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Lipocortin 1 Protects Against Splanchnic Artery Occlusion and Reperfusion Injury by Affecting Neutrophil Migration
Abstract
Splanchnic artery occlusion and reperfusion (SAO/R) shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (60-min reperfusion). Following this reperfusion period, rats developed a fall in mean arterial blood pressure, associated with a significant increase in tissue myeloperoxidase (MPO) activity in the intestine and a marked histologic injury to the distal ileum. Treatment of rats with a lipocortin-1 (LC1)-derived N-terminal peptide, peptide Ac(2-26), dose-dependently (0.125-0.5 mg/kg s.c.) reduced the progressive fall in blood pressure and prevented the infiltration of neutrophils into the reperfused intestine (reduced MPO activity). The LC1 peptide also reduced the degree of ischemia/reperfusion injury in the bowel as evaluated by histologic examination. The glucocorticoid dexamethasone (0.1 mg/kg s.c., -1 h) also produced a marked improvement in SAO/R shock (i.e., maintained mean arterial blood pressure and reduced tissue MPO activity), and this was reversed by pretreatment with two different antisera raised against the LC1 pharmacophore. Peptide Ac(2-26) (0.5 mg/kg s.c., -30 min) reduced (>60%) the extent of IL-1beta-induced cell emigration and significantly attenuated (approximately 45%) the number of adherent leukocytes in the rat mesenteric vascular bed, as assessed by video microscopy. These results suggest that LC1 inhibits neutrophil migration and accumulation into reperfused tissues, thereby ameliorating the outcome of SAO/R shock.
... Besides affecting PMN recruitment in the peritoneal compartment after stimulation with classical inflammogens (20,21), LC1 also modulates neutrophil accumulation into the rat small intestine in a model of reperfusion injury (22). This effect was linked to a reduction in PMN recruitment to the damaged tissue. ...
... This effect was linked to a reduction in PMN recruitment to the damaged tissue. Finally, in most, if not all, of these experimental conditions, LC1 appeared to act as a mediator of the protective effect displayed by glucocorticoids (e.g., dexamethasone [DEX]), inasmuch as anti-LC1 antibodies prevented the inhibition afforded by the steroid (20,22). Therefore, the present study investigated whether myocardial injury could be modulated by systemic treatment with LC1. ...
... Other groups have confirmed these observations (17,21). In addition, peptides derived from the LC1 NH 2 terminus region, which contains the pharmacophore responsible for the antimigratory activity displayed by the entire protein (41), are effective in reducing the extent of tissue damage produced in the splanchnic region by occlusion and reopening of the superior mesenteric artery (22). ...
... Studies have suggested that AnxA1 is an important endogenous mediator, which is activated in response to tissue injury (Damazo et al., 2005;Gil et al., 2006), glucocorticoids (Perretti and D'Acquisto 2009), inflammation (Damazo et al., 2005), heat or chemical insults (Rhee et al., 2000) and pro-inflammatory cytokines (Yang et al., 2009). The ultimate aim of treatments based on AnxA1 is to retain the anti-inflammatory properties of glucocorticoids that signal for the resolution of inflammatory events, while avoiding the highly detrimental potential metabolic side-effects of long-term use of exogenous glucocorticoids (Cuzzocrea et al., 1997;D'Amico et al., 2000;Perretti and Gavins 2003;Gavins and Hickey 2012;Perretti and D'Acquisto, 2009). ...
... In a recent study, this group described a protective role for AnxA1 in the haemodynamic changes and inflammation in cyclosporine-treated animals after ischaemia/reperfusion injury (Araujo et al., 2012). Moreover, there is evidence demonstrating the important and protective role of AnxA1 in conditions of ischaemia/reperfusion (I/R) injury in the mesentery, heart, brain and kidney (Cuzzocrea et al., 1997;La et al., 2001;Gavins et al., 2005Gavins et al., , 2007Facio et al., 2010;Araujo et al., 2012). Also, by promoting phagocytosis of apoptotic neutrophils, and restoring the pre-inflammatory state of the tissue or organ (Serhan et al., 2007). ...
... This result was corroborated by quantification of the cells in the blood, where an increased number of circulating neutrophils was observed after Ac2-26 treatment. Similar results were also obtained in models of uveitis and mesenteric, cardiac, cerebral and renal ischaemia/reperfusion injury, in which a reduction in tissue damage induced by leukocyte extravasation could be observed after treatment with Ac2-26 (Cuzzocrea et al., 1997;La et al., 2001;Gavins et al., 2007;Facio et al., 2010;Silva et al., 2011). Furthermore, in the peripheral blood, the results showed that treatment with CsA and AnxA1 diminished the number of lymphocytes on days 3 and 5 compared with the control group after transplant. ...
Immunosuppressive drugs have a critical role in inhibiting tissue damage and allograft rejection. Studies have demonstrated the anti-inflammatory effects of the annexin A1 (AnxA1) in the regulation of transmigration and apoptosis of leucocytes. In the present study, an experimental skin allograft model was used to evaluate a potential protective effect of AnxA1 in transplantation survival. Mice were used for the skin allograft model and pharmacological treatments were carried out using either the AnxA1 mimetic peptide Ac2-26, with or without cyclosporine A (CsA), starting 3 days before surgery until rejection. Graft survival, skin histopathology, leucocyte transmigration and expression of AnxA1 and AnxA5 post-transplantation were analysed. Pharmacological treatment with Ac2-26 increased skin allograft survival related with inhibition of neutrophil transmigration and induction of apoptosis, thereby reducing the tissue damage compared with control animals. Moreover, AnxA1 and AnxA5 expression increased after Ac2-26 treatment in neutrophils. Interestingly, the combination of Ac2-26 and cyclosporine A showed similar survival of transplants when compared with the cyclosporine A group, which could be attributed to a synergistic effect of both drugs. Investigations in vitro revealed that cyclosporine A inhibited extracellular-signal-regulated kinase (ERK) phosphorylation induced by Ac2-26 in neutrophils. Overall, the results suggest that AnxA1 has an essential role in augmenting the survival of skin allograft, mainly owing to inhibition of neutrophil transmigration and enhancement of apoptosis. This effect may lead to the development of new therapeutic approaches relevant to transplant rejection. Copyright © 2013 John Wiley & Sons, Ltd.
... These actions result in potent immunoregulatory effects, especially on inflammatory phagocytes and neutrophils by inhibiting their accumulation and migration. After splanchnic artery occlusion and reperfusion injury, treatment with ANXA1 N-terminal peptidomimetic (Ac2-26) reduced MPO activity and neutrophil infiltration into the reperfused tissue and thus, improved outcome after shock (38). ...
Resolution agonists, including lipid mediators and peptides such as annexin A1 (ANXA1), are providing novel approaches to treat inflammatory conditions. Surgical trauma exerts a significant burden on the immune system that can affect and impair multiple organs. Perioperative cerebral injury after cardiac surgery is associated with significant adverse neurological outcomes such as delirium and postoperative cognitive dysfunction. Using a clinically relevant rat model of cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA), we tested the pro-resolving effects of a novel bioactive ANXA1 tripeptide (ANXA1sp) on neuroinflammation and cognition. Male rats underwent 2 h CPB with 1 h DHCA at 18°C, and received vehicle or ANXA1sp followed by timed reperfusion up to postoperative day 7. Immortalized murine microglial cell line BV2 were treated with vehicle or ANXA1sp and subjected to 2 h oxygen–glucose deprivation followed by timed reoxygenation. Microglial activation, cell death, neuroinflammation, and NF-κB activation were assessed in tissue samples and cell cultures. Rats exposed to CPB and DHCA had evident neuroinflammation in various brain areas. However, in ANXA1sp-treated rats, microglial activation and cell death (apoptosis and necrosis) were reduced at 24 h and 7 days after surgery. This was associated with a reduction in key pro-inflammatory cytokines due to inhibition of NF-κB activation in the brain and systemically. Treated rats also had improved neurologic scores and shorter latency in the Morris water maze. In BV2 cells treated with ANXA1sp, similar protective effects were observed including decreased pro-inflammatory cytokines and cell death. Notably, we also found increased expression of ANXA1, which binds to NF-κB p65 and thereby inhibits its transcriptional activity. Our findings provide evidence that treatment with a novel pro-resolving ANXA1 tripeptide is neuroprotective after cardiac surgery in rats by attenuating neuroinflammation and may prevent postoperative neurologic complications.
... Furthermore, immunoreactive bands of ~36 kDa, consistent with molecular mass of Annexin A1, were obtained by co-immunoprecipitation with anti-FPRL1 antibody (Perretti, Chiang et al. 2002), thus confirming the interaction between Annexin A1 and the FPRL1 in human cells. (Flower 1988), inflammatory and infectious diseases (Goulding, Jefferiss et al. 1992;Cuzzocrea, Tailor et al. 1997) and tissue or cell damage (Rescher, Goebeler et al. 2006). In such cases the released Annexin A1 is proteolytically cleaved generating the active N-terminal peptides. ...
... 7 At the cellular level, Anx-A1 is implicated in the control of cell growth 8 and differentiation, 9 signal transduction and arachidonic acid release, 10,3 as well as vesicle trafficking. [11][12][13] In vivo, Anx-A1 is implicated in the glucocorticoid regulation of leukocyte migration, 14 acute 15 and chronic 16 inflammation, ischaemic damage, [17][18][19] pain, 20 fever, 21 HPA activity 22 and other aspects of neuroendocrine function, notably the control of growth hormone and prolactin. 23,24 There have been suggestions that Anx-A1 is involved in human disease. ...
Annexin-A1 (AnxA1) and its N-terminal derived peptide Ac2-26 regulate the inflammatory response in several experimental models of disorders. This study evaluated the effect of endogenous AnxA1 and its N-terminal peptide Acetyl 2-26 (Ac2-26) on allergic asthma triggered by house dust mite (HDM) extract in mice. ANXA1−/− and wildtype (WT) mice were exposed to intranasal instillation of HDM every other day for 3 weeks, with analyses performed 24 h following the last exposure. Intranasal administration of peptide Ac2-26 was performed 1 h before HDM, beginning 1 week after the initial antigen application. ANXA1−/− mice stimulated with HDM showed marked exacerbations of airway hyperreactivity (AHR), eosinophil accumulation, subepithelial fibrosis, and mucus hypersecretion, all parameters correlating with overexpression of cytokines (IL-4, IL-13, TNF-α, and TGF-β) and chemokines (CCL11/eotaxin-1 and CCL2/MCP-1). Intranasal treatment with peptide Ac2-26 decreased eosinophil infiltration, peribronchiolar fibrosis, and mucus exacerbation caused by the allergen challenge. Ac2-26 also inhibited AHR and mediator production. Collectively, our findings show that the AnxA1-derived peptide Ac2-26 protects against several pathological changes associated with HDM allergic reaction, suggesting that this peptide or related AnxA1-mimetic Ac2-26 may represent promising therapeutic candidates for the treatment of allergic asthma.
Crush syndrome (CS) is characterized by ischemia/reperfusion-induced rhabdomyolysis and subsequent systemic inflammation and has a high mortality rate, even when treated with conventional therapy. In previous studies, we demonstrated that treatment of rats with acute lethal CS using dexamethasone (DEX) had therapeutic effects in laboratory findings and improved the clinical course of CS. However, because the application of DEX in CS therapy is unknown, evaluation of the pharmacokinetic parameters of DEX was considered essential to support its clinical use. Here, we investigated the pharmacokinetic characteristics of DEX in a rat model of CS. Anesthetized rats were subjected to bilateral hind limb compression using rubber tourniquets for 5 h, followed by reperfusion for 0 to 24 h. Rats were divided randomly into 4 groups: saline-treated sham (S) and CS groups and 5.0 mg/kg DEX-treated S (S-DEX) and CS (CS-DEX) groups. Blood and tissue samples were collected for HPLC analysis. In the CS-DEX group, the pharmacokinetic parameters of the area under the concentration-time curve, mean residence time, and distribution volume levels increased significantly compared to the S-DEX group, whereas total body clearance, elimination rate constant, and renal clearance levels decreased significantly. Moreover, decrease of muscle tissue DEX concentration and of CYP3A activity were observed in the CS-DEX group. These results show the pharmacokinetic characteristics of DEX in the rat CS model and support the potential use of DEX in disaster medical care.
Since primeval times, the inflammatory process has been described in many different ways. Several anti-inflammatory therapies have been used in different biological models. However, in a recent "back to nature move", modern man is searching for natural products with medicinal properties, particularly those obtained from plants and bees. Propolis has been used in folk medicine for a very long time. The many compounds present in propolis require investigation.Physical-chemical analysis studies have not sufficiently established quality standards of propolis containing products. These standards should depend especially on their different pharmacological activities. There are few studies reporting on the in vitro anti-inflammatory activity of propolis containing products. It is necessary to evaluate the anti-inflammatory potential of commercial products containing propolis.
The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I–mediated antiinflammatory effects.
To evaluate the influence of interleukin-1 on leukocyte-endothelial cell interactions and the microcirculation in the liver after hemorrhagic shock by means of intravital microscopy using an interleukin-1 receptor antagonist (IL-1ra).
Prospective, randomized, blinded, controlled study.
University research laboratory.
Anesthetized female Sprague Dawley rats weighing 200 to 230 g.
Hypovolemic shock was induced and maintained for 1 hr (mean arterial pressure 40 mm Hg; cardiac output 50% of baseline). After adequate resuscitation and 5 hrs of reperfusion (mean arterial pressure > 100 mm Hg; cardiac output > 120% of baseline), the microcirculation in liver sinusoids was examined by intravital fluorescence microscopy. Continuous administration of IL-1ra (5 mg/kg/hr) was started at different times in a prospective, randomized, blinded fashion, either as pretreatment 5 mins before shock induction (n = 6), or as therapy at the time of resuscitation (n = 6). An additional bolus injection of 5 mg/kg of IL-1ra was given to the latter group.
Mean arterial pressure, cardiac output, heart rate, and blood gases were comparable in all shock groups during the experiments. The percentage of permanently adherent leukocytes (adhesion time of > 20 secs) in the pretreated group was significantly decreased in comparison with the control group (pretreatment group 16.9 +/- 1.9% vs. control group 42.1 +/- 5.4%; p < .001 by analysis of variance; sham group 9.1 +/- 1.1%). Administration of IL-1ra at the time of resuscitation also reduced firm adhesion of leukocytes to sinusoidal endothelium (treated group 28.8 +/- 3.6%, p < .01). Temporary adhesion rates of leukocytes (adhesion time of < 20 secs) were unaffected by pretreatment or treatment with IL-1ra with respect to control values. Liver microcirculation was impaired after hemorrhagic shock but not improved by IL-1ra.
The results show that adhesion of leukocytes to hepatic sinusoidal endothelium is at least partly regulated by interleukin-1. Adherence was attenuated by the application of IL-1ra, which might be due to diminished expression of adhesion receptors by endothelial cells or leukocytes. Even administration of IL-1ra at the time of resuscitation reduces the early inflammatory response in the liver after shock, thus offering a potentially important therapeutic approach.
Pulmonary complications are a major cause of death in patients in various forms of shock. The exact causes of the pulmonary complications are unknown. This study evaluates the functional characteristics of intralobar pulmonary arteries (IPA) and veins (IPV) obtained from swine subjected to splanchnic arterial occlusion (SAO) shock ans subsequent cardiovascular collapse and sham-shocked swine subjected to surgery but no occlusion. The contractile response of pulmonary arteries to norepinephrine (NE) and serotonin (5-HT) were depressed when obtained from pigs subjected to SAO shock. The depression in the sensitivity to 5-HT and maximal tension development to 5-HT and NE was selective, since the responses to potassium ion were not depressed. IPA obtained from swine with SAO shock were more sensitive to the relaxant actions of arachidonic acid, a precursor for bisenoic prostaglandins, than were IPA from sham-shocked swine. This was not observed when prostaglandins were used as agonists. This suggests that synthesis of prostaglandin differs in the pulmonary vasculature of sham-shocked and SAO-shocked animals. IPV obtained from swine in SAO shock were less sensitive to 5-HT and NE but more sensitive to arachidonic acid and 9 alpha, 11 alpha-epoxymethano-PGH2, a thromboxane like compound. IPV obtained from swine in SAO shock converted more arachidonic acid into a substance with the chromatographic mobility of thromboxane B2. The data suggest that alterations in the prostaglandin system within the pulmonary artery and vein may contribute to the pulmonary complications of SAO shock. The alterations appear to include an enhanced synthesis of prostacyclin as well as thromboxane-like substance. Because the veins were more sensitive than the arteries to 9 alpha, 11 alpha-epoxymethano-PGH2, thromboxanes or endoperoxides may elevate venous tone and contribute to the pulmonary edema associated with shock states.
Splanchnic arteries were clamped for 45 min to induce splanchnic artery occlusion (SAO) shock in anesthetized rats. Sham-operated animals were used as controls. Survival time, serum tumor necrosis factor-alpha (TNF-alpha), white blood cell (WBC) count, mean arterial blood pressure, myeloperoxidase (MPO) activity, and serum levels of soluble E-selectin (sE-selectin) were investigated. SAO-shocked rats exhibited decreased survival time (95 +/- 11 min, whereas sham-shocked rats survived for > 5 h), reduced mean arterial blood pressure, increased serum levels of TNF-alpha (185 +/- 8 U/ml) and MPO activity in the ileum (0.11 +/- 0.03 U x 10(-3)/g tissue) and lung (1.5 +/- 0.4 U x 10(-3)/g tissue), leukopenia, and enhanced serum levels of sE-selectin. Furthermore SAO-shocked rats showed histological alterations in the ileum and lung. Administration of cloricromene (2 mg/kg i.v.), an inhibitor of TNF-alpha, significantly increased survival time (225 +/- 10 min), decreased serum levels of TNF-alpha and sE-selectin, reduced leukopenia and MPO activity in the ileum (0.035 +/- 0.003 U x 10(-3)/g tissue) and lung (0.3 +/- 0.005 U x 10(-3)/g tissue), improved the cardiovascular changes, and reduced the histological changes in the ileum and lung. Finally, an anti-E-selectin antibody protected rats against SAO shock. Our findings are consistent with an involvement of E-selectin, "in vivo," in the pathogenesis of SAO shock.
It has been suggested that leukocytes play a key role in the pathogenesis of splanchnic artery occlusion shock. Intercellular adhesion molecule 1 (ICAM‐1) is an adhesion molecule of crucial importance in the phenomenon of leukocyte accumulation.
We investigated the involvement of ICAM‐1 in the pathogenesis of splanchnic artery occlusion shock. Splanchnic artery occlusion (SAO) shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min. Sham‐operated animals were used as controls. Survival time, serum tumour necrosis factor‐α (TNF‐α), white blood cell (WBC) count, mean arterial blood pressure, myeloperoxidase activity (MPO; studied as a quantitative means to assess leukocyte accumulation) and the responsiveness to acetylcholine of aortic rings were investigated. SAO shocked rats had a decreased survival time (90 ± 9.5 min, while sham‐shocked rats survived more than 4 h), reduced mean arterial blood pressure, increased serum levels of TNF‐α (201 ± 10 u ml ⁻¹ ) and MPO activity in the ileum (0.15 ± 0.03 u × 10 ⁻³ per g tissue) and in the lung (1.9 ± 0.8 u × 10 ⁻³ per g tissue), leukopenia and reduced responsiveness to acetylcholine (ACh, 10nM‐10 μ m ) of aortic rings.
Administration of monoclonal antibody raised against rat ICAM‐1 significantly increased survival time (225 ± 9 min), reduced leukopenia and MPO activity both in the ileum (0.031 ± 0.003 u × 10 ⁻³ per g tissue) and in the lung 0.23 ± 0.03 u × 10 ⁻³ per g tissue), improved the cardiovascular changes and restored the responsiveness to ACh of aortic rings.
Our findings are consistent with an involvement of adhesion mechanisms in vivo in the pathogenesis of SAO shock and suggest that specific adhesion mechanisms, which support leukocyte accumulation, may represent potentially important therapeutic targets in circulatory shock.