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
3
.
1
. Chronological changes of fibrofatty lesion formation without and after stent implantation
3
.
1
.
1
. General findings After the rabbits had been fed the cholesterol diet for
3 months, their serum cholesterol levels rose from 1.240 9 0.36 – 28.62 9 12.29 mmoll, while triglyceride
levels showed no significant changes 0.5770 9 227 – 0.243 9 0.236 mmoll. On gross examination, the tho-
racic aortas of the cholesterol-fed animals sacrificed without stenting presented extensive atherosclerotic le-
sions on the luminal surface. These atherosclerotic le- sions were considered to be an advanced stage of fatty
streaks which are called ‘fibrofatty lesions’ [18].
The process of neointima formation after stent im- plantation in the aortas of the atherosclerotic group
was compared with that of the control animals which is described in our previous paper [5]. In the control
animals, the intimal index increased gradually until day 14, and no further increase was seen thereafter, whereas
in the atherosclerotic group, the intima expanded markedly and became circumferential, covering all of
the luminal surface of the aorta by day 14, continuing to thicken up to day 56. The intimal areas of both
groups were measured and expressed as the intimal index, and the chronological changes of both groups
are compared in Fig. 1. The intimal index at the stent-implanted site was much greater than that of the
internal controls measured at the immediately distal portion of the stent-implanted site at each point, and
the differences became greater as time elapsed.
Cell type-specific immunohistochemistry revealed that fibrofatty lesions without stenting were made up of
an intimal accumulation of lipid-laden macrophages mologies of 94.4 and 89.1 compared to the human
sequences, respectively. Each plasmid was amplified in Escherichia coli and
obtained using CsCl ultracentrifuge methods, then lin- earized with restriction enzymes. Digoxigenin-labeled
antisense and sense RNA probes were generated by T3 or T7 RNA polymerase, respectively.
2
.
7
. Messenger RNA in situ hybridization Messenger RNA in situ hybridization was performed
on the serial 3 – 4-mm thick frozen sections of stent implanted tissues essentially as described by Hirota et
al. [17]. In brief, the sections were fixed with phosphate- buffered 4 paraformaldehyde for 10 min, acidified
with 0.2 M HCl for 10 min, and acetylated with 0.25 acetic anhydride in 0.1 M triethanolamine, pH 8.0, for
10 min. They were air-dried after dehydration with a graded series of ethanol, and hybridized with both
antisense and sense probes at 50°C for 16 h. Hybridized digoxigenin-labeled probes were detected using a nu-
cleic
acid detection
kit Boehringer
Mannheim, Mannheim, Germany. After the color reaction, the
slides were rinsed with 10 mM Tris – HCl, pH 8.0, 1 mM
EDTA, fixed
with phosphate-buffered
4 paraformaldehyde for 15 min, dehydrated through a
graded ethanol series, permeated with xylene, and cov- ered with coverslips. The sense probes were used in-
stead of antisense probes for negative control analysis. These probes have the same GC ratio as the comple-
mentary antisense probes, and thus have a propensity for nonspecific hybridization. The control hybridization
using these sense probes was uniformly negative data not shown. The same process of hybridization with the
omission of the cRNA probe was performed, and the signal was negative except for a nonspecific reaction in
the adventitia.
positive for RAM11, and intermingled with a-actin- positive SMCs. In the lesions, macrophages were domi-
nant Fig. 2A. By day 14 following the stent implantation in the atherosclerotic group, the stent wire
was covered by a fibrocellular layer containing dense a-actin-positive SMCs, and the layer thickened further
after day 56. A large number of RAM-11-positive macrophages accumulated adjacent to the stent wires,
and some of them were scattered in the layer above the fibrocellular tissue composed of SMCs on days 14 and
56 Fig. 2B.
3
.
1
.
2
. Immunohistological findings The immunostaining for MHC isoforms in the frozen
sections of thoracic aorta without stent implantation showed that medial SMCs were stained positive for
SM1 and SM2, and negative for SMemb except for weak staining in the superficial media, while the intimal
SMCs were SMemb-positive and SM2-negative, indi- cating that embryonic phenotype SMCs predominate in
the fibrofatty lesions Fig. 3A and B. Endothelial cells were also stained positive for SMemb, as in the normal
aorta [4], whereas, in the atherosclerotic thoracic aorta 56 days after implantation, the intimal SMCs remained
SMemb-positive and SM2-negative throughout the ex- perimental period Fig. 3C and D.
3
.
2
. Chronological changes in extracellular matrix — immunohistochemistry and mRNA in situ hybridization
3
.
2
.
1
. Control group without stent implantation By immunohistochemistry, the media were not
stained for decorin, while the adventitia were strongly stained. With in situ hybridization, neither biglycan nor
decorin mRNAs were detected in the intima or media of the control animals. Detection of mRNA signals in
the adventitia was not possible because of a nonspecific reaction data not shown.
3
.
2
.
2
. Control group after stent implantation By immunohistochemistry, decorin was not stained in
the neointima or media throughout the experimental period; it showed strong and constant staining only in
the adventitia Fig. 4. In an mRNA in situ hybridization study, biglycan
and TGF-b mRNA were found to be maximally ex- pressed in the media around the sites injured by the
stent wires on day 7 Fig. 5A and B, whereas they were expressed slightly on day 7 and maximally on day 14 in
the fibrocellular neointima Fig. 5D and E. These expressions were transient and had almost disappeared
on day 56 Fig. 5G and H. These mRNA expressions were closely associated with the distribution of SMemb-
positive embryonic type SMCs Fig. 5C, F and I.[4] The gene expressions of decorin and IL-1b were unde-
tectable in the intima and the media throughout the experimental period.
3
.
2
.
3
. Atherosclerotic group without stent implantation In the atherosclerotic group without stent implanta-
tion, decorin immunostaining was faintly positive in the intima, in addition to strong staining in the adventitia,
as observed in the control animals. With in situ hy- bridization, however, biglycan and decorin mRNAs
were undetectable in the intima and media data not shown.
3
.
2
.
4
. Atherosclerotic group after stent implantation As described above, stent implantation induced accel-
erated and extensive neointima formation. Decorin was not recognized in the newly-formed intima until day 14,
but it was deposited in such a manner that it concealed the macrophages that accumulated around the stent
wire on day 56 Fig. 6A and B.
The in situ hybridization showed that both biglycan and TGF-b mRNAs were expressed in the cells around
the injured area of media on day 4, and newly formed intima around the stent wire was seen on day 7 Fig. 7A
Fig. 2. Triple-immunostained rabbit thoracic aortas without stent implantation A and on day 56 after stent implantation B in the
atherosclerotic group. Red staining shows macrophages, brown stain- ing shows SMCs, and black staining shows PCNA-positive nuclei,
respectively. Bar, 30 mm, and applies to both panels.
Fig. 3. Immunohistochemical stainings for the myosin heavy chain isoforms SM2 A and C and SMemb B and D in frozen sections of thoracic aortas. Aortic tissues were dissected from animals sacrificed without stent implantation A and B and on day 56 after stent implantation C and
D. Bar in panel B, 20 mm, and applies to panels A and B. Bar in panel D, 40 mm, and applies to panels C and D. Asterisks in C and D indicate vacuities due to stent wires.
and B similar to normal aorta. The expression of the IL-1b gene was also detected in the neointima around
the stent wire on day 7 Fig. 7C unlike the result in the control group. Biglycan and TGF-b mRNA expressions
continued to be widely present in the neointima throughout the experimental period Fig. 8A and B.
The IL-1b expression was detected strongly around the stent wire, where macrophages were observed Fig. 8C.
Decorin mRNA was not detected until days 7 and 14 data not shown. On day 56, its expression was ob-
served in SMCs which surrounded the macrophages accumulating near the stent wires Fig. 8D.
4. Discussion
