Accumulation of extracellular matrix ECM is an- other major event in the intimal hyperplasia following
arterial injury, as are the migration and proliferation of SMCs [5]. Atherosclerotic lesions also contain a great
amount of various ECM components. Recent studies revealed that ECM not only maintains the structural
integrity of the tissues, but also interacts with cellular components and regulates their functions [6,7].
Proteoglycans are a family of noncollagenous glyco- proteins constituting ECM. The functions of proteogly-
cans are varied, and some of them are known to interact with particular cytokines [8]. Recent studies on
fibroproliferative lesions such as in liver fibrosis, [9] pulmonary fibrosis [10] and atherosclerosis [11] have
focused on two kinds of leucine-rich small proteogly- cans, biglycan and decorin, which can bind to trans-
forming growth factor-beta TGF-b, the most potent inducer of ECM synthesis [12,13]. Decorin, in particu-
lar, neutralizes the effect of TGF-b activities in vitro and in vivo [14,15]. These proteoglycans may therefore
have a role in the modulation of ECM synthesis and accumulation.
Atherosclerotic lesions also contain a large number of macrophages secreting various kinds of cytokines
such as TGF-b and interleukin-1 IL-1. Recently IL-1 was reported to enhance the production of decorin in
arterial SMCs in vitro, [16] suggesting the dynamic interactions of macrophages with ECM formation in
atherosclerotic lesions.
The above findings prompted us to examine the spatial and chronological distribution of the expressions
of decorin and biglycan in the association of TGF-b and IL-1 expression in normal and atherosclerotic rab-
bit aortas after vascular stent implantation in order to clarify the ECM kinetics in the process of neointima
formation.
2. Materials and methods
2
.
1
. Stent description The stent used was the same as that used in our
previous study [4], a 12-mm-long self-expandable device with an expanded diameter of 7.0 mm, made of 0.2-mm
stainless steel wire SUS304 with five bends in a zigzag pattern known as Gianturco’s Z type stent.
2
.
2
. Animals Thirty-six adult male New Zealand White rabbits
weighing 3.5 – 4.0 kg Kbl-NZW, Nagano, Japan were used in this study and divided into two groups. Eigh-
teen rabbits had been fed a diet containing 0.5 choles- terol ORC4, Oriental Yeast, Tokyo, Japan to induce
atherosclerotic lesions atherosclerotic group, and the others had been fed regular chow control group. The
stents were implanted in the aortas of 12 rabbits from the atherosclerotic group after 3 months on the choles-
terol diet and in 15 animals from the control group. The diets did not change until the time of sacrifice. The
procedures were in accordance with the Guide for Care and Use of Laboratory Animals issued by the US
Institute of Laboratory Resources.
2
.
3
. Stent implantation The stents were implanted as described in our preced-
ing paper [4]. In brief, general anesthesia was achieved with an intramuscular injection of xylazine 5 mgkg
and ketamine 5 mgkg, followed by an additional intravenous administration of 5 mg xylazine. A midline
incision was made in the ventral area of the neck after infiltration with 1 xylocaine, and the right common
carotid artery was exposed and used for arterial access in all animals. After heparin administration 300 Ukg,
a 4F intra-arterial sheath was placed in the descending thoracic aorta, slightly beyond the area of interest, over
a 0.028-in. tight J-wire. The stent was loaded into the sheath just before deployment in the contracted
configuration, and pushed to the distal end of the sheath by an inner catheter. Finally, the sheath was
withdrawn with the inner catheter being fixed to release the stent and allow it to expand at the proper place.
This procedure was repeated to implant another stent at a site just proximal to the first site. The right
common carotid artery was ligated, and the neck inci- sion was closed with interrupted sutures. Angiography
was performed before and immediately after the stent implantation with contrast medium Iohexol, Daiichi
Pharmaceutical, Tokyo, Japan to confirm the stent location. No anticoagulant agents were administered
except for the initial bolus injection of heparin.
2
.
4
. Animal sacrifice and tissue preparation Three animals from each group were sacrificed by
exsanguination while under deep sodium pentobarbitu- rate anesthesia 10 mgkg 4, 7, 14, and 56 days after
the stent implantation. In the control group, additional three animals were sacrificed after 28 days. For com-
parison, three animals without stent implantation were sacrificed in the control group, and also in the
atherosclerotic group after 3 and 5 months on the cholesterol diet. The aorta implanted with two stents
was dissected, briefly rinsed in 0.9 saline solution, and divided into two parts. Half of the specimen with one
stent was immersion-fixed in methanol-Carnoy’s fixa- tive 60 methanol, 30 chloroform, 10 acetic acid
overnight,
dehydrated through
three changes
of methanol, and embedded in paraffin. The metallic stent
wires were carefully removed under a dissecting micro-
scope, and the aorta was divided into three parts, each 4-mm long, before being embedded in paraffin. The
paraffin sections were stained with hematoxylin eosin as the routine stain, with Elastica van Gieson as the
elastin stain, and immunohistochemistry as described below. The specimen with the other stent was frozen in
an OCT compound Miles Scientific, Naperville, IL in a cryomold in liquid nitrogen after removal of the stent
wires, and used for the immunohistochemical staining for myosin heavy chain MHC isoforms, and mRNA
in situ hybridization as described below. The adjoining sites proximal and distal to the stent implantation site
were also examined in paraffin-embedded and frozen sections.
2
.
5
. Immunohistochemistry on paraffin-embedded sections and frozen sections
Paraffin-embedded sections were immunostained us- ing the immunoperoxidase streptavidin – biotin complex
system with nickel chloride NiCl color modification as previously described [4]. Monoclonal antibodies against
smooth muscle-specific a-actin 1A4, DAKO, Glostrup, Denmark, rabbit macrophages RAM11, DAKO,
proliferating
cell nuclear
antigen PCNA:
PC10, DAKO, and decorin 6B6, Seikagaku, Tokyo, Japan
were used on the paraffin sections as specific markers for
SMCs, macrophages,
proliferating cells,
and decorin, respectively. Triple immunostaining was per-
formed to identify the cell types of proliferating cells with an application of the immunoalkalinephosphatase
streptavidin – biotin complex system DAKO in combi- nation with the enhanced polymer one-step staining
EPOS system DAKO with and without NiCl color modification. This immunostaining procedure enabled
us to discriminate three immunostaining reaction prod- ucts in three different colors as follows; macrophages in
red, SMCs in brown, and PCNA-positive nuclei in black.
For the identification of SMC phenotypes, MHC isoforms SM1, SM2, SMemb were immunostained on
frozen sections as described previously [4]. SM1 is expressed in both adult and embryonic phenotypes of
SMCs, whereas SM2 has been observed only in adults, and SMemb only in embryonic SMCs. Thus SM2 can
be used as a marker for the adult phenotype and SMemb is specific for the embryonic phenotype of
SMCs. These antibodies were generated and kindly provided by Dr R. Nagai Gumma University, Japan.
2
.
6
. cDNA cloning and digoxigenin-labeling of cRNA probes
Each cDNA was cloned using the reverse transcrip- tion-polymerase chain reaction RT-PCR method. In
brief, total cellular RNA was extracted from the ho- mogenized rabbit aorta with an acid guanidine thio-
cyanate – phenol – chloroform extraction method using ISOGEN Nippon Gene, Tokyo, Japan. Single-
stranded cDNA was then synthesized with random ninemer oligonucleotide as a primer. One microgram of
the total cellular RNA was incubated at 30°C for 10 min and at 42°C for 30 min with AMV reverse tran-
scriptase XL TAKARA Shuzo, Otsu, Japan and the random ninemer in a total volume of 20 ml. After
incubation, the total reaction mixture was diluted with 100 ml of a PCR buffer. Respective antisense and sense
primers were designed for the targeted cDNAs; decorin, biglycan, TGF-b, and IL-1b; the nucleotide sequences
of the primers are listed in Table 1. Rabbit-specific cDNA of TGF-b and biglycan were not released.
Therefore, they were derived from the human sequence entry.
The reaction mixture for PCR contained 100 pmol each of the sense and antisense primers, and the reac-
tion was started by adding 2.5 U of Taq DNA poly- merase TAKARA Shuzo. The conditions for PCR
were 1 min at 95°C, 1 min at 55°C, and 1.5 min at 72°C for 35 cycles. Each PCR product was digested with two
proper restriction enzymes Table 1 and ligated into a plasmid pBluescript SKII
+
TOYOBO, Osaka, Japan. The obtained sequences of decorin and IL1-b cDNAs
were identical to published sequences. TGF-b and biglycan cDNAs were sequenced and we found ho-
Table 1 Strategy of cDNA cloning by RT-PCR
a
and subcloning into plasmids Fragment size bp
Insertion of plasmid Insert fragment
Antisense primer Sense primer
Eco-RIPst-I ACCAGGTGGTTCTTGGA
Eco-RIPst-I Biglycan
GMTTCTGGGACTTCAC 360
CTTGGA GATGTA
TTATTCAGTCCTTTCAGG 370
Hinc-IIBan-III Decorin
Hinc-IIBan-III GACCTGCAAAACMCAA
CT AAT
CACTGCAGTTGTGCGG 445
CACGTAGTAGACGATGG TGF-b
Pst-ISma-I Pst-ISma-I
CAGTGG GCAG
TGMTGGCAGAGGTMG IL-1b
GGTCCCMTTACATGMGA 550
Sma-IEco-RI Aat-IEco-RI
AGAG GC
a
RT-PCR, reverse transcription-polymerase chain reaction.
Fig. 1. Chronological changes in intimal thickening expressed as the intimal index intimal areamedial area after stent implantation. The
stent-implanted site of control animals
; the stent-implanted site of the atherosclerotic animals ; the immediately distal portion of
the stent-implanted site of atherosclerotic animals .
2
.
8
. Measurement of the intimal area The measurement of the intimal area was performed
on the middle part of the three paraffin sections taken from the aorta with a stent, because the stent wires
aligned in almost the same distance in these sections. The sections of the aorta just distal to the stent implan-
tation sites were also measured as an internal control of primary atherosclerotic lesions. Intimal and medial ar-
eas were measured in each section stained with Elastica van Gieson stain using a computerized image-analysis
system Image Command 5098, Olympus, Tokyo, Japan. The area of the stent wires was excluded from
the measurement. In the atherosclerotic group, we mea- sured the intimal areas including both newly-formed
intima after stenting, and the primary atherosclerotic lesion that had been formed prior to stenting, since
their boundaries were undelineable.
3. Results
