From the Cardiovascular Biology Laboratory, Harvard School of Public
Health (J.K., T.G.-J., M.E.R.), Cardiovascular Medicine Division, Brigham and
Women's Hospital (M.E.R.), and Harvard Medical School (M.E.R.), Boston,
Mass, and Merck Research Laboratories, Rahway, NJ (J.S.M.).
Correspondence to Mary E. Russell, MD, Cardiovascular Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave, Boston, MA 02115. E-mail russell{at}cvlab.harvard.edu
Methods and Results—To determine whether NOS2-mediated pathways
protect against or promote transplant
arteriosclerosis, we used NOS2-deficient mice as
recipients in our vascularized chronic cardiac rejection model. The
severity of vascular thickening in 55-day grafts placed into NOS2 -/-
recipients (n=13) was compared with that in wild-type recipients
(n=15). Computer-assisted analysis of all elastin-stained
vessels (n=283) showed significantly increased luminal occlusion
(77.1±9.4% versus 40.8±13.6%, P<.0001) and
intima/media ratios in allografts from NOS2 -/- recipients (1.9±1.3
versus 0.4±0.3, P=.0002). To elucidate potential
mechanisms, we studied NOS2 effects on T-cell differentiation
(Th1/Th2) and neointimal smooth
muscle cell accumulation. Normalized mRNA levels for Th1-
(signal transducer and activator of transcription [STAT]
4, interleukin [IL]-2, interferon-
Conclusions—NOS2 plays a protective role in the development of
transplant arteriosclerosis, suppressing
neointimal smooth muscle cell accumulation.
NO has been implicated as a potential regulator in each of these
arteriosclerotic stages.4 NO
is formed from L-arginine by the enzyme NO
synthase, which exists in different isoforms.5
The cytokine-inducible isoform of NO synthase (NOS2 or iNOS)
responds to inflammatory stimuli producing large amounts of
NO.6 7 NO is a pleiotropic signaling molecule
involved in numerous processes.7 Among its
effector functions, those pertinent to
arteriosclerotic lesion development in transplanted
organs could regulate immune cells8 9 and/or
SMCs.7 In addition to the well-described role of
macrophage-derived NO in antimicrobial processes, NO is a
mediator of T-cell responses.8
Th1-differentiated lymphocytes, a T-cell subset
associated with an accelerated alloimmune
response,10 have been shown to express high
levels of NOS2 comparable to levels produced by
macrophages.11 NO inhibits the expansion
of cloned Th1 but not Th2
cells,11 a subset that has been hypothesized to
be associated with allograft acceptance. NOS2 deficiency
produced by gene targeting results in preferential expansion of
Th1 lymphocytes.12
Conversely, NO exhibits potential antiproliferative effects in the
vasculature, including inhibition of VSMC
migration13 and
proliferation.14 15 In balloon injury models,
treatment with different NO donors reduces neointimal
formation, an effect that can be blocked by coadministration of an
inhibitor of NOS.16 17 18 19 Hence, NO
generated by NOS2 may play a role in the early immune system–driven
and/or later SMC stage of transplant
arteriosclerosis.
In transplanted organs, NOS2 expression shows an early and persistent
upregulation resulting in increased levels of
NO.4 20 21 Development of transplant
arteriosclerosis is associated with induction of
NOS2 expression in many cell types involved in lesion formation, such
as macrophages, lymphocytes, VSMCs, and
endothelial cells.4 22 In vivo
measures that attenuate the alloimmune response
(immunosuppressives)23 or inhibit
arteriosclerotic lesion development in
allografts4 24 25 reduce NOS2 expression.
However, the exact role of NOS2 in chronic cardiac rejection and
development of transplant arteriosclerosis has yet
to be defined.
The purpose of this study was to determine whether NOS2-mediated
pathways promote or protect against transplant
arteriosclerosis. Mice with targeted disruption of
the NOS2 gene provide a valuable tool to study the functional role of
NOS2 deficiency in the alloimmune response to MHC-mismatched grafts.
NOS2 -/- knockout mice are viable, fertile, and without phenotypic
evidence of histopathological abnormalities.12 26
However, when challenged, NOS2-deficient mice show altered immune
responses to lipopolysaccharides and bacterial or parasitic
infections.12 26 To study the role of NOS2 in
arteriosclerosis initiated by an alloimmune injury,
we used our vascularized heterotopic mouse cardiac transplantation
model of attenuated rejection.25 This T
cell–dependent model permits evaluation of effector pathways that
mediate the early immune and late myointimal stages of
arteriosclerotic lesion development in response to
alloimmune injury. To evaluate the effect of NOS2 on transplant
arteriosclerosis, we compared frequency and
severity of arteriosclerotic lesion development in
grafts placed into NOS2-deficient and wild-type recipients. To study
alterations in the early immune or late myointimal stages of transplant
arteriosclerosis, we characterized T-cell
differentiation and neointimal SMC accumulation.
Heterotopic Cardiac Transplantation
Morphometric Analysis
Immunohistological Analysis
Reverse Transcriptase–Polymerase Chain Reaction
Statistical Analysis
A total of 283 vessels (10.1±5.5 vessels per graft) were subjected to
computer-assisted analysis to establish severity and frequency
of arteriosclerotic lesion development (Fig 1
NOS2 Expression in Cardiac Transplants
Immunostaining was performed to determine the cellular
localization of NOS2. In allografts from wild-type recipients, both
infiltrating recipient mononuclear cells and donor parenchymal cells
(myocytes, SMCs, and endothelial cells) expressed NOS2
antigen (Fig 2B
T-Cell Differentiation in Allografts From NOS2 -/- and
Wild-type Recipients
SMC Contribution to Neointimal Formation
NOS2 deficiency aggravated the degree of luminal occlusion in part by
altering the SMC characteristics of the expanded neointima.
Specifically, we show that the area of
NOS2 and VSMCs
In this and earlier studies,4 we show that there
is a differentiated or activated subset of SMCs within the
media and neointima expressing NOS2. The presence of these
differentiated sets of VSMCs expressing NO during more advanced stages
of chronic cardiac rejection in this mouse and the LEW to F344 rat
model4 has suggested that vascular forms of NOS2
may serve a compensatory role to inhibit further
arteriosclerotic thickening when the vessel reaches
a quiescent stage. Our present results provide further support that
one of the roles of NOS2 is probably to promote direct
inhibitory actions of NO on VSMCs, as previously suggested
by in vitro studies.14
NOS2 and T-Cell Differentiation
The expansion of Th1 cells in NOS2-deficient mice
after a strong microbial challenge shows that in some circumstances, NO
can regulate T-cell differentiation.12 In
addition to regulating the differentiation of T cells, NO
production itself is regulated by Th1-
and Th2-type cytokines. For example,
Th1-derived IL-2 and IFN-
We had hypothesized that an exaggerated Th1
response produced by loss of NO-mediated inhibition of IL-2 and IFN-
NOS2 and Arteriosclerosis
NO is believed to maintain the integrity of coronary vessels
through a wide range of physiological
effects.7 Hence, disrupted NO production
has been hypothesized to promote various aspects of
arteriosclerotic lesion development. NO could
affect the leukocyte contribution to endothelial cell
injury or vessel thickening in a number of ways. First, NO can inhibit
leukocyte adhesion to the
endothelium.39 Impaired NO
production in the vessel produces increased leukocyte adhesion
and emigration. Thus, NO deficiency could promote the
leukocyte-endothelium interaction that culminates in
endothelial injury. Second, NO and its redox forms
could control leukocyte accumulation within the expanded intima through
apoptotic mechanisms. In vitro, the activity of NO synthase in
macrophages has been shown to correlate inversely with their
life span,40 and this effect appears to be
mediated through NO-triggered apoptosis. NO also modulates
platelet-endothelium interactions. Ex vivo, NO
inhibits platelet function by reducing ADP-induced platelet
aggregation and effectively disaggregating already activated
platelets.41 This points to potential
antithrombotic effects of NO in the preinjured
endothelium. Besides its effects on VSMCs,
endogenous NO has been demonstrated to effectively inhibit
proliferation of fibroblasts.42 In later stages
of lesion development, NO could play an important role by mediating
antiproliferative effects in different cell types that are considered
essential components of late vascular remodeling.
Conclusions
Received September 12, 1997;
revision received December 9, 1997;
accepted December 19, 1997.
© 1998 American Heart Association, Inc.
Basic Science Reports
Exacerbated Transplant Arteriosclerosis in Inducible Nitric Oxide–Deficient Mice
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Inducible NO synthase
(NOS2, or iNOS) is upregulated in grafts with transplant
arteriosclerosis. However, the functional role of
NOS2 in the pathogenesis of transplant
arteriosclerosis remains unclear. NOS2 may regulate
lesion development by modulating the early alloimmune response and/or
late myointimal thickening. 
) and Th2- (STAT 6,
IL-4, and IL-5) associated factors were comparable in both groups. In
contrast, quantitative analysis of the 
-actin–positive area
showed a significant increase in the contribution of smooth muscle
cells within the neointima in allografts from NOS2 -/-
recipients (28.2±2.0%) compared with wild-type controls (13.2±2.3%;
P<.0001).
Key Words: transplantation • immunology • lymphocytes • remodeling • muscle, smooth
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Current
immunosuppressive regimens control acute rejection of transplanted
organs.1 However, transplant
arteriosclerosis continues to be a primary obstacle
to long-term survival of the transplanted heart. Despite a rapidly
growing body of data concerning the involved immunological events, the
exact pathogenetic mechanisms for this immunologically mediated form of
arteriosclerosis remain unclear. Experimental
models show that vascular thickening develops in
stages.2 3 An initial vascular injury is
established by an acute alloimmune response involving recipient-derived
T cells, B cells, and macrophages and graft
endothelial cells. This initial injury is followed by
infiltration of activated macrophages and lymphocytes
within the vessels and initiation of an inflammatory activation
cascade. Finally, this develops into a late stage with
neointimal SMC expansion, extracellular matrix formation,
and late intimal fibrosis. Understanding key regulators of these
arteriosclerotic stages and their precise mode of
action may lead to development of more selective interventions.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Mice
Male CBA/CaJ (H-2k) mice 6 to 8 weeks old
were used as heart donors. As organ recipients, male mice deficient in
inducible NO synthase (NOS2 -/-) on a C57BL/6Jx129/Sv
(H-2b) background26 were
compared with NOS2-competent wild-type mice. Two wild-type strains,
B6Jx129/Sv (H-2b), hereafter referred to as
B6/129, and C57BL/6J (H-2b), hereafter referred
to as B6, were used as comparative control groups. The genotype
of the knockout mice was confirmed by PCR as previously
described.26 NOS2-deficient mice were obtained in
collaboration with Dr Carl Nathan (Cornell University Medical College,
New York, NY). CBA/CaJ (stock number 000654), C57BL/6J (stock number
000664), and B6Jx129/Sv mice (stock number 101045) were obtained from
Jackson Laboratory, Bar Harbor, Me.
Vascularized, heterotopic abdominal cardiac transplantation was
performed as described by Corry et al.27
Postoperatively, all recipients were prophylactically
treated with trimethoprim/sulfamethoxazole (Bactrim, Roche
Pharmaceuticals) in the drinking water (trimethoprim 80 mg/L,
sulfamethoxazole 400 mg/L). Transplanted hearts were harvested as
described previously.25 Allografts from NOS2
-/- recipients (n=13) were compared with those from allogeneic
wild-type recipients (B6 recipients, n=8; B6/129 recipients, n=7).
Anti-CD4 and CD8 antibodies (GK1.5 and 2.43; 2.0 mg IP, days 1 to 4,
and then weekly to day 28) were used as T-cell–depleting
immunosuppressive therapy. This program of immunosuppression has been
shown to produce cardiac allografts that undergo chronic
rejection.28 Native hearts from transplant
recipients (NOS2 -/- and wild-type) exposed to the same circulation
were used as one control group. Syngeneic isografts placed into
untreated recipients (NOS2 -/- and wild-type) exposed to the same
surgical procedure were used as a second control group. Graft function
was monitored daily by measurement of the force of palpable heart beat.
After perfusion with PBS, cardiac allografts were harvested at 55 days
after transplantation.
Transverse heart sections were fixed in methyl Carnoy's
solution and embedded in paraffin. Sections (4 µm) were stained
with Verhoeff's elastin for histological evaluation of
cellular rejection and vascular analysis. Microscopic images of
all elastin-positive vessels were captured. The captured images were
used to trace the lumen, internal elastic lamina, and external elastic
lamina (NIH 1.6 software). The intimal area was determined by
subtracting the area of the lumen from the area enclosed by the
internal elastic lamina. The medial area was determined by subtracting
the area enclosed by the internal elastic lamina from the area enclosed
by the external elastic lamina. From these data, we derived percent
luminal occlusion25 and intima/media ratios.
Values are calculated as the mean from all captured vessels per heart
and reported as the mean±SD for all grafts in each recipient
group.
As a marker of more advanced
arteriosclerotic stages,29 we
estimated the contribution of SMCs to the expanded
neointima. Immunohistochemical staining for -SMC actin
was compared in paraffin sections (4 µm) from allografts
transplanted into NOS2 -/- recipients (n=5) and B6 wild-type
recipients (n=5). Duplicate sections were prestained with Verhoeff's
elastin to detect the internal elastic lamina. -SMC actin staining
was performed as described previously4 with a
monoclonal mouse IgG2a antibody (1:20 000, overnight at 4°C)
directed against -actin (Sigma Chemical Co). Microscopic images of
each vessel were captured separately. Differences in immunopositive
cells were estimated by image analysis with NIH Image 1.6. For
each vessel, the neointima was defined by tracing the
internal elastic lamina and the lumen. The area stained specifically
for -SMC actin was detected by measurement of the area encompassed
by pixels of the color intensity of immunopositive cells and tabulated
as the percentage of the total neointimal area. A mean from
all captured vessels per heart was calculated and is reported as mean
value for all grafts in each recipient group. To localize NOS2
expression within the rejecting heart, immunohistochemical staining for
NOS2 was compared in paraffin sections (4 µm) from chronically
rejecting allografts transplanted into NOS2 -/- and B6 wild-type
recipients. Polyclonal rabbit anti-NOS2 was prepared by Jeffrey R.
Weidner and Richard A. Mumford (Merck Research Laboratories) and kindly
provided to us by Carl Nathan. Immunostaining was
performed as described previously,4 with a
incubation time with polyclonal antiserum against NOS2 (1:250) of 60
minutes at room temperature. Negative controls included omission of
primary or secondary antibody and staining of native heart sections
from NOS2 -/- mice.
Semiquantitative 32P RT-PCR to measure
relative transcript levels was performed as described
previously.25 Briefly, total RNA was extracted
from ventricular sections with RNAzol B (Tel-Test).
First-strand cDNA synthesis using 2.5 µg of total RNA per reaction
was completed on all samples at the same time to ensure comparability
between samples (cDNA kit, GIBCO BRL). Given the large number of
grafts, the cDNA panel was restricted to the analysis of cDNA
prepared from the following hearts: allografts placed in NOS2 -/-
recipients (n=13) and B6 wild-type recipients (n=8) and native host
hearts from NOS2 -/- (n=13) and B6 wild-type recipients (n=8). PCR
primers designed by use of MacVector 5.0 (Oxford Molecular Scientific)
were synthesized on an Oligo 1000 DNA Synthesizer (Beckman). Primer
sequences, sequence accession numbers, annealing temperatures, and
cycle numbers are listed in the Table
. For each primer pair, conditions
were optimized to generate a single specific band, and the identity was
confirmed by restriction analysis. Triplicate samples were
amplified with 0.625 U AmpliTaq Gold DNA polymerase (Perkin Elmer) in a
total volume of 25 µL. After initial activation of this specific
polymerase at 95°C for 9 minutes, the thermal cycling
parameters were denaturation at 94°C for 30 seconds,
annealing at a primer-optimized temperature for 20 seconds, and
extension at 72°C for 60 seconds (increased by 2 seconds per cycle),
followed by a final extension of 7 minutes at the end of all cycles.
[32P]dCTP (150 000 cpm per reaction) was
included for quantitative PCR studies. The amount of incorporated
32P dCTP in amplified product bands from
dried agarose gels was measured by volume integration (Molecular
Dynamics). Corrected levels of the specific product were derived by
dividing the amplified product value by the mean value of the
control gene G3PDH in the respective sample. Corrected levels for
T-cell transcription factors were standardized for CD4 (corrected
leveltranscription factor/corrected
levelCD4).
View this table:
[in a new window]
Table 1. PCR Primer Sequences, Accession Numbers, Annealing
Temperatures, and Cycle
For comparison of two groups, an unpaired t test was
used. A value of P<.05 was considered significant. For
comparison of more than two groups, ANOVA was used. If the ANOVA was
significant, the Bonferroni/Dunn procedure was used as a post hoc test.
Group data are expressed as mean±SD.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Analysis of Transplant Arteriosclerosis
In this transplant model of attenuated rejection, all grafts
transplanted in NOS2 -/- (n=13) or wild-type (total n=15) recipients
survived to the end point of 55 days after transplantation. Native
hearts and isografts had no significant vessel thickening. Allografts
at 55 days from wild-type as well as NOS2 -/- recipients had typical
circumferential thickening of the vessels with perivascular mononuclear
cell infiltration. In diseased vessels, the internal elastic lamina was
intact, with occasional breaks. The expanded neointima
contained cells with features of mononuclear inflammatory cells and
SMCs. ). The mean luminal area was
significantly decreased in allografts from NOS2 -/- recipients
(587±217 µm2) compared with those placed
into both wild-type controls (B6, 1168±322
µm2, P<.0006; B6/129,
1248±546 µm2, P<.003). The
mean intimal area was significantly increased in allografts from NOS2
-/- recipients (2005±883 µm2) compared
with both wild-type controls (B6, 932±850
µm2, P<.007; B6/129, 740±513
µm2, P<.003). In contrast, the mean
medial area was not significantly different between the three groups
(NOS2 -/-, 1834±376 µm2; B6,
2321±1053 µm2; B6/129, 2329±621
µm2; P<.003). As shown in Fig 1, this resulted in a significant increase in severity of
arteriosclerotic thickening in allografts from NOS2
-/- recipients, as indicated by significantly higher mean percent
luminal occlusion (77.1±9.4%) and mean intima/media ratio
(1.91±1.26) compared with those in both wild-type recipient groups
(luminal occlusion: B6, 40.3±14.8%, P<.0001; B6/129,
41.3±13.1%, P<.0001; intima/media ratio: B6, 0.45±0.24,
P<.002; B6/129, 0.42±0.24, P<.002). Vessel
thickening developed in small, medium, and large myocardial allograft
vessels in both NOS2 -/- recipients (97±8% of all vessels) and
wild-type recipients (78±26% in B6, P<.03 versus NOS
-/-, and 79±17% in B6/129, P<.01 versus NOS2 -/-).
The widespread involvement confirmed the diffuse nature of this
process. There was similarity in frequency (78% versus 79%) and
severity (40.3% versus 41.3% luminal occlusion, 0.45 versus 0.42
intima/media ratio) between both of the two control groups (B6 and
B6/129), indicative of comparable arteriosclerotic
responses to alloimmune injury, whereas NOS2 -/- recipients showed
exacerbation in the magnitude of lesion development.
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Figure 1. Morphometric analysis of transplant
arteriosclerosis. Allografts placed into
NOS2-deficient recipients (n=13) have decreased mean luminal areas (A),
increased mean intimal areas (B), and unchanged mean medial areas (C)
compared with those placed into B6 (n=8) or B6/129 (n=7) wild-type
controls. This results in a significantly increased mean percent
luminal occlusion (D) and mean intima/media ratio (E) in allografts
from NOS2-deficient recipients. Mean areas (µm2) and
percent luminal occlusions from all captured vessels per heart are
reported as mean±SD for all grafts in each recipient group.
*P<.005 NOS2 -/- vs B6/129, 
P<.01
NOS -/- vs B6.
Corrected NOS2 transcript levels were evaluated by
32P RT-PCR (Fig 2).
In wild-type recipients, the expected allograft-specific induction of
NOS2 was confirmed by an increase in mean mRNA transcript levels in
allografts (0.51±0.40 RU) compared with native hearts (0.17±0.19 RU,
P=.0014). However, there was a significant reduction in NOS2
transcript levels in allografts placed in NOS2 -/- recipients
compared with grafts placed in wild-type recipients
(P<.0001, Fig 2A). NOS2 transcript levels were also
increased in cardiac grafts placed in NOS2 -/- recipients (0.25±0.11
RU) compared with the paired native hearts (0.05±0.01 RU). This
fivefold increase did not reach statistical significance with the
present sample numbers of 13 allografts and 8 native hearts.
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Figure 2. NOS2 mRNA levels. RT 32P PCR
amplifications normalized against G3PDH were compared in allografts and
native host hearts of NOS2 -/- and wild-type recipients. NOS2 levels
were higher in allografts than in native hearts. Allografts placed in
NOS2-deficient recipients (n=13) are compared with those placed in
wild-type control animals (n=8). A, Each bar represents
mean±SD from all cDNAs analyzed in triplicate in each
subgroup. B, NOS2 antigen expression detected by
immunostaining (red) localizes to graft-infiltrating
inflammatory cells (focal staining of lymphocyte-like cells with small
cytoplasmic rim and macrophage-like cells, open arrowheads) and
parenchymal cells (focal staining of cardiac myocytes and sporadic
staining of endothelial cells and VSMCs, solid arrows)
in grafts placed in wild-type recipients, whereas expression is
restricted to parenchymal cells (solid arrows) in NOS2 -/- recipients
(C). 
), whereas in allografts from NOS2 -/- recipients,
NOS2 antigen was absent in graft-infiltrating inflammatory cells and
present only within donor parenchymal cells (myocytes, VSMCs, and
endothelial cells; Fig 2C). This coexistence of both
donor and recipient sources of NOS2 in wild-type recipients underlines
the chimeric nature of the myocardial microenvironment within the
transplanted heart. In the setting of recipient NOS2 deficiency, there
was attenuated NOS2 induction in allografts, with expression in the
donor parenchyma but not graft-infiltrating recipient cells.
Given that NO under some conditions is known to regulate
Th1/Th2 differentiation of
CD4+ lymphocytes, we used
32P RT-PCR from graft tissue to compare the
allograft-specific mRNA expression for the respective transcription
factors and signature cytokines between the two recipient
groups. As shown in Fig 3, there were no
significant differences in the Th1 response
between grafts from either NOS2 -/- or wild-type recipients. There
were comparable transcript levels for STAT 4 (normalized for CD4:
0.043±0.01 versus 0.043±0.03 RU, P=.98), IL-2
(0.025±0.015 versus 0.018±0.010 RU, P=.27), and IFN-
(0.043±0.025 versus 0.042±0.023 RU, P=.89). The
Th2 response was also similar between
NOS2-deficient and -competent recipients, as assessed by measurement of
transcript levels for STAT 6 (normalized for CD4: 0.036±0.01 versus
0.049±0.02 RU, P=.10), IL-4 (0.033 ±0.013 versus
0.024±0.010 RU, P=.12), and IL-5 (0.026±0.013 versus
0.019±0.006 RU, P=.16). Exacerbation of transplant
arteriosclerosis was not associated with
alterations in the Th1/Th2
balance when RT-PCR was used to assess expression of transcription
factors and signature cytokines.
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Figure 3. Immunohistological
analysis of neointimal VSMC proliferation.
Increased contribution of -actin–positive VSMCs (red/brown) to
formation of neointima in allografts of NOS2-deficient
recipients (C) compared with allografts from wild-type controls (A).
Comparison of immunohistochemical staining of all vessels (n=66) in
grafts from NOS2 -/- recipients (n=5) with grafts from wild-type
recipients (n=5) (B). Area stained specifically for -SMC actin is
depicted as percentage of total neointimal area. A mean±SD
from all captured vessels per heart was calculated for all grafts in
each recipient group.
To study whether NOS2 mediates its protective effects by
attenuation of the later stage of myointimal expansion, we estimated
the SMC contribution to the expanded neointima using
-SMC actin as a marker. Immunohistochemical staining for -SMC
actin was compared in a total of 66 vessels from 5
representative grafts each placed into NOS2 -/- and
B6 wild-type recipients. Computer-assisted image analysis
showed that grafts placed into NOS2 -/- recipients had a significant
increase in the contribution of -SMC actin–positive cell area
within the expanded neointima (28.2±2.0%) compared with
the neointimal area in control allografts (13.2±2.3%,
P<.0001).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Protracted alloimmune injury to the donor vasculature is believed
to propagate the accelerated form of vascular thickening that
accompanies chronic rejection. Here, we demonstrate that NOS2 plays a
protective role in transplant arteriosclerosis by
using NOS2 -/- mice as recipients in a heterotopic cardiac transplant
model. We show that NOS2 deficiency results in significant increases in
severity and frequency of intimal thickening in response to alloimmune
injury. The medial areas were conserved among the groups. This
indicates that the increased vessel occlusion resulted from increased
intimal thickening as opposed to significant vessel shrinkage. Compared
with previous studies using pharmacological NOS inhibitors,
this experimental approach offers the advantage of ongoing and more
specific NOS2 inhibition through targeted gene deletion. Hence, we
establish that NOS2 has a causal role in the cascade that culminates in
vascular remodeling in this alloimmunologically initiated form of
arteriosclerosis. -SMC actin–positive cells
within the expanded neointima is significantly larger in
grafts from NOS2-deficient recipients than wild-type recipients. Given
that SMCs are characteristic of more advanced
lesions,29 this suggests that NOS2 interferes
with the late stage of transplant arteriosclerosis
by reducing neointimal SMC accumulation. This
proarteriosclerotic effect seen in cardiac
allografts from NOS2-deficient recipients might have arisen from
changes in the temporal progression of this disease, the actual manner
in which vascular remodeling occurs, or both.
There are a number of ways in which increased myointimal
thickening might develop in this arteriosclerotic
model. The SMC component of the neointima could increase
from migration of VSMCs into the intima or proliferation of these
immigrated cells.30 Alternatively, there might
also be decreased loss of intimal cells (ie, apoptosis),
resulting in the accumulation of SMCs within the expanded
intima.31 In vitro evidence shows that
endogenous NO can directly modulate VSMC migration as well
as proliferation. First, addition of different NO donors reduced the
number of migrating cultured VSMCs and the maximal distance migrated in
a concentration-dependent manner.13 Second, SMC
migration resumed after removal of NO donors. Third, addition of NO
donors also reduced serum-induced thymidine incorporation and
significantly decreased proliferation of rat aortic
SMCs.14 15 Conversely, there is evidence that NO
can induce apoptosis. In vitro stimulation of rat VSMCs with
IFN- and tumor necrosis factor- or IL-1ß produced
apoptotic cell death associated with increased elaboration of
nitrite, an end product of NO.31 Second,
apoptosis is partially blocked by L-NMMA. Hence, at least in
vitro, NO-dependent mechanisms can promote apoptosis in
VSMCs.
One might also speculate that NO serves a role in regulating
early immune system–mediated forces that lead to later myointimal
expansion. The immune response to a specific stimulus has been
hypothesized to involve the differentiation of
CD4+ T lymphocytes into Th1
or Th2 programs,10 32 which
differ in their cytokine secretion patterns and immunological
effector functions.10 NO could play a role by
modulating the differentiation of T cells into distinct subsets of T
helper cells (Th1 and Th2),
altering the functions of these differentiated T cells, or both. alter
macrophage effector functions by induction of NO, whereas
Th2-derived IL-4 and IL-10 are able to inhibit
the Th1-mediated induction of
NOS.33 34 In vivo, it has been demonstrated that
Th1-mediated protective immunity in a rodent
malaria model involved NO-dependent mechanisms.35
This was exemplified when L-NMMA reversed the protective effect on
parasitemia seen in Th1-reconstituted mice.
would result in accelerated chronic rejection and transplant
arteriosclerosis. The present results,
demonstrating unchanged expression of the program-specific
Th1 and Th2 transcription
factors and intragraft cytokine levels, are
inconsistent with this hypothesis. Instead, these findings
suggest that exacerbation of transplant
arteriosclerosis in NOS2-deficient recipients may
arise as a CD4+-cell–independent process.
The present study is the first to use NOS2 knockout animals to
demonstrate a protective, antiarteriosclerotic role
for NO in an in vivo model of arteriosclerotic
lesion development initiated by immune injury. Our findings confirm and
extend studies showing reduced neointimal thickening after
augmentation of NO production in various
arteriosclerotic models. For example,
administration of different NO donors17 18 19 or
gene transfer–mediated restoration of endothelial
NOS36 have been shown to suppress intimal
thickening after carotid balloon injury. In a model of atherosclerotic
lesion development in hypercholesterolemic rabbits,
lesion development was attenuated by dietary supplementation of
L-arginine, the precursor for
NO.37 38 These
antiarteriosclerotic effects could be blocked by
coadministration of an inhibitor of NO synthases
(NG-nitro-L-arginine methyl
ester) in a rabbit model after balloon catheter–induced
injury.18 The fact that NO-related
antiarteriosclerotic effects are demonstrated in
models based on an immune system–mediated injury (reported here) as
well as in models of mechanically induced lesion development provides
evidence for a immune cell–independent mode of action for NOS in
arteriosclerotic syndromes.
The present study establishes that endogenous NOS2
participates in the antiarteriosclerotic response
that regulates intimal thickening in chronically rejecting mouse
cardiac allografts. The antiarteriosclerotic effect
was independent of T-cell differentiation. Instead, the augmented
intimal thickening in NOS2 -/- recipients correlated with an
increased contribution of -SMC actin–positive cells to the
formation of the neointima. This suggests that NOS2 may
mediate its protective effects by controlling neointimal
SMC migration or proliferation. Ongoing exploration of the mechanisms
through which NOS2 regulates vessel responses to alloimmune injury in
this mouse model may provide insight into its role in this and other
forms of arteriosclerosis.
Selected Abbreviations and Acronyms
IFN
=
interferon
IL
=
interleukin
L-NMMA
=
NG-monomethyl-L-arginine
NOS
=
NO synthase
NOS2
=
inducible NO synthase
PCR
=
polymerase chain reaction
RT
=
reverse transcription
RU
=
relative unit
SMC
=
smooth muscle cell
STAT
=
signal transducer and activator of transcription
VSMC
=
vascular smooth muscle cell
Acknowledgments
This work was supported by a grant from the Milton Foundation. Dr
Koglin was supported by a fellowship grant from the Deutsche
Forschungsgemeinschaft, Bonn, Germany.
References
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Abstract
Introduction
Methods
Results
Discussion
References
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