1690
Allograft-Induced Arterial Wall Injury
and Response in Normotensive and
Spontaneously Hypertensive Rats
Didier Plissonnier, Bernard I. Levy, Jean-Loup Salzmann,
Dominique Nochy, Jacques Watelet, and Jean-Baptiste Michel
The role of genetically determined immune attack and blood pressure in graft rejectioninduced arterial wall injury and response was assessed by studying the compliance and changes
in wall structure of aortic isografts and allografts in normotensive (Wistar-Kyoto [WKY]) and
hypertensive (spontaneously hypertensive [SHR]) rats. Six groups of 8-week-old rats were
compared: sham-operated in both strains, isografts, and allografts between the two strains
(SHR aortas grafted in WKYs, designated SWs; WKY aortas grafted in SHRs, designated WSs;
isografts in SHRs, designated SSs; and isografts in WKYs, designated WWs). Each arterial
graft was studied 8 weeks after transplantation for volume and compliance (pressures of 75-175
mm Hg) under basal conditions. The amounts of collagen, elastin, and nuclei in the media and
intima of the walls of control and grafted aortas were quantified morphometrically. Isografts
and controls had the same mechanical characteristics under basal conditions: the arterial
volume and arterial compliance of hypertensive rats were lower than those of normotensive rats
(/?<0.001). Allografts had a greater initial volume (/><0.001) and a lower compliance
(p< 0.001) than did isografts. Allografts in SHRs (SSs) were initially dilated, whereas
allografted WKYs (WWs) were not There was intimal proliferation in hypertensive isografts
(14±0.77 /tin) and in both types of allografts (WS, 69±1.55 /tm; SW, 44±1.81 /tin); nucleus
density was higher in hypertensive allografts (WS) than in normotensive allografts (SW); and
collagen density was also higher in SW than in WS allografts. Allografts had decreased medial
thickness and decreased smooth muscle cell density. Medial thickness and the absolute
amounts of elastin and collagen were significantly lower in SWs than in WSs. Rejection of the
arterial graft had major effects on arterial wall injury and response in both strains. The
thickness and composition of the intimal proliferative response was mainly correlated with the
genetically determined blood pressure and smooth muscle cell proliferative potential. In
contrast, differences in the medial structural changes of SWs and WSs were correlated mainly
with the genetically determined immune process. Functional changes were therefore related
to changes in both intimal and medial structure. (Arteriosclerosis and Thrombosis
1991;ll:1690-1699)
T
he arterial system is one of the main targets of
chronic graft rejection.1-5 In particular,
chronic rejection-induced coronary artery
disease is a major impediment of long-term survival
in human heart transplantation.6 High blood pressure is frequently one of the factors involved in graft
rejection, especially in the rejection of renal and
cardiac grafts. However, despite their clinical impor-
From INSERM Unit 36 (J.-B.M.), INSERM Unit 141, Lariboisiere Hospital (B.I.L.), and INSERM Unit 28, Broussais Hospital
(J.-L.S., D.N.), and the Department of Surgery, Charles Nicole
Hospital (D.P., J.W.), Rouen, France.
Address for correspondence: Jean-Baptiste Michel, MD, PhD,
INSERM Unit 36-17, rue du Fer-a-Moulin, 75005 Paris, France.
Received October 8, 1990; revision accepted May 28, 1991.
tance, the mechanisms of injury and response in
vascular graft rejection remain poorly documented.
Vascular graft rejection between two allogenic
strains of rats (Brown Norway and Lewis) has been
reported by Schmitz-Rixen et al,7 but to our knowledge there have been no published studies of the
effects of high blood pressure on allograft rejection.
Spontaneously hypertensive rats (SHRs) differ
from their normotensive controls (Wistar-Kyoto rats,
WKYs) in several ways, particularly in their vascular
smooth muscle mass and immune response. The
SHR strain has been genetically selected for its
abnormally high blood pressure. This genetic selection is associated with hyperplasia and hypertrophy
of smooth muscle cells in the arterial wall in vivo,8
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Plissonnier et al
and with an increase in smooth muscle cell proliferative potential in vitro (in response to growth factors).9-14 SHRs are also immunologically depressed
because they possess fewer than normal thymodependent lymphocytes, which are involved in a delayed type of hypersensitivity and allograft rejection.15"18 SHRs and WKYs have different haplotypes
in the major histocompatibility complex (SHR, RTlk
and WKY, RTl1*)*9-20 and are thus histoincompatible.
We therefore designed an experimental allograft
model with SHRs and WKYs to study the interaction
of genetically determined immune response, high
blood pressure, and smooth muscle cell proliferation
with arterial wall injury and response during graft
rejection without immunosuppression. In this model,
segments of abdominal aortas from SHRs were
grafted into WKYs and vice versa. The injury and
response of these arterial allografts were compared
with those of isografts and sham-operated controls.
Graft functional and mechanical properties were analyzed in terms of the structural changes induced by
graft rejection, with and without hypertension.
Methods
Experimental Design
One hundred fifty 8-week-old male rats (IffaCredo, Lyon, France) were used in this study: 75
SHRs (body weight, 190±27 g) and 75 normotensive
control WKYs (body weight, 200±27 g). All animals
were cared for in compliance with the European
Community standards on the care and use of laboratory animals (Ministere de l'agriculture, France, autorisation No. 00577, 1989).
Aortas were transplanted into 60 rats by use of
microsurgical techniques. Rats were anesthetized
with pentobarbital (5 mg/100 g body wt i.p.). Two
animals were operated on simultaneously, one as the
donor of the aortic graft and the other as the
recipient. With the aid of the operating microscope,
the infrarenal aorta was exposed from the left renal
vein to the aortic bifurcation via a midline laparotomy. Any aortic branches in this segment were
identified and ligated. Two microclips were placed,
one below the renal arteries and the second above
the aortic bifurcation. A 1-cm segment of aorta was
removed from the donor animal and washed with
saline serum. A similar resection of the infrarenal
aorta was performed in the recipient rat. The donor
segment was inserted in the aorta of the recipient
with end-to-end interrupted anastomoses with 9-0
nonabsorbable monofilament nylon sutures. The total time of warm ischemia was 30-50 minutes, corresponding to the time of aortic clamping. Each
animal's graft patency was evaluated. No acute secondary graft thrombosis occurred during any of these
experiments.
The laparotomy was closed, and the rat was returned to its cage. All rats were fed a standard diet,
and water was provided ad libitum. They were investigated 8 weeks later (at 16 weeks old).
Allograft Injury in SHRs and WKYs
1691
Fifteen SHRs (SS) and 15 WKYs (WW) were
isografted with an aortic graft from the same strain.
Fifteen SHRs were allografted with aortic grafts
from WKYs (WS), and 15 WKYs were allografted
with an aortic graft from SHRs (SW). Fifteen rats
of both strains were sham operated under similar
conditions, including ligature of collateral vessels,
without gTafts (control SHRs [SCs] and control
WKYs [WCs]). No immunosuppressive treatment
was used in the present study.
Measurements of Aortic Graft
Static-Mechanical Properties
Eight weeks after graft microsurgery, animals were
investigated while they were under Inactin anesthesia
(0.1 mg/100 g body wt i.p.). A Teflon catheter (0.6-mm
i.d.) connected to a Statham P23 pressure transducer
(Gould Instruments, Qeveland, Ohio) was introduced
into the right carotid artery for blood pressure recording. The left femoral artery was catheterized with a
Teflon catheter (0.6-mm i.d.), and the tip of the
catheter was positioned in the infrarenal aorta. The
infrarenal grafted aorta and the position of the catheter were checked via a midline laparotomy.
The aortic catheter was filled with a solution of
Evans blue dye in 0.9% saline and 4% bovine serum
albumin and connected to an adjustable manometer.
A three-way tap was placed between the manometer
and the nylon tube, permitting part of the tube to be
filled to allow observation of the position of the
meniscus. The root of the infrarenal aorta was dissected free, and a removable clamp was positioned
just above the upper anastomosis. This preparation
allowed us to isolate about 10 mm of abdominal aorta
in situ (Figure 1, upper panel).
To start the measurements, the segment of isolated
artery was first exposed to atmospheric pressure for 5
minutes, and the position of the meniscus was recorded. The artery was then submitted to stepwise
increases in pressure of 75 mm Hg each. The movement of the meniscus, representing changes in the
volume contained within the arteries, was observed and
noted every 10 seconds for 5 minutes. Inflow was rapid
during the first 30-45 seconds and then became linear
with time. The initial transient increase in volume with
pressure was assumed to result from viscoelastic behavior of the tissue and relaxation of vascular smooth
muscle.21 The later linear inflow within the aortic artery
after this initial increase in arterial volume could be
attributed tofluidfiltration through the vascular wall.22
The initial increase in volume free of viscoelastic effects
was estimated by extrapolating the linear portion of the
inflow curve to the time when the pressure step was
applied. These measurements were repeated for pressures ranging from 75 to 175 mm Hg in 25 mm Hg
increments (Figure 1, lower panel). Each pressure was
maintained for 5 minutes. The volumes of the aortic
segment at each pressure were used to construct a
pressure-volume relation for each rat, for which the
slope of the curve represented the aortic compliance.23
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1692
Arteriosclerosis and Thrombosis
Vol 11, No 6 November/December 1991
Morphological Studies
Time (min)
FIGURE 1. Upper panel: Schematic drawing of experimental system for measuring pressure-volume relation in the
abdominal aorta in situ: catheterization of the left femoral
artery, Evans blue dye filling, meniscus deplacement measurement, and adjustable manometer. P, pressure. Lower panel:
Curves showing pressure-volume relations in the abdominal
aorta, with pressure steps of 75 mm Hg each for 5 minutes,
including the effect of water filtration through the arterial wall.
Initial increase in volume free of viscoelastic effects (dashed
lines) was estimated by extrapolating the linear portion of the
inflow curve to the time when the pressure step was applied.
At the end of the pressure-volume measurements,
the 1 cm of grafted abdominal aorta was fixed in situ
by infusion of Duboscq Brazil (formol, ethanol, and
picric acid) solution at mean arterial pressure, removed, dehydrated, and longitudinally embedded in
paraffin for light microscopy. Three successive sagittal sections (5 p,m) were cut and stained to obtain a
monochromatic color for each of the various structures studied in the aortic wall. Collagen fibers were
stained with Sirius red, elastin was stained with
orcein, and nuclei were stained with hematoxylin
after periodic acid oxidation. Slides were analyzed in
an automatic image processor (NS 1500, NachetVision, Paris, France).24 An additional section was
stained with Masson's trichrome for standard histological examination. Algorithms were developed to
analyze structures stained in each of the three successive sections. The first algorithm analyzed the
mean medial thickness by measuring the distance
between the internal and external elastic laminae (20
measurements on each section). The medial elastin
network was analyzed in terms of relative area, and
measurements and calculations were made for 10
fields in each section. The second algorithm analyzed
the collagen matrix by measuring the relative area,
density, and mean thickness of collagen fibers in 20
contiguous fields in each Sirius red-stained section.
The third algorithm counted the number of nuclei
within 20 fields in each section and measured the
mean area of each nucleus. Repetitive measurements
were performed, pooled, and averaged for the three
algorithms on the corresponding stained sections of
the aortic wall media of each animal. Similar measurements of nucleus density, collagen, and elastin
were performed for intimal proliferation. In controls
and normotensive isografts, the intimal thickness in
these vessels was below the resolution of the measuring system and hence, was expressed as zero.
Statistical Analysis
Results are expressed as mean±SEM. The experimental design allowed two-way repeated or factorial
analysis of variance to be used to show the effect of
blood pressure, the type of the graft (isograft or
FIGURE 2. Arteriographic appearance, 8 weeks after allograft, of the
abdominal aorta in recipient spontaneously hypertensive rats (SHRs) (right
panel) and recipient Wistar-Kyoto rats
(WKYs) (left panel). The SHR to
WKY allograft was well calibrated,
whereas the WKY to SHR allograft
remained dilated.
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Plissonnier et al
Allograft Injury in SHRs and WKYs
TABLE 1. Blood Pressures and Body Weights of Each Experimental Group (n=15)
1693
WS
1WW
Recipient
Systolic blood pressure (mm Hg)
Controls («=30)
Isografts (/i=30)
Allografts (n=30)
Diastolic blood pressure (mm Hg)
Controls (n=30)
Isografts (n=30)
AJlografts (n=30)
Body weight (g)
Controls (n=30)
Isografts (n=30)
Allografts (/i=30)
WKY
SHR
(n=45)
(n=45)
131±5.8
118±7.9
130±2.6
210±8.2
211±8.5
204±10.2
105±6.15
95 ±7.94
105±333
151±7.91
158±6.15
155±9.23
363 ±3.84
365±4.10
359±2.82
365 ±333
371 ±1.79
367 ±230
Values are mean±SEM.
WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats.
allograft), and the recipient effect on the functional
and structural parameters. The pressure-volume
relations were analyzed by repeated two-way analysis of variance (effect of incremental pressure is the
repeated measure, the effect of the experimental
model and the interaction between the two representing the differential effect of the model, on
aortic compliance). When this first statistical analysis demonstrated significant interaction, pressurevolume relations were treated as linear regression
curves (least-squares method), thereby permitting
comparisons of slope (compliance).
Results
Blood Pressure
All SHR recipients had significantly higher blood
pressures than did the WKYs (Table 1) (F=86.74,
/?<0.001). Allografts did not change blood pressure
in either SHRs or WKYs.
Macroscopic Aspect
There were macroscopic differences between the
different types of grafts. Immediately after uriclamping, the isograft diameter appeared to be similar to
that of the recipient aorta. Allografts initially appeared not to be calibrated to the recipient: aortas
from normotensive rats grafted onto hypertensive
recipients (WS) were initially dilated, whereas aortas
from hypertensive rats grafted onto normotensive
recipients (SW) were contracted.
Eight weeks later at the time the animals were
killed, WS allografts remained dilated, whereas SW
allografts seemed to be well calibrated. A typical
example is shown in Figure 2.
Pressure-Volume Relation
There was no significant difference between controls and isografts. The pressure-volume relations
ISO
1X0
80
120
-no
Pressure (mmHg)
FIGURE 3. Pressure-volume relations in allografts (right
panel) and isografts (left panel) (mean±SEM, n=15 per
group). By analysis of variance, time effect p < 0.002; strain
effect p<0.001; interaction p<0.001; difference in slope
p<0.05. WW, Wistar-Kyoto isografts; SS, spontaneously
hypertensive rat isografts; WS, Wistar-Kyoto to spontaneously
hypertensive allografts; SW, spontaneously hypertensive to
Wistar-Kyoto allografts.
were significantly shifted toward the pressure axis in
hypertensive rats.
The normalized arterial volumes of SHRs (SC and
SS) were significantly lower than those of WKYs
(WC and WW; F=38.25,/><0.001), and the arterial
compliance was lower in hypertensive rats, as shown
by the significant interaction between strain and the
pressure-volume relation (F =11.25, /><0.001). This
result was confirmed by the difference in pressurevolume slopes (f=2.08,p<0.05).
Allografts produced both a significant increase in
initial volume (F=44.4, /><0.001) and a significant
decrease in compliance (F=11.9, /?<0.001) (Figure
3). There were also functional differences between
WSs and SWs. The aortic volumes of WSs were
higher than those of SWs at all blood pressures
(F=51.42,/?<0.001), and the compliance of WSs was
significantly higher than that of SWs (interaction
F= 10.02, p< 0.001). These results were confirmed by
the pressure-volume slopes of SWs and WSs (f=4.35,
/xO.001) (Table 2).
Morphological Results
The isografts appeared normal by light microscopy,
whereas the allografts were the sites of rejection. In
our study all these WS and SW aortic allografts
showed evidence of acute rejection. The histological
signs of rejection at 8 weeks were adventitial cellular
infiltration, intimal thickening, and medial necrosis.
There were two patterns of mononuclear inflammatory infiltrates: 1) diffuse and massive adventitial and
periadventitial infiltrates and 2) focal cellular infiltration of the thickened intima. In most cases, the
thickened intima showed some focal fibrinoid necrosis. At this time a few mononuclear cells in the media
were associated with pronounced smooth muscle cell
necrosis. The media of all specimens had lost the
normal population of smooth muscle cells, and the
elastic fiber network was disrupted. There were few
isolated mononuclear cells (Figure 4). The lumina of
the vasa vasorum was often plugged with fibrin,
platelets, and leukocytes. A monolayer of endothelial
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1694
Arteriosclerosis and Thrombosis
Vol 11, No 6 November/December 1991
TABLE 2. Pairwise Comparisons of Parameters of Pressure-Volume Relations in Experimental Groups Under
Basal Conditions
Recipient
WKY
(»=45)
SHR
(»=45)
t
Slope (P/V)
A
Controls (n=30)
NS
}
*
0.024 ±0.001
0.031 ±0.002
*
0.023 ±0.002
1
NS
Isografts (n=30)
Allografts (n=30)
0.034±0.002
NS
J
t
0.018+0.0015
0.024+0.001
J
}
NS
Values are mean±SEM.
WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats.
0.01; tp<0.001.
cells was present in all the experimental group,
including the allografts.
Morphometric Results
Intima. There was intimal proliferation in hypertensive isografts (SS) and both types of allografts but
not in controls (WC and SC) or normotensive
isografts (WW). The intimal thicknesses of hypertensive SSs and WSs were significantly greater than that
of normotensive WWs and SWs (F=16.75,/?<0.001).
The allografts in WSs and SWs also had significantly
thicker intimas than did the isografts (F=105,
/?<0.001) (Figure 5). Morphometric analysis of intimal content was only performed on allografts (WS
and SW).
The numbers of myointimal cells (nucleus density) in
WSs and SWs were significantly different (Table 3)
(F=118.5, p<0.00l). Nucleus density was significantly
higher in WSs than in SWs. The intimal cell populations were heterogeneous, containing mainly smooth
muscle cells and some mononuclear cells. Intimal elastin was not different (F=1.7,p=NS) in the two strains
(WS and SW). Collagen density was significantly higher
in SWs than in WSs (F=59.2,/><0.001).
Media (Table 4). Hypertension was associated with
a significantly thicker media in controls (SC) and
isografts (SS) than in WCs and WWs (F=6.51,
p<0.05). The smooth muscle cell nucleus density and
size (F=32.17, p<0.001) (hyperplasia and hypertrophy) (F=11.25,p<0.01) were significantly increased
in the hypertensive group. Elastin density was not
significantly changed by hypertension, but hypertension increased the collagen content of controls and
isografts.
Medial thickness (F= 10.29, p<0.01) and smooth
muscle cell density (F=5.47, p<0.05) were significantly increased in isografts, and there was a decrease in nucleus size (F=9.21,/?<0.01). The extracellular matrix was not significantly modified in
isografts (F=2.99,/>=NS).
The medial thickness of allografts was significantly
less than that of isografts (F=33.56, p<0.001) because of a large decrease in smooth muscle cell
density in allografts (F=439,p<0.001). The nuclear
dimensions of the remaining smooth muscle cells in
allografts were unchanged (F=1.51, p=NS). The
extracellular matrix of allografts was different from
that of isografts, with greater relative amounts of
elastin and collagen (density) in allografts (F=6.87,
p<0.01).
These effects on extracellular matrix were mainly
seen in SWs. The medial thickness (F=54.8,/?<0.001),
FIGURE 4. Photomicrograph showing morphological
appearance of graft rejection (Masson's trichrome in
spontaneously hypertensive to Wistar-Kyoto allografts. I,
intima; M, media; A, adventitia. x.32.
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Plissonnier et al Allograft Injury in SHRs and WKYs
/
WKY
• Recipient
1695
N
SHR
Isograft
Allograft
Elastin staining
.
•
-
•
•
.
.
FIGURE 5. Photomicrographs showing morphological appearance after orcein staining for elastin in
the walls of isografts and allografts (upper panel),
morphological appearance after Sirius red staining
for collagen (middle panel), and periodic acidhematoxylin staining of nuclei for measuring the
density of smooth muscle cells in media and intima
(lower panel). WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats. x32.
Isograft
Allograft
Collagen staining
Isograft
Allograft
^
Nucleus staining
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1696
Arteriosclerosis and Thrombosis
Vol 11, No 6 November/December 1991
TABLE 3. Pairwise Comparisons of Morphometric Parameters
of the Aortic Allograft Intima
TABLE 4. Pairwise Comparisons of Morphometric Parameters of
the Aortic Wall Media in Each Experimental Group
Recipient
Recipient
Parameter
Intimal thickness (tun)
Controls (n=30)
1
Isografts (n=30)
J
Allografts (n=30)
}
Nucleus density (NoVfields)
Allografts (n=30)
Elastin density (%)
Allografts (n=30)
Collagen density (%)
Allografts (n=30)
SHR
("=45)
WKY
("=45)
t
0
0
44±1.81
A
0
14±0.77
69±1.55
*
WKY
(n=45)
Parameter
Medial thickness (fim)
}
Controls (n=30)
Isografts (n=30) 1
Allografts (71=30)
SHR
("=45)
A
t
71^5±2.56
79^0 ±2.89
53.97±1.96v _±
77.15±3.19
92.04±2.63
81.63±2.45
J
4.3±0.23
t
16.1 ±0.18
Nucleus density (No./fields)
1
Controls (TJ=30)
19.4±0.7
NS
13.2±1.03
35.47±0.77
t
10.42±0.40
Isografts (71 =30)
Allografts (n=30)
j
I
A
I
t
I
t
}
NS
23.49±0.62 ±_J18.37±0.S4
25.30±0.66 J 29.81 ±0.85
8.72±0.78 v —v—' 6.51 ±0.64
Nucleus area (/AHI2)
Values are mean±SEM. Elastin and collagen densities are the
relative black areas in each field (24; 15 rats per group).
WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive
rats.
.Ol; 1p<0.00l.
number of elastic laminae, and absolute amounts of
elastin and collagen (density times medial thickness)
were all significantly lower in SWs than in WSs
(F=5.25,p<0.05).
Controls (n=30)
Isografts (TI=30)
Allografts (TJ=30)
Elastic laminae (No.)
Controls (TI=30)
5
Isografts (n=30)
5
Allografts (TI=30)
3
5
5
5
NS
Elastin density (%)
Controls (TI=30)
Isografts (TI=30)
Discussion
Most published studies have concentrated on vascular graft rejection in allotransplants of arterial
tissue to arterial locations. These experiments have
been performed in humans, dogs, rabbits, and
rats,7'25-32 and they describe the pathological process
of rejection. Halpert et al25 described the alteration
of elastic fibers in the media of human allograft
aorta. Arterial grafts or aorto-aortic allografts were
used in dogs and rats. Arterial rejection in dogs was
described by different authors.26-27-30 It involved predominantly inflammatory infiltration, medial necrosis, and intimal proliferation. Azathioprine alone26-32
or azathioprine plus prednisolone27-31 did not completely suppress the rejection injury in these early
models of outbred animals. Williams et al28 and more
recently Schmitz-Rixen et al,7 using inbred strains,
described the three components of arterial allograft
rejection in the rat aorta model: intimal thickening,
including smooth muscle cells and inflammatory
cells; necrosis of the media, with loss of medial
smooth muscle cells and a time-dependent attack on
the extracellular matrix; and inflammatory cell infiltration, mostly in the adventitia. The results of the
present study show similar changes in rat aortic
allografts. However, this study compares the effects
of arterial allograft rejection in two genetically different host strains, quantifies the phenomenon, and
describes the functional changes in terms of structural modifications.
A
7.02±0.29 v _ t v 8.17±0.24
6.22±0.23 •
7.21±0.19
7.03±0.31 V ~ v — ' 7.17±0.28
Allografts (n =30)
Collagen density (%)
Controls (TI=30)
Isografts (n=30)
Allografts (TI=30)
A
|
}
J
}
NS
T
54.47±3.51
53.15±1.39
65.15±2.16
56.55 ±1.41
% 52.72±1.78
'
53.04±2.05
A
NS
j .
T
8.28±1.17 •f 15.75±1.57
8.62±0.48 T~^ 14.85±0.68
14.82±2.16 ^ ~ T ^ 10.74±0.87
Values are mean±SEM. Elastin and collagen densities are the
relative black areas in each field.
WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive
rats.
•p<0.05; 1p<0.0l; ±/><0.001.
Isografts and Controls
In agreement with earlier results,7 isografts in
syngeneic strains were normal, with no signs of
rejection. Nevertheless, isografts differed structurally
from controls in having increased medial thickness
due to increased smooth muscle cell density. These
phenomena were independent of the strain and are
probably related to the decrease in longitudinal wall
stress induced by the surgery itself. The grafted aortic
segments were slightly longer than the segment removed from the recipient. Therefore, the reduced
longitudinal stress could lead to increased numbers
of smooth muscle cells per area unit and decreased
stress on each cell, resulting in lower cell biosynthetic
activity and nuclear volume.
There is also a small but constant intimal proliferation in some groups of SHRs. The response to
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Plissonnier et al
endothelial surgical injury in normotensive and
hypertensive strains was also different: endothelial
injury was associated with platelet activation and
growth factor secretion.33 There was no intimal proliferation in the normotensive WKY strain, whereas
surgery induced myointimal proliferation in SHRs.
As previously described8 and demonstrated in our
present work, there was significant smooth muscle
cell hyperplasia in the wall of the abdominal aortas in
SHRs, leading to a significant increase in medial
thickness. Several recent studies have reported that
SHR arterial smooth muscle cells are significantly
hyperresponsive, particularly to platelet-derived
growth factor in vitro.9"12 This hyperresponsiveness
could be due to abnormally high levels of phospholipase C activity and the Na+/H+ exchanger.3414
Smooth muscle cells from SHRs are also less susceptible to growth inhibition by heparin.13 Qowes and
Clowes35 and Krupski et al36 have reported greater
intimal proliferation in SHRs than in WKYs after
balloon-induced arterial wall injury. Nevertheless,
blood pressure could also play a role in this hyperresponsiveness. Similar results have been reported for
deoxycorticosterone acetate salt-induced hypertension, in which environmental conditions rather than
genetics lead to an increase in smooth muscle cell
proliferative potential.37 This difference in the physiological activity of smooth muscle cells in primary
and secondary hypertension could explain the greater
intimal proliferation in SHRs.
Other structural parameters, including the extracellular matrix, did not differ in controls and isografts.
Nevertheless, there were still structural and functional
differences between normotensive and hypertensive
strains. As reported earlier by us38 and others,39 SHRs
have a smaller initial arterial volume and lower compliance than do WKYs. The hypertensive strain has
increased smooth muscle density and nuclear size,
which probably reflect greater protein biosynthesis.
The extracellular matrix components of SHRs and
WKYs were also different: collagen density was greater
in SHRs, but their elastin densities were the same. This
difference in smooth muscle cell density and function
could also explain the lower values of aortic volume at
low pressure in SHRs, whereas the decreased compliance may be due to both the smooth muscle cells and
collagen enrichment.
AUografts and Isografts
Allografts unlike isografts were significantly different from controls in terms of both structure and
function. These differences could be caused by the
rejection process. Aortic grafts between two histoincompatible strains of rats have the structural features
of cellular immunological rejection. Rejection mainly
involves the adventitial layer,7 where both specific
lymphocytes and nonspecific macrophage cells are
present. In contrast, there is no cell proliferation in
the medial layer. The arterial wall allograft rejection
seen in this study and in others results in necrosis of
smooth muscle cells in the media, while the extracel-
Allograft Injury in SHRs and WKYs
1697
lular matrix is largely preserved. The exact reason
why medial smooth muscle cells disappear is unclear,
as there are no major direct signs of rejection within
the media. It could be due to an early direct rejection
process that is not seen 2 months later, to a medial
layer infarction that follows adventitial inflammation
and destroys the vasa vasorum, to intimal proliferation that could impair direct oxygenation of the
media from the aortic lumen, or to a combination of
one or more of these factors.
Intimal proliferation associated with endothelial
regeneration is the most prominent feature of allografts. Most of the proliferating cells are myofibroblasts, but some are inflammatory cells. The source of
the intimal proliferation is not clear, but there are
indications that these cells are from the host (absence of direct rejection signs). This point has been
discussed by others.40 Myointimal proliferation is
always significantly greater in allografts than in
isografts. The initial loss of endothelium that occurs
in both isografts and allografts cannot alone account
for the importance of the phenomenon in allografts.
Growth factors such as platelet-derived growth factor
are secreted by activated inflammatory cells.33 Raines
et al41 reported that interleukin-1 has mitogenic
activity for smooth muscle cells due to plateletderived growth factor. This study points out that, as
in endothelial injury or atherosclerosis,42 activated
inflammatory cells have a significant role in myointimal proliferation in this model of chronic arterial
wall rejection process.
The structural changes in the three layers of the
arterial wall of allografts can account for the functional changes in these grafts: allografts have a
greater initial volume and decreased compliance.
The lower smooth muscle tone and higher initial
volume are probably due to the fewer smooth muscle
cells in the medial layers and to the blood pressureinduced arterial wall stress-strain relation. The increased stiffness of the aortic wall may be caused by
the increased fibrosis, particularly intimal fibrosis.
There were significant functional and structural
differences between graft rejection in SHRs and
WKYs over and above the common effect of allograft
rejection. Allograft rejection is more severe in normotensive recipients than in hypertensive ones. Medial smooth muscle cell loss and medial fibrosis were
less pronounced in SHR recipients, and the elastic
tissue was less attacked, whereas rejection involved
considerable medial smooth muscle cell destruction,
medial fibrosis, and attacks on elastic laminae in
WKY recipients. These differences could be due to
some immunological incompetence in the SHR
strain. Several studies have reported thymodependent failure in SHRs.15-18 Thymus grafts in young
SHRs prevent the increase in blood pressure and
restore cellular immunological competence.18 This
cellular immunodeficiency could lead to a depressed
delayed type of hypersensitivity and allograft rejection. This genetic immunological deficiency may un-
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1698
Arteriosclerosis and Thrombosis
Vol 11, No 6 November/December 1991
derlie the less pronounced allograft rejection in
recipient SHRs.
Myointimal proliferation is also different in the
two recipient strains. The intima of SHR recipients is
thicker than that of WKY recipients. The myointimal
proliferation of SHR recipients contains more
smooth muscle cells than fibrosis. The differences
may be due to the biologic activity of smooth muscle
cells in SHRs and to hypertension as discussed
above. The intimal response of the SHR recipients
appears to be more cellular, and rejection, less
pronounced, indicating that intimal proliferation is of
host origin, whereas intimal proliferation is less cellular and more fibrotic in WKY recipients. Lian and
Chan43 have recently reported that collagen biosynthesis in vitro increases after the phase of proliferation during the quiescent stage. Our in vivo data
seem to be in agreement. Smooth muscle cells from
SHRs proliferate more and could thus be less quiescent than smooth muscle cells from WKYs, which
could be more quiescent 2 months after surgery and
secrete more collagen. The structural differences in
the media and intima of SHR and WKY recipients
can explain the observed differences in function.
Allografts in SHR recipients have a larger initial
volume and a higher compliance. The macroscopic
and arteriographic appearance of WKY allografts in
SHR recipients shows that the high blood pressure in
SHR recipients initially increases the luminal volume
of the graft, and this remains elevated 2 months later
and is not completely compensated for by intimal
smooth muscle cell proliferation. In contrast, the
increased rigidity in WKY recipients could be due to
increased fibrosis in the neointimal layer.
In conclusion, aortic grafts between SHRs and
WKYs are rejected because of their histoincompatibility, arising from haplotype differences in the major
histocompatibility complex.1920 Certain major functional and structural consequences of allograft rejection are common to the two strains, leading to
chronic medial injury and myointimal proliferative
response, initial dilatation, and increased stiffness.
There are also structural and functional differences
between the two recipient strains that are more
related to the genetic selection of the strain. Genetic
determination of the cellular immune process seems
to be the most important factor in the injury of the
medial layer, whereas the genetic determination of
blood pressure and smooth muscle cell proliferative
potential are the major factors governing the intimal
response.
Arterial graft rejection offers a model of chronic
arterial wall injury in which the response of the
recipient can be studied in terms of the immune
response, the level of blood pressure, and the biology
of smooth muscle cell, as in the model of provoked
deendothelization in rats.44 It is characterized by the
chronicity of the arterial wall injury and response
phenomena and by the presence of the different
types of specific and nonspecific inflammatory cells.
It also represents an in vivo model for studying the
relation between chronic arterial rejection and the
development of secondary atheromatous disease,45 as
observed in human cardiac transplantation.46
No form of immunosuppressive treatment was used
in the present work, but the model would be appropriate for testing the specific effects of various types of
treatment such as cyclosporine,47 which limits the rejection process; heparin, which inhibits both smooth
muscle cell proliferation48 and lymphocyte tissue traffic49; and converting enzyme inhibitors, which decrease
blood pressure and inhibit smooth muscle cell proliferation50; on the prevention of arterial wall injury and
response due to graft rejection.
Acknowledgments
We thank Daniele Gentric and Martine Douhare
for expert technical assistance and Florence Lopez,
Nicole Braure, and Annie Boisquillon for preparing
the manuscript.
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KEY WORDS • spontaneously hypertensive rats • Wistar-Kyoto
rats • aortic transplantation • blood pressure • arterial
compliance • myointimal proliferation • arterial media •
thymodependent lymphocytes
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Allograft-induced arterial wall injury and response in normotensive and spontaneously
hypertensive rats.
D Plissonnier, B I Levy, J L Salzmann, D Nochy, J Watelet and J B Michel
Arterioscler Thromb Vasc Biol. 1991;11:1690-1699
doi: 10.1161/01.ATV.11.6.1690
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