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From the Department of Medicine, Section of Cardiovascular Sciences and Cardiology, the DeBakey Heart Center, Baylor College of Medicine Houston, and the Methodist Hospital, Houston, Texas
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or by postischemic cardiac lymph containing
TNF-. However, the release of TNF- during early
reperfusion did not explain the venular localization of MCP-1
induction. To better understand the factors mediating MCP-1
induction, we examined the role of ischemia/reperfusion in a
model of brief coronary occlusion in which no necrosis or inflammatory
response is seen. Adult mongrel dogs were subjected to 15 minutes of
coronary occlusion and 5 hours of reperfusion. Ribonuclease protection
assay revealed up-regulation of MCP-1 mRNA only in ischemic segments of
reperfused canine myocardium. Pretreatment with the reactive oxygen
scavenger N-(2-mercaptopropionyl)-glycine completely
inhibited MCP-1 induction. In situ hybridization
localized MCP-1 message to small venular endothelium in ischemic areas
without myocyte necrosis. Gel shift analysis of nuclear extracts from
the ischemic area showed enhanced DNA binding of the transcription
factors AP-1 and nuclear factor (NF)-
B, crucial for MCP-1
expression, in ischemic myocardial regions. Immunohistochemical
staining demonstrated reperfusion-dependent nuclear translocation of
c-Jun and NF-B (p65) in small venular endothelium, only in the
ischemic regions of the myocardium, that was inhibited by
N-(2-mercaptopropionyl)-glycine. In
vitro, treatment of cultured canine jugular vein
endothelial cells with the reactive oxygen intermediate
H2O2 induced a concentration-dependent increase
in MCP-1 mRNA levels, which was inhibited by the antioxidant
N-acetyl-L-cysteine, a precursor of
glutathione, but not pyrrolidine dithiocarbamate, an
inhibitor of NF-B and activator of AP-1. In contrast to our studies
with infarction, incubation of canine jugular vein endothelial
cells with postischemic cardiac lymph did not induce MCP-1 mRNA
expression suggesting the absence of cytokine-mediated MCP-1 induction
after a sublethal ischemic period. These results suggest that reactive
oxygen intermediate generation, after a brief ischemic
episode, is capable of inducing MCP-1 expression in venular
endothelium through AP-1 and NF-B. Short periods of
ischemia/reperfusion, insufficient to produce a myocardial
infarction, induce MCP-1 expression, potentially
mediating angiogenesis in the ischemic noninfarcted
heart.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, as
well as phorbol esters, which regulate its induction at the
transcriptional level through the cooperative interaction of the
transcription factors AP-1 and nuclear factor
(NF)-B.2-4
Specifically, in endothelial cells,
IL-1ß, TNF-, and phorbol esters mediate human MCP-1 gene
expression through the cooperative interaction of the NF-B binding
site at -90 bp (relative to the start site) and the AP-1 binding site
at -68 bp.2,3
Early reperfusion of the ischemic myocardium is a key clinical
intervention after myocardial infarction and is attended by a robust
inflammatory reaction involving both neutrophils and mononuclear
cells.5,6
It has been shown that late reperfusion, when
myocardial salvage is no longer possible, improves myocardial repair
and obviates infarct expansion.7
One of the early events
after reperfusion of a myocardial infarct is the dramatic induction of
MCP-1 in the cardiac venules.8
We have demonstrated that
this molecule is a chemotactic signal for mononuclear cell migration in
the reperfused myocardial infarction8,9
and others have
demonstrated its potential role in angiogenesis.10
In our
previous studies with reperfused myocardial infarcts we demonstrated an
important role for TNF- as an upstream cytokine that was released
from a preformed store in mast cells during ischemia11
and
showed that TNF- could also induce endothelial cell MCP-1
expression.8
Our studies also showed that cardiac lymph
collected after reperfusion induced MCP-1 mRNA. This led us to
hypothesize that TNF- was responsible, at least in part, for MCP-1
induction after reperfusion of the infarcted myocardium; however, the
assignment of this role to TNF- did not explain the highly specific
cellular localization of MCP-1 induction to the venular endothelium
during early reperfusion. Our studies demonstrated that released
TNF-11
bound to other cell types throughout the same
time period in which localization of MCP-1 mRNA was confined to the
venular endothelium.8
These observations led to the
current study that examines the hypothesis that the highly localized
induction of MCP-1 is induced by reactive oxygen generated from
ischemic myocardium.12-14
Because both leukocyte
transmigration and angiogenic budding occur at the postcapillary
venular level, understanding this highly localized induction is an
important issue.
At subcytolytic concentrations, reactive oxygen intermediates (ROIs)
can induce chemokine and cytokine gene expression. In A549 and HS-24
epithelial cell lines, ROIs were shown to induce chemokines such as
interleukin-8 (IL-8) and MCP-1.15-17
The observation that
ROIs may act as transcriptional activators is complicated by the fact
that transcriptional regulation of ROI-mediated chemokine genes varies
greatly with cell type.17-19
For example, the ROI
H2O2 differentially
regulates chemokine expression in a cell-type, gene-type-specific
manner in A549 epithelial cells through the transcription factor AP-1,
however, it does not induce IL-8 in endothelial
cells.17,18
Similarly, generation of ROIs with
xanthine/xanthine oxidase induced IL-8 and MCP-1 production from
monocytes, but not U937 cells, and this induction is associated with
increased NF-B DNA binding.20
Thus the potential for a
role of ROIs in induction of MCP-1 in venular endothelium was
considered.
In this study, we have elected to use the model of brief ischemia
followed by reperfusion that results in the release of ROIs from the
myocardium but is not associated with infarction or after reperfusion
inflammation (see Results).12-14
The absence of
postinfarction inflammation obviates the complications associated with
inflammatory cell adhesion to the postcapillary venular network that
might induce MCP-1 in these venules via a paracrine- or
adhesion-dependent signal mechanism. The results demonstrate that MCP-1
expression after 15 minutes of ischemia and 5 hours of reperfusion
occurs specifically in the small venular endothelium and is inhibited
by pretreatment with the anti-oxidant,
N-(2-mercaptopropionyl)-glycine (MPG). The
oxidative-dependent induction of MCP-1 was attended by histological
demonstration of nuclear translocation of NF-B and c-Jun exclusively
in the nuclear proteins of the venules of the reperfused myocardium;
gel shift studies of nuclear proteins isolated from the reperfused area
correlated with these observations by demonstrating activation of
NF-B and AP-1. In vitro,
H2O2 induced MCP-1 mRNA and
NF-B and AP-1 translocation in venular endothelium, however,
postischemic cardiac lymph collected during reperfusion after 15
minutes of ischemia did not. The potential significance of MCP-1
induction by ROIs in a noninfarcted ischemia-reperfusion (I/R) model
will be discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mongrel dogs (15 to 25 kg) were surgically instrumented with a hydraulic occluder around the circumflex coronary artery and cardiac lymph duct cannulation as previously described.11,12,21 After instrumentation, animals were allowed to recover for 72 hours before occlusion. Coronary artery occlusion was initiated by inflating the hydraulic occluder until mean blood flow in the coronary artery was zero as determined by Doppler flow probe. After 15 minutes of occlusion, the coronary occluder was deflated and the heart was reperfused for 5 hours. To investigate the potential role of ROIs as mediators of gene induction, one treatment group of instrumented dogs (n = 4) was infused with the cell soluble antioxidant, MPG13 (100 mg/kg), administered beginning 15 minutes before onset of occlusion (15 minutes) and reperfusion (5 hours). After reperfusion, the heart was stopped with a rapid infusion of KCl, removed, and sectioned. Myocardial sections were fixed in neutral-buffered formalin, 10% paraformaldehyde, B*5 or frozen in tissue-freezing medium (OCT) for histological studies. Adjacent tissue sections were flash-frozen in liquid N2, then homogenized and processed for either RNA or nuclear protein extraction. Additional sections were used for determination of blood flow with radiolabeled microspheres as previously described.11,21 Blood flow in regions deemed ischemic had a reduction in blood flow >80% relative to control sections. Blood flow data were collected from 15 animals (n = 7 for 15 minutes occlusion/0 hours reperfusion, and n = 8 for 15 minutes occlusion/5 hours reperfusion, data not shown).
Cell Culture and Treatments
Isolation of canine jugular vein endothelial cells (CJVECs) was
performed as previously described.8
Before agonist
administration cell culture groups were washed twice with 1x
phosphate-buffered saline (PBS) and incubated for 24 hours in 0.5%
growth media (complete media diluted 1/40 with Dulbecco’s modified
Eagle’s medium).17,18,22
CJVECs were treated with
H2O2 or TNF- using a
protocol adapted from Lakshminarayanan and
colleagues.17,18
CJVECs were washed twice with 1x PBS and
then incubated in serum-free, phenol red-free Dulbecco’s modified
Eagle’s medium (Life Technologies, Inc., Gaithersburg, MD) with
H2O2 or TNF- diluted to
their appropriate final concentrations.
Immunostaining and Histology
Immunostaining of canine myocardial tissue sections was performed
as described by Frangogiannis and colleagues11
using the
Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Tissue
sections were incubated with the primary antibodies c-Fos/p62 (K-25),
c-Jun/p39 (H-79), NF-B/p65 (H-286) (Santa Cruz Biotechnology, Santa
Cruz, CA), SG8H6 anti-neutrophil,23
anti-CD3124
and Mac387 anti-myeloid cell antibody (DAKO,
Carpinteria, CA) for 2 hours at room temperature, rinsed three times in
1x PBS and transferred into the corresponding secondary antibody.
Tissue sections were then rinsed three times in 1x PBS, and incubated
with avidin-biotin-peroxidase solution for 30 minutes. The
peroxidase-based reaction was developed with diaminobenzidine.
In Situ Hybridization
All solutions used for in situ hybridization were treated with 0.1% diethyl pyrocarbonate (Sigma Chemical Co., St. Louis, MO) and sterilized, or prepared with diethyl pyrocarbonate-treated water. In situ hybridization was performed as described by Kumar and colleagues.8 After deparaffinization and rehydration, tissue sections were rinsed in 2x standard saline citrate (SSC) for 5 minutes, incubated in prehybridization buffer (50% deionized formamide, 4x SSC, 1x Denhardt’s reagent, 0.5 mg/ml salmon sperm DNA, 0.25 mg/ml yeast tRNA, 10% dextran sulfate) at room temperature for 1 hour, rinsed in 2x SSC, then incubated with hybridization buffer (prehybridization buffer containing 150 ng of digoxigenin-labeled MCP-1 riboprobe) at 42°C for 24 hours in a humidified incubator. After hybridization, tissue slides were washed twice in 1x SSC at room temperature for 30 minutes each. To reduce nonspecific hybridization, wash stringency was increased by reducing the concentration of SSC to 0.2x and increasing the wash temperature to 55°C. For immunological detection an anti-digoxigenin antibody and nitro blue tetrazolium staining of the alkaline phosphatase reaction were used.
RNA Isolation
All solutions used for RNA isolation were treated with 0.1%
diethyl pyrocarbonate and sterilized, or prepared with diethyl
pyrocarbonate-treated water. T75 cultures of CJVECs or sections of
whole canine myocardium (5 mg) were solubilized in 2 ml or 5 ml of
Trizol (Life Technologies, Inc.), respectively. Total RNA was isolated
according to the manufacturer’s protocol then lysate was extracted
with phenol-chloroform. Samples were mixed vigorously, incubated at
room temperature for 2 to 3 minutes, and then spun at 
12,000 x
g for 15 minutes. The aqueous phase was collected and total
RNA was precipitated by mixing with equal volumes of isopropyl alcohol
and storing at -70°C for 1 hour.
Ribonuclease Protection Assays (RPAs)
The RPA was adapted from previously published protocols.17,25 The canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plasmid was a generous gift from Dr. T. Rosol (Ohio State University, Columbus, OH). Canine GAPDH and MCP-1 plasmids were linearized with the restriction enzymes HindIII and SAC I (Promega, Madison, WI), respectively. Radiolabeled cRNA templates were generated by in vitro transcription of canine GAPDH25 and MCP-18 cDNAs in the presence of [32P]-uridine triphosphate (UTP) (Amersham, Pharmacia Biotechnology, Piscataway, NJ) and T7 RNA polymerase (Pharmingen, La Jolla, CA). The radiolabeled templates were combined, precipitated with 3 mol/L ammonium acetate/100% ethanol, and 4 x 105 cpm were hybridized to total RNA for 16 hours at 56°C. For negative control and experimental samples, 30 µg of total RNA was used. Because of the robust nature of the lipopolysaccharide controls, only 20 µg of total RNA was used. mRNA probe complexes were treated with RNase A+T1, phenol-chloroform extracted, resolved on a 6% denaturing polyacrylamide-sequencing gel and exposed to radiographic film. Determination of protected complex size was made by linear regression comparison to unprotected probe, and comparison to [32P]-adenosine triphosphate (ATP) end-labeled 1.77-kb RNA ladder (Life Technologies, Inc.). The resulting protected cRNA products for MCP-1 and GAPDH complexes were 282 bp and 191 bp, respectively. Phosphate incorporation analysis was performed using the Storm 860 phosphorimager (Molecular Dynamics, Sunnyvale, CA) and quantitated with ImageQuant software.
DNA-Binding Studies from Nuclear Extracts
Electrophoretic mobility shift assays were performed essentially
as described.18,22
Briefly, nuclear protein extracts (10
µg) were prepared from homogenized canine myocardial sections (5 mg)
or CJVECs by a variation of the methods of Muller and
colleagues26
and Roebuck and colleagues22
and
were incubated with 5 x 104
cpm (0.1 ng)
of [32P]-end-labeled oligonucleotide probes for
consensus AP-1 or consensus NF-B sequences (Promega) for 20 to 30
minutes at room temperature in binding buffer containing 12% glycerol,
12 mmol/L HEPES-NaOH (pH 7.9), 60 mmol/L KCl, 5 mmol/L
MgCl2, 4 mmol/L Tris-Cl (pH 7.9), 0.6 mmol/L
ethylenediaminetetraacetic acid (pH 7.9), 0.6 mmol/L dithiothreitol,
and 0.25 µg of poly(dI-dC). To demonstrate binding specificity, a
100-fold molar excess of either specific or nonspecific
oligonucleotides was included in the binding reaction. Protein-DNA
complexes were resolved in 5% polyacrylamide gels in 0.25x TBE
buffer. Gels were dried and exposed to radiographic film with an
intensifying screen at -70°C. Gel shifts were performed at least
three times using nuclear extracts prepared from different animals or
cell culture experiments. Similar results were obtained and a
representative gel is shown in each of the figures.
Statistics
Graphs are presented as the mean (±SEM) of the MCP-1/GAPDH mRNA relative densitometry ratio with significance defined as (P < 0.05). Statistical analysis was performed using a one-way, two-tailed analysis of variance using InStat (GraphPad Software).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Comparison of acute, ischemia alone (15 minutes/0 hours), brief
I/R (15 minutes/5 hours), and lipopolysaccharide myocardium revealed
that MCP-1 mRNA was induced in the ischemic region of myocardium
subjected to I/R but not in the corresponding acute control (Figure 1A)
. Relative densitometry of MCP-1 mRNA
increased significantly in the ischemic regions compared to the acute
control (Figure 1B)
. No significant increase was observed in the
nonischemic control or ischemic regions of myocardium subjected to
ischemia alone, or in the control region of myocardium subjected to
I/R, suggesting MCP-1 mRNA induction is dependent on reperfusion of an
ischemic bed. When MPG was administered before the I/R protocol, MCP-1
mRNA induction was eliminated suggesting that induction relied on the
presence of ROIs (Figure 1, A and B)
.
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In situ hybridization was performed on control and
ischemic tissue sections from hearts subjected to brief I/R. Kumar and
colleagues8
previously demonstrated MCP-1 expression
specifically in small venular endothelium after I/R (1 hour/3 hours).
Similarly, we show brief I/R also induced MCP-1 mRNA expression that
was localized to the venular endothelium (Figure 2B)
, but not in hearts subjected to
ischemia alone (Figure 2A)
. Serial sections were immunostained with the
neutrophil-specific antibody SG8H6,23
the myeloid cell
marker Mac 387 and the anti-CD-31 endothelial-specific
antibody,24
and showed in situ MCP-1 staining
was specific for microvascular endothelium in the heart in the absence
of neutrophil or monocyte infiltration, indicating that MCP-1 induction
was independent of an inflammatory response.
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B, but Not c-Fos, that Are
Localized in the Nuclei of Small Venular Endothelium
To further characterize the expression of AP-1 and NF-B after
brief I/R, control and ischemic tissue sections from hearts subjected
to ischemia alone or brief I/R were immunohistochemically stained with
antibodies specific for the DNA-binding factors NF-B, c-Jun, and
c-Fos. Ischemic tissue sections from brief I/R myocardium, but not
nonischemic control myocardium, showed significant positive nuclear
staining for the DNA binding factor NF-B and the AP-1 component
c-Jun, but not c-Fos (Figure 3; A to C
).
Staining serial sections of the control and ischemic tissue with the
endothelial-specific antibody CD-31 confirmed that NF-B and c-Jun
protein expression were specifically found in venular endothelium
(Figure 3D)
. Tissue sections from animals that received MPG before the
I/R protocol did not show nuclear translocation of c-Jun and NF-B in
the venular endothelium. No AP-1 or NF-B nuclear translocation
occurred in ischemic, nonreperfused samples.
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B-Binding Activity in Whole
Myocardium
Two of the transcription factors intricately involved in the
regulation of MCP-1 are AP-1 and NF-B.3,4
Because AP-1
and NF-B are redox-sensitive transcription
factors,27,28
we investigated their role in activation of
MCP-1 gene expression associated with brief I/R using gel shift
analysis of nuclear extracts prepared from ischemic and control canine
myocardium subjected to ischemia alone or brief I/R. In hearts
subjected to brief I/R, enhanced DNA binding to consensus AP-1 and
NF-B oligonucleotides was observed in nuclear extracts of ischemic
tissue sections (Figure 4, A and B
; lane
5) relative to the nonischemic control sections (Figure 4, A and B
;
lane 4). Minimal DNA binding was apparent in nuclear extracts from the
control and ischemic tissue sections from hearts subjected to ischemia
alone, without reperfusion (Figure 4, A and B
; lanes 2 and 3).
Competition with 100-fold molar excess of nonlabeled oligonucleotides
to AP-1 and NF-B abrogated DNA binding with respect to each probe,
whereas the nonspecific oligonucleotide OCT-1 did not inhibit binding,
showing specificity of the DNA binding reaction. May-Newman and
colleagues29
demonstrated that canine myocardium contains
a high vascular endothelial density, comprising a potential source for
the enhanced AP-1 and NF-B DNA binding induced by ROIs in myocardial
nuclear extracts.
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MCP-1 is an oxy-sensitive chemokine induced by
H2O2 in endothelial
cells.30
To further examine the potential mechanism of
oxidant induction of MCP-1 in venular endothelium, we used CJVECs as an
in vitro endothelial cell culture model. ROI scavenger
experiments by Bolli and colleagues12
used a
glucose/glucose oxidase reaction to generate
H2O2 and measured its rate
of production to be 11 µmol/L/min. Experiments by Wung and
colleagues31
induced MCP-1 expression in bovine artery
endothelial cells with exogenous
H2O2 at a concentration of
100 µmol/L. Therefore, we stimulated CJVECs with equivalent
concentrations of H2O2.
CJVECs were isolated as previously described8
and
stimulated with H2O2 (10 to
200 µmol/L) or TNF- (100 U/ml) for 3 hours, and MCP-1 induction
was measured by RPA. Figure 5A
shows
MCP-1 message increased in a dose-dependent manner with maximal
expression at 100 to 200 µmol/L
H2O2. Phosphorimager
analysis (Figure 5B)
indicates 100 µmol/L
H2O2 induced MCP-1 mRNA
2.8-fold relative to unstimulated CJVECs. To address the question of
a stable soluble agonist (eg, cytokines) secreted into the cardiac
lymph stimulating MCP-1 expression, canine cardiac lymph was isolated
at 1-hour intervals of reperfusion after brief ischemia and used to
stimulate CJVECs. Previous studies from Kumar and
colleagues8
showed postischemic canine cardiac lymph (1
hour/3 hours) was capable of stimulating MCP-1
message in CJVECs. In contrast, postischemic canine cardiac lymph after
brief I/R was unable to induce MCP-1 mRNA in CJVECs (data not shown).
Thus, in the absence of infarction, there is no apparent
cytokine-stimulated MCP-1 induction; this contrasts with the study by
Kumar and colleagues8
(see Introduction).
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B-Binding
Activity in CJVECs
To further understand the role of ROI-mediated AP-1 and
NF-B DNA binding, nuclear extracts were prepared from CJVECs
stimulated with H2O2 (100
µmol/L) or TNF- (100 U/ml), then incubated with consensus
oligonucleotides against AP-1 and NF-B. As shown in Figure 6, A and B
, nuclear extracts from
H2O2-stimulated CJVECs increased AP-1
and NF-B binding compared to unstimulated CJVECs. The increase in
DNA binding activity in
H2O2-stimulated CJVECs
correlated with the enhanced binding seen in the nuclear extracts
prepared from ischemic myocardial tissue sections from brief I/R
experiments (Figure 3, A and B)
and the increased c-Jun and NF-B
nuclear translocation (Figure 4B)
seen in histological sections from
brief I/R hearts.
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Because oxidant-induced MCP-1 expression in CJVECs is potentially
mediated by AP-1 and NF-B, we investigated the induction of MCP-1
mRNA in the presence of the antioxidants NAC, a precursor of
glutathione (GSH), and PDTC, an anti-oxidant, which has been shown to
inhibit NF-B binding.32-34
CJVECs were pretreated with
NAC (5 mmol/L) or PDTC (1 mmol/L) for 1 hour before stimulation with
H2O2 (100 µmol/L) for 3
hours (Figure 7A)
. These concentrations
of NAC (5 mmol/L) and PDTC (1 mmol/L) were previously shown to inhibit
the oxidant-induced expression of ICAM-1 in human umbilical vein
endothelial cells.22
Total RNA was isolated and analyzed
for MCP-1 mRNA by RPA from
H2O2-stimulated CJVECs.
Densitometry indicated H2O2
induced MCP-1 mRNA approximately twofold relative to unstimulated
CJVECs (Figure 7B
, lane 4). Pretreatment with NAC abrogated the
H2O2 induction of MCP-1
(lane 5) but had no effect on MCP-1 mRNA in untreated controls (lane
2). This effect is similar to the ICAM-1 studies.22
In
contrast, PDTC alone (lane 3) increased MCP-1 message 2.6-fold in the
absence of H2O2 treatment,
and pretreatment with PDTC further enhanced the
H2O2 induction of MCP-1
mRNA (lane 6). These studies are in contrast to those described for
ICAM-1.22
To examine these observations further, we
investigated the effects of NAC and PDTC on the binding activity of
AP-1 and NF-B.
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B Binding
Previous work from Munoz and colleagues35
showed PDTC
alone, or in concert with TNF-, enhanced AP-1 DNA-binding activity
while inhibiting TNF--mediated NF-B activity in human umbilical
vein endothelial cells. Because of the oxy-sensitive nature of the
transcription factors AP-1 and NF-B28
and their
potential role in mediating the induction of MCP-1 by oxidant
stress,31
we investigated the effects of anti-oxidants NAC
and PDTC on oxidant-induced AP-1 and NF-B DNA binding (Figure 8)
. Nuclear proteins isolated from CJVECs
pretreated with NAC (5 and 10 mmol/L) or PDTC (1 and 10 mmol/L) for 1
hour before stimulation with
H2O2 (100 µmol/L) were
incubated with consensus oligonucleotides against AP-1 (Figure 8A)
and
NF-B (Figure 8B)
. In Figure 8A
, NAC alone (lanes 2 and 3) failed to
induce AP-1 DNA binding relative to unstimulated CJVECs (lane 1). In
contrast, PDTC (lanes 4 and 5) and
H2O2 (lane 6) enhanced AP-1
DNA binding. Pretreatment with 10 mmol/L NAC significantly reduced
H2O2-mediated AP-1-binding
activity (lane 8) whereas 1 mmol/L PDTC pretreatment enhanced
H2O2-induced AP-1 binding
(lane 9). In Figure 8B
, H2O2 induced NF-B DNA
binding (lane 6) that was inhibited by NAC (5 and 10 mmol/L) (lanes 7
and 8) as well as PDTC (1 and 10 mmol/L) (lanes 9 and 10) pretreatment.
The inhibitory effect of PDTC on oxidant-induced NF-B binding was
more pronounced at higher concentrations, reducing NF-B binding at 1
mmol/L and abrogating the response at 10 mmol/L (Figure 8B
, lanes 9 and
10). Taken as a whole, these studies show that PDTC effectively
inhibited ROI-mediated NF-B DNA-binding activity while activating
AP-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in
the reperfused infarction during early reperfusion and the ability of
both postischemic cardiac lymph and TNF- to induce MCP-1 identified
TNF- as a major candidate for MCP-1 induction.8
However, cellular distribution of TNF- is far more extensive than
merely the cardiac venules and would not completely explain such an
exquisite localization9
of MCP-1 induction to cardiac
venules.8 Extensive evidence supports a role for reactive oxygen in chemokine induction chemokines in various cell types; however, this observation is confounded by significant variability of response to ROI with regard to cell type and chemokine species.17,19 Nevertheless, the possibility that reactive oxygen might be responsible for the induction of MCP-1 localized in the venules was considered because: 1) H2O2 is capable of inducing MCP-1 mRNA in endothelial cells,1,36 2) ROI formation is induced during early reperfusion of the myocardium,12,14,37 and 3) ROIs from the reperfused myocardium egress to the capillary vascular bed.
We also considered the fact that the postcapillary venular endothelium represented the site to which leukocytes adhere and through which leukocytes transmigrate. Because leukocyte adhesion and transmigration might induce genes in the endothelium either through paracrine cytokine secretion mechanisms or through direct stimulation associated with adhesion, we chose to examine MCP-1 induction in a model using brief, nonlethal ischemia followed by reperfusion that has been well characterized in the past as inducing ROI formation and secretion from myocytes associated with dysfunction but no cell death.5,12-14 We have also confirmed that there is no significant leukocyte infiltration associated with this protocol.
Spin-trapping techniques have allowed quantitation of ROI generation in
models of stunned myocardium, which are characterized by reperfusion of
ischemic noninfarcted hearts. Administration of
-phenyl-N-tert-butylintrone and analysis of coronary
vessel blood by electron paramagnetic resonance has been used to assess
the time course of ROI generation during brief ischemia (15 minutes) in
a canine model,14,38
demonstrating a release of ROIs at
the onset of reperfusion after ischemia, peaking within the first 2 to
3 minutes, but sustained for 3 hours of reperfusion.38
Thus, in this model of stunned myocardium and reversible cardiac
dysfunction, ROIs are potential candidates to induce expression of
oxy-sensitive chemokines such as MCP-1.
In this report, we demonstrate that MCP-1 mRNA is expressed exclusively
in the postcapillary venules of the ischemic myocardium during
reperfusion after brief sublethal periods of ischemia (15 minutes).
In situ hybridization (Figure 2)
showed
reperfusion-dependent-specific localization of MCP-1 mRNA expression in
the endothelium of small venules, similar to that described in the
infarction protocol.8
In contrast to the infarction model
however, the 15 minutes/5 hours protocol demonstrated no significant
inflammatory infiltrate, as determined by immunostaining with the
anti-neutrophil-specific monoclonal antibody SG8H623
or
Mac 387, which identifies infiltrating neutrophils and
monocytes.24
MCP-1 mRNA induction was accompanied by
nuclear translocation of NF-B and c-Jun in the same postcapillary
venules, and nuclei isolated from ischemic myocardium demonstrated both
NF-B and AP-1 activation by gel retardation assays. A brief ischemic
insult induced prolonged expression of NF-B and c-Jun, as previously
described in the isolated perfused rat heart.39
Finally,
MCP-1 mRNA induction and venular nuclear translocation of NF-B and
c-Jun were not seen in animals in whom MPG infusion preceded the I/R
protocol. Taken as a whole the data suggest that ROI production is
necessary for transcriptional activation of the MCP-1 gene in the
absence of inflammation or infarction. The methods used in
vivo do not allow dissection of other factors in the extracellular
fluid that might contribute to this induction. To examine whether ROI
was sufficient for MCP-1 induction and activation of NF-B and AP-1,
we examined an in vitro model of venular endothelial
cultures in crystalloid solution.
In the in vitro study, we used two antioxidants (NAC and
PDTC) with distinct mechanisms of inhibition to investigate the
up-regulation of MCP-1 transcription by
H2O2. NAC readily diffuses
into cells and serves as a precursor to GSH.34
Pretreatment of CJVECs with 5 mmol/L NAC abrogated induction of MCP-1
mRNA by H2O2 (Figure 7)
.
The anti-oxidant PDTC specifically inhibits NF-B activation by
preventing I-B separation from the active NF-B cytoplasmic
domain, thereby precluding nuclear translocation and regulation of gene
transcription.33
In contrast to the inhibitory effects by
NAC on H2O2-induced MCP-1
mRNA, PDTC was ineffective as an inhibitor of MCP-1 mRNA (Figure 7)
. In
fact, PDTC alone (Figure 7A
, lane 3) and in combination with
H2O2 (Figure 7A
, lane 6)
induced MCP-1 message relative to
H2O2 alone (Figure 7A
, lane
4). This apparent discrepancy was reconciled by the observation that
PDTC also strongly enhanced DNA binding to the consensus AP-1
oligonucleotide (Figure 8A
, lanes 4 and 5). This suggests that PDTC may
induce AP-1 gene expression in addition to inhibiting NF-B in
endothelial cells, which has been well described by
others.35
Meyer and colleagues40
showed PDTC
had strikingly different effects on NF-B and AP-1 DNA binding under
oxidative stress. Similarly, in our in vitro system PDTC
inhibited oxidant-induced NF-B activity and had the secondary effect
of activating AP-1 in CJVECs. Thus, it seems that AP-1 activation is
sufficient to support MCP-1 induction in cultured adult endothelial
cells, which is in contrast to observations on ICAM-1
stimulation.22
The in vivo demonstration in our
experiments of concomitant nuclear translocation of NF-B suggests
that, at least in cardiac venules, the oxidant response involves both
factors.
To our knowledge, this represents the first demonstration of reperfusion-dependent chemokine expression induced by ROI release in vivo after an acute ischemic episode in the absence of injury. It is interesting to speculate what the role of MCP-1 induction might be in a physiological circumstance in which it is induced in the absence of injury. Ito and co-workers10 demonstrated that injection of MCP-1 into ischemic skeletal muscle beds results in angiogenesis and collateral blood flow. Because angiogenesis begins with endothelial budding at the venular level, locally generated venular MCP-1 might be involved in an angiogenic response to reversible ischemia. The mechanism by which MCP-1 can induce angiogenesis remains unclear, but recent reports demonstrate the presence of a CCR2 receptor in endothelial cells.41 In addition, the recent experiments42 demonstrated that repeated brief occlusions of the coronary artery resulted in the formation of collateral circulation without inducing myocardial injury. It is interesting to speculate whether similar mechanisms might result from repeated bouts of myocardial ischemia distal to a critical coronary lesion and result in collateral blood flow formation. It is possible that ROIs generated by these reversible ischemic episodes induce MCP-1 in venules facilitating the angiogenic response.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grant HL-42550 and a grant from the Methodist Hospital Foundation (to N. G. F.).
Accepted for publication June 20, 2001.
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