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* Institute of Pharmacology and
Department of Biomorphology School of Medicine, University of Messina, Italy School of Medicine, University of Messina, Italy; G. D Searle,
Discovery Pharmacology, St. Louis, Missouri 63167, USA;
§ Department of Pharmacological and Physiological Sciences, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA;
|| Metaphore Pharmaceuticals, St. Louis, Missouri 63110, USA
1Correspondence: Institute of Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario Via C. Valeria-Gazzi, 98100 Messina, Italy. E-mail: salvator{at}www.unime.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: SAO • polymorphonuclear leukocyte • cytokine • nitrate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. An important
component of SAO shock is endothelial dysfunction (1
, 3
4
5)
. In fact, a reduction in the ecNOS activity in reperfused
intestine has been demonstrated by direct enzyme measurements by Kanwar
and co-workers (6)
.
The development of endothelial dysfunction was originally attributed to
oxygen-derived free radicals released from both the reperfused
endothelium (1
, 7)
and activated adherent
polymorphonuclear leukocytes (PMNs) (8
9
10
11)
. The
endothelial dysfunction (i.e., a deficit of endothelial nitric oxide
production) predisposes to vasospasm, platelet deposition, and
increased neutrophil adherence, which exacerbate the shock state.
Ischemia-reperfusion is a stimulus for leukocyte–endothelial
interaction (12)
. Loss of endothelial-derived nitric oxide
(NO) is known to be one of the earliest manifestations of
ischemia-reperfusion injury (13)
, and this reduced NO is
no longer able to significantly reduce leukocyte–endothelial
interaction (14)
. Leukocyte–endothelial interaction
involves a complex system of adhesion molecules including the
selectins, ß2 integrins, and the immunoglobulin
superfamily (15
16
17)
. Leukocyte interaction with the
endothelium begins with leukocyte rolling, followed by adherence and
transendothelial migration. P-selectin, a member of the selectin family
of adhesion molecules, is believed to play a major role in the initial
phase of leukocyte emigration, which is characterized by the rolling of
leukocytes along the vascular endothelial surface. PMN rolling serves
to tether the unstimulated neutrophil to activated endothelium, thus
bringing the neutrophil in closer contact with adhesion-promoting
substances such as platelet-activating factor (18)
.
Although P-selectin is necessary for early neutrophil contact with the
endothelium, P-selectin-mediated leukocyte–endothelial interaction is
not sufficient to allow neutrophil emigration from the vessel. A firmer
adherence of the neutrophil to the endothelial surface is required for
transendothelial migration (19)
. This firm adherence
involves the interaction of ß2 integrins (i.e.,
CD11/CD18) on the PMN surface and intercellular adhesion molecule 1
(ICAM-1) on the endothelial cell surface (19
, 20)
. ICAM-1
has a molecular mass of 95 kDa (21)
. The binding molecules
for ICAM-1 in neutrophils and monocytes are the integrin ligands LFA-1
and Mac-1 (21
, 22)
. De novo expression of
ICAM-1 has been described in different cell types in inflammatory
lesion, during rejection, and on melanoma cells (23
, 24)
.
Experimental studies have also showed that antibodies against ICAM-1
inhibit leukocyte adhesion to endothelial cells, granulocyte migration
through endothelium, and mixed lymphocyte reactions in vitro
(25
, 26)
. In addition, in vivo administration
of antibodies raised against ICAM-1 reduces neutrophil infiltration
into the inflamed lungs in the rabbit and protects the development of
SAO-induced injury.
Recent evidence suggests that peroxynitrite
(ONOO-), a toxic oxidant formed from the
reaction of NO and superoxide, is present in the reperfused heart
(27
, 28)
, liver (29)
, kidney
(30)
, intestine (31)
, brain
(32)
, and lung (33)
. The biological activity
and decomposition of peroxynitrite is dependent on the cellular or
chemical environment (presence of proteins, thiols, glucose, the ratio
of NO and superoxide, carbon dioxide levels, and other factors), and
these factors influence its toxic potential (34)
.
The cytotoxic effect of peroxynitrite toward the vascular
endothelium has been directly demonstrated in reperfused hearts
(27
, 28)
and cultured human umbilical vein endothelial
cells (35)
. Therefore, in the presence of superoxide
generators, agonist-induced release of NO from ecNOS exerts autocrine
cytotoxic effects via the generation of peroxynitrite, which can be
prevented by inhibition of endothelial NOS in vitro
(36)
. Last, data from in vivo/ex
vivo experiments suggest that under certain conditions, e.g.,
during hypercholesterolemia and atherosclerosis (37)
and
during endotoxic shock (38)
, endothelium-derived NO can
combine with superoxide, and the resulting formation of peroxynitrite
causes an impairment of the endothelial function. To date, the data
generated to support the role of ONOO in vivo are indirect
and have relied mainly on pharmacological (presumed inhibition of ONOO
through reduction of NO by NOS inhibitors) and biochemical (measurement
of rhodamine oxidation or nitrotyrosine formation as markers for
ONOO-mediated reaction) approaches. To explore directly the role(s) of
peroxynitrite in disease, we have used a class of porphyrin-containing
compounds that catalytically decompose peroxynitrite to nitrate
(39)
. These catalysts do not react directly with either
O2 or NO (39)
and therefore can be
used to assess the direct contributions of ONOO (39
40
41)
.
Catalysis is proposed to proceed via an oxo-Fe (IV) intermediate
generated from the metal-promoted cleavage of the O-O bond. Subsequent
recombination with NO2 regenerates the Fe(III)
state and produces nitrate. These catalysts thus dramatically increase
the rate of ONOO isomerization, preempting the formation of oxidizing
radical species and generating the harmless nitrate anion. This mode of
catalysis manifests itself by dramatic shifts in the resulting
nitrite-to-nitrate ratio when compared with the proton-catalyzed
decomposition (39
40
41
42)
. Two of these catalysts,
5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrinato
iron (III) (FeTMPS) and
5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrin
iron (III) (FeTMPyP), were found to be effective both in
vitro and in vivo at removing ONOO and preventing its
cytotoxic effects resulting in potent antiinflammatory effects
(40
, 41)
. More important, pharmacological use of these
catalysts allowed us to demonstrate for the first time that
peroxynitrite indeed plays a key role in vivo in the
development of ischemia and reperfusion. In the present study, we
examined the protective effect of FeTMPS against oxidative stress
during reperfusion of the splanchnic region after induced ischemia
using both biochemical and morphological parameters.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Surgical procedures
Male Sprague-Dawley rats weighing 250–300 g were allowed access
to food and water ad libitum. The rats were anesthetized
with sodium pentobarbital (45 mg/kg, intraperitoneal). After
anesthesia, catheters were placed in the carotid artery and jugular
vein as described previously (43)
. Blood pressure was
monitored continuously by a Maclab A/D converter (AD Instruments, Ugo
Basile, Comerio (Ua), Italy), and stored and displayed on a Macintosh
personal computer. After midline laparotomy, the celiac and superior
mesenteric arteries were isolated near their aortic origins. During
this procedure, the intestinal tract was maintained at 37°C by
placing it between gauze pads soaked with warmed 0.9% NaCl solution.
Rats were observed for a 30 min stabilization period before either
splanchnic ischemia or sham ischemia. SAO shock was induced by clamping
both the superior mesenteric artery and the celiac trunk, resulting in
a total occlusion of these arteries for 45 min. After this period of
occlusion, the clamps were removed. In one study, the various groups of
rats were killed at 60 min for histological examination of the bowel
and for biochemical studies, as described below.
Experimental groups
In the treated group of animals, ONOO decomposition catalyst was
given as an intravenous (i.v.) bolus 10 mg/kg i.v injection 30 min
before reperfusion (SAO+FeTMPS group). In a vehicle-treated group of
rats, vehicle (saline) was given instead of ONOO decomposition catalyst
(SAO group). In separate groups of rats, surgery was performed
identically to the SAO group, except that the blood vessels were not
occluded (time-controlled sham group, Sham). In an additional group of
animals, SAO shock was combined with the administration of ONOO
inactive catalyst compound (dose as above) (SAO+H2TMPS
inactive decomposition catalyst).
Measurement of nitrite/nitrate in the plasma
Nitrite/nitrate levels, an indicator of NO synthesis, were
measured in plasma samples from sham or SAO-shocked rats at 60 min
after reperfusion as described previously (31)
. First,
nitrate in the plasma was reduced to nitrite by incubation with nitrate
reductase (670 mU/ml) and NADPH (160 µM) at room temperature for
3 h. After 3 h, nitrite concentration in the samples was
measured by the Griess reaction by adding 100 µl of Griess reagent
(0.1% naphthalethylenediamine dihydrochloride in
H2O and 1% sulfanilamide in 5% concentrated
H3PO4; vol. 1: 1) to 100
µl samples. The optical density at 550 nm
(OD550) was measured using a Spectramax 250
microplate reader (Molecular Devices Sunnyvale, Calif.). Nitrate
concentrations were calculated by comparison with
OD550 of standard solutions of sodium nitrate
prepared in saline solution.
Measurement of peroxynitrite production
The formation of peroxynitrite was estimated by the
peroxynitrite-dependent oxidation of dihydrorhodamine 123 to rhodamine,
using a previously described method (31)
. In separate
groups, animals were injected with dihydrorhodamine 123 (2 µmol/kg in
0.3 ml saline i.v.) 40 min after reperfusion. Twenty minutes later,
rats were killed and plasma samples were taken for rhodamine
fluorescence evaluation using a Perkin-Elmer fluorometer (Model LS50B;
Perkin-Elmer, Norwalk, Conn.) at an excitation wavelength of 500 nm,
emission wavelength of 536 nm (slit widths 2.5 and 3.0 nm,
respectively). The rate of rhodamine formation, an index of
peroxynitrite production, was calculated using a standard curve
obtained with authentic rhodamine (1–30 nM) prepared in plasma
obtained from untreated rats. Background plasma fluorescence was
subtracted from all samples.
Immunohistochemical localization of nitrotyrosine
Tyrosine nitration, an index of the nitrosylation of proteins by
peroxynitrite and/or oxygen-derived free radicals, was determined by
immunohistochemistry as described previously (31)
. After
reperfusion, tissues were fixed in 10% buffered formalin and 8 µm
sections were prepared from paraffin-embedded tissues. After
deparaffinization, endogenous peroxidase was quenched with 0.3%
H2O2 in 60% methanol for
30 min. The sections were permeabilized with 0.1% Triton X-100 in
phosphate-buffered saline (PBS) for 20 min. Nonspecific adsorption was
minimized by incubating the section in 2% normal goat serum in PBS for
20 min. Endogenous biotin or avidin binding sites were blocked by
sequential incubation for 15 min with avidin and biotin (DBA, Milan,
Italy). The sections were then incubated overnight with 1:1000 dilution
of primary anti-nitrotyrosine antibody (DBA) or with control solutions.
Controls included buffer alone or nonspecific purified rabbit
immunoglobulin G (IgG). Specific labeling was detected with a
biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase
complex (DBA).
Immunohistochemical analysis of P-selectin and ICAM-1
P-selectin and ICAM-1 localization was detected as described
previously (44)
in ileum sections by immunohistochemistry.
After reperfusion, tissues were fixed in 10% buffered formalin;
sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min
and incubated in 2% normal rat serum (for P-selectin evaluation) or
hamster serum (for ICAM-1) for 2 h in order to minimize
nonspecific adsorption. Sections were then incubated overnight 4°C
with monoclonal biotinylated antibodies directed at P-selectin (rat
anti-mouse CD62P) or ICAM-1 (hamster anti-mouse CD54) at dilution
1:500. Controls included buffer alone or nonspecific purified IgG.
Antibody binding sites were visualized by an avidin-biotin peroxidase
complex immunoperoxidase technique (Vector Laboratories, Burlingame,
Calif.) using diaminobenzidine.
Immunocytochemistry photographs (n=5) were assessed by densitometry. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3–266).
Myeloperoxidase (MPO) activity
MPO activity, an index of PMN accumulation, was determined as
described previously (10)
. Intestinal tissues, collected
60 min after reperfusion, were homogenized in a solution containing
0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM
potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000
g at 4°C. An aliquot of the supernatant was then allowed
to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM
H2O2. The rate of change in
absorbance was measured by a spectrophotometer at 650 nm. MPO activity
was defined as the quantity of enzyme degrading 1 µmol of peroxide
min-1 at 37°C and was expressed in µunits
per gram weight of wet tissue.
Leukocyte count
Tail vein blood samples for leukocyte count were taken at
60 min after reperfusion. The number of leukocytes
(WBCx103/mm3) is shown as mean ±
SD.
Malonaldehyde (MDA) measurement
Levels of MDA in the intestinal tissues were determined as an
index of lipid peroxidation, as described by Okhawa et al.
(45)
. Intestinal tissues, collected 60 min after
reperfusion, were homogenized in 1.15% KCl solution. An aliquot (100
µl) of the homogenate was added to a reaction mixture containing 200
µl of 8.1% sodium dodecyl sulfate, 1500 µl of 20% acetic acid (pH
3.5), 1500 µl of 0.8% thiobarbituric acid, and 700 µl distilled
water. Samples were then boiled for 1 h at 95°C and centrifuged
at 3000 g for 10 min. The absorbance of the supernatant was
measured by spectrophotometry at 650 nm.
Measurement of cytokines
Tumor necrosis factor 
(TNF-) and interleukin 1ß
(IL-1ß) levels were evaluated in plasma samples at 60 min after
reperfusion. The assay was carried out by using a colorimetric
commercial kit (Calbiochem-Novabiochem Corporation, San Diego, Calif.).
The enzyme-linked immunoassay has a lower detection of 30 pg/ml.
Light microscopy
For histopathological examination, biopsies of small intestine
were taken 60 min after reperfusion. The tissue were fixed in Dietric
solution (14.25% ethanol, 1.85% formaldehyde, 1% acetic acid) for 1
wk at room temperature, dehydrated by graded ethanol, and embedded in
Paraplast (Sherwood Medical, Mahwah, N.J.). From each biopsy, 7 µm
thick sections were obtained, then stained with trichromic Van Gieson
and studied using light microscopy (Dialux 22 Leitz).
Evaluation of survival
The various groups of rats were monitored for 4 h
after SAO and reperfusion, and survival rates and survival times were
evaluated.
Reagents
Biotin blocking kit, biotin-conjugated goat anti-rabbit IgG, and
avidin-biotin peroxidase complex were obtained from Vector
Laboratories. Primary anti-nitrotyrosine antibody was purchased from
Upstate Biotech (Saranac Lake, N.Y.). Dihydrorhodamine 123 and
rhodamine 123 were purchased from Molecular Probes (Eugene Oreg.).
Peroxynitrite decomposition catalysts were prepared as described
previously (39)
.
Data analysis
All values in the figures and text are expressed as
mean ± standard error of the mean of n observations, where
n represents the number of animals studied. In experiments
involving histology or immunohistochemistry, the figures shown are
representative of at least three experiments performed on different
experimental days. Data sets were examined by one- and two-way analysis
of variance and individual group means were then compared with
Student’s unpaired t test. Nonparametric data were analyzed
with the Fisher’s exact test. A P value of less than 0.05
was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The mean survival time was found to be 90 ± 5 min
(n=27) whereas control sham animals survived for the entire
period of observation (4 h, n=24; Table 1
). Having established the survival time, in another series of
experiments animals were killed either after the period of ischemia or
60 min post reperfusion in order to collect blood and tissues for
biochemical analysis. Reperfusion of the ischemic splanchnic
circulation led to a substantial increase in plasma lipid peroxidation
products, as determined by increased levels of MDA (Fig. 2
), TNF-, and IL1ß (Fig. 3
), and a profound infiltration of neutrophils into the intestine (the
degree of infiltration was much more significant in the lungs than in
the ileum by at least 10- to 15-fold) (Fig. 4
). These inflammatory events were triggered by the reperfusion phase
since no changes were observed when blood or tissues were removed after
the period of ischemia alone (not shown).
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When given i.v 30 min prior to reperfusion, the active peroxynitrite
decomposition catalyst FeTMPS (1–10 mg/kg, n=6), but not
the inactive catalyst H2TMPS (10 mg/kg,
n=6), inhibited in a dose-dependent manner the increased
plasma and ileum levels of MDA (Fig. 2A, B
) as well as
TNF- and IL-1ß (Fig. 3)
. FeTMPS significantly reduced the
neutrophils infiltration into the ileum (Fig. 4)
.
Therefore, FeTMPS (n=6) but not H2TMPS
(n=6), both at 10 mg/kg, prevented the fall in blood
pressure (Fig. 1)
seen after reperfusion and increased the survival
time (90±5% survival at 4 h in FeTMPS-treated rats vs. 0%
survival at 4 h in untreated rats; Table 1
).
NO and peroxynitrite production in splanchnic artery occlusion
shock
There was no change in the plasma levels of nitrate/nitrite at 60
min of the reperfusion period (Fig. 5A
), in agreement with previous observations where we have
demonstrated that the current protocol of ischemia and reperfusion does
not trigger the expression of the inducible isoform of NOS (31
, 46)
. Treatment with FeTMPS or H2TMPS did
not affect baseline nitrite/nitrate levels (Fig. 5A
).
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In agreement with previous observations (31
, 46)
, SAO
shock caused a significant increase in the rhodamine fluorescence of
plasma, indicative of peroxynitrite-induced oxidation of
dihydrorhodamine during the reperfusion phase (Fig. 5B
).
In vivo treatment with FeTMPS reduced the oxidation of
dihydrorhodamine 123 during reperfusion (Fig. 5B
).
At 60 min after reperfusion, ileum sections were taken from sham or
shocked rats in order to determine the immunohistological staining for
nitrotyrosine. Although there was negligible staining in the intestinal
sections of control animals (Fig. 6A
), immunohistochemical analysis, using a specific
anti-nitrotyrosine antibody, revealed a positive staining (14.23±1.2%
of total tissue area) in ileum from SAO-shocked rats (see arrows; Fig. 6B
). In agreement with its effect on plasma dihydrorhodamine
oxidation (Fig. 5B
), FeTMPS reduced the degree of
immunostaining for nitrotyrosine (1.2±0.9% and 1.4±0.6 respectively
of total tissue area) in the reperfused intestine (Fig. 6C
).
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The inactive catalyst H2TMPS did not affect
dihydrorhodamine oxidation (Fig. 6B
) or nitrotyrosine
formation (data not shown).
Leukocyte count
The administration of FeTMPS did not modify the leukocyte count in
sham-shocked rats. In contrast, SAO shock produced a marked leukopenia.
Our data show that the leukocyte count was markedly decreased at 60 min
after reperfusion. The administration of FeTMPS significantly
ameliorated leukopenia (Table 2
). In contrast, H2TMPS did not affect leukopenia
(Table 2)
.
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Immunohistochemical localization of P-selectin and ICAM-1 in the
reperfused intestine
Ileum tissue section obtained from SAO-shocked rats undergoing 45
min of ischemia, followed by 1 h reperfusion, showed positive
staining (3.026±0.04% of total tissue area) for P-selectin localized
in the vascular endothelial cells of microvessels (see arrows
Fig. 7B
). No staining was observed in sham-operated rats (Fig. 7A
). In tissue obtained from FeTMPS-treated rats, no
up-regulation of P-selectin was found during ischemia and reperfusion
(Fig. 7C
). Staining of ileum tissue sections obtained from
sham-operated rats with anti-ICAM-1 antibody showed a specific staining
along vessels, demonstrating that ICAM-1 is constitutively expressed
(1.117±0.035% of total tissue area) in endothelial cells (see arrows;
Fig. 8A
). After 1 h of reperfusion, the staining intensity
increased substantially along vessels and in the necrotic tissues
(6.07±0.052% of total tissue area) (Fig. 8B
, arrows).
Sections from FeTMPS-treated rats did not reveal any up-regulation of
the constitutive ICAM-1 (Fig. 8C
, arrow), which normally was
expressed in the endothelium along the vascular wall (2.11±0.09%; of
total tissue area). H2TMPS (inactive catalyst)
did not affect adhesion molecule expression (data not shown).
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Histological change
Histological examinations of the small intestine at 60 min of
reperfusion (see Fig. 9
) revealed pathological changes. Ileum section showed inflammatory
infiltration (see arrows) by neutrophil and lymphocytes extending
through the wall and concentrated below the epithelial layer (Fig. 9B
). FeTMPS-treated rats show a significant reduction in
organ injury (Fig. 9C
). H2TMPS
(inactive catalyst) did not affect ileum injury (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Peroxynitrite is
also stable enough to cross several cell diameters to reach target
cells before becoming protonated and decomposing (48)
. It
is therefore not surprising that evidence is increasing for a major
role for peroxynitrite in the development of tissue damage during
inflammation (49
, 50)
as well as in human subjects during
sepsis (51)
. Nevertheless, as indicated earlier,
implications for the participation of peroxynitrite in disease have
relied on indirect evidence, mainly through the measurement of
nitrotyrosine. The presence of nitrotyrosine at the site of injury does
not prove, however, that peroxynitrite caused the damage, but simply
that it is formed. Nitrotyrosine formation, along with its detection by
immunostaining, was initially proposed as a relatively specific means
for detection of the footprint of peroxynitrite (52)
.
Recent evidence indicates, however, that certain other reactions can
also induce tyrosine nitration: the reaction of nitrite with
hypochlorous acid and the reaction of MPO with hydrogen peroxide can
lead to the formation of nitrotyrosine (53)
. Increased
nitrotyrosine staining is thus considered an indication of increased
nitrosative stress rather than a specific marker of peroxynitrite. Our
results using pharmacological agents specific for peroxynitrite
decomposition (40
41
42)
clearly indicate that peroxynitrite
is the major cause of the intestinal damage after ischemia and
reperfusion. FeTMPS and FeTMPyP, which have been shown to be active
peroxynitrite decomposition catalysts (39
40
41)
, protected
against microvascular injury, lipid peroxidation, and epithelial cell
injury. In addition, we have recently evidenced the efficacy of ONOO
decomposition catalysts in vitro and in vivo
(40
41
42)
. At the cellular level, the catalyst FeTMPS was
cytoprotective against exogenously added ONOO with an
EC50 of 
3.5 µM, a concentration almost
1/60th of the ONOO added. This protection correlated well with a
reduction in the nitrotyrosine content of cellular proteins released
after ONOO insult, an observation consistent with the proposed
mechanism of action for these compounds. Moreover, we report that
neither TMPS, an inactive structurally related compound, nor free Fe
was cytoprotective (40
41
42)
. Our data demonstrate that FeTMPS treatment exerts an important protective effect against SAO shock. In fact, the present study provides evidence that 1) FeTMPS reduced the development of SAO-induced shock, 2) FeTMPS reduced morphological injury in SAO shock, 3) FeTMPS reduced lipid peroxidation, 4) in FeTMPS-treated rats subjected to occlusion and reperfusion of the splanchnic artery, up-regulation of P-selectin and ICAM-1 in the ileum was completely abolished, 5) FeTMPS reduced neutrophil infiltration, and 6) FeTMPS reduced cytokine production.
Endothelial cells appear to be major regulators of neutrophil
traffic, regulating the process of neutrophil chemoattraction,
adhesion, and emigration from the vasculature to the tissue. During the
early phase of reperfusion, P-selectin is rapidly released to the cell
surface from preformed storage pools after exposure to certain stimuli,
such as hydrogen peroxide, thrombin, histamine, or complement, and
allows the leukocytes to roll along the endothelium (16
, 20)
. ICAM-1 constitutively expressed on the surface of
endothelial cells is then involved in neutrophil adhesion (16
, 54)
. Hypoxic endothelial cells synthesize proinflammatory
cytokines, which can up-regulate endothelial expression of the
constitutive adhesion molecule ICAM-1 in autocrine fashion
(55)
. Significant expression of ICAM-1 in microvessels of
previously ischemic tissues occurs within 1 h after reperfusion
(56)
. The expression of P-selectin and ICAM-1 corresponds
with the induction of neutrophil recruitment, which is maximal within
the first hour of reperfusion and persists at a lower rate in the late
phase of reperfusion (43
, 57)
. In accordance with these
findings, we observed that a 45 min occlusion of the splanchnic artery,
followed by 1 h reperfusion, induced the synthesis of
proinflammatory cytokines (TNF- and IL-1ß) and the appearance of
P-selectin on the endothelial vascular wall, and up-regulated the
surface expression of ICAM-1 on endothelial cells. FeTMPS treatment
reduced proinflammatory cytokines production (Fig. 3)
and abolished the
expression of P-selectin and the up-regulation of ICAM-1 (Fig. 4C
, Fig. 5C
) while not affecting the constitutive
levels of ICAM-1 on endothelial cells (data not shown). Numerous cell
types release superoxide anions during the inflammatory response and
these include endothelial cells, epithelial cells, macrophages, and
neutrophils (58
, 59)
. As seen here, neutrophils do
infiltrate into the intestine and their activation at the site of
injury may contribute to O2- production and
subsequent damage. Recent evidence supports a damaging role of
superoxide in the gastrointestinal tract (60)
. Neutrophils
are probably the main source for the release of superoxide and
presumably play a major role in the generation of peroxynitrite.
Evidence that reactive nitrogen and oxygen species can exert
proinflammatory properties includes recruitment of neutrophils at sites
of inflammation, formation of chemotactic factors (58
, 59)
, DNA damage, depolymerization of hyaluronic acid and
collagen (61
, 62)
, lipid peroxidation, and release of
cytokines such as TNF- and IL-1ß.
We can hypotheses that the mechanism of this peroxynitrite
decomposition catalyst may be related to a prevention of
peroxynitrite-induced endothelial injury as well as to the inhibition
of reactive nitrogen and oxygen intermediates proinflammatory
properties. In other words, we propose a positive feedback cycle in SAO
shock (Fig. 10
). Peroxynitrite decomposition catalyst would intercept this cycle at
the level of endothelial injury and cytokine release. This model would
explain the reduction of cytokine formation and the reduction of
P-selectin and ICAM-1 and PMNs infiltration during reperfusion in the
FeTMPS-treated rats.
|
In conclusion, we have directly demonstrated that peroxynitrite is associated with the intestinal damage evoked by ischemia and reperfusion. The peroxynitrite decomposition catalysts may offer a novel approach for manipulating the pathological sequelae that are associated with ischemia and reperfusion. The further use of these catalysts as pharmacological tools in animals models of human disease may lead to a better understanding of when and where peroxynitrite plays a key role(s) in the development of inflammatory diseases such as ischemia and reperfusion. This, in turn, should provide more effective treatment strategies in the clinic for disease. Peroxynitrite decomposition catalysts are not only useful tools for the pharmacological dissection of free radical-mediated pathology, but also offer promise as disease-modifying therapeutic agents capable of preserving the positive aspect of the double-edged sword of nitric oxide action.
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FOOTNOTES |
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REFERENCES |
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  P. Pacher, J. S. Beckman, and L. Liaudet Nitric Oxide and Peroxynitrite in Health and Disease Physiol Rev, January 1, 2007; 87(1): 315 - 424. [Abstract] [Full Text] [PDF]
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