Kuliszewski, et al.
SUPPLEMENTARY MATERIALS AND METHODS
Animal Preparation
The study protocol was approved by the Animal Care and Use Committee at St.
Michael’s Hospital Research Centre, University of Toronto. Proximal hindlimb adductor
muscle ischemia was produced in 126 Fisher F344 rats. Rats were anesthetized with
intraperitoneal injection of ketamine hydrochloride (90 mg·kg-1) and xylazine (10 mg·kg1
). Under sterile conditions, the left common iliac artery and small proximal branches
were exposed and ligated with 4-0 silk sutures. The incision was closed in layers and
animals were recovered. In this model, while flow is immediately reduced to ~25%
normal, endogenous angiogenesis occurs over the subsequent 2 weeks, after which
perfusion remains chronically reduced in the proximal adductor muscles at ~40-50% of
normal.[1-3] In this model of proximal hindlimb ischemia, we do not observe any limb
necrosis or auto-amputation.
Cell Preparation and Labeling
Endothelial progenitor cells (EPCs) used for all our experiments were isolated
from the tibias and femurs of 3-5 week old syngeneic Fisher 344 rats (Charles River).
The aspirated marrow was centrifuged and plated on fibronectin coated flasks at a density
of >1x106 cells/ml and grown in endothelial cell basal medium 2 (EBM-2) (Clonetics)
supplemented with 5% fetal bovine serum, VEGF-A, FGF-2, epidermal growth factor,
insulin-like growth factor-1, and ascorbic acid, to promote differentiation into an
endothelial phenotype.[4] EPCs were labeled the viable fluorophore chloromethyl
trimethyl rhodamine (CMTMR; Invitrogen). CMTMR provides an accurate method of
detecting ex vivo-labeled cells because the molecule undergoes irreversible esterification
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and glucoronidation after passing into the cytoplasm of a cell and thereby generates a
membrane-impermeable final product.[5] This active fluorophore is then unable to
diffuse from the original labeled cell into adjacent cells or structures, and may be
detected in vivo for several months. The labeling solution (25M) was prepared with
serum free EBM-2 media and the cells were incubated for 45min at 37C. Cells were
then washed and the labeling solution was replaced with fresh EBM-2 medium. The cells
were allowed to recover for 24 hours after labeling, before being injected.
EPC Characterization and Functional Analysis
Unlabeled and CMTMR-labeled EPCs were stained for the presence of mature
endothelial markers, endothelial specific lectin, UEA-1 (1:200) (Sigma), VEGFR-II
(1:50) (R&D Systems) and CXCR4 (1:150) (abcam). Percent marker positivity was
determined using FACS analysis.
In vitro EPC function was assessed by Boyden Migration and Matrigel Tubule
formation assays.[6] Unlabeled and CMTMR-labeled cells were serum starved for 30
min, after which time they were trypsinized, centrifuged at 360 RCF at 18°C for 10
minutes and re-suspended in 1mL of 10% EBM-2 medium diluted to a desired
concentration of 500,000 cells/mL. Two chemo-attractants were used, human stromal
cell-derived factor (SDF-1) (Sigma) and human vascular endothelial growth factor
(VEGF) (R&D Systems) at 100 ng/mL and placed in each well of the Boyden companion
plate. An 8 µm (pore size) insert was placed in each well containing 500µL of the cell
suspension. After a 4-hour incubation period, each Boyden chamber insert was gently
washed with 10% medium, and non-adherent cells were removed. Cells were fixed and
stained using DiffQuik (Sigma) and allowed to dry overnight. The membrane was
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subsequently removed and mounted on a slide for quantification using light microscopy
with a 20X objective.
Unlabeled and CMTMR-labeled EPCs were plated on BD BioCoat Matrigel
Basement Membrane coated 6-well plates (BD BioSciences, USA) at a density of 75,000
cells per well. Cells were suspended in 2mL of medium supplemented with 100ng/mL of
recombinant vascular endothelial growth factor (rVEGF) (R&D Systems) and grown for
24 hours. Tube formation was assessed by computer software (Image J).
Time Course of Exogenous EPC Circulation
The time course of circulating exogenous EPCs after an intravenous injection was
studied in 12 rats: 6 control non-ligated rats, and 6 rats at 2 weeks after induction of
hindlimb ischemia by left iliac artery ligation. CMTMR labeled EPCs (1x106 in 1 mL
sterile saline) were injected via the right jugular vein over the course of one minute.
Blood samples (100uL) were serially taken at 5, 30, and 60 minutes for short-term
tracking. For longer term tracking 100uL samples were taken at 1, 3, 7, 14 and 28 days
post injection. Whole blood samples were lysed with BD FACS lysing solution (BD
Biosciences), which provides an alternative to density gradient centrifugation, thus
making analysis of small volumes of blood feasible. Samples were analyzed by FACS to
quantify circulating CMTMR labeled cells and endogenous EPC numbers. EPCs were
characterized based on VEGFR-II, CD34 and CD133 marker positivity. Based on this
criterion we are able to assay for endogenous (triple marker positivity without CMTMR)
and exogenous (triple marker positivity + CMTMR) EPCs at the various time points and
determined what quantity of cells continue to circulate.
Microbubble and DNA Preparation and Assembly
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Microbubbles with a cationic lipid shell were created by sonicating an aqueous
dispersion of 1 mg•ml-1 polyethyleneglycol-40 stearate (Sigma), 2 mg•ml-1 distearoyl
phophatidylcholine (Avanti) and 0.4 mg•ml-1 1,2- distearoyl-3trimethylammoniumpropane (Avanti) with decafluorobutane gas.[2, 3] These
microbubbles have a zeta potential of +60 mV, and when incubated with plasmid DNA,
approximately 6700 plasmids are charge-coupled to the surface of each microbubble. For
perfusion imaging, lipid-shelled decafluorobutane microbubbles were used. Microbubble
concentrations were determined using a Coulter Multisizer IIe (Beckman-Coulter), prior
to intravenous administration.
Perfusion Imaging
Contrast-enhanced ultrasound (CEU) imaging of the proximal hindlimb adductor
muscles was performed with pulse inversion imaging (HDI 5000, Philips Ultrasound) at a
mechanical index of 1.0 and a transmit frequency of 3.3 Mhz.[1-3] Data were recorded
digitally, saved to magnetic-optical disk and transferred to a computer workstation for
off-line analysis. Perfusion in the adductor muscles was assessed during i.v. infusion of
lipid microbubbles (1•107 min-1) using a syringe pump (Baxter). Background images
were acquired prior to microbubble infusion. Intermittent imaging was then performed by
progressive prolongation of the pulsing interval (PI) from 0.2 to 20 s, using an internal
timer. Averaged background frames were digitally subtracted from averaged contrastenhanced frames at each PI. PI versus signal intensity (SI) data were fit to the function, y
= A (1-e-βt), where y is SI at the pulsing interval t, A is the plateau SI which is an index
of microvascular blood volume (MBV), and β is the rate constant reflecting
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microvascular blood velocity. Microvascular blood flow (MBF) was calculated by the
product of A and β.[7]
Gene Delivery
For ultrasound-mediated (UM) gene delivery, ultrasound transmission was
performed with a phased array transducer (Sonos 5500, Philips Ultrasound) at 1.3 MHz
and a transmit power of 0.9W (120 V, 9 mA), using a pulsing interval (PI) of 5 s. The
focus was positioned at the mid muscle.[2, 3] Cationic microbubbles (1.5 mL; 1x109)
charge-coupled with 500 μg of human SDF-1 cDNA (InvivoGen, San Diego, CA) was
infused over 10 minutes intravenously. To allow for a wider field of delivery, ultrasound
was transmitted during a slow sweep along the length of the proximal ischemic hindlimb,
for a total of 20 minutes.[2]
Fluorescent Microangiogaphy (FMA)
Fluorescent microangiography was performed as previously described.[2, 3]
Immediately prior to sacrifice, the distal hindlimbs were flushed with heparinized saline
via an abdominal aortic cannula, until clear venous return was seen. A 10% solution of a
50/50 mixture of fluorescent microspheres (0.2 μm and 0.02 μm) (Sigma) mixed with a
1% solution of low melting point agarose at 45oC was injected at constant rate into the
aortic cannula until seen on venous return. The animal was euthanized and placed in an
ice bath to facilitate rapid cooling and solidification of the casting agent. Tissue from
both hindlimbs were placed in 10% buffered formalin, sectioned (200 μm) using a
vibratome and visualized under confocal microscopy. A series of stacked images (4μm
slices) were taken and the middle 25 slices (100 μm total thickness) were projected in
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order to quantify the vessel density using automated software (IPTK analysis software,
Reindeer Graphics Inc.).[2]
Capillary and Arteriole Density by Branch Order
Sections from FMA were digitized and rendered in 3D using Neurolucida
Software package (MBF Bioscience). Rendered 3D image data sets were comprised of
skeletal muscle slices, each measuring 512μm x 512μm x 100μm. Vascular
reconstruction was performed and branch order analysis performed based on the Strahler
Method. [8-9] Briefly, the Strahler method (modified from the original Horton model)
classifies the vascular structure starting at terminal end points (Branch order 0). The
labeling of segments progresses toward segment origins or nodes. At each node, the
parent segment is labeled with an order number one larger than the daughter segment.
This ordering system is independent of branch length and results in the following
breakdown according to branch order; Distal Segments = 0, i.e. capillaries, while
arterioles carry higher branch orders of 1-3). (See Supplementary Figure 2). Based on
this ordering system a determination of branch order ratio (capillary-to-arteriolar ratio),
capillary density and arteriolar density can be automatically calculated. The vascular
density is measured by the software and expressed as number of vessels per volume (mm3
).
Immunohistochemistry
In vivo EPC engraftment and spatial localization was determined using
immunohistochemistry. Explanted tissue was cryo-embedded in OCT (Sakura, Japan)
and stored at -80oC. Cryo-blocks were sectioned (15 μm thick) every 25 μm and rehydrated in phosphate buffered saline (PBS), fixed in 2% paraformaldehyde (Sigma) in
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PBS, and washed with PBS. Cell surface antigens were identified using: mouse antihuman CD31 (Alpha Diagnostics Inc.), rabbit anti-human SDF-1 (Upstate
Biotechnology), mouse anti-rat alpha Actin (abcam) and an endothelial cell specific
Lectin (UEA-1) (Sigma). The presence of antibody was confirmed by exposure to a
phycoerythrin (PE) or FITC conjugated secondary antibody. TO-PRO-3 (Sigma) was
used as a nuclear marker.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Quantitative real-time RT-PCR for exogenous (human) and endogenous (rat)
SDF-1 transcript was performed as previously described.[2, 3] Hindlimb tissue was
homogenized using Trizol (Sigma), and total RNA was isolated using the GenElute
Mammalian total RNA kit (Sigma) and quantified by absorbance at 260 nm. Total RNA
was reverse transcribed in 20 μl volumes using Omniscript RT kit (Qiagen) with 1 μg of
random primers. For each RT product, aliquots (2-10 μl) of the final reaction volume
were amplified by real-time PCR reactions using standardized concentrations of RNA.
SDF-1 [ FWD 5’-TGGTCGTGCTGGTCCTC-3’, REVERESE 5’GGCAACATGGCTTTCGAAG-3’] and Cyclophilin [FWD 5’TGATCCAGGGTGGAGACTTC-3’, REVERSE 5 -GCCCATAGTGCTTCAGCTTG3’] specific primers and SYBR green (Applied BioSystems) were then used to detect
amplicon production using an ABI system.
Experimental Protocol
CEU perfusion imaging of the ischemic and contralateral control hindlimb
adductor muscles was performed 14 days after ligation, at a time when endogenous
angiogenesis was complete. Delivery was then performed, according to assigned
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treatment groups: group 1: control group, no treatment; group 2: ultrasound mediated
SDF-1 plasmid microbubble delivery; group 3: intravenous EPC delivery; group 4: SDF1 plasmid microbubble delivery plus intravenous EPC delivery (n=30 per group). CEU
imaging was performed at days 3, 7, and 14 post-delivery (n=10 per group). In 5 rats per
group (day 14) FMA was performed prior to sacrifice. Tissue for immunohistochemistry
and rt-PCR was obtained from the remaining rats’ ischemic and non-ischemic hindlimbs,
lungs, heart and liver.
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Kobulnik, J., Kuliszewski, M. A., Stewart, D. J., Lindner, J. R., and Leong-Poi, H.
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