Serine Protease Inhibition Reduces Post-Ischemic Granulocyte Recruitment in Mouse Intestine

INSERM, U1043, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.
American Journal Of Pathology (Impact Factor: 4.59). 11/2011; 180(1):141-52. DOI: 10.1016/j.ajpath.2011.09.031
Source: PubMed
ABSTRACT
Proteases and proteinase-activated receptor (PAR) activation are involved in several intestinal inflammatory conditions. We hypothesized that serine proteases and PAR activation could also modulate the intestinal injury induced by ischemia-reperfusion (I-R). C57Bl/6 mice were subjected to 90 minutes of intestinal ischemia followed or not by reperfusion. Sham-operated animals served as controls. After ischemia, plasma and tissue serine protease activity levels were increased compared to the activity measured in plasma and tissues from sham-operated mice. This increase was maintained or further enhanced after 2 and 5 hours of reperfusion, respectively. Trypsin (25 kDa) was detected in tissues both after ischemia and 2 hours of reperfusion. Treatment with FUT-175 (10 mg/kg), a potent serine protease inhibitor, increased survival after I-R, inhibited tissue protease activity, and significantly decreased intestinal myeloperoxidase (MPO) activity and chemokine and adhesion molecule expression. We investigated whether serine proteases modulate granulocyte recruitment by a PAR-dependent mechanism. MPO levels and adhesion molecule expression were significantly reduced in I-R groups pre-treated with the PAR(1) antagonist SCH-79797 (5 mg/kg) and in Par(2)(-/-)mice, compared, respectively, to vehicle-treated group and wild-type littermates. Thus, increased proteolytic activity and PAR activation play a pathogenic role in intestinal I-R injury. Inhibition of PAR-activating serine proteases could be beneficial to reduce post-ischemic intestinal inflammation.

Full-text

Available from: Martin Steinhoff, Dec 19, 2015
Gastrointestinal, Hepatobiliary, and Pancreatic Pathology
Serine Protease Inhibition Reduces Post-Ischemic
Granulocyte Recruitment in Mouse Intestine
Thomas Gobbetti,*
†‡§
Nicolas Cenac,*
†‡
Jean-Paul Motta,*
†‡
Corinne Rolland,*
†‡
Laurence Martin,*
†‡
Patricia Andrade-Gordon,
Martin Steinhoff,
Elisabetta Barocelli,
§
and
Nathalie Vergnolle*
†‡
**
From INSERM,* U1043, CNRS,
U5282, and the Université de
Toulouse,
UPS, Centre de Physiopathologie de Toulouse Purpan
(CPTP), Toulouse, France; the Department of Pharmacological,
Biological and Applied Chemical Sciences,
§
University of Parma,
Parma, Italy; RW Johnson & Johnson,
Spring House,
Pennsylvania; the Departments of Dermatology and Surgery,
University of California San Francisco, San Francisco,
California; and the Department of Physiology and
Pharmacology,
ⴱⴱ
University of Calgary, Calgary, Alberta, Canada
Proteases and proteinase-activated receptor (PAR) ac-
tivation are involved in several intestinal inflamma-
tory conditions. We hypothesized that serine pro-
teases and PAR activation could also modulate the
intestinal injury induced by ischemia-reperfusion
(I-R). C57Bl/6 mice were subjected to 90 minutes of
intestinal ischemia followed or not by reperfusion.
Sham-operated animals served as controls. After isch-
emia, plasma and tissue serine protease activity levels
were increased compared to the activity measured in
plasma and tissues from sham-operated mice. This
increase was maintained or further enhanced after 2
and 5 hours of reperfusion, respectively. Trypsin (25
kDa) was detected in tissues both after ischemia and 2
hours of reperfusion. Treatment with FUT-175 (10
mg/kg), a potent serine protease inhibitor, increased
survival after I-R, inhibited tissue protease activity,
and significantly decreased intestinal myeloperoxi-
dase (MPO) activity and chemokine and adhesion
molecule expression. We investigated whether serine
proteases modulate granulocyte recruitment by a
PAR-dependent mechanism. MPO levels and adhesion
molecule expression were significantly reduced in I-R
groups pre-treated with the PAR
1
antagonist SCH-
79797 (5 mg/kg) and in Par
2
/
mice, compared, re-
spectively, to vehicle-treated group and wild-type lit-
termates. Thus, increased proteolytic activity and PAR
activation play a pathogenic role in intestinal I-R in-
jury. Inhibition of PAR-activating serine proteases
could be beneficial to reduce post-ischemic intestinal
inflammation.
(Am J Pathol 2012, 180:141–152; DOI:
10.1016/j.ajpath.2011.09.031)
Acute mesenteric ischemia is a potentially fatal abdomi-
nal emergency implicated in a large array of pathological
conditions that involve a critical reduction of blood flow to
the gut as consequence of surgical states (abdominal
aortic aneurism surgery, cardiopulmonary bypass), ves-
sel occlusions (embolism or thrombosis), septic or hemo-
dynamic shock, intestinal hernias, and also transplants of
the small intestine.
1–3
Although acute mesenteric isch-
emia accounts for only 1% to 2% of gastrointestinal ill-
nesses, it still causes a high in-hospital mortality rate
(60% to 80%).
3
The pathogenesis of ischemic injury is related to the
interruption of blood supply to the gut, which results in
rapid metabolic damage to active tissues, particularly to
the labile cells of mucosa. Paradoxically, reoxygenation,
depending on the time and intensity of the ischemia,
further increases the damage, causing an additional cell
injury known as reperfusion injury. This acute inflamma-
tory response causes a rapid deterioration of the intesti-
nal barrier, leading to intestinal bacterial translocation
through the epithelial mucosa to extra-intestinal sites
(mesenteric lymph nodes, liver, and spleen).
3,4
The sub-
sequent sepsis and production of proinflammatory mole-
cules leads to the development of a systemic inflamma-
tory response syndrome, which can progress to multiple
organ failure involving organs such as liver, heart, kid-
neys, and lungs.
2,5,6
Proteases, particularly serine proteases, act as signal-
ing molecules that are able to send specific signals to
cells involved in intestinal inflammatory responses
through the activation of a subclass of four G protein–
Supported by INSERM-Avenir program, Agence Nationale de la Re-
cherche, Fondation Bettencourt-Schueller, and Fondation Schlum-
berger (N.V.).
Accepted for publication September 20, 2011.
Address reprint requests to Nathalie Vergnolle, Ph.D., INSERM U1043,
CHU Purpan, BP3028, 31024 Toulouse-Cedex, France, Tel: (33)
562744500. E-mail: nathalie.vergnolle@inserm.fr.
The American Journal of Pathology, Vol. 180, No. 1, January 2012
Copyright © 2012 American Society for Investigative Pathology.
Published by Elsevier Inc. All rights reserved.
DOI: 10.1016/j.ajpath.2011.09.031
141
Page 1
coupled proteinase-activated receptors (PAR
1
, PAR
2
,
PAR
3
, and PAR
4
).
7–12
To activate those receptors, which
are largely expressed in the gut, proteases cleave at
specific sites within the extracellular N-terminus domain
of PARs. This cleavage unmasks a new N-terminal se-
quence that acts as a tethered ligand, which binds to the
receptor to initiate multiple signaling cascades.
8
Several
findings report that direct injection of proteases such as
thrombin, trypsin, tryptase, or selective agonist for PAR
1
and PAR
2
into the paw of rodents produces inflamma-
tion.
13–15
In addition, we have demonstrated that luminal
administration of selective peptides, agonists for PAR
1
,
PAR
2
, and PAR
4
provoked a colonic inflammatory re-
sponse.
16 –21
All these observations provide the background for the
hypothesis that proteases and PARs could play a major
role in the pathogenesis of intestinal inflammation asso-
ciated with ischemia-reperfusion (I-R) injury. Although a
role for proteases in ischemic tissues has been sug-
gested in kidney,
22
liver,
23,24
heart,
25,26
and digestive
tract,
27,28
it is not clear what role proteases may play in
ischemic disorders of the intestine, which is, above all,
the organ the most exposed to a variety of proteases.
We used a model of small intestine ischemia devel-
oped in mice by reversible occlusion of the superior
mesenteric artery for 90 minutes, followed by 0, 2, or 5
hours of reperfusion. By combining pharmacological and
gene-deletion approaches, we demonstrate here the piv-
otal role of serine proteases, through the activation of
PAR
1
and PAR
2
, in inflammatory damage associated with
intestinal ischemia-reperfusion.
Materials and Methods
Animals
C57Bl/6 male mice (8 weeks old) were obtained from
Janvier (Le Genest Saint Isle, France). PAR
2
/
and wild-
type littermates (Par
2
/
) were originally provided by
Johnson & Johnson Pharmaceutical Research Insti-
tute.
29,30
Animals were kept under pathogen-free condi-
tions and were given free access to food and water. All of
the experimental protocols were approved by local ani-
mal care and ethics committees and followed the guide-
lines of French Councils on Animal Care.
Surgical Procedure and Treatments
Mice were anesthetized with sodium pentobarbital (Pen-
tobarbital sodique, Ceva Santé Animale, Libourne,
France) (50 mg/kg intraperitoneally). After abdominal lap-
arotomy, the small bowel was retracted to the left and the
superior mesenteric artery was temporarily occluded us-
ing a microvascular clip to cause ischemia. After 90 min-
utes, the clip was gently removed, allowing reperfusion.
Mice were sacrificed right after the ischemic period (time
0), 2, or 5 hours after reperfusion. Sham-operated (SO)
animals, in which abdominal laparotomy and artery iso-
lation were performed without occlusion of the vessel,
served as controls. After the surgical procedure, the mid-
line incision of the abdominal wall was closed by two-
layer sutures. After recovering from anesthesia, animals
were returned to their cages. At the end of the experi-
ments, animals were anesthetized with sodium pentobar-
bital to collect blood through cardiac puncture. Euthana-
sia was performed by cervical dislocation. Heparinized
blood samples were centrifuged at 7500 g for 10 min-
utes to obtain plasma. For biochemical analysis, ileal
tissue was excised and processed.
Mice were treated as follows: the serine protease in-
hibitor FUT-175 (10 mg/kg dissolved in saline; Santa Cruz
Biotechnology, tebu-bio, Le Perray en Yvelines Cedex,
France) was administered intravenously at the beginning
of ischemia and repeated at the moment of reperfusion;
the PAR
1
antagonist SCH-79797 (5 mg/kg dissolved in
carboxymethylcellulose; Tocris Bioscience, Bristol, UK)
was administered intraperitoneally twice, at 18 hours and
2 hours before the surgery. A similar surgical procedure
was used for both the Par
2
/
and Par
2
/
mice.
Protease Activity Assay
Trypsin-like proteolytic activity was measured both in in-
testinal tissue and plasma using a microplate reader
NOVOstar (BMG Labtech, Champigny s/Marne, France).
On sacrifice, plasma samples were collected and a piece
of ileal tissue was excised and rinsed in 1 PBS to
remove all intraluminal content and was then homoge-
nized in 1 mL of 1 PBS (pH 7.2) with 1% NP-40, 0.5%
sodium deoxycholate, 0.1% SDS using Precellys 24 ho-
mogenizer in Precellys lysing CK14 tubes (Bertin Tech-
nologies, Ozyme, France). The homogenized tissues
were centrifuged at 5000 g for 5 minutes. The trypsin-
like activity was measured using tosyl-Gly-Pro-Arg p-ni-
troanilide (150
mol/L; Sigma, Saint Quentin Fallavier,
France) as substrate in 100 mmol/L Tris/HCl, 1 mmol/L
CaCl
2
buffer (pH 8). The hydrolysis rate was measured at
37°C over a 30-minute period in absorbance at 405 nm.
31
Activity was standardized to the rate generated by trypsin
of known concentration from porcine pancreas (Sigma).
Plasma activity was expressed as U/mL, whereas tissue
activity was normalized to the homogenate protein con-
centrations determined with a BCA kit (Pierce, Thermo
Scientific, Courtaboeuf, France) and expressed as U/mg
protein. When required, protease inhibitors were added
to tissue supernatants or plasma samples: E-64 10
mol/L (tebu-bio), Pepstatin A 1
mol/L (Sigma), AESBF
10 mmol/L (tebu-bio), FUT-175 50
mol/L (tebu-bio). Pro-
tease inhibitor doses were chosen according to previous
data reported in the literature.
32–37
Myeloperoxidase Activity
Myeloperoxidase (MPO) activity was measured as an
index of granulocyte infiltration as previously described in
ileal tissues harvested at the time of sacrifice.
38
Briefly,
ileal tissue samples were homogenized in a solution of
0.5% hexadecyltrimethylammonium bromide dissolved in
phosphate buffer solution (pH 6) using Precellys 24 ho-
mogenizer in Precellys lysing CK14 tubes (Bertin Tech-
nologies). The homogenized tissues were centrifuged at
13,000 g for 5 minutes (4°C), and the supernatants
142 Gobbetti et al
AJP January 2012, Vol. 180, No. 1
Page 2
were placed on 96 well plates. Buffer, supplemented with
1% hydrogen peroxide/O-dianisidine dihydrochloride
(Sigma), was added to each well. Optical density read-
ings were taken for 1 minute at 30-second intervals at 450
nm using a microplate reader NOVOstar (BMG Labtech).
Activity was normalized to the sample protein concentra-
tion determined with a BCA kit (Pierce) and expressed as
mU/mg protein.
Chemokines Protein Expression
Ileal tissue samples harvested at the time of sacrifice
were homogenized in 700
L of cell lysis buffer [20
mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L
Na
2
EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L
sodium pyrophosphate, 1 mmol/L
-glycerophosphate, 1
mmol/L Na
3
VO
4
,1
g/mL leupeptin; Sigma] supple-
mented with antiprotease cocktail (SIGMAFAST; Sigma)
using Precellys 24 homogenizer in Precellys lysing CK14
tubes (Bertin Technologies). After centrifugation (10,000
g 10 minutes, 4°C), supernatants were filtered on
QIAshredder columns (Qiagen, Courtaboeuf Cedex,
France). Fifty microliters of this homogenate were used
for simultaneous measurement of keratinocyte chemoat-
tractant (KC) and monocyte chemoattractant protein
(MCP-1), using cytometric bead array on fluorescent cell
sorter FACSCalibur, according to the manufacturer’s in-
structions (BD Biosciences, Le Pont de Claix, France).
39
Raw values were normalized to the sample protein con-
centration determined with a BCA kit (Pierce). Chemokine
concentrations were extrapolated from standard curves
with the help of FCAP Array software (Soft Flow, St. Louis
Park, MN) and expressed as pg/mg protein. In accor-
dance with the manufacturer’s information, only values
above the limit of cytokine detection were considered.
Immunohistochemistry
Samples of mouse small intestines were formalin fixed,
embedded in paraffin, and cut into 5-
m sections. To
reveal antigenic motifs, slides were boiled for 40 minutes
at pH 6 after deparaffinization and incubated in sodium
borohydride 1% (Sigma) to remove autofluorescence.
Slides were then washed three times in phosphate-buff-
ered saline (PBS; Invitrogen, Cergy Pontoise, France)
with 1% bovine serum albumin (Sigma) and 0.5% Triton
X-100, and were incubated overnight with primary anti-
body directed against PAR
1
(rabbit; AbCam, Paris,
France), PAR
2
(rabbit; Santa Cruz Biotechnology, tebu-
bio), or CD45 (goat; R&D Systems, Lille, France).
40
After
three washes with 1 PBS, slides were incubated with
appropriate secondary antibody (Alexa Fluor 680, or Al-
exa Fluor 555; Invitrogen) for 30 minutes. Slides were
mounted with Prolong (Invitrogen), and intestinal samples
were visualized using a Zeiss 710 Meta confocal fluores-
cence microscope (Le Pecq, France) with a 20 or 63
objective.
41
Survival Rates
The survival rates in each group were monitored from the
beginning of the surgery to the end of the reperfusion
times.
Western Blot Analysis
Western blot analysis was performed on ileal samples
collected from SO animals and mice subjected to 90
minutes of ischemia followed by 2 hours of reperfusion.
Proteins were extracted by homogenizing the tissues in 1
mL of 1 PBS (pH 7.2) with 1% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS, supplemented with antipro-
tease cocktail (SIGMAFAST; Sigma), using Precellys 24
homogenizer in Precellys lysing CK14 tubes (Bertin Tech-
nologies). The homogenized tissues were centrifuged at
5000 g for 5 minutes. Samples were normalized for
total protein concentration using the BCA kit (Pierce) and
resuspended in Laemmli buffer (Bio-Rad, Marnes-la-Co-
quette, France). Proteins (40
g/30
L) were resolved by
SDS-polyacrylamide gel electrophoresis on 8% gels and
were transferred to nitrocellulose membranes (Bio-Rad).
The membranes were probed with antibodies against
actin (ab1801, 1:1000; Abcam, Paris, France), trypsin
(sc-67388, 1:200; Santa Cruz Biotechnology, Heidelberg,
Germany), intracellular adhesion molecule 1 (ICAM-1)
(sc-1511; Santa Cruz Biotechnology), vascular cell adhe-
sion molecule I (VCAM-1) (sc-1504; Santa Cruz Biotech-
nology), and
-2 macroglobulin (
-2M) (sc-28870; Santa
Cruz Biotechnology). Western blots were probed with
donkey anti-goat secondary antibody conjugated to
IRdye 800 (1:1000; LI-COR, Cergy-Pontoise-Cedex,
France) and donkey anti-rabbit Alexa Fluor 555 (Invitro-
gen). Blotted proteins were detected and quantified us-
ing the Odyssey infrared imaging system (LI-COR NE).
Statistical Analysis
Data are presented as mean SEM. Analyses were
performed using GraphPad Prism 5 software (GraphPad
Software, La Jolla, CA). All data were normally distrib-
uted. Between-group comparisons were performed by
Student’s unpaired 2-tailed t-test. Multiple compari-
sons for within group I-R were performed by repeated-
measures one-way analysis of variance followed by
Tukey’s procedure. Statistical significance was ac-
cepted at P 0.05.
Results
Intestinal Ischemia-Reperfusion Injury Is
Associated with Protease Release
Intestinal I-R caused inflammation to ischemic tissues as
observed by increased intestinal MPO activity, a marker
of granulocyte infiltration, and mortality (Figure 1, A and
B). Specifically, superior mesenteric artery occlusion
without reperfusion period (0hR) did not significantly in-
crease MPO activity. However, 90 minutes of ischemia
Proteases and Intestinal Ischemia 143
AJP January 2012, Vol. 180, No. 1
Page 3
followed by 2 hours (2hR) or 5 hours (5hR) of reperfusion
caused a significant increase in MPO activity, compared
to the corresponding SO group (71.6 6.9 versus 23.4
2.3 mU/mg protein, P 0.001, and 134.1 48.1 versus
18.0 2.7 mU/mg protein, P 0.01) (Figure 1A). The
experimental condition of ischemia alone allowed 100%
animal survival rate. Mortality only appeared after reper-
fusion, and its rate increased with time of reperfusion (I-R
2hR 25% and I-R 5hR 70%) (Figure 1B). We investigated
trypsin-like proteolytic activity balance associated with
intestinal ischemic insult in ileal tissues and in plasma, by
measuring the hydrolysis of the substrate tosyl-Gly-Pro-
Arg p-nitroanilide. Proteolytic activity released in plasma
after 90 minutes of ischemia (0hR) was twofold higher
than the activity in the corresponding SO group (2.8
0.4 versus 1.4 0.11 U/mL, P 0.05). This increase was
not only maintained, but also further enhanced after rep-
erfusion, compared to SO group (2hR 5.3 0.74 versus
1.3 0.26 U/mL, P 0.001 and 5hR 12.5 6.2 versus
1.5 0.24 U/mL, P 0.05) (Figure 1C). Similarly, isch-
emia caused a significant increase of tissue proteolytic
activity (9.4 2.56 versus 0.9 1.4 U/mL, P 0.01), and
this activity was significantly maintained after 2 and 5
hours of reperfusion compared to the corresponding SO
group (7.4 2.2 versus 1.5 0.7 U/mL, P 0.01 and
19.9 10.6 versus 1.4 0.7 U/mL, P 0.01) (Figure
1D). These findings show that ischemia, as well as rep-
erfusion, is associated with an intense release of proteo-
lytic activity (directed at an arginine site), both within
ischemic tissues and in plasma.
Serine Proteases Are the Major Proteases
Dosed in Plasma and Ischemic Tissues on
Ischemia-Reperfusion Injury
To identify the proteases responsible for increasing pro-
teolytic activity during I-R, different specific protease in-
hibitors were incubated in vitro (20 minutes at room tem-
perature) with tissue homogenates or plasma samples
from animals subjected to 90 minutes of ischemia, fol-
lowed by 2 hours of reperfusion. Proteolytic activity assay
using the same substrate as in Figure 1 was then per-
formed on those samples.
Cysteine protease inhibitor E-64 (10
mol/L) and as-
partate proteases inhibitor Pepstatin A (1
mol/L) signif-
icantly reduced plasma proteolytic activity respectively to
23% and 34% (Figure 2A), whereas the same inhibitors
did not affect tissue proteolytic activity (Figure 2B), even
when tested at 100 and 1000 times higher concentrations
(data not shown). These data suggest that cysteine and
aspartate proteases are most likely not responsible for
the proteolytic activity measured in tissues after I-R,
whereas in plasma, some cysteine and aspartate pro-
teases are active after I-R. Incubation with two different
serine protease inhibitors, compound FUT-175 (50
mol/L) and AEBSF (10 mmol/L), almost completely abol-
ished the proteolytic activity induced by I-R both in intes-
tinal tissue (87% for AESBF and 94% for FUT-175) and
plasma samples (respectively, 93% and 96%) (Figure 2,
A and B).
Figure 1. Effects of 90 minutes of ischemia followed by different times of
reperfusion (0 hours, 2 hours, 5 hours) on intestinal MPO activity (A),
survival rate (B), plasma protease activity (C), and tissue protease activity (D)
in sham-operated (SO) (white bars) or I-R (black bars) mice. In B, SO groups
showed 100% survival rate (data not shown). In A, C, and D, data represent
means SEM of 8 to 12 animals per group. *P 0.05, **P 0.01, and
***P 0.001 versus the corresponding SO group;
P 0.05,
††
P 0.01, and
†††
P 0.001 versus the indicated I-R group.
144 Gobbetti et al
AJP January 2012, Vol. 180, No. 1
Page 4
Taken together, these data suggest that the arginine-
directed proteolytic activity detected during I-R injury in
tissues and in plasma was, for the most part, due to
serine proteases.
Serine Protease Inhibitor Prevents Intestinal
Ischemia-Reperfusion Injury
Based on these results, we then investigated in vivo the
effects of serine protease inhibition in the I-R model by
injecting, intravenously, the serine protease inhibitor FUT-
175 (10 mg/kg in saline) at the moment of ischemia and
at the beginning of the 2-hour reperfusion period.
Systemic treatment with FUT-175 did not significantly
reduce the level of proteolytic activity in plasma (Figure
3A), but completely abolished tissue proteolytic activity
(0.35 0.1 versus 4.5 1.2 U/mg protein, P 0.01;
Figure 3B) released after I-R. Interestingly, basal proteo-
lytic activity in plasma of SO groups was significantly
reduced by FUT-175 treatment (1.3 0.2 versus 0.4
0.1 U/mL, P 0.05; Figure 3A). Systemic treatment with
the serine protease inhibitor FUT-175, significantly de-
creased granulocyte recruitment induced by I-R, as ob-
served by reduced intestinal MPO activity (74.6 6.9
versus 40.6 3.7 mU/mg protein, P 0.01, Figure 3C).
In addition, the survival rate was significantly increased in
FUT-175-treated mice, compared to I-R saline group
(71% versus 95%, P 0.05; Figure 3D).
Because serine protease inhibition significantly de-
creased leukocyte recruitment to ischemic and reper-
fused tissues, the possibility that this lack of inflammatory
cell recruitment was due to deficient expression of
chemokines or adhesion molecules was investigated. I-R
markedly increased KC and MCP-1 intestinal concentra-
tions compared to the sham-operated group. The treat-
ment of mice submitted to I-R with FUT-175 significantly
reduced the expression of both KC and MCP-1 in jejunal
tissues, compared to vehicle-treated mice (KC: 31.1
4.7 versus 59.3 10.1, P 0.05, and MCP-1: 89.4
15.3 versus 181.3 37.9, P 0.05) (Figure 3, E and F).
I-R also modulated the expression of endothelial adhe-
sion molecules such as ICAM and VCAM. The expression
levels of both ICAM-1 and VCAM-1 were increased in the
I-R group compared to SO mice; the treatment with FUT-
175 significantly decreased the expression of both
ICAM-1 and VCAM-1 in I-R intestinal tissue, compared to
I-R vehicle-treated mice (Figure 3, G and H).
These data demonstrate that the administration of a
serine protease inhibitor at the moment of ischemia and
at the beginning of reperfusion protects from granulocyte
infiltration into tissues, through the reduction of chemo-
kine protein expression and through a down-regulation of
adhesion molecules.
PAR
1
and PAR
2
Expression in the Mouse Small
Intestine
As shown in Figure 4, PAR
1
was weakly expressed in the
mucosa of mouse small intestine: faintly in cells at the
base of villi, and clearly on blood vessels. In contrast,
PAR
2
was strongly detected in epithelial cells and colo-
calized with 88.1% of CD45-positive cells, whereas PAR
1
did not colocalize with the CD45 marker in the small
intestine. The surgical procedure (SO animals) and the
condition of ischemia alone or ischemia followed by 2
hours of reperfusion did not change the pattern of cell
types expressing PAR
1
and PAR
2
(not shown).
Ischemia-Reperfusion–Induced Granulocyte
Recruitment Is PAR
1
and PAR
2
Dependent
As shown in Figure 5, A and B, neither the intraperitoneal
administration of a selective PAR
1
antagonist (compound
SCH-79797, 5 mg/kg in carboxymethylcellulose), nor
PAR
2
deficiency, modified plasma and tissue proteolytic
activity induced by 90 minutes of ischemia followed by 2
hours of reperfusion, compared to I-R vehicle. However,
SCH-79797 treatment and PAR
2
deficiency caused a sig-
nificant decrease in MPO activity (40.58 6.4 versus
89.5 13.8 mU/mg protein, P 0.05, and 65.0 8.0
Figure 2. Effects on proteolytic activity of the addition of specific protease
inhibitors to samples collected from plasma (A) and intestinal tissue (B).
Different protease inhibitors (E-64 10
mol/L, Pepstatin A 1
mol/L, AESBF
10 mmol/L, and FUT-175 50
mol/L) were incubated 20 minutes at room
temperature with plasma samples or tissue homogenates from animals sub-
jected to ischemia followed by 2 hours of reperfusion. Proteolytic activity was
then performed on those samples using tosyl-Gly-Pro-Arg p-nitroanilide as
substrate. Data represent means SEM of 8 to 12 animals per group.
P
0.05,
††
P 0.01,
†††
P 0.001 versus vehicle.
Proteases and Intestinal Ischemia 145
AJP January 2012, Vol. 180, No. 1
Page 5
versus 106.8 12.7 mU/mg protein, P 0.05; Figure 5,
C and D) and significantly reduced the levels of both
ICAM-1 and VCAM-1 (Figure 5, E, F, G, and H) in I-R
mice, compared to the vehicle I-R group and I-R Par
2
/
mice (Figure 5, C and D). Interestingly, I-R mice treated
with the PAR
1
antagonist SCH-79797 (KC: 33.9 6.4
versus 79.1 14.7, P 0.05, and MCP-1: 76,5 11.6
versus 140.5 26.8; P 0.05; Figure 5, I and J), but not
I-R PAR
2
-deficient mice (KC: 73.6 26.6 versus 73.2
26.3 and MCP-1: 145.3 54.9 versus 245.1 92.1;
Figure 5, K and L) showed a significant reduction in
protein expression levels of both KC and MCP-1 com-
pared to I-R vehicle and I-R Par
2
/
mice. These findings
suggest that serine protease release induced by I-R oc-
curred independently of PAR
1
or PAR
2
activation, but
both receptors contribute to post-ischemic intestinal neu-
trophil recruitment, although through distinct mecha-
nisms involving differential regulation of adhesion mole-
cules or chemokine expression.
Ischemia-Reperfusion Induces Trypsin and
Decreases
-2M Expression in Ileal Tissue
Levels of the serine protease trypsin, thrombin, plasmin,
and chymotrypsin were measured using Western blotting
technique in ileal tissues of SO mice and mice subjected
to 90 minutes of ischemia alone or followed by 2 hours of
Figure 3. In vivo effects of systemic treatment with the serine protease inhibitor FUT-175 (10 mg/kg i.v.) on proteolytic activity dosed in plasma (A) and in
intestinal tissue (B), on MPO activity (C), survival rate (D), chemokine (KC and MCP-1) protein expression (E and F), and adhesion molecules (ICAM-1 and
VCAM-1) (G and H). Sham-operated (SO) group is represented by white bars and I-R group (90 minutes 2 hours reperfusion) by black bars. Data in A, B, C,
E, and F represent means SEM of 8 to 12 animals per group. **P 0.01, ***P 0.001 versus the corresponding SO group;
P 0.05,
††
P 0.01 versus the
indicated I-R group.
146 Gobbetti et al
AJP January 2012, Vol. 180, No. 1
Page 6
reperfusion (Figure 6A). In tissues after ischemia alone or
I-R, the protein levels of 25-kDa trypsin were significantly
higher compared to SO mice. A trypsin 32-kDa isoform
was also detected in intestinal tissues from I-R animals
(data not shown). The treatment with FUT-175 did not
modify trypsin expression in I-R mice (data not shown).
Thrombin, plasmin, and chymotrypsin were not detected
in intestinal tissues, whether mice were SO or subjected
to I-R. Finally, no difference in plasma levels of trypsin
was found between I-R and SO mice (data not shown).
Levels of the pan-proteinase inhibitor
-2M were mea-
sured using Western blotting technique. As shown in
Figure 6B, the
-2M tissue levels were reduced after 90
minutes of ischemia and significantly decreased by 2
hours of reperfusion after the ischemic period. No differ-
ence in plasma levels of
-2M was found between I-R
and SO mice (data not shown).
Discussion
In the present study, we provide evidence of a significant
increase of serine protease activity in mouse plasma and
ileal samples as a consequence of intestinal I-R injury.
We also provide experimental evidence that this proteo-
lytic activity plays an important role in the lethality and
inflammatory response characterized by granulocyte re-
cruitment to ischemic tissues. This effect appears to be
mediated through the activation of PAR
1
and PAR
2
.
Trypsin, which was found to be up-regulated in tissues
just after the ischemic period and which can activate
both PAR
2
and PAR
1
, could be the protease responsi-
ble for the IRI-associated PAR-dependent granulocyte
recruitment.
Our results not only provide definitive insights into the
pathogenetic role for arginine-specific cleavage proteo-
lytic activity in intestinal IRI, but also point to the impor-
tance of proteases during the hypoxic period. Ischemia
alone clearly increased proteolytic activity both in tissues
and in plasma, demonstrating that this activity is thus not
directly linked to the release of proteases from inflamma-
tory cells, which infiltrate the tissues solely during reper-
fusion. Tissue proteolytic activity was maintained signifi-
cantly higher, and it was further increased in plasma after
2 hours of reoxygenation after intestinal hypoxia. During
that period, it is possible that proteolytic activity is in-
creased by the release of infiltrated inflammatory cells
proteases. If this is the case, a different pattern of pro-
teases might be released during ischemia, versus reper-
fusion. Our results also showed that the expression of the
pan-proteinase inhibitor
-2 macroglobulin was signifi-
cantly reduced in intestinal tissues after 2-hour reperfu-
sion, but not after ischemia only, suggesting again that
the type of active proteases during ischemia and during
reperfusion might be different. However, one cannot rule
out that common proteases are produced during isch-
emia and reperfusion periods. Indeed, we found that
trypsin expression was up-regulated in intestinal tissues
both after ischemia and after 2 hours of reperfusion (Fig-
ure 6). Above all, we provide evidence here that the
inhibition of tissue protease activity during the ischemic
Figure 4. PAR
1
and PAR
2
protein expression in mouse small intestine detected by immunohistochemistry. Upper panels: anti- PAR
1
antibody (blue) and
anti-CD45 antibody, an immune cell marker (red) staining; Lower panels: anti-PAR
2
antibody (blue) and anti-CD45 antibody (red) staining. Arrowheads showed
PAR
1
and PAR
2
staining, respectively, in the upper and lower panels. Arrows indicate CD45 staining, and double arrows indicate colocalization of CD45
and PAR
2
.
Proteases and Intestinal Ischemia 147
AJP January 2012, Vol. 180, No. 1
Page 7
period is important to prevent post-ischemic intestinal
granulocyte recruitment. The compound FUT-175, a syn-
thetic inhibitor of “trypsin-like” serine proteases such as
factor VIIa, factor XIIa, kallikrein, thrombin, plasmin, tryp-
sin, and tryptase
42
administered just before ischemia and
at the beginning of the reperfusion period significantly
reduced intestinal MPO and tissue proteolytic activity. In
contrast, a single administration of FUT-175 performed
Figure 5. In A and B, effects of treatment with the PAR
1
antagonist SCH-79797 (5 mg/kg i.p.), effects of PAR
2
deficiency (Par
2
/
), and effects of treatment with the
serine protease inhibitor FUT-175 in I-R mice (90 minutes 2 hours reperfusion), compared, respectively, to vehicle or littermate mice (Par
2
/
). Effects of treatment
with the PAR
1
antagonist SCH-79797 and effects of PAR
2
deficiency (Par2
/
) on MPO activity in sham-operated (SO) or I-R mice (C and D), chemokines (KC and MCP-1)
protein expression (E, F, G, and H) and adhesion molecules (ICAM-1 and VCAM-1) (I, J, K, and L) compared to vehicle or littermate mice (Par
2
/
). Data represent
means SEM of 8 to 12 animals per group. *P 0.05, **P 0.01 versus the corresponding sham group);
P 0.05 versus the indicated I-R group.
148 Gobbetti et al
AJP January 2012, Vol. 180, No. 1
Page 8
either at the moment of ischemia or at the beginning of
the reperfusion period did not show the same protective
effects on intestinal granulocyte infiltration (data not
shown).
The inflammatory leukocyte recruitment is first set by
the release of chemokines, such as KC and MCP-1,
which act as chemoattractants to guide the migration of
white blood cells to the site of ischemia.
43
Complex in-
teractions between leukocytes and endothelium at the
site of injury then lead to the rolling, adhesion, and trans-
migration of leukocytes, through processes involving var-
ious adhesion molecules expressed on the endothelium
(such as ICAM and VCAM), and integrins expressed on
immune cells. We showed that treatment with the large-
spectrum serine protease inhibitor FUT-175 significantly
reduced intestinal granulocyte recruitment after I-R and
concomitantly decreased the expression levels of
chemokines KC and MCP-1, as well as adhesion mole-
cules ICAM-1 and VCAM-1. Our data thus provide in-
sights into the mechanisms by which proteases might
induce granulocyte recruitment on intestinal I-R. Pro-
teases seem to be able to regulate both the chemoattrac-
tant signal and the leukocyte/endothelium adhesion mol-
ecule signals.
Our results showed a proteolytic balance in plasma
and intestinal tissues disrupted toward an increased pro-
teolytic activity after intestinal IRI. This can be due to the
increased expression of proteases, but also to the de-
creased expression of protease inhibitors. As a matter of
fact, we show here that the 25-kDa trypsin content was
significantly higher in I-R tissue, compared to SO, thereby
feeding the concept that some proteases are up-regu-
lated after IRI. Trypsin could derive from pancreatic
source and then enter intestinal tissues through the dam-
aged epithelial barrier, but we cannot exclude that, as
already reported, trypsin could be generated directly
from epithelial or endothelial cells subjected to patholog-
ical stimuli.
44–46
The proteolytic imbalance observed
during reperfusion could also be due, at least in part, to
the inactivation of endogenous protease inhibitors. It has
been established that oxidants, massively produced dur-
ing reperfusion, can inactivate physiological antipro-
teases.
47–51
Our study showed decreased intestinal lev-
els of
-2M, a well-known pan-protease inhibitor, after IRI
compared to the sham-operated group. Endogenous
protease inhibitors are important protective mechanisms
against the potential deleterious effects of proteases, and
the decreased expression or inhibition of
-2M or other
inhibitors such as antithrombin III (coagulation system),
2-antiplasmin (fibrinolytic system) could also be respon-
sible for the disrupted proteolytic activity observed on
intestinal ischemia-reperfusion.
52,53
Intestinal I-R not only damage gut tissues, but also
causes distant organ failure, rapidly leading to death.
Compromised cardiac functions have been reported as
the major cause of lethality in the intestinal I-R model.
54
Since we report here that intravenous administration of
the protease inhibitor FUT-175 strongly inhibited mortality
in that model, it can be speculated that proteases are
involved in mechanisms leading to cardiac failure. In-
deed, it was reported that myocardial depressant factor
(MDF) is released through the proteolytic action of serine
proteases.
55
Treatment with FUT-175 could inhibit some
of the proteases responsible for the release of MDF,
thereby decreasing MDF-related cardiac failure mortality.
Interestingly, we observed that neither PAR
1
antagonist
nor PAR
2
depletion was able to significantly inhibit intes-
tinal I-R–induced mortality (data not shown), suggesting
that the systemic consequences of intestinal I-R, possibly
on multiple organ failure, occur independently of PAR
1
or
PAR
2
activation. Regardless of its causes, restoration of
the proteolytic unbalance by administration of a broad,
large-spectrum serine protease inhibitor such as FUT-
175 has been shown to be therapeutically useful in our
model of intestinal IRI in mice.
Using different proteases inhibitors in vitro, we showed
that arginine-directed proteolytic activity detected during
I-R injury in tissues and in plasma was, for the most part,
due to serine proteases. However, the profile of pro-
teases dosed in plasma versus intestinal tissues seems
to be slightly different, with a significant activity of cys-
teine and aspartate proteases detected in plasma,
whereas those levels were not significant in intestinal
tissues. The in vivo approach aiming at inhibiting arginine-
cleavage proteolytic activity demonstrated that inhibition
Figure 6. Protein expression of trypsin (A) and
-2 macroglobulin (
-2M)
(B) measured by Western blot analysis in ileal tissue obtained from mice
subjected to 90 minutes of ischemia alone (without reperfusion) (0hR) or
mice subjected to ischemia followed by 2 hours of reperfusion, compared to
sham-operated (SO) mice. SO group is represented by white bars and I-R
group by black bars. The densitometry data of bands obtained by Western
blot analysis is shown as a bar graph and represents means SEM of six to
eight animals per group. All densitometry readings are standardized to actin.
*P 0.05 versus the corresponding sham group.
Proteases and Intestinal Ischemia 149
AJP January 2012, Vol. 180, No. 1
Page 9
of this activity in plasma was not necessary to obtain a
significant protection against lethality or inflammation,
whereas inhibition of proteolytic activity in intestinal tis-
sues by FUT-175 treatment was associated with protec-
tive effects. This demonstrates that tissue protease activ-
ity more than plasma activity is crucial for the dramatic
issue of intestinal IRI.
This study indicates that endogenous proteases regu-
late the post-ischemic granulocyte recruitment. Over the
most recent years, a number of studies have demon-
strated that the proinflammatory effects of proteases, and
particularly serine proteases, are mediated, in large part,
by the activation of PARs.
8
We showed by immunohisto-
chemistry that in mouse small intestine, PAR
1
is mostly
expressed on blood vessels and weakly expressed in
other cell types, whereas PAR
2
is expressed on epithelial,
endothelial, and immune intestinal cells. Mice treated
with the PAR
1
antagonist SCH-79797 or Par
2
/
pheno-
type mice submitted to ischemia followed by 2 hours of
reperfusion were protected from the post-ischemic gran-
ulocyte recruitment. Logically, in those mice, although the
inflammation was reduced, the proteolytic activity levels
in plasma and tissues were still significantly elevated.
These data suggest that both PAR
1
and PAR
2
could be
responsible for the proinflammatory effects of proteases
in this model, having a primary role in the recruitment of
granulocytes. Indeed, various in vitro experiments have
shown up-regulated levels of adhesion molecules on
PAR
1
or PAR
2
activation on endothelial cells. PAR
1
and
PAR
2
also regulate the release of chemokines such as
IL-8 from gut epithelial cells in vitro
56,57
Here, we dem-
onstrated that PAR
1
blockade caused a significant de-
crease in adhesion molecules (ICAM-1 and VCAM-1) and
in chemokine expression (KC and MCP-1) compared to
the I-R vehicle group. The effects of PAR
1
blockade were
thus similar to the effects of protease inhibition by FUT-
175 (Figure 3), suggesting that these effects of proteases
on adhesion molecules and chemokine expression might
be mediated, at least in part, by PAR
1
activation. PAR
2
activation might also be implicated in the protease-in-
duced adhesion molecule expression, but the fact that
PAR
2
deficiency failed to modify the increased expres-
sion of KC and MCP-1 showed that the regulation of the
expression of those two chemokines by proteases is not
a PAR
2
-dependent mechanism. PAR
1
and PAR
2
are both
expressed on endothelial cells where they are known to
induce adhesion molecule expression.
58–61
Indeed, our
results demonstrated a common role for PAR
1
and PAR
2
in inducing ICAM-1 and VCAM-1 overexpression on isch-
emia-reperfusion, facilitating granulocyte recruitment.
Because PAR
2
is expressed both on endothelial cells and
on CD45-positive cells, it is difficult to conclude on the
cellular origin responsible for PAR
2
-associated leukocyte
recruitment. Hyun et al
59
have recently shown that PAR
2
plays an important role in up-regulating the expression of
adhesion molecules ICAM-1 and V-CAM-1 in three differ-
ent models of colitis, and further demonstrated that PAR
2
expression both on bone marrow– derived cells and in
host-derived tissues was important for regulating granu-
locyte recruitment.
58
A similar role for PAR
2
could be
hypothesized in our model of ischemia-reperfusion. In
contrast, PAR
1
activation could be restricted to endothe-
lial cells as no colocalization was observed for PAR
1
with
the CD45 marker.
Taken together, these findings suggest that the serine
protease release induced by I-R occurs independently of
PAR
1
or PAR
2
activation, but that PAR
1
and PAR
2
activa-
tion represents a most important step upstream from
protease-dependent post-ischemic granulocyte recruit-
ment. Those different subtypes of receptors seem to
have distinct but complementary roles in granulocyte
recruitment, having distinct mechanisms of action and
distinct patterns of expression in the small intestine.
Finally, it is important to consider that PAR
1
and PAR
2
are not necessarily activated by the same serine pro-
teases. For instance, PAR
1
, but not PAR
2
, is activated
by thrombin whereas PAR
2
, but not PAR
1
, is activated
by tryptase. In contrast, trypsin, which we have shown
to be significantly present in I-R tissues, can activate
both receptors.
In conclusion, the present study suggests that serine
proteases and PAR activation are involved in the
pathogenesis of intestinal IRI. These findings feed our
understanding of the IRI pathogenesis, pointing to a
role for arginine-specific cleavage proteases, and
PAR
1
/PAR
2
activation. Re-equilibrating the tissue pro-
tease/antiprotease balance in ischemia-reperfusion
processes actively protects from injury and death, and
the use of a broad, large-spectrum protease inhibitor
such as FUT-175, also available commercially as Na-
famostat, could represent an interesting therapeutic
option to prevent local and systemic consequences of
intestinal IRI.
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    • "of oxygen supply to the tissue (ischemia), which in turn causes cellular dysfunction, protease and 88 phospholipases activation (Gobbetti et al., 2012; Otamiri et al., 1987; Vollmar et al., 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: Background and purpose: Long-term intake of dietary fatty acids is known to predispose to chronic inflammation, but their effects on acute intestinal ischaemia/reperfusion (I/R) injury is unknown. The aim of this study was to determine the consequences of a diet rich in n-3 or n-6 polyunsaturated fatty acids (PUFA) on intestinal I/R-induced damage. Experimental approach: Mice were fed three different isocaloric diets: a balanced diet used as a control and two different PUFA-enriched diets, providing either high levels of n-3 or of n-6 PUFA. Intestinal injury was evaluated after intestinal I/R. PUFA metabolites were quantitated in intestinal tissues by LC-MS/MS. Key results: In control diet-fed mice, intestinal I/R caused inflammation and increased COX and lipoxygenase-derived metabolites compared with sham-operated animals. Lipoxin A4 (LxA4 ) was significantly and selectively increased after ischaemia. Animals fed a high n-3 diet did not display a different inflammatory profile following intestinal I/R compared with control diet-fed animals. In contrast, intestinal inflammation was decreased in the I/R group fed with high n-6 diet and level of LxA4 was increased post-ischaemia compared with control diet-fed mice. Blockade of the LxA4 receptor (Fpr2), prevented the anti-inflammatory effects associated with the n-6 rich diet. Conclusions and implications: This study indicates that high levels of dietary n-6, but not n-3, PUFAs provides significant protection against intestinal I/R-induced damage and demonstrates that the endogenous production of LxA4 can be influenced by diet.
    Full-text · Article · Oct 2014 · British Journal of Pharmacology
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    • "Among the variety of events affecting post-ischemic reperfused tissues, oxygen free radical formation, alteration of the microvasculature integrity, and neutrophil recruitment are pivotal: they sustain a local organ damage preceding the development of a systemic inflammatory response syndrome, followed, in its turn, by a serious multiple organ failure [5,6]. injury caused by mesenteric I/R789101112. More recently, we focused our attention on 5-HT, a known paracrine mediator and neurotransmitter involved in the physiological regulation of gut motility, perception and secretion [13] . "
    [Show abstract] [Hide abstract] ABSTRACT: Intestinal ischemia and reperfusion (I/R) is a potentially life-threatening disease, ensuing from various clinical conditions. Experimentally, either protective or detrimental roles have been attributed to 5-HT in the functional and morphological injury caused by mesenteric I/R. Recently, we proved the involvement of 5-HT2A receptors in the intestinal dysmotility and leukocyte recruitment induced by 45min occlusion of the superior mesenteric artery (SMA) followed by 24hours reperfusion in mice. Starting from these premises, the aim of our present work was to investigate the role played by endogenous 5-HT in the same experimental model where 45min SMA clamping was followed by 5hours reflow. To this end, we first observed that ischemic preconditioning before I/R injury (IPC+I/R) reverted the increase in 5-HT tissue content and in inflammatory parameters induced by I/R in mice. Second, the effects produced by intravenous administration of 5-HT1A ligands (partial agonist buspirone 10 mg·kg(-1), antagonist WAY100135 0.5-5 mg·kg(-1)), 5-HT2A antagonist sarpogrelate (10mg·kg(-1)), 5-HT3 antagonist alosetron (0.1mg·kg(-1)), 5-HT4 antagonist GR125487 (5mg·kg(-1)) and 5-HT re-uptake inhibitor fluoxetine (10mg·kg(-1)) on I/R-induced inflammatory response were investigated in I/R mice and compared to those obtained in sham-operated animals (S). Our results confirmed the significant role played by 5-HT2A receptors not only in the late but also in the early I/R-induced microcirculatory dysfunction and showed that blockade of 5-HT1A receptors protected against the intestinal leukocyte recruitment, plasma extravasation and reactive oxygen species formation triggered by SMA occlusion and reflow. The ability of α7 nicotinic receptor (α7nAchR) antagonist methyllycaconitine (5mg·kg(-1)) to counteract the beneficial action provided by buspirone on I/R-induced neutrophil infiltration suggests that the anti-inflammatory effect produced by 5-HT1A receptor antagonism could be partly ascribed to the indirect activation of α7nAch receptors.
    Full-text · Article · Feb 2014 · Pharmacological Research
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    • "Microscopic histological damage score was evaluated by a person unaware of the treatments and was based on a semiquantitative scoring system in which the following features were graded: extent of destruction of normal mucosal architecture (0, normal; 1, 2, and 3, mild, moderate, and extensive damage, respectively), presence and degree of cellular infiltration (0, normal; 1, 2, and 3, mild, moderate, and transmural infiltration), extent of muscle thickening (0, normal; 1, 2, and 3, mild, moderate, and extensive thickening), presence or absence of crypt abscesses (0, absent; 1, present), and presence or absence of goblet cell depletion (0, absent; 1, present). The scores for each feature were then summed with a maximum possible score of 11 as previously described [9], [10]. "
    [Show abstract] [Hide abstract] ABSTRACT: Polyunsaturated fatty acid (PUFA) metabolites are bioactive autoacoids that play an important role in the pathogenesis of a vast number of pathologies, including gut diseases. The induction and the resolution of inflammation depend on PUFA metabolic pathways that are favored. Therefore, understanding the profile of n-6 (eicosanoids)/n-3 (docosanoids) PUFA-derived metabolites appear to be as important as gene or protein array approaches, to uncover the molecules potentially implicated in inflammatory diseases. Using high sensitivity liquid chromatography tandem mass spectrometry, we characterized the tissue profile of PUFA metabolites in an experimental model of murine intestinal ischemia reperfusion. We identified temporal and quantitative differences in PUFA metabolite production, which correlated with inflammatory damage. Analysis revealed that early ischemia induces both pro-inflammatory and anti-inflammatory eicosanoid production. Primarily, LOX- (5/15/12/8-HETE, LTB4, LxA4) and CYP- (5, 6-EET) metabolites were produced upon ischemia, but also PGE3, and PDx. This suggests that different lipids simultaneously play a role in the induction and counterbalance of ischemic inflammatory response from its onset. COX-derived metabolites were more present from 2 to 5 hours after reperfusion, fitting with the concomitant inflammatory peaks. All metabolites were decreased 48 hours post-reperfusion except for to the pro-resolving RvE precursor 18-HEPE and the PPAR-γαμμα agonist, 15d-PGJ2. Data obtained through the pharmacological blockade of transient receptor potential vanilloid-4, which can be activated by 5, 6-EET, revealed that the endogenous activation of this receptor modulates post-ischemic intestinal inflammation. Altogether, these results demonstrate that different lipid pathways are involved in intestinal ischemia-reperfusion processes. Some metabolites, which expression is severely changed upon intestinal ischemia-reperfusion could provide novel targets and may facilitate the development of new pharmacological treatments.
    Full-text · Article · Sep 2013 · PLoS ONE
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