ANXA1 Protein Research Articles

A meta-analysis of genome-wide association studies of childhood wheezing phenotypes identifies ANXA1 as a susceptibility locus for persistent wheezing.

Many genes associated with asthma explain only a fraction of its heritability. Most genome-wide association studies (GWASs) used a broad definition of 'doctor-diagnosed asthma', thereby diluting genetic signals by not considering asthma heterogeneity. The objective of our study was to identify genetic associates of childhood wheezing phenotypes. We conducted a novel multivariate GWAS meta-analysis of wheezing phenotypes jointly derived using unbiased analysis of data collected from birth to 18 years in 9568 individuals from five UK birth cohorts. Forty-four independent SNPs were associated with early-onset persistent, 25 with pre-school remitting, 33 with mid-childhood remitting, and 32 with late-onset wheeze. We identified a novel locus on chr9q21.13 (close to annexin 1 [ANXA1], p<6.7 × 10-9), associated exclusively with early-onset persistent wheeze. We identified rs75260654 as the most likely causative single nucleotide polymorphism (SNP) using Promoter Capture Hi-C loops, and then showed that the risk allele (T) confers a reduction in ANXA1 expression. Finally, in a murine model of house dust mite (HDM)-induced allergic airway disease, we demonstrated that anxa1 protein expression increased and anxa1 mRNA was significantly induced in lung tissue following HDM exposure. Using anxa1-/- deficient mice, we showed that loss of anxa1 results in heightened airway hyperreactivity and Th2 inflammation upon allergen challenge. Targeting this pathway in persistent disease may represent an exciting therapeutic prospect. UK Medical Research Council Programme Grant MR/S025340/1 and the Wellcome Trust Strategic Award (108818/15/Z) provided most of the funding for this study.

In-depth quantitative proteomics analysis revealed C1GALT1 depletion in ECC-1 cells mimics an aggressive endometrial cancer phenotype observed in cancer patients with low C1GALT1 expression

BackgroundEndometrial cancer (EC) is the most common cancer of the female reproductive organs. Despite the good overall prognosis of most low-grade ECs, FIGO I and FIGO II patients might experience tumor recurrence and worse prognosis. The study of alterations related to EC pathogenesis might help to get insights into underlying mechanisms involved in EC development and progression.MethodsCore tumoral samples were used to investigate the role of C1GALT1 in EC by immunohistochemistry (IHC). ECC-1 cells were used as endometrioid EC model to investigate the effect of C1GALT1 depletion using C1GALT1 specific shRNAs. SILAC quantitative proteomics analyses and cell-based assays, PCR, qPCR, WB, dot-blot and IHC analyses were used to identify, quantify and validate dysregulation of proteins.ResultsLow C1GALT1 protein expression levels associate to a more aggressive phenotype of EC. Out of 5208 proteins identified and quantified by LC-MS/MS, 100 proteins showed dysregulation (log2fold-change ≥ 0.58 or ≤-0.58) in the cell protein extracts and 144 in the secretome of C1GALT1 depleted ECC-1 cells. Nine dysregulated proteins were validated. Bioinformatics analyses pointed out to an increase in pathways associated with an aggressive phenotype. This finding was corroborated by loss-of-function cell-based assays demonstrating higher proliferation, invasion, migration, colony formation and angiogenesis capacity in C1GALT1 depleted cells. These effects were associated to the overexpression of ANXA1, as demonstrated by ANXA1 transient silencing cell-based assays, and thus, correlating C1GALT and ANXA1 protein expression and biological effects. Finally, the negative protein expression correlation found by proteomics between C1GALT1 and LGALS3 was confirmed by IHC.ConclusionC1GALT1 stably depleted ECC-1 cells mimic an EC aggressive phenotype observed in patients and might be useful for the identification and validation of EC markers of progression.

E2F1-initiated transcription of PRSS22 promotes breast cancer metastasis by cleaving ANXA1 and activating FPR2/ERK signaling pathway

Breast cancer (BC) is the most common malignant tumor in women worldwide. Metastasis is the main cause of BC-related death. The specific mechanism underlying BC metastasis remains obscure. Recently, PRSS22 was discovered to be involved in tumor development, however, its detailed biological function and regulatory mechanism in BC are unclear. Here, we characterized that PRSS22 expression is upregulated in BC tissues compared with non-tumorous breast tissues. Dual luciferase assays, bioinformatics analyses and chromatin immunoprecipitation (ChIP) assays indicated that transcription factor E2F1 directly binds to the PRSS22 promoter region and activates its transcription. Functionally, upregulation of PRSS22 promoted invasion and metastasis of BC cells in vitro and in vivo, whereas knockdown of PRSS22 inhibited its function. Mechanistically, the combination of PRSS22 and ANXA1 protein in BC cells was first screened by protein mass spectrometry analysis, and then confirmed by co-immunoprecipitation (Co-IP) and western blot assays. Co-overexpression of PRSS22 and ANXA1 could promote BC cell migration and invasion. We further demonstrated that PRSS22 promotes the cleavage of ANXA1 and in turn generates an N-terminal peptide, which initiates the FPR2/ERK signaling axis to increase BC aggressiveness.

The role of annexin A1-derived peptide Ac2–26 on liver and kidney injuries induced by cisplatin in rats

AimsIn this study we evaluated the effect of pharmacological treatment with AnxA1-derived peptide Ac2–26 in an experimental model of toxicity induced by cisplatin. Main methodsMale rats were divided into Sham (control), Cisplatin (received intraperitoneal injections of 10 mg/kg/day of cisplatin for 3 days) and Ac2–26 (received intraperitoneal injections of 1 mg/kg/day of peptide, 15 min before cisplatin) groups. Key findingsAfter 6 h of the last dose of cisplatin, an acute inflammatory response was observed characterized by a marked increase in the number of neutrophils and GM-CSF, IL-β, IL-6, IL-10 and TNF-α plasma levels. These findings were associated with increased AnxA1 protein levels in liver and kidneys, as well as positive AnxA1/Fpr2 circulating leukocytes. Treatment with Ac2–26 produced higher levels of GM-CSF, corroborating the high numbers of neutrophils, and the anti-inflammatory cytokine IL-4. Ac2–26 preserved the morphology of liver structures and increased Fpr1 expression, preventing the damage caused by cisplatin. In the kidneys, Ac2–26 caused downregulation of renal Fpr1 and Fpr2 levels and abrogated the increased levels of the CLU and KIM-1 biomarkers of kidney damage induced by cisplatin. However, no effect of peptide treatment was detected in cisplatin-induced kidney morphology injury. SignificanceDespite activation of the anti-inflammatory AnxA1/Fpr axis during cisplatin administration, treatment with Ac2–26 did not efficiently prevent its deleterious effects on the liver and kidneys.

HA/CD44 Regulates the T Helper 1 Cells Differentiation by Activating Annexin A1/Akt/mTOR Signaling to Drive the Pathogenesis of EAP.

CD44 partcipates in multiple inflammatory reactions. Here, we aimed to investigate the role of CD44 and the ligand, hyaluronan (HA), on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) pathogenesis. We found that CD44 was universally expressed in CD4+ lymphocytes in the peripheral blood of CP/CPPS patients. After silencing CD44 expression or delivering 4-methylumbelliferone (4-MU), the pain severity and prostatic inflammation were significantly relieved. In vitro assay found that HA/CD44 was able to regulate T helper 1 (Th1) cells differentiation, the deficiency of which diminished experimental autoimmune prostatitis (EAP) susceptibility. Bioinformatic analysis suggested that after HA or 4-MU treatment, mTOR signaling was significantly altered, and these results were confirmed by subsequent Western blotting assay. Besides, mass spectrometry and co-immunoprecipitation assays found that CD44 was able to interact with Annexin A1 (ANX A1), and this kind of interaction stabilized ANX A1 protein and maintained the activation of Akt/mTOR pathway. Meanwhile, HA-treatment-enhanced prostatic inflammation, Th1 cell differentiation, and Akt/mTOR pathway activation were reversed after silencing the expression of ANX A1 using shANX A1-lentivirus. The present study systematically investigates the functional role of HA/CD44 in CP/CPPS and identifies novel mechanisms for HA/CD44 promoting Th1 cell differentiation. Targeting the HA/CD44/ANX A1/Akt/mTOR signaling represents novel potential therapeutic strategies for patients with CP/CPPS.

Specificity of Antibodies for the Identification of Annexin 1 (ANXA1) Protein in Various Types of Cancer

Annexins are a well‐characterized multigene family of phospholipid‐binding and membrane‐bound proteins that are Ca2+‐regulated. ANXA1 expression is variable in tumor cells, ranging from high levels to none, which is why it is helpful to know the staining potential of various antibodies against the protein. Our research aims to evaluate the specificity of five antibodies used to detect ANXA1 expression in selecting different types of cancer and normal tissue and the differential specificity between the two groups. We examined a data set composed of 2177 samples from The Human Protein Atlas (HPA) as mentioned, cancer patients:1904 samples, and healthy persons: 273 samples. We examined the specificity of five different antibody types of detecting ANXA1 expression (HPA011271, HPA011272, CAB013023, CAB035987, and CAB058693). The specificity of each antibody against ANXA1 protein was evaluated using a staining scale of 0 ‐ 3.00, where 0 indicates no staining, 0.01‐1.0 low staining, 1.01 ‐ 2.0 medium staining, and 2.01 ‐ 3.00 high staining.ResultsCAB013023 had the highest staining values for both cancer and healthy samples; thyroid cancer and endometrial cancer had the highest staining values (2.75 and 2.80, respectively). Breast, head and neck, and bladder normal tissues stained the most intensely in healthy samples (3.00 in all three cases). The antibody's specificity for identifying ANXA1 expression suggests that this may be used as a prognosis and treatment marker in cancer.

Inhibition of STAT6 Activation by AS1517499 Inhibits Expression and Activity of PPARγ in Macrophages to Resolve Acute Inflammation in Mice.

Signal transducer and activator of transcription 6 (STAT6) promotes an anti-inflammatory process by inducing the development of M2 macrophages. We investigated whether modulating STAT6 activity in macrophages using AS1517499, the specific STAT6 inhibitor, affects the restoration of homeostasis after an inflammatory insult by regulating PPARγ expression and activity. Administration of AS1517499 suppressed the enhanced STAT6 phosphorylation and nuclear translocation observed in peritoneal macrophages after zymosan injection. In addition, AS1517499 delayed resolution of acute inflammation as evidenced by enhanced secretion of pro-inflammatory cytokines, reduced secretion of anti-inflammatory cytokines in PLF and supernatants from peritoneal macrophages, and exaggerated neutrophil numbers and total protein levels in PLF. We demonstrate temporal increases in annexin A1 (AnxA1) protein and mRNA levels in peritoneal lavage fluid (PLF), peritoneal macrophages, and spleen in a murine model of zymosan-induced acute peritonitis. In vitro priming of mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages with AnxA1 induced STAT6 activation with enhanced PPARγ expression and activity. Using AS1517499, we demonstrate that inhibition of STAT6 activation delayed recovery of PPARγ expression and activity, as well as impaired efferocytosis. Taken together, these results suggest that activation of the STAT6 signaling pathway mediates PPARγ expression and activation in macrophages to resolve acute inflammation.

Evaluation of Colocasia esculenta Schott in anti-cancerous properties with proximity extension assays.

BackgroundColocasia esculenta Schott (called as Xiangshayu in Chinese) is an excellent local cultivar of the genus polymorpha in Jiangsu Province, China.ObjectiveIn the present study, we have performed a comparative study before and after dietary consumption with Colocasia esculenta Schott to evaluate its anti-cancerous properties.DesignForty-two healthy volunteers were recruited, and dietary consumption with 200 g of tap water cooked Colocasia esculenta Schott daily was conducted for 1 month. Plasma samples from the subjects before and after dietary consumption with Colocasia esculenta Schott were analyzed with proximity extension assays for the alteration of 92 proteins in relation with cancers, while blood samples were examined for physiological parameters with an automatic biochemical analyzer. Bioinformatic analyses were conducted using MalaCards and GEPIA.ResultsAfter taking dietary consumption with Colocasia esculenta Schott, circulating CYR61, ANXA1, and VIM protein levels in the subjects was found to be most significantly downregulated, while for ITGB5, EPHA2, and CEACAM1, it was upregulated. Alternation of these proteins was predicted to be associated with the development of tumors such as pancreatic adenocarcinoma and breast and prostate cancers.ConclusionThe present study provides evidence that Colocasia esculenta Schott, as a healthy food, has anti-cancerous properties. Further investigation of phytochemistry in Colocasia esculenta Schott has been taken into our consideration.

2-Methoxyestradiol Protects Against Lung Ischemia/Reperfusion Injury by Upregulating Annexin A1 Protein Expression.

Background: 2-Methoxyestradiol (2ME), a natural 17-β estradiol metabolite, is a potent anti-inflammatory agent, but its effect on ischemia/reperfusion (IR)-induced acute lung inflammation remains unknown. Annexin A1 (AnxA1), a glucocorticoid-regulated protein, is effective at inhibiting neutrophil transendothelial migration by binding the formyl peptide receptors (FPRs). We aimed to investigate whether 2ME upregulates the expression of AnxA1 and protects against IR-induced lung damage.Methods: IR-mediated acute lung inflammation was induced by ischemia for 40 min followed by reperfusion for 60 min in an isolated, perfused rat lung model. The rat lungs were randomly treated with vehicle or 2ME, and the functional relevance of AnxA1 was determined using an anti-AnxA1 antibody or BOC2 (a pan-receptor antagonist of the FPR). In vitro, human primary alveolar epithelial cells (HPAECs) and rat neutrophils were pretreated with 2ME and an AnxA1 siRNA or anti-AnxA1 antibody and subjected to hypoxia-reoxygenation (HR).Results: 2ME significantly decreased all lung edema parameters, neutrophil infiltration, oxidative stress, proinflammatory cytokine production, lung cell apoptosis, tight junction protein disruption, and lung tissue injury in the IR-induced acute lung inflammation model. 2ME also increased the expression of the AnxA1 mRNA and protein and suppressed the activation of nuclear factor-κB (NF-κB). In vitro, 2ME attenuated HR-triggered NF-κB activation and interleukin-8 production in HPAECs, decreased transendothelial migration, tumor necrosis factor-α production, and increased apoptosis in neutrophils exposed to HR. These protective effects of 2ME were significantly abrogated by BOC2, the anti-AnxA1 antibody, or AnxA1 siRNA.Conclusions: 2ME ameliorates IR-induced acute lung inflammation by increasing AnxA1 expression. Based on these results, 2ME may be a promising agent for attenuating IR-induced lung injury.

Maintenance of cellular annexin A1 level is essential for PI3K/AKT/mTOR-mediated proliferation of pancreatic beta cells.

Annexin A1 (AnxA1, also known as lipocortin-1), is a calcium-dependent phospholipid binding protein with diverse functions. Previous studies have indicated that AnxA1 is associated with age-related β-cell dysfunction and aging, which lead to decreased β-cell proliferation capacity. However, it has been uncertain whether AnxA1 affects the proliferation of pancreatic beta (β) cells. In the present study, we reduced AnxA1 expression in the MIN6 islet β-cell line using small interfering RNA (AnxA1-siRNA), then measured cell cycle distribution and cellular proliferation. We also measured the expression levels of cell cycle-related proteins such as cyclin D1, cyclin E, and cyclin-dependent kinase 2 (CDK2) by Western blot analysis. We investigated the phosphatidylinositol 3-kinase (PI3K)/ serine/threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway to explore the potential mechanism underlying the observed effects. Knockdown of AnxA1 expression using siRNA reduced the rates of MIN6 cell proliferation. The proportions of cells in S and G2/M phases also decreased upon inhibition of AnxA1. Moreover, AnxA1 protein expression in MIN6 cells was positively related to the protein levels of cyclin D1, cyclin E, and CDK2. Activation of the PI3K/Akt/mTOR signaling pathway by AnxA1 may be involved in the signaling cascade to regulate cell proliferation. This study identified a positive correlation between AnxA1 protein and pancreatic β-cell proliferation. AnxA1 protein expression might affect the proliferation of MIN6 cells via regulation of cyclin D1, cyclin E, and CDK2 proteins, as well as the PI3K/Akt/mTOR signaling pathway.

Methanolic extract of Cariniana rubra Gardner ex Miers stem bark negatively regulate the leukocyte migration and TNF-α and up-regulate the annexin-A1 expression

Ethnopharmacological relevanceCariniana rubra Gardner ex Miers (Lecythidaceae), is a native and endemic tree in Brazil, whose inner stem bark decoction preparation is used in folk medicine to treat various inflammatory disorders. Previous scientific reports confirmed its popular use as an anti-inflammatory, without, however, evaluating its action mechanisms. AimThe objective of this study was to determine the cytotoxicity and anti-inflammatory mechanism of action of the methanolic extract of Cariniana rubra (MECr), using experimental models in vivo and in vitro, as well as to identify secondary metabolites present in the extract. Material and methodsThe MECr was prepared by maceration of inner stem bark powder in methanol (1:10 w/v). The in vitro cytotoxicity effect was evaluated in CHO-k1 cells. The Hippocratic screening test was conducted to evaluate the acute toxicity of MECr in mice. The actions of MECr on leukocyte migration, cytokine levels (IL-1β and TNF-α) and annexin-A1 (AnxA1) expression, were carried out on lambda-type carrageenan air pouch inflammation model in Swiss mice. Additionally, the phytochemical analysis of MECr was carried out by thin-layer chromatography (TLC) and spectrometric mass analysis with electrospray ionization ESI(−)/MS and gas chromatography-mass spectrometry (GC-MS). ResultsTreatment of CHO-k1 cells for 24 h with MECr did not cause cytotoxicity (IC50 > 200 μg/mL), however, the MECr was shown to be cytotoxic after 72 h of cell exposure (IC50 = 19.90 ± 3.51 μg/mL). In the Hippocratic test, oral treatment of mice with 750, 1500, or 3000 mg/kg of MECr did not show any histopathological changes and mortality during the 14 days of observation. In the carrageenan air pouch inflammation model, MECr reduced (p < 0.001) polymorphonuclear migration (57.7% and 57.8%), leukocyte monocyte migration (74.5% and 61.8%) in the air pouch cavity and in the skin tissue, respectively. MECr also inhibited TNF-α concentration in the air cavity wash (3.2%, p < 0.01) and increased expression of the AnxA1 protein (26.9%, p < 0.01) in the skin tissue, particularly in neutrophils. β-sitosterol (1.95%), gallic acid (1.24%), β-amyrin (0.87%) and stigmasterol (0.66%) were identified as the major constituents in methanolic extract. ConclusionMECr exhibits significant anti-inflammatory action at least by increasing AnxA1 expression and by inhibiting the release of TNF-α pro-inflammatory cytokine and leukocyte migration, which is probably linked to the presence of identified biologically active compounds, especially gallic acid and terpenes. We believe that the results of this study provide a pharmacological basis for the MECr to be considered as a potential therapeutic agent for the treatment of inflammatory diseases.

Immunoproteomics approach revealed elevated autoantibody levels against ANXA1 in early stage gallbladder carcinoma

BackgroundEarly diagnosis is important for the timely treatment of gallbladder carcinoma (GBC) patients and may lead to increased survival outcomes. Here, we have applied serological proteome analysis (SERPA), an immunoproteomics approach, for the detection of ‘tumor-associated antigens (TAAs) that elicit humoral response’ in early stage GBC patients.MethodsTotal protein from pooled tumor tissue of GBC patients (n = 7) was resolved by two-dimensional gel electrophoresis (2-DE) followed by immunoblotting using pooled blood plasma from healthy volunteers (n = 11) or gallstone disease (GSD) cases (n = 11) or early stage GBC (Stage I and II) (n = 5) or GBC stage IIIA (n = 9). 2-D gel and immunoblot images were acquired and analyzed using PDQuest software to identify immunoreactive spots in GBC cases in comparison to controls. Proteins from immunoreactive spots were identified by liquid chromatography- tandem mass spectrometric analysis (LC-MS/MS). Autoantibody levels for two of the functionally relevant proteins were investigated in individual plasma samples (52 cases and 89 controls) by dot blot assay using recombinant proteins.ResultsImage analysis using PDQuest software identified 25 protein spots with significantly high or specific immunoreactivity in GBC cases. Mass spectrometric analysis of 8 corresponding protein spots showing intense immunoreactivity (based on densitometric analysis) in early stage GBC or GBC stage IIIA cases led to the identification of 27 proteins. Some of the identified proteins include ANXA1, HSPD1, CA1, CA2, ALDOA and CTSD. Among the two proteins, namely ANXA1 and HSPD1 verified using a cohort of samples, significantly elevated autoantibody levels against ANXA1 were observed in early stage GBC cases in comparison to healthy volunteers or GSD cases (unpaired t-test, p < 0.05). Receiver operating characteristic (ROC) curve analysis for ANXA1 showed an Area under the Curve (AUC) of 0.69, with 41.7% sensitivity against a specificity of 89.9% for early stage GBC. IHC analysis for ANXA1 protein showed ‘high’ expression levels in 72% of GBC cases whereas all the controls showed ‘low’ expression levels.ConclusionsThe study suggests that the ANXA1 autoantibody levels against ANXA1 may be potentially employed for early stage detection of GBC patients. Other proteins could also be explored and verified in a large cohort of clinical samples.

Cellular Function of Annexin A1 Protein Mimetic Peptide Ac2-26 in Human Skin Keratinocytes HaCaT and Fibroblast Detroit 551 Cells.

Inflammation of the skin is the most common dermatological problem in human. The anti-inflammatory mediated responses of the skin cells provide a mechanism for combating these conditions. Annexin A1 (AnxA1) is one of the proteins that has been shown to have a potent anti-inflammatory effect. However, the effects and mechanisms of AnxA1 in skin keratinocyte and fibroblast have not been reported yet. In the current study, we hypothesized that Ac2-26, AnxA1 mimetic peptide, ameliorates inflammation and wrinkle formation in human skin cells. Therefore, we aimed to identify whether Ac2-26 has anti-inflammatory and anti-wrinkle effects in human keratinocyte (HaCaT) and fibroblast (Detroit 551) cells, respectively. Human HaCaT cells were stimulated by TNF-α/IFN-γ with or without Ac2-26, to identify the anti-inflammatory effect. Human Detroit 551 cells were treated with Ac2-26 to verify the anti-wrinkle effect. Initially, cell cytotoxicity was carried out in each cell line treated using Ac2-26 by MTT assay. Human MDA, IL-8, and procollagen secretion were detected by ELISA assay. The inflammatory chemokines were measured by qRT-PCR analysis. To demonstrate the mechanism, MAPK, NF-κB, JAK/STAT, and MMPs were analyzed by Western blotting. As a result, we identified that Ac2-26 significantly decreased the expression of TNF-α/IFN-γ-stimulated pro-inflammatory chemokines, including IL-1β, IL-6, IL-8, MDC, TARC, and TNF-α, by inhibiting the activation of MAPK, NF-κB, and JAK/STAT pathway in TNF-α/IFN-γ-stimulated HaCaT human keratinocytes. In addition, we also identified that Ac2-26 significantly induced collagen synthesis by generating pro-collagen, and suppressed collagen degradation by inhibiting the collagenase MMP-1 and MMP-8 expression. Collectively, these results suggest that Ac2-26 shows anti-inflammatory and anti-wrinkling effect. These effects may lead to the development of preventive and therapeutic application for inflammation-related skin disease and wrinkle formation.

Upregulation of annexin A1 protein expression in the intratumoral vasculature of human non-small-cell lung carcinoma and rodent tumor models.

Annexin A1 (anxA1) is an immunomodulatory protein that has been proposed as a tumor vascular target for antitumor biologic agents, yet to date the vascular expression of anxA1 in specific tumor indications has not been systematically assessed. Attempts to evaluate vascular anxA1 expression by immunohistochemistry are complicated by a lack of available antibodies that are both specific for anxA1 and bind the N-terminal–truncated form of anxA1 that has previously been identified in tumor vasculature. To study the vascular expression pattern of anxA1 in non–small-cell lung carcinoma (NSCLC), we isolated an antibody capable of binding N-terminal–truncated anxA127-346 and employed it in immunohistochemical studies of human lung specimens. Lung tumor specimens evaluated with this antibody revealed vascular (endothelial) anxA1 expression in five of eight tumor samples studied, but no vascular anxA1 expression was observed in normal lung tissue. Tumor microarray analysis further demonstrated positive vascular staining for anxA1 in 30 of 80 NSCLC samples, and positive staining of neoplastic cells was observed in 54 of 80 samples. No correlation was observed between vascular and parenchymal anxA1 expression. Two rodent tumor models, B16-F10 and Py230, were determined to have upregulated anxA1 expression in the intratumoral vasculature. These data validate anxA1 as a potential vascular anti-tumor target in a subset of human lung tumors and identify rodent models which demonstrate anxA1 expression in tumor vasculature.

Annexin A1 Regulates NLRP3 Inflammasome Activation and Modifies Lipid Release Profile in Isolated Peritoneal Macrophages.

Annexin A1 (AnxA1) is a potent anti-inflammatory protein that downregulates proinflammatory cytokine release. This study evaluated the role of AnxA1 in the regulation of NLRP3 inflammasome activation and lipid release by starch-elicited murine peritoneal macrophages. C57bl/6 wild-type (WT) and AnxA1-null (AnxA1-/-) mice received an intraperitoneal injection of 1.5% starch solution for macrophage recruitment. NLRP3 was activated by priming cells with lipopolysaccharide for 3 h, followed by nigericin (1 h) or ATP (30 min) incubation. As expected, nigericin and ATP administration decreased elicited peritoneal macrophage viability and induced IL-1β release, more pronounced in the AnxA1-/- cells than in the control peritoneal macrophages. In addition, nigericin-activated AnxA1-/- macrophages showed increased levels of NLRP3, while points of co-localization of the AnxA1 protein and NLRP3 inflammasome were detected in WT cells, as demonstrated by ultrastructural analysis. The lipidomic analysis showed a pronounced release of prostaglandins in nigericin-stimulated WT peritoneal macrophages, while ceramides were detected in AnxA1-/- cell supernatants. Different eicosanoid profiles were detected for both genotypes, and our results suggest that endogenous AnxA1 regulates the NLRP3-derived IL-1β and lipid mediator release in macrophages.

Endogenous Annexin-A1 Negatively Regulates Mast Cell-Mediated Allergic Reactions.

Mast cell stabilizers like cromoglycate and nedocromil are mainstream treatments for ocular allergy. Biochemical studies in vitro suggest that these drugs prevent mast cell degranulation through the release of Annexin-A1 (Anx-A1) protein. However, the direct effect of Anx-A1 gene deletion on mast cell function in vitro and in vivo is yet to be fully investigated. Hence, we aim to elucidate the role of Anx-A1 in mast cell function, both in vivo and in vitro, using a transgenic mouse model where the Anx-A1 gene has been deleted. Bone marrow-derived mast cells (BMDMCs) were cultured from wild-type animals and compared throughout their development to BMDMCs obtained from mice lacking the Anx-A1 gene. The mast cell differentiation, maturity, mediator, and cytokine release were explored using multiple biochemical techniques, such as Western blots, ELISA, and flow cytometry analysis. Electron microscopy was used to identify metachromatic granules content of cells. For in vivo studies, Balb/C wild-type and Anx-A1-deficient mice were divided into the following groups: group 1, a control receiving only saline, and group 2, which had been sensitized by prior exposure to short ragweed (SRW) pollen by topical contact with the conjunctival mucosae. Allergic conjunctivitis was evaluated blind after 24 h by trained observers scoring clinical signs. Electron micrographs of BMDMCs from Anx-A1-null mice revealed more vacuoles overall and more fused vacuoles than wild-type cells, suggesting enhanced secretory activity. Congruent with these observations, BMDMCs lacking the Anx-A1 gene released significantly increased amounts of histamine both spontaneously as well as in response to Ig-E-FcεRI cross-linking compared to those from wild-type mice. Interestingly, the spontaneous release of IL-5, IL-6, IL-9, and monocyte chemoattractant protein-1 (MCP-1) were also markedly increased with a greater production observed upon IgE cross-linking. This latter finding is congruent with augmented calcium mobilization in BMDMCs lacking the Anx-A1 gene. In vivo, when compared to wild-type animals, Anx-A1-deficient mice exposed to SRW pollen displayed exacerbated signs and symptoms of allergic conjunctivitis. Taken together, these results suggest Anx-A1 is an important non-redundant regulator of mast cell reactivity and particularly in allergen mediated allergic reactions.

Modulation of the endogenous Annexin A1 in a cigarette smoke cessation model: Potential therapeutic target in reversing the damage caused by smoking?

BackgroundSmoking cessation may help in the reversal of inflammation and damage caused by smoking. The endogenous annexin A1 (AnxA1) protein has anti-inflammatory effects which instigates the understanding of its role in the attenuation of inflammatory processes caused by smoking. Material and methodsWistar rats were exposed to cigarette smoke for 8 weeks. After the exposure period, one of the groups remained other 8 weeks in the absence of smoke. Animals not exposed to smoke were used as control. Blood, trachea and lungs were obtained for histopathological, immunohistochemical and biochemical analyses. ResultsLoss of cilia of the tracheal lining epithelium was found by smoke exposure, but smoking cessation led to recovery of the tracheal epithelium. Similarly, chronically exposed-to-smoke animals showed increased lymphocytes and macrophages in bronchoalveolar lavage and higher levels of glucose and gamma-GT in their blood. Reduction of lymphocytes, glucose and gamma-GT occurred after smoking cessation. In addition, IL-1β, IL-6, IL-10, TNF-α and MCP-1 levels were elevated by smoke exposure. Smoking cessation significantly reduced the levels of IL-1β, IL-6 and MCP-1 but increased the IL-10 concentration. Numerous mast cells and macrophages were observed in the lung of chronically exposed-to-smoke animals with reduction by smoking cigarette abstinence. AnxA1 increased expression and concomitant NF-κB reduction were found in the smoking cessation group. ConclusionOur results showed that cigarette abstinence promoted partial recovery of the inflammatory process. The attenuation of the inflammatory profile may be associated with the overexpression of AnxA1 protein.

Annexin a1 and formyl peptide receptor 2/3 like: Interaction between hypothalamic–pituitary–adrenal axis and hippocampal memory in a model of corticosterone‐induced depressive like behavior

The activity of the hypothalamic‐pituitary‐adrenal axis is commonly disregulated in stress‐related psychiatric disorders. Formyl peptide receptors (FPR) belong to a family of sensors of the immune system that detect microbe‐associated molecules. It was demonstrated that Fpr2/3 deficient mice show a distinct profile of behaviour characterised by reduced anxiety. Annexin 1 (ANXA1), an endogenous ligand of Formyl Peptide receptor 2/3 like (FPRL 2/3) is a member of the family of phospholipids and calcium annexes proteins with a well‐defined role in the delayed early inhibitory feedback of glucocorticoids in the pituitary gland. The aim of study was first, to evaluate the potential role of ANXA1 and FPRL 2/3 as mediator cell‐mediator of the inhibitory effects of glucocrticoids on corticotrophin secretion (ACTH), subsequently the possible correlation between depression like behaviour and memory impairment in the hippocampus studying Ras/ERK pathway. C57BL/6 (CORT +), Anx a1 −/− and Fpr 2/3 −/− mice were exposed to corticosterone (35 μ/ml/day) to induce depression like state via drinking water for 28 days and then behavioural test was performed to measure immobility time in the forced swimming test, locomotor activity in the open field test, sucrose consumption in the sucrose preference test, anxiety in the novelty suppressed feeding and short‐term memory, intermediate‐term memory and long‐term memory in the novel object recognition test. Anx a1−/− mice showed an improvement in depression‐like behavior compared to Fpr 2/3 like −/− and CORT + mice. Histological analyses were performed to evaluate the number of neurons in the dentate gyrus, as well as the degree of neurogenesis through the neurotrophic factors of BDNF and GDNF. In conclusion, we can state that the absence of the anx a1 protein, even more than the absence of FPR 2/3, improves the depressive behavior induced by chronic administration of glucocorticoids, as it appears to have a neuroprotective role in the hippocampus following a damage caused from a chronic condition of depressionSupport or Funding Informationno fundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Low expression of CRISP3 predicts a favorable prognosis in patients with mammary carcinoma.

The discovery of cysteine-rich secretory protein 3 (CRISP3) has been made in human neutrophils for the first time. Cloning of the complementary DNA (cDNA) for CRISP3 was performed from a cDNA library of human bone marrow. In patients with mammary carcinoma, we found that lower expression of CRISP3 was connected to a significantly improved DFS (disease-free survival) and OS (overall survival). Furthermore, the CRISP3 protein level was significantly associated with negative ANXA1 protein level. In addition, the heterogeneous expression of CRISP3 had been exhibited in diverse mammary carcinoma cells. A significant higher mRNA and the protein level of CRISP3 were seen in T-47D as well as SK-BR-3 cells compared with those in other types of mammary carcinoma cells. Knockdown of CRISP3 in T-47D or SK-BR-3 cells resulted in the weakened migration or invasion abilities. Furthermore, CRISP3 knockdown significantly inhibited the ERK1/2 MAPK signaling pathway in T-47D or SK-BR-3 cells. Research results indicated that the lowering in the expression of CRISP3 is likely to serve as an unprecedented approach for the treatment of mammary carcinoma.

Genetic polymorphism of miRNA-196a and its target gene annexin-A1 expression based on ethnicity in Pakistani female breast cancer patients.

Objective:To evaluate the association of miR-196a rs11614913 C/T genetic variation and its target gene annexin A1 mRNA expression with breast cancer risk in Pakistani female ethnicities.Methods:This case control study, conducted from March 2017 to November 2018 included 295 breast cancer patients, 295 controls of three Pakistani ethnicities and archived 100 samples of cohort group for genotyping and expression profiling. Genotyping of miR-196a (rs11614913 C/T) was done by ARMS PCR technique. Annexin-A1 (ANXA1) mRNA expression was measured with qRT-PCR and detection of protein expression of ANXA1 was done by immunohistochemistry.Results:CC homozygous genotype of miR-196a rs11614913 was present in 81.4% of cases and 73.9% controls. C/T polymorphism was found to be significantly associated with decrease risk of breast cancer (OR=0.25 (0.11- 0.58, p <0.05). Similar trend was seen with the minor T allele (OR=0.55 (0.39-0.77, p <0.05, and both dominant and recessive models (OR=0.64; p=0.02 and OR=0.26, p=0.00). In the KPK ethnic group significant decrease association with breast cancer risk was observed (OR= 0.22 (0.09-0.53, p < 0.05). Immunohistochemical staining showed loss of ANXA1 protein expression in 72 samples, and significant association was observed with pathological type p=0. 00 and triple negative receptor status p=0.03 and with genotypes of miR-196a p=0.00. Increase relative expression of 2.81± .88 by qPCR analysis of ANXA1 mRNA was noted with TT genotype.Conclusions:Our results demonstrate that miR-196a rs11614913 C/T polymorphism is associated with a decreased risk and loss of protein expression in breast cancer in the Pakistani population.