Supplement (Figure Legends)
Figure I: Tissue distribution of transplanted BM-MNCs
Tissue distribution of BM-MNCs (kidney, spleen, liver, heart, pancreas, brain) 3 weeks after targeted cell delivery to the ischemic limbs by Bubble+US+BM-MNCs-i.v. was examined. Small numbers of transfused BM-MNCs (labeled with red-fluorescence; yellow arrows) were detected in the kidney (mainly in the tubule) and spleen but not in other tissues. Capillaries were immunostained by anti-factor VIII antibody followed by incubation with FITC-conjugated secondary antisera (labeled with green-fluorescence). Bar=50 mm.
Figure II. Angiographic analysis of collateral vessel formation
Representative angiograms obtained on postoperative Day 28 (n=6 in each group). Arrows indicate the ligated ends of femoral arteries. Numerous collateral vessels were observed in Bubble+US+BM-MNC-i.v. and BM-MNC-i.m. groups compared with Bubble+US, BM-MNC-i.v. or control groups. Bar=1 cm.
Figure III. Regional delivery of microbubbles to ishemic tissues
Microbubbles was injected 3 days after ligation of femoral artery via contralateral femoral vein. The echo prove was placed on the thigh muscle and the regional delivery of microbubbles was recorded by the optical disk (echogarphy; Sonos-5500, Phillips, MA). An increase in contrast densities in thigh muscles (indicated by arrows) was detected in both control (normal) and ischemic limbs compared with the pre-images before injection of microbubbles. The images at ~15 seconds after injection of microbubbles were shown. An increase in contrast densities diminished after 1 min of US stimulation (Bubble+US).
Supplement (Methods)
Adhesion assay under laminar flow conditions and immunofluorescence study
HUVEC were positioned in the flow chamber (shear stresses of 1 dyne/cm2). The entire period was recorded on videotape and captured images were transferred to a PC computer (10 randomly selected x20 microscopic fields for each experiment). Cells were considered to be adherent after 10 seconds of stable contact with the monolayer.
Laser doppler perfusion image (LDPI) and angiography
The recovery of blood flow in the lower limbs was evaluated by a LDPI analyzer (Moor Instruments, Devon, UK) on Day 0, 3, 7, 14, 21, and 28 as described (3,18). After scanning blood flow three times, stored images were subjected to computer-assisted quantification of blood flow, and the average flows of ischemic and nonischemic feet were quantitatively calculated. To minimize data variables due to ambient light and temperature, the LDPI index was expressed as a ratio of ischemic (left) to nonischemic (right) limb blood flow (n=8, each group).
Vessel density was evaluated using a microfocus X-ray television device (HITEX CO. LTD) at Day 28 as described (3). Longitudinal laparotomy was performed to introduce a catheter into the abdominal aorta followed by injection of contrast medium (lipiodol). Angiography was performed for 2 seconds after the injection. We quantitatively analyzed collateral vessel numbers (3, 18). Briefly, numbers of vessels in the thigh area were counted using 5 mm2 grids by two radiologists who were unaware of the group identity of the angiographic film (n=6 in each group). Interobserver variation was <5%.