Supplemental Methods
Histology
For immunofluorescent staining, tissues were embedded in OCT (Sakura
Finetechnical Co., Tokyo, Japan), snap frozen in liquid nitrogen, and stored at -80°C until
use. Cryosections (5 m) were prepared, fixed in acetone and stored at -20°C until use.
For digestion of tissue sections, heparinase I (0.25 units) was brought up in digestion
buffer [0.03M tris(hydroxymethyl)aminomethane (Tris)-HCl, 3.3 mM calcium acetate,
6mM sodium acetate, pH 7.0] containing 1mM phenylmethanesulphonylfluoride
(PMSF). The heparinase solution was applied to the tissue sections for 2 hours at 37°C,
after which the sections were washed and stained per routine. All primary antibodies
(chicken anti-human IL-2, rabbit anti-
-perlecan, mouse anti- HS,
and biotinylated anti-CD31, goat anti-human IL-2R), their appropriate secondary
antibodies [biotinylated rabbit anti-chicken (Abcam), goat anti-rabbit IgG, biotinylated
goat anti-mouse IgG, and biotinylated donkey anti-goat IgG (Jackson ImmunoResearch
Labs, West Grove, PA)], and UEA-1-FITC (Sigma) were each diluted in
immunohistochemistry buffer (BupH Modified Dulbecco’s PBS, 0.01% Tween-20, 0.1%
saponin). Streptavidin-horseradish peroxidase was applied to the biotinylated primary or
secondary antibodies (anti-IL-2, anti-CD31, and anti- HS) followed by tyramide
amplification (Invitrogen, Carlsbad, CA). Tissue sections were mounted in Permount
(Biomeda, Foster City, CA) and visualized using a Nikon Eclipse 80i microscope.
Chicken anti-IL-2 antibodies were pre-adsorbed with human IL-2 by incubating
the anti-IL-2 antibodies at 4°C for 18 hours with a 5M excess of human IL-2 (BD
Pharmingen).
Permanent sections were prepared by preserving pieces of murine aortas in 4%
formalin and then embedding them in paraffin blocks. Five m sections were cut,
deparaffinated, and stained with Verhoeff van Gieson stain.
Images were captured using a Nikon 80i Eclipse camera equipped with
Metamorph image acquisition software. Confocal images were generated using a Zeiss
510 Meta confocal microscope utilizing AIM software. The signal for each fluor was
maximized without over-saturation of the image and identical settings were used to
capture all images.
Western blot analysis
Western blot analysis of cell lysates and supernatants was performed as follows.
CTLL-2 cells (1 x 105) were cultured for 72 h in RPMI 1640 media with 5% FBS. The
cells were pelleted by centrifugation, and the supernatant was collected. An equal
volume of 2X Laemmli running buffer (Bio-Rad, Hercules, CA) containing 5% v/v mercaptoethanol (Bio-Rad) was added to the media which was then separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels (Bio-Rad),
transferred to 0.2 m nitrocellulose (Bio-Rad), blocked with Tris-buffered saline (TBS)
with 0.25% Tween-20 (TTBS) then probed overnight with an anti-heparanase antibody
(Platt antibody) in TTBS. After washing with TTBS, bound antibody was detected with a
horseradish peroxidase conjugated anti-rabbit secondary antibody (Abcam) in TTBS.
The blots were developed using enzyme chemiluminescence (Pierce Biotechnology,
Rockford, IL) and blue X-ray film (Phenix, Hayward, CA). Select media from CTLL-2
cultures was passed through a heparin-sepharose column. Bound proteins were eluted
with 2M NaCl and then assessed for the presence of heparanase as described above.
Lysates of CTLL-2 cells were prepared by resuspending the cell pellet in 50mM
Tris-HCl, pH 7.5, containing 4M urea and 1mM PMSF and sonicated on ice. An equal
volume of 2X Laemmli running buffer containing 5% -mercaptoethanol was then added
to the resulting cell lysate preparation. The cell lysates (5 l) were then assessed for
mammalian heparanase expression by Western blot analysis as described above.
Western blot analysis of arterial tissues was performed by first mincing aortic
tissue into pieces, rinsing the pieces in PBS, and then homogenizing the minced tissue in
ice-cold 4M urea buffer (100 l buffer/g tissue) containing 0.05% Triton X-100 v/v and
1 mM PMSF. The homogenate was sonicated on ice followed by centrifugation to
remove insoluble material. The resulting supernatant (extract) was collected, and the
protein concentration measured by DC protein assay (Bio-Rad). An 85 g aliquot was
then mixed with an equal volume of 2X Laemmli running buffer containing 5% v/v mercaptoethanol. The samples were then analyzed by Western blot as previously
described.
Heparinase I digestion of tissues was performed by incubating 6, 2 x 2 mm, pieces
of human iliac artery in digestion buffer (see Immunohistochemistry) containing 2 U
heparinase I for approximately 18 h at 37°C. The vessel tissue was pelleted by
centrifugation and the resultant supernatant collected and total protein present determined
by DC protein assay (Bio-Rad). A 50 g aliquot was mixed with an equal volume of 2X
Laemmli running buffer (Bio-Rad) containing 5% v/v -mercaptoethanol and separated
by SDS-PAGE on a 10-20% gradient gel. Following transfer to nitrocellulose paper, the
resulting blot was developed as described above.
RNA Extraction and reverse-transcriptase PCR
Total RNA was extracted from pulverized spleen, aorta, or cultured smooth
muscle cells using RNeasy Mini kit (Qiagen) per manufacturer’s instruction. Aortic
tissues were prepared for extraction by removing the adventitia and then rinsing minced
pieces of aorta in PBS. The final concentration of RNA was determined by a
spectrophotometer. Reverse transcription was preformed using 250ng of RNA by firststrand cDNA synthesis using Superscript II (Invitrogen) per manufacturer’s instruction.
Reverse transcriptase-PCR was preformed using 2µl of cDNA template using Dream Taq
DNA polymerase (Fermentas, Glen Burnie, MD). PCR reactions were performed with
10x Dream Taq pH 8.4 buffer, 2.0mM MgCl2, 0.2mM dNTPs, 0.3µM primers, 5 units/µl
Dream Taq. The primer sequences are: murine IL-2 sense 5´-ATG TAC AGC ATG CAG
TCT GCA TC-3´, anti-sense 5´-GGC TTG TTG AGA TGA TGC TTT GAC A-3´
(product size 502 bp); murine -actin sense 5´-AAA TCG TGC GTG ACA TCA AA-3´,
anti-sense 5´- ATG CCA CAG GAT TCC ATA CC-3´ (product size 202bp). Human IL2R sense 5´ – CCG TGG CTC GGC CAC CTC-3´; anti-sense 5´-TAG GGG TCG 5´TAA GTA AAG TAC ACC-3´ (product size 437 bp). Human  actin sense 5´-CGT
GGA CAT CCG TAA AGA CC-3´; anti-sense 5´-ACA TCT GCT GGA AGG TGG AC-
3´ (product size 209 bp). PCR products were analyzed by electrophoresis on 2% agarose
gels stained with ethidium bromide and visualized using Bio-Rad gel doc system.
Statistical analysis – aortic morphometry
For the aortic morphometry, eighty independent measurements were performed
on tissue sections from 2 mice/group (IL-2 WT and KO) for a total of 320 measurements.
Preliminary analysis and descriptive statistics did not reveal the presence of outliers,
although it was discovered that the data did not follow a normal distribution. After
transformation of the original measurements to a logarithmic scale, the goodness–of-fit
tests did not reject the hypothesis of normality. The transformed data was then analyzed
with a general linear mixed model (SAS procedure “Mixed”) to test the null hypothesis
that the average percentage interlaminar space of the wild type was not significantly
different from the average percentage of the knockout type. The output from SAS
procedure MIXED leads to the rejection of the null hypothesis and the conclusion that the
average percentage interlaminar space of the wild type is significantly higher than the
average percentage of the knockout type with a p-value of < 0.0001.