(Circulation. 1999;100:1983-1991.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Serum From Patients With Severe Heart Failure Downregulates eNOS and Is Proapoptotic
Role of Tumor Necrosis Factor-
From the Salvatore Maugeri Foundation, Institute for Care and Research, Cardiovascular Pathophysiology Research Centre (L.A., T.B., G.G., L.C.), and the Departments of Cardiology (S.C., P.B.), Statistics (G.P.), and Pathologic Anatomy (M.C., P.G.G.), University of Brescia; the Immunology Department, Spedali Civili, Brescia (F.M.); the Cardiology Department, Gussago, Brescia (M.V.); and the Department of Cardiology, University of Ferrara (R.F.), Italy.
Correspondence to Prof Roberto Ferrari, Cardiovascular Pathophysiology Research Center, Salvatore Maugeri Foundation, Via Pinidolo, 23, 25064 Gussago, Brescia, Italy. E-mail ferrari{at}master.cci.unibs.it
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Abstract |
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Background—Cytokine activation and endothelial dysfunction are typical phenomena of congestive heart failure (CHF). We tested the hypothesis that incubating human umbilical vein endothelial cells with serum from patients with CHF will downregulate endothelial constitutive nitric oxide synthase (eNOS) and induce apoptosis.
Methods and Results—We studied 21 patients with severe CHF.
Levels of tumor necrosis factor-
(TNF-) and several
neuroendocrine parameters were assessed. eNOS was measured
by Western Blot analysis and apoptosis by optical
microscopy and flow cytometry. We observed (1) eNOS downregulation
(difference versus healthy subjects at 24 hours
[P<0.05] and 48 hours [P<0.001]),
(2) nuclear morphological changes typical of apoptosis; and (3)
a high apoptotic rate with propidium iodide (increasing from
2.1±0.4% to 11.3±1.2% at 48 hours; P<0.001 versus
healthy subjects) and annexin V. An anti-human TNF- antibody did not
completely counteract these effects. A strong correlation existed
between eNOS downregulation and apoptosis
(r=-0.89; P<0.001).
Conclusions—Serum from patients with severe CHF downregulates
eNOS expression and increases apoptosis. High levels of TNF-
likely play a role, but they cannot be the only factor responsible.
Key Words: heart failure • tumor necrosis factor • endothelium • nitric oxide synthase • apoptosis
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Congestive heart failure (CHF) is a syndrome characterized by neuroendocrine and cytokine activation.1 2 Among cytokines, tumor necrosis factor-
(TNF-) and its soluble receptors I (sTNF-RI) and II
(sTNF-RII) are increased in patients with severe
CHF.3 4 5 6
In in vitro experiments, high doses of TNF- induce
endothelial dysfunction as a result of the
downregulation of endothelial constitutive nitric oxide
synthase (eNOS)7 expression and the increase of
apoptosis, a mechanism of physiological
cell death. Whether or not the negative effects of TNF- on eNOS and
apoptosis also occur in CHF is not known. However,
endothelial dysfunction does occur in CHF, as
demonstrated in studies in dogs and humans.8 9 This may be
due to the activation of several circulating
neurohormones.9 Because of the poor correlation between
neurohormones and systemic vascular resistances,10 other
systems are necessarily involved in this pathological
process.11
To evaluate the potential role of TNF- in
endothelial dysfunction, we studied the protein
expression of eNOS and the rate of apoptosis of human umbilical
vein endothelial cells (HUVECs) after incubation for
48 hours with serum from patients with CHF in New York Heart
Association (NYHA) class IV. eNOS was evaluated by Western blot
analysis and apoptosis by both optical microscopy and
flow cytometry.
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Methods |
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Populations Studied
This study was performed at the Cardiology Departments of the Universities of Brescia and Ferrara and at the Heart Failure Unit of the Salvatore Maugeri Foundation, Gussago, Brescia, Italy. A total of 21 male patients with severe CHF (NYHA class IV) and 21 healthy subjects (normal controls) participated in the study. All gave informed consent, and local Ethics Committee approval was obtained. The Table
shows the
clinical characteristics of the populations studied.
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The average age of the patients was 56±9 years. The causes of CHF were coronary artery disease (n=14), idiopathic dilated cardiomyopathy (n=5), or valvular disease (n=2). The mean left ventricular ejection fraction, as assessed by 2D echocardiogram, was 21±5%. Hemodynamic parameters were measured in the postabsorptive state with a Swan-Ganz catheter. Cardiac output was determined by thermodilution with a Gould cardiac output computer (model SP 1445). All patients were treated with angiotensin-converting enzyme inhibitors, diuretics, and digoxin; some were also taking vasodilators, low-dose ß-blockers, and positive inotropic agents. Thirteen patients were receiving hepatic hydroxymethylglutaryl coenzyme A reductase inhibitors. Anti-inflammatory drugs were not allowed during the 2 weeks preceding the study. Exclusion criteria included infections; renal failure; pulmonary, thyroid, and collagen vascular diseases; and malignancy.
The normal control group was sex- (male) and age-matched (57±4 years) to the patients. No subjects in this group had any clinical sign of acute or chronic illnesses or any symptoms related to the cardiovascular system.
Blood Processing
After 30 minutes of supine rest, peripheral venous
blood was taken and centrifuged within 1 hour to measure serum
electrolytes, norepinephrine (NE), aldosterone,
plasma renin activity (RA), atrial natriuretic peptide
(ANP), TNF-, sTNF-RI, and sTNF-RII. All serum samples were stored at
-80°C.
Plasma Hormones
Plasma NE levels were measured by high-performance
liquid chromatography with electrochemical
detection.4 12 Levels of RA, aldosterone, and
ANP were measured by radioimmunoassay (DiaSorin, Radim).
TNF- Immunoactivity
Antigenic TNF- was determined by a sandwich ELISA with a
commercially available kit (Medgenix Diagnostic). The
mixture of monoclonal antibodies used does not neutralize TNF-,
which allows the measurement of total circulating TNF-, even if
bound to soluble TNF receptors. The sensitivity of the assay is 3
pg/mL.
sTNF-RI and sTNF-RII
Serum sTNF-RI and sTNF-RII levels were assessed by a sandwich
ELISA with a commercially available kit (Amersham). The minimum
detectable doses for sTNF-RI and sTNF-RII were 2.5 and 5 pg/mL,
respectively.
Cell Culture
HUVECs were isolated from umbilical cords using the method of
Jaffe et al.13 No additional growth factor was added to
propagate the cultures. HUVECs were characterized by
immunofluorescence for von Willebrand
factor and by microscope observation of typical cobblestone
morphology.
Between splits 2 and 4, the cells were treated with 40 ng/mL TNF-
(1x108 units/mg) for 6, 12, 24, and 48 hours to
establish a positive control group. Moreover, HUVECs were incubated for
the same times with 20% serum from either the normal controls or
patients with CHF. To test the specific role of TNF-, we used 1
µg/mL monoclonal anti-human TNF- antibody (Genzyme).
Nitric Oxide Synthase Analysis
eNOS immunoblotting was performed as previously
described.14 Mouse monoclonal anti-human eNOS (Affiniti)
and peroxidase-conjugated rabbit anti-mouse IgG (Dako) were used as
primary and secondary antibodies, respectively. The specific signal was
detected with an enhanced chemiluminescence system (ECL, Amersham) and
quantified by densitometry. Each sample was processed 3 times.
Qualitative Assessment of Apoptosis by Optical
Microscopy
Optical microscopy assessment was performed by the
ApopTag-peroxidase kit and by hematoxylin-eosin and Feulgen
staining.
ApopTag-Peroxidase Kit
Cytological samples, fixed in 95% alcohol, were stained with
the ApopTag-peroxidase kit (Oncor): the reaction is referred to as
TUNEL detection. In this process, residues of digoxigenin
nucleotide are catalytically added to DNA by terminal
deoxinucleotidal transferase.15
Hematoxylin-Eosin Staining
The cytological slides were placed in Harris hematoxylin
(Bio-Optica) for 1 minute and contrasted in bidistilled water. They
were dehydrated through the increasing alcohol scale and fixed by
xylene with Eukitt balsam.16
Feulgen Staining
The cytological slides were treated with HCl at 60°C for 7 to
10 minutes and then placed in Schiff reactive medium (Sigma) for 45
minutes at room temperature, washed in tap water, dehydrated through
the decreasing alcohol scale, and fixed by xylene with Eukitt
balsam.17
Quantitative Assessment of Apoptosis by Flow
Cytometry
Apoptosis was quantified by flow cytometry using a
slightly modified version of the method of Nicoletti et
al.18 Endothelial cells fixed in 1 mL of
cold 70% ethanol were then incubated with a solution of 100 µg/mL
propidium iodide (PI) and immediately analyzed. The correct
threshold was selected using the apoptotic model in murine
thymocytes after 72 hours of culture with dexamethasone
10-7 mol/L. Apoptotic cell nuclei, which
were easily distinguishable from debris by the condensation of nuclear
chromatin, emitted red fluorescence in red rodamine
fluorescence channels 46 to 146. The subdiploid population was
detected using the fluorescein isothiocyanate
(FITC)–annexin V/PI double-staining method of Vermes et
al19 ; it was then analyzed with a flow cytometer
(Cytoron Absolute; Ortho) using ABS software. Dead cells were excluded
by setting an appropriate threshold trigger on the low forward light
scatter parameter, and nonspecific staining was assessed
using FITC-conjugated nonimmune mouse IgG (Coulter).
Statistical Analysis
The significant difference in means of the studied variables
was tested by Student’s t test. The correlation between
variables was tested by Spearman’s rank or Pearson’s correlation
methods, depending on the type of distribution of each variable.
Multiple linear regression analysis was used to study the
possible association between apoptosis and eNOS with TNF-
and hormones. The correlation between variables and the multiple
linear regression analysis were performed only on data from
patients with CHF. The comparison between patients with CHF and normal
controls was analyzed by the linear mixed effects model, with
adjustment for multiple comparisons. The tests were considered
statistically significant at P<0.05.
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Results |
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The Table
shows the clinical
characteristics, plasma hormones, and TNF- data of the populations
studied. In patients with CHF, the cardiac index was severely reduced
(2±0.5 L · min-1 ·
(m2)-1), the right
atrial and pulmonary pressures were high (16±7 and 41±10
mm Hg, respectively), and the systemic vascular resistances (1968±453
dyne · s-1 ·
cm5) and serum levels of sodium were lowered
(135±5 mmol/L). Mean values for RA and aldosterone
were 34±14 ng · mL-1 ·
h-1 and 505±323 pg/mL, respectively. Plasma
concentrations of NE and ANP were 3.4 and 17 times greater,
respectively, than those of the normal controls. The mean values of
antigenic TNF- in patients with CHF and in normal controls were
47.1±14.4 and 21.6±4.1 pg/mL, respectively (P<0.001). The
same patterns were recorded for the soluble receptors of the
cytokine (P<0.001 for both sTNF-RI and sTNF-RII
versus normal controls).
eNOS
Figures 1 and 2, respectively, show a
representative Western blot of eNOS and the mean data
for each group obtained after incubation with exogenous TNF- 40
ng/mL (positive control) and serum from normal controls and patients
with CHF, both with and without the anti-human TNF- antibody. As
expected, the incubation of the HUVECs with TNF- resulted in a
downregulation of eNOS expression: after 6 hours, a 23% reduction
versus normal control was observed (P<0.05), which reached
85% after 48 hours (P<0.001). The addition of the
anti-human TNF- antibody completely counteracted this decrease.
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In normal controls, the incubation of HUVECs had no effect on eNOS,
whereas in patients with CHF, it resulted in a time-dependent
downregulation of the protein expression (P<0.05 and
P<0.001 versus normal controls after 24 and 48 hours,
respectively). The TNF- antibody partially counteracted the
inhibitory effect of the serum from patients with CHF.
Multiple linear regression analysis was performed with the
variables from patients with CHF that significantly correlated with
eNOS expression. After step-wise selection, TNF- levels showed a
mild correlation to the reduction of eNOS expression
(r=0.51; P<0.05).
Qualitative Assessment of Apoptosis by Optical
Microscopy
The apoptotic pattern, as observed with optical
microscopy, is shown in Figure 3. HUVECs
from normal controls did not undergo relevant changes (A through C),
whereas those from patients with CHF showed changes in nuclear profile
and alterations of chromatin structure (D through F). The
immunocytochemical ApopTag-peroxidase staining (brown positive nuclei)
showed the increase of the DNA 3'OH terminals that was generated by the
internucleosomic rupture, which precedes the detachment of the
apoptotic bodies. With this method, in the normal controls, a
positive nucleus could be occasionally detected in some fields;
conversely, in patients with CHF, 
1 positive nucleus was detectable
in each field (D). This finding was also confirmed by the
hematoxylin-eosin and Feulgen stains (E and F). The most frequent
alteration was nuclear membrane expansion (sometimes ovoidal), which
was associated with hyperchromatic zones; these nuclei appeared as
pyknotic nuclei later evolving in apoptotic bodies.
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Quantitative Assessment of Apoptosis by Flow
Cytometry
Figure 4 shows apoptosis
over 6, 12, 24, and 48 hours as measured by flow cytometry using a
solution of PI as the fluorochrome after membrane ethanol
permeabilization. The top panels show representative
cytofluorometric recordings of HUVECs in positive and normal
controls and patients with CHF. As expected, a proportional induction
of apoptosis occurred over time, which reached a maximum of
33.7±2.6% at 48 hours. Obviously, the induced apoptosis was
completely counteracted by adding the anti-human TNF- antibody
(P<0.001) (Figure 4, lower left). The lower right
panels show the mean data obtained from the incubation of HUVECs with
the serum from either normal controls or patients with CHF, with and
without the anti-human TNF- antibody. Over 48 hours, the percentage
of apoptotic cells in normal controls increased from 1.8±0.8%
to 4.2±0.5%. In patients with CHF, a higher rate of apoptosis
existed when compared with normal controls; this higher rate could be
seen at 6 hours, and it increased from 2.1±0.4% to 11.3±1.2% at 48
hours (P<0.001 at both 24 and 48 hours). The addition of
the anti-human TNF- antibody to the serum from patients with CHF
reduced, but did not counteract, its apoptotic effect
(P<0.001 at both 24 and 48 hours versus CHF without
antibody).
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To better quantify apoptosis, we also performed flow cytometry
using annexin V/PI double staining (Figure 5). Annexin V binds with a high affinity
to phosphatidylserine, which is normally located in
the inner part of the cell membrane. In the early apoptotic
states, phosphatidylserine translocates from the
inner to the outer layer of the cell membrane, thus allowing its
binding to annexin V. PI used without ethanol acts as a cellular
viability marker. Therefore, annexin V reveals the early
apoptotic processes of still-viable cells, whereas PI, which
binds to DNA as a consequence of end-stage apoptosis or
ongoing necrosis, reveals signs of membrane permeability impairment.
The upper panel of Figure 5 shows 3 dot-plots of HUVECs relevant
to positive controls, normal controls, and patients with CHF. The
dot-plot of the positive controls clearly identified 3 different
clusters of cells: (1) viable ones that were not stained by either of
the 2 markers, (2) apoptotic ones that were positively stained
only by annexin V, and (3) end-stage apoptotic or necrotic
cells that were stained by both markers. The dot-plot from the normal
controls did not show relevant positivity to annexin V or PI, whereas
incubation with serum from patients with CHF showed a similar cell
distribution to that of the positive controls. The mean data are shown
in the lower panels and resemble the data presented in Figure 4, although annexin V revealed higher apoptosis than PI
at each evaluation time.
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Correlation Analyses
A strong negative correlation exited between the
downregulation of eNOS expression and apoptosis
(r=-0.89; P<0.001), suggesting a link between
the 2 phenomena. In addition, multiple linear regression
analysis performed on patients with CHF, including all the
parameters studied, showed significant correlation with
apoptosis. After step-wise selection, only TNF- blood levels
were significantly related to apoptosis (r=0.56;
P<0.05). A strong correlation also existed between TNF-
and its receptors (r=0.76; P<0.05 for both
sTNF-RI and sTNF-RII) and between the 2 receptors (r=0.87;
P<0.01). Conversely, no correlation was found among
apoptosis, neurohormones, or any of the clinical
parameters measured.
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Discussion |
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We investigated some aspects of the endothelial function of patients with CHF by applying an original experimental approach. We tested the serum of patients with CHF on the in vitro function of HUVECs. Because we used the serum of patients as the "real" clinical mean, we avoided the artificial use of single or multiple exogenous inducers of endothelial dysfunction, as occurs in the classical in vitro/ex vivo experiments. Moreover, by determining HUVEC eNOS expression and apoptosis, we overcame the limitations of the indirect evaluation of endothelial dysfunction that are typical of clinical studies. HUVECs (human origin) resemble a homologous milieu, although they are not strictly indicative of endothelial cells in other systemic blood vessels.
Our data show that serum from patients with severe CHF downregulates
eNOS expression and increases apoptosis, indirectly suggesting
endothelial dysfunction. Because no correlation existed
between neurohormones and the 2 end points relevant to the
endothelial function measured in the current study, we
anticipate a possible role of another system: the system of
cytokines and, in particular, TNF-. Although our findings
cannot be fully extrapolated to in vivo clinical conditions, they do
represent the consequences of abnormal interaction between the
bloodstream of patients with CHF and human endothelium.
Obviously, our data cannot demonstrate any organ/tissue dysfunction
because endothelium is only 1 of the involved
components.
Serum from Patients With CHF Downregulates the Expression of eNOS.
Is This a TNF-–Mediated Process?
The endothelium regulates vascular tone by
producing vasodilating and vasoconstrictive
substances.20 In normal vessels, acetylcholine induces NO
synthesis by activating eNOS.20 Conversely, in patients
with CHF, acetylcholine results in a blunted vasodilating response,
whereas nitric oxide–donor administration exerts a vasodilating
response similar to that observed in normal controls, suggesting the
integrity of the vascular muscle cell. One explanation for this
apparent paradox is that eNOS expression is impaired in CHF. A recent
study in dogs with severe CHF indicates that the impaired response to
acetylcholine is due to reduced eNOS expression, confirming that the 2
phenomena are closely linked.8
Cytokines, when used in vitro, inhibit eNOS
expression.21 Our data, which were obtained by directly
administering TNF- to HUVECs expressing TNF receptors, confirm this
finding. Thus, the direct administration of TNF- to HUVECs can be
referred to as an alternative cytotoxicity assay because it resembles
the physiopathological conditions.
The incubation of the HUVECs with serum from patients with CHF also
resulted in a time-dependent downregulation of the protein expression,
showing a weak correlation only with TNF-. This weak correlation
could be due to the small sample size and to the difference in
sensitivity between the Western blot technique and the immunoenzymetric
test. Moreover, TNF- does not completely account for eNOS
downregulation, because the addition of the anti-human TNF- antibody
partially counteracts the effect of CHF serum on eNOS after 48 hours.
Because the effects of TNF- on the expression and activity of eNOS
in vitro are enhanced by interferon 
and interleukin 1ß, it is
possible that this cytokine mixture, present in the blood
of patients with CHF, is responsible for eNOS downregulation.
Interestingly, the incubation of the HUVECs with 40 pg/mL TNF-,
which corresponds to the mean plasma concentration of our patients,
only had a minor effect on eNOS expression (data not shown). This
suggests that the combination of TNF- with other factors is
responsible for eNOS downregulation.
Our data must also be considered in view of the actual treatment the patients were receiving, including angiotensin-converting enzyme inhibitors and hepatic hydroxymethylglutaryl coenzyme A reductase inhibitors, which increase the expression and activity of eNOS through a bradykinin-mediated22 and a post-transcriptional mechanism,23 respectively.
Serum from Patients with CHF Induces Apoptosis. Is This a
TNF-–Mediated Process?
Apoptosis is a mechanism by which the cell actively
participates in its own death by activating enzymes that induce
morphological and structural changes on cell components (eg, nucleus,
membrane, etc).24 An abnormal apoptotic rate is
possibly involved in the pathogenesis of several
cardiovascular diseases, including
atherosclerosis, restenosis, conduction-system
defects, and several pathological features of CHF, such as myocardial
and endothelial dysfunction. Measurement of
apoptosis, however, is not an easy process; therefore, >1
method should be used, particularly in studies on human
tissues,19 25 as we have done.
The initial description of apoptosis was based on morphological features16 17 26 ; therefore, we examined the HUVECs by optical microscopy using the ApopTag-peroxidase kit and the hematoxylin-eosin and Feulgen stains. Typical apoptotic nuclei were shown in the HUVECs treated with serum from patients with CHF. We also found evidence of specific DNA fragmentation (in the form of fragments of 180 bp or multiples) in all HUVECs treated with serum from patients with CHF (data not shown).
Both these techniques are subject to criticism because they are mainly qualitative. Therefore, to properly quantify apoptosis, we also used flow cytometry with 2 different fluorochromes: PI and annexin V-FITC. Flow cytometry allows the detection of events occurring in thousands of cells when compared with microscopy, which can only explore limited fields. Because PI detects apoptosis only in the advanced stages, when the loss of fragmented DNA is already an ongoing process, we also used annexin V to quantify the early phases of apoptosis. In addition, we combined annexin V with PI but without ethanol, thus using PI as a dye-exclusion test to establish the integrity of cell membranes. The calculation of the percentage of apoptosis did not include the cells with marked membrane alterations because, at end-stage apoptosis, membrane permeability changes in apoptotic and necrotic cells are indistinguishable.
Our data confirm that, in vitro, the endothelium has a physiological rate of apoptosis that is not influenced by cytokines; they also show that serum from patients with CHF is proapoptotic. More than 30 inducers of apoptosis have been identified, including neurohormones, cytokines, and newly discovered genes and second messengers,27 28 29 thus making it difficult to identify the primum movens.
We tested the hypothesis that TNF- plays a crucial role in the
complex biochemistry of apoptosis signaling.30
This cytokine induces its cytotoxic effects by binding to
specific receptors present in almost all cells.31
TNF-RI has a cytoplasmic signaling motif called the death
domain,30 which induces caspases activation,
resulting in inactivation of several proteins controlling different
functions.32 All these changes induce the morphological
characteristics that differentiate apoptosis from necrosis.
Interestingly, we found a 5-fold increase in sTNF-RI, suggesting that
TNF- interacted with this receptor.
TNF- may increase the inducible type of nitric oxide synthase
(iNOS) expression, and the resulting peroxinitrite would induce
apoptosis. However, this sequence of events is expected in
bovine aortic endothelial cells but not in
HUVECs.33 We did not observe any induction of iNOS (data
not shown), thus ruling out the possibility of TNF-–induced,
high-output nitric oxide production in human
endothelial cells.
Although TNF- is one of the main regulators of
endothelial cell life/death equilibrium, other factors
may play a major role in the maintenance of this ratio. Among
these, NF
B, whose activation can be stimulated by TNF- itself
under specific conditions, increases the resistance to the
apoptotic stimuli; angiotensin II, by a different
modulation of the receptors AT1 and
AT2, can have a pro- or antiapoptotic
effect; and Bcl-2 family members can also promote or inhibit
apoptosis induced by certain triggers.
Conclusions
Our results support the hypothesis that the circulating
blood of patients with CHF causes endothelial
dysfunction. TNF- likely plays a role, but it is not the only
responsible factor.
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Acknowledgments |
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This work was supported by the National Research Council targeted project 9100156 PF41, "Prevention and Control of Disease Factors"; by the European Commission project PL 950838 "The New Ischemic Syndromes"; and by the Local Grant for Scientific Research "TNF-
in Endothelial Dysfunction in
Patients With Severe Heart Failure," which was issued by the
University of Ferrara. The authors thank Michela Palmieri and Daniela
Bastianon for technical assistance, Alessandro Bettini for editing the
text, and Roberta Bonetti for secretarial assistance. Received February 18, 1999; revision received June 15, 1999; accepted July 8, 1999.
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