© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Effect of Cyclosporine on Arterial Balloon Injury Lesions in Cholesterol-Clamped Rabbits
T Lymphocyte–Mediated Immune Responses Not Involved in Balloon Injury–Induced Neointimal Proliferation
From the Department of Thoracic Surgery (H.Ø.A.), Rigshospitalet, National University Hospital, Copenhagen; the Department of Pathology (B.F.H.), Hvidovre University Hospital, Hvidovre; the Department of Women's Health Care Biology (P.H.), Novo Nordisk; the Department of Clinical Biochemistry (S.S.), Gentofte University Hospital, Gentofte; and the Department of Clinical Biochemistry (B.G.N.), Glostrup University Hospital, Glostrup, Denmark.
Correspondence to Dr H.Ø. Andersen, Department of Thoracic Surgery, 2152 Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Restenosis after balloon dilatation of stenosed coronary arteries is a major clinical problem. Because T lymphocytes occur in neointima and because cyclosporine inhibits T-lymphocyte proliferation, we tested the hypothesis that cyclosporine would attenuate neointimal proliferation after balloon dilation injury. Rabbits with a balloon-injured aorta, randomized to cyclosporine in the human therapeutic range (n=13) or vehicle (n=14) were followed up for 5 weeks; as a control for the effect of cyclosporine, half the rabbits received in addition an aorta allograft. Rabbits were clamped at a human plasma cholesterol level of 5 to 7 mmol/L. Cyclosporine did not affect aorta cholesterol accumulation or neointimal proliferation in balloon-injured aortas; however, it attenuated both in transplanted aortas. Likewise, cyclosporine had no effect on endothelial cells at balloon-injured sites, but protected these cells in the transplanted aortas. Infiltration of smooth muscle cells, T lymphocytes, and macrophages were unaffected by cyclosporine in balloon-injured aortas; however, in transplanted aortas, cyclosporine reduced the relative number of T lymphocytes and macrophages but increased the relative number of smooth muscle cells. Finally, in balloon-injured aortas, cyclosporine did not affect expression of vascular adhesion molecule-1, intercellular adhesion molecule-1, or major histocompatibility complex II, but all these cellular activation markers were attenuated by cyclosporine in transplanted aortas. These results suggest that cyclosporine does not attenuate neointimal proliferation after balloon dilatation, and that T lymphocyte—mediated immune responses are not involved in neointimal proliferation after balloon dilatation.
Key Words: balloon injury • restenosis • cyclosporine • endothelial activation • chronic rejection
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Balloon dilatation of stenosed arteries, ie, percutaneous transluminal coronary angioplasty is used more and more in the treatment of ischemic heart disease. Although implantation of stents at the site of percutaneous transluminal coronary angioplasty reduces the restenosis rate1 long-term complications from this treatment are yet unresolved and restenosis still occurs. It would be of great clinical importance if a harmless medical treatment given around the time of percutaneous transluminal coronary angioplasty could attenuate or prevent restenosis.
One candidate for such a beneficial effect is cyclosporine,
an immunosuppressive agent that inhibits T-lymphocyte
proliferation.2 In experimental studies,
cyclosporine has been shown to inhibit early
atherosclerosis in the cholesterol-fed
rabbit3 as well as the development of transplant
arteriosclerosis after immune
injury.4 5 6 As the histopathological events occurring
after a balloon dilatation injury have many common features with that
seen after cholesterol feeding and immune
injury,7 8 9 10 11 12 13 including the presence of T
lymphocytes,10 11 it could be hypothesized that
cyclosporine would also inhibit neointimal
proliferation after a balloon dilatation injury. Previous studies of
this hypothesis gave equivocal results14 15 16 ; however,
these studies were all performed in animal models with plasma
cholesterol at 
0.5 to 1.5 mmol/L, levels below that
seen in most humans, and in only 1 was a clinically relevant dose of
cyclosporine used.15
We tested the hypothesis that a clinically relevant dose of cyclosporine would attenuate aortic neointimal proliferation after a balloon dilatation injury in rabbits with plasma cholesterol levels clamped at a human level of 5 to 7 mmol/L; as a positive control of the beneficial effect of cyclosporine on neointimal proliferation,4 5 half the rabbits also received an aorta allograft4 in addition to the balloon injury. To test this hypothesis, we measured aortic cholesterol accumulation, neointimal proliferation, integrity of the endothelial cell layer, and intimal infiltration with smooth muscle cells, T lymphocytes, macrophages, and mast cells, as well as upregulation of the cellular activation markers vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and major histocompatibility complex II (MHCII).
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals and Surgical Procedures
Outbred male White rabbits of the Danish Country Strain from Statens Seruminstitut, Copenhagen, Denmark, were used. Donors and recipients of aortic allografts belonged to different litters from this strain. All experimental procedures were performed in accordance with Danish regulations for experiments on animals.
Animals were randomized to either (1) balloon injury of the descending
thoracic aorta alone or (2) balloon injury combined with an aorta
allograft of a thoracic aorta as a bypass to the abdominal aorta; the
allograft was included as a positive control for the effect of
cyclosporine to attenuating the neointimal
proliferation in transplanted aortas.4 5 17 18 To exclude
that the allograft was influencing events seen at the balloon-injured
site, only half of the rabbits received an allograft. All rabbits were
anesthetized with repeated small doses of an
intravenous 30% pentobarbital solution. On average, a
total dose of 50 mg/kg of body weight was given to each rabbit.
Balloon Injury Alone
After systemic heparinization (100 IU/kg), a 4-F embolectomy
catheter (Baxter Health Care Corp) was introduced through the
superficial femoral artery to the descending thoracic aorta. The
placement was verified by x-ray. The balloon was inflated with 0.6 mL
of saline (distention, 9.0 mm) and the catheter retracted 3 cm.
Finally, the balloon was deflated and withdrawn.
Balloon Injury Combined With an Aorta Allograft
The abdomen of the recipient rabbit was opened through a midline
incision and the 4-F embolectomy catheter was inserted into the aorta
via an incision prepared for the proximal anastomosis between the donor
thoracic aorta and the recipient abdominal aorta. Balloon injury was
performed exactly as described above. The thoracic aorta of the donor
rabbit was removed and transplanted as a bypass graft onto the
abdominal aorta of the recipient.4 Finally, the abdominal
aorta of the recipient rabbit was ligated between the 2 anastomoses,
thereby directing the entire aortic blood flow through the graft.
Cyclosporine Treatment
Rabbits with a balloon injury and rabbits with balloon injury
combined with an aorta allograft were randomly assigned to
intramuscular injections of cyclosporine or vehicle.
Cyclosporine (10 mg/kg) (Sandimmun, Sandoz), or an
equivalent volume of the cyclosporine vehicle
(cremofor/ethanol, 66%:33%), was administered intramuscularly
at the completion of the balloon injury/transplantation and
subsequently once daily in individualized doses designed to give whole
blood trough levels in the human therapeutic range of 0.08 to 0.33
µmol/L; blood cyclosporine concentrations were determined
once per week (Emit Cyclosporine Assay, Syva Company).
Cholesterol Feeding
After a recovery period of 2 weeks when the rabbits were fed
ordinary chow, each rabbit was fed individually adjusted
cholesterol-enriched pellets (0 to 1 g of
cholesterol per rabbit per day) to achieve a mean plasma
cholesterol concentration in the average human range of 5
to 7 mmol/L; plasma cholesterol concentrations were
determined twice before, and every second day during, the
cholesterol feeding period. Lipoprotein
cholesterol concentrations were determined at the start and
end of the cholesterol feeding period. Plasma and
lipoprotein cholesterol concentrations were measured with
an enzymatic kit (CHOD-PAP, Boehringer Mannheim). HDL (HDL,
d>1.063 g/mL), LDL (1.063>d>1.019 g/mL), IDL
(1.019>d>1.006 g/mL), and VLDL (d<1.006 g/mL)
were separated by using ultracentrifugation as
described previously.19
Neointimal Proliferation
Five weeks after the surgical procedures, rabbits were killed
with intravenous pentobarbital (50 to 100 mg/kg). To
delineate the region of the balloon injury, rabbits were injected
intravenously with 5 mL of Evans Blue, 5 minutes before
they were killed; this permitted the native thoracic aorta to be
divided into 2 segments, a blue-stained balloon-injured part and the
remaining normal aorta. A catheter was introduced into the left
ventricle of the heart, and the vascular system perfused with 500 mL of
saline; blood and perfusate left through an incision in the
inferior vena cava. After perfusion, the entire aorta was
dissected free and a 3- to 5-mm-long specimen of unopened aorta was
taken from the native aorta (central part of white area above the
blue-stained area), the balloon-injured aorta (central part of blue
stained area), and the transplanted aorta (central part); after
fixation in formalin, these specimens were embedded in paraffin, and 2
serial sections were stained with elastic van Gieson and elastic
hematoxylin and eosin, respectively. Another 3- to 5-mm-long specimen
from native, balloon-injured, and transplanted aortas (next to the
specimen already taken) was immediately frozen in isopentane cooled
with dry ice, and kept at -80°C until further processing. Of the
remaining native, balloon-injured, and transplanted aortas, the luminal
surface areas were outlined, and the tissues divided into intima inner
media layers and outer media layers. Each of these parts were weighed
and the tissues stored at -20°C until further processing. Total,
free, and esterified cholesterol content was determined as
described.19
Histomorphometric studies were performed blinded in all animals by 1 of the investigators (H.Ø.A.); neointimal proliferation and medial area were quantified by point counting.20 In addition, cross sections were evaluated independently and blinded for qualitative morphological features by 1 of the authors (H.Ø.A.).
Immunohistochemistry
Immunohistochemical demonstration of macrophages, T
lymphocytes, smooth muscle cells, and endothelial cells
was performed by the avidin-biotin method on formalin-fixed,
paraffin-embedded aortic tissue, using the following monoclonal
antibodies: RAM11 (DAKO Corporation), which recognizes an
uncharacterized cytoplasmic antigen expressed by rabbit alveolar
macrophages,21 L11/135 (Serotec), which is
a pan-T-lymphocyte marker that recognizes rabbit T lymphocytes in blood
and tissues but does not cross-react with other leukocytes or any other
cell type,22 HHF35 (DAKO A/S), which is a specific
marker for smooth muscle cell–specific actin in
rabbits,22 and CD31 (DAKO A/S), a monoclonal antibody that
reacts with a 100-kDa glycoprotein in
endothelial cells. Because this latter antibody can
cross-react with other cell types, it was a requirement that the
staining should be found luminally, before the presence of
endothelial cells could be accepted. Mast cells were
visualized by an enzymatic stain (LEDER, naphthol
AS-D-chloracetate).
In cryostat sections of aortas, the 2C4 antibody (Serotec), which binds to the rabbit homolog of the class II MHC antigen, was used as a marker of inflammation with activation of the immune system.22 Furthermore, VCAM-1 and ICAM-1 were recognized by using the monoclonal antibodies Rb1/9 (mouse IgG, hybridoma supernatant)9 and Rb2/3 (mouse IgG, hybridoma supernatant),9 respectively. Both antibodies were kindly provided by Myron I. Cybulsky, Brigham and Women's Hospital, Harvard Medical School.
To quantify the immunohistochemical changes in the different groups of rabbits the following method, performed "blind" by 1 of the investigators (H.Ø.A.), was used for the detection of macrophages (RAM11), T lymphocytes (L11/135), smooth muscle cells (HHF35), and mast cells (LEDER): In a representative field (magnification, x400), antibody-positive cells were expressed as percentages of the total number of cells (counted as nuclei) in the intimal layer. The number of a given cell type in the field chosen for counting varied from 0 to 127 cells, whereas the highest total number of cells in 1 field was 336. The mean bias±standard error values for the relative number of smooth muscle cells, T lymphocytes, and macrophages, based on counting in 2 different representative fields of the same 13 aortic allografts, were 2.2±1.5%, 1.2±0.8%, and 0.1±0.2%.
For class II MHC (2C4), VCAM-1 (Rb1/9), ICAM-1 (Rb2/3), and endothelial cells (CD31), the intensity of staining was graded numerically on a scale from 0 to 5 as follows: grade 0, no staining; grade 1, patchy and weak staining; grade 2, uniform and weak staining; grade 3, patchy and moderate staining; grade 4, uniform and less intense staining; and grade 5, uniform and intense staining.23
Statistics
All results are given as mean±SEM values. Wilcoxon's
test for paired samples was used to evaluate the change in body weight
during the experiment. For comparison between 2 groups, the
Mann–Whitney U test was used. For comparison between 3 or
more groups, the Kruskal–Wallis analysis of
variance24 was used. In case of a significant
analysis of variance, post hoc analysis was performed
with the Mann–Whitney U test. For categorical data,
Fisher's exact test was used. No correction for multiple comparisons
was performed. P<0.05, on 2-sided tests was chosen as the
level of significance.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
All rabbits thrived and the body weight stayed constant throughout the 5-week study period (data not shown). Plasma cholesterol concentration curves were similar in all 4 groups of rabbits throughout the study (Figure 1
). Mean plasma cholesterol
concentrations during the entire study period as well as during the
cholesterol feeding period were similar in all 4 groups
(data not shown). Lipoprotein cholesterol levels were
similar in all 4 groups except LDL cholesterol between
cyclosporine- and vehicle-treated rabbits from the groups
of rabbits with the combined procedure (2.6±0.2 versus 1.7±0.2
mmol/L, P=0.01). To achieve the desired plasma
cholesterol level, cyclosporine-treated
animals needed significantly less dietary cholesterol than
did vehicle-treated animals (balloon injury, 0.10±0.01 versus
0.04±0.01 g · kg-1 ·
d-1, P=0.001; balloon
injury+transplantation, 0.08±0.01 versus 0.04±0.01 g ·
kg-1 · d-1,
P=0.001), which is in agreement with findings in earlier
studies.4 5
|
Doses of cyclosporine in rabbits with balloon injury alone and in rabbits with balloon injury combined with an aorta allograft were 6.9±0.1 and 7.5±0.4 g · kg-1 · d-1. Mean trough levels of cyclosporine were held within the human therapeutic level of 0.08 to 0.33 µmol/L in both cyclosporine-treated groups (0.17±0.01 and 0.14±0.01 µmol/L).
Aortic Cholesterol Accumulation
Cyclosporine had no effect on intimal
cholesterol accumulation in native or balloon-injured
aortas (Figure 1
). In transplanted aortas, however,
cyclosporine reduced intimal cholesterol
accumulation (P=0.005).
Neointimal Proliferation
Cyclosporine had no effect on neointimal
proliferation in native or balloon-injured aortas (Figures 1 and 2). However, in transplanted aortas,
cyclosporine inhibited neointimal proliferation
(P=0.04).
|
Endothelial Cells
In 25 of 27 native aortas, we found a thin luminal staining
representing endothelial cells in the
entire circumference. There was no difference between vehicle- and
cyclosporine-treated rabbits (Figure 3) (data not shown).
|
In balloon-injured aortas, luminal staining was only seen in areas
without neointimal proliferation, reaching and often
creeping only a little way up the slope of the neointimal
proliferation humps (Figure 3). There was no difference between
cyclosporine- and vehicle-treated groups in staining
intensity (Figure 3) (data not shown).
In transplanted aortas, however, only 1 of 7 transplants from the
vehicle-treated group had staining, whereas all 6 from the
cyclosporine-treated group had
endothelial staining (Figure 3)
(P<0.005, Fisher's exact test).
Smooth Muscle Cells, T Lymphocytes, and Macrophages
Neointimal proliferation in balloon-injured aortas
from vehicle-treated rabbits mainly consisted of smooth muscle cells
and only a low number of macrophages and T lymphocytes were
demonstrated (Figures 3 and 4).
There was no difference between cyclosporine- and
vehicle-treated groups in the relative number of smooth muscle cells, T
lymphocytes, or macrophages.
|
In transplanted aortas from vehicle-treated rabbits,
macrophages, smooth muscle cells, and T lymphocytes were all
abundant (Figures 3 and 4). The numbers of both
macrophages and T lymphocytes were significantly reduced in
aortic allografts by cyclosporine, whereas
cyclosporine caused a relative, but not absolute, increase
in the number of smooth muscle cells.
Mast Cells
Only 2 mast cells were seen in the adventitia of 1 of the
balloon-injured aortas, but none in the intimas of native,
balloon-injured, or transplanted aortas.
VCAM-1, ICAM-1, and MHCII
In the intima of native and balloon-injured aortas, staining
intensity for VCAM-1, ICAM-1, and MHCII did not differ between vehicle-
and cyclosporine-treated rabbits (Figures 5 and 6).
In transplanted aortas, however, staining intensities for VCAM-1,
ICAM-1, and MHCII were lower in cyclosporine- than in
vehicle-treated rabbits.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In the rabbit, neointimal proliferation in arteries occurs after intimal balloon injury,10 13 25 26 where the process is accelerated by increased plasma cholesterol levels,25 26 as well as in cholesterol-induced atherogenesis,7 9 and after transplantation of solid organs or arteries.4 6 12 T lymphocytes, macrophages, and smooth muscle cells, and upregulation of VCAM-1, ICAM-1, and MHCII, are all components of these 3 types of neointimal proliferation.7 8 9 10 11 12 13 Cyclosporine has been shown to inhibit early atherosclerosis in the cholesterol-fed rabbit.3 Furthermore, cyclosporine has been shown to inhibit development of transplant arteriosclerosis4 5 18 and to reduce an otherwise increased occurrence of T lymphocytes, macrophages, and smooth muscle cells and upregulation of VCAM-1, ICAM-1, and MHCII in transplanted arteries.5 6 27 We therefore hypothesized that cyclosporine would also inhibit neointimal proliferation occurring after aortic balloon dilatation injury. However, in the present study, using a clinically relevant dose of cyclosporine, rabbits with plasma cholesterol clamped at a mean level of 5 to 7 mmol/L, and a 5-week experimental period, cyclosporine had no effect on any of these changes in balloon-injured aortas, but significantly inhibited all these changes in the transplanted aortas, the positive controls.
To ensure that rabbits with a combined balloon injury and aorta had fully recovered from the surgical intervention and regained a normal gastrointestinal function before the start of the individualized cholesterol feeding, cholesterol feeding was not initiated before 2 weeks after surgery. It could be suggested that many of the proliferation and migration processes leading to intimal hyperplasia after balloon injury may have already been completed at this time. However, the significant upregulation of cellular activation parameters such as VCAM-1, ICAM-1, and MHCII as well as proliferation of smooth muscle cells and macrophages were seen 4 to 5 weeks after balloon injury in the present study as well as in earlier studies.10 13 Furthermore, intimal proliferation has been reported to proceed from 4 weeks up to as much as 20 weeks after balloon injury.10 14 28 29 30 31 32 Finally, cholesterol accumulation in both deendothelialized and reendothelialized areas has been reported to be an ongoing process from day 2,33 through to day 30,34 and up to day 119 after balloon injury.35 We therefore found it justifiable to delay the initiation of the cholesterol feeding until 2 weeks after the balloon injury and transplantation.
Upregulation of VCAM-1, ICAM-1, and MHCII in
balloon-injured10 and transplanted
aortas5 12 36 have been described earlier.
Cyclosporine had no significant effect on VCAM-1, ICAM-1,
and MHCII in the balloon-injured aorta, but a significant
inhibitory effect on the very same cellular activation
markers in the transplanted aorta. The inhibitory effect of
cyclosporine on these markers in allografted arteries has
been observed by others,6 27 36 37 whereas the lack of
effect of cyclosporine on upregulation of VCAM-1, ICAM-1,
and MHCII in arteries after a balloon injury has not been observed
previously. This difference suggests that different mechanisms are
responsible for upregulation of these cellular activation markers in
the 2 forms of neointimal proliferation. In transplanted
arteries from rabbits receiving no immunosuppression, the main stimulus
for the upregulation is supposed to come from a high number of
activated T lymphocytes38 and
macrophages38 excreting the cytokines that
induces upregulation. Among these cytokines are interferon-
(IFN-) and tumor necrosis factor-
(TNF-). IFN- increases
upregulation of the adherence proteins VCAM-18 38 and
ICAM-1,38 39 as well as upregulation of
MHCII.38 Furthermore, IFN- stimulates the activation of
macrophages38 and augments the effects of
TNF-.40 TNF- itself induces upregulation of
adherence molecules on endothelial
cells.38 39 40 Cyclosporine inhibits the
excretion of IFN- indirectly by an inhibition of T-lymphocyte
proliferation and via a direct inhibitory effect on the
production.41 Thus, a lowered presence of IFN-
per se would reduce the upregulation of VCAM-1, ICAM-1, and MHCII in
the transplanted artery. In the balloon-injured aorta, however, other
mechanisms such as increased levels of plasma
cholesterol,8 9 altered shear
stress,42 or local paracrine induction by TNF- from
smooth muscle cells43 may be more important stimuli for
upregulation of at least VCAM-1 and ICAM-1. This could explain why
cyclosporine is without effect on the expression of VCAM-1
and ICAM-1 as well as on the neointimal proliferation after
a balloon injury. Upregulation of MHCII is normally believed to be
induced by stimulation with IFN-.38
Cholesterol feeding in rabbits, however, also induces
upregulation of MHCII8 22 ; the exact mechanism for this is
not known. That cyclosporine had no effect on upregulation
of MHCII in balloon-injured aortas suggests that IFN- may not be the
stimulus for upregulation after a balloon injury.
We have shown in previous studies that cyclosporine has a powerful inhibitory effect on the development of transplant arteriosclerosis in aorta allografts in rabbits with plasma cholesterol clamped at a human level of 5 to 7 mmol/L.4 5 17 The cells seen in connection with neointimal proliferation in transplanted aortas from rabbits were immunological competent cells, ie, T lymphocytes and macrophages, as well as smooth muscle cells.5 18 Transmission electron microscopic investigations of allografted aortas within 2 weeks after transplantation showed that although cyclosporine exerted a powerful inhibition of the occurrence of T lymphocytes and macrophages, there still was some smooth muscle cell proliferation in the allograft.18 In the present study, we found a relative, but not an absolute, increase of smooth muscle cells in aortic allografts from cyclosporine-treated rabbits. The response of the artery to balloon injury consists mainly of proliferation of smooth muscle cells and, to a lesser extent, of infiltration of T lymphocytes and macrophages.10 26 In vitro studies investigating whether cyclosporine may have an inhibitory effect on smooth muscle cell proliferation have given equivocal results; in 1 study cyclosporine exhibited a dose-dependent inhibitory effect on smooth muscle cell proliferation,15 in another study the effect was either stimulatory or inhibitory, dependent on the dose of cyclosporine,43 and in a third study cyclosporine was without any effect.16 Finally, in a fourth study, cyclosporine was reported to inhibit smooth muscle cell proliferation indirectly via endothelial cell–derived factors.44 The in vitro results from the 2 latter studies16 44 are compatible with our present and most,4 5 18 but not all,45 former in vivo findings supporting the notion that cyclosporine has none or only a minor inhibitory effect on smooth muscle cell proliferation in vivo.
Ferns et al,15 using rabbits with plasma
cholesterol at 2 mmol/L and
cyclosporine in a clinically relevant dose, likewise found
no effect on carotid neointimal proliferation 2 weeks after
balloon injury, which is in agreement with our results. In a similar
manner, Gregory et al,14 using very low doses of
cyclosporine intraperitoneally in rats
for a period of 2 weeks, found that cyclosporine had no
effect on balloon injury–induced neointimal proliferation
in carotid arteries. Jonasson et al,16 using supraclinical
doses for only 2 days followed by 12 days without any treatment in
rats, however, found that cyclosporine inhibited
neointimal proliferation in balloon-injured carotid
arteries examined 2 weeks after injury. They found no direct effect of
cyclosporine on smooth muscle cell proliferation in vitro
and therefore suggested that the inhibitory effect of
cyclosporine seen in vivo was mediated through an
inhibition of T-lymphocyte proliferation.16 The
present study, however, could not demonstrate any difference in the
occurrence of T lymphocytes in balloon-injured aortas between
cyclosporine- and vehicle-treated rabbits. In addition, the
scarce presence of T lymphocytes in the neointima formed as
a consequence of balloon injury would not suggest that these cells
would have a major impact on this type of lesion. Results from studies
using athymic nude rats46 47 or T lymphocyte–depleted
rats46 have given equivocal results concerning the role of
T lymphocytes in the pathogenic process of balloon injury–induced
neointimal proliferation. Thus, present and previous
results taken together do not support a significant influence of T
lymphocytes on neointimal proliferation in the artery after
balloon injury.
In balloon-injured rats, Hancock et al48 reported that mononuclear phagocytes, but not T lymphocytes, had a major impact on the development of intimal proliferation. Mononuclear leukocytes also seem to be the principal cell type when neointimal proliferation is induced by a perivascular electrical injury. In this latter model, an inflammatory reaction with mononuclear leukocytes eventually leads to smooth muscle cell migration and proliferation.49 50
Although the lesions formed after balloon injury and transplantation have several common features, it is quite possible that the mechanism at work differs in the 2 situations. This is illustrated clearly in the present study by the inhibitory effect of cyclosporine on neointimal proliferation in the transplanted aorta, but not in the balloon-injured aorta. In the transplanted artery, alloimmunity involving T lymphocytes and their consequent recruitment of macrophages seem to be of major importance51 ; cyclosporine inhibits T lymphocytes. In the balloon-injured artery, smooth muscle cell proliferation10 26 in response to wound healing and possibly macrophages under conditions with hypercholesterolemia13 25 30 48 may be of more importance.
In native and balloon-injured aortas, cyclosporine had no effect on endothelial coverage. In the transplanted aorta, however, cyclosporine significantly inhibited the disappearance of the endothelial coverage, in accordance with previous results.18 The mechanism behind this effect is not completely clear. However, it is believed that cyclosporine inhibits T cell–mediated endothelial injury.18 In a recent study by Walter et al,52 cyclosporine reduced oxidized LDL-induced apoptosis of human endothelial cells. In the present study, cholesterol feeding may have led to endothelial cell exposure, to increased levels of oxidized LDL, and thereby to an increased endothelial cell apoptosis. In native and balloon-injured aortas, this kind of injury may not have reached a significant or measurable extent, whereas this mechanism in concert with the immunological injury may have been substantial in transplanted aortas.
In conclusion, the present data suggest that (1) cyclosporine does not influence the response of arteries to balloon injury, whereas it reduces allograft arteriosclerosis; (2) the expression of VCAM-1, ICAM-1, and MHCII after arterial balloon injury is not influenced by cyclosporine; (3) T lymphocytes do not play a crucial role in the arterial response to balloon injury; and (4) this response is quite distinct from allograft arteriosclerosis. In the present study, balloon injury was performed in a healthy artery, which is not the case when balloon dilatation is performed on an atherosclerotic stenosis of a human artery. Therefore, it cannot be ruled out entirely that cyclosporine may influence human restenosis, which is a far more complex process than the response of a normal rabbit aorta to balloon injury.53
|
Acknowledgments |
|---|
Børge G. Nordestgaard was supported by the Danish Heart Foundation. We thank technicians Lone Christensen, Hanne Damm, and Kurt Svarre Jensen for their help during the study.
Received November 19, 1998; accepted December 10, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Fischman DL, Leon MB, Baim DS, Schatz RA, Savage MP, Penn I, Detre K, Veltri L, Ricci D, Nobuyoshi M, Cleman M, Heuser R, Almond D, Teirstein PS, Fish D, Colombo A, Brinker J, Moses J, Shaknovich A, Hirsfeld J, Baily S, Ellis S, Rake R, Goldberg S. A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease. N Engl J Med. 1994;331:496–501.
2. Kunz J, Hall MN. Cyclosporin A, FK506 and rapamycin: more than just immunosuppression. Trends Biochem Sci. 1993;18:334–338.[Medline] [Order article via Infotrieve]
3. Drew AF, Tipping PG. Cyclosporine treatment reduces early atherosclerosis in the cholesterol-fed rabbit. Atherosclerosis. 1995;116:181–189.[Medline] [Order article via Infotrieve]
4.
Andersen HO, Madsen G, Nordestgaard BG, Hansen
BF, Kjeldsen K, Stender S. Cyclosporin suppresses transplant
arteriosclerosis in the aorta-allografted,
cholesterol-clamped rabbit: suppression preceded by
decrease in arterial lipoprotein permeability.
Arterioscler Thromb. 1994;14:944–950.
5.
Andersen HO, Holm P, Stender S, Hansen BF,
Nordestgaard BG. Dose-dependent suppression of transplant
arteriosclerosis in aorta-allografted,
cholesterol-clamped rabbits: suppression not eliminated by
the cholesterol-raising effect of cyclosporine.
Arterioscler Thromb Vasc Biol. 1997;17:2515–2523.
6. Molossi S, Clausell N, Sett S, Rabinovitch M. ICAM-1 and VCAM-1 expression in accelerated cardiac allograft arteriopathy and myocardial rejection are influenced differently by cyclosporine A and tumour necrosis factor-alpha blockade. J Pathol. 1995;176:175–182.[Medline] [Order article via Infotrieve]
7.
Drew AF, Tipping PG. T helper cell infiltration and
foam cell proliferation are early events in the development of
atherosclerosis in cholesterol-fed rabbits.
Arterioscler Thromb Vasc Biol. 1995;15:1563–1568.
8.
Li H, Cybulsky MI, Gimbrone MA Jr, Libby P. An
atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable
mononuclear leukocyte adhesion molecule, in rabbit aortic
endothelium. Arterioscler Thromb. 1993;13:197–204.
9.
Cybulsky MI, Gimbrone MA. Endothelial
expression of a mononuclear leucocyte adhesion molecule during
atherogenesis. Science. 1991;251:788–791.
10.
Tanaka H, Sukhova GK, Swanson SJ, Clinton SK, Ganz P,
Cybulsky MI, Libby P. Sustained activation of vascular cells and
leukocytes in the rabbit aorta after balloon injury.
Circulation. 1993;88:1788–1803.
11. Gregory CR, Huie P, Billingham ME, Morris RE. Rapamycin inhibits arterial intimal thickening caused by both alloimmune and mechanical injury: its effect on cellular, growth factor, and cytokine response in injured vessels. Transplantation. 1993;55:1409–1418.[Medline] [Order article via Infotrieve]
12.
Tanaka H, Sukhova GK, Libby P. Interaction of the
allogeneic state and hypercholesterolemia in
arterial lesion formation in experimental cardiac
allografts. Arterioscler Thromb. 1994;14:734–745.
13.
Stadius ML, Gown AM, Kernoff R, Collins CL. Cell
proliferation after balloon injury of iliac arteries in the
cholesterol-fed New Zealand White rabbit.
Arterioscler Thromb. 1994;14:727–733.
14. Gregory CR, Huang X, Pratt RE, Dzau VJ, Shorthouse R, Billingham ME, Morris RE. Treatment with rapamycin and mycophenolic acid reduces arterial intimal thickening produced by mechanical injury and allows endothelial replacement. Transplantation. 1995;59:655–661.[Medline] [Order article via Infotrieve]
15. Ferns G, Reidy M, Ross R. Vascular effects of cyclosporine A in vivo and in vitro. Am J Pathol. 1990;137:403–413.[Abstract]
16.
Jonasson L, Holm J, Hansson GK. Cyclosporin A inhibits
smooth muscle proliferation in the vascular response to injury.
Proc Natl Acad Sci U S A. 1988;85:2303–2306.
17. Andersen HO, Holm P, Stender S, Hansen BF, Kjeldsen K, Nordestgaard BG. Relative importance of ischemic injury and immunological injury on the development of transplant arteriosclerosis in rabbit aortic allografts. Transplantation. 1995;60:631–638.[Medline] [Order article via Infotrieve]
18. Andersen HO, Qvortrup K, Rostgaard J, Nordestgaard BG. Effect of cyclosporine during initiation of transplant arteriosclerosis: an ultrastructural study in the aorta-transplanted rabbit. Atherosclerosis. 1997;133:171–181.[Medline] [Order article via Infotrieve]
19. Larsen BA, Nordestgaard BG, Stender S, Kjeldsen K. Effect of testosterone on atherogenesis in cholesterol-fed rabbits with similar plasma cholesterol levels. Atherosclerosis. 1993;99:79–86.[Medline] [Order article via Infotrieve]
20. Andersen HO, Holm P, Nordestgaard BG, Hansen BF, Kjeldsen K, Elinder LS, Stender S. Effect of the antioxidant probucol on transplant arteriosclerosis in aorta-allografted rabbits. J Mol Cell Cardiol. 1995;27:1561–1571.[Medline] [Order article via Infotrieve]
21. Tsukada T, Rosenfeld M, Ross R, Gown AM. Immunocytochemical analysis of cellular components in atherosclerotic lesions: use of monoclonal antibodies with the Watanabe and fat-fed rabbit. Arteriosclerosis. 1986;6:601–613.[Abstract]
22.
Hansson GK, Seifert PS, Olsson G, Bondjers G.
Immunohistochemical detection of macrophages and T lymphocytes
in atherosclerotic lesions of cholesterol-fed rabbits.
Arterioscler Thromb. 1991;11:745–750.
23. Tanaka H, Sukhova GK, Swanson SJ, Cybulsky MI, Schoen FJ, Libby P. Endothelial and smooth muscle cells express leukocyte adhesion molecules heterogeneously during acute rejection of rabbit cardiac allografts. Am J Pathol. 1994;144:938–951.[Abstract]
24. Altman DG. Comparing groups—continuous data. In Altman DG, ed. Practical Statistics for Medical Research. London, UK: Chapman & Hall, 1991:179–228.
25.
Weidinger FF, McLenachan JM, Cybulsky MI, Fallon JT,
Hollenberg NK, Cooke JP, Ganz P.
Hypercholesterolemia enhances
macrophage recruitment and dysfunction of regenerated
endothelium after balloon injury of the rabbit iliac
artery. Circulation. 1991;84:755–767.
26. Ferns GAA, Stewart-Lee AL, Änggård EE. Arterial response to mechanical injury: balloon catheter de-endothelialization. Atherosclerosis. 1992;92:89–104.[Medline] [Order article via Infotrieve]
27. Koskinen PK, Lemstrom KB, Hayry PJ. How cyclosporine modifies histological and molecular events in the vascular wall during chronic rejection of rat cardiac allografts. Am J Pathol. 1995;146:972–980.[Abstract]
28. Nikkari ST, Järveläinen HT, Wight TN, Ferguson M, Clowes AW. Smooth muscle cell expression of extracellular matrix genes after arterial injury. Am J Pathol. 1994;144:1348–1356.[Abstract]
29. Kocher O, Gabbiani F, Gabbiani G, Reidy MA, Cokay MS, Peters H, Hüttner I. Phenotype features of smooth muscle cells during the evolution of experimental carotid artery intimal thickening: biochemical and morphological studies. Lab Invest. 1991;65:459–470.[Medline] [Order article via Infotrieve]
30.
Stadius ML, Rowan R, Fleischhauer F, Kernoff R,
Billingham M, Gown AM. Time course and cellular characteristics of the
iliac artery response to acute balloon injury: an angiographic
morphometric, and immunocytochemical analysis in the
cholesterol-fed New Zealand White rabbit.
Arterioscler Thromb. 1992;12:1267–1273.
31. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest. 1983;49:208–215.[Medline] [Order article via Infotrieve]
32. Koletsky S, Snajdar RM. Atherosclerosis following balloon catheter injury to the carotid artery and the aorta of hypertensive rats with normolipidemia or hyperlipidemia. Am J Pathol. 1981;103:105–115.[Abstract]
33. Schwenke DC, Zilversmit. The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit. 1. Studies during the first two days after mild aortic injury. Atherosclerosis. 1989;77:91–103.[Medline] [Order article via Infotrieve]
34. Schwenke DC, Zilversmit. The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit. 2. Long-term studies in deendothelialized and reendothelialized aortas. Atherosclerosis. 1989;77:105–115.[Medline] [Order article via Infotrieve]
35. Moore S, Belbeck LW, Richardson M, Taylor W. Lipid accumulation in the neointima formed in normally fed rabbits in response to one or six removals of the aortic endothelium. Lab Invest. 1982;47:37–42.[Medline] [Order article via Infotrieve]
36.
Clausell N, Molossi S, Sett S, Rabinovitch
M. In vivo blockade of tumor necrosis factor-alpha in
cholesterol-fed rabbits after cardiac transplant inhibits
acute coronary artery neointimal formation.
Circulation. 1994;89:2768–2779.
37. De Caterina R, Tanaka H, Nakagawa T, Hauptman PJ, Libby P. The direct effect of injectable cyclosporine and its vehicle, cremophor, on endothelial vascular cell adhesion molecule-1 expression: ricinoleic acid inhibits coronary artery endothelial activation. Transplantation. 1995;60:270–275.[Medline] [Order article via Infotrieve]
38. Libby P, Salomon RN, Payne DD, Schoen FJ, Pober JS. Functions of vascular wall cells related to development of transplantation-associated coronary arteriosclerosis. Transplant Proc. 1989;21:3677–3684.[Medline] [Order article via Infotrieve]
39. Springer TA. Adhesion receptors of the immune system. Nature. 1990;346:425–434.[Medline] [Order article via Infotrieve]
40. Azuma H, Heemann UW, Tullius SG, Tilney NL. Cytokines and adhesion molecules in chronic rejection. Clin Transplant. 1994;8:168–180.[Medline] [Order article via Infotrieve]
41. Reem GH, Cook LA, Palladino MA. Cyclosporine inhibits interleukin-2 and interferon gamma synthesis by human thymocytes. Transplant Proc. 1983;15:2387–2389.
42.
Walpola PL, Gotlieb AI, Cybulsky MI, Langille L.
Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries
exposed to altered shear stress. Arterioscler Thromb Vasc
Biol. 1995;15:2–10.
43. Tavares P, Martínez-Salgado C, Eleno N, Teixeira F, Novoa JML. Effect of cyclosporin A on rat smooth-muscle cell proliferation. J Cardiovasc Pharmacol. 1998;31:46–49.[Medline] [Order article via Infotrieve]
44. Leszczynski D, Zhao Y, Yeagley TJ, Foegh ML. Direct and endothelial cell-mediated effect of cyclosporin A on the proliferation of rat smooth muscle cells in vitro. Am J Pathol. 1993;142:149–155.[Abstract]
45. Wengrovitz M, Selassie LG, Gifford RRM, Thiele BL. Cyclosporine inhibits the development of medial thickening after experimental arterial injury. J Vasc Surg. 1990;12:1–7.[Medline] [Order article via Infotrieve]
46.
Hansson GK, Holm J, Holm S, Fotev Z, Hedrich HJ,
Fingerle J. T lymphocytes inhibit the vascular response to injury.
Proc Natl Acad Sci U S A. 1991;88:10530–10534.
47. Ferns GAA, Reidy MA, Ross R. Balloon catheter de-endothelialization of the nude rat carotid: response to injury in the absence of functional T lymphocytes. Am J Pathol. 1991;138:1045–1057.[Abstract]
48. Hancock WW, Adams DH, Wyner LR, Sayegh MH, Karnovsky MJ. CD4+ mononuclear cells induce cytokine expression, vascular smooth muscle cell proliferation, and arterial occlusion after endothelial injury. Am J Pathol. 1994;145:1008–1014.[Abstract]
49.
Kling D, Fingerle J, Harlan JM, Lobb RR, Lang F.
Mononuclear leucocytes invade rabbit arterial intima during
thickening formation via CD18- and VLA-4-dependent mechanisms and
stimulate smooth muscle migration. Circ Res. 1995;77:1121–1128.
50. Carmeliet P, Moons L, Stassen JM, De Mol M, Bouché A, van den Oord JJ, Kockx M, Collen D. Vascular wound healing and neointima formation induced by perivascular electric injury in mice. Am J Pathol. 1997;150:761–776.[Abstract]
51. Hosenpud JD. Immune mechanisms of cardiac allograft vasculopathy: an update. Transpl Immunol. 1993;1:237–249.[Medline] [Order article via Infotrieve]
52.
Walter DH, Haendeler J, Galle J, Zeiher AM, Dimmeler S.
Cyclosporin A inhibits apoptosis of human
endothelial cells by preventing release of cytochrome C
from mitochondria. Circulation. 1998;98:1153–1157.
53.
Schwartz SM, deBlois D, O'Brien ERM. The intima: soil
for atherosclerosis and restenosis. Circ
Res. 1995;77:445–465.
54. Campbell GR, Campbell JH, Manderson JA, Horrigan S, Rennick RE. Arterial smooth muscle: a multifunctional mesenchymal cell. Arch Pathol Lab Med. 1988;112:977–986.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  P. Religa, K. Bojakowski, M. Bojakowska, Z. Gaciong, J. Thyberg, and U. Hedin Allogenic immune response promotes the accumulation of host-derived smooth muscle cells in transplant arteriosclerosis Cardiovasc Res, February 1, 2005; 65(2): 535 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Satonaka, E. Suzuki, H. Nishimatsu, S. Oba, R. Takeda, A. Goto, M. Omata, T. Fujita, R. Nagai, and Y. Hirata Calcineurin Promotes the Expression of Monocyte Chemoattractant Protein-1 in Vascular Myocytes and Mediates Vascular Inflammation Circ. Res., March 19, 2004; 94(5): 693 - 700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. Visner, F. Lu, H. Zhou, J. Liu, K. Kazemfar, and A. Agarwal Rapamycin Induces Heme Oxygenase-1 in Human Pulmonary Vascular Cells: Implications in the Antiproliferative Response to Rapamycin Circulation, February 18, 2003; 107(6): 911 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. D. Danenberg, F. G. P. Welt, M. Walker III, P. Seifert, G. S. Toegel, and E. R. Edelman Systemic Inflammation Induced by Lipopolysaccharide Increases Neointimal Formation After Balloon and Stent Injury in Rabbits Circulation, June 18, 2002; 105(24): 2917 - 2922. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Zhu, C. A. Reardon, G. S. Getz, and D. Y. Hui Both Apolipoprotein E and Immune Deficiency Exacerbate Neointimal Hyperplasia After Vascular Injury in Mice Arterioscler Thromb Vasc Biol, March 1, 2002; 22(3): 450 - 455. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||