© 1999 American Heart Association, Inc.
Molecular Medicine |
Induction of Heme Oxygenase-1 Suppresses Venular Leukocyte Adhesion Elicited by Oxidative Stress
Role of Bilirubin Generated by the Enzyme
From the Departments of Biochemistry (R.T., T.Y., N.M., Y.I., M.S.) and Department of Surgery (S.H., K.M., M.K.), School of Medicine, Keio University, Tokyo; Pharmaceutical Frontier Research Laboratories (T.T.), JT Inc, Kanagawa; and Research Center (S.J.T.), Sumitomo Pharmaceutical Co Ltd, Osaka, Japan.
Correspondence to Makoto Suematsu, MD, PhD, Associate Professor, Department of Biochemistry, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. E-mail msuem{at}mc.med.keio.ac.jp
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Abstract |
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Abstract—This study aimed to examine whether an elevated activity of heme oxygenase (HO)-1 in the tissue attenuates endothelial cell–leukocyte interactions microvessels in vivo. When rats were pretreated with an intraperitoneal injection of hemin, an HO-1 inducer, mesenteric tissues, including their microvessels, displayed a marked induction of HO-1 concurrent with an increase in plasma concentrations of bilirubin-IX
, the product
of HO-catalyzed degradation of protoheme IX. In these rats, oxidative
stress such as superfusion with H2O2 and
ischemia-reperfusion of the tissues neither induced rolling nor
exhibited adherent responses of leukocytes in venules. In contrast, the
oxidative stresses evoked marked rolling and adhesion of leukocytes in
the control rats without HO-1 induction. The HO-1 induction also
downregulated leukocyte adhesion elicited by other pro-oxidant stimuli
such as N
-nitro-L-arginine
methyl ester. The decreases in the oxidant-elicited leukocyte adhesive
responses under HO-1–inducing conditions were restored by perfusion
with zinc protoporphyrin-IX, an HO inhibitor, but not with
copper protoporphyrin-IX, which did not inhibit the enzyme.
Furthermore, the effects of zinc protoporphyrin-IX were repressed by
superfusion with bilirubin or biliverdin at the micromolar level, but
not by the same concentration of carbon monoxide, another product
of HO. These results indicate that induction of the HO-1 activity
serves as a potential stratagem to prevent oxidant-induced
microvascular leukocyte adhesion through the action of bilirubin, a
product of HO reaction.
Key Words: heme oxygenase • bilirubin • carbon monoxide • oxidative stress • endothelial cell
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Introduction |
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Heme oxygenase (HO) is the enzyme that oxidatively degrades protoheme IX to biliverdin and carbon monoxide (CO). In mammals, biliverdin is further converted into bilirubin, an endogenous radical scavenger, through the action of biliverdin reductase.1 HO responsible for heme degradation exists in 3 distinct isoforms, HO-1 and HO-2.2 3 HO-3 has recently been found, although its catalytic activity appears to be smaller than other isozymes.4 HO-2 is thought to be constitutive, whereas HO-1 is inducible by various stimuli such as cytokines, heavy metals, oxidants, and protoheme IX, the substrate for HO by itself.5 Such a diversity of stimuli that induce HO-1 expression led several investigators to speculate that HO-1 could play a role in maintaining homeostasis of organ functions. When tissues are preexposed to the HO-1 inducers, their damage and/or acute inflammatory responses are markedly attenuated in a variety of models, such as carrageenan-induced pleuritis,6 oxidant-induced lung injury,7 and endotoxin shock.8 9 However, the mechanisms by which pretreatment with HO-1 induction attenuates inflammatory responses have not fully been elucidated.
It has been widely accepted that tissue leukocyte recruitment is an important factor that determines the severity of tissue injury in acute inflammation or in ischemia-reperfusion.10 11 12 The entry of leukocytes at sites of inflammation is known to be regulated by multistep processes involving the actions of several different adhesion molecules on both leukocytes and endothelial cells; among these, endothelial cell–associated adhesion molecules such as P-selectin participate in the initial step for leukocyte recruitment, as documented by a number of previous studies.10 11 12 13 This adhesion molecule is rapidly expressed on the plasma membrane and triggers leukocyte rolling and adhesion by proinflammatory agonists such as thrombin,14 histamine,15 16 and oxygen free radicals,17 18 or by inhibition of nitric oxide synthase.19 20 This study aimed to examine whether HO-1 induction could attenuate inflammatory responses through downregulation of endothelial cell–leukocyte interactions in vivo. Our findings have provided evidence that tissue pretreatment with the HO-1 induction antagonizes oxidant-elicited leukocyte rolling and adhesion through a mechanism involving generation of HO-derived bile pigments such as biliverdin and bilirubin.
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Materials and Methods |
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Experimental protocols to care and use animals in the present study were approved by the institutional guidelines of Keio University School of Medicine. Male Wistar rats were anesthetized and treated with an intraperitoneal injection of hemin, a potent inducer of HO-1. Mesenteric tissues were collected for Western blotting analysis and immunohistochemistry to examine the HO-1 expression.21 Alterations in plasma bilirubin concentrations were determined by an ELISA using an anti–bilirubin-IX
monoclonal antibody (MoAb) 24G7, as described
previously.22 23
Leukocyte rolling and adhesion in mesenteric microvessels were examined
by playback of video images recorded under an intravital microscope
monitoring erythrocyte velocity.18 20 Venular leukocyte
adhesion was elicited by superfusion with
H2O2 on the mesentery of
the control and the HO-1–induced rats. The relative leukocyte rolling
velocity versus erythrocyte velocity
(VW/VR) and density of
adherent cells were determined. In other experiments, one of the
following interventions was superfused from 10 minutes before the start
of the H2O2 application
until the end of the experiments: zinc protoporphyrin IX (ZnPP) or
copper protoporphyrin IX (CuPP),24 or unconjugated
bilirubin or CO.25 We also examined the effects of an iron
chelator, desferrioxamine mesilate (DFO), on the
H2O2-elicited leukocyte
adhesion.20 When necessary, anti–rat P-selectin (ARP2.4,
Sumitomo Pharmaceutical, Inc) was injected before the
H2O2
superfusion.26 Effects of
N-nitro-L-arginine
methyl ester (L-NAME),20 tert-butyl hydroperoxide
(BHPOx),18 histamine dihydrochloride,16 18
and folmyl methionyl leucyl phenylalanine (FMLP)27 were
also tested. Separately, microvessels of rats pretreated with or
without hemin were subjected to hemorrhagic shock followed by
reperfusion, and adhesive responses were examined according to the
previous protocols.28 We also analyzed expression
of adhesion molecules expressed on neutrophils using MoAbs recognizing
adhesion molecules.18 The MoAbs were WT-3 (anti-rat CD18),
WT-5 (anti-rat CD11b), and HRL-3 (anti-rat L-selectin), generous gifts
from Prof Masayuki Miyasaka, Osaka University School of
Medicine,18 and 2H5 (anti–sialyl Lewis X-like
carbohydrate structure [SLeX]).29 Separately,
secretagogue activation of mast cells by
H2O2 was examined by
measuring the release of the enzyme ß-hexosaminidase (ß-hex), as
described in the previous method,30 31 with modifications.
The density of degranulated and undegranulated mast cells in the
mesenteric tissue was also studied using the toluidine blue staining
method as described previously.32
Expression of P-selectin in venules was examined in vivo using real-time laser confocal video microscopy.33 34 A MoAb against rat P-selectin (ARP2.4) was labeled with FITC and injected into the femoral vein. Before, and 10 minutes after, the start of superfusion of H2O2 on the mesentery, fluorescence images were captured and digitally processed. Data calibration was carried out according to our previous method, with modifications.34 On the other hand, effects of pretreatment with the HO-1 induction by hemin or with application of bilirubin on oxidative changes were examined in cultured human umbilical venous endothelial cells (HUVECs) using carboxydihydrofluorescein (CDCFH) diacetate bis-acetoxymethyl ester, an oxidant-sensing fluorochrome precursor.20 27 Differences in the H2O2-induced fluorescence elevation were determined among the groups by digital microfluorography.34 Statistical significance was determined by 1-way ANOVA with the Fisher multiple comparison test. All data were expressed as mean±SD, and P<0.05 was considered significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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Effects of HO-1 Induction on the Interaction of Leukocytes With Venular Endothelium
Figure 1
illustrates alterations in
expression of the HO-1 protein in the mesentery of hemin-treated rats.
As illustrated in Western blotting analysis in Figure 1A, an intraperitoneal injection of hemin
induced a marked increase in the protein expression, which became
evident at 6 hours and peaked at 12 hours followed by a recovery to the
control level at 18 hours. We therefore performed fluorescence
immunohistochemistry using the same MoAb labeled with FITC in the
mesenteric tissue of the 12-hour hemin-treated rats. As seen in the
left portion of Figure 1B, the hemin-untreated mesentery
exhibited little immunoreactivity of HO-1, if any, and only
counterstaining with KiM2R-associated phycoerythrin on tissue
macrophages was evident. On the other hand, the hemin-treated
mesentery displayed marked immunoreactivities of HO-1 both in vascular
regions and in the interstitial space; among these
HO-1–positive cells, arteriolar and venular endothelia constituted a
major site of prominent HO-1 expression. Such HO-1–associated
fluorescence activities were not evident in the
mesentery of the 18-hour hemin-treated rats (data not
shown).
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The Table illustrates differences in venular leukocyte
adhesion elicited by topical superfusion of
H2O2 or by hemorrhagic
shock followed by reperfusion among varied time intervals after the
hemin treatment. Adherent responses of leukocytes time-dependently
decreased and became smallest at 12 hours in both experimental groups,
suggesting that pretreatment with hemin downregulates oxidant-elicited
venular leukocyte adhesion. It should be noted that plasma
concentrations of bilirubin-IX increased at 6 hours, reached a
maximum level at 12 hours, and decreased at 18 hours. On the other
hand, plasma concentrations of at 6, 12, and 18 hours after treatment
with the hemin-untreated vehicle control were 1.0±0.3, 0.9±0.2, and
0.9±0.3 µmol/L (n=5), respectively, indicating no significant
changes. These results indicated that the decrease in oxidative
stress-induced adhesive changes in the HO-1–induced mesentery became
most prominent at a time when the HO-1 induction and actual heme
degradation reached a maximum level.
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Attenuation of H2O2-Elicited Leukocyte
Rolling and Adhesion by the Induction of HO-1
Figure 2 showed temporal alterations
in rolling and adherent responses of leukocytes in venules of the
mesentery elicited by the superfusion of
H2O2 at 500 µmol/L.
In the control rats in which HO-1 was not induced, the
H2O2 superfusion evoked a
marked decrease in the
VW/VR values, which reached
a minimum at 10 minutes after the start of superfusion, were sustained
for 20 minutes, and then increased to restore the baseline level.
During these events, venular shear rates did not exhibit any
significant reduction (data not shown). The density of adherent
leukocytes (closed circles) also elevated time dependently in response
to the H2O2 superfusion and
kept high levels even at 40 minutes after the start of superfusion. On
the other hand, the mesenteric microvessels undergoing the 12-hour
hemin treatment exhibited quite different pictures for the adhesive
changes in response to the
H2O2 superfusion; as
indicated by open squares, the rolling velocity was not significantly
reduced on the application of 500 µmol/L
H2O2, suggesting little
increase in the adhesion capacity, if any, between venular
endothelium and leukocytes. At the same time, little
elevation of the adhesion density was observed in these rats during the
entire observation period. We further examined effects of different
concentrations of H2O2 on
the mesentery. At concentrations >1 mmol/L,
H2O2 induced a significant
reduction of venular shear rates, which coincided with an increase in
the density of adherent cells. On the other hand, at those <500
µmol/L, we observed a transient reduction of the rolling velocity but
not stationary adhesion, as was observed at 500 µmol/L. These
results were consistent with previous data by Suzuki et
al.35 On the basis of these findings, we used 500
µmol/L of H2O2 as a
maximum concentration that did not alter microvascular shear rates and
that allowed us to rule out hemodynamic effects on
leukocyte adhesion in these and later experiments.
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and 
denote
VW/VR (%). • and 
indicate number of
adhesions per 100-µm venular segment (adhesion density).
*P<0.05 as compared with 
P<0.05
as compared with
Requirement of HO Activities for Downregulation of Leukocyte
Adhesion
We then inquired whether the enzyme activity of HO-1 could be
necessary to attenuate the oxidant-elicited adhesive responses in the
12-hour hemin-treated rats. To that end, effects of local superfusion
with ZnPP, an HO inhibitor, were examined. At 20 minutes
after the start of superfusion of 0.5 µmol/L ZnPP with
H2O2, the
VW/VR value was
significantly decreased as compared with that measured under the
superfusion with H2O2 alone
(1.1±0.6 versus 2.7±0.2%, P<0.05, mean±SD of 7
experiments). At the same time, the treatment with the same
concentration of ZnPP alone did not significantly alter the baseline
VW/VR value in the group
(2.5±0.4%, n=5). On the other hand, treatment with 0.5 µmol/L
CuPP, a metalloprotoporphyrin that did not inhibit HO, did not mimic
the effects of ZnPP, which could restore the
H2O2-elicited
VW/VR reduction
(2.4±0.5%, n=5). Because no significant differences in venular shear
rates were observed among these groups (data not shown), these results
suggest that the HO activity in situ could downregulate the
H2O2-induced enhancement of
adhesion between leukocytes and venular endothelium.
The ZnPP-induced restoration of the rolling responses was abolished by
pretreatment with an anti–P-selectin MoAb ARP2.4 (2.6±0.5%, n=5),
suggesting that P-selectin is involved in the rolling response elicited
by H2O2 plus ZnPP in the
12-hour hemin-treated mesenteric venules.
Figure 3 illustrates effects of ZnPP, an
HO inhibitor, and the anti–P-selectin MoAb on the density
of venular leukocyte adhesion in control and 12-hour hemin-treated
rats. As already illustrated in Figure 2
, the
H2O2 superfusion elicited a
significant increase in the density of leukocyte adhesion in the
control mesentery. This change was not altered by cosuperfusion with
0.5 µmol/L ZnPP or CuPP. At the same time, treatment with ZnPP
or with CuPP at this concentration per se did not elicit leukocyte
adhesion per se (data not shown). The
H2O2-induced elevation of
the adhesion density was attenuated completely by pretreatment with an
anti–P-selectin MoAb, ARP 2.4. The adhesive changes were also
inhibited significantly by pretreatment of DFO, an iron chelator,
suggesting that iron-mediated oxyradical propagation is necessary to
evolve the H2O2-elicited
adhesion, as will be discussed later. These results suggest that the
HO-induced leukocyte adhesion is not altered by inhibition of the HO
activity and is mediated by P-selectin in the control rats.
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On the other hand, a reduction of the H2O2-induced leukocyte adhesion in the hemin-treated rats was significantly reversed by cosuperfusion with 0.5 µmol/L ZnPP, but not with the same concentration of CuPP. Again, the ZnPP-induced recovery of the H2O2-induced adhesive changes was attenuated either by pretreatment with ARP2.4 or in part by that with DFO. These results collectively suggest that the enzyme activity of HO plays a crucial role in acquisition of resistance against the H2O2-elicited, and P-selectin-mediated, rolling and adhesion of leukocytes in the HO-1–induced mesenteric microcirculation.
Effects of Exogenously Applied Bilirubin and CO on Leukocyte
Adhesion In Vivo
The data showing the ZnPP-induced recovery of adhesive responses
of leukocytes in the hemin-treated mesentery tempted us to examine
whether bilirubin and/or CO could alter the
H2O2-induced rolling and
adhesion of leukocytes (Figure 4). The
upper and lower left panels illustrate alterations by coperfusion with
varied concentrations of bilirubin in the
H2O2-induced changes in
leukocyte rolling and adhesion in the hemin-untreated control group,
respectively. As indicated by the open circles, the bilirubin
superfusion dose-dependently attenuated both rolling and adherent
responses, and 5 µmol/L of the reagent was sufficient to fully
inhibit the H2O2-elicited
changes. On the other hand, the superfusion with biliverdin at 10
µmol/L but not with the same concentration of CO significantly
attenuated the rolling and adherent responses elicited by
H2O2, as shown in closed
circles and open squares, respectively.
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The upper and lower right panels of Figure 4 depict alterations
in rolling and adherent responses of leukocytes in the hemin-treated
rats (HO-1 induction [+]). As shown above and also as indicated by
the open circles in these panels, these rats were characterized by a
marked reduction of the adhesive responses to the oxidative impacts,
and the inhibition of the HO activity by ZnPP was necessary to restore
the H2O2-induced adhesive
responses, as indicated by shaded circles plotted at no bilirubin
treatment. Bilirubin dose-dependently attenuated the ZnPP-dependent
restoration of the
H2O2-elicited rolling and
adherent changes. Again, such effects of bilirubin were mimicked by
biliverdin (closed circles), but not by CO (shaded squares). These
results indicate that supplement of bilirubin at micromolar levels can
inhibit the H2O2-induced
rolling and adhesion of leukocytes in the venules, and downregulation
of adhesive responses in the HO-1-induced rats appears to be ascribable
to the effects of biliverdin and/or bilirubin but not to those of
CO.
Reduction of L-NAME– or Hydroperoxide-Induced Leukocyte Adhesion
in the Hemin-Treated Rats
We further inquired whether downregulation of leukocyte
adhesion in the HO-1–induced mesentery could occur on application of
other stimuli that are known to induce adherent changes. Four different
stimuli, 500 µmol/L BHPOx, 100 µmol/L L-NAME, 10
µmol/L histamine, and 100 nmol/L FMLP, were chosen to examine
differences in venular leukocyte adhesion between the control and the
12-hour hemin-treated rats; as seen in the open bars of the left panel
of Figure 5, superfusion with
these reagents under the given concentrations evoked a marked increase
in the adherent cells. These results were also consistent with
the previous observations.18 20 In the 12-hour
hemin-treated rats, the adhesive responses elicited by BHPOx or
by L-NAME were significantly attenuated, whereas those elicited by
histamine or by FMLP superfusion were still evident and comparable with
the responses in the hemin-untreated group. On the other hand, as shown
by flow cytometric analysis in the right panels of Figure 5, the hemin treatment did not significantly alter the
expression of adhesion molecules on circulating neutrophils such as
SLeX-like carbohydrate structure, L-selectin, and CD11b/CD18. The
expression of L-selectin and CD11b/CD18 on lymphocytes was also
examined but with no significant differences (data not shown). These
results suggest that downregulation of the leukocyte adhesion in the
HO-1–overexpressed microvessels occurs through leukocyte-independent
mechanisms.
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Considering that degranulation of mast cells is known to evoke venular leukocyte rolling and adhesion,36 we next examined whether a sensitivity of the cells to H2O2 could differ between the hemin-treated and -untreated mesenteric tissues. The percentage densities of degranulated mast cells in the hemin-untreated tissues before and after H2O2 application at 500 µmol/L were 8.0±3.8% and 8.1±5.4%, whereas those in the hemin-treated tissues were 7.7±4.2% and 7.2±4.3% (mean±SD of 5 separate experiments), respectively, indicating that this concentration of H2O2 did not evolve notable degranulation of mast cells in both groups. Using the isolated mast cells, we also examined effects of 500 µmol/L H2O2 on the degranulation in vitro but were unable to detect measurable levels of the ß-hex release (6.1±0.9% in no treatment versus 6.6±0.8% in the H2O2 treatment; n=5). On the other hand, the same cells exhibited significant ß-hex release, at 60.2±5.2% (P<0.05, n=5), on the application of 1 µg/mL compound 48/80, a stimulator of mast cell degranulation. These results suggest that the hemin treatment did not evoke measurable alterations in the population of degranulated mast cells under basal and H2O2-stimulated conditions. Thus, the mechanism for downregulation of leukocyte adhesion in the hemin-treated mesentery is unlikely to be ascribable to the altered sensitivity of mast cells, at least under the current experimental conditions.
Reduction of H2O2-Elicited Venular
P-Selectin Expression in the Hemin-Treated Mesentery
Figure 6 illustrates
representative pictures of the P-selectin–associated
laser confocal fluorescence images captured before (baseline)
and 10 minutes after the start of the
H2O2 superfusion at
500 µmol/L (H2O2) in
the hemin-untreated and -treated preparations. On the
H2O2 application, the
fluorescence intensities along venular
endothelium in the hemin-untreated mesentery
(HO-1[–]) became markedly increased. Under these circumstances, the
H2O2 superfusion evoked
only little leukocyte adhesion if any, as shown in the right upper
panel. These results suggest that the MoAb ARP2.4 administered into the
circulation mainly binds to P-selectin expressed on the
H2O2-treated venular
endothelium and thereby blocks leukocyte adhesion in
situ. On the other hand, the mesenteric venules in the hemin-treated
rats (HO-1[+]) exhibited only little elevation of the
fluorescence on the
H2O2 superfusion at the
same concentration.
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We attempted to quantify
H2O2-elicited changes in
the venular FITC labeling by calibrating the fluorescence
activities. Figure 7A illustrates the net
increase in the venular fluorescence intensities on a 10-minute
exposure to 500 µmol/L
H2O2. As seen, in the
hemin-untreated control, the
H2O2 exposure induced a
marked increase in the ARP2.4-associated fluorescence. The
H2O2-induced
fluorescence elevation was attenuated almost completely by
pretreatment with the bilirubin superfusion at 10 µmol/L. On the
other hand, the venules of the hemin-pretreated rats exhibited only
small elevation of the fluorescence on the application of
H2O2. The absence of
H2O2-induced elevation of
fluorescence was markedly restored by supplementation with
0.5 µmol/L ZnPP, reaching the level equivalent to that observed
in the hemin-untreated venules undergoing
H2O2 exposure. It should be
noted that pretreatment with either bilirubin or DFO significantly
repressed the ZnPP-elicited restoration of the fluorescence
elevation. Most importantly, as shown in the upper portion of Figure 7A, the H2O2-induced
elevation of the fluorescence in the hemin-untreated mesenteric
preparations or in those pretreated with hemin plus ZnPP was not
evident when the fluorescence-labeled ARP2.4 was replaced by
FITC-labeled nonspecific IgG. These results suggest that the
differences in the
H2O2-induced alterations
among these groups occurred specifically because of those in the
P-selectin labeling in vivo. Collectively, downregulation of the
H2O2-induced rolling and
adherent responses of leukocytes in the hemin-treated rats is likely to
be ascribable to decreased responses of the P-selectin translocation.
Effects of the HO-1 induction or bilirubin on the
H2O2-induced oxidative
changes were also examined. As seen in Figure 7B, HUVECs
undergoing the HO-1 induction exhibited lesser oxidative responses on
H2O2 application. Different
effects of 500 µmol/L
H2O2 on CDCFH
oxidation among the groups are summarized in Figure 7C. The data
show that the H2O2-induced
oxidative changes were attenuated significantly by the HO-1 induction
or by the bilirubin application, and that ZnPP restored the
H2O2-induced changes in the
HO-1–induced HUVECs. Furthermore, the ZnPP-elicited restoration of
oxidative changes was again cancelled by application of bilirubin or
DFO. These results suggest that the HO-1 induction, as well as
bilirubin supplementation, attenuates intracellular oxidant generation
elicited by H2O2.
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) in CDCFH oxidation among the groups. Groups
correspond to those in the lower portion of panel A.
*P<0.05 as compared with the data collected from rats
untreated with H2O2; |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current study demonstrates that microvascular endothelial cells overexpressing HO-1 become resistant to oxidative stress and resultant upregulation of leukocyte adhesion and suggests the role of bilirubin generated through the enzyme reaction. Recent studies, including those using HO-1–knockout mice, have shown that the induction of HO-1 helps to ameliorate tissue injury or inflammatory changes in a variety of experimental models.37 38 39 40 However, these previous studies did not fully address molecule(s) responsible for the HO-1–dependent attenuation of inflammatory responses. Our results provide for the first time in vivo evidence showing that pretreatment with the HO-1 induction helps reduce tissue leukocyte recruitment through mechanisms involving bilirubin, which is capable of inhibiting the very initial process of cell attachment, that is, leukocyte rolling on the endothelial surface.
There are several possibilities through which HO activity could contribute to a reduction of the tissue sensitivity to oxidative stress. First, the HO reaction is attributable to a decrease in the substrate protoheme IX, which is known to enhance oxidant-induced cell injury.41 Second, the HO reaction can exert its ability to reduce oxidative stress through biological actions of its reaction products, CO and biliverdin. Biliverdin and its reduced product bilirubin scavenge various oxidants, such as hydroxyl radical, singlet oxygen, and lipid hydroperoxides and are thus considered endogenous antioxidants that protect cells from oxidative insults.1 42 On the other hand, CO is known to function as a vasorelaxing mediator that could guarantee ample blood supply to microvascular beds.26 Furthermore, CO has the ability to inhibit cytochrome P-450, which can promote oxidation of fatty acids.23 43 Among these putative mechanisms by which HO-1 activity can attenuate the impact of oxidative stress, our study shed light on the importance of bilirubin, which was actually elevated in plasma and downregulated the H2O2-induced P-selectin translocation, as well as rolling and adhesion of leukocytes in venules. On the other hand, the current study did not show any antioxidative or antiadhesive properties of CO, another product that is generated stoichiometrically through the HO reaction, as topical application of this gaseous monoxide at concentrations comparable with bilirubin did not repress the leukocyte adhesion elicited by H2O2 and ZnPP in hemin-treated rats. Our results thus provided evidence in vivo for such a product-specific antioxidative mechanism under the HO-1–inducing conditions that has not fully been addressed in previous results obtained in experiments using HO-1 gene-targeting mice,40 whereas such inhibitory actions of bilirubin on locomotion of inflammatory cells were reported previously in cultured systems.44
It should be noted in the current study that tissue pretreatment with HO-1 downregulated venular leukocyte adhesion elicited by H2O2, BHPOx, and L-NAME, but did not attenuate that elicited by histamine or FMLP. These results are not surprising, in that H2O2, BHPOx, and L-NAME share the oxidant-propagating mechanism on endothelial cells. When topically applied on the mesentery, the former 3 reagents can induce overproduction of hydroperoxides in microvascular endothelial cells that is followed by P-selectin-mediated leukocyte adhesion, as shown by previous studies.18 20 28 On the other hand, histamine or FMLP can elicit receptor-mediated P-selectin expression or directly upregulate expression of CD11/CD18 on marginating neutrophils, respectively, and does not primarily induce oxidative changes in the venular endothelium. In this context, pretreatment with HO-1 induction serves as a potential stratagem to ameliorate venular leukocyte adhesion specifically promoted by direct oxidative impacts (eg, BHPOx) and/or by pro-oxidant reagents (eg, L-NAME), but not that induced by oxidant-independent mechanisms (eg, histamine, FMLP). Further investigation on quantitative determination of the P-selectin expression45 is needed to resolve the question of whether the inhibitory action of the HO-1 induction or bilirubin on the oxidant-elicited and P-selectin–mediated leukocyte adhesion in vivo, shown in the current study, is applicable to other organ microvascular systems.
It is not known what kinds of cells in the HO-1–overexpressed
mesentery were responsible for bilirubin generation. However, several
lines of evidence lead us to suggest that microvascular
endothelial cells serve as an important site for the
enzyme reaction of HO-1. First, as seen in Figure 1, the
microvascular endothelium exhibited a most prominent
response of the enzyme induction in the hemin-treated rats. Second, at
least under the current experimental conditions, mast cells were
unlikely to display any visible degranulation on stimulation with
H2O2 in both the control
and the hemin-treated groups. In addition, tissue macrophages
in the mesenteric interstitial space appear to express
little HO-1 expression, if any, in the both groups. Third, local
superfusion with ZnPP, an HO inhibitor, on the mesentery
significantly restored the
H2O2-induced P-selectin
translocation in the venular endothelium overexpressing
HO-1. Finally, and most importantly, bilirubin at
physiologically reasonable concentrations
repressed the ZnPP-elicited recovery of the
H2O2-induced leukocyte
adhesion and mimicked effects of the HO-1 induction. Because the plasma
bilirubin concentrations in the hemin-pretreated rats were still
greater than the control, even under the local ZnPP superfusion (data
not shown), these findings suggest that a continuous generation of this
heme-degrading product in microvessels is necessary to maintain
their antiadhesive property.
Of interest is that only 5 to 10 µmol/L bilirubin is sufficient to ameliorate adhesive responses elicited by as much as 500 µmol/L H2O2. Considering that bilirubin tends to accumulate in the cell membrane and efficiently captures superoxide, hydroxyl radicals, and lipid hydroperoxide radicals but not H2O2 directly,1 46 it is not unreasonable to hypothesize that most of the H2O2 applied on the mesentery in vivo could be degraded rapidly by antioxidant enzymes that abundantly occur in intravascular space (eg, catalase and glutathione peroxidase in circulating erythrocytes), and the rest could participate in propagation of oxyradical species responsible for endothelial P-selectin translocation. Such a hypothesis is supported by the current findings showing inhibitory effects of an iron chelator, DFO, on H2O2-induced P-selectin expression and leukocyte adhesion.17 Detailed kinetics of the bilirubin generation in and around the endothelial cells in the HO-1–pretreated tissue should also be examined in the future. However, the present results suggest that microvascular endothelial cells constitute an important cellular component sensing tissue overloading of protoheme IX: When exposed to the stimulus, regional tissues undergo oxidative injury and increase their ability to decompose the heme molecules by inducing HO-1, and finally become less sensitive to oxidative stress through bilirubin-dependent mechanisms. Such a notion is well supported by a previous study in vitro showing that transfection of the HO-1 gene renders endothelial cells less susceptible to heme-mediated oxidative stress.38
We utilized overloading of free heme molecules as a tool to induce HO-1. Clinical situations in which tissues are exposed to excessive amounts of heme involve rhabdomyolysis,47 intravascular hemolysis, and endotoxemia,48 which are known to trigger the release of myoglobin or hemoglobin as a source of protoheme IX. Under these circumstances, microvascular endothelium serves as a primary site exposed to circulating heme molecules. In this context, one may speculate that the HO-1 induction in the endothelial cells could not only detoxify excessive heme molecules in circulation but also help reduce unnecessary leukocyte recruitment into damaged tissues under aforementioned disease conditions. On the other hand, besides the overloading of heme or ischemia-reperfusion,49 there are several alternate procedures to obtain the HO-1 induction, such as exposure to endotoxin.21 Such previous results raise an important question as to whether pretreatment with these non-heme stimuli could also render microvessels less adhesive to circulating leukocytes. Further investigation is obviously required to examine whether the tissue HO-1 induction and subsequent downregulation of leukocyte adhesion are involved in the mechanisms for tolerance against endotoxin or shock conditions.33
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Acknowledgments |
|---|
We thank Prof Masayuki Miyasaka for his generous gifts of MoAbs and Prof Makoto Murakami for instruction of mast cell isolation. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan; by grants from Keio University School of Medicine; by Advanced Medical Technology in Health Science Research Grants from the Ministry of Health and Welfare in Japan; and in part by Takeda Science Foundation.
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Footnotes |
|---|
A portion of this work was presented at the Experimental Biology 98 meeting, April 1998, San Francisco, Calif.
Received February 11, 1999; accepted August 10, 1999.
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References |
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A. Almolki, C. Taille, G. F. Martin, P. J. Jose, C. Zedda, M. Conti, J. Megret, D. Henin, M. Aubier, and J. Boczkowski Heme oxygenase attenuates allergen-induced airway inflammation and hyperreactivity in guinea pigs Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L26 - L34. [Abstract] [Full Text] [PDF]
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M. P. Soares, M. P. Seldon, I. P. Gregoire, T. Vassilevskaia, P. O. Berberat, J. Yu, T.-Y. Tsui, and F. H. Bach Heme Oxygenase-1 Modulates the Expression of Adhesion Molecules Associated with Endothelial Cell Activation J. Immunol., March 15, 2004; 172(6): 3553 - 3563. [Abstract] [Full Text] [PDF]
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N. Lindenblatt, R. Bordel, W. Schareck, M.D. Menger, and B. Vollmar Vascular Heme Oxygenase-1 Induction Suppresses Microvascular Thrombus Formation In Vivo Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 601 - 606. [Abstract] [Full Text]
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F. Zhang, J. I. Kaide, L. Yang, H. Jiang, S. Quan, R. Kemp, W. Gong, M. Balazy, N. G. Abraham, and A. Nasjletti CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H137 - H144. [Abstract] [Full Text] [PDF]
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 R. Zegdi, O. Fabre, N. Lila, P. Fornes, M. Cambillau, M. Shen, P. Herve, A. Carpentier, and J.-N. Fabiani Exhaled carbon monoxide and inducible heme oxygenase expression in a rat model of postperfusion acute lung injury J. Thorac. Cardiovasc. Surg., December 1, 2003; 126(6): 1867 - 1874. [Abstract] [Full Text] [PDF]
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A. M. Vicente, M. I. Guillen, A. Habib, and M. J. Alcaraz Beneficial Effects of Heme Oxygenase-1 Up-Regulation in the Development of Experimental Inflammation Induced by Zymosan J. Pharmacol. Exp. Ther., December 1, 2003; 307(3): 1030 - 1037. [Abstract] [Full Text] [PDF]
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X. Liu and Z. Spolarics Methemoglobin is a potent activator of endothelial cells by stimulating IL-6 and IL-8 production and E-selectin membrane expression Am J Physiol Cell Physiol, November 1, 2003; 285(5): C1036 - C1046. [Abstract] [Full Text] [PDF]
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N. G. Abraham, T. Kushida, J. McClung, M. Weiss, S. Quan, R. Lafaro, Z. Darzynkiewicz, and M. Wolin Heme Oxygenase-1 Attenuates Glucose-Mediated Cell Growth Arrest and Apoptosis in Human Microvessel Endothelial Cells Circ. Res., September 19, 2003; 93(6): 507 - 514. [Abstract] [Full Text] [PDF]
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F. A. D. T. G. Wagener, H.-D. Volk, D. Willis, N. G. Abraham, M. P. Soares, G. J. Adema, and C. G. Figdor Different Faces of the Heme-Heme Oxygenase System in Inflammation Pharmacol. Rev., September 1, 2003; 55(3): 551 - 571. [Abstract] [Full Text] [PDF]
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 F. A. D. T. G. Wagener, H. E. van Beurden, J. W. von den Hoff, G. J. Adema, and C. G. Figdor The heme-heme oxygenase system: a molecular switch in wound healing Blood, July 15, 2003; 102(2): 521 - 528. [Abstract] [Full Text] [PDF]
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 T. D. Blydt-Hansen, M. Katori, C. Lassman, B. Ke, A. J. Coito, S. Iyer, R. Buelow, R. Ettenger, R. W. Busuttil, and J. W. Kupiec-Weglinski Gene Transfer-Induced Local Heme Oxygenase-1 Overexpression Protects Rat Kidney Transplants From Ischemia/Reperfusion Injury J. Am. Soc. Nephrol., March 1, 2003; 14(3): 745 - 754. [Abstract] [Full Text] [PDF]
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S. K. R. Kanakiriya, A. J. Croatt, J. J. Haggard, J. R. Ingelfinger, S.-S. Tang, J. Alam, and K. A. Nath Heme: a novel inducer of MCP-1 through HO-dependent and HO-independent mechanisms Am J Physiol Renal Physiol, March 1, 2003; 284(3): F546 - F554. [Abstract] [Full Text] [PDF]
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M. AMON, M. D. MENGER, and B. VOLLMAR Heme oxygenase and nitric oxide synthase mediate cooling-associated protection against TNF-{alpha}-induced microcirculatory dysfunction and apoptotic cell death FASEB J, February 1, 2003; 17(2): 175 - 185. [Abstract] [Full Text] [PDF]
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X.-L. Chen, S. E. Varner, A. S. Rao, J. Y. Grey, S. Thomas, C. K. Cook, M. A. Wasserman, R. M. Medford, A. K. Jaiswal, and C. Kunsch Laminar Flow Induction of Antioxidant Response Element-mediated Genes in Endothelial Cells. A NOVEL ANTI-INFLAMMATORY MECHANISM J. Biol. Chem., January 3, 2003; 278(2): 703 - 711. [Abstract] [Full Text] [PDF]
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S. Kashiwagi, M. Kajimura, Y. Yoshimura, and M. Suematsu Nonendothelial Source of Nitric Oxide in Arterioles But Not in Venules: Alternative Source Revealed In Vivo by Diaminofluorescein Microfluorography Circ. Res., December 13, 2002; 91 (12): e55 - e64. [Abstract] [Full Text] [PDF]
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P. Naughton, R. Foresti, S. K. Bains, M. Hoque, C. J. Green, and R. Motterlini Induction of Heme Oxygenase 1 by Nitrosative Stress. A ROLE FOR NITROXYL ANION J. Biol. Chem., October 18, 2002; 277(43): 40666 - 40674. [Abstract] [Full Text] [PDF]
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R. Takamiya, M. Murakami, M. Kajimura, N. Goda, N. Makino, Y. Takamiya, T. Yamaguchi, Y. Ishimura, N. Hozumi, and M. Suematsu Stabilization of mast cells by heme oxygenase-1: an anti-inflammatory role Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H861 - H870. [Abstract] [Full Text] [PDF]
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C. Wunder, R. W Brock, S. D McCarter, A. Bihari, K. Harris, O. Eichelbronner, and R. F Potter Inhibition of haem oxygenase activity increases leukocyte accumulation in the liver following limb ischaemia-reperfusion in mice J. Physiol., May 1, 2002; 540(3): 1013 - 1021. [Abstract] [Full Text] [PDF]
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 S. C. Stoica, M. Goddard, and S. R. Large The endothelium in clinical cardiac transplantation Ann. Thorac. Surg., March 1, 2002; 73(3): 1002 - 1008. [Abstract] [Full Text] [PDF]
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 F. TAMION, V. RICHARD, G. BONMARCHAND, J. LEROY, J.-P. LEBRETON, and C. THUILLEZ Induction of Heme-oxygenase-1 Prevents the Systemic Responses to Hemorrhagic Shock Am. J. Respir. Crit. Care Med., November 15, 2001; 164(10): 1933 - 1938. [Abstract] [Full Text] [PDF]
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K. Ishikawa, D. Sugawara, J. Goto, Y. Watanabe, K. Kawamura, M. Shiomi, H. Itabe, and Y. Maruyama Heme Oxygenase-1 Inhibits Atherogenesis in Watanabe Heritable Hyperlipidemic Rabbits Circulation, October 9, 2001; 104(15): 1831 - 1836. [Abstract] [Full Text] [PDF]
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F. A. D. T. G. Wagener, A. Eggert, O. C. Boerman, W. J. G. Oyen, A. Verhofstad, N. G. Abraham, G. Adema, Y. van Kooyk, T. de Witte, and C. G. Figdor Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase Blood, September 15, 2001; 98(6): 1802 - 1811. [Abstract] [Full Text] [PDF]
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W. P. Wang, X. Guo, M. W. L. Koo, B. C. Y. Wong, S. K. Lam, Y. N. Ye, and C. H. Cho Protective role of heme oxygenase-1 on trinitrobenzene sulfonic acid-induced colitis in rats Am J Physiol Gastrointest Liver Physiol, August 1, 2001; 281(2): G586 - G594. [Abstract] [Full Text] [PDF]
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M. Nakayama, K. Takahashi, T. Komaru, M. Fukuchi, H. Shioiri, K.-i. Sato, T. Kitamuro, K. Shirato, T. Yamaguchi, M. Suematsu, et al. Increased Expression of Heme Oxygenase-1 and Bilirubin Accumulation in Foam Cells of Rabbit Atherosclerotic Lesions Arterioscler. Thromb. Vasc. Biol., August 1, 2001; 21(8): 1373 - 1377. [Abstract] [Full Text] [PDF]
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T. J. Rabelink and E. Stroes Atherosclerosis : Defeat of the Defense? Circ. Res., March 16, 2001; 88(5): 456 - 457. [Full Text] [PDF]
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K. A. Nath, J. P. Grande, J. J. Haggard, A. J. Croatt, Z. S. Katusic, A. Solovey, and R. P. Hebbel Oxidative Stress and Induction of Heme Oxygenase-1 in the Kidney in Sickle Cell Disease Am. J. Pathol., March 1, 2001; 158(3): 893 - 903. [Abstract] [Full Text] [PDF]
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 M Cavicchi, L Gibbs, and B J R Whittle Inhibition of inducible nitric oxide synthase in the human intestinal epithelial cell line, DLD-1, by the inducers of heme oxygenase 1, bismuth salts, heme, and nitric oxide donors Gut, December 1, 2000; 47(6): 771 - 778. [Abstract] [Full Text] [PDF]
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 W. Gonzalez, V. Fontaine, M. E. Pueyo, N. Laquay, D. Messika-Zeitoun, M. Philippe, J.-F. Arnal, M.-P. Jacob, and J.-B. Michel Molecular Plasticity of Vascular Wall During NG-Nitro-L-Arginine Methyl Ester-Induced Hypertension : Modulation of Proinflammatory Signals Hypertension, July 1, 2000; 36(1): 103 - 109. [Abstract] [Full Text] [PDF]
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T. Katayama, Y. Ikeda, M. Handa, T. Tamatani, S. Sakamoto, M. Ito, Y. Ishimura, and M. Suematsu Immunoneutralization of Glycoprotein Ib{alpha} Attenuates Endotoxin-Induced Interactions of Platelets and Leukocytes With Rat Venular Endothelium In Vivo Circ. Res., May 26, 2000; 86(10): 1031 - 1037. [Abstract] [Full Text] [PDF]
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T. J. Vachharajani, J. Work, A. C. Issekutz, and D. N. Granger Heme oxygenase modulates selectin expression in different regional vascular beds Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1613 - H1617. [Abstract] [Full Text] [PDF]
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C. Wunder, R. W. Brock, S. D. McCarter, A. Bihari, K. Harris, O. Eichelbroanner, and R. F. Potter Inhibition of haem oxygenase activity increases leukocyte accumulation in the liver following limb ischaemia-reperfusion in mice J. Physiol., March 15, 2002; (2002) 2001015446. [Abstract] [PDF]
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T. Imai, T. Morita, T. Shindo, R. Nagai, Y. Yazaki, H. Kurihara, M. Suematsu, and S. Katayama Vascular Smooth Muscle Cell-Directed Overexpression of Heme Oxygenase-1 Elevates Blood Pressure Through Attenuation of Nitric Oxide-Induced Vasodilation in Mice Circ. Res., July 6, 2001; 89(1): 55 - 62. [Abstract] [Full Text] [PDF]
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M. Sano, K. Fukuda, T. Sato, H. Kawaguchi, M. Suematsu, S. Matsuda, S. Koyasu, H. Matsui, K. Yamauchi-Takihara, M. Harada, et al. ERK and p38 MAPK, but not NF-{kappa}B, Are Critically Involved in Reactive Oxygen Species-Mediated Induction of IL-6 by Angiotensin II in Cardiac Fibroblasts Circ. Res., October 12, 2001; 89(8): 661 - 669. [Abstract] [Full Text] [PDF]
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