Pro-Angiogenic Macrophage Phenotype to Promote Myocardial Repair.

BACKGROUND: Heart failure following myocardial infarction (MI) remains one of the major causes of death worldwide, and its treatment is a crucial challenge of cardiovascular medicine. An attractive therapeutic strategy is to stimulate endogenous mechanisms of myocardial regeneration. OBJECTIVES: This study evaluates the potential therapeutic treatment with annexin A1 (AnxA1) to induce cardiac repair after MI. METHODS: AnxA1 knockout (AnxA1-/-) and wild-type mice underwent MI induced by ligation of the left anterior descending coronary artery. Cardiac functionality was assessed by longitudinal echocardiographic measurements. Histological, fluorescence-activated cell sorting, dot blot analysis, and in vitro/ex vivo studies were used to assess the myocardial neovascularization, macrophage content, and activity in response to AnxA1. RESULTS: AnxA1-/- mice showed a reduced cardiac functionality and an expansion of proinflammatory macrophages in the ischemic area. Cardiac macrophages from AnxA1-/- mice exhibited a dramatically reduced ability to release the proangiogenic mediator vascular endothelial growth factor (VEGF)-A. However, AnxA1 treatment enhanced VEGF-A release from cardiac macrophages, and its delivery in vivo markedly improved cardiac performance. The positive effect of AnxA1 treatment on cardiac performance was abolished in wild-type mice transplanted with bone marrow derived from Cx3cr1creERT2Vegfflox/flox or in mice depleted of macrophages. Similarly, cardioprotective effects of AnxA1 were obtained in pigs in which full-length AnxA1 was overexpressed by use of a cardiotropic adeno-associated virus. CONCLUSIONS: AnxA1 has a direct action on cardiac macrophage polarization toward a pro-angiogenic, reparative phenotype. AnxA1 stimulated cardiac macrophages to release high amounts of VEGF-A, thus inducing neovascularization and cardiac repair.

Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells.

BACKGROUND: Annexin-1 (ANXA1) plays pivotal roles in regulating various physiological processes including inflammation, proliferation and apoptosis, and deregulation of ANXA1 functions has been associated with tumorigenesis and metastasis events in several types of cancer. Though ANXA1 levels correlate with breast cancer disease status and outcome, its distinct functional involvement in breast cancer initiation and progression remains unclear. We hypothesized that ANXA1-responsive kinase signaling alteration and associated phosphorylation signaling underlie early events in breast cancer initiation events and hence profiled ANXA1-dependent phosphorylation changes in mammary gland epithelial cells. METHODS: Quantitative phosphoproteomics analysis of mammary gland epithelial cells derived from ANXA1-heterozygous and ANXA1-deficient mice was carried out using stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry. Kinase and signaling changes underlying ANXA1 perturbations were derived by upstream kinase prediction and integrated network analysis of altered proteins and phosphoproteins. RESULTS: We identified a total of 8110 unique phosphorylation sites, of which 582 phosphorylation sites on 372 proteins had ANXA1-responsive changes. A majority of these phosphorylation changes occurred on proteins associated with cytoskeletal reorganization spanning the focal adhesion, stress fibers, and also the microtubule network proposing new roles for ANXA1 in regulating microtubule dynamics. Comparative analysis of regulated global proteome and phosphoproteome highlighted key differences in translational and post-translational effects of ANXA1, and suggested closely coordinated rewiring of the cell adhesion network. Kinase prediction analysis suggested activity modulation of calmodulin-dependent protein kinase II (CAMK2), P21-activated kinase (PAK), extracellular signal-regulated kinase (ERK), and IκB kinase (IKK) upon loss of ANXA1. Integrative analysis revealed regulation of the WNT and Hippo signaling pathways in ANXA1-deficient mammary epithelial cells, wherein there is downregulation of transcriptional effects of TEA domain family (TEAD) suggestive of ANXA1-responsive transcriptional rewiring. CONCLUSIONS: The phosphoproteome landscape uncovered several novel perspectives for ANXA1 in mammary gland biology and highlighted its involvement in key signaling pathways modulating cell adhesion and migration that could contribute to breast cancer initiation.

Protective Aptitude of Annexin A1 in Arterial Neointima Formation in Atherosclerosis-Prone Mice-Brief Report.

OBJECTIVE: Restenosis as a consequence of arterial injury is aggravated by inflammatory pathways. Here, we investigate the role of the proresolving protein annexin A1 (AnxA1) in healing after wire injury. APPROACH AND RESULTS: Apoe-/- and Apoe-/-Anxa1-/- mice were subjected to wire injury while fed a high-cholesterol diet. Subsequently, localization of AnxA1 and AnxA1 plasma levels were examined. AnxA1 was found to localize within endothelial cells and macrophages in the neointima. Levels of AnxA1 in the plasma and its lesional expression negatively correlated with neointima size, and in the absence of AnxA1, neointima formation was aggravated by the accumulation and proliferation of macrophages. In contrast, reendothelialization and smooth muscle cell infiltration were not affected in Apoe-/-Anxa1-/- mice. CONCLUSIONS: AnxA1 is protective in healing after wire injury and could, therefore, be an attractive therapeutic compound to prevent from restenosis after vascular damage.

Role of the protein annexin A1 on the efficacy of anti-TNF treatment in a murine model of acute colitis.

TNF-α is involved in the mechanisms that initiate inflammatory bowel diseases (IBDs). Anti-TNF-α drugs, such as infliximab (IFX), cause non-responsiveness and side effects, indicating the need to investigate alternative therapies for these diseases. The anti-inflammatory protein, annexin A1 (AnxA1), has been associated with the protection of the gastrointestinal mucosa. To further address the role of endogenous AnxA1 on the TNF-α blockade efficacy in a murine model, we assessed colitis induced by Dextran Sulfate Sodium (DSS) in wild-type (WT) and AnxA1(-/-) Balb/c mice treated with IFX. We consistently observed endogenous AnxA1 prevented clinical and physiological manifestations of experimental colitis treated with IFX, additionally the manifestation of the disease was observed earlier in AnxA1(-)(/-) mice. Rectal bleeding, diarrhea, histological score, epithelial damages and collagen degradation caused by DSS were prevented following IFX treatment only in WT mice. IL-6 increased during colitis in WT and AnxA1(-)(/-) mice, decreasing under IFX treatment in WT. The influx of neutrophils and TNF-α secretion were largely elevated in AnxA1(-)(/-) mice when compared to WT mice. In the group WT/DSS+IFX, phagocytes were more susceptible to apoptosis following treatment with IFX. Endogenous expression of AnxA1 increased after DSS and decreased with IFX treatment, demonstrating an attenuated inflammatory response. The data indicate that AnxA1 contributes to the establishment of intestinal homeostasis after blocking of TNF-α was used as a treatment of IBD, constituting a key molecule in the mechanism of action and a potential biomarker of therapeutic efficacy.

Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis.

Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.

Annexin A1 Deficiency does not Affect Myofiber Repair but Delays Regeneration of Injured Muscles.

Repair and regeneration of the injured skeletal myofiber involves fusion of intracellular vesicles with sarcolemma and fusion of the muscle progenitor cells respectively. In vitro experiments have identified involvement of Annexin A1 (Anx A1) in both these fusion processes. To determine if Anx A1 contributes to these processes during muscle repair in vivo, we have assessed muscle growth and repair in Anx A1-deficient mouse (AnxA1-/-). We found that the lack of Anx A1 does not affect the muscle size and repair of myofibers following focal sarcolemmal injury and lengthening contraction injury. However, the lack of Anx A1 delayed muscle regeneration after notexin-induced injury. This delay in muscle regeneration was not caused by a slowdown in proliferation and differentiation of satellite cells. Instead, lack of Anx A1 lowered the proportion of differentiating myoblasts that managed to fuse with the injured myofibers by days 5 and 7 after notexin injury as compared to the wild type (w.t.) mice. Despite this early slowdown in fusion of Anx A1-/- myoblasts, regeneration caught up at later times post injury. These results establish in vivo role of Anx A1 in cell fusion required for myofiber regeneration and not in intracellular vesicle fusion needed for repair of myofiber sarcolemma.

Targeting the annexin 1-formyl peptide receptor 2/ALX pathway affords protection against bacterial LPS-induced pathologic changes in the murine adrenal cortex.

Hypothalamo-pituitary-adrenocortical dysfunction contributes to morbidity and mortality in a high proportion of patients with sepsis. Here, we provide new insights into the underlying adrenal pathology. Using a murine model of endotoxemia (LPS injection), we demonstrate that adrenal insufficiency is triggered early in the disease. LPS induced a local inflammatory response in the adrenal gland within 4 hours of administration, coupled with increased expression of mRNAs for annexin A1 (AnxA1) and the formyl peptide receptors [(Fprs) 1, 2, and 3], a loss of lipid droplets in cortical cells (index of availability of cholesterol, the substrate for steroidogenesis), and a failure to mount a steroidogenic response to ACTH. Deletion of AnxA1 or Fpr2/3 in mice prevented lipid droplet loss, but not leukocyte infiltration. LPS increased adrenal myeloid differentiation primary response gene 88 and TLR2 mRNA expression, but not lymphocyte antigen 96 or TLR4. By contrast, neutrophil depletion prevented leukocyte infiltration and increased AnxA1, Fpr1, and Fpr3 mRNAs but had no impact on lipid droplet loss. Our novel data demonstrate that AnxA1 and Fpr2 have a critical role in the manifestation of adrenal insufficiency in this model, through regulation of cholesterol ester storage, suggesting that pharmacologic interventions targeting the AnxA1/FPR/ALX pathway may provide a new approach for the maintenance of adrenal steroidogenesis in sepsis.

The regulation of TNFα production after heat and endotoxin stimulation is dependent on Annexin-A1 and HSP70.

Febrile temperatures can induce stress responses which protect cells from damage and can reduce inflammation during infections and sepsis. However, the mechanisms behind the protective functions of heat in response to the bacterial endotoxin LPS are unclear. We have recently shown that Annexin-1 (ANXA1)-deficient macrophages exhibited higher TNFα levels after LPS stimulation. Moreover, we have previously reported that ANXA1 can function as a stress protein. Therefore in this study, we determined if ANXA1 is involved in the protective effects of heat on cytokine levels in macrophages after heat and LPS. Exposure of macrophages to 42 °C for 1 h prior to LPS results in an inhibition of TNFα production, which was not evident in ANXA1(-/-) macrophages. We show that this regulation involves primarily MYD88-independent pathways. ANXA1 regulates TNFα mRNA stability after heat and LPS, and this is dependent on endogenous ANXA1 expression and not exogenously secreted factors. Further mechanistic studies revealed the possible involvement of the heat shock protein HSP70 and JNK in the heat and inflammatory stress response regulated by ANXA1. This study shows that ANXA1, an immunomodulatory protein, is critical in the heat stress response induced after heat and endotoxin stimulation.

Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.

Annexin A1 counteracts chemokine-induced arterial myeloid cell recruitment.

RATIONALE: Chemokine-controlled arterial leukocyte recruitment is a crucial process in atherosclerosis. Formyl peptide receptor 2 (FPR2) is a chemoattractant receptor that recognizes proinflammatory and proresolving ligands. The contribution of FPR2 and its proresolving ligand annexin A1 to atherosclerotic lesion formation is largely undefined. OBJECTIVE: Because of the ambivalence of FPR2 ligands, we here investigate the role of FPR2 and its resolving ligand annexin A1 in atherogenesis. METHODS AND RESULTS: Deletion of FPR2 or its ligand annexin A1 enhances atherosclerotic lesion formation, arterial myeloid cell adhesion, and recruitment. Mechanistically, we identify annexin A1 as an endogenous inhibitor of integrin activation evoked by the chemokines CCL5, CCL2, and CXCL1. Specifically, the annexin A1 fragment Ac2-26 counteracts conformational activation and clustering of integrins on myeloid cells evoked by CCL5, CCL2, and CXCL1 through inhibiting activation of the small GTPase Rap1. In vivo administration of Ac2-26 largely diminishes arterial recruitment of myeloid cells in a FPR2-dependent fashion. This effect is also observed in the presence of selective antagonists to CCR5, CCR2, or CXCR2, whereas Ac2-26 was without effect when all 3 chemokine receptors were antagonized simultaneously. Finally, repeated treatment with Ac2-26 reduces atherosclerotic lesion sizes and lesional macrophage accumulation. CONCLUSIONS: Instructing the annexin A1-FPR2 axis harbors a novel approach to target arterial leukocyte recruitment. With the ability of Ac2-26 to counteract integrin activation exerted by various chemokines, delivery of Ac2-26 may be superior in inhibition of arterial leukocyte recruitment when compared with blocking individual chemokine receptors.

A formyl peptide receptor agonist suppresses inflammation and bone damage in arthritis.

BACKGROUND AND PURPOSE: Annexin A1 (AnxA1) is an endogenous anti-inflammatory protein and agonist of the formyl peptide receptor 2 (FPR2). However, the potential for therapeutic FPR ligands to modify immune-mediated disease has been little explored. We investigated the effects of a synthetic FPR agonist on joint disease in the K/BxN model of rheumatoid arthritis (RA) and RA fibroblast-like synoviocytes (FLS). EXPERIMENTAL APPROACH: Arthritis was induced by injection of K/BxN serum at day 0 and 2 in wild-type (WT) or AnxA1(-/-) mice and clinical and histopathological manifestations measured 8-11 days later. WT mice were given the FPR agonist compound 43 (Cpd43) (6 or 30 mg·kg(-1) i.p.) for 4 days. Effects of AnxA1 and Cpd43 on RANKL-induced osteoclastogenesis were assessed in RAW 264.7 cells and human RA FLS and macrophages. KEY RESULTS: Treatment with Cpd43 before or after the onset of arthritis reduced clinical disease severity and attenuated synovial TNF-α and osteoclast-associated gene expression. Deletion of AnxA1 in mice exacerbated arthritis severity in the K/BxN model. In vitro, Cpd43 suppressed osteoclastogenesis and NFAT activity elicited by RANKL, and inhibited IL-6 secretion by mouse macrophages. In human RA joint-derived FLS and monocyte-derived macrophages, Cpd43 treatment inhibited IL-6 release, while blocking FPR2 or silencing AnxA1 increased this release. CONCLUSIONS AND IMPLICATIONS: The FPR agonist Cpd43 reduced osteoclastogenesis and inflammation in a mouse model of RA and exhibited anti-inflammatory effects in relevant human cells. These data suggest that FPR ligands may represent novel therapeutic agents capable of ameliorating inflammation and bone damage in RA.

Increased adiposity in annexin A1-deficient mice.

Production of Annexin A1 (ANXA1), a protein that mediates the anti-inflammatory action of glucocorticoids, is altered in obesity, but its role in modulation of adiposity has not yet been investigated. The objective of this study was to investigate modulation of ANXA1 in adipose tissue in murine models of obesity and to study the involvement of ANXA1 in diet-induced obesity in mice. Significant induction of ANXA1 mRNA was observed in adipose tissue of both C57BL6 and Balb/c mice with high fat diet (HFD)-induced obesity versus mice on chow diet. Upregulation of ANXA1 mRNA was independent of leptin or IL-6, as demonstrated by use of leptin-deficient ob/ob mice and IL-6 KO mice. Compared to WT mice, female Balb/c ANXA1 KO mice on HFD had increased adiposity, as indicated by significantly elevated body weight, fat mass, leptin levels, and adipocyte size. Whereas Balb/c WT mice upregulated expression of enzymes involved in the lipolytic pathway in response to HFD, this response was absent in ANXA1 KO mice. A significant increase in fasting glucose and insulin levels as well as development of insulin resistance was observed in ANXA1 KO mice on HFD compared to WT mice. Elevated plasma corticosterone levels and blunted downregulation of 11-beta hydroxysteroid dehydrogenase type 1 in adipose tissue was observed in ANXA1 KO mice compared to diet-matched WT mice. However, no differences between WT and KO mice on either chow or HFD were observed in expression of markers of adipose tissue inflammation. These data indicate that ANXA1 is an important modulator of adiposity in mice, with female ANXA1 KO mice on Balb/c background being more susceptible to weight gain and diet-induced insulin resistance compared to WT mice, without significant changes in inflammation.

Identification of an essential endogenous regulator of blood-brain barrier integrity, and its pathological and therapeutic implications.

The blood-brain barrier (BBB), a critical guardian of communication between the periphery and the brain, is frequently compromised in neurological diseases such as multiple sclerosis (MS), resulting in the inappropriate passage of molecules and leukocytes into the brain. Here we show that the glucocorticoid anti-inflammatory messenger annexin A1 (ANXA1) is expressed in brain microvascular endothelial cells, where it regulates BBB integrity. In particular, ANXA1(-/-) mice exhibit significantly increased BBB permeability as a result of disrupted interendothelial cell tight junctions, essentially related to changes in the actin cytoskeleton, which stabilizes tight and adherens junctions. This situation is reminiscent of early MS pathology, a relationship confirmed by our detection of a selective loss of ANXA1 in the plasma and cerebrovascular endothelium of patients with MS. Importantly, this loss is swiftly restored by i.v. administration of human recombinant ANXA1. Analysis in vitro confirms that treatment of cerebrovascular endothelial cells with recombinant ANXA1 restores cell polarity, cytoskeleton integrity, and paracellular permeability through inhibition of the small G protein RhoA. We thus propose ANXA1 as a critical physiological regulator of BBB integrity and suggest it may have utility in the treatment of MS, correcting BBB function and hence ameliorating disease.

Reperfusion-induced myocardial dysfunction is prevented by endogenous annexin-A1 and its N-terminal-derived peptide Ac-ANX-A1(2-26).

BACKGROUND AND PURPOSE: Annexin-A1 (ANX-A1) is an endogenous, glucocorticoid-regulated anti-inflammatory protein. The N-terminal-derived peptide Ac-ANX-A1(2-26) preserves cardiomyocyte viability, but the impact of ANX-A1-peptides on cardiac contractility is unknown. We now test the hypothesis that ANX-A1 preserves post-ischaemic recovery of left ventricular (LV) function. EXPERIMENTAL APPROACH: Ac-ANX-A1(2-26) was administered on reperfusion, to adult rat cardiomyocytes as well as hearts isolated from rats, wild-type mice and mice deficient in endogenous ANX-A1 (ANX-A1(-/-)). Myocardial viability and recovery of LV function were determined. KEY RESULTS: Ischaemia-reperfusion markedly impaired both cardiomyocyte viability and recovery of LV function by 60%. Treatment with exogenous Ac-ANX-A1(2-26) at the onset of reperfusion prevented cardiomyocyte injury and significantly improved recovery of LV function, in both intact rat and wild-type mouse hearts. Ac-ANX-A1(2-26) cardioprotection was abolished by either formyl peptide receptor (FPR)-nonselective or FPR1-selective antagonists, Boc2 and cyclosporin H, but was relatively insensitive to the FPR2-selective antagonist QuinC7. ANX-A1-induced cardioprotection was associated with increased phosphorylation of the cell survival kinase Akt. ANX-A1(-/-) exaggerated impairment of post-ischaemic recovery of LV function, in addition to selective LV FPR1 down-regulation. CONCLUSIONS AND IMPLICATIONS: These data represent the first evidence that ANX-A1 affects myocardial function. Our findings suggest ANX-A1 is an endogenous regulator of post-ischaemic recovery of LV function. Furthermore, the ANX-A1-derived peptide Ac-ANX-A1(2-26) on reperfusion rescues LV function, probably via activation of FPR1. ANX-A1-based therapies may thus represent a novel clinical approach for the prevention and treatment of myocardial reperfusion injury.

Deficiency of annexin A1 in CD4+ T cells exacerbates T cell-dependent inflammation.

Annexin A1 (AnxA1) is recognized as an endogenous anti-inflammatory molecule. However, its effects on the adaptive immune response and, in particular, on T cells remain unclear. In this study, we investigated the actions of AnxA1 in three distinct models of T cell-mediated inflammation. In contact hypersensitivity, collagen-induced arthritis, and inflammation induced by OT-II TCR transgenic T cells responding to OVA, AnxA1 deficiency significantly increased Ag-induced T cell proliferation and the resultant level of inflammation. In the contact hypersensitivity model, this was associated with increased adhesion of CD4(+) T cells, CD8(+) T cells, and neutrophils in the dermal microvasculature, as well as increased T cell expression of RORγt and IL-17A. In collagen-induced arthritis, deficiency of endogenous AnxA1 increased susceptibility to arthritis and Ag-specific T cell activation. Deficiency of AnxA1 also increased OVA-induced cutaneous delayed-type hypersensitivity and IFN-γ and IL-17 release. Transfer experiments using CD4(+) T cells from AnxA1(-/-) mice demonstrated that the absence of AnxA1 solely in T cells resulted in increased inflammatory responses in wild-type recipients. Similarly, experiments using AnxA1(-/-) OT-II CD4(+) T cells demonstrated that the absence of AnxA1 in T cells was sufficient to induce increased Ag-specific CD4(+) T cell proliferation in vivo, augment T cell production of IFN-γ, IL-17, TNF, and IL-6, and increase Akt, ERK, and p38 activation. Together, these findings indicate that T cell-expressed AnxA1 functions to attenuate T cell-driven inflammatory responses via T cell-intrinsic effects on intracellular signaling, proliferation, and Th1/Th17 cytokine release.

Annexin-1-deficient mice exhibit spontaneous airway hyperresponsiveness and exacerbated allergen-specific antibody responses in a mouse model of asthma.

BACKGROUND: Glucocorticoids are the mainstream drugs used in the treatment and control of inflammatory diseases such as asthma. Annexin-1 (ANXA1) is an anti-inflammatory protein which has been described as an endogenous protein responsible for some anti-inflammatory glucocorticoid effects. Previous studies have identified its importance in other immune diseases such as rheumatoid arthritis and cystic fibrosis. ANXA1-deficient ((-/-)) mice are Th2 biased, and ANXA1 N-terminus peptide exhibits anti-inflammatory activity in a rat model of pulmonary inflammation. OBJECTIVE: ANXA1 protein is found in bronchoalveolar lavage fluid from asthmatics. However, the function of ANXA1 in the pathological development of allergy or asthma is unclear. Thus, in this study we intended to examine the effect of ANXA1 deficiency on allergen-specific antibody responses and airway responses to methacholine (Mch). METHODS: ANXA1(-/-) mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA. Airway resistance, lung compliance and enhanced pause (PenH) were measured in naïve, sensitized and saline or allergen-challenged wild-type (WT) and ANXA1(-/-) mice. Total and allergen-specific antibodies were measured in the serum. RESULTS: We show that allergen-specific and total IgE, IgG2a and IgG2b levels were significantly higher in ANXA1(-/-) mice. Furthermore, naïve ANXA1(-/-) mice displayed higher airway hypersensitivity to inhaled Mch, and significant differences were also observed in allergen-sensitized and allergen-challenged ANXA1(-/-) mice compared with WT mice. CONCLUSIONS: In conclusion, ANXA1(-/-) mice possess multiple features characteristic to allergic asthma, such as airway hyperresponsiveness and enhanced antibody responses, suggesting that ANXA1 plays a critical regulatory role in the development of asthma. CLINICAL RELEVANCE: We postulate that ANXA1 is an important regulatory factor in the development of allergic disease and dysregulation of its expression can lead to pathological changes which may affect disease progression.

Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NF-κB: implication in breast cancer metastasis.

The molecular mechanisms underlying constitutive nuclear factor-κB (NF-κB) activation in solid tumors has not been elucidated. We show that Annexin-1 (ANXA1) is involved in this process, and suppression of ANXA1 in highly metastatic breast cancer cells impedes migration and metastasis capabilities in vitro and in vivo. ANXA1 expression correlates with NF-κB activity, suggesting that ANXA1 may be required for the constitutive activity of IκB kinase (IKK) and NF-κB in highly metatstatic breast cancer. Gel-filtration analysis demonstrated that ANXA1 co-elutes with the members of the IKK complex and NF-κB signaling pathway, and immunoprecipitation confirmed that ANXA1 can bind to and interact with IKKγ or NEMO, but not IKKα or IKKß. Importantly, silencing of ANXA1 prevents the interaction of NEMO and RIP1, which indicates that ANXA1 is required for the recruitment of RIP1 to the IKK complex, which may be important for the activation of NF-κB. Downstream targets of NF-κB include uPA and CXCR4, which can be modulated by ANXA1 silencing. CXCR4-mediated migration of breast cancer cell lines in response to CXCL12 was significantly modulated by ANXA1, indicating its importance in the tissue-specific migration of breast cancer cells. Chromatin immunoprecipitation experiments confirmed that in ANXA1 overexpressed cells, NF-κB was recruited to CXCR4 promoter without external stimulation, indicating that ANXA1 is critical for the constitutive activation of NF-κB in breast cancer to promote metastasis. Finally, we show that ANXA1 overexpression enhances metastasis and reduces survival in an intracardiac metastasis model, while ANXA1-deficient mice crossed with MMTV-PyMT mice display significantly less metastasis than their heterozygous littermates, indicating that ANXA1 is an important gene in breast cancer metastasis. Our data reveal that ANXA1 can constitutively activate NF-κB in breast cancer cells through the interaction with the IKK complex, and suggests that modulating ANXA1 levels has therapeutic potential to suppress breast cancer metastasis.

Antiallergic cromones inhibit neutrophil recruitment onto vascular endothelium via annexin-A1 mobilization.

OBJECTIVE: To determine whether the inhibitory action of the antiallergic cromone "mast cell stabilizing" drugs on polymorphonuclear leukocyte (PMN) trafficking is mediated through an annexin-A1 (Anx-A1) dependent mechanism. METHODS AND RESULTS: Intravital microscopy was used to monitor the actions of cromones in the inflamed microcirculation. Reperfusion injury provoked a dramatic increase in adherent and emigrated leukocytes in the mesenteric vascular bed, associated with augmented tissue levels of myeloperoxidase. Nedocromil, 2 to 20 mg/kg, significantly (P<0.05) inhibited cell adhesion and emigration, as well as myeloperoxidase release, in wild-type but not Anx-A1(-/-) mice. Short pretreatment of human PMNs with nedocromil, 10 nmol/L, inhibited cell adhesion (P<0.05) in the flow chamber assay, and this effect was reversed by specific anti-AnxA1 or a combination of antiformyl peptide receptors 1 and 2, but not irrelevant control, antibodies. Western blotting experiments revealed that cromones stimulate protein kinase C-dependent phosphorylation and release Anx-A1 in human PMNs. CONCLUSIONS: We propose a novel mechanism to explain the antiinflammatory actions of cromones on PMN trafficking, an effect that has long puzzled investigators.

Impaired tumor growth, metastasis, angiogenesis and wound healing in annexin A1-null mice.

Despite 2 decades of research, no clear function for annexin A1 (AnxA1) has been established. Using AnxA1-KO mice, we show that tumor growth and metastasis are significantly decreased, whereas rodent survival and tumor necrosis are greatly increased when tumors grow in AnxA1-KO mice. Systems analysis of gene expression in these tumors specifically implicates 2 related vascular functions, angiogenesis and wound healing, in this impairment. Both tumor vascular development and wound healing are greatly retarded in KO tissues. Aortic ring assays reveal induced AnxA1 expression on sprouting endothelial cells of normal mice whereas KO aortas exhibit impaired endothelial cell sprouting that is rescued by adenoviral expression of AnxA1. Key differences in specific gene regulation may define new molecular pathways mediating angiogenesis, including a reset profile of pro- versus anti-angiogenic factors, apparently distinct for physiological versus pathological angiogenesis. These studies establish novel pro-angiogenic functions for AnxA1 in vascular endothelial cell sprouting, wound healing, and tumor growth and metastasis, thereby uncovering a new functional target for repairing damaged tissue and treating diseases such as cancer. They also provide critical new evidence that the tumor stroma and its microenvironment can greatly affect tumor progression and metastasis.

Annexin A1 and glucocorticoids as effectors of the resolution of inflammation.

Glucocorticoids are widely used for the management of inflammatory diseases. Their clinical application stems from our understanding of the inhibitory effect of the corticosteroid hormone cortisol on several components of the immune system. Endogenous and exogenous glucocorticoids mediate their multiple anti-inflammatory effects through many effector molecules. In this Opinion article, we focus on the role of one such effector molecule, annexin A1, and summarize the recent studies that provide insight into its molecular and pharmacological functions in immune responses. In addition, we propose a model in which glucocorticoids regulate the expression and function of annexin A1 in opposing ways in innate and adaptive immune cells to mediate the resolution of inflammation.