(Circulation. 2000;101:2518.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Soluble Tumor Necrosis Factor Receptor Abrogates Myocardial Inflammation but Not Hypertrophy in Cytokine-Induced Cardiomyopathy
From the Cardiovascular Institute (T. Kubota, G.S.B., T. Kadokami, C.F.M., A.M.F.), Department of Surgery (M.M.), Division of Neuropathology (V.J.S.), and Department of Molecular Genetics and Biochemistry (C.B., P.D.R.), University of Pittsburgh Medical Center, Pittsburgh, Pa.
Correspondence to Arthur M. Feldman, MD, PhD, 200 Lothrop St, S 572 Scaife Hall, Pittsburgh, PA 15213. E-mail feldmanam{at}msx.upmc.edu
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Abstract |
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Background—Transgenic mice with cardiac-specific overexpression of tumor necrosis factor (TNF)-
develop dilated cardiomyopathy. The present
study was designed to evaluate therapeutic effects of
adenovirus-mediated neutralization of TNF- on this model.
Methods and Results—An adenovirus encoding the 55-kDa TNF
receptor–IgG fusion protein (AdTNFRI) was injected
intravenously into 6-week-old transgenic mice, which
resulted in high levels of TNFRI in both plasma and
myocardium. AdTNFRI did not reverse cardiomegaly but
abrogated myocardial inflammation. Furthermore, AdTNFRI blocked the
myocardial expression of intercellular adhesion molecule-1 and
downstream cytokines, including interleukin-1ß and monocyte
chemotactic protein-1. Downregulation of -myosin heavy chain was
restored by the treatment, whereas upregulation of ß-myosin heavy
chain was not reversed. In contrast, the downregulation of sarcoplasmic
reticulum Ca2+-ATPase and phospholamban was normalized by
AdTNFRI. Echocardiographic measurements showed that
left ventricular end-systolic diameter was
significantly larger in transgenic mice than in control mice, and this
increase was reversed by the AdTNFRI treatment. However, left
ventricular wall thickening was not reversed.
Conclusions—These results suggest that anti-TNF therapy may hold promise in the treatment of end-stage heart failure.
Key Words: viruses • genes • hormones
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Introduction |
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Tumor necrosis factor (TNF)-
is a proinflammatory
cytokine with pleiotropic biological effects.1
Elevated plasma levels of TNF- occur in a variety of
cardiovascular diseases, including acute myocarditis,
cardiac allograft rejection, myocardial infarction, and congestive
heart failure (see review in Reference 2 ). Recent
studies demonstrated that the heart itself can produce TNF- in these
disorders.3 To investigate the
pathophysiological importance of myocardial
production of TNF-, we created 2 lines of transgenic mice
that overexpress TNF- in the heart under the control of -myosin
heavy chain (MHC) promoter.4 5 When TNF- was robustly
overexpressed, all mice developed severe lymphohistiocytic myocarditis
and died in the neonatal period.4 When TNF-
overexpression was more modest, most mice survived the neonatal period;
however, this line displayed a 6-month mortality of nearly
25%.5 Transgenic mice developed ventricular
hypertrophy and dilatation, interstitial
infiltrates and fibrosis, attenuation of adrenergic responsiveness,
reexpression of atrial natriuretic factor (ANF) in the
ventricle, and overt congestive heart failure.
Suppression of TNF- bioactivity ameliorates the severity of
myocarditis induced by injection of myosin6 or
encephalomyocarditis virus.7 Furthermore, soluble
TNF-binding proteins reverse the negative inotropic effects of TNF-
in vitro.8 The present study was undertaken to assess
the effects of TNF- suppression in mice having myocardial
inflammation secondary to TNF- overexpression using a
replication-deficient recombinant adenovirus encoding an extracellular
domain of human 55-kDa TNF receptor coupled with a mouse IgG heavy
chain (AdTNFRI).9
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Methods |
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Animals
Previously characterized transgenic mice (TNF1.6)5 were used under protocols approved by the Institutional Animal Care and Use Committee, University of Pittsburgh.
Adenoviruses
A replication-deficient recombinant adenovirus
(AdTNFRI)9 encoding the extracellular domain of the human
55-kDa TNF receptor coupled with a mouse IgG heavy chain9
was generously provided by Dr Bruce Beutler, University of Texas
Southwestern Medical Center, Dallas. An adenovirus encoding LacZ
(AdLacZ) served as a control. Viruses were propagated in 293 cells and
purified as previously described.9 The virus titer was
equal to OD260 divided by
9.09x10-13 (in particles/mL). A hundred
particles were assumed to be 1 pfu.
Experimental Design
Six-week-old TNF1.6 transgenic mice were injected through the
retro-orbital venous plexi with 109 pfu of
adenovirus and were euthanized for analysis 2 or 6 weeks later.
After determination of body and ventricular weights,
excised ventricles were snap-frozen in liquid nitrogen for biochemical
analysis or processed for histological
analysis. Plasma was collected for assessment of soluble TNFRI.
Hearts from age- and sex-matched transgenic (TNF1.6) or wild-type (FVB)
mice served as untreated controls. An additional 3 groups of mice (wild
type, untreated TNF1.6, and TNF1.6 treated with AdTNFRI for 2 weeks)
were used for echocardiography.
Echocardiography
Echocardiographic studies were performed with an
Acuson ultrasonograph system (Sequoia 512, Acuson). Mice were
anesthetized10 and lightly secured in a shallow
left lateral decubitus position. A 13.0-MHz transducer was applied to
the left hemithorax. 2D targeted M-mode imaging was obtained from the
short-axis view at the level of the largest left ventricular
diameter.
M-mode measurements of left ventricular end-diastolic and end-systolic diameters and left ventricular anterior and posterior wall thickness were made by use of the leading-edge convention of the American Society of Echocardiography. Three to 5 beats were averaged for each measurement. End diastole was determined at the maximal left ventricular diastolic dimension, and end systole was taken at the peak of posterior wall motion. Heart rate was determined from 3 to 5 consecutive RR intervals. Left ventricular fractional shortening (%) was calculated as LVFS (%)=[(LVEDD-LVESD)/LVEDD]x100, where LVEDD and LVESD indicate left ventricular end-diastolic and end-systolic diameters, respectively.
Quantification of Myocarditis
Myocardial infiltration was quantified in hematoxylin and
eosin–stained sections by determination of nuclear density
(nuclei/mm2). Because it is difficult to
differentiate inflammatory cells from myocytes and/or fibroblasts, all
nuclei were included. In each animal, 6 independent high-power fields
(0.233x0.312 mm; 0.0729-mm2 area) were
analyzed and averaged by investigators blinded to the treatment
group.
RNase Protection
Total RNA was extracted from frozen tissues as
described.4 5 RNase protection assays (RPAs) were
performed according to the manufacturer’s protocol (RiboQuant,
PharMingen; template sets mCK-1b, mCK-2b, mCK-3b, and mCK-5) with 5
µg of total RNA. After RNAase digestion, protected probes were
resolved on denaturing polyacrylamide gels and quantified by
PhosphoImager (ImageQuant software, Molecular Dynamics). The value of
each hybridized probe was normalized to that of GAPDH included in each
template set as an internal control.
Slot Blot
Total RNA (2.5 µg each sample) denatured with formaldehyde was
vacuum-blotted onto nitrocellulose. Hybridization probes were prepared
from oligomers complementary to murine - or ß-MHC11
and other probes (18S ribosomal RNA, ANF, sarcoplasmic/endoplasmic
reticulum Ca2+-ATPase [SERCA], and
phospholamban) as previously described.5 12 MHC oligomer
probes were hybridized (55°C in 4x SSC, 5x Denhardt’s, 0.1% SDS,
0.05% sodium pyrophosphate, tRNA 20 µg/mL,
32P-labeled oligomer 3 ng/mL), washed (2x
SSC–0.1% SDS at 60°C), and exposed to a storage phosphor screen,
and radioactive images were quantified as described above. Filters were
rehybridized with the 18S oligonucleotide, reexposed,
and quantified.5 12 Hybridization signals were normalized
to that of the 18S probe and in turn normalized to the mean of the
control samples.
Reverse Transcription–Polymerase Chain Reaction
Reverse transcription–polymerase chain reaction (RT-PCR)
reactions were performed as described.4 5 12
Oligonucleotide primer pairs included (1) intercellular
adhesion molecule-1 (ICAM-1) sense primer corresponding to base pairs
1112 to 1135 and antisense primer complementary to base pairs 1525 to
1547 (GenBank Accession No. X52264) and (2) total TNF- (both
transgene and endogenous gene) sense primer corresponding
to base pairs 164 to 187 and antisense primer complementary to base
pairs 833 to 855 (GenBank accession No. M13049). PCR was performed for
30 cycles (94°C for 60 seconds, 55°C for 30 seconds, and 72°C for
60 seconds), and products were visualized on 2% agarose gels by
ethidium bromide staining.
Enzyme-Linked Immunosorbent Assay
Cytokine protein levels were assessed by ELISA (mouse
TNF-, mouse interleukin [IL]-1ß, mouse monocyte chemotactic
protein [MCP]-1, and human TNFRI, Quantikine, R&D Systems). Plasma
samples (assayed in duplicate) were measured at a dilution of
10-1 to 10-6.
Cytokines in the myocardium were measured as
previously reported,4 5 with 100 µg of protein for
TNF-, IL-1ß, and MCP-1 and 1 µg for TNFRI. Standard reference
cytokines were provided by the manufacturer. Values are
reported as pg/mg or ng/mg protein. Because this study used a different
vendor for the TNF- ELISA kits, the values of TNF- protein in the
myocardium were substantially lower than those previously
reported.5 This is attributable to differences in the
standard reference cytokines as well as a substantially lower
background of TNF- in tissue homogenates.
Immunohistochemistry
Mouse hearts were fixed in ice-cold 2%
paraformaldehyde, immersed in ice-cold 30% sucrose,
and flash-frozen in OCT (Miles) medium with isopentane cooled by liquid
nitrogen. Blocks were cut at 10 µm, and sections were mounted,
immersion-fixed in acetone, rinsed in PBS, and treated for 30 minutes
with 5% normal rabbit serum. Primary antibodies used were polyclonal
goat anti-murine TNF- (1:100, Santa Cruz Biotechnology), goat
anti-murine IL-1ß, and goat anti-murine MCP-1 (both 1:100, R&D
Systems). Samples were treated with primary antibody (24 hours at
4°C), rinsed with PBS, treated (2 hours) with multilink biotinylated
anti-goat secondary antibody (1:4 dilution, Biogenex Laboratories),
rinsed in PBS, and treated with avidin-biotin complex (45 minutes;
Vector Laboratories). Reactions were visualized with
3-amino-9-ethyl-carbazole in 0.1 mol/L acetate buffer, pH 5.2. Sections
were weakly counterstained with Mayer’s hematoxylin.
Statistics
Results are presented as mean±SD. Statistical
comparisons were performed by ANOVA with Student-Newman-Keuls post hoc
test. Differences were considered significant at a value of
P<0.05.
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Results |
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Production of TNF Receptor Fusion Protein
Pilot studies in male wild-type mice determined that intravenous injections of 109 and 108 pfu of AdTNFRI produced a substantial amount of TNFRI in plasma after 1 week, whereas intraperitoneal injection did not (Figure 1
). Plasma levels of TNFRI declined
thereafter but remained in the µg/mL range for as long as 6 weeks.
Attempts to reinoculate mice to boost TNFRI levels were unsuccessful
(data not shown). Subsequent studies used a single
intravenous inoculation with 109 pfu
of adenovirus.
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Six-week-old female transgenic (TNF1.6) mice were injected with
109 pfu of either AdTNFRI or AdLacZ and
euthanized for analysis after an additional 2 or 6 weeks.
Age-matched transgenic or wild-type mice were euthanized as untreated
controls. As in wild-type mice, AdTNFRI produced substantial amounts of
soluble TNFRI in plasma (96.2±61.0 µg/mL) as well as in
myocardium (82.5±21.5 ng/mg protein) after 2 weeks.
Because the amounts of TNF- protein in the myocardium of
AdTNFRI-treated transgenic mice was 419±137 pg/mg protein, there was a
large excess (
200-fold) of TNFRI protein in the heart.
Cardiomegaly
Transgenic mice presented significant cardiomegaly, as
indicated by the increased ventricular weight/body weight
ratio, and 2 weeks of anti-TNF treatment was insufficient to ameliorate
cardiomegaly (Table 1).
Furthermore, cardiomegaly remained unchanged 6 weeks after injection of
AdTNFRI, although plasma levels of TNFRI remained high (32.3±8.8
µg/mL).
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Myocardial Inflammation
Eight-week-old untreated transgenic mice presented a
diffuse interstitial infiltration in the
myocardium consisting of mostly histiocytes with
some lymphocytes (Figure 2). Two weeks
after treatment with AdTNFRI, interstitial infiltration was
substantially reduced, as evidenced by nuclear density (Figure 3), an effect not observed by treatment
with AdLacZ. Myocardial infiltrates remained suppressed for 
6
weeks (data not shown), reflecting the persistent elevation in plasma
levels of TNFRI.
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P<0.05
TG+AdTNFRI vs TG+AdLacZ.
To assess the potential mechanism by which TNFRI overexpression reduces
myocardial infiltration, we analyzed expression of ICAM-1 by
RT-PCR in wild-type mice, TNF1.6 mice, and TNF1.6 mice 2 weeks after
inoculation with AdTNFRI. RT-PCR analysis demonstrated
increased ICAM-1 transcript levels in TNF1.6 mice, which were markedly
reduced in animals treated with AdTNFRI (Figure 4). However, TNF- transcript levels
(both transgene-driven and endogenous gene transcripts)
remained elevated in TNF1.6 mice regardless of TNF-soluble-receptor
therapy, consistent with RPA analyses (see below,
Figure 5).
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Cytokine Expression
Because TNF- can induce the expression of other proinflammatory
cytokines and chemokines that contribute to TNF-–induced
pathophysiology,1 we examined the expression of additional
cytokines by use of multiprobe RPAs.
Representative images of RPAs are shown in Figure 5
, and quantitative results are summarized in Table 2. Although overexpression of TNF-
induced a large number of cytokines, the induction of IL-1ß
and MCP-1 was particularly robust. With the exception of transforming
growth factor-ß induction, all of the changes in cytokine and
chemokine expression evident in the TNF- transgenic mice were
reversed after 2 weeks of treatment with soluble TNF receptor.
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To confirm that changes in mRNA reflected alterations at the protein
level, we selected 3 cytokines for ELISA: TNF-, IL-1ß, and
MCP-1. IL-1ß was chosen because of its synergistic effects with
TNF-, as well as its independent effects on
cardiomyocyte function and gene
expression.12 13 MCP-1, a prominent signal for the
accumulation of monocytes, is expressed in response to proinflammatory
cytokines14 and may be an important mediator of
myocardial infiltration.15 All 3 cytokine proteins
were not found in wild-type mice but were abundant in the TNF-
transgenic mice (Figure 6). Two weeks of
anti-TNF treatment abrogated the induction of IL-1ß and MCP-1
proteins, consistent with changes in the transcript levels. The
induction of these downstream cytokines remained inhibited 6
weeks after treatment (data not shown). In contrast, despite a reversal
of myocardial infiltration and a reduction in other cytokine
expression, anti-TNF treatment actually increased the immunodetectable
levels of TNF- in myocardium >2-fold.
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Immunohistochemical stains were performed to identify the cell source
of these cytokines (Figure 7).
Anti–TNF- resulted in diffuse background staining with some
positive-stained interstitial cells, suggesting that both
cardiomyocytes and infiltrating cells produced TNF- in
the myocardium. In contrast, the staining for IL-1ß and
MCP-1 was confined to interstitial cells, including
inflammatory infiltrates and fibroblasts.
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Cardiac-Specific Gene Expression
Figure 8 summarizes changes in the
expression of cardiac-specific genes in TNF1.6 mice. Relative to
wild-type untreated controls, -MHC, SERCA, and phospholamban
transcripts were downregulated, whereas ß-MHC and ANF were
upregulated, in the ventricles of transgenic mice. Treatment with
AdLacZ had no significant effects on the expression of these genes,
whereas anti-TNF treatment with AdTNFRI reversed changes in -MHC,
SERCA, and phospholamban, partially reversed ANF, and had no effect on
ß-MHC expression. Gene transcript levels after the 6-week treatment
were similar to those observed after the 2-week treatment. -MHC,
SERCA, and phospholamban transcripts were restored to wild-type levels
(0.89±0.11, 0.95±0.25, and 0.96±0.20, n=4, respectively), whereas
ß-MHC and ANF transcripts remained elevated (4.21±0.80 and
4.00±1.68, n=4). The trend toward further reduction in ANF transcripts
was not statistically significant.
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Anti-TNF Therapy and Cardiac Function
Examples of M-mode echocardiographic measurements
in TNF- transgenic mice with or without AdTNFRI treatment and
wild-type controls are presented in Figure 9 and quantitative results in Table 3. At this age, the left
ventricular fractional shortening in transgenic mice was
comparable to that in controls, whereas left ventricular
end-systolic diameter was significantly larger in transgenic
mice than in control mice, suggesting a latent left
ventricular dysfunction in young transgenic mice. This
increase in the diameter was reversed by the AdTNFRI treatment. In
contrast, significant left ventricular wall thickening in
the transgenic mice was not reversed after the TNF neutralizing
therapy.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Soluble TNF receptor was achieved by intravenous injection with a recombinant adenovirus encoding a fusion protein containing the extracellular domain of human TNFRI (p55) coupled with a mouse IgG heavy chain.9 The virus transfects the liver, which then produces soluble TNFRI. A sustained secretion of TNFRI into the plasma permeates the extracellular space of multiple organs, including the myocardium.16 The principal limitation of using recombinant adenovirus is that endogenous immunological activity reduces AdTNFRI expression and significant levels of TNFRI cannot be found after several months. However, beneficial biological effects of TNFRI in mice overexpressing proinflammatory cytokines can be clearly demonstrated within 2 weeks of therapy and persist through 6 weeks.
Transgenic mice with cardiac-specific overexpression of TNF-
present lymphohistiocytic myocarditis, cardiomegaly, cardiac
dysfunction, and congestive heart failure.5 Whereas some
of the phenotypic changes that characterize the TNF- transgenic mice
could be attributed to TNF- alone, the TNF- induction of
"downstream" cytokines and chemokines may contribute to
TNF-–induced pathophysiology.1 Indeed, overexpression
of TNF- induced the expression of a large number of
cytokines and chemokines. Nearly all of the induced
cytokines showed reversal of expression after AdTNFRI
treatment, suggesting their activation downstream of TNF-.
Both transcript and protein levels of TNF-, IL-1ß, and MCP-1 were
increased in the myocardium of transgenic mice and reversed
by anti-TNF treatment. Immunohistochemical analysis suggested
that IL-1ß and MCP-1 were produced predominantly by
nonmyocytes. In contrast, TNF- expression was found in both
myocytes and nonmyocytes, and the protein level, elevated in
transgenic animals, was further increased after the treatment with
AdTNFRI. Because TNF- transcripts were not significantly higher in
AdTNFRI-treated mice, the increase in TNF- protein is unlikely to
arise from enhanced transcriptional activity of the TNF- genes. More
likely, although TNF- loses its bioactivity when bound by TNFRI, it
may also gain stability as soluble TNF receptors stabilize TNF-
protein despite blockade of its bioactivity.17 Because the
ELISA measures both receptor-bound and free TNF-, the major
contribution to the increase might be due to soluble receptor-bound
TNF-. Regardless of increased TNF- protein in AdTNFRI-treated
mice, the biological effects of TNF- were attenuated, as shown by
the abrogation of myocarditis and expression of downstream
cytokines.
Although anti-TNF treatment with AdTNFRI abrogated the expression of
inflammatory mediators and the development of interstitial
infiltrates, treatment had no effect on ventricular
hypertrophy. Furthermore, treatment had no effect on the
increased ventricular expression of ß-MHC in TNF-
transgenic mice and only a partial suppression of the enhanced ANF
expression. Because upregulation of ß-MHC expression is an integral
part of the development of myocardial
hypertrophy,18 the failure of soluble TNF
receptor to attenuate either ß-MHC expression or
ventricular hypertrophy is internally
consistent. Because - and ß-MHC are thought to be
regulated reciprocally, it is of interest that anti-TNF treatment
reversed the TNF-–induced downregulation of -MHC but not the
upregulation of ß-MHC. Gupta and Zak19 demonstrated,
however, that isoform shifts in the pressure-overload–induced
hypertrophy model were not temporally related; -MHC
expression was normalized within 2 weeks after the removal of the
aortic band, whereas ß-MHC expression required 7 weeks to be
normalized.
Although we cannot exclude the possibility that a more prolonged
exposure to anti-TNF therapy will reverse hypertrophy and
normalize ß-MHC upregulation, these results suggest that the
hypertrophic process may persist regardless of apparent neutralization
of myocardial TNF-. Indeed, the hypertrophic program may be
initiated early in the development of heart failure and be
self-sustaining and thus unresponsive to anti–TNF- intervention at
6 weeks of age. Alternatively, a very low level of biologically active
TNF- may persist despite therapy with TNFRI, allowing continued
stimulation of a hypertrophic signaling pathway.
-MHC, SERCA, and phospholamban transcripts are downregulated in
end-stage heart failure in both human20 21 and
animal22 models. IL-1ß inhibits expression of these
genes in cultured neonatal cardiomyocytes,12
and both IL-1ß and TNF- alter cardiomyocyte
contractile activity.13 Thus, the observation that
cardiac-specific overexpression of TNF- and induction of IL-1ß is
associated with downregulation of these genes is consistent
with in vitro studies.
The potential utility of anti-TNF therapy in the management of
congestive heart failure is currently an area of great
interest.23 Infusion of a dimeric soluble TNF receptor has
been shown to antagonize the negative inotropic effects of chronic
TNF- infusion in rats.24 This model is not accompanied
by myocardial infiltrates or cardiac-specific overexpression of
TNF-, suggesting a beneficial role of TNF- blockade in
noninflammatory models of heart failure. However, the complete role of
TNF- in heart failure that does not arise as a consequence of
TNF- administration remains to be fully elucidated. Because anti-TNF
therapies reduce the severity of autoimmune6 or
virus-induced myocarditis,7 one might hypothesize that
anti-TNF therapies would prove to be of benefit in cardiac inflammatory
diseases. However, anti–TNF- antibodies exacerbate the pathology in
murine models of Chagas
cardiomyopathy25 ; hence, the full
utility of anti-TNF therapy in the regulation of inflammatory heart
disease and heart failure remains promising yet incompletely explored.
Additional information regarding the contribution of TNF- to heart
failure may come from ongoing investigations of the efficacy of soluble
TNF receptor therapy in patients with chronic congestive heart
failure.
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Acknowledgments |
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This work was supported by NIH grant HL-60032-01. Dr Kubota is the recipient of a Japan Heart Foundation and Bayer Yakuhin Research Grant Abroad. Dr Kadokami is supported by the Bill Hilgrove Fellowship. The authors thank Tracey Barry for editorial and Carole Frye for technical assistance.
Received August 19, 1999; revision received December 15, 1999; accepted December 15, 1999.
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 Y. Monden, T. Kubota, T. Tsutsumi, T. Inoue, S. Kawano, N. Kawamura, T. Ide, K. Egashira, H. Tsutsui, and K. Sunagawa Soluble TNF receptors prevent apoptosis in infiltrating cells and promote ventricular rupture and remodeling after myocardial infarction Cardiovasc Res, March 1, 2007; 73(4): 794 - 805. [Abstract] [Full Text] [PDF]
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K. Nishikawa, M. Yoshida, M. Kusuhara, N. Ishigami, K. Isoda, K. Miyazaki, and F. Ohsuzu Left ventricular hypertrophy in mice with a cardiac-specific overexpression of interleukin-1 Am J Physiol Heart Circ Physiol, July 1, 2006; 291(1): H176 - H183. [Abstract] [Full Text] [PDF]
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 S. S Schleithoff, A. Zittermann, G. Tenderich, H. K Berthold, P. Stehle, and R. Koerfer Vitamin D supplementation improves cytokine profiles in patients with congestive heart failure: a double-blind, randomized, placebo-controlled trial. Am. J. Clinical Nutrition, April 1, 2006; 83(4): 754 - 759. [Abstract] [Full Text] [PDF]
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Y. Xu, I. A. Arenas, S. J. Armstrong, W. C. Plahta, H. Xu, and S. T. Davidge Estrogen improves cardiac recovery after ischemia/reperfusion by decreasing tumor necrosis factor-{alpha} Cardiovasc Res, March 1, 2006; 69(4): 836 - 844. [Abstract] [Full Text] [PDF]
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H. Matsusaka, M. Ikeuchi, S. Matsushima, T. Ide, T. Kubota, A. M. Feldman, A. Takeshita, K. Sunagawa, and H. Tsutsui Selective disruption of MMP-2 gene exacerbates myocardial inflammation and dysfunction in mice with cytokine-induced cardiomyopathy Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1858 - H1864. [Abstract] [Full Text] [PDF]
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N. Kawamura, T. Kubota, S. Kawano, Y. Monden, A. M. Feldman, H. Tsutsui, A. Takeshita, and K. Sunagawa Blockade of NF-{kappa}B improves cardiac function and survival without affecting inflammation in TNF-{alpha}-induced cardiomyopathy Cardiovasc Res, June 1, 2005; 66(3): 520 - 529. [Abstract] [Full Text] [PDF]
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 T. Bachetti, L. Comini, E. Pasini, and R. Ferrari Anti-cytokine therapy in chronic heart failure: new approaches and unmet promises Eur. Heart J. Suppl., November 1, 2004; 6(suppl_F): F16 - F21. [Abstract] [Full Text] [PDF]
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Y. Higuchi, C. F. McTiernan, C. B. Frye, B. S. McGowan, T. O. Chan, and A. M. Feldman Tumor Necrosis Factor Receptors 1 and 2 Differentially Regulate Survival, Cardiac Dysfunction, and Remodeling in Transgenic Mice With Tumor Necrosis Factor-{alpha}-Induced Cardiomyopathy Circulation, April 20, 2004; 109(15): 1892 - 1897. [Abstract] [Full Text] [PDF]
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 T. Shiomi, H. Tsutsui, M. Ikeuchi, H. Matsusaka, S. Hayashidani, N. Suematsu, J. Wen, T. Kubota, and A. Takeshita Streptozotocin-induced hyperglycemia exacerbates left ventricular remodeling and failure after experimental myocardial infarction J. Am. Coll. Cardiol., July 2, 2003; 42(1): 165 - 172. [Abstract] [Full Text] [PDF]
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E. S. Chung, M. Packer, K. H. Lo, A. A. Fasanmade, and J. T. Willerson Randomized, Double-Blind, Placebo-Controlled, Pilot Trial of Infliximab, a Chimeric Monoclonal Antibody to Tumor Necrosis Factor-{alpha}, in Patients With Moderate-to-Severe Heart Failure: Results of the Anti-TNF Therapy Against Congestive Heart failure (ATTACH) Trial Circulation, July 1, 2003; 107(25): 3133 - 3140. [Abstract] [Full Text] [PDF]
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 A. W. Ashton, G. M. Ware, D. K. Kaul, and J. A. Ware Inhibition of Tumor Necrosis Factor alpha -mediated NFkappa B Activation and Leukocyte Adhesion, with Enhanced Endothelial Apoptosis, by G Protein-linked Receptor (TP) Ligands J. Biol. Chem., March 28, 2003; 278(14): 11858 - 11866. [Abstract] [Full Text] [PDF]
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 J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential Pharmacol. Rev., March 1, 2003; 55(1): 133 - 166. [Abstract] [Full Text] [PDF]
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B. London, L. C. Baker, J. S. Lee, V. Shusterman, B.-R. Choi, T. Kubota, C. F. McTiernan, A. M. Feldman, and G. Salama Calcium-dependent arrhythmias in transgenic mice with heart failure Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H431 - H441. [Abstract] [Full Text] [PDF]
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Y. Machida, T. Kubota, N. Kawamura, H. Funakoshi, T. Ide, H. Utsumi, Y. Y. Li, A. M. Feldman, H. Tsutsui, H. Shimokawa, et al. Overexpression of tumor necrosis factor-alpha increases production of hydroxyl radical in murine myocardium Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H449 - H455. [Abstract] [Full Text] [PDF]
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 P A Henriksen and D E Newby Therapeutic inhibition of tumour necrosis factor {alpha} in patients with heart failure: cooling an inflamed heart Heart, January 1, 2003; 89(1): 14 - 18. [Abstract] [Full Text] [PDF]
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T. Shiomi, H. Tsutsui, S. Hayashidani, N. Suematsu, M. Ikeuchi, J. Wen, M. Ishibashi, T. Kubota, K. Egashira, and A. Takeshita Pioglitazone, a Peroxisome Proliferator-Activated Receptor-{gamma} Agonist, Attenuates Left Ventricular Remodeling and Failure After Experimental Myocardial Infarction Circulation, December 10, 2002; 106(24): 3126 - 3132. [Abstract] [Full Text] [PDF]
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P. C. Brum, J. Kosek, A. Patterson, D. Bernstein, and B. Kobilka Abnormal cardiac function associated with sympathetic nervous system hyperactivity in mice Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H1838 - H1845. [Abstract] [Full Text] [PDF]
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 H. Funakoshi, T. Kubota, N. Kawamura, Y. Machida, A. M. Feldman, H. Tsutsui, H. Shimokawa, and A. Takeshita Disruption of Inducible Nitric Oxide Synthase Improves {beta}-Adrenergic Inotropic Responsiveness but Not the Survival of Mice With Cytokine-Induced Cardiomyopathy Circ. Res., May 17, 2002; 90(9): 959 - 965. [Abstract] [Full Text] [PDF]
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G. Wright, I. S. Singh, J. D. Hasday, I. K. Farrance, G. Hall, A. S. Cross, and T. B. Rogers Endotoxin stress-response in cardiomyocytes: NF-kappa B activation and tumor necrosis factor-alpha expression Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H872 - H879. [Abstract] [Full Text] [PDF]
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W. S. Bradham, B. Bozkurt, H. Gunasinghe, D. Mann, and F. G. Spinale Tumor necrosis factor-alpha and myocardial remodeling in progression of heart failure: a current perspective Cardiovasc Res, March 1, 2002; 53(4): 822 - 830. [Abstract] [Full Text] [PDF]
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C G Densem, I V Hutchinson, N Yonan, and N H Brooks Tumour necrosis factor {alpha} gene polymorphism: a predisposing factor to non-ischaemic myocardial dysfunction? Heart, February 1, 2002; 87(2): 153 - 155. [Abstract] [Full Text] [PDF]
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D. Hilfiker-Kleiner, A. Hilfiker, B. Schieffer, D. Engel, D. L Mann, K. C Wollert, and H. Drexler TNF{alpha} decreases {alpha}MHC expression by a NO mediated pathway: role of E-box transcription factors for cardiomyocyte specific gene regulation Cardiovasc Res, February 1, 2002; 53(2): 460 - 469. [Abstract] [Full Text] [PDF]
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 P. O. Iversen, P. R. Woldbaek, T. Tonnessen, and G. Christensen Decreased hematopoiesis in bone marrow of mice with congestive heart failure Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2002; 282(1): R166 - R172. [Abstract] [Full Text] [PDF]
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P. O. Iversen, G. Nicolaysen, and M. Sioud DNA enzyme targeting TNF-alpha mRNA improves hemodynamic performance in rats with postinfarction heart failure Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H2211 - H2217. [Abstract] [Full Text] [PDF]
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T. Kadokami, C. Frye, B. Lemster, C. L. Wagner, A. M. Feldman, and C. F. McTiernan Anti-Tumor Necrosis Factor-{alpha} Antibody Limits Heart Failure in a Transgenic Model Circulation, September 4, 2001; 104(10): 1094 - 1097. [Abstract] [Full Text] [PDF]
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T. Kadokami, C. F. McTiernan, T. Kubota, C. S. Frye, G. S. Bounoutas, P. D. Robbins, S. C. Watkins, and A. M. Feldman Effects of soluble TNF receptor treatment on lipopolysaccharide-induced myocardial cytokine expression Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2281 - H2291. [Abstract] [Full Text] [PDF]
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B. Bozkurt, G. Torre-Amione, M. S. Warren, J. Whitmore, O. Z. Soran, A. M. Feldman, and D. L. Mann Results of Targeted Anti-Tumor Necrosis Factor Therapy With Etanercept (ENBREL) in Patients With Advanced Heart Failure Circulation, February 27, 2001; 103(8): 1044 - 1047. [Abstract] [Full Text] [PDF]
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D. L. Mann Tumor Necrosis Factor and Viral Myocarditis: The Fine Line Between Innate and Inappropriate Immune Responses in the Heart Circulation, February 6, 2001; 103(5): 626 - 629. [Full Text] [PDF]
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 Y. Y. Li, Y. Q. Feng, T. Kadokami, C. F. McTiernan, R. Draviam, S. C. Watkins, and A. M. Feldman Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor necrosis factor alpha therapy PNAS, November 7, 2000; 97(23): 12746 - 12751. [Abstract] [Full Text] [PDF]
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H. Funakoshi, T. Kubota, Y. Machida, N. Kawamura, A. M. Feldman, H. Tsutsui, H. Shimokawa, and A. Takeshita Involvement of inducible nitric oxide synthase in cardiac dysfunction with tumor necrosis factor-alpha Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2159 - H2166. [Abstract] [Full Text] [PDF]
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