From the Departments of Medicine (S.R., A.A., M.M., A.B., P.J.C.),
Pathology (M.S.), and Surgery (R.E.M.), Columbia University College of
Physicians and Surgeons, New York, NY.
Correspondence to Paul J. Cannon, MD, Department of Medicine, Division of Cardiology, Columbia University, 630 W 168th St, New York, NY 10032.
Methods and Results—We studied 15 patients with TCAD and 10 with
normal coronary arteries. In situ hybridization and
immunohistochemistry were used in tissue sections to localize iNOS mRNA
and protein, respectively. The presence of peroxynitrite was indirectly
assessed by immunostaining with an anti-nitrotyrosine
antibody. Normal coronary arteries had no evidence of iNOS
expression. In contrast, 30 of 36 coronary artery segments with
TCAD (83%) were immunostained by the iNOS antibody. The
presence of iNOS mRNA was demonstrated in these vessels by in situ
hybridization. Specific cell markers identified iNOS-positive cells as
neointimal macrophages and smooth muscle cells.
Nitrotyrosine immunoreactivity colocalized with iNOS expression in
arteries with TCAD, distributed in macrophages and smooth
muscle cells.
Conclusions—iNOS mRNA and protein are expressed in human arteries
with TCAD, where they are associated with extensive nitration of
protein tyrosines. These findings indicate that the high-output nitric
oxide pathway and possibly the oxidant peroxynitrite might be involved
in the process leading to the development of TCAD.
The oxidation of the amino acid L-arginine by the family
of enzymes known as NOS is important in a large number of
physiological and pathological processes. Three
isoforms of NOS have been identified and cloned: neuronal,
cytokine-inducible (iNOS), and endothelial.
Endothelial NOS, found predominantly in
endothelial cells, is calcium-calmodulin
dependent and produces small amounts of NO in response to shear stress
or to agonists like bradykinin.7 8 9 iNOS, induced
in macrophages, SMCs, endothelial cells, and
other cells by cytokines, is not dependent on increased calcium
and calmodulin and produces large amounts of NO for long
periods of time.7 8 9 NO, by activating soluble
guanylyl cyclases in target cells, can initiate responses such as
vasodilation,10 inhibition of platelet
aggregation and adhesion to the vessel wall,11
and inhibition of SMC proliferation.12 NO, when
produced in higher concentrations by iNOS, has other effects, some of
which are cytotoxic. It can cause nitration and nitrosylation of
proteins, inhibit enzymes involved in oxidative metabolism
and mitochondrial respiration, bind to FeS clusters in proteins, cause
ADP ribosylation, and damage DNA.8 9 When NO
combines with equimolar amounts of superoxide, peroxynitrate is formed,
which (although an NO donor at low concentrations) can at high
concentrations cause nitration of cell proteins and decompose to form
cytotoxic hydroxyl radicals.13 14
In addition to its established function in the
cardiovascular system, NO participates in immune
responses15 and has been implicated in allograft
rejection. Using a rat model of heterotopic cardiac transplantation,
Yang et al16 reported that iNOS mRNA, protein,
and enzyme activity were induced in rejecting allografts in association
with reduced contractility and death of cardiac
myocytes. Immunoreactivity for iNOS was present in
macrophages infiltrating the myocardium and in
cardiac myocytes in the rejecting allografts. Using a similar model,
Worrall et al17 confirmed these results and
demonstrated that treatment with aminoguanidine, a relatively selective
inhibitor of iNOS, prolonged allograft survival, improved
contractility, and reduced the intensity of rejection.
In studies of human endomyocardial biopsies,
induction of iNOS mRNA was correlated with a reduced ejection fraction
of the transplanted hearts.18 In rat cardiac
allografts, Szabolcs and coworkers19 reported
that apoptotic death of infiltrating macrophages and of
cardiac myocytes in the rejecting hearts was correlated in time and
intensity with the induction of iNOS mRNA, protein, and enzyme
activity.
iNOS is expressed in human atherosclerotic coronary
arteries.20 Russel et al21
and Akyurek et al22 also reported recently that
iNOS expression is upregulated in macrophages and SMCs in the
intimal lesions of TCAD in rat transplantation models. The purpose of
the present study was to investigate, with in situ hybridization
and immunohistochemistry, the expression and cellular distribution of
iNOS and nitrated proteins in the human lesions of TCAD.
Tissue Preparation
Immunohistochemistry
Sections were deparaffinized, rehydrated in sequential alcohol baths,
and then washed in PBS. Endogenous peroxidase was
inactivated with 3% hydrogen peroxide in ethanol and
nonspecific antibody binding was suppressed with 20% nonimmune serum
in PBS. Sections were incubated in a humidified chamber overnight at
4°C with the anti-iNOS antibody or for 1 hour at room temperature
with the other antibodies. With intervening washes in PBS, sections
were then incubated with a biotinylated secondary antibody. A goat
anti-rabbit IgG (Vector Laboratories) was used for the anti-iNOS and
von Willebrand antibodies, and a horse anti-mouse IgG (Vector)
was used for the monoclonal anti-nitrotyrosine, CD-68, and
Double Immunofluorescence
Histological/Immunohistochemical Analysis
In Situ Hybridization
Sections were rehydrated in PBS containing 5mmol/L
MgCl2 and then digested in 5 µg/mL proteinase K
(Boehringer Mannheim) for 15 minutes at 37°C. The tissue was
postfixed in 4% paraformaldehyde in PBS for 10
minutes, washed in diethylpyrocarbonate (DEPC)-treated distilled water,
and acetylated in triethanolamine-acetic anhydride (TEA) buffer
(100 mmol/L triethanolamine with 0.25% acetic anhydride, pH 8.0).
After being rinsed in DEPC-treated distilled water, sections were
prehybridized in a humid chamber for 1 hour at 53°C in hybridization
buffer consisting of 50% formamide, 5x SSC, 2% blocking reagent
(Boehringer Mannheim), 0.02% SDS, and 0.1%
N-lauroylsarcosine. Hybridization was then performed
overnight in a humid chamber at 56°C with digoxigenin-labeled sense
and antisense probes in hybridization buffer. Subsequently, the
sections were washed in 2x SSC at 65°C and 1x SSC and 0.1x SSC at
72°C for 30 minutes each; then they were incubated in maleic acid
buffer (100 mmol/L maleic acid, 150 mmol/L NaCl, pH 7.5) plus
1.5% wt/vol blocking reagent (Boehringer Mannheim) for 30
minutes with slow agitation. To detect the hybridization signal, the
sections were incubated with an anti-digoxigenin antibody
(Boehringer Mannheim), diluted 1:500, followed by rabbit
anti-mouse immunoglobulin and standard immunoalkaline phosphatase
reaction, with nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Boehringer
Mannheim) as a substrate. After development, the slides were washed in
TE buffer (10 mmol/L TRIS-HCl, pH 8.0, containing 1 mmol/L
EDTA) for 5 minutes and mounted with coverslips by use of aqueous
mounting medium (Innovex Biosciences).
Inflamed human tonsils, rich in activated macrophages,
were used as positive controls for both in situ hybridization and
immunohistochemistry. Labeling the tissue sections with the sense probe
and replacing the primary antibody with normal rabbit serum provided
negative controls for in situ hybridization and immunohistochemistry,
respectively.
Statistical Analysis
iNOS
Coronary arteries with TCAD were classified as
atheromatous in 8 of 15 patients (53%) and
proliferative in 7 of 15 (47%) on the basis of preestablished criteria
that included lesion focality (ie, eccentric versus diffuse), the
presence or absence of extracellular lipid deposits in the expanded
intima, the integrity or disruption of the internal elastic lamina, and
the presence or absence of calcifications. Foam cells and an intense
neointimal lymphocytic infiltrate were present in both
types of lesion. iNOS immunoreactivity was equally distributed among
the two histological types of TCAD. The mean
semiquantitative grade, ie, the intensity of iNOS
immunostaining, was 1.9 for blood vessels with
atheromatous features and 1.75 for those with
proliferative ones (P=NS).
Nitrotyrosine
In Situ Hybridization
Russell and coworkers21 have previously
demonstrated upregulation of iNOS in rat cardiac allografts with
chronic rejection and transplant arteriosclerosis.
In their study, iNOS mRNA was found to be markedly increased in
coronary arteries of the transplanted hearts compared with
paired host hearts and syngeneic grafts. iNOS protein immunoreactivity
was demonstrated to be localized to medial and neointimal
SMCs and macrophages in blood vessels with graft
arteriosclerosis. Akyurek et
al22 reported that iNOS mRNA and protein were
expressed in rat aortic allografts. iNOS-positive SMCs and
macrophages were found in the thickened neointima
and in the media of the transplanted aortas. Recently, Lafond-Walker
and coworkers23 have demonstrated the presence of
iNOS in coronary arteries of human hearts with accelerated
graft arteriosclerosis. By
immunostaining of serial sections, these authors found
iNOS in CD68-positive macrophages but not in
neointimal SMCs. Our study therefore confirms previous
findings in the experimental transplant model and demonstrates, for the
first time in humans, that iNOS is also produced by
neointimal SMCs in arteries with TCAD.
The expression of iNOS has also been established in numerous
other human tissues. Nicholson et al24
demonstrated that iNOS mRNA and protein are expressed by alveolar
macrophages in patients with tuberculosis. iNOS enzyme
activity, protein, and mRNA have been found in myocardial cells of
patients with cardiomyopathy and cardiac allograft
rejection19 25 and in human
megakaryocytes.26 The demonstration of iNOS in
human cells in vivo contrasts with the difficulties previously
encountered by many investigators in inducing its expression in human
cells in vitro.27 Recently, however, several
laboratories have reported the induction of iNOS in human cells in
vitro by appropriate cytokine combinations. Kolb et
al28 have demonstrated that IFN-
In animal models of TCAD, activated lymphocytes interact with
macrophages and vascular endothelial cells to
produce a variety of growth factors and cytokines, including
IL-1, IL-2, and IL-6; IFN-
In contrast to the marked iNOS upregulation we found in the epicardial
coronary arteries of patients with TCAD, no expression was
noted in intramyocardial arterioles. In this respect, our findings in
humans differ from those of Russell and
coworkers,21 who reported iNOS
immunostaining in very small vessels, including
intramyocardial arterioles, in a rat model of transplant
arteriosclerosis. It is currently unknown whether
the factors that have been demonstrated to regulate iNOS expression in
large arteries such as the aorta or the epicardial coronary
arteries also control iNOS production in small resistance
vessels such as the intramyocardial arterioles. Clausell and
coworkers34 have shown that
histological abnormalities of small arterioles observed
in endomyocardial biopsies specimens, albeit
frequent, do not correlate with intravascular ultrasound findings and
with endothelial dysfunction of large epicardial
arteries. Taken together, these observations suggest that TCAD might be
a more heterogeneous disease than usually thought in which
different pathogenic factors contribute to disease development in
different segments of the coronary artery tree.
The role that iNOS activation might play in the atherogenic
process, in both native vessels and transplanted hearts, has yet to be
elucidated. NO clearly has numerous potential antiatherogenic
properties: it inhibits the proliferation of vascular SMCs in
vitro,12 decreases platelet adhesion and
aggregation,11 35 and reduces the
endothelial expression of adhesion molecules and
chemotactic factors.36 37 Inhibition of NOS with
NG-nitro-L-arginine methyl
ester increases the extent of atherosclerotic lesions in the
experimental animal,38 39 40 whereas administration
of the NO precursor L-arginine has clear antiatherogenic
effects in both native atherosclerosis and
TCAD.40 41 42 NO has also been shown to induce
apoptosis in vascular SMCs and macrophages in vitro
through cGMP-dependent and independent
mechanisms.43 44 Thus, iNOS- and NO-mediated
apoptosis of macrophages and SMCs might decrease the
cellularity of the atherosclerotic plaque and participate in the
process of vascular remodeling. In a recent report, TCAD was
exacerbated in iNOS-deficient mice, a finding consistent with a
protective role for iNOS in this situation.45
Some of the effects deriving from the production of large
amounts of NO by iNOS may nonetheless be proatherogenic. NO reacts with
the free radical superoxide to form
peroxynitrite,13 a strong oxidant that damages
cellular proteins by nitration of tyrosine residues to form
nitrotyrosine.46 In the present study, using
a highly specific antibody developed by Beckman et
al,47 we demonstrated extensive protein nitration
in coronary arteries with TCAD, in concordance with previous
observations in native
atherosclerosis.20 47 Although
little is known about the effects of tyrosine nitration on protein
function in vivo,47 it has been shown that
peroxynitrite or its decomposition products induce membrane lipid
peroxidation48 and can initiate lipid
peroxidation in human LDL.49 By altering the
cellular redox state, it can also induce the expression of
redox-sensitive genes (such as vascular cell adhesion molecule-1) that
participate in the recruitment of inflammatory cells to the
endothelial surface.50 The
generation of large amounts of peroxynitrite consequent to the
increased levels of NO and superoxide in the atherosclerotic lesion
might therefore be a factor contributing to the progression of the
disease.
In summary, the present study provides evidence that iNOS is
expressed in macrophages and SMCs in the lesions of TCAD, where
it is associated with extensive nitration of cellular proteins. Whether
an increased local synthesis of NO is beneficial or detrimental in this
process remains unclear and requires further investigation.
Received December 16, 1997;
revision received January 20, 1998;
accepted February 4, 1998.
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
Inducible Nitric Oxide Synthase Expression in Smooth Muscle Cells and Macrophages of Human Transplant Coronary Artery Disease
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—The inducible isoform of
the nitric oxide synthase (iNOS) produces large amounts of nitric oxide
in response to cytokine stimulation. Previous investigations
have demonstrated iNOS expression in the setting of acute and chronic
rejection in experimental cardiac transplant models. The goal of this
study was to investigate whether iNOS is upregulated in human
transplant coronary artery disease (TCAD), a major cause of
late mortality after cardiac transplantation.
Key Words: arteriosclerosis • endothelium-derived factors • transplantation
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Long-term survival of
cardiac transplant recipients has been limited by the development of a
vasculopathy in the coronary arteries of the
allograft.1 The pathogenesis of this
vasculopathy, called TCAD, is not fully understood but is thought to
result from the interaction of immunological and nonimmunological
factors leading to the migration and proliferation of SMCs,
T-lymphocytes, and macrophages in an expanded
neointima.2 3 4 5 6 The inflammatory
reaction leads to luminal narrowing, myocardial ischemia and
infarction, and the development of heart failure and malignant
arrhythmias.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Patient Characteristics
Patients demographics are summarized in Table 1
. Written informed consent for the use
of cardiac tissue samples was obtained from all subjects.
Coronary arteries were obtained from the explanted allografts
of 15 patients (12 men) undergoing retransplantation for severe TCAD.
Allograft survival ranged from 13 to 126 months.
Cyclosporine, corticosteroids, and
azathioprine were used in combination for immunosuppression. Normal
coronary arteries were obtained for comparison from the native
hearts of 10 patients undergoing transplantation for idiopathic
cardiomyopathy or congenital heart disease.
View this table:
[in a new window]
Table 1. Characteristics of the Patient Population
Coronary arteries were fixed in 10% buffered formalin,
paraffin-embedded, sectioned at 4-µm intervals, and stained with
hematoxylin and eosin. Parallel sections were applied to
organosilane-coated slides and used for immunohistochemistry. For in
situ hybridization, unfixed segments of coronary arteries were
placed in OCT embedding compound (Miles Laboratory) and immediately
snap-frozen. Cryosections were cut under RNase-free conditions, mounted
on RNase-free glass slides (Fisher Scientific), air-dried, and fixed in
4% paraformaldehyde in PBS for 20 minutes in a
humidified chamber. Sections were then dehydrated in a graded series of
ethanol (2 minutes each), air-dried, and stored at -70°C until
processing.
The source of each antibody used and the optimal working
dilution are summarized in Table 2. The
affinity-purified rabbit antibody to a synthetic peptide from mouse
iNOS (amino acid residues 961 through 1144) was purchased from
Transduction Laboratories. The presence of nitrated proteins was
determined by a monoclonal anti-nitrotyrosine antibody (Upstate
Biotechnology). Antibodies directed against CD-68 (Dako) and 
-smooth
muscle actin (Bio Genex) were used to identify macrophages and
SMCs, respectively.
View this table:
[in a new window]
Table 2. Antibodies Used for Immunohistochemistry -smooth
muscle actin antibodies. The avidin-biotin-immunoperoxidase complex
(ABC Elite, Vector) was then applied. Peroxidase activity was
visualized with diaminobenzidine (Sigma Chemical Co).
Deparaffinized, rehydrated sections were
simultaneously incubated with either anti-iNOS antibody and
anti–smooth muscle actin or anti-iNOS and anti-CD68 antibody. After
rinsing with PBS, both secondary antibodies, tetramethylrhodamine
isothiocyanate (TRITC)-labeled goat anti-rabbit IgG and fluorescein
isothiocyanate (FITC)-labeled goat anti-mouse, were applied for 30
minutes, demonstrating the presence of iNOS by red
immunofluorescence labeling and the presence of
smooth muscle actin or CD-68 by green
immunofluorescence labeling. Sections were
analyzed with a fluorescence microscope (Olympus BX 40)
equipped with a blue filter for FITC (green
immunofluorescence) and a green filter for TRITC
(red immunofluorescence). Double-labeled sections
in which one of the primary antibodies was replaced with an irrelevant
isotype-specific antibody (Sigma) served as controls.
Sections were independently examined by two observers for the
presence of iNOS immunoreactivity and its distribution in the vessel
wall. The intensity of staining was graded numerically on a scale from
1 to 3 as follows: 1=weak, 2=moderate, and 3=intense. Coronary
arteries with TCAD were classified as atheromatous if
they had at least three of the following histological
features: (1) eccentric plaque, (2) disrupted internal elastic lamina,
(3) large lipid deposits ("cholesterol clefts"), or (4)
calcifications. Conversely, lesions were defined as proliferative if
they consisted of a concentric accumulation of myointimal cells, with a
mostly intact internal elastic lamina, without lipid deposits and
calcifications.
A fragment of the mouse iNOS cDNA corresponding to the
nucleotides 524 through 869 (85% homology with the human
iNOS) was amplified by polymerase chain reaction from 1 µg of mouse
iNOS cDNA (generous gift of Dr Carl Nathan, Cornell University Medical
College) and cloned into the polymerase chain reaction II vector (TA
Cloning Kit, Introgen). Sense and antisense DNA templates were
synthesized from this gene construct by polymerase chain reaction.
Digoxigenin-labeled sense and antisense RNA probes were generated from
the corresponding DNA templates by use of the SP6 and T7 RNA
polymerase, respectively.
All data are reported as mean±SEM. The rank-sum test was used
for statistical comparison of the intensity of
immunostaining. A value of P<0.05 was
considered statistically significant.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Immunohistochemistry
A total of 36 epicardial coronary artery segments from 15
patients with TCAD (2.4 segments per patient) and 22 segments from 10
patients with normal coronary arteries (2.2 segments per
patient) were analyzed for iNOS and nitrotyrosine
immunoreactivity.
Immunostaining for iNOS was not present
in any of the normal coronary arteries studied. In contrast, 30
of 36 coronary artery segments with TCAD (83%) were labeled by
the iNOS antibody. Representative sections of a normal
coronary artery and of an artery with advanced TCAD labeled
with the iNOS antibody are shown in Figure 1. Immunoreactivity for iNOS was found in
the neointimal layer of epicardial coronary
arteries, localized to spindle-shaped mesenchymal cells, mononuclear
cells, and foam cells, morphologically consistent with SMCs and
macrophages, respectively (Figure 1). The intensity of iNOS
immunostaining, assessed by a semiquantitative
microscopic analysis, varied considerably not only from patient
to patient but also in different vessels of the same patient. Labeling
was confined to the cytoplasm of individual cells in the expanded
neointima and was not observed in the extracellular space.
To identify with certainty the cell types immunoreactive for iNOS, we
used two different immunohistochemical techniques. In one, parallel
sections were immunostained with the antibodies CD-68 and
-smooth muscle actin to recognize macrophages and SMCs,
respectively. In the second, the same primary antibodies were incubated
with TRITC- and FITC-labeled secondary antibodies, yielding red and
green immunofluorescence, respectively, when viewed
by fluorescence microscopy. The results are shown in Figure 2 (Figure 2A through 2E for
immunohistochemistry; Figure 2F through 2I for double
immunofluorescence). It is evident from the
representative sections that iNOS expression was
present in both neointimal SMCs and
macrophages. Vasa vasorum, small blood vessels located in the
thickness of the adventitia, consistently showed intense
immunoreactivity for iNOS (Figure 3);
intramyocardial arterioles, on the other hand, were
consistently negative, even in cases with advanced occlusive
disease (Figure 3). In all cases, the immunoreactivity was abolished
when the primary antibody was replaced with nonimmune serum, confirming
the specificity of the reaction (Figure 2).
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Figure 1. Sections of a normal coronary artery (A)
and of a coronary artery with TCAD (B)
immunostained with the anti-iNOS antibody. Diaminobenzidine
was used as a chromogen in the immunoperoxidase reaction, yielding a
brown reaction product in positive cells. The normal artery has no
immunoreactivity. In contrast, the artery with graft
arteriosclerosis has intense immunoreactivity
localized to spindle and foam cells in the thickened
neointima. Hematoxylin counterstain. Magnification
x400.
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Figure 2. Identification of cell types expressing iNOS in
TCAD. Single-label immunohistochemistry is shown in A through E. Serial
sections were immunostained with the anti-iNOS antibody,
-smooth muscle actin, and CD-68. Diaminobenzidine was used as a
chromogen, yielding a brown reaction product in positive cells.
iNOS-positive cells (A and C) express SMC (B) and macrophage
(D) markers in serial sections. No immunoreactivity was seen in the
negative controls (E). Double immunofluorescence is
shown in F through I. The same section was simultaneously
incubated with iNOS (F and H), recognized by red
immunofluorescence, and -smooth muscle actin (G)
or CD-68 (I), recognized by green
immunofluorescence. In numerous
neointimal cells, iNOS colocalizes with the SMC and the
macrophage markers. Magnification x400.
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Figure 3. Representative sections from two
cases of TCAD immunostained for iNOS. Several vasa vasorum
located in the thickness of the inflamed adventitia are intensely
labeled (brown reaction product) with the iNOS antibody (A).
Conversely, intramyocardial arterioles, such as the one depicted (B),
were consistently negative, even in cases with advanced
occlusive disease. Hematoxylin counterstain. A, Magnification x400; B,
magnification x100.
Representative examples of two
coronary arteries with severe TCAD immunostained
with a monoclonal anti-nitrotyrosine antibody are shown in Figure 4. Extensive immunoreactivity is shown in
both cases. A large number of nitrotyrosine-positive cells were seen
throughout the markedly thickened neointima. The labeling
was found in cells morphologically consistent with
macrophages and vascular SMCs in a distribution similar to that
observed for iNOS. No labeling was present when the sections were
incubated with control mouse IgG.
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Figure 4. Protein nitration in TCAD. Coronary
arteries were immunostained with an anti-nitrotyrosine
antibody by use of the immunoperoxidase technique. Brown reaction
product indicates specific antibody binding. Low magnification (A)
shows diffuse immunoreactivity in the markedly expanded
neointima. At high magnification (B), staining is seen in
cells morphologically consistent with macrophages and
SMCs, with a distribution that parallels that of iNOS. Hematoxylin
counterstain. A, Magnification x100; B, magnification x400.
iNOS mRNA expression in coronary arteries with TCAD was
investigated with antisense riboprobes labeled with digoxigenin and
detected with an immunoalkaline phosphatase method. Inflamed human
tonsils, rich in activated macrophages, were used as
positive controls (Figure 5).
Hybridization of the antisense iNOS probe was found in all arteries
that immunostained for iNOS protein and was localized to
the superficial and deep layers of the vessels neointima
(Figure 6A through 6C) and occasionally
to the SMCs of the tunica media (not shown). Comparison with parallel
sections immunostained with the cell-specific antibodies
CD-68 and -smooth muscle actin indicated that iNOS mRNA-positive
cells were macrophages and SMCs. Tissue sections from the same
coronary arteries incubated with the sense probe, used as
negative controls, did not show any hybridization signal (Figure 6D).
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Figure 5. In situ hybridization for iNOS mRNA in inflamed
human tonsils. Staining with the antisense probe showed intense
labeling for iNOS mRNA in activated macrophages (A).
Sections incubated with the sense probe, used as negative controls, did
not show any hybridization signal (B). No counterstaining.
Magnification x400.
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[in a new window]
Figure 6. In situ hybridization for iNOS mRNA in TCAD.
Low-power microphotograph (A) of a coronary artery with
advanced TCAD incubated with the antisense probe shows intense labeling
for iNOS mRNA in the superficial and deep layers of a markedly
thickened neointima. At higher magnification (B and C),
labeling can be seen in cells morphologically consistent with
SMCs and macrophages. The same artery incubated with the sense
probe did not show any hybridization signal (D). No counterstaining. A,
Magnification x25; B, magnification x100; C and D, magnification
x400.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The present study demonstrates that iNOS is expressed in
coronary arteries of patients with TCAD. iNOS immunoreactivity
was found in 30 of 36 arterial segments with TCAD (83%)
and was localized to neointimal foam cells, SMCs, and
macrophages. The presence of iNOS mRNA was demonstrated in the
same vessels by in situ hybridization. We also showed that iNOS
expression is associated with the presence of nitrated cellular
proteins in the thickened neointima of coronary
arteries with TCAD. 
alone was not
able to induce iNOS in human monocytes, unlike its effect in murine
macrophages, but that its activation was triggered by the
combination of IFN- and IL-4. Human epithelioid cells of the A549
cell line transfected with the human iNOS promoter were recently found
to produce NO only when stimulated with a combination of IL-1ß and
IFN- but not with either cytokine
alone.29 Additional stimulation was obtained by
adding TNF-. Thus, despite the marked similarity between the human
and murine iNOS genes, differences exist in the control of their
activation. ; and TNF-.30 31
The presence of a cytokine-rich milieu in the vessel wall might
therefore account for the expression of iNOS in this setting. Another
immunologically mediated pathway might play an important role in the
induction of iNOS in the cardiac allograft, namely the interaction
between CD40, present on the surface of antigen presenting
cells, endothelial cells, and SMCs, and the CD40 ligand
(gp39) expressed by activated T-lymphocytes. Recent animal
studies have shown that treatment of allograft recipients with an
antibody directed against gp39 prolonged survival and markedly reduced
the intragraft expression of iNOS.32 Preliminary
data from our laboratory indicate that CD40 and CD40 ligand-positive
cells are present in human lesions of
atherosclerosis and TCAD.33
Selected Abbreviations and Acronyms
IFN
=
interferon
IL
=
interleukin
iNOS
=
inducible isoform of NOS
NO
=
nitric oxide
NOS
=
nitric oxide synthase
SMC
=
smooth muscle cell
SSC
=
standard saline citrate
TCAD
=
transplant-associated coronary artery disease
TNF
=
tumor necrosis factor
Acknowledgments
We thank Virginia A. Capo, MD, PhD, and Ramona I. Polvere, MPH,
for their invaluable help with the in situ hybridization. This work was
supported in part by National Heart, Lung, and Blood Institutes grants
HL-56984 and HL-54764.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
-activated human monocytes: involvement of the
nitric oxide synthase pathway. J Biol Chem. 1994;269:9811–9816.
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 M. J. Szabolcs, J. Sun, N. Ma, A. Albala, R. R. Sciacca, G. B. Philips, J. Parkinson, N. Edwards, and P. J. Cannon Effects of Selective Inhibitors of Nitric Oxide Synthase-2 Dimerization on Acute Cardiac Allograft Rejection Circulation, October 29, 2002; 106(18): 2392 - 2396. [Abstract] [Full Text] [PDF]
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M. H. Yen, G. Pilkington, R. C. Starling, N. B. Ratliff, P. M. McCarthy, J. B. Young, G. M. Chisolm, and M. S. Penn Increased Tissue Factor Expression Predicts Development of Cardiac Allograft Vasculopathy Circulation, September 10, 2002; 106(11): 1379 - 1383. [Abstract] [Full Text] [PDF]
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Z. Qian, R. Gelzer-Bell, S.-x. Yang, W. Cao, T. Ohnishi, B. A. Wasowska, R. H. Hruban, E. R. Rodriguez, W. M. Baldwin III, and C. J. Lowenstein Inducible Nitric Oxide Synthase Inhibition of Weibel-Palade Body Release in Cardiac Transplant Rejection Circulation, November 6, 2001; 104(19): 2369 - 2375. [Abstract] [Full Text] [PDF]
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S. M. Wildhirt, M. Weis, C. Schulze, N. Conrad, S. Pehlivanli, G. Rieder, G. Enders, W. von Scheidt, and B. Reichart Expression of Endomyocardial Nitric Oxide Synthase and Coronary Endothelial Function in Human Cardiac Allografts Circulation, September 18, 2001; 104(90001): I-336 - 343. [Abstract] [Full Text] [PDF]
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 S. M. Wildhirt, M. Weis, C. Schulze, N. Conrad, S. Pehlivanli, G. Rieder, G. Enders, W. von Scheidt, and B. Reichart Coronary flow reserve and nitric oxide synthases after cardiac transplantation in humans Eur. J. Cardiothorac. Surg., June 1, 2001; 19(6): 840 - 847. [Abstract] [Full Text] [PDF]
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 S. Sasu, A. L. Cooper, and D. Beasley Juxtacrine effects of IL-1{alpha} precursor promote iNOS expression in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1615 - H1623. [Abstract] [Full Text] [PDF]
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 U. Weiland, J. Haendeler, C. Ihling, U. Albus, W. Scholz, H. Ruetten, A. M. Zeiher, and S. Dimmeler Inhibition of endogenous nitric oxide synthase potentiates ischemia-reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway Cardiovasc Res, February 1, 2000; 45(3): 671 - 678. [Abstract] [Full Text] [PDF]
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C. Metais, J. Li, J. Li, M. Simons, and F. W. Sellke Serotonin-Induced Coronary Contraction Increases After Blood Cardioplegia-Reperfusion : Role of COX-2 Expression Circulation, November 9, 1999; 100(90002): II-328 - 334. [Abstract] [Full Text] [PDF]
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M. Kibbe, T. Billiar, and E. Tzeng Inducible nitric oxide synthase and vascular injury Cardiovasc Res, August 15, 1999; 43(3): 650 - 657. [Abstract] [Full Text] [PDF]
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 C. L. Glenn, W. Y. S. Wang, and B. J. Morris Different Frequencies of Inducible Nitric Oxide Synthase Genotypes in Older Hypertensives Hypertension, April 1, 1999; 33(4): 927 - 932. [Abstract] [Full Text] [PDF]
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