2) Physical examination. A general as well as specific vascular examination will be performed. As part 
of the latter, auscultation of the lower extremities will be performed for bruits, and palpation will be 
performed for femoral, popliteal, and pedal pulses; grading of each will be done on a 0-4+ scale. 
Status of ischemic ulcers will be documented by gross photographs. The location, size, depth and 
necrosis characteristics of all ischemic ulcers will be recorded after cleansing and debridement (if any). The 
area of the ulcer(s) will be estimated as the product of the longest length times the longest perpendicular 
width. For inaccessible sites such as interdigital locations, an estimate of surface area will be made. The 
depth of the ulcer(s) will be graded as l=superficial defect; 2=involvement of subcutaneous tissue; 
3=exposure of tendon or bone; 4=necrosis of tendon or bone. The ulcer(s) will be further characterized as 
wet or dry. 
3) Clinical Laboratory Studies. 
a) Hemoglobin, hematocrit , white blood cell count, white blood cell differential count, platelet 
count, Westergren sedimentation rate, serum electrolytes (sodium, potassium, chloride, bicarbonate), 
BUN, creatinine, glucose, uric acid, total protein, albumin, calcium, phosphate, total bilirubin, conjugated 
bilirubin, AST, ALT, alkaline phosphatase, LDH, CEA, PSA. 
b) Urine analysis: qualitative protein, blood, glucose, ketones, pH, microscopic examination. 
c) Stool: hemoccult 
d) Chest x-ray 
e) CT scan of chest and abdomen 
f) Head MRI scan 
g) Sigmoidoscopy and prostate examination. 
4) Indices, pressures, and pulse volume recordings (PVRs). The ankle-brachial index ( ABI) and great 
toe index (GTT) will be performed with the patient supine, as previously described. A continuous wave 
Doppler ultrasonic instrument detects the arterial pulse, and standard cuffs are used to measure systolic 
pressures in both the dorsalis pedis and posterior tibial arteries in each leg, as well as in each arm. The 
highest pressure of the two arms will be used for calculating ABI. At rest, systolic pressure is determined 
in both the dorsalis pedis and posterior tibial arteries of each ankle, and the ABI is calculated from these 
values. GTI will be measured using a digital blood pressure cuff placed at base of the great toe and 
brachial blood pressure as described above. 
Provided that the results of these examinations are consistent with the inclusion and exclusion criteria 
cited above, the opportunity to participate in this study will be discussed with the patient. 
III. CONSTRUCTION OF PLASMID 
III. A. Plasmid Structure 
The cDNA to be used in this protocol encodes the 165 amino acid isoform of VEGF and has 
been described previously 26,58 
The plasmid into which the VEGF cDNA has been inserted, phVEGFigs, is a simple 
eucaryotic expression plasmid that utilizes the 763-base-pair cytomegalovirus (CMV) promoter/enhancer to 
drive VEGF expression. This promoter/enhancer has been used to express reporter genes in a variety of 
cell types and can be considered to be constitutive. Downstream from the VEGF cDNA is the SV40 
polyadenylation sequence. Also included in this plasmid is a fragment containing the SV40 origin of 
replication that includes the 72 base pair repeat, but this sequence is not functionally relevant (for 
autonomous replication) in the absence of SV40 T-antigen. These fragments occur in the pUC118 vector 
which includes an E.coli origin of replication and the 6-lactamase gene for ampicillin resistance. 
The VEGF expression plasmid, phVEGFi 65 , was constructed by David Leung while working 
at Genentech. The entire phVEGF^ sequence (5651 bp) has been determined (designated VEGF V1-V2) 
using 24 sequencing primers. This determined sequence is compared with the deduced sequence 
(designated 1. VEGF) provided to us by Dr. Buffer Fennie at Genentech. The structure of the double- 
stranded DNA was determined by the cycle sequencing method using fluorescent dideoxy terminator 
nucleotides with an Applied Biosystem 373A Automated sequencer. Sequences were analyzed on 
Macintosh Quadra computers with MacVector and Sequence Navigator software. The quality control for 
this sequencing analysis consists of parallel sequence analyses of Bluescript and Ml 3 controls. 
Our sequencing found 3 regions that were missing from the sequence predicted by Genentech: 
i) -4 bp (of a repeat) at the junction of the SV40 early polyA and origin sequences 
ii) -35 bps within the M13 region 
iii) -14 bps at the junction between M13 and pUC. 
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Recombinant DNA Research, Volume 20 
