure phasic blood flow velocity in the rabbit's internal iliac artery, was used to investigate resting and 
maximum flow following therapeutic angiogenesis with a single, intra-arterial bolus of VEGF165. By 30 
days post-VEGFi65, flow at rest, as well as maximum flow velocity and maximum blood flow provoked 
by 2 mg papaverine were all significantly higher in the VEGF-treated group (see attached manuscript) 39. 
More recently, we considered that one of the distinguishing features of VEGF mentioned above 
- the fact that the VEGF gene encodes a secretory signal sequence - might be exploited as part of a strategy 
designed to accomplish therapeutic angiogenesis by arterial gene transfer. We had previously observed that 
site-specific transfection of rabbit ear arteries with the plasmid pXGH5 encoding the gene for human 
growth hormone - a secreted protein - yields local levels of human growth hormone equivalent to what has 
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Recombinant DNA Research, Volume 20 
