Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.-Dalli, J., Jones, C. P., Cavalcanti, D. M., Farsky, S. H., Perretti, M., Rankin, S. M. Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow. FASEB J. 26, 387-396 (2012). www.fasebj.org

Carboxypeptidases A1 and A2 from the perfusate of rat mesenteric arterial bed differentially process angiotensin peptides

Here we report the isolation of carboxypeptidases A1 and A2 (CPA1 and CPA2) from the rat mesenteric arterial bed perfusate, which were found to be identical with their pancreatic counterparts. Angiotensin (Ang) I, Ang II, Ang-(1-9) and Ang-(1-12) were differentially processed by these enzymes, worthy mentioning the peculiar CPA1-catalyzed conversion of Ang II to Ang-(1-7) and the CPA2-mediated formation of Ang I from Ang-(1-12). We detected gene transcripts for CPA1 and CPA2 in mesentery and other extrapancreatic tissues, indicating that these CPAs might play a role in the renin-angiotensin system in addition to their functions as digestive enzymes. (C) 2011 Elsevier Inc. All rights reserved.

Leishmania Promastigotes Lack Phosphatidylserine but Bind Annexin V upon Permeabilization or Miltefosine Treatment

The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.

RegCM3 nested in HadAM3 scenarios A2 and B2: projected changes in extratropical cyclogenesis, temperature and precipitation over the South Atlantic Ocean

The RegCM3 (Regional Climate Model-version 3) was nested in HadAM3 model to simulate present (1975-1989, referred hereafter as RegHad) and two future climate scenarios (A2 and B2 from 2071 to 2085, referred as RegA2 and RegB2) over the South America (SA) and South Atlantic Ocean (SAO). Projected changes in the air temperature, precipitation, low level circulation and cyclogenesis climatology were investigated. The cyclogenesis were identified using an automatic scheme for tracking based on the minimum of relative vorticity (zeta) from 10-m height wind. During summer, a general decrease (increase) in the precipitation is projected by RegA2 and RegB2 over the northeastern SA (center-west and south Brazil, north Argentina and Uruguay). For winter, an anomalous low level anticyclonic circulation is associated with the reduction in the rainfall over the central part of southern Brazil in RegA2 and RegB2 scenarios. Similar to HadAM3, RegCM3 projects larger warming in A2 scenario. For the present climate, when compared to HadAM3, RegHad defines better both the location of the main cyclogenetic areas and its annual cycle near southwestern SAO. The projections indicate a reduction in the total number of cyclones of -7.2% and -4.7% for RegA2 and RegB2, respectively, while HadAM3 reduction is -4.5% for both scenarios. The decrease is larger for initially intense cyclones (zeta <=-<= 2.5 x 10(-5) s-(1)): -20.9% (RegA2) and -11.3% (RegB2). For the lifetime, distance traveled and mean velocity of the cyclones, the A2 and B2 scenarios present mean values close to the present climate ( 3 days, 1900 km, and 9 m s(-1), respectively). Regarding the initial mean vorticity of the systems, RegB2 simulates values similar to the present climate, but they are initially weaker in RegA2. In general, RegA2 and RegB2 show a large decrease in the number of cyclones over the southern SAO due to an anticyclonic anomaly covering SAO between 30-55A degrees S. The reduction is larger in the scenario with higher concentrations of greenhouse gases (RegA2).

Production of Human Antibody Fragments Binding to Melittin and Phospholipase A2 in Africanised Bee Venom: Minimising Venom Toxicity

The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.

Annexin A1 protein attenuates cyclosporine-induced renal hemodynamics changes and macrophage infiltration in rats

Cyclosporine (CsA) remains an important immunosuppressant for transplantation and for treatment of autoimmune diseases. The most troublesome side effect of CsA is renal injury. Acute CsA-induced nephrotoxicity is characterized by reduced renal blood flow (RBF) and glomerular filtration rate (GFR) due to afferent arteriole vasoconstriction. Annexin A1 (ANXA1) is a potent anti-inflammatory protein with protective effect in renal ischemia/reperfusion injury. Here we study the effects of ANXA1 treatment in an experimental model of acute CsA nephrotoxicity. Salt-depleted rats were randomized to treatment with VH (vehicles 1 mL/kg body weight/day), ANXA1 (Ac2-26 peptide 1 mg/kg body weight/day intraperitoneally), CsA (20 mg/kg body weight/day subcutaneously) and CsA + ANXA1 (combination) for seven days. We compared renal function and hemodynamics, renal histopathology, renal tissue macrophage infiltration and renal ANXA1 expression between the four groups. CsA significantly impaired GFR and RBF, caused tubular dilation and macrophage infiltration and increased ANXA1 renal tissue expression. Treatment with ANXA1 attenuated CSA-induced hemodynamic changes, tubular injury and macrophage infiltration. ANXA1 treatment attenuated renal hemodynamic injury and inflammation in an acute CsA nephrotoxicity model.

Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom

Two myotoxic and noncatalytic Lys49-phospholipases A2 (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A2 (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.562.05 angstrom and belonged to space groups P3121 (braziliantoxin-II), P6522 (braziliantoxin-III) and P21 (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A2 (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A2 braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A2.

Antioxidant and inflammatory aspects of lipoprotein-associated phospholipase A2 (Lp-PLA2 ): a review

The association of cardiovascular events with Lp-PLA2 has been studied continuously today. The enzyme has been strongly associated with several cardiovascular risk markers and events. Its discovery was directly related to the hydrolysis of the platelet-activating factor and oxidized phospholipids, which are considered protective functions. However, the hydrolysis of bioactive lipids generates lysophospholipids, compounds that have a pro-inflammatory function. Therefore, the evaluation of the distribution of Lp-PLA2 in the lipid fractions emphasized the dual role of the enzyme in the inflammatory process, since the HDL-Lp-PLA2 enzyme contributes to the reduction of atherosclerosis, while LDL-Lp-PLA2 stimulates this process. Recently, it has been verified that diet components and drugs can influence the enzyme activity and concentration. Thus, the effects of these treatments on Lp-PLA2 may represent a new kind of prevention of cardiovascular disease. Therefore, the association of the enzyme with the traditional assessment of cardiovascular risk may help to predict more accurately these diseases.

Influence of obesity and cardiometabolic makers on lipoprotein-associated phospholipase A2 (Lp-PLA2) activity in adolescents: the healthy young cross-sectional study

Abstract Background Lipoprotein-associated phospholipase A2 activity (Lp-PLA2) is a good marker of cardiovascular risk in adults. It is strongly associated with stroke and many others cardiovascular events. Despite this, the impact of obesity on this enzyme activity and its relation to biomarkers of cardiovascular disease in adolescents is not very well investigated. The purpose of this article is to evaluate the influence of obesity and cardiometabolic markers on Lp-PLA2 activity in adolescents. Results This cross-sectional study included 242 adolescents (10–19 years) of both gender. These subjects were classified in Healthy Weight (n = 77), Overweight (n = 82) and Obese (n = 83) groups. Lipid profile, glucose, insulin, HDL size, LDL(−) and anti-LDL(−) antibodies were analyzed. The Lp-PLA2 activity was determined by a colorimetric commercial kit. Body mass index (BMI), waist circumference and body composition were monitored. Food intake was evaluated using three 24-hour diet recalls. The Lp-PLA2 activity changed in function to high BMI, waist circumference and fat mass percentage. It was also positively associated with HOMA-IR, glucose, insulin and almost all variables of lipid profile. Furthermore, it was negatively related to Apo AI (β = −0.137; P = 0.038) and strongly positively associated with Apo B (β = 0.293; P < 0.001) and with Apo B/Apo AI ratio (β = 0.343; P < 0.001). The better predictor model for enzyme activity, on multivariate analysis, included Apo B/Apo AI (β = 0.327; P < 0.001), HDL size (β = −0.326; P < 0.001), WC (β = 0.171; P = 0.006) and glucose (β = 0.119; P = 0.038). Logistic regression analysis demonstrated that changes in Apo B/Apo AI ratio were associated with a 73.5 times higher risk to elevated Lp-PLA2 activity. Conclusions Lp-PLA2 changes in function of obesity, and that it shows important associations with markers of cardiovascular risk, in particular with waist circumference, glucose, HDL size and Apo B/Apo AI ratio. These results suggest that Lp-PLA2 activity can be a cardiovascular biomarker in adolescence.

Annexin-A1 peptide down-regulates the leukocyte recruitment and up-regulates interleukin-10 release into lung after intestinal ischemia-reperfusion in mice

Abstract Background Intestinal ischemia/reperfusion (IR) injury is a serious and triggering event in the development of remote organ dysfunction, from which the lung is the main target. This condition is characterized by intense neutrophil recruitment, increased microvascular permeability. Intestinal IR is also responsible for induction of adult respiratory distress syndrome, the most serious and life-threatening form of acute lung injury. The purpose of this study was to investigate the effect of annexin-A1 protein as an endogenous regulator of the organ remote injury induced by intestinal ischemia/reperfusion. Male C57bl/6 mice were subjected to intestinal ischemia, induced by 45 min occlusion of the superior mesenteric artery, followed by reperfusion. Results The intestinal ischemia/reperfusion evoked a high intensity lung inflammation as indicated by the number of neutrophils as compared to control group. Treatment with annexin-A1 peptidomimetic Ac2-26, reduced the number of neutrophils in the lung tissue and increased its number in the blood vessels, which suggests a regulatory effect of the peptide Ac2-26 in the neutrophil migration. Moreover, the peptide Ac2-26 treatment was associated with higher levels of plasma IL-10. Conclusion Our data suggest that the annexin-A1 peptidomimetic Ac2-26 treatment has a regulatory and protective effect in the intestinal ischemia/reperfusion by attenuation of the leukocyte migration to the lung and induction of the anti-inflammatory cytokine IL-10 release into the plasma. The anti-inflammatory action of annexin-A1 and its peptidomimetic described here may serve as a basis for future therapeutic approach in mitigating inflammatory processes due to intestinal ischemia/reperfusion.

Hepatocyte nuclear factors 1α/4α and forkhead box A2 regulate the solute carrier 2A2 (Slc2a2) gene expression in the liver and kidney of diabetic rats

AIMS: Solute carrier 2a2 (Slc2a2) gene codifies the glucose transporter GLUT2, a key protein for glucose flux in hepatocytes and renal epithelial cells of proximal tubule. In diabetes mellitus, hepatic and tubular glucose output has been related to Slc2a2/GLUT2 overexpression; and controlling the expression of this gene may be an important adjuvant way to improve glycemic homeostasis. Thus, the present study investigated transcriptional mechanisms involved in the diabetes-induced overexpression of the Slc2a2 gene. MAIN METHODS: Hepatocyte nuclear factors 1α and 4α (HNF-1α and HNF-4α), forkhead box A2 (FOXA2), sterol regulatory element binding protein-1c (SREBP-1c) and the CCAAT-enhancer-binding protein (C/EBPβ) mRNA expression (RT-PCR) and binding activity into the Slc2a2 promoter (electrophoretic mobility assay) were analyzed in the liver and kidney of diabetic and 6-day insulin-treated diabetic rats. KEY FINDINGS: Slc2a2/GLUT2 expression increased by more than 50% (P<0.001) in the liver and kidney of diabetic rats, and 6-day insulin treatment restores these values to those observed in non-diabetic animals. Similarly, the mRNA expression and the binding activity of HNF-1α, HNF-4α and FOXA2 increased by 50 to 100% (P<0.05 to P<0.001), also returning to values of non-diabetic rats after insulin treatment. Neither the Srebf1 and Cebpb mRNA expression, nor the SREBP-1c and C/EBP-β binding activity was altered in diabetic rats. SIGNIFICANCE: HNF-1α, HNF-4α and FOXA2 transcriptional factors are involved in diabetes-induced overexpression of Slc2a2 gene in the liver and kidney. These data point out that these transcriptional factors are important targets to control GLUT2 expression in these tissues, which can contribute to glycemic homeostasis in diabetes.

Annexin-A1 peptide down-regulates the leukocyte recruitment and up-regulates interleukin-10 release into lung after intestinal ischemia-reperfusion in mice

BACKGROUND: Intestinal ischemia/reperfusion (IR) injury is a serious and triggering event in the development of remote organ dysfunction, from which the lung is the main target. This condition is characterized by intense neutrophil recruitment, increased microvascular permeability. Intestinal IR is also responsible for induction of adult respiratory distress syndrome, the most serious and life-threatening form of acute lung injury. The purpose of this study was to investigate the effect of annexin-A1 protein as an endogenous regulator of the organ remote injury induced by intestinal ischemia/reperfusion. Male C57bl/6 mice were subjected to intestinal ischemia, induced by 45 min occlusion of the superior mesenteric artery, followed by reperfusion. RESULTS: The intestinal ischemia/reperfusion evoked a high intensity lung inflammation as indicated by the number of neutrophils as compared to control group. Treatment with annexin-A1 peptidomimetic Ac2-26, reduced the number of neutrophils in the lung tissue and increased its number in the blood vessels, which suggests a regulatory effect of the peptide Ac2-26 in the neutrophil migration. Moreover, the peptide Ac2-26 treatment was associated with higher levels of plasma IL-10. CONCLUSION: Our data suggest that the annexin-A1 peptidomimetic Ac2-26 treatment has a regulatory and protective effect in the intestinal ischemia/reperfusion by attenuation of the leukocyte migration to the lung and induction of the anti-inflammatory cytokine IL-10 release into the plasma. The anti-inflammatory action of annexin-A1 and its peptidomimetic described here may serve as a basis for future therapeutic approach in mitigating inflammatory processes due to intestinal