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TRANSLATIONAL PHYSIOLOGY
cardioprotection from myocardial infarction
Departments of Medicine and Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10021
Submitted 18 February 2003 ; accepted in final form 29 April 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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(TNF-),
suggesting that its receptor(s) may be downregulated in older cardiac
endothelial cells. Immunostaining demonstrated that TNF-receptor 1 (TNF-R1)
density was significantly lower in the subendocardial endothelium of the aging
murine heart. Functional studies confirmed the senescent dysregulation of
TNF- receptor pathways, demonstrating that TNF- induced PDGF-B
expression in cardiac microvascular endothelial cells of 4-mo-old, but not
24-mo-old, rats. Moreover, TNF- mediated cardioprotective pathways were
impaired in the aging heart. In young rat hearts, injection of TNF-
significantly reduced the extent of myocardial injury after coronary ligation:
TNF-, 7.9 ± 1.9% left ventricular injury (n = 4) versus
PBS, 16.2 ± 7.9% (n = 10; P < 0.05). The addition
of PDGF-AB did not augment the cardioprotective action of TNF-. In
myocardial infarctions of older hearts, however, TNF- induced
significant postcoronary occlusion mortality (TNF- 80% vs. PBS 0%;
n = 10 each, P < 0.05) that was reversed by the
coadministration of PDGF-AB. Overall, these studies demonstrate that
aging-associated alterations in TNF- receptor cardiac microvascular
pathways may contribute to the increased cardiovasular pathology of the aging
heart. Strategies targeted at restoring TNF- receptor-mediated
expression of PDGF-B may improve cardiac microvascular function and provide
novel approaches for treatment and possible prevention of cardiovascular
disease in older individuals.
aging; heart; endothelial; phage display; functional genomics
Previous studies (31,
34,
40) have shown that aging is
associated with significant alterations in the expression patterns of cell
surface cytokine receptors, thus suggesting a potential mechanism that may
contribute to senescent changes in cardiac microvasculature activity. Our
study is aimed at defining the molecular pathways that underlie the
age-associated impairment of cardiovascular function. Here we report the
results of in vivo cyclic peptide phage display library biopanning studies
that identified an age-associated alteration in cardiac microvascular
endothelial tumor necrosis factor- (TNF-) receptor pathways that
can contribute to the senescent impairment in PDGF-B induction and loss of
cardioprotection.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Phage-Display Peptide Library In Vivo Cardiac Vascular Biopanning.
The age-associated changes in cardiac microvascular surface receptors were
probed by in vivo phage-display biopanning with a cyclic peptide pSKAN
phagemid library (6 amino acid variable; 
107 total complexity,
Mo Bio Tech). Young adult (3 mo old) and aging (18 mo old) C57B61/L mice were
anesthetized with 0.015 ml/g Avertin and injected with phage peptide library
phage [1012 colony forming units (CFU)/200 µl PBS] via the tail
veins. Four minutes after injection, the mice were euthanized, the hearts
explanted, and the phage recovered with WK6
mutS Escherichia
coli. Age-specific phage pools were amplified and titrated for two
additional rounds of biopanning enrichment. The phagemid DNA of the resultant
clones were sequenced and translated amino acid motifs determined employing
read through of all codons, as previously described for phage display library
analysis (6,
13). Translated motifs were
analyzed for homology to known cytokines (FASTA3), as determined by the first
homologous mammalian sequence identified with E value <1. In addition, to
probe the structural relevance of the phage motif cytokine binding epitopes,
the regions of homology were mapped in the tertiary models and labeled by
Cn3D3.0 software.
Individual phage clone in vivo cardiac vascular biopanning. To
confirm the age-associated differential cardiac binding capacity of a
candidate phage with homology to TNF-, phage clone 
Y12, as well as
helper phage without the insert, were prepared with TG1 E. coli to
generation of phage motifs with supE suppression, as previously described
(6). The Y12 phage
(1012 CFU in 200 µl PBS) was injected into both 3- and 18-mo-old
mice, as described above (n = 3, each group). The phages were
recovered from the explanted hearts, as described above, with WK6mutS
E. coli, which were then quantified by serial dilution titration.
In situ cardiac TNF- receptor analysis. On the
basis of the homology of Y12 to TNF-, the potential age-associated
changes in TNF- receptor patterns were analyzed in situ. Cardiac
sections of 3- and 18-mo-old wild-type mice were probed with goat antibodies
directed against TNF-receptors 1 and 2 (TNF-R1 and -R2; Santa Cruz, Sc1079 and
1074) and developed with an anti-goat ABC kit with diaminobenzidine (Vector).
Immunostained vascular density was quantified in the subendocardial tissue, as
previously described: 16 high-power fields magnified x40 per heart
(n = 3, each group)
(10). Counts were performed by
two investigators in a blinded fashion.
TNF- induction of PDGF-B in CMECs. The potential
age-associated impairment in TNF--mediated induction of cardiac
endothelial PDGF-B expression was measured in vitro. CMECs of 4- and 24-mo-old
F344 rats were isolated and cultured, as previously described
(26), with minor modification.
Briefly, the hearts were removed and minced in endothelial cell medium; DMEM
containing 20% FBS, 1% endothelial cell growth factor, 1% endothelial cell
growth supplement, 1% basal medium Eagle, 10–4%
heparin, and 1% penicillin (10,000 IU/ml streptomycin 10,000 µg/ml) and
digested with the addition of 0.2% collagenase, 0.005% DNAse, and 5% FBS for
45 min with subsequent endothelial cells isolation by platelet endothelial
cell adhesion molecule-mediated magnetic particle collection (Dynabeads), as
detailed earlier (7). The
resultant CMECs (Dil-LDL uptake >95%) were cultured for two passages and
seeded into 12-well culture dish (105 cell/well) and grown to
confluence. The medium was changed to serum-free DMEM for 1 h and then to DMEM
with 2% FBS ± TNF- (30 ng/ml) for 3 h. Total RNA was then
isolated and RT-PCR performed, as previously described
(10). The primers for PCR were
listed as follows: rat PDGF-B (sense) 5'-GATCCGCTCCTTTGATGATC-3';
rat PDGF-B (antisense) 5'-GTCTCACACTTGCATGCCAG-3'; rat
-actin (sense) 5'-ATTGGCAATGAGCGGTTCCGC-3'; and rat
-actin (antisense) 5'-CTCCTGCTTGCTGATCCACATC-3'.
Age-dependent in vivo response to TNF-. To probe the
potential age-dependent effects of TNF- on PDGF-B expression and
protection from myocardial injury, sets of 4- and 24-mo-old F344 rats received
intramyocardial injections of the growth factor, as previously described
(10). Briefly, the rats were
anesthetized with xylazine (5–10 mg/kg ip) and ketamine (80–90
mg/kg ip) and underwent a left intercostal thoracotomy. After the left
anterior descending coronary artery (LAD) was identified, 100 ng of
TNF- in 50 µl PBS or PBS alone were injected through a 30 gauge
needle, using a 250-µl Hamilton syringe. Two injections (25 µl/injection
2 mm apart) were made at the middle left ventricular anterior wall. The chest
wall was then closed, the lungs were inflated, the rat was extubated, and the
tracheotomy was closed.
Age-dependent TNF--mediated induction of PDGF-B.
Rats receiving pretreatments alone (TNF- or PBS control, 4- and
24-mo-old, n = 3 each group) were euthanized 24 h postinjection. The
hearts were excised, fixed, sectioned, and immunostained for PDGF-B (murine
anti-PDGF-B, 376M, BioGenex) and visualized with a Texas red conjugated donkey
anti-mouse antibody (Sc2785, Santa Cruz). PDGF-B staining was quantified in
the subendocardial tissue in the anterior left ventricular wall at the level
of the midpapillary muscles from each heart by identifying all PDGF-B-stained
luminal structures, as previously described
(10) (8 high-power fields
magnified x40 per heart). Two investigators performed quantification
independently in a blinded fashion.
Age-specific TNF--mediated protection from myocardial
infarction. The potential cardioprotective effects of TNF-
pretreatments were studied in a myocardial infarction model. One day after
TNF- (4 mo old, n = 4; 24 mo old, n = 10) or control
(4 mo old, n = 4; 24 mo old, n = 10) intramyocardial
injections, the rats were anesthetized, the heart was exposed, and the LAD was
ligated just below (4 mo old) or 2 mm below (24 mo old) the left atrial
appendage with 8-0 nylon sutures. Pallor and regional wall motion abnormality
of the left ventricle confirmed occlusion. The chest wall was closed, and,
after recovery, the rats were returned to the animal facility and kept for 14
days. The rats were killed and the hearts were harvested, fixed, and
sectioned. Myocardial infarction size measured at the level of the
midpapillary heart muscles was scored by Masson's trichrome staining
(20,
30), and the images were
analyzed in a blinded fashion employing NIH Image Software version J1.22
(27,
38). Infarction size was
expressed as a percentage of the total left ventricle myocardial area. Cardiac
samples from aging rats that expired within 72 h of TNF- injection were
sectioned and analyzed for apoptotic induction by terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling staining (in situ cell death kit
no. 684817, Roche) (n = 3). To measure the potential interactions of
TNF- and PDGF-AB, sets of rats were treated with a combination of
TNF- (100 ng/heart) and PDGF-AB (100 ng/heart) in 4-mo-old rats
(n = 5) and 24-mo-old rats (n = 5), as well as by PDGF-AB
alone (100 ng/heart) in 4-mo-old rats (n = 8) and 24-mo-old rats
(n = 10).
Survival assay of TNF--treated old hearts. To
investigate TNF- induced mortality after LAD occlusion in the 24-moold
rats, additional sets of older rats received intramyocardial injection of
TNF- (100 ng/heart; n = 5) without subsequent coronary
occlusion. These TNF--treated old rats were observed for 2 wk, and the
survival number was recorded.
Statistical analysis. Comparisons of categorical variables were conducted using the binomial distribution or Fisher's exact test, as appropriate. For nonnormally distributed continuous variables, a Wilcoxon rank-sum test was used. A two-tailed P value <0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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in
the younger but not older cardiachoming phage clones (2/101 vs. 0/100,
P < 0.05) in Fig. 1, A
and B. In vivo injection with individual phage colonies
confirmed the diminished cardiac homing of Y12 in the old murine heart
(Fig. 1C), suggesting
that the binding sites for the TNF--like phage motif may be decreased
in the aging cardiac microvasculature. Immunostaining of 3- and 18-mo-old
hearts revealed age-associated changes in one of the TNF receptors. TNF-R2
patterning was similar in the microvasculature throughout the young and old
murine hearts (Fig. 2, A and
B). Similarly, TNF-R1 was present in the microvasculature
throughout the younger hearts; however, in the older hearts, the receptor was
restricted to the subepicardial microvasculature, with a significantly lower
density of TNF-R1 in the senescent subendocardium.
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The functional significance of the changes in TNF- receptor pathways
in the aging cardiac microvaculature was then examined. On the basis of
previous reports (12,
15) demonstrating that
TNF- promotes the expression of PDGF-B in endothelial cells in vitro,
we hypothesized that TNF- induction of PDGF-B would be impaired in the
aging CMECs. In vitro studies confirmed that TNF- induced the
expression of PDGF-B in CMECs of the 4-mo-old rat heart but not in cells from
the 24-mo-old hearts, Fig.
3A. In vivo TNF- specifically induced the increase
of PDGF-B in the younger hearts, while having minimal effect on the aging
tissue (Fig. 3, B and
C).
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The functional impact of the senescent dysregulation of cardiac TNF-
receptor pathways resulted in a loss of cardioprotection in the aging heart.
In the young heart, intramyocardial injection of TNF-, like PDGF-AB,
markedly reduced the extent of myocardial injury induced by ligation of the
LAD (Fig. 4, A and
B). In the old rats, TNF- was deleterious,
resulting in a high mortality rate within 72 h of coronary occlusion, which
was associated with extensive myocardial apoptosis
(Fig. 5, A and
B). Coinjection with PDGF-AB restored viability after
coronary occlusion in the old rats (Fig.
5A) with a reduction in myocardial infarction size
compared with control injections (Fig.
4). Combined injection of TNF- and PDGF-AB in the younger
rats, however, provided no increase in cardioprotection beyond injection of
the individual growth factors.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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receptor pathways contribute to cardiac microvascular dysfunction
and loss of cardioprotection in the aging rodent heart. In vivo phage display
studies identified a cardiac microvascularhoming TNF- homologous
peptide in young hearts that subsequently revealed an age-associated decrease
of TNF-R1 in cardiac subendocardial microvasculature. Functional studies
(10) demonstrated that this
dysregulation in cardiac microvascular endothelial TNF- receptor
pathways contributes to the age-associated impairment in cardioprotective
PDGF-B induction. Indeed, the senescent alterations in the cardiac actions of
TNF- resulted in increased mortality after coronary occlusion, which
was reversed by the restoration of PDGF.
Previous studies (5,
8,
9,
11,
25,
35) have revealed that
TNF- mediates a diverse array of both beneficial and deleterious
molecular and cellular cardiovascular responses. Potential protective actions
of TNF- include preconditioning of the ischemic heart
(35), reducing hypoxic injury
of cardiac myocytes (24),
enhancing angiogenic activity
(23,
42), and inducing PDGF
pathways in endothelial cells
(12,
15). The pathways governing
these complex multicellular responses as well as TNF--mediated
pathophysiology in the heart have not been fully elucidated but likely involve
TNF- secreted from cardiac myocytes
(18,
39) regulating the function of
endothelial cells expressing TNF receptors
(44). The present studies
demonstrate that short-term stimulation of intact TNF- receptor
pathways in the young cardiac microvasculature can induce expression of
PDGF-B, and, more importantly, reduce the extent of myocardial injury after
coronary occlusion. Indeed, the lack of synergistic actions by coinjection of
TNF- and PDGF in the young hearts suggests that PDGF may mediate
similar or overlapping downstream signal pathways in the cardioprotection of
young hearts. The age-associated dysregulation of these pathways contributes
to the impairment in endothelial cell function and increases
TNF--induced mortality after coronary occlusion. Restoration of the
PDGF-dependent downstream pathways reverses this age-associated mortality,
potentially modulating apoptotic signaling cascades to promote
cardioprotection in the senescent heart.
Age-associated alterations of receptor pathways and increases in cytokine
levels may underlie the diminished expression of TNF-R1 and the senescent
switch in the actions of TNF- from cardioprotection to pathophysiology.
Intracellular cofactors regulating the conformation and activity of TNF-R1
(22) may govern the function
of the subpopulation of endothelial cells recognized by the Y12 motif.
Senescent changes in these cofactors, with increases in
TNF-receptor-associated death domain proapoptotic pathways
(1,
14), may contribute to
TNF-induced apoptosis in aging endothelial cells
(17) and loss of
cardioprotective effects on cardiac myocytes
(19,
24). Moreover, aging is
associated with chronic increases in systemic levels of TNF-
(4,
28) and thus could compound
vascular dysfunction due to apoptosis of older endothelial cells with altered
TNF-R1 signaling pathways
(17). Indeed, experimental
models with sustained overexpression of TNF- in the rodent heart result
in marked left ventricular dysfunction and heart failure
(5,
11), and the mortality after
coronary occlusion observed in the TNF--treated older hearts may be due
to the further enhancement of the senescent receptor pathways mediating this
cardiovascular pathophysiology.
Therapies based on the cardioprotective role of TNF- may have
clinical utility in the younger heart, but novel strategies are required to
optimize treatments of cardiovascular disease in older persons. Restoration of
TNF-R1 expression in the aging cardiac microvasculature through gene therapy
approaches has limited utility because the receptor itself can mediate
viral-induced apoptotic pathways
(16,
21,
43). Moreover, reexpression of
the receptors may enhance the TNF- proapoptotic pathways in the aging
heart. Repopulation of the TNF-R1 endothelial cells with autologous or
genetically matched endothelial precursor cells could overcome these
limitations and potentially restore senescent vascular function.
Alternatively, approaches aimed a decreasing or reversing the age-associated
alterations in TNF- and its receptor pathways may have significant
applicability. To this end, previous studies
(36) have demonstrated that
estrogen protects endothelial cells from TNF--induced apoptosis and may
be useful in preventing the senescent loss of TNF-R1 in cardiac endothelial
cells. Similarly, identification of the critical genes downstream from
TNF-, potentially acting with PDGF-B induction, may allow for the
development of cardiac-specific therapies that could have benefit for persons
of all ages. Small molecules, including cyclic peptides, based on the
structure and sequence of Y12, could provide novel strategies to
selectively target cardioprotective pathways, potentially without the
concomitant induction of apoptosis in older hearts. Moreover, such therapies
could be combined with molecule approaches that are based on pathways that are
upregulated in the senescent heart to develop therapies specifically tailored
for the treatment and possible prevention of cardiovascular disease in older
persons.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2000.This article has been cited by other articles:
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