Complementary DNA clones for recombinant human VEGF 165 , isolated from cDNA libraries 
prepared from HL60 leukemia cells, were assembled into a mammalian expression vector containing the 
cytomegalovirus promoter 26. The biological activity of VEGF 165 secreted from cells transfected with this 
construct (phVEGFi 65 ) was previously confirmed by the evidence that media conditioned by transfected 
human 293 cells promoted the proliferation of capillary cells 26. 
To evaluate expression of phVEGFi 65 in vascular smooth muscle cells (SMCs), the target cells 
for our gene transfer strategy, rabbit arterial SMCs were transfected in vitro 4. Cells were cultured by 
explant outgrowth from the thoracic aorta of New Zealand White rabbits. The identity of vascular SMCs 
was confirmed morphologically using phase contrast microscopy and by positive immunostaining using a 
monoclonal antibody to smooth muscle a-actin. Cells were grown in media supplemented with 10 % 
FBS. In vitro transfection was performed by incubating SMCs (1.48xl0 6 cells/10 cm plate) with 11.5 p.g 
of the plasmid DNA and 70 (ig of liposomes (Transfection-reagent, Boehringer Mannheim, Indianapolis, 
IN) as previously described (28). After completion of transfection, media was changed to 10% FBS. 
Culture supernatant was sampled at 3 days post-transfection, and was analyzed by ELISA assay for VEGF 
protein (29). The media of VEGF-transfected SMCs contained an average of 1.5 pg of VEGF protein 
(n=3). In contrast, culture media of 6-galactosidase-transfected SMCs (n=3) or non-transfected SMCs 
(n=3) did not contain detectable levels of VEGF protein. 
The angiogenic response to transfection of the gene for VEGF was investigated in vivo using 
the rabbit ischemic hindlimb model 7,21 All protocols were approved by St. Elizabeth's Institutional 
Animal Care and Use Committee. Male New Zealand White rabbits weighing 4-4.5 kg (Pine Acre 
Rabbitry, Norton, MA) were anesthetized with a mixture of ketamine (50 mg/kg) and acepromazine (0.8 
mg/kg) following premedication with xylazine (2.5 mg/kg). A longitudinal incision was then performed, 
extending inferiorly from the inguinal ligament to a point just proximal to the patella. Through this 
incision, the femoral artery was dissected free along its entire length; all branches of the femoral artery, 
including the inferior epigastric, deep femoral, lateral circumflex and superficial epigastric arteries, were 
also dissected free. After further dissecting the popliteal and saphenous arteries distally, the external iliac 
artery as well as all of the above arteries were ligated. Finally, the femoral artery was completely excised 
from its proximal origin as a branch of the external iliac artery, to the point distally where it bifurcates into 
the saphenous and popliteal arteries. Once the femoral artery is excised, thrombotic occlusion of the 
external iliac artery extends retrograde to its origin from the common iliac. As a result, the blood supply to 
the distal limb is dependent on the collateral arteries which may originate from the internal iliac artery. 
Accordingly, in the current study, direct arterial gene transfer of VEGF was performed into the internal 
iliac artery of the ischemic limb. 
An interval of 10 days between the time of surgery and gene transfer was allowed for post- 
operative recovery of rabbits and development of endogenous collateral vessels. Beyond this time-point, 
studies performed up to 90 days post-operatively 8 have demonstrated no significant collateral vessel 
augmentation. At 10 days post-operatively (day 0), after performing a baseline angiogram, the internal 
iliac artery of the ischemic limb of 8 animals was transfected with phVEGFi 65 percutaneously using a 2.0 
mm hydrogel-coated balloon catheter. The angioplasty balloon was prepared (ex vivo) by first advancing 
the deflated balloon through a 5 Fr. teflon sheath, applying 400 jig of phVEGFi 65 to the 20 |im-thick 
layer of hydrogel on the external surface of the inflated balloon, and then retracting the inflated balloon 
back into the protective sheath. The sheath and angioplasty catheter were then introduced via the right 
carotid artery, and advanced to the lower abdominal aorta using a 0.014 in. guidewire under fluoroscopic 
guidance. The balloon catheter was then advanced into the internal iliac artery of the ischemic limb, 
inflated for 1 min at 6 atmospheres, deflated, and withdrawn. An identical protocol was employed to 
transfect the internal iliac artery of 9 control animals with the plasmid pGSVLacZ containing a nuclear 
targeted (i-galactosidase sequence. Heparin was not administered at the time of transfection or at any point 
subsequently. 
To confirm expression of human VEGF gene in transfected rabbit iliac arteries in vivo, we 
analyzed transfected arteries for the presence of human VEGF mRNA by RT-PCR. To ensure the 
specificity of RT-PCR for human VEGF mRNA resulting from successful transfection (versus 
endogenous rabbit VEGF mRNA), primers employed were selected from a region which is not conserved 
among different species. Arteries were harvested at 5 days post-transfection. The presence of human 
VEGF mRNA was readily detected in rabbit SMC culture (n=3) and rabbit iliac arteries (n=3) transfected 
with phVEGFi 65 - Rabbit iliac arteries transfected with pGSVLacZ (n=3) were negative for human VEGF 
mRNA. Southern blot analysis was used to further confirm that the 258 bp bands obtained by RT-PCR 
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Recombinant DNA Research, Volume 20 
