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-independent manner: role of oxygen
radicals
1 Section of Cardiovascular Sciences and Cardiology, Department of Medicine, the DeBakey Heart Center, Baylor College of Medicine, and the Methodist Hospital; 2 Veterans Administration Medical Center, Winters Center for Heart Failure Research, Houston, Texas 77030; and 3 Immunex Corporation, Seattle, Washington 98101
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Early chemokine induction in
the area at risk of an ischemic-reperfused (I/R) myocardium is
first seen in the venular endothelium. Reperfusion is associated with
several induction mechanisms including increased extracellular
tumor necrosis factor (TNF)-
, reactive oxygen intermediate (ROI)
species formation, and adhesion of leukocytes to the venular
endothelium. To test the hypothesis that chemokine induction in cardiac
venules can occur by ROIs in a TNF--independent manner, and in the
absence of leukocyte accumulation, we utilized wild-type (WT) and
TNF- double-receptor knockout mice (DKO) in a closed-chest mouse
model of myocardial ischemia (15 min) and reperfusion (3 h), in
which there is no infarction. We demonstrate that a single brief period
of I/R induces significant upregulation of the chemokines macrophage
inflammatory protein (MIP) -1, -1
, and -2 at both the mRNA and
protein levels. This induction was independent of TNF-, whereas
levels of these chemokines were increased in both WT and DKO mice.
Chemokine induction was seen predominantly in the endothelium of
small veins and was accompanied by nuclear translocation of nuclear
factor-
B and c-Jun (AP-1) in venular endothelium. Intravenous
infusion of the oxygen radical scavenger
N-2-mercaptopropionyl glycine (MPG) initiated 15 min before
ischemia and maintained throughout reperfusion obviated chemokine induction, but MPG administration after reperfusion had begun
had no effect. The results suggest that ROI generation in the
reperfused myocardium rapidly induces C-C and C-X-C chemokines in the
venular endothelium in the absence of infarction or irreversible cellular injury.
macrophage inflammatory protein; endothelium; transgenic animals
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IT HAS BEEN RECOGNIZED FOR years that inflammation plays a critical role in the pathophysiology of many cardiovascular disease states. The activation of the inflammatory response may in some circumstances lead to inappropriate tissue destruction thus extending cellular injury, as in the case of reperfusion of ischemic myocardium (10). The precipitation of the inflammatory cascade during reperfusion causes the induction of many inflammatory mediators, such as early response cytokines and chemokines, oxygen radicals, and adhesion molecules (13, 14, 26, 44) that tightly coordinate the sequence of events, which ultimately lead to tissue injury and organ dysfunction.
Chemokines are chemoattractant cytokines that share homologous
sequences and a highly conserved cysteine motif in the amino terminus
of their primary amino acid structure (28). Interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1, important in
regulating early neutrophil and monocyte trafficking, are significantly induced in myocardial infarction in animals (4, 19, 22, 23) and humans (29). Induction of chemokines has
been affected in other systems by both tumor necrosis factor-
(TNF-) and reactive oxygen intermediate (ROI) species (3, 35,
43, 46), which are pertinent to ischemia-reperfusion
(I/R) (10, 14, 26). The independent role of each of these
chemokine-induction mechanisms in I/R is difficult to study because of
the following confounding features: 1) ROIs are invariably
induced by I/R, but quenching them also alters the signaling by TNF-
(7); 2) TNF- is stored in cardiac mast cells
and released during I/R (12); and 3) the role
of ROIs in chemokine induction is tissue, cell, and chemokine specific
(25, 37).
In this study, we investigate the hypothesis that C-X-C and C-C
chemokines are induced by a single brief period of ischemia (15 min) and reperfusion (3 h) and ROI generation is necessary and
sufficient to induce these chemokines independent of TNF-. This
brief period of ischemia was selected because it is known to
significantly increase oxygen radical production in the myocardium and
induce reversible cellular injury (6). In addition, the brief ischemic period avoids the confounding effects of
necrotic cell death and leukocyte infiltration on chemokine production, which occur with longer periods of ischemia.
The data in this report show for the first time that a single brief
period of myocardial I/R markedly upregulate macrophage inflammatory
protein (MIP)-2, -1, and -1-mRNA and protein expression in both
wild-type (WT) mice and mice with genetic deletion of both TNF-
receptors (p55 and p75). The increased chemokine expression and
increased nuclear factor (NF)-B, as well as c-Jun protein nuclear
translocation occurred only in the venular endothelium of the
previously ischemic myocardium. In addition, we demonstrate that the chemokine expression, and NF-B, and c-Jun nuclear
translocation were markedly blunted by infusion of the oxygen radical
scavenger in both sets of mice. Taken together, these data indicate
that after brief periods of myocardial I/R, oxygen radicals induce marked upregulation of chemokine expression in the venular endothelium of the reperfused myocardium in a TNF--independent manner.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Transgenic mice. The generation of the TNF double-receptor knockout (DKO) mice has been previously described (24). Briefly, the p55 [TNF-receptor 1 (TNFR1)]-deficient and p75 [TNF-receptor 2 (TNFR2)]-deficient mice were generated through targeted gene disruption of the p55 or p75 alleles by using homologous recombination, as previously reported (36). Appropriate crosses of TNFR1- and TNFR2-deficient mice were performed to generate the double TNFR1-TNFR2-deficient mice, and these deficiencies were maintained on a random C57BL/6 × 129 SVEV hybrid (F1) background (36). Parental C57BL/6 and 129 SVEV mice were obtained from Harlan Sprague Dawley and from Taconic Farms, respectively.
Preparation and surgery of animals. Male and female wild-type (WT) C57BL/6 × SVEV, and TNF DKO (8- to 12-wk of age, 18.0-22.0 g body wt) were anesthetized by an intraperitoneal injection of pentobarbital sodium (10 µl/g). A closed-chest mouse model of myocardial I/R was utilized in this investigation as previously described (34), to avoid as much as possible the confounding effects of surgical trauma and inflammation, which may influence the baseline levels of chemokines and cytokines. Briefly, after thoracotomy, the pericardium was dissected, and an 8-0 Surgipro monofilament polypropylene suture with the U-shaped tapered needle was passed under the left anterior descending coronary artery (LAD). The needle was then cut from the suture, and the two ends of the 8-0 suture were then threaded through a 0.5-mm piece of polyethylene (PE)-10 tubing, forming a loose snare around the LAD. The PE-10 tubing was previously soaked for 24 h in 100% ethanol. Each end of the suture was then threaded through the end of a size 3 Kalt suture needle (Fine Science Tools), and exteriorized through each side of the chest wall. The chest was closed with four interrupted stitches utilizing 6-0 suture, carefully avoiding pneumothorax. The ends of the exteriorized 8-0 suture were then tucked under the skin, which was then also closed with 6-0 suture. The animal was removed from the respirator and the endotracheal tube was withdrawn. The animal was kept warm with a heat lamp and it was allowed to breathe 100% oxygen via nasal cone until full recovery of consciousness.
Ten days postinstrumentation, the animals were reanesthetized with pentobarbital sodium, and the extremities were taped to a lead II elecrocardiogram (ECG) board to measure S-T elevations during the I/R protocol. The skin above the chest wall was then reopened. For animals randomized to the myocardial ischemia (15 min) and reperfusion (3 h) groups, the left jugular vein was cannulated to allow infusion of either normal saline (WT mice, n = 9; DKO mice, n = 7) or the oxygen radical scavenger N-2-mercaptopropionyl glycine (MPG) (WT mice, n = 9; DKO mice, n = 7). The intravenous infusion was started 15 min before the initiation of ischemia, and continued throughout both I/R periods. In another group of mice, the intravenous infusion of MPG was started 5 min after reperfusion, and continued for the rest of the experimental protocol. The 8-0 suture, which had been previously exteriorized outside the chest wall and placed under the skin, was cleared of all debris from the skin and chest, and was carefully taped to heavy metal picks. Ligation of the LAD was accomplished by gently pulling the heavy metal picks apart until an S-T elevation appeared on the ECG. The ECG was constantly monitored throughout the entire ischemic interval to ensure persistent ischemia. At the end of ischemia, 3 h of reperfusion was accomplished by pushing the metal picks towards the animal and by cutting the suture close to the chest wall. Reperfusion was confirmed by resolution of the S-T elevation, which usually occurred very quickly. During this procedure, the animal was not reintubated and breathed room air under normal ventilation. At the end of the experiment (i.e., 3 h of reperfusion), the chest was opened and the heart was immediately excised, snap-frozen, and stored at
80°C until mRNA isolation. Sham animals (10-day sham group) were
prepared identically without undergoing the I/R protocol
(n = 9, WT mice; n = 6, DKO mice).
Additional mice (n = 9 for both groups) were utilized
as a control naïve heart group, to assess constitutive levels
of MIP-1, MIP-1, and MIP-2. These mice were anesthetized with
pentobarbital sodium, as was done for all other mice. With no prior
instrumentation, these hearts were immediately excised, snap-frozen,
and stored at 80°C until mRNA isolation, as described below.
Measurement of S-T elevation. The S-T elevation was measured in microvolts from baseline to the top of the T wave for each animal. Because there is no consistently distinguishable S-T segment in mice due to their extremely high heart rates, the S-T elevation was measured to the top of the T wave as a consistent approximation of the S-T segment. This was done at the end of 15 min of ischemia for the animals undergoing the I/R protocol. The S-T segment elevation was not significantly different among any groups of mice undergoing the I/R protocol.
MPG. MPG (Sigma; St. Louis, MO) is a low molecular weight synthetic analogue of glutathione that is freely diffusible across cell membranes (41), and has been found to scavenge oxygen-derived free radicals such as hydroxyl (49) and hydrogen peroxide (8, 21). The MPG solution was prepared fresh every morning before use as previousy described (17). MPG, which is highly soluble in aqueous solutions, was dissolved in normal saline. Because an aqueous solution of MPG is highly acidic (pH ~2.0), the pH was adjusted to 7.4 by the addition of sodium hydroxide (0.1 N). Because MPG has a half-life in plasma of ~15 min (41), it had to be continuously infused intravenously throughout the entire experimental protocol, starting either 15 min before ischemia, or 5 min after reperfusion.
mRNA isolation. All solutions for RNA analysis were treated with 0.1% diethylpyrocarbonate and sterilized or prepared in diethylpyrocarbonate-treated water. Glassware was baked at 240°C for 5 h to remove trace RNases. Total RNA was isolated from whole heart according to the acid guanidium thiocyanate-phenol-chloroform extraction method developed by Chomczynski and Sacchi (9). Briefly, whole hearts were homogenized in RNA STAT-60 solution (Tel-Test; Friendswood, TX). For RNA extraction, 0.2 volumes of R-chloroform were then added per volume homogenate. This mixture was incubated on ice for 15 min, and then spun at 12,000 g for 15 min at 4°C. The supernatant was transferred to another tube, and an equal volume of isopropanol was added for RNA precipitation overnight at 4°C. The tubes were then spun at 12,000 g for 15 min at 4°C, and the supernate was then decanted. The pellet was washed twice with 75% ethanol, briefly dried, and dissolved in 0.1% diethylpyrocarbonate-treated water. Quantification and purity of RNA was assessed by A260/A280 ultraviolet absorption, and RNA samples with ratios >1.9 were utilized for further analysis.
Ribonuclease protection assay and quantitation.
The expression levels of MIP-1, MIP-1, MIP-2 mRNA
interferon-
inducible protein (IP)-10 and monocyte
chemoattractant protein-1 (MCP-1) were determined using a ribonuclease
protection assay (RPA). A commercially available kit (Riboquant,
Pharmingen; San Diego, CA) and antisense RNA probe were utilized (mck-5
probe, Pharmingen) according to the manufacturer's protocol as
previously described (34). Phosphorimaging of the RPA gels
was performed with a Storm 860 phosphorimager (Molecular Dynamics).
Signals were quantified using ImageQuaNT software and normalized to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Immunohistochemistry.
For immunohistochemistry, mouse hearts from the control
naïve group, 10-day sham group, and the I/R groups (with and
without MPG infusion) were isolated from both WT and DKO mice. After
the experimental protocol, hearts were excised and immediately fixed in
zinc formalin buffer (Anatech; Battle Creek, MI), dehydrated by
incubation in increasing concentrations of ethanol with a standard protocol, and then embedded in paraffin; 4-µm sections were obtained with microtomy. For immunostaining, the slides were rehydrated and
incubated with 3% hydrogen peroxide. After being rinsed in phosphate-buffered saline, sections were blocked from nonspecific antibody staining with 10% goat serum in phosphate-buffered saline. Immunostaining with antibodies to MIP-1, MIP-1, MIP-2, the p65 subunit of NF-B, c-Fos, and c-Jun (all from Santa Cruz
Biotechnology; Santa Cruz, CA), was performed with the Elite kit
(Vector; Burlingame, CA), which has a peroxidase-based detection
system. The color reaction was developed with a diaminobenzidine kit
(Vector) as a substrate. For positive control of MIP-1, MIP-1,
and MIP-2 staining, mouse spleen was utilized from an LPS-stimulated
mouse according to the method of Rovai et al.
(38). Endothelial cell labeling was performed
using lectin histochemistry with Griffonia simplicifolia-I
(Vector) as previously described (1). Sections from the
spleen of a normal mouse were utilized as negative controls for
chemokine staining.
Statistical analysis.
All values are means ± SE. Statistical significance among control
naïve heart, 10-day sham, and the I/R groups (with and without
MPG infusion) for MIP-1, MIP-1, and MIP-2 mRNA were analyzed by
two-tailed analysis of variance with Bonferroni correction. Results
were considered significant at P < 0.05.
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RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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I/R induced chemokine mRNA expression in WT mice.
To determine chemokine expression in myocardial tissue, mRNA from whole
hearts of WT mice was isolated in the control naïve group
(i.e., no instrumentation), 10-day sham group, and the 15-min ischemia and 3-h reperfusion groups (i.e., with and without MPG infusion). Figure 1A
illustrates a representative autoradiograph of mRNA from each group of
mice obtained by RPA of MIP-1, MIP-1, and MIP-2. The
densitometric analysis of each mRNA normalized to GAPDH is shown in
Fig. 1, B-D.
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mRNA
in the myocardium compared with both control (i.e., noninstrumented)
and sham-operated groups (P < 0.001). In addition, infusion
of the oxygen radical scavenger MPG starting 15 min before
ischemia and continuing throughout reperfusion caused a
significant decrease in MIP-1 mRNA levels vs. I/R (P < 0.001).
Figure 1C shows that 15 min of ischemia and 3 h
of reperfusion caused a pronounced upregulation of MIP-1 mRNA
compared with the control (i.e., noninstrumented) group (P
< 0.001), but not compared with the sham-operated group (not
significant). As with MIP-1, the MPG infusion caused a
decrease of MIP-1 mRNA that came back down to control (i.e.,
noninstrumented levels; P < 0.05 vs. I/R).
Figure 1D demonstrates that 15 min of ischemia and
3 h of reperfusion significantly increased MIP-2 mRNA levels
compared with both control (P < 0.001) and
sham-operated (P < 0.01) groups. Similar to MIP-1,
infusion of the oxygen radical scavenger MPG caused a decrease of MIP-2
mRNA, which came back down to control levels (NS vs. control, and
P < 0.05 vs. I/R).
The experiments with MPG described in Fig. 1 involved pretreatment of
the animals with the ROI scavenger. We evaluated the efficacy of MPG in
subsequent experiments by starting the MPG infusion 5 min after
reperfusion. In contrast with pretreatment, MPG infusion initiated
after reperfusion caused levels of MIP-1, MIP-1, and MIP-2 that
were similar to those observed in the untreated I/R groups (data not
shown, NS vs. I/R). Taken together, these data demonstrate that brief
periods of ischemia cause a significant induction of chemokine
mRNA, which require only short exposure to ROIs. Finally, permanent
occlusion of 15 min or 3 h of ischemia did not induce
measureable MIP-1, MIP-1 or MIP-2.
I/R does not induce IP-10 or MCP-1.
In contrast with MIP-1, MIP-1, and MIP-2 induction, there was no
detectable induction of IP-10 or MCP-1 after 15 min of ischemia
and 3 h of reperfusion in any of the experimental groups (data not shown).
Localization of chemokine protein in I/R.
We then determined the cellular source of the chemokine upregulation
observed in the various groups of WT mice. Figure
2, A-C,
demonstrates immunolocalization of MIP-1, MIP-1, and MIP-2 protein to myocardial microvascular endothelial cells, identified by
Griffonia simplicifolia-I lectin histochemistry (Fig.
2D), in animals subjected to 15 min of ischemia and
3 h of reperfusion. Staining for these chemokines was much reduced
in sham-operated mice and in MPG-treated animals (Fig. 2,
E-G). Control murine hearts showed minimal
chemokine immunoreactivity.
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I/R induced chemokine mRNA expression in TNF- DKO
mice.
To determine chemokine expression in myocardial tissue, mRNA from whole
hearts of TNF- DKO mice was isolated in the control group (i.e., no
instrumentation), 10-day sham group, and the 15 min of ischemia
and 3 h of reperfusion groups (i.e., with and without MPG
infusion). Figure 3A
illustrates a representative autoradiograph of mRNA from each group of
mice obtained by RPA of MIP-1, MIP-1, and MIP-2. The
densitometric analysis of each mRNA normalized to GAPDH is shown in
Fig. 3, B-D.
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mRNA compared with control
(P < 0.05) and sham-operated (P < 0.05) groups. MPG infusion initiated 15 min before ischemia,
and, continuing throughout reperfusion, markedly reduced MIP-1 mRNA
levels (P < 0.05 vs. I/R) to control values (NS).
Figure 3C shows marked upregulation of MIP-1 mRNA in the
15 min of ischemia and 3 h of reperfusion group compared
with control (i.e., noninstrumented, P < 0.001), but
not compared with the sham-operated group (NS). Similarly to MIP-1,
MPG infusion initiated 15 min before ischemia, and continuing
throughout reperfusion blunted MIP-1 mRNA levels (P < 0.01 vs. I/R) to control values (NS).
Figure 3D indicates that 15 min of ischemia and
3 h of reperfusion caused a significant increase of MIP-2 mRNA
compared with both control (P < 0.01) and
sham-operated groups (P < 0.05). As was the case for
both MIP-1 and MIP-1 infusion of MPG caused a marked
downregulation of MIP-2 mRNA, which was not different than that of
control (NS).
As with WT mice, when the MPG infusion was started 5 min after
reperfusion, thus not quenching ROI production on reperfusion, the
levels of MIP-1, MIP-1, and MIP-2 were comparable to those observed in the I/R groups (data not shown, NS). Taken together, these
data demonstrate that brief periods of ischemia, which induce neither irreversible cellular injury nor leukocyte infiltration, cause
a marked increase in chemokine levels that are mediated by a short
burst of oxygen radical production and are TNF- independent.
Localization of chemokine protein in DKO mice with
I/R.
We then determined the cellular source of the chemokine upregulation
observed in the groups of DKO mice. MIP-1, MIP-1, and MIP-2
protein were localized in venular endothelial cells in the mouse
myocardium of the groups subjected to 15 min of ischemia and
3 h of reperfusion (data not shown), identical to the staining found in WT mice (Fig. 2, A-C). Similar to WT
mice, staining for these chemokines was much reduced in sham-operated
and MPG-treated mice. Control hearts showed minimal chemokine staining.
Mechanism of oxygen radical-induced chemokine expression.
It is well known that nuclear translocation (activation) of the
transcription factors NF-B and AP-1 (i.e., c-Fos and c-Jun) are
primary mechanisms for oxygen radical mediated upregulation of
inflammatory mediators, and that many chemokines contain NF-B and
AP-1 sites in their promoters. Therefore, we sought to
determine the mechanism of TNF--independent chemokine production
induced by oxygen radicals by staining sections of mouse myocardium
with antibodies directed against NF-B, c-Fos, and c-Jun. In WT mice, 15 min of ischemia and 3 h of reperfusion caused a marked
nuclear translocation of NF-B and c-Jun (Fig.
4, A and B),
proteins in the same venular endothelial cells that demonstrated
chemokine expression (Fig. 2). A similar pattern of staining was
observed in DKO mice that underwent the I/R protocol (data not shown). NF-B and c-Jun staining was virtually absent in WT mice receiving MPG infusion initiated 15 min before ischemia (Fig. 4,
D and E). MPG infusion in DKO mice that were
subjected to the I/R protocol also showed virtual absence of NF-B,
and c-Jun staining (data not shown). C-Fos was not stained in any of
the groups. These data suggest that oxygen radicals modulate chemokine
induction via NF-B and AP-1 sensitive mechanisms.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In our previous work (22, 23) using a
reperfused infarcted model in the dog, we demonstrated early induction
of MCP-1 and IL-8 on reperfusion and that this induction occurred
almost exclusively in the small venules of the myocardium. The
observation that these chemokines could be induced in venular
endothelium by TNF- led us to hypothesize that TNF- was
responsible for this induction (22, 23). Subsequently, we
noted that TNF- activity in early reperfusion arose from preformed
TNF- in cardiac mast cells (12). This observation led
us to question the role of TNF- in chemokine induction because it
was difficult to explain the very localized chemokine induction when
the TNF- release was quite general (present in cardiac extracellular
fluid). The literature demonstrating a role for reactive oxygen in
induction of chemokine synthesis (35, 42, 43, 46) led us
to consider the alternative hypothesis that reactive oxygen might be
responsible for this localized induction.
Previous work (4) in the area of chemokine induction had
been done in an infarct model where leukocyte adhesion and
transendothelial migration to the venules was occurring during early
reperfusion and being driven, at least in part, by generation of C5a.
Because leukocyte adhesion in early reperfusion occurs at the cardiac venular level, signals from these adherent leukocytes to the
endothelium were also considered as possible candidates of endothelial
gene induction. Another potential confounding problem in delineating the role of reactive oxygen was the fact that reactive oxygen scavengers may disrupt TNF- signal transduction (7). We
elected, therefore, to take advantage of the fact that short periods of coronary occlusion, followed by reperfusion, result in reactive oxygen
formation (2, 5, 15), but in the absence of infarction. A
significant ROI burst occurs during the first 5 min of reperfusion (2, 5, 27, 47, 48). In addition, substantial literature (17, 33, 41, 45) exists suggesting that free-radical
scavengers can obviate the functional effects of reactive oxygen
generated by short periods of ischemia, followed by
reperfusion. These investigators demonstrated a significant reduction
of infarct size when the animals were subjected to long periods of
ischemia and marked improvement of myocardial function after a
brief period of ischemia concomitant with a blunting of the ROI
induction during I/R. Thus we elected to examine the role of a single
short period of occlusion (i.e., 15 min), followed by reperfusion on
chemokine induction and its potential transcriptional mechanisms in WT
mice. To assess the role of reactive oxygen, we investigated the effect
of the free-radical scavenger MPG that has been used in numerous
experiments with I/R (6, 33, 45). In addition, to obviate
the difficulty with regard to the potential effects of free-radical
scavengers on TNF- signal transduction, we repeated the experiments
in a mouse model with genetic deletion of p55 and p75. We have utilized this model in the past to demonstrate the role of TNF- as an anti-apoptotic molecule in similar experiments (24).
To examine the role of ROIs in chemokine induction, we utilized our previously described model of I/R in the mouse with a shorter occlusion time (i.e., 15 min) (30, 31). We soon found that the experiments had to be modified because of the high levels of chemokine induction in the sham animals. Therefore, we (34) developed a technique by which the animal could recover from surgical trauma resulting from the initial instrumentation, and I/R protocols could be carried out at later periods of time. In our study describing this model (34), we point out that failure to use this chronic model results not only in higher sham levels of cytokines compared with naïve hearts, but also in exaggerated responses of cytokine upregulation after an I/R insult. On a pragmatic basis, we chose 10 days of recovery as an ultimate period of time to reduce our shams to an acceptable level of chemokine expression.
The present study demonstrates induction of MIP-1, MIP-1, and
MIP-2 utilizing RPA to assess mRNA levels (Figs. 1,
A-D, and 3, A-D), and
immunocytochemistry to demonstrate the presence of the protein confined
to cardiac venules (Fig. 2). The chemokine induction correlated with
histological demonstration of nuclear translocation of NFB, c-Fos,
and c-Jun in cardiac venules (Fig. 4). Induction of all three of these
chemokines (i.e., MIP-1, MIP-1, and MIP-2), was suppressed by MPG
infusion when it was begun before reperfusion and continued throughout
the 3-h reperfusion time. Because there was some residual chemokine
expression may be because MPG does not efficiently scavenge superoxide
radicals. However, when MPG was initiated 5 min after reperfusion, it
did not suppress any of these signals (NS vs. I/R). Myers et al.
(33) have shown that MPG administration initiated after
reperfusion resulted in loss of functional improvement of the
myocardium after a single brief ischemic episode. This suggests
that a short burst of reactive oxygen occurring immediately on
reperfusion is necessary and sufficient to induce transcription of
these three genes. Similar experiments were done in the TNF- DKO
mice with identical results. There was no statistical difference
between the WT and DKO animals. It is to be noted that the MIP-1
induction consistently observed histologically was attended in RPA
studies by a trend toward increased mRNA concentration, which was not
statistically different than the sham-operated animals. However, MPG
markedly suppressed the MIP-1 mRNA and protein expression. We
presume that the failure to find statistical significance relates to a
greater variability in the level of MIP-1 in sham-operated animals.
The balance of the data appears to support induction of MIP-1.
In our infarction studies in dogs (22, 23), we found that
the chemokine induction was primarily in the endothelial cells of the
venules, and this also appeared to be so in this noninfarction short
occlusion model in mice. One of the potential mechanisms proposed for
venular induction was cytokine or ROI secretion from leukocytes
adhering to these venules after infarction (10). This
noninfarction model suggests that venular chemokine induction is not
dependent on leukocyte products because the absence of infarction
obviates significant leukocyte infiltration into the reperfused
myocardium (10). Interestingly, MCP-1 and IP-10 do not
appear to be induced after a single brief ischemic insult in
mice, whereas other C-C and C-X-C chemokines are upregulated. The
reason for the difference in induction of these chemokines, despite
containing similar NF-B and AP-1 sites in their promoter (39,
51) is not exposed by the present data. On balance, these experiments support a critical role for reactive oxygen in induction of
chemokines in the small venules even in the absence of infarction. These venules are strategically placed with regard to two known actions
of the chemokines: 1) the postcapillary venular network is
the site of transendothelial migration in the heart (16), and 2) angiogenesis begins in the venular endothelium
(11).
Perhaps more intriguing is the potential role of chemokines as angiogenic agents under circumstances of transient ischemia without tissue infarction. IL-8 has been demonstrated to be an angiogenic agent (20) and endothelial cells contain a CCR-2 receptor (32). Ito and co-workers (18) suggested that MCP-1 can also function in the induction of collateral circulation to ischemic skeletal muscle. Furthermore, Salcedo et al. (40) recently demonstrated that endothelial cells do indeed contain a CCR-2 receptor, which may be pertinent to its potential angiogenic role. A study by Weihrauch et al. (50), which utilized a model of repeated short occlusions of dog coronary arteries over a 21-day period, demonstrates a significant increase in collateral circulation in a model in which no infarction occurred. It is interesting to speculate that ROIs may be one of the mediators of the formation of collateral circulation in a compromised vascular bed by virtue of their induction of chemokines in the postcapillary venules.
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ACKNOWLEDGEMENTS |
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We thank Sharon Malinowski and Concepcion Mata for editorial assistance with the manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-42550, the Medallion Foundation, and a grant from the Methodist Hospital Foundation (to N. G. Frangogiannis). T. O. Nossuli is a postdoctoral fellow supported by National Heart, Lung, and Blood Institute Grant T32 HL-07747-06.
Address for reprint requests and other correspondence: M. L. Entman, Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza, M/S F-602, Houston, TX 77030 (E-mail: mentman{at}bcm.tmc.edu).
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.
Received 9 February 2001; accepted in final form 22 August 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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