Serine protease inhibition reduces post-ischemic granulocyte recruitment in mouse intestine.
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.
SourceAvailable from: Thomas Gobbetti[Show abstract] [Hide abstract]
ABSTRACT: Background and purposeLong-term intake of dietary fatty acids is known to predispose to chronic inflammation, but their potential influence on acute intestinal ischemia/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 approachMice were fed 3 different isocaloric diets: a balanced diet used as a control, and two different PUFA-enriched diets, providing either high n-3 or high n-6 intake. Intestinal injury was evaluated after intestinal I/R. PUFA metabolite quantification was performed in intestinal tissues by liquid chromatography tandem mass spectrometry.Key resultsIn control diet-fed mice, intestinal I/R caused inflammation and increased cyclooxygenase (COX) and lipoxygenase (LOX)-derived metabolites, compared to sham-operated animals. Lipoxin A4 (LxA4) was significantly and selectively increased after ischemia. Animals fed a high n-3 diet did not display a different inflammatory profile following intestinal I/R, compared to control diet-fed animals. In contrast, intestinal inflammation was decreased in I/R group fed a high n-6 diet and level of LxA4 was increased post-ischemia, compared to control diet-fed mice. Blockade of formyl-peptide receptor-2, the LxA4 receptor, abrogated n-6 rich diet-associated anti-inflammatory effects.Conclusions and ImplicationsThis study indicates that high n-6, but not n-3, PUFAs leads to significant protection against intestinal I/R-induced damage and demonstrates that LxA4 endogenous production can be influenced by diet.British Journal of Pharmacology 10/2014; 172(3). DOI:10.1111/bph.12957 · 4.99 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Proteinase-activated receptor 2 (PAR2) is a G-protein-coupled receptor for trypsin and mast cell tryptase; it is highly expressed at the intestinal level with multiple functions, such as epithelial permeability and intestinal motility. Many proteases activate PAR2 during tissue damage, suggesting a role of the inflammatory response receptors. The aim of the study was to evaluate the distribution and expression of PAR2 in the jejunum, the ileum and the pelvic flexure, using samples collected from healthy adult horses after slaughter. Proteinase-activated receptor 2 immunoreactivity (PAR2-IR) was observed in the enterocytes, intestinal glands, the smooth muscle of the muscularis mucosae, and the longitudinal and circular muscle layers; there were no differences in the distribution of PAR2-IR in the different sections of the intestinal tract. The protein expression level showed that the relative amount of the PAR2 content in the mucosa of the intestinal tract decreased from the small to the large intestine while the PAR2 mRNA analysed showed similar values. This study provides relevant findings concerning the distribution of the PAR2 in the intestines of healthy horses and represents the starting point for evaluating the role of the PAR2 during strangulative intestinal disease and consequent systemic intestinal reperfusion/injury complications in horses in order to identify and employ antagonist PAR2 molecules.Research in Veterinary Science 06/2014; DOI:10.1016/j.rvsc.2014.03.006 · 1.51 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.The Journal of Immunology 08/2014; 193(7). DOI:10.4049/jimmunol.1400334 · 5.36 Impact Factor