(Circulation. 1996;93:720-729.)
© 1996 American Heart Association, Inc.
Articles |
Induction of Nitric Oxide Synthase in the Human Cardiac Allograft Is Associated With Contractile Dysfunction of the Left Ventricle
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.
From the Divisions of Cardiovascular Medicine (N.P.L., P.S.T., P.R.R., G.A.H., H.v.d.L., P.T.T., J.P.C., S.A.H., H.A.V., M.B.F.) and Pathology (M.E.B.), Stanford (Calif) University School of Medicine; and Department of Anesthesiology, University of Virginia School of Medicine, Charlottesville (C.X., R.A.J.).
Correspondence to Dr Neil P. Lewis, Division of Cardiovascular Medicine, Cardiopulmonary Transplant Center, Box 191, University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail nlewis@virginia.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMethods Results Discussion References |
|---|
Background The mechanisms underlying cardiac contractile dysfunction after transplantation remain poorly defined. Previous work has revealed that inducible nitric oxide synthase (iNOS) is expressed in the rat heterotopic cardiac allograft during rejection; resultant overproduction of nitric oxide (NO) might cause cardiac contractile dysfunction via the negative inotropic and cytotoxic actions of NO. In this investigation, we tested the hypothesis that induction of iNOS may occur and be associated with cardiac allograft contractile dysfunction in humans.
Methods and Results We prospectively studied 16 patients in the first year after cardiac transplantation at the time of serial surveillance endomyocardial biopsy. Clinical data, the results of biopsy histology, and echocardiographic and Doppler evaluation of left ventricular systolic and diastolic function were recorded. Total RNA was extracted from biopsy specimens, and mRNA for ß-actin, detected by reverse transcription–polymerase chain reaction (RT-PCR) using human specific primers, was used as a constitutive gene control; iNOS mRNA was similarly detected by RT-PCR using human specific primers. iNOS protein was detected in biopsy frozen sections by immunofluorescence. Myocardial cGMP was measured by radioimmunoassay, and serum nitrogen oxide levels (NOx=NO2+NO3) were measured by chemiluminescence. iNOS mRNA was detected in allograft myocardium at some point in each patient and in 59 of 123 biopsies (48%) overall. In individual patients, iNOS mRNA expression was episodic and time dependent; the frequency of expression was highest during the first 180 days after transplant (P=.0006). iNOS protein associated with iNOS mRNA was detected by immunofluorescence in cardiac myocytes. iNOS mRNA expression was not related to the ISHLT histological grade of rejection or to serum levels of NOx but was associated with increased levels of myocardial cGMP (P=.01) and with both systolic (P=.024) and diastolic (P=.006) left ventricular contractile dysfunction measured by echocardiography and Doppler.
Conclusions These data support a relation between iNOS mRNA expression and contractile dysfunction in the human cardiac allograft.
Key Words: transplantation • rejection • nitric oxide • heart failure
|
Introduction |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Cardiac transplantation is an effective treatment for end-stage heart failure, leading to improved functional status and 1- and 5-year survival >80% and >65%, respectively.1 After transplantation, episodic cardiac contractile dysfunction is frequently detected on routine echocardiography2 3 ; in certain patients, this dysfunction can markedly deteriorate and adversely affect outcome. The mechanisms underlying cardiac contractile dysfunction after transplantation are poorly understood. Despite the sparse cellular infiltrates and infrequent myocyte necrosis often encountered with histological rejection, contractile dysfunction is nevertheless usually attributed to cellular rejection. In many instances, however, severe allograft contractile dysfunction occurs in the presence of very minor histological change,4 suggesting that other poorly understood mechanisms may be responsible for allograft contractile dysfunction.
NO is a potent and widely distributed autacoid whose complex biological actions include the control of blood vessel wall function, neuronal transmission, and immune targeting. NO is enzymatically generated from its precursor L-arginine by three structurally distinct isoforms of NOS: endothelial, neuronal, and inducible NOS.5 These enzymes also differ functionally. The endothelial and neuronal NOS isoforms, referred to as constitutive, are regulated by intracellular calcium concentration and generate small amounts of NO in response to physical (eg, shear stress) and pharmacological stimulation.6 In contrast, the inducible isoform is not expressed in normal tissue but is transcriptionally upregulated in several tissues by LPS and cytokine stimulation, where it generates the sustained release of large amounts of NO.5 NO overproduction by iNOS in the systemic vascular bed is thought to underlie the vasodilatation and resistance to vasoconstrictors characteristic of sepsis.7 8 9 10 11 NO has also been shown to have profound effects on cardiac myocyte contractile function.12 13 14 15 After administration of LPS or cytokines, iNOS is induced in the myocardium of experimental animals8 16 ; in experimental sepsis models, myocardial iNOS induction is considered to contribute to impaired ventricular contractile performance via increased local NO generation.8 15 In humans, cytokine induction of myocardial iNOS with reduced cardiac contractility may also be present in sepsis17 18 and after the use of cytokines as antitumor therapy.19 20
Cardiac allograft rejection is associated with increased cytokine expression,21 22 and several cytokines have been shown to induce iNOS expression in myocardium.8 16 23 24 25 Acute rejection of the rat heterotopic cardiac allograft has recently been shown to be associated with induction of iNOS mRNA and protein in cardiac myocytes, cardiac microvascular endothelium, and infiltrating macrophages.26 These changes are associated with increased myocardial cGMP, the putative second messenger for the effects of NO.26 During rejection, NO formation is markedly increased in the rat heterotopic cardiac allograft, causing allograft protein nitrosylation27 and increased levels of NO degradation products in the systemic circulation.28 In the present study, we prospectively investigated whether induction of iNOS mRNA and protein occurs in human cardiac allografts and whether expression of iNOS mRNA is associated with cardiac contractile dysfunction. Our findings demonstrate induction of iNOS mRNA associated with protein expression, increased myocardial cGMP levels, and LV contractile dysfunction. We postulate that iNOS expression may contribute to contractile dysfunction of the human cardiac allograft by increased myocardial generation of NO, leading to increased cGMP levels, and that myocardial iNOS may represent a new target for therapeutic agents in the treatment of allograft contractile dysfunction.
|
Methods |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Patients
The study protocol was approved by the Committee for the Protection of Human Subjects at Stanford University Medical Center, and written informed consent was obtained from all subjects before they were included in the study. Between January 1993 and January 1994, 16 consecutive adult patients undergoing cardiac transplantation who gave consent were recruited. Data were collected prospectively from all patients until June 1994. All patients were managed with a standard immunosuppressive regimen that included prophylactic antilymphocyte therapy (OKT3) during the early postoperative period and maintenance treatment with cyclosporin A, azathioprine, and tapering doses of prednisone. Episodes of moderate (grade 3) rejection, according to the ISHLT grading system,29 were treated with increased doses of corticosteroids, and in refractory cases, with antilymphocyte antibody. Adjunctive therapy with methotrexate was used in one patient for recurrent histological rejection. Full clinical data, posttransplant complications, and the results of routine laboratory investigations were documented. Samples of normal myocardium were obtained from 11 subjects at necropsy performed within 6 hours of sudden noncardiac death (intracranial hemorrhage 2, asphyxia 2, pulmonary embolus 1, carcinoma bronchus 1, butane overdose 1, murder 1, unknown 3).
Biopsy and Histological Grading
Surveillance right
ventricular
endomyocardial biopsy was performed via the right
internal jugular vein with a modified Stanford-Caves-Schultz bioptome.
Histological evaluation of rejection was performed by
an experienced pathologist (M.E.B.) using the ISHLT grading system. One
or two additional myocardial samples were immediately frozen in liquid
nitrogen, after any adherent thrombus had been carefully removed, and
stored at -80°C for subsequent experimental analysis. A
10-mL blood sample was drawn from the introducing sheath during the
biopsy procedure, and serum was separated and stored at -80°C
for measurement of serum nitrogen oxides
(NOx=NO2-+NO3-).
The routine surveillance biopsy regimen used at Stanford is weekly for
the first 4 weeks after transplant, then every 2 weeks for 1 month,
reducing gradually to once every 3 months, determined by the results of
biopsy histology; biopsy was performed more frequently in the presence
of histological rejection (ie, ISHLT grades 1 to
4).
RNA Preparation
Biopsies were homogenized in TRIzol reagent
(Life Technologies Inc); total cellular RNA was extracted by the
single-step acid
guanidinium–thiocyanate-phenol-chloroform
method.30
Reverse Transcription of mRNA and Generation of First-Strand
cDNA
RNA (2 µg) was reverse transcribed to give cDNA in a final
volume of 30 µL containing (final concentrations) Tris-HCl 10 mmol/L
(pH 8.3), KCl 50 mmol/L, MgCl2 5 mmol/L, random hexamers
1.7 µmol/L, 0.5 mmol/L each of dATP, dTTP, dCTP, and dGTP, RNase
inhibitor 0.5 U/µL, and Moloney murine leukemia virus
reverse transcriptase 3.3 U/µL (Perkin-Elmer). The reaction was
carried out at 42°C for 1 hour, followed by heat inactivation of the
enzyme at 75°C for 10 minutes. cDNA was stored at -80°C.
Polymerase Chain Reaction
First-strand cDNA copies were
amplified with Taq
polymerase (Perkin-Elmer) and human-specific primers for
ß-actin cDNA31 (Table 1
), selected to
control for adequate isolation of mRNA and conversion to cDNA or for
iNOS cDNA based on the sequences reported for human hepatic and
chondrocyte cDNAs32 33 and the iNOS chromosomal
gene34 (Table 1). Reactions were performed in a
PTC 100-60
thermal cycler (MJ Research Inc) for 40 cycles. Annealing temperatures
used were 50°C for ß-actin, 60°C for iNOS (Clontech) and iNOS
1, and 63°C for iNOS 2 (Table 1). Reactions were carried
out in a
final volume of 50 µL with 3 µL cDNA in (final concentrations)
Tris-HCl 10 mmol/L (pH 8.3), KCl 50 mmol/L, 0.2 mmol/L each of dATP,
dTTP, dCTP, and dGTP, 0.5 µmol/L primers, and Taq
polymerase 0.02 U/µL, and MgCl2 concentration for each
primer was set as shown in Table 1.
|
Reproducibility of RT-PCR
Reproducibility of iNOS RT-PCR was
examined in 10 biopsies (5
with iNOS mRNA expression and 5 without) using the second sample taken
at the same surveillance biopsy. Because of the inherent nature of
biopsy, the second sample is obtained from a different part of the
interventricular septum.
PCR Product Resolution and Verification of
Nucleotide Sequence
After amplification, 15 µL PCR products per
lane was
resolved on a 1.5% agarose gel containing ethidium bromide 0.5 µg/mL
in 1x TBE buffer (Tris 0.45 mol/L, boric acid 0.45 mol/L, and EDTA 10
mmol/L). Bands were confirmed by two observers in blinded fashion after
photography under UV fluorescence. PCR product band
identity was determined in every case by Southern blot hybridization
using a [
-32P]ATP 5' terminus-labeled
(5' DNA
terminus labeling system; Gibco BRL) internal sequence probe for human
specific iNOS (5'-CCCTCCTGTAGGCCCTC-3'). To probe for iNOS cDNA,
the
nylon membranes were prehybridized for 10 minutes in a solution
containing 5x Denhardt's solution (in wt/vol: Ficoll 0.1%, PVP
0.1%, BSA 0.1%), 0.5% SDS, and 5x SSPE (in mmol/L: NaCl 0.75,
NaH2PO4 50, EDTA 5 [pH 7.4]) at 42°C and
then hybridized in the same medium overnight at 42°C with the added
radiolabeled DNA probe. After hybridization, the nylon membranes were
washed twice in 2x SSC for 10 minutes at room temperature. The
membranes were exposed to x-ray film (Kodak, X-OMAT AR) with
intensifying screens for 16 hours at -70°C.
Exclusion of Genomic DNA Contamination of RNA Samples
To
exclude contamination of RNA samples by genomic DNA (since
the iNOS [Clontech] primers do not encompass an intron), 18 RNA
samples with a positive iNOS PCR-amplified product were incubated
at 37°C for 30 minutes with RNase A 1.8 µg/µL (Life Technologies
Inc) before reverse transcription and PCR for iNOS and ß-actin.
Additionally, in 48 samples, PCR for iNOS was repeated with primers
that encompassed two exon-intron junctions (iNOS 1, Table 1
),
and
the results of the two assays were compared in blinded fashion by two
observers. The RNA content of each biopsy was also recorded to
determine whether variations in RNA content influenced the result of
the RT-PCR for iNOS mRNA.
iNOS Immunostaining
Myocardial samples were fixed in
paraformaldehyde (4% wt/vol) in PBS for 90 minutes,
then dehydrated in an increasing gradient of sucrose in PBS, rapidly
frozen by immersion in liquid nitrogen, and embedded in a 1:2 solution
containing OCT compound (Miles Inc) and 20% sucrose in PBS. Sections
(2 to 4 µm) were cut and thaw-mounted onto precleaned Superfrost
Plus slides (Fisher Scientific Inc). After preincubation with 20%
horse serum (Sigma Chemical Co) for 15 minutes, tissue cryostat
sections were washed (2x10 minutes in PBS) and incubated at 4°C
overnight with a rabbit anti-iNOS polyclonal antibody raised to a
synthetic peptide derived from the C-terminal end of the mouse
macrophage iNOS sequence (dilution 1:500; Affinity Bioreagents
Inc) or rabbit IgG as negative control. Specific iNOS antibody binding
was detected with a biotin-conjugated goat anti-rabbit IgG and
strepavidin-conjugated Texas Red (Vector Laboratories, Inc). The
slides were mounted and examined under an Olympus Vanox
fluorescence microscope.
Western Blot
Specificity of the anti-iNOS antibody for human
iNOS was
determined by Western blot. Crude protein fractions (150 µg each)
were separated on denaturing 7.5% SDS-PAGE gels, followed by blotting
onto nitrocellulose filters. The blot was blocked with buffer
(composition: 50 mmol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl, 2% BSA,
0.1% Tween-20) for 1 hour at room temperature, then incubated with the
iNOS antibody (1:2000 dilution; Affinity Bioreagents Inc) for 1 hour at
room temperature. The blot was washed six times with Tris-buffered
saline (5 minutes each) and then incubated for 1 hour with
anti-rabbit IgG antibody conjugated with HRP (Vector Laboratories
Inc) at room temperature. The blot was washed six times (5 minutes
each) with PBS, followed by detection of immunoreactive proteins by
enhanced chemiluminescence (ECL, Amersham).
Myocardial cGMP Content
cGMP content was assayed in 10
biopsies (5 positive and 5
negative for iNOS mRNA expression). Biopsy tissue was
homogenized in ice-cold 6% trichloroacetic acid (500
µL) and centrifuged (10 minutes, 4°C, 12 000 rpm); protein
content in the pellet was measured with BioRad reagent (Bio-Rad
Laboratories), and cGMP in the supernatant was measured by
radioimmunoassay according to the manufacturer's instructions (Cyclic
GMP [125I] RIA kit, Du Pont).
Measurement of Serum Nitrogen Oxides
Serum nitrogen oxides
(NO and one-electron oxidation
products of NO [NOx]) were measured with a
commercially available chemiluminescence detector (model 2108, Dasibi)
after sample deproteinization using ethanol (1:3 vol/vol) and reduction
in boiling acidic vanadium (III) chloride.35 By this
technique, NO2- and
NO3- are both quantitatively reduced to
NO; NO is quantified by the chemiluminescence detector after reaction
with ozone. Signals from the detector were analyzed by a
computerized integrator and recorded as areas under the curve.
Standard curves for
NO2-/NO3-
were linear over the range 100 pmol to 5 nmol, and serum was diluted to
fall within this range. The assay was extensively validated against the
Griess reaction, after reduction of serum
NO3- to
NO2- by nitrate reductase (data not
shown).
Echocardiographic Assessment of LV
Function
The echocardiographic examination included
recordings of M-mode and two-dimensional images and
pulsed-wave Doppler flow velocity signals across the mitral
valve. All studies were performed with a Hewlett-Packard ultrasonograph
using 2.5- or 3.5-MHz combined imaging and Doppler transducer. All
studies were analyzed without prior knowledge of the
endomyocardial biopsy findings or other clinical
features of the patient. Standard recording techniques,
recorded in detail elsewhere,36 37 were used. The
following parameters of diastolic function were
recorded: isovolumic ventricular relaxation time, peak
early diastolic mitral flow velocity, and rate of peak
early mitral flow deceleration, expressed as deceleration half-time
as previously described.36 Intraobserver and interobserver
correlation coefficients were .85 and .95, respectively, for
measurements of isovolumic relaxation time, deceleration half-time,
and peak early diastolic mitral flow velocity, as
previously reported.37 Changes from baseline in any given
patient were used as the index for a Doppler diagnosis of LV
diastolic dysfunction; stable values are regarded as those
that do not change beyond the threshold of spontaneous variations of
the method, as previously reported.37 In these previous
studies, a statistically significant change in isovolumic relaxation
time or deceleration half-time has been determined to be one that
exceeds 15%, and for peak early diastolic mitral flow
velocity, one that exceeds 20%. In the present study, a
significant decrease in isovolumic relaxation time or deceleration
half-time formed the basis of a diagnosis of LV
diastolic dysfunction. An increase in peak early
diastolic mitral flow velocity in the presence of stable
isovolumic relaxation time and deceleration half-time measurements
was not regarded as indicative of diastolic dysfunction,
owing to the propensity for this parameter to be influenced
by heart rate. In the rare event of deceleration half-time
and isovolumic relaxation time changing in opposite directions, an
increase of 
20% in peak early diastolic mitral flow
velocity was used as an indicator of LV diastolic
dysfunction.
Measurements of LV dimension were made in end systole and end diastole by the conventional American Society of Cardiography criteria. Percent fractional shortening derived from these measurements was compared in serial studies. Based on an intraobserver variation of 10%, systolic dysfunction was diagnosed when a decrease in percent fractional shortening >10% was observed between two serial studies. With these variables, LV diastolic and systolic function were described as categorical variables, ie, diastolic function normal or impaired, systolic function normal or impaired. Echocardiographic recordings were obtained on the day of surveillance biopsy in 74 of 123 cases (60%); systolic function was determined in all of these (60% of 123 biopsy data points). Diastolic parameters were measured in 58 (47%) of 123 biopsy data points; in 14 cases, diastolic parameters were not obtained because of equipment failure, and in 2 cases because of inadequate technical recordings. The frequency of abnormal echocardiographic LV function was analyzed in relation to iNOS mRNA expression as follows: systolic dysfunction alone, diastolic dysfunction alone, and either systolic or diastolic dysfunction (in this latter category, the 58 cases [47%] in which both parameters were recorded were analyzed).
Statistical Analysis
Data are reported as group
mean±SD. The significance of
differences between groups was tested by unpaired Student's
t test or by contingency table analysis as
appropriate. The association of multiple variables with iNOS
expression was assessed by multiple regression analysis. A
value of P<.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Clinical Data
Sixteen male patients formed the study group (mean age, 48±14 years; range, 18 to 65 years). The indications for transplantation were ischemic heart disease in 10, dilated cardiomyopathy in 3, allograft coronary artery disease in 2, and acute graft failure in 1. One hundred thirty study biopsies (1 or 2 samples per biopsy) were obtained in these patients (4 to 12 serial samples per patient; range, 12 to 353 days after transplant). Of 130 biopsies, 123 (95%) were positive for ß-actin mRNA expression (see below) and formed the group for further study. The donor organs for these 16 patients were obtained from 13 men and 3 women, age 28±10 years, ischemic time 186±48 minutes; 6 donors and 8 recipients were CMV positive, and CMV mismatch (negative recipient, positive donor status) was present in 1 patient. Complications encountered in this group were gastrointestinal CMV infection in 1 patient, pulmonary aspergillosis in 1, and jaundice of undetermined cause in 2. Two patients died: 1 of mucormycosis and 1 of chronic renal and multisystem failure. The ISHLT grades of rejection of this group of 123 biopsies were grade 0, 60 (49%); grade 1, 35 (28%); grade 2, 13 (11%); and grade 3, 15 (12%). The distribution of echocardiographic results within this group were systolic dysfunction, 23 of 74 (31%); diastolic dysfunction, 39 of 58 (67%); either systolic or diastolic dysfunction, 44 of 58 (76%); and normal function, 14 of 58 (24%). The mean age of subjects from whom normal myocardium was obtained at necropsy was 42±23 years.
RT-PCR
PCR bands identifying the presence of ß-actin mRNA
were
present in 123 of 130 transplant biopsies (95%); the 7 samples
without a positive band (indicating inadequate RNA extraction or
inadequate conversion of RNA to cDNA) were excluded from further
analysis (Fig 1). PCR bands indicating
expression of iNOS mRNA were present in 59 of 123 biopsies (48%).
iNOS mRNA was detected on at least one occasion in every patient, and
expression was episodic as a function of time after transplantation
(Fig 1). iNOS mRNA expression was not detected in the 11 normal
myocardial samples, which all showed positive ß-actin bands (Fig
1).
|
x174 RF DNA/Hae III); lane 2, negative control; lane 3,
positive control; and lanes 4 through 14, sequential samples obtained
at surveillance biopsy. The iNOS product size is 259 bp; the
ß-actin product 532 bp. b, Data from panel a shown as a
function of time after transplant (Tx) (corresponding to timing of
surveillance biopsy). Multiple time points are shown between 14 and 282
days after cardiac transplantation. ß-Actin expression was absent
at day 98 in this patient, and data from this time point were excluded
from the analysis (see text). Note the episodic expression of
iNOS present to day 183 after transplantation; expression is
unrelated to the ISHLT histological rejection grade.
Note also the association between episodic iNOS expression and episodic
diastolic echocardiographic dysfunction (n
indicates normal systolic and diastolic function;
D, impaired diastolic function; and -,
echocardiographic data not of satisfactory quality/no
record). c, Amplification of iNOS (top) and ß-actin (bottom)
mRNA by RT-PCR. PCR products were analyzed by agarose gel
electrophoresis. In each panel, lane 1 is the molecular mass marker
(
Reproducibility of RT-PCR
Reproducibility of the iNOS RT-PCR
was confirmed in 10 transplant
biopsies (5 expressing iNOS and 5 not) from RNA preparation through
iNOS RT-PCR, showing agreement in every case. Since the two biopsies
were taken from slightly different sites in the
interventricular septum, this finding also suggests
that expression of iNOS mRNA in transplant myocardium is
homogeneous, at least within the
interventricular septum where the biopsies were
obtained.
RT-PCR Product Band Verification
PCR product band identity
was confirmed in every case (59 of
59) by Southern blot hybridization using the 5' end-labeled
internal probe.
Exclusion of Genomic DNA Contamination of RNA
Samples
Predigestion of RNA with RNase A before RT-PCR eliminated the
PCR
product band in 17 of 18 samples; in 1 sample, repeat PCR did not
show a product band before or after RNase A digestion. iNOS RT-PCR
was repeated using a primer set encompassing 2 introns (Table
1, iNOS 1
primer set) showing agreement in samples with a positive band in 16 of
18 and in samples without a band in 24 of 30. These results suggest
that significant genomic DNA contamination, which might influence the
results of PCR, was not present. The mean amount of RNA isolated
per biopsy was 5.5±4.7 µg in the 123 samples; RNA isolation was
similar (P=.44) in the group without iNOS mRNA expression
(5.8±5.6 µg, n=59) and in the group with iNOS mRNA
expression
(5.1±3.5 µg, n=64), suggesting that RNA isolation did not
influence
the results of RT-PCR.
iNOS Protein Detection by
Immunofluorescence
The anti-iNOS antibody identified a 130-kD protein,
representing rat iNOS, isolated from crude protein extracts
of LPS-induced rat lung that was used as a positive control (lane 4,
Fig 2B, inset). The antibody also recognized a similar
130-kD protein isolated from the cytosol of
cytokine-stimulated human colonic carcinoma cells,
representing human iNOS (lanes 1 and 2, Fig 2B, inset),
kindly provided by Dr Paula Sherman.38 The antibody did
not cross-react with endothelial NOS isolated from
the particulate fraction of bovine aortic endothelial
cells (lane 3, Fig 2B, inset). With this antibody, iNOS protein
was
identified by immunostaining in 4 of 5 biopsies that
showed iNOS mRNA expression by RT-PCR; no
immunostaining for iNOS was detected in 3 of 4 biopsies
without iNOS mRNA expression, and results were equivocal in the other 2
samples. The results obtained from representative
biopsies with and without iNOS mRNA expression are shown in Fig
2. iNOS
protein immunostaining was detected in cardiac myocytes
in all samples that expressed iNOS protein (myocytes labeled M, Fig
2A and 2B). In 3 of 4 biopsies expressing iNOS
protein, there was, in
addition, staining of vascular smooth muscle cells in small
intramyocardial vessels (indicated by arrows in Fig 2A and
2B). These
patterns of staining were not detected in samples that did not express
iNOS mRNA (Fig 2C and 2D).
|
Myocardial cGMP Content
Myocardial cGMP content was
significantly increased
(P=.01) in biopsies with iNOS mRNA expression compared with
biopsies without iNOS mRNA expression (Fig 3).
|
Serum Nitrogen Oxide Levels
Systemic venous serum samples
from patients without iNOS mRNA
expression showed NOx concentrations (48.7±35.4 µmol/L,
n=38) similar to those from patients with iNOS mRNA expression
(39.8±21.4 µmol/L, n=49; P=.15).
Relations of iNOS mRNA Expression to Clinical
Variables
The relations of iNOS mRNA expression to clinical variables
are shown in Table 2 and in Figs 1b and
4. Expression of iNOS mRNA was time dependent, the
frequency of expression being highest in the first 180 days after
transplant (Figs 1 and 4). iNOS mRNA expression
was associated with
higher prednisone dosage and with worse renal function (BUN and
creatinine, Table 2); however, multiple regression
analysis showed that prednisone dosage, BUN, and
creatinine were related to iNOS mRNA expression only
through their relation to time after transplantation. The progressive
steroid dose taper that is part of standard clinical care after
transplantation and the time-dependent cumulative nephrotoxic
effects of cyclosporin A, which progressively worsen renal function,
appear to account for these associations.
|
|
Relation of iNOS mRNA Expression to Biopsy Histology
The mean
ISHLT histological rejection grade was
the same in the presence and absence of iNOS mRNA expression (Table
2).
The frequency of iNOS mRNA expression was similar in all grades of
rejection (0 to 3, Fig 5) and similar when rejection was
analyzed as no rejection (grade 0) compared with rejection
(grades 1 to 3, Fig 5).
|
Relation of iNOS mRNA Expression to
Echocardiographic LV Function
The relations of iNOS mRNA expression to
LV function evaluated by
echocardiography and Doppler are shown in Fig 6. The
frequencies of both abnormal systolic and
diastolic functions were significantly increased in the
group with iNOS mRNA expression. Echocardiographic LV
function was completely normal (systolic and
diastolic functions normal) in only 6% of the biopsies
expressing iNOS mRNA. In contrast, echocardiographic LV
function was completely normal in 46% of biopsies in which iNOS mRNA
expression was absent.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Summary of Findings
The results of this study demonstrate, for the first time in humans, induction of iNOS mRNA and protein in the cardiac allograft associated with increased levels of myocardial cGMP and LV contractile dysfunction. The salient findings of the study are as follows. (1) iNOS mRNA expression was detected in
50% of routine surveillance
biopsies after transplant; reproducibility of this finding was
demonstrated in 10 biopsies, suggesting that iNOS mRNA expression is
homogeneously distributed in transplant
myocardium (at least within the
interventricular septum). (2) iNOS mRNA expression
occurred in every patient at some stage after transplant; expression
was episodic after transplant and occurred most frequently during the
first 180 days after transplant. (3) iNOS mRNA expression was
associated with iNOS protein expression in cardiac myocytes and in the
vascular smooth muscle cells of small intramyocardial vessels; iNOS
expression was associated with increased myocardial levels of the
intracellular second messenger of NO, cGMP. iNOS mRNA expression was
not, however, associated with increased systemic serum levels of
NOx (serum products of NO). (4) iNOS mRNA expression
was unrelated to the presence of histological
rejection. (5) iNOS mRNA expression was associated with increased
frequencies of systolic and diastolic LV
contractile dysfunction by echocardiography and
Doppler. In the presence of iNOS mRNA expression,
echocardiographic LV function was completely normal in
only 6% of cases; conversely, LV function was completely normal in
46% of biopsies that did not express iNOS mRNA.
Relation of iNOS Expression to Cardiac
Contractility
The mechanisms underlying contractile changes in the
intact heart
after cardiac transplantation are complex. Immune
response–mediated vascular injury may influence cardiac myocyte
function by altering vascular permeability and causing edema, by
causing ischemia, or by altering the release of
endothelium-derived substances, such as NO, that
influence myocyte contractile function. Components of the immune system
may also directly injure cardiac myocytes, leading to cell necrosis or
dysfunction through the action of cytotoxic T lymphocytes,
macrophages, and neutrophils; through pathways of humoral
injury; and via the action of cytokines.2 Several
studies have demonstrated that NO can modulate myocardial
contractility. Exogenous NO and its second messenger,
cGMP, reduce myocardial
contractility.12 13 14 15
Cytokines
reduce myocardial contractility acutely by stimulating
constitutive NOS and increasing NO formation.13 Chronic
exposure to cytokines has been shown to stimulate the
transcriptional upregulation of iNOS and impair myocyte
contractility through increased NO
formation.8 12 13 16 In
humans, myocardial dysfunction
during sepsis and the therapeutic use of cytokines (as
antitumor agents) may be mediated by induction of myocardial
iNOS.17 18 19 20 Our data are
consistent with the
hypothesis that iNOS mRNA and protein expression occurs after cardiac
transplantation and that this leads to increased myocardial NO
generation within the allograft, causing increased myocardial cGMP
levels and reduced myocardial contractility. The
association of iNOS expression with diastolic dysfunction
in our study is at variance with the results of a recent
study39 showing improved diastolic
distensibility during bicoronary infusion of sodium
nitroprusside in patients with structurally normal hearts. However, it
is important to recognize that the findings of Paulus et
al39 are likely to reflect the
physiological effects of small concentrations of NO
on the heart at the concentrations of sodium nitroprusside infused. The
effects of large amounts of NO on myocardial
contractility, produced under pathological
circumstances by the inducible isoform of NOS, are likely to be
different. Furthermore, our data do not address the possibility that
iNOS induction might adversely affect myocyte contractile function via
multiple actions, such as the known inhibitory effects of
NO on Fe2+-containing enzymes of the Krebs cycle and
mitochondrial respiration5 ; nor have we addressed the
possibility that iNOS expression might be associated with upregulation
of other substances that have been shown to exert effects on myocyte
contractility.40 Furthermore, the vascular
expression of iNOS that we have described might result from a vascular
injury whose effects on cardiac function are mediated by other
mechanisms, such as the formation of interstitial edema or
ischemia.2 It is also possible that iNOS induction
could occur secondary to the reduction in myocardial
contractility rather than contribute to it; preliminary
data (by activity assay) showing induction of iNOS in inflammatory
cardiac muscle disorders but not in ischemic heart disease with
similar contractile dysfunction suggests that this is unlikely to be
the case.41 Confirmation of the proposed direct effects of
iNOS and NO on myocardial contractility in the human
cardiac allograft awaits the development of specific
inhibitors of iNOS. However, the use of
inhibitors of iNOS in this situation could theoretically be
detrimental; NO produced by graft-infiltrating macrophages
has been shown to inhibit the cytotoxic T-lymphocyte response to
alloantigen; inhibition of iNOS might thus promote T
lymphocyte–mediated cytotoxicity.42
Relation of iNOS Expression to Biopsy Histology
Although
biopsy histology has evolved as an invaluable tool for
determining the level of immunosuppression to be used after transplant,
it is nevertheless associated with certain biological and clinical
inconsistencies. Histology is poorly correlated with markers of
cytotoxic T-cell activation in the allograft43 and with
cytokine gene expression.21 22 There is also a
poor correlation between binding of immunoglobulin to the
coronary microvasculature and biopsy
histology.44 45 Last, there is limited association
between
the development of graft vascular disease, which is considered to have
an immune basis, and antecedent histological
rejection.46 The absence of a relation between iNOS
expression and histology is therefore not without precedent. We
speculate that histology represents one facet of the biology of
cardiac allograft rejection, which coincides to an extent with other
biological changes during the allograft immune response;
histological change, however, may be present
without evidence of concurrent activation of other immune components,
eg, cytokines,21 22 and likewise, certain immune
components may be activated without a concurrent change in
histology.42 No single parameter can be
regarded as sole mediator in the biology of rejection.
Comparison With Data Obtained in Animal Models
In a recent
study examining iNOS expression in the rat heterotopic
cardiac transplant model, iNOS mRNA and protein were identified in
cardiac myocytes, in the microvasculature, and in infiltrating
macrophages.26 These changes were associated with
evidence of iNOS protein expression by activity assay and with
increased myocardial cGMP levels. In earlier studies using the same
model, rejection was accompanied by increased levels of serum oxides of
nitrogen28 and evidence of graft protein
nitrosylation,27 indicative of increased intragraft NO
formation. It is not clear why the responses to cardiac allograft
rejection differ somewhat in the rat compared with the human; however,
the rat model is one of acute untreated rejection in a nonworking
heart, in which induction of abnormal biochemical processes might be
more marked, leading to larger increases in NO formation and thus
NOx. Furthermore, iNOS induction in macrophages has
been more readily demonstrable in several animal models than in
humans32 47 ; infiltrating macrophages may be the
major source of allograft NO generation in rats, leading to increases
in serum NOx and graft protein nitrosylation. Last, in the
present study it was not possible to regulate dietary nitrate
intake during prolonged posttransplant follow-up, so that
differences in nitrate intake may have caused alterations in serum
NOx that would conceal smaller changes in
endogenous NO production by the allograft.
Differences in allograft NO generation related to myocardial iNOS
expression may be more readily detected by sampling across the
coronary vascular bed in future studies.
In animal models, steroid pretreatment generally inhibits the expression of iNOS induced by cytokines and LPS in several tissues.5 In apparent contrast, in the present study we found that iNOS mRNA expression was associated with higher steroid dosage. However, the association between iNOS expression and steroid dose was not an independent one; multiple regression analysis revealed that iNOS expression and steroid dose were correlated only through their independent associations with time after transplantation. iNOS expression itself exhibited marked time dependency after transplant: the highest frequency of expression was in the first 180 days after transplantation. Because high steroid doses are used early after transplantation and subsequently tapered, there is an apparent but spurious association between iNOS expression and higher steroid dosage. On the basis of these data, we cannot confirm or exclude a relation between steroid dosage and iNOS expression in human cardiac transplantation.
Study Limitations
A limitation of this study, in common with
any clinical study
involving limited amounts of tissue, is the inability to measure every
parameter in every biopsy. The amounts of mRNA and protein
present in such small specimens (5 mg) precluded application of
quantitative methods such as Northern and Western blotting. We adopted
the approach of using iNOS RT-PCR as a screening technique to detect
iNOS gene transcription in small samples of allograft
myocardium, after demonstrating that iNOS mRNA is
homogeneously expressed in myocardium. We
subsequently performed other tissue measurements and categorized
samples according to iNOS mRNA expression by RT-PCR. A further
limitation in the interpretation of our results is that the biopsy
samples assessed in this study did not include any samples taken at the
time of a clinically severe reduction in myocardial contractile
function. Accordingly, we cannot be certain that our extrapolations
relating iNOS induction to graft contractile dysfunction apply to the
development of cardiogenic shock occurring as a consequence of immune
injury after transplantation.
Summary
We have shown episodic, time-dependent expression of
iNOS mRNA
in the human cardiac allograft associated with iNOS protein expression,
elevated myocardial cGMP concentrations, and ventricular
contractile dysfunction. We postulate that iNOS expression and
contractile dysfunction are causally related via the local effects of
NO and cGMP on cardiac myocyte function. Future studies directed at an
understanding of the factors responsible for iNOS expression in the
allograft and the long-term consequences of iNOS expression will be
of interest.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
Dr Lewis is the recipient of a British Heart Foundation International Fellowship. Dr Tsao is the recipient of a National Service Research Award (1F32-HL-08779). Dr Rickenbacher is the recipient of a grant from the Margarete and Walther Lichtenstein Foundation. Dr Haywood is the recipient of a grant from the Wessex Cardiothoracic Center Research Trust. Dr von der Leyen is the recipient of an award from the Deutsche Forschungsgemeinschaft (Le 567/3-1 and 3-2). Dr Cooke is a recipient of the Vascular Academic Award from the National Heart, Lung, and Blood Institute. Dr Valantine is supported by NIH Clinical Investigator grant 5K08-HL-02477.
Received April 3, 1995; revision received September 12, 1995; accepted September 25, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
1. The Registry of the International Society for Heart and Lung Transplantation: Tenth Official Report. J Heart Lung Transplant. 1993;12:543-548.
2.
Barry WH. Mechanisms of immune-mediated
myocyte injury. Circulation. 1994;89:2421-2432.
3. Valantine HA. Rejection surveillance by Doppler echocardiography. J Heart Lung Transplant. 1993;12:422-426. [Medline] [Order article via Infotrieve]
4. Stevenson LW, Lewis W, McAlpin WR, Clark S. Acute reversible cardiogenic shock: immune-mediated with mild histologic change. Am Heart J. 1986;111:611-613. [Medline] [Order article via Infotrieve]
5. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Physiol Rev. 1991;43:109-142.
6. Cooke JP, Rossitch E Jr, Andon NA, Loscalzo J, Dzau VJ. Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator. J Clin Invest. 1991;88:1663-1671.
7. Knowles RG, Merrett M, Salter M, Moncada S. Differential induction of brain, lung and liver nitric oxide synthase by endotoxin in the rat. Biochem J. 1990;270:833-836. [Medline] [Order article via Infotrieve]
8. Salter M, Knowles RG, Moncada S. Widespread tissue distribution, species distribution and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide synthases. FEBS Lett. 1991;291:145-149. [Medline] [Order article via Infotrieve]
9. Nava E, Palmer RJM, Moncada S. Inhibition of nitric oxide synthesis in septic shock: how much is beneficial? Lancet. 1991;338:1555-1557. [Medline] [Order article via Infotrieve]
10. Petros A, Bennett D, Valance P. Effect of nitric oxide synthase inhibitors on hypotension in patients with septic shock. Lancet. 1991;338:1557-1558. [Medline] [Order article via Infotrieve]
11. Nava E, Palmer RMJ, Moncada S. The role of nitric oxide in endotoxic shock: effects of NG-monomethyl-L-arginine. J Cardiovasc Pharmacol. 1992;20(suppl):S132-S134.
12. Balligand JL, Ungureanu D, Kelly RA, Kobzik L, Pimental D, Michel T, Smith T. Abnormal contractile function due to induction of nitric oxide synthase in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest. 1993;91:2314-2319.
13.
Finkel MS, Odis CV, Jacob TD, Watkins SC, Hattler BG,
Simmons RL. Negative inotropic effects of cytokines on
the heart mediated by nitric oxide. Science. 1992;257:387-389.
14.
Brady AJB, Warren JB, Poole-Wilson PA, Williams TJ,
Harding SE. Nitric oxide attenuates cardiac myocyte
contraction. Am J Physiol. 1993;265:H176-H182.
15.
Brady AJB, Poole-Wilson PA, Harding SE, Warren
JB. Nitric oxide production within cardiac myocytes
reduces their contractility in endotoxemia.
Am J Physiol. 1992;263:H1963-H1966.
16. Schulz R, Nava E, Moncada S. Induction and potential biological relevance of a Ca2+-independent nitric oxide synthase in the myocardium. Br J Pharmacol. 1992;105:575-580. [Medline] [Order article via Infotrieve]
17. Suffredini AF, Fromm RF, Parker MM, Brenner M, Kovacs JA, Wesley RA, Parrillo JE. The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med. 1989;321:280-287. [Abstract]
18. Abel FL. Does the heart fail in endotoxin shock? Circ Shock. 1990;30:5-13. [Medline] [Order article via Infotrieve]
19. Dayton LR, Walker RE, Kovacs JA, Herpin B, Parker M, Masur H, Fauci AS, Lane HC. Reversible cardiac dysfunction associated with interferon alfa therapy in AIDS patients with Kaposi's sarcoma. N Engl J Med. 1989;321:1246-1249. [Medline] [Order article via Infotrieve]
20.
Nora R, Abrams JS, Tait NS, Hiponia DJ, Silverman
HJ. Myocardial toxic effects during recombinant interleukin-2
therapy. J Natl Cancer Inst. 1989;81:59-63.
21. Wu CJ, Lovett M, Wong-Lee J, Moeller F, Kitamura M, Goralski TJ, Billingham ME, Starnes VA, Clayberger C. Cytokine gene expression in rejecting cardiac allografts. Transplantation. 1992;54:326-332. [Medline] [Order article via Infotrieve]
22. Cunningham DA, Dunn MJ, Yacoub MH, Rose ML. Local production of cytokines in the human cardiac allograft. Transplantation. 1994;57:1333-1337. [Medline] [Order article via Infotrieve]
23.
Kinugawa K, Takahashi T, Kohmoto O, Yao A, Aoyagi T,
Momomura S, Hirata Y, Serizawa T. Nitric oxide–mediated
effects of interleukin-6 on [Ca2+]i and
cell
contraction in cultured chick ventricular myocytes.
Circ Res. 1994;75:285-295.
24.
Roberts AB, Vodouotz Y, Roche NS, Sporn MB, Nathan
FC. Role of nitric oxide in antagonistic effects of
transforming growth factor ß and interleukin-1 ß on the beating
rate of cultured cardiac myocytes. Mol Endocrinol. 1992;6:1921-1930.
25. Balligand JL, Ungureanu D, Pimental D, Kelly RA, Smith TW, Michel T. Detection of transcripts for a cytokine-inducible nitric oxide synthase isoform in adult rat ventricular myocytes. Circulation. 1993;88(pt 2):II-1-II-384.
26. Yang X, Chowhury N, Cai B, Marboe S, Sciacca RR, Michler RE, Cannon PJ. Induction of myocardial nitric oxide synthase by cardiac allograft rejection. J Clin Invest. 1994;94:714-721.
27.
Lancaster JR, Langrehr JM, Bergonia HA, Murase N,
Simmons RL, Hoffman RA. EPR detection of heme and nonheme
iron-containing protein nitrosylation by nitric oxide during
rejection of the rat heart allograft. J Biol
Chem. 1992;267:10994-10998.
28. Langrehr JM, Murase N, Markus PM, Cai X, Neuhaus P, Schraut W, Simmons RL, Hoffman RA. Nitric oxide production in host-versus-graft and graft-versus-host reactions in the rat. J Clin Invest. 1992;90:679-683.
29. Billingham ME, Carey NRB, Hammond ME, Kemnitz J, Marboe C, McAllister HA. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group. J Heart Lung Transplant. 1990;9:587-592.
30. Chomozynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]
31.
Shi Y, Hutchinson H, Hall DJ, Zalewski A.
Downregulation of c-myc expression by antisense
oligonucleotides inhibits proliferation of human smooth
muscle cells. Circulation. 1993;88:1190-1195.
32.
Geller DA, Lowenstein CJ, Shapiro RA, Nussler AK, Di
Silvio M, Wang SC, Nakayama DK, Simmons RL, Snyder SH, Billiar
TR. Molecular cloning and expression of inducible nitric oxide
synthase from human hepatocytes. Proc Natl
Acad Sci U S A. 1993;90:3491-3495.
33.
Charles IG, Palmer RMJ, Hickery MS, Bayliss MT, Chubb
AP, Hall VS, Moss DW, Moncada S. Cloning, characterization and
expression of a cDNA encoding an inducible nitric oxide synthase from
the human chondrocyte. Proc Natl Acad Sci U S A. 1993;90:11419-11423.
34.
Chartrain NA, Geller DA, Koty PP, Sitrin NF, Nussler
AK, Hoffman EP, Billiar TR, Hutchinson NI, Mudgett JS. Molecular
cloning, structure and chromosomal localization of the human inducible
nitric oxide synthase gene. J Biol Chem. 1994;269:6765-6772.
35.
Tsao PS, McEvoy LM, Drexler H, Butcher EC, Cooke
JP. Enhanced endothelial adhesiveness in
hypercholesterolemia is attenuated by
L-arginine. Circulation. 1994;89:2176-2182.
36. Valantine HA, Appleton CP, Hatle LK, Hunt SA, Stinson EB, Popp RL. Influence of recipient atrial contraction on left ventricular filling dynamics on the transplanted heart assessed by Doppler echocardiography. Am J Cardiol. 1987;59:1159-1163. [Medline] [Order article via Infotrieve]
37. Valantine HA, Appleton CP, Hatle LK, Hunt SA, Stinson EB, Popp RL. Variability of Doppler echocardiographic indices of left ventricular filling in transplant recipients and normal subjects. J Am Soc Echocardiogr. 1990;3:276-284. [Medline] [Order article via Infotrieve]
38. Sherman PA, Laubach VE, Reep BR, Wood ER. Purification and cDNA sequence of an inducible nitric oxide synthase from a human tumor cell line. Biochemistry. 1993;32:11600-11605. [Medline] [Order article via Infotrieve]
39.
Paulus WJ, Vantrimpont PJ, Shah AM. Acute
effects of nitric oxide on left ventricular relaxation and
diastolic distensibility in humans: assessment by
bicoronary sodium nitroprusside infusion.
Circulation. 1994;89:2070-2078.
40.
Shah AM, Mebazaa A, Wetzel RC, Lakatta EG. Novel
cardiac myofilament desensitizing factor released by endocardial and
vascular endothelial cells.
Circulation. 1994;89:2492-2497.
41. De Belder A, Radomski M, Why H, Richardson P, Martin J, Moncada S. Expression of an inducible NO synthase in heart tissue from patients with inflammatory heart disease. J Am Coll Cardiol. 1994;(suppl):263A. Abstract.
42. Langrehr JM, Hoffman RA, Billiar TR, Lee KKW, Schraut WH, Simmons RL. Nitric oxide synthesis in the in vivo allograft response: a possible regulatory mechanism. Surgery. 1991;110:335-342. [Medline] [Order article via Infotrieve]
43. Griffiths GM, Namikawa R, Mueller C, Liu C, Young JD, Billingham ME, Weissman I. Granzyme A and perforin as markers for rejection in cardiac transplantation. Eur J Immunol. 1991;21:687-692. [Medline] [Order article via Infotrieve]
44. Miller LW, Wesp A, Jennison SH, Graham MA, Martin TW, McBride LR, Pennington DG, Peigh P. Vascular rejection in heart transplant recipients. J Heart Lung Transplant. 1993;12(pt 2):S147-S153.
45. Bonnaud EN, Lewis NP, Billingham ME. The reliability and usefulness of immunofluorescence in cardiac transplantation. J Heart Lung Transplant. 1995;14:163-171. [Medline] [Order article via Infotrieve]
46. Gao SZ, Shroeder JS, Hunt SA, Valantine HA, Hill IR, Stinson EB. Influence of graft rejection on incidence of accelerated graft coronary artery disease: a new approach to analysis. J Heart Lung Transplant. 1993;12:1029-1035. [Medline] [Order article via Infotrieve]
47. Hibbs JB, Westenfelder C, Taintor R, Vavrin Z, Kablitz C, Baranowski RL, Ward JH, Menlove RL, McMerry MP, Kushner JP, Baranowski WE. Evidence for cytokine-induced nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy. J Clin Invest. 1992;89:867-877.
This article has been cited by other articles:
 |
|
 |
|
  N. Goren, J. Cuenca, P. Martin-Sanz, and L. Bosca Attenuation of NF-{kappa}B signalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes Cardiovasc Res, November 1, 2004; 64(2): 289 - 297. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. FOGLI, P. NIERI, and M. C. BRESCHI The role of nitric oxide in anthracycline toxicity and prospects for pharmacologic prevention of cardiac damage FASEB J, April 1, 2004; 18(6): 664 - 675. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Pieper, V. Nilakantan, G. Hilton, X. Zhou, A. K. Khanna, N. L. N. Halligan, C. C. Felix, B. Kampalath, O. W. Griffith, M. A. Hayward, et al. Variable efficacy of N6-(1-iminoethyl)-L-lysine in acute cardiac transplant rejection Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H525 - H534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Stoica, D. K. Satchithananda, C. Atkinson, S. Charman, M. Goddard, and S. R. Large Heat shock protein, inducible nitric oxide synthase and apoptotic markers in the acute phase of human cardiac transplantation Eur. J. Cardiothorac. Surg., December 1, 2003; 24(6): 932 - 939. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Pieper, V. Nilakantan, G. Hilton, N. L. N. Halligan, C. C. Felix, B. Kampalath, A. K. Khanna, A. M. Roza, C. P. Johnson, and M. B. Adams Mechanisms of the protective action of diethyldithiocarbamate-iron complex on acute cardiac allograft rejection Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1542 - H1551. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. W. Cohen, D. S. Park, S. E. Woodman, T. M. Williams, M. Chandra, J. Shirani, A. Pereira de Souza, R. N. Kitsis, R. G. Russell, L. M. Weiss, et al. Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts Am J Physiol Cell Physiol, February 1, 2003; 284(2): C457 - C474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 D. M. Flanagan, C. D. Jennings, S. W. Goes, B. E. Caywood, R. Gross, A. M. Kaplan, and J. S. Bryson Nitric oxide participates in the intestinal pathology associated with murine syngeneic graft-versus-host disease J. Leukoc. Biol., October 1, 2002; 72(4): 762 - 768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Linke, W. Li, H. Huang, Z. Wang, and T. H. Hintze Role of cardiac eNOS expression during pregnancy in the coupling of myocardial oxygen consumption to cardiac work Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H1208 - H1214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G.F Bronzwaer, C. Zeitz, C. A Visser, and W. J Paulus Endomyocardial nitric oxide synthase and the hemodynamic phenotypes of human dilated cardiomyopathy and of athlete's heart Cardiovasc Res, August 1, 2002; 55(2): 270 - 278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 D. Sarkar, P. Vallance, and S. E. Harding Nitric oxide: not just a negative inotrope Eur J Heart Fail, October 1, 2001; 3(5): 527 - 534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hayashi, Y. Sawa, N. Fukuyama, H. Nakazawa, and H. Matsuda Inducible nitric oxide production is an adaptation to cardiopulmonary bypass-induced inflammatory response Ann. Thorac. Surg., July 1, 2001; 72(1): 149 - 155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Aziz, R. A. Saad, M. Burgess, N. Yonan, P. Hasleton, and I. V. Hutchinson Transforming growth factor beta and myocardial dysfunction following heart transplantation Eur. J. Cardiothorac. Surg., July 1, 2001; 20(1): 177 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Szabolcs, N. Ma, E. Athan, J. Zhong, M. Ming, R. R. Sciacca, J. Husemann, A. Albala, and P. J. Cannon Acute Cardiac Allograft Rejection in Nitric Oxide Synthase-2-/- and Nitric Oxide Synthase-2+/+ Mice : Effects of Cellular Chimeras on Myocardial Inflammation and Cardiomyocyte Damage and Apoptosis Circulation, May 22, 2001; 103(20): 2514 - 2520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Geller, S. H. Chia, Y. Takahashi, G. P. Yagnik, G. Tsoulfas, and N. Murase Protective Role of the L-Arginine-Nitric Oxide Synthase Pathway on Preservation Injury after Rat Liver Transplantation JPEN J Parenter Enteral Nutr, May 1, 2001; 25(3): 142 - 147. [Abstract] [PDF]
|
|
|
|
|
|
H. Itano, W. Zhang, J. H. Ritter, T. J. McCarthy, N. S. Yew, T. Mohanakumar, and G. A. Patterson Endobronchial transfection of naked viral interleukin-10 gene in rat lung allotransplantation Ann. Thorac. Surg., April 1, 2001; 71(4): 1126 - 1133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Wort, J. A. Mitchell, and T. W. Evans Inducible nitric oxide synthase: a tissue-specific affair? Am J Physiol Lung Cell Mol Physiol, March 1, 2001; 280(3): L387 - L389. [Full Text] [PDF]
|
|
|
|
 |
|
 P. Andreka, J. Zang, C. Dougherty, T. I. Slepak, K. A. Webster, and N. H. Bishopric Cytoprotection by Jun Kinase During Nitric Oxide-Induced Cardiac Myocyte Apoptosis Circ. Res., February 16, 2001; 88(3): 305 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sarkar, P. Vallance, C. Amirmansour, and S. E. Harding Positive inotropic effects of NO donors in isolated guinea-pig and human cardiomyocytes independent of NO species and cyclic nucleotides Cardiovasc Res, December 1, 2000; 48(3): 430 - 439. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. F. Soto, C.-X. Jia, D. G. Rabkin, J. P. Hart, Y. M. Carter, M. J. Sardo, D. T. Hsu, P. E. Fisher, D. J. Pinsky, and H. M. Spotnitz Improvement of rejection-induced diastolic abnormalities in rat cardiac allografts with inducible nitric oxide synthase inhibition J. Thorac. Cardiovasc. Surg., July 1, 2000; 120(1): 39 - 46. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. L. Skarsgard, X. Wang, P. McDonald, A. H. Lui, E. K. Lam, B. M. McManus, C. van Breemen, and I. Laher Profound Inhibition of Myogenic Tone in Rat Cardiac Allografts Is Due to eNOS- and iNOS-Based Nitric Oxide and an Intrinsic Defect in Vascular Smooth Muscle Contraction Circulation, March 21, 2000; 101(11): 1303 - 1310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Yang, N. Ma, M. J. Szabolcs, J. Zhong, E. Athan, R. R. Sciacca, R. E. Michler, G. D. Anderson, J. F. Wiese, K. M. Leahy, et al. Upregulation of COX-2 During Cardiac Allograft Rejection Circulation, February 1, 2000; 101(4): 430 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M Shah Inducible nitric oxide synthase and cardiovascular disease Cardiovasc Res, January 1, 2000; 45(1): 148 - 155. [Full Text] [PDF]
|
|
|
|
 |
|
 R R Chaturvedi, V E Hjortdal, E V Stenbog, H B Ravn, P White, T D Christensen, A B Thomsen, J Pedersen, K E Sorensen, and A N Redington Inhibition of nitric oxide synthesis improves left ventricular contractility in neonatal pigs late after cardiopulmonary bypass Heart, December 1, 1999; 82(6): 740 - 744. [Abstract] [Full Text]
|
|
|
|
|
|
M. A. Arstall, D. B. Sawyer, R. Fukazawa, and R. A. Kelly Cytokine-Mediated Apoptosis in Cardiac Myocytes : The Role of Inducible Nitric Oxide Synthase Induction and Peroxynitrite Generation Circ. Res., October 29, 1999; 85(9): 829 - 840. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. de Frutos, L. S. de Miguel, M. Garcia-Duran, F. Gonzalez-Fernandez, J. A. Rodriguez-Feo, M. Monton, J. Guerra, J. Farre, S. Casado, and A. Lopez-Farre NO from smooth muscle cells decreases NOS expression in endothelial cells: role of TNF-alpha Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1317 - H1325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Pinsky, W. Aji, M. Szabolcs, E. S. Athan, Y. Liu, Y. M. Yang, R. P. Kline, K. E. Olson, and P. J. Cannon Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture Am J Physiol Heart Circ Physiol, September 1, 1999; 277(3): H1189 - H1199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. J Paulus and A. M Shah NO and cardiac diastolic function Cardiovasc Res, August 15, 1999; 43(3): 595 - 606. [Full Text] [PDF]
|
|
|
|
|
|
C. Heymes, M. Vanderheyden, J. G. F. Bronzwaer, A. M. Shah, and W. J. Paulus Endomyocardial Nitric Oxide Synthase and Left Ventricular Preload Reserve in Dilated Cardiomyopathy Circulation, June 15, 1999; 99(23): 3009 - 3016. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Flesch, H. Kilter, B. Cremers, U. Laufs, M. Sudkamp, M. Ortmann, F. U. Muller, and M. Bohm Effects of endotoxin on human myocardial contractility involvement of nitric oxide and peroxynitrite J. Am. Coll. Cardiol., March 15, 1999; 33(4): 1062 - 1070. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. K. Worrall, T. P. Misko, M. D. Botney, P. M. Sullivan, J.-J Hui, G. M. Suau, P. T. Manning, and T. B. Ferguson Jr Time course and cellular localization of inducible nitric oxide synthases expression during cardiac allograft rejection Ann. Thorac. Surg., March 1, 1999; 67(3): 716 - 722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ravalli, A. Albala, M. Ming, M. Szabolcs, A. Barbone, R. E. Michler, and P. J. Cannon Inducible Nitric Oxide Synthase Expression in Smooth Muscle Cells and Macrophages of Human Transplant Coronary Artery Disease Circulation, June 16, 1998; 97(23): 2338 - 2345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Shimizu, K.-i. Kinugawa, Y. Sugishita, K. Sugishita, K. Harada, H. Matsui, O. Kohmoto, T. Serizawa, and T. Takahashi Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes Cardiovasc Res, May 1, 1998; 38(2): 405 - 413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Cannon, X. Yang, M. J. Szabolcs, S. Ravalli, R. R. Sciacca, and R. E. Michler The role of inducible nitric oxide synthase in cardiac allograft rejection Cardiovasc Res, April 1, 1998; 38(1): 6 - 15. [Full Text] [PDF]
|
|
|
|
|
|
J. Igarashi, M. Nishida, S. Hoshida, N. Yamashita, H. Kosaka, M. Hori, T. Kuzuya, and M. Tada Inducible nitric oxide synthase augments injury elicited by oxidative stress in rat cardiac myocytes Am J Physiol Cell Physiol, January 1, 1998; 274(1): C245 - C252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-i. Kinugawa, T. Shimizu, A. Yao, O. Kohmoto, T. Serizawa, and T. Takahashi Transcriptional Regulation of Inducible Nitric Oxide Synthase in Cultured Neonatal Rat Cardiac Myocytes Circ. Res., December 19, 1997; 81(6): 911 - 921. [Abstract] [Full Text]
|
|
|
|
|
|
W. J. Paulus, S. Kastner, P. Pujadas, A. M. Shah, H. Drexler, and M. Vanderheyden Left Ventricular Contractile Effects of Inducible Nitric Oxide Synthase in the Human Allograft Circulation, November 18, 1997; 96(10): 3436 - 3442. [Abstract] [Full Text]
|
|
|
|
|
|
K. Graf, Y. S. Do, N. Ashizawa, W. P. Meehan, C. M. Giachelli, C. C. Marboe, E. Fleck, and W. A. Hsueh Myocardial Osteopontin Expression Is Associated With Left Ventricular Hypertrophy Circulation, November 4, 1997; 96(9): 3063 - 3071. [Abstract] [Full Text]
|
|
|
|
 |
|
 J.-L. Balligand and P. J. Cannon Nitric Oxide Synthases and Cardiac Muscle : Autocrine and Paracrine Influences Arterioscler Thromb Vasc Biol, October 1, 1997; 17(10): 1846 - 1858. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Flesch, H. Kilter, B. Cremers, O. Lenz, M. Südkamp, F. Kuhn-Regnier, and M. Böhm J. Pharmacol. Exp. Ther., June 1, 1997; 281(3): 1340 - 1349. [Abstract] [Full Text]
|
|
|
|
 |
|
 W. W. Simmons, D. Ungureanu-Longrois, G. K. Smith, T. W. Smith, and R. A. Kelly Glucocorticoids Regulate Inducible Nitric Oxide Synthase by Inhibiting Tetrahydrobiopterin Synthesis and L-Arginine Transport J. Biol. Chem., September 27, 1996; 271(39): 23928 - 23937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Kelly, J.-L. Balligand, and T. W. Smith Nitric Oxide and Cardiac Function Circ. Res., September 1, 1996; 79(3): 363 - 380. [Full Text]
|
|
|
|
|
|
N. K. Worrall, T. P. Misko, P. M. Sullivan, J.-J. Hui, and T. B. Ferguson Jr Inhibition of Inducible Nitric Oxide Synthase Attenuates Established Acute Cardiac Allograft Rejection Ann. Thorac. Surg., August 1, 1996; 62(2): 378 - 385. [Abstract] [Full Text]
|
|
|
|
|
|
J. Heger, A. Godecke, U. Flogel, M. W. Merx, A. Molojavyi, W. N. Kuhn-Velten, and J. Schrader Cardiac-Specific Overexpression of Inducible Nitric Oxide Synthase Does Not Result in Severe Cardiac Dysfunction Circ. Res., January 11, 2002; 90(1): 93 - 99. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||