Perfil da proteína anexina a1 na colite, adenoma e adenocarcinoma de cólon annexin a1 / Protein profile in colitis, adenoma and denocarcinoma of the colon / Perfil proteico de anexina a1 en colitis, adenoma y adenocarcinoma de colon

Introdução: Investigações têm mostrado que a proteína anti-inflamatória Anexina A1 (ANXA1) está associada ao processo de invasão tumoral e que suas ações podem ser mediadas pelo receptor para peptídeos formilados (FPR), metaloproteinases (MMP-2 e MMP-9) e enzima de ciclooxigenase (COX-2). Por essas razões o objetivo do trabalho foi verificar a expressão da ANXA1 e dos receptores FPR1 e FPR2 em processos inflamatórios e tumorais do cólon. Material e Métodos: A detecção das proteínas foi realizada, por imuno-histoquímica, de biópsias de inflamação de cólon (colite) e tumores benignos (adenomas) e malignos (adenocarcinomas) de cólon, provenientes dos arquivos do Serviço de Patologia do Centro Universitário Padre Albino (UNIFIPA). As secções foram incubadas com os anticorpos primários policlonais anti-ANXA1 (1:1000), anti-FPR1, anti-FPR2 (1:500), anti-COX-2 (1:500), anti-MMP-2 (1:350) e anti-MMP-9 (1:350), reveladas com DAB e contracoradas com Hematoxilina. As quantificações foram feitas por densitometria e as análises estatísticas realizadas pela ANOVA e post test de Bonferroni. Resultados: Nossas análises imuno-histoquímicas e densitométricas mostraram aumento da expressão de ANXA1, FPR1 e COX-2 nos casos de colite, adenoma e adenocarcinoma, comparados a margem normal. Não houve imunorreatividade para FPR2 em nenhuma das condições clínicas estudadas. Também, foi observado aumento de MMP-2 nos casos de colite e adenocarcinoma e aumento de MM 9 no adenocarcinoma. Os dados mostram a correlação positiva da proteína ANXA1 com o receptor FPR1, COX-2 e MMP 2 nas biópsias de colite. Conclusões: A ANXA1 pode interagir de maneira específica com o receptor FPR1, COX-2 e MMP 2 nos processos inflamatórios e no desenvolvimento tumoral do cólon

Introduction: Investigations have shown that the anti-inflammatory protein Annexin A1 (ANXA1) is associated with the process of tumor invasion and that its actions can be mediated by the receptor for formylated peptides (FPR), metalloproteinases (MMP-2 and MMP-9) and cyclooxygenase enzyme (COX-2). For these reasons, the objective of the work was to verify the expression of ANXA1 and the FPR1 and FPR2 receptors in inflammatory and tumor processes of the colon. Material and Methods: Detection of proteins was carried out, by immunohistochemistry, from biopsies of colon inflammation (colitis) and benign (adenomas) and malignant (adenocarcinomas) colon tumors. The material was obtained from the archives of the Pathology Service of Padre Albino University Center (UNIFIPA). The sections were incubated with the primary polyclonal antibodies anti-ANXA1 (1:1000), anti-FPR1, anti-FPR2 (1:500), anti-COX-2 (1:500), anti-MMP-2 (1: 350) and anti-MMP-9 (1:350), revealed with DAB and counterstained with Hematoxylin. Quantifications were made by densitometry and statistical analyzes were carried out by ANOVA and Bonferroni post test. Results: Our immunohistochemical and densitometric analyzes showed increased expression of ANXA1, FPR1 and COX-2 in cases of colitis, adenoma and adenocarcinoma, compared to normal margins. There was no immunoreactivity for FPR2 in any of the clinical conditions studied. Also, an increase in MMP-2 was observed in cases of colitis and adenocarcinoma and an increase in MM-9 in adenocarcinoma. The data show the positive correlation of the ANXA1 protein with the FPR1, COX 2 and MMP-2 receptor in colitis biopsies. Conclusions: ANXA1 can specifically interact with the FPR1, COX-2 and MMP-2 receptor in inflammatory processes and colon tumor development

Introducción: Investigaciones han demostrado que la proteína antiinflamatoria Anexina A1 (ANXA1) está asociada al proceso de invasión tumoral y que sus acciones pueden ser mediadas por el receptor de péptidos formilados (FPR), metaloproteinasas (MMP-2 y MMP-9) y la enzima ciclooxigenasa (COX-2). Por estos motivos, el objetivo del trabajo fue verificar la expresión de ANXA1 y de los receptores FPR1 y FPR2 en procesos inflamatorios y tumorales del colon. Material y Métodos: La detección de proteínas se realizó, mediante inmunohistoquímica, a partir de biopsias de inflamación de colon (colitis) y tumores benignos (adenomas) y malignos (adenocarcinomas) de colon, del archivo del Servicio de Patología del Centro Universitario Padre Albino (UNIFIPA). Las secciones se incubaron con los anticuerpos policlonales primarios anti-ANXA1 (1:1000), anti-FPR1, anti-FPR2 (1:500), anti-COX-2 (1:500), anti-MMP-2 (1:350) y anti-MMP-9 (1:350), revelados con DAB y contrateñidos con hematoxilina. Las cuantificaciones se realizaron mediante densitometría y los análisis estadísticos se realizaron mediante ANOVA y post test de Bonferroni. Resultados: Nuestros análisis inmunohistoquímicos y densitométricos mostraron una mayor expresión de ANXA1, FPR1 y COX-2 en casos de colitis, adenoma y adenocarcinoma, en comparación con los márgenes normales. No hubo inmunorreactividad para FPR2 en ninguna de las condiciones clínicas estudiadas. Además, se observó un aumento de MMP-2 en casos de colitis y adenocarcinoma y un aumento de MM-9 en adenocarcinoma. Los datos muestran la correlación positiva de la proteína ANXA1 con los receptores FPR1, COX-2 y MMP-2 en biopsias de colitis. Conclusiones: ANXA1 puede interactuar específicamente con los receptores FPR1, COX-2 y MMP-2 en procesos inflamatorios y desarrollo de tumores de colon

Annexin A1 as a potential prognostic biomarker for COVID-19 disease: Case-control study.

BACKGROUND: Annexin A1 (AnxA1) is an important endogenous glucocoticoid protein that contributes to the suppression of inflammation by limiting the production of neutrophil and pro-inflammatory cytokines. This study aims to determine the clinical predictivity value of blood AnxA1 levels in patients with mild and severe-critical pneumonia induced by COVID-19. METHODS: This study employed a prospective, case-control study design and was conducted at Ankara Training and Research hospital between 10 February 2021 and 15 March 2021. A total of 74 patients (42 of whom had moderate and 32 of whom had severe/critical cases of COVID-19 disease according to World Health Organization guidelines) and 50 nonsymptomatic healthy volunteers participated in the study. Blood samples were taken from patients at the time of hospital admission, after which serum was isolated. Following the isolation of serum, AnxA1 levels were evaluated using the enzyme-linked immunosorbent assay method. RESULTS: The serum AnxA1 levels were measured as 25.5 (18.6-38.6) ng/ml in the control group, 21.2 (14.7-32) ng/ml in the moderate disease group, and 14.8 (9.7-26.8) ng/ml in the severe/critical disease group. Serum AnxA1 levels were significantly lower in the severe/critical disease group compared with the control and moderate disease groups (P = .01 and P = .0001, respectively). Using receiver operating characteristic analysis, a larger area under the curve (AUC) for the serum AnxA1 levels of the control group (AUC = 0.715, 95% CI = 0.626-0.803; P = .0001) was calculated compared with the COVID-19 patient group for the diagnosis of COVID-19 disease. The AnxA1 level was found to be 80% sensitive and 54.1% specific at a cut-off level of 18.5 ng/ml for the diagnosis of COVID-19 disease. Moreover, the AnxA1 level was found to be 69.8% sensitive and 58.1% specific at a cut-off level of 17.2 ng/ml in predicting the need for intensive care unit (ICU) treatment. CONCLUSION: AnxA1 levels may be a beneficial biomarker in the diagnosis of COVID-19 pneumonia and in predicting the need for ICU treatment in patients with COVID-19 pneumonia at the time of admission to the emergency department.

Low Annexin A1 level in HTLV-1 infected patients is a potential biomarker for the clinical progression and diagnosis of HAM/TSP.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is etiologically associated with the chronic inflammatory neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) Annexin A1 (AnxA1) is an anti-inflammatory protein with proposed neuroprotective and anti-neuroinflammatory functions. We hypothesized that ANXA1 gene expression may be dysregulated in HTLV-1-infected HAM/TSP patients. METHODS: This study involved 37 individuals infected with HTLV-1, including 21 asymptomatic (AS) carriers and 16 with HAM/TSP, and a control group of 30 individuals negative for HTLV-1 and HTLV-2. For AS HTLV-1-positive and HAM/TSP patients, ANXA1 and formyl peptide receptor (FPR1, FPR2 and FPR3) expression and HTLV-1 proviral load (PVL) in peripheral blood cells were evaluated by real-time quantitative PCR (qPCR), and plasma AnxA1 levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: ANXA1 gene expression was increased in the AS group compared with the HAM/TSP and control groups, but the differences were not statistically significant. FPR1 gene expression was higher in patients with HTLV-1 than in controls (AS, p = 0.0032; HAM/TSP, p < 0.0001). Plasma AnxA1 levels were higher in the AS group than in the HAM/TSP group (p = 0.0045), and PVL was higher in patients with HAM/TSP than in AS individuals (p = 0.0162). The use of a combined ROC curve using Annexin 1 levels and proviral load significantly increased the sensitivity and specificity to predict progression to HAM/TSP (AUC = 0.851 and AUC = 0.937, respectively, to AUC = 1000). CONCLUSIONS: Our results suggest that AnxA1 may be dysregulated in HAM/TSP patients. Serological detection of AnxA1 in association with proviral load may provide a prognostic biomarker for HTLV-1-associated neurodegenerative disease.

Decreased serum Annexin A1 levels in Kawasaki disease with coronary artery aneurysm.

BACKGROUND: Kawasaki disease (KD) is an acute and systemic vasculitis whose etiology remains unclear. The most crucial complication is the formation of coronary artery aneurysm (CAA). Annexin A1 (ANXA1) is an endogenous anti-inflammatory agent and pro-resolving mediator involved in inflammation-related diseases. This study sought to investigate the serum ANXA1 levels in KD patients and further explore the relationship between ANXA1 and CAA, as well as additional clinical parameters. METHODS: Serum samples were collected from 95 KD patients and 39 healthy controls (HCs). KD patients were further divided into two groups: KD with CAAs (KD-CAAs) and KD non-CAAs (KD-NCAAs). Serum levels of ANXA1 and interleukin-6 (IL-6) were determined using enzyme-linked immunosorbent assays. RESULTS: Serum ANXA1 levels in the KD group were significantly lower than in the HC group. In particular, serum ANXA1 levels were substantially lower in the KD-CAA groups. Moreover, serum ANXA1 levels were positively correlated with N%, C-reactive protein (CRP), and IL-6 but negatively correlated with L% in the KD group. Positive correlations between serum ANXA1 levels and erythrocyte sedimentation rate (ESR), IL-6, and D-dimer (DD) were observed in the KD-CAA group. CONCLUSIONS: ANXA1 may be involved in the development of KD, and downregulation of ANXA1 may lead to the hypercoagulability seen in KD. IMPACT: For the first time, it was demonstrated that serum ANXA1 levels were significantly decreased in Kawasaki disease with coronary artery aneurysms. ANXA1 might be involved in the acute phase of Kawasaki disease. Low serum concentrations of ANXA1 might lead to the hypercoagulability stage in Kawasaki disease. ANXA1 might be a potential therapeutic target for patients with Kawasaki disease.

Leucine-rich alpha-2 glycoprotein 1, high mobility group box 1, matrix metalloproteinase 3 and annexin A1 as biomarkers of ulcerative colitis endoscopic and histological activity.

OBJECTIVE: The LRG, HMGB1, MMP3 and ANXA1 proteins have been implicated in different inflammatory pathways in ulcerative colitis (UC), but their role as specific biomarkers of both endoscopic and histological activity has yet to be elucidated. In the present study, we aimed to evaluate the LRG1, HMGB1, MMP3 and ANXA1 as potential serum biomarkers for UC endoscopic and histological activity. METHODS: This cross-sectional study included UC patients under 5-ASA, and healthy controls (HC) undergoing colonoscopy. Blood and biopsy samples were obtained and endoscopic Mayo sub-score (Ms) was recorded for the UC patients. Intramucosal calprotectin as a marker of histologic activity was evaluated in all biopsy samples and serum LRG1, HMGB1, MMP3 and ANXA1 levels were measured in the blood samples. RESULTS: The HCs ANXA1 level was lower compared to that of the UC group [P = 0.00, area under the curve (AUC) = 0.881] and so was the HCs MMP3 level compared to that of patients (P = 0.00, AUC = 0.835). The HCs ANXA1 levels were also lower compared to these of the independent Ms groups, even to the Ms = 0 (P = 0.00, AUC = 0.913). UC endoscopic activity was associated with MMP3 levels (r = 0.54, P = 0.000) but not with ANXA1, LRG1 and HMGB1 levels CONCLUSION: Serum ANXA1 is a potential diagnostic biomarker of UC and serum MMP3 is a potential biomarker of UC endoscopic and histological activity.

Evaluation of miR-196a2 expression and Annexin A1 level in children with bronchial asthmaEvaluation of miR-196a2 expression and Annexin A1 level in children.

BACKGROUND: Annexin A1 (ANXA1) is an important anti-inflammatory mediator that may play a significant role in bronchial asthma. MiR-196a2 can target ANXA1 and therefore may play a role in the pathogenesis of asthma. AIM OF STUDY: This is the first study which aimed to evaluate the expression of miR-196a2 in the serum of asthmatic children and correlate its expression with ANXA1 serum level and asthma severity. SUBJECTS AND METHODS: The study included 100 asthma patients who were subdivided into three groups (mild, moderate and severe) and 50 healthy control subjects. Assessment of miR-196a2 expression and ANXA1 serum level were done using quantitative reverse transcriptase PCR (RT qPCR) and Elisa techniques, respectively. RESULTS: Compared to the control group, asthmatic children showed an increased ANXA1 serum level and decreased expression of miR-196a2 (p=0.001). However, ANXA1 serum level was lower and miR-196a2 expression was higher in severe asthmatic patients compared to moderate asthmatic ones (p=0.01, 0.03). Pearson's correlation coefficient revealed no significant correlations between ANXA1 serum level and miR-196a2 expression in the patient group (p=0.9). CONCLUSIONS: Altered miR-196a2 expression and serum ANXA1 concentration may play a role in the pathogenesis of asthma. In addition, ANXA1 and miR-196a2 may represent potential diagnostic biomarkers for asthma and future targets for therapy.

Altered Levels of mRNAs for Calcium-Binding/Associated Proteins, Annexin A1, S100A4, and TMEM64, in Peripheral Blood Mononuclear Cells Are Associated with Osteoporosis.

BACKGROUND: Osteoporosis is the most common metabolic bone disease in the world. Since osteoporosis is clinically symptomless until the first fracture occurs, early diagnosis is critical. Calcium, along with calcium-binding and calcium-associated proteins, plays an important role in homeostasis, maintaining healthy bone metabolism. This study is aimed at investigating the level of calcium-binding/associated proteins, annexin A1, S100A4, and TMEM64, in peripheral blood mononuclear cells associated with osteoporosis and its clinical significance. METHODS: The levels of mRNAs of annexin A1, S100A4, and TMEM64 in human peripheral blood mononuclear cells were evaluated among 48 osteopenia and 23 osteoporosis patients compared to 17 nonosteoporotic controls. Total RNAs were isolated from clinical samples, and quantitation of mRNA levels was performed using real-time quantitative PCR. RESULTS: The levels of mRNAs for calcium-binding proteins, annexin A1 and S100A4, and calcium-associated protein, TMEM64, in human peripheral blood mononuclear cells were significantly reduced in osteopenia and osteoporosis patients compared with nonosteoporotic controls (one-way ANOVA, P < 0.0001, P = 0.039, and P = 0.0195, respectively). Annexin A1 and TMEM64 mRNAs were also significantly reduced in female osteoporosis patients over the age of 50 years compared to nonosteoporotic controls (one-way ANOVA, P = 0.004 and P = 0.0037, respectively). ROC analysis showed that the reduction in the level of mRNA for annexin A1, S100A4, or TMEM64 in the patients' peripheral blood mononuclear cells has a good diagnostic value for osteoporosis. CONCLUSIONS: The results show for the first time that calcium-binding/associated proteins, annexin A1 and TMEM64, could be future diagnostic biomarkers for osteoporosis.

Inflammatory and Pro-resolving Mediators in Frontotemporal Dementia and Alzheimer's Disease.

Chronic inflammation contributes to neuronal death in Alzheimer's disease (AD) and frontotemporal dementia (FTD). Here we evaluated inflammatory and pro-resolving mediators in AD and behavioural variant of FTD (bvFTD) patients compared with controls, since neuroinflamamtion is a common feature in both diseases. Ninety-eight subjects were included in this study, divided into AD (n = 32), bvFTD (n = 30), and control (n = 36) groups. The levels of hsCRP, IL-1ß, IL-6, TNF, and TGF-ß1, as well as annexin A1 (AnxA1) and lipoxin A4 (LXA4) were measured in blood and cerebrospinal fluid (CSF). The expression profile of AnxA1 was evaluated in peripheral blood mononuclear cells (PBMCs) as well the distribution of ANXA1 rs2611228 polymorphism. We found reduced peripheral levels of hsCRP and TNF in AD compared with bvFTD patients and controls, and increased levels of TGF-ß1 in AD compared to controls. Moreover, reduced plasma levels of AnxA1 were observed in bvFTD compared to AD and controls. There was a significant cleavage of AnxA1 in PBMCs in both dementia groups. The results suggest differential regulation of inflammatory and pro-resolving mediators in bvFTD and AD, while AnxA1 cleavage may impair pro-resolving mechanisms in both groups.

Identification of AnnexinA1 as an Endogenous Regulator of RhoA, and Its Role in the Pathophysiology and Experimental Therapy of Type-2 Diabetes.

Annexin A1 (ANXA1) is an endogenously produced anti-inflammatory protein, which plays an important role in the pathophysiology of diseases associated with chronic inflammation. We demonstrate that patients with type-2 diabetes have increased plasma levels of ANXA1 when compared to normoglycemic subjects. Plasma ANXA1 positively correlated with fatty liver index and elevated plasma cholesterol in patients with type-2 diabetes, suggesting a link between aberrant lipid handling, and ANXA1. Using a murine model of high fat diet (HFD)-induced insulin resistance, we then investigated (a) the role of endogenous ANXA1 in the pathophysiology of HFD-induced insulin resistance using ANXA1-/- mice, and (b) the potential use of hrANXA1 as a new therapeutic approach for experimental diabetes and its microvascular complications. We demonstrate that: (1) ANXA1-/- mice fed a HFD have a more severe diabetic phenotype (e.g., more severe dyslipidemia, insulin resistance, hepatosteatosis, and proteinuria) compared to WT mice fed a HFD; (2) treatment of WT-mice fed a HFD with hrANXA1 attenuated the development of insulin resistance, hepatosteatosis and proteinuria. We demonstrate here for the first time that ANXA1-/- mice have constitutively activated RhoA. Interestingly, diabetic mice, which have reduced tissue expression of ANXA1, also have activated RhoA. Treatment of HFD-mice with hrANXA1 restored tissue levels of ANXA1 and inhibited RhoA activity, which, in turn, resulted in restoration of the activities of Akt, GSK-3ß and endothelial nitric oxide synthase (eNOS) secondary to re-sensitization of IRS-1 signaling. We further demonstrate in human hepatocytes that ANXA1 protects against excessive mitochondrial proton leak by activating FPR2 under hyperglycaemic conditions. In summary, our data suggest that (a) ANXA1 is a key regulator of RhoA activity, which restores IRS-1 signal transduction and (b) recombinant human ANXA1 may represent a novel candidate for the treatment of T2D and/or its complications.

Identification of an activated neutrophil phenotype in polymyalgia rheumatica during steroid treatment: a potential involvement of immune cell cross-talk.

We have reported the existence of a distinct neutrophil phenotype in giant cell arteritis (GCA) patients arising at week 24 of steroid treatment. In the present study, we investigated whether longitudinal analysis of neutrophil phenotype in patients with polymyalgia rheumatica (PMR) could reveal a novel association with disease status and immune cell cross-talk. Thus, we monitored PMR patient neutrophil phenotype and plasma microvesicle (MV) profiles in blood aliquots collected pre-steroid, and then at weeks 1, 4, 12 and 24 post-steroid treatment.Using flow cytometric and flow chamber analyses, we identified 12-week post-steroid as a pivotal time-point for a marked degree of neutrophil activation, correlating with disease activity. Analyses of plasma MVs indicated elevated AnxA1+ neutrophil-derived vesicles which, in vitro, modulated T-cell reactivity, suggesting distinct neutrophil phenotypic and cross-talk changes at 24 weeks, but not at 12-week post-steroid.Together, these data indicate a clear distinction from GCA patient neutrophil and MV signatures, and provide an opportunity for further investigations on how to 'stratify' PMR patients and monitor their clinical responses through novel use of blood biomarkers.

Resolution of inflammation in neuromyelitis optica spectrum disorders.

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) are a spectrum of neuroinflammatory disorders associated with autoimmune antibodies against aquaporin-4 (AQP4). Accumulating evidence suggests that inflammation is involved in NMOSD pathogenesis. Resolution of inflammation, which is a highly regulated process mediated by specialized pro-resolving lipid mediators (SPMs) is important to prevent over-responsive inflammation. Deficiency in resolution of inflammation may lead to or accelerates inflammatory diseases. However, whether resolution of inflammation is impaired in NMOSD is not known. The objective of this study was to analyze the levels of SPMs in the serum and cerebrospinal fluid (CSF) of NMOSD patients, and to explore the roles of SPMs in clinical features of NMOSD. METHODS: Thirty-five patients with NMOSD, 34 patients with multiple sclerosis, and 36 patients with non-inflammatory neurological diseases were enrolled in this study. Pro-resolving mediators including Annexin A1 (ANXA1) and resolvin D1 (RvD1), as well as pro-inflammatory lipid mediator leukotriene B4 (LTB4) levels were analyzed by enzyme-linked immunosorbent assay. Pro- and anti-inflammatory cytokines as well as chemokine levels were analyzed using cytometric beads array (CBA). RESULTS: Our results showed RvD1 levels were significantly decreased, whereas LTB4 levels were significantly increased in the CSF of NMOSD patients. AQP4-IgG titer was negatively correlated with RvD1 levels in the CSF of NMOSD patients. CONCLUSIONS: Decreased RvD1 levels indicate impaired resolution of inflammation in NMOSD patients. AQP4-IgG may contribute to increased inflammation and lead to unresolved inflammation in NMOSD.

Reciprocal regulation of pro-inflammatory Annexin A2 and anti-inflammatory Annexin A1 in the pathogenesis of rheumatoid arthritis.

Annexin A2 has been implicated in several immune modulated diseases including Rheumatoid arthritis (RA) pannus formation. The most relied treatment option for RA pathogenesis is glucocorticoids. Glucocorticoids regulate the synthesis, phosphorylation and cellular deposition of Annexin A1. This annexin mediates the anti-inflammatory actions of glucocorticoids. These two first characterized members of annexin superfamily proteins acts reciprocally, one as an anti-inflammatory and the other proinflammatory in nature. The possibility of these molecules as soluble biomarkers and as an upstream regulator of major cytokine devastation at RA microenvironment has not been previously explored. Current study elucidates the reciprocal regulation of these two annexins in RA pathogenesis. These Annexin A2/A1 and downstream cytokines in RA serum were analysed by ELISA. Western blot, Immunocytochemistry, immunoprecipitation and Immunohistochemistry were adapted to analyse these molecules in tissue and synovial fibroblasts and also in different experimental conditions. Significant increase in the level of Annexin A2 was noticed in naïve RA patients compared to controls (14.582 ± 1.766 ng/ml vs. 7.37 ± 1.450 ng/ml; p ≤ 0.001). In remission cases significant low levels was detected. On the contrary, significant decrease in the level of Annexin A1 was noticed in naïve RA patients compared to healthy controls (12.322 ± 2.91 vs. 16.998 ± 4.298 ng/ml; p ≤ 0.001), wherein remission cases serum Annexin A1 was significantly high. The knockdown of proinflammatory Annexin A2 by siRNA/antibody treatment could mimic the glucocorticoid treatment as which induced cellular Annexin A1 and membrane translocation resulting in the terminal action. Current data elucidating the regulatory interplay between Annexin A2 and Annexin A1 in RA pathogenesis.

Association of serum annexin A1 with treatment response and prognosis in patients with esophageal squamous cell carcinoma.

OBJECTIVE: Annexin A1 (ANXA1), a calcium-dependent phospholipid binding protein, is known to be regulated by microRNA-196a (miR-196a) in esophageal adenocarcinoma, and its high expression in tumor tissue is correlated with the poor prognosis of esophageal squamous cell carcinoma (ESCC). However, the role of ANXA1 in the serum of patients with ESCC remains unclear. MATERIALS AND METHODS: In this study, we used enzyme-linked immunosorbent assay to evaluate the levels of ANXA1 and real-time polymerase chain reaction to detect the expression of miR-196a in the serum of ESCC patients (healthy donors as controls) and evaluated the relationship between ANXA1 and clinical outcomes. RESULTS: The results showed that the level of serum ANXA1 in ESCC patients was significantly lower than that in controls (P = 0.001) but increased after chemoradiotherapy (P = 0.001). There was no correlation between the baseline level of serum ANXA1 and the short-term efficacy of treatment (P = 0.26) as well as the 1-year progression-free survival (PFS) (P = 0.094). However, there existed a significant correlation between the increases of serum ANXA1 expression and the 1-year PFS (P = 0.04). A higher increase (>2-fold of baseline) in the serum ANXA1 levels was correlated with a poorer PFS (hazard ratio = 3.096, 95% confidence interval 1.239-7.861). There was an inverse correlation between the expressions of miR-196a and ANXA1 in serum (Pearson's correlation of -0.54, P = 0.021). CONCLUSION: Our data revealed that the expression of serum ANXA1 in ESCC patients increases after chemoradiotherapy and the increased fold change in serum ANXA1 confers independent negative prognostic impact in ESCC. The higher the increase in serum ANXA1 levels, the poorer the outcome.

Defective formyl peptide receptor 2/3 and annexin A1 expressions associated with M2a polarization of blood immune cells in patients with chronic obstructive pulmonary disease.

BACKGROUND: Controversy exists in previous studies on macrophage M1/M2 polarization in chronic obstructive pulmonary disease (COPD). We hypothesized that formyl peptide receptor (FPR), a marker of efferocytosis and mediator of M1/M2 polarization, may be involved in the development of COPD. METHODS: We examined FPR 1/2/3 expressions of blood M1/M2a monocyte, neutrophil, natural killer (NK) cell, NK T cell, T helper (Th) cell, and T cytotoxic (Tc) cell by flowcytometry method in 40 patients with cigarette smoking-related COPD and 16 healthy non-smokers. Serum levels of five FPR ligands were measured by ELISA method. RESULTS: The COPD patients had lower M2a percentage and higher percentages of NK, NK T, Th, and Tc cells than the healthy non-smokers. FPR2 expressions on Th/Tc cells, FPR3 expressions of M1, M2a, NK, NK T, Th, and Tc cells, and serum annexin A1 (an endogenous FPR2 ligand) levels were all decreased in the COPD patients as compared with that in the healthy non-smokers. FPR1 expression on neutrophil was increased in the COPD patient with a high MMRC dyspnea scale, while FPR2 expression on neutrophil and annexin A1 were both decreased in the COPD patients with a history of frequent moderate exacerbation (≥ 2 events in the past 1 year). In 10 COPD patients whose blood samples were collected again after 1-year treatment, M2a percentage, FPR3 expressions of M1/NK/Th cells, FPR2 expression on Th cell, and FPR1 expression on neutrophil were all reversed to normal, in parallel with partial improvement in small airway dysfunction. CONCLUSIONS: Our findings provide evidence for defective FPR2/3 and annexin A1 expressions that, associated with decreased M2a polarization, might be involved in the development of cigarette smoking induced persistent airflow limitation in COPD.

Annexin A1 is elevated in patients with COPD and affects lung fibroblast function.

Purpose: Fibrosis in peripheral airways is responsible for airflow limitation in chronic obstructive pulmonary disease (COPD). Annexin A1 modulates several key biological events during inflammation. However, little is known about its role in airway fibrosis in COPD. We investigated whether levels of Annexin A1 were upregulated in patients with COPD, and whether it promoted airway fibrosis. Methods: We quantified serum Annexin A1 levels in never-smokers (n=12), smokers without COPD (n=11), and smokers with COPD (n=22). Correlations between Annexin A1 expression and clinical indicators (eg, lung function) were assessed. In vitro, human bronchial epithelial (HBE) cells were exposed to cigarette smoke extract (CSE) and Annexin A1 expression was assessed. Primary human lung fibroblasts were isolated from patients with COPD and effects of Annexin A1 on fibrotic deposition of lung fibroblasts were evaluated. Results: Serum Annexin A1 was significantly higher in patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines stage III or IV than in those with GOLD stages I or II (12.8±0.8 ng/mL versus 9.8±0.7 ng/mL; p=0.016). Annexin A1 expression was negatively associated with airflow obstruction (forced expiratory volume in one second % predicted; r=-0.72, p<0.001). In vitro, Annexin A1 was significantly increased in CSE-exposed HBE cells in a time- and concentration-dependent manner. Annexin A1 promoted lung fibroblasts proliferation, migration, differentiation, and collagen deposition via the ERK1/2 and p38 mitogen-activated protein kinase pathways. Conclusion: Annexin A1 expression is upregulated in patients with COPD and affects lung fibroblast function. However, more studies are needed to clarify the role of Annexin A1 in airway fibrosis of COPD.

Annexin A1 in plasma from patients with bronchial asthma: its association with lung function.

BACKGROUND: Annexin-A1 (ANXA1) is a glucocorticoid-induced protein with multiple actions in the regulation of inflammatory cell activation. The anti-inflammatory protein ANXA1 and its N-formyl peptide receptor 2 (FPR2) have protective effects on organ fibrosis. However, the exact role of ANXA1 in asthma remains to be determined. The aim of this study was to identify the role of ANXA1 in bronchial asthma. METHODS: In mice sensitized and challenged with ovalbumin (OVA-OVA mice) and mice sensitized with saline and challenged with air (control mice), we investigated the potential links between ANXA1 levels and bronchial asthma using ELISA, immunoblotting, and immunohistochemical staining. Moreover, we also determined ANXA1 levels in blood from 50 asthmatic patients (stable and exacerbated states). RESULTS: ANXA1 protein levels in lung tissue and bronchoalveolar lavage fluid were significantly higher in OVA-OVA mice compared with control mice. FPR2 protein levels in lung tissue were significantly higher in OVA-OVA mice compared with control mice. Plasma ANXA1 levels were increased in asthmatic patients compared with healthy controls. Plasma ANXA1 levels were significantly lower in exacerbated patients compared with stable patients with bronchial asthma (p < 0.05). The plasma ANXA1 levels in controlled asthmatic patients were correlated with forced expiratory volume in 1 s (FEV1) (r = - 0.191, p = 0.033) and FEV1/forced vital capacity (FVC) (r = -0.202, p = 0.024). CONCLUSION: These results suggest that ANXA1 may be a potential marker and therapeutic target for asthma.

Expression correlates between Annexin A1 and A2 autoantibodies in patients with Behçet's disease.

The autoantibodies profile of Behçet's disease (BD) is yet incompletely understood. Annexins are a family of highly conserved proteins which are involved in some human autoimmune diseases. Autoantibodies directed toward Annexin A1 and A2 are involved in BD pathology, but correlation in their clinical role is controversial. The aim of our study is to estimate and evaluate the expression correlation between Annexin A1 and A2 autoantibodies in BD patients. We have designed and implemented different technical approaches to prove the hypothesis. First, bioinformatics tools such as amino acid sequence alignment, epitope prediction analysis, and 3D structural comparison were performed to find out the correlation between Annexin A1 and A2. Second, amplification of the corresponding gene by RT-PCR, then cloning, and purification techniques were applied to acquire the recombinant Annexin A1. Third, the target protein band was excised from gel electrophoresis, digested with trypsin, and analyzed by MALDI-TOF/TOF. Finally, in-house ELISA was developed to determine the induced anti-Annexin A1 autoantibodies in BD patients. Obtained results demonstrated that the BD serum reactivity against recombinant Annexin A1 was significantly higher as compared with healthy control (HC) (P<0.001). Moreover, bioassay results of Annexin A1 and A2 also showed the presence, absence, and independent coexistence of autoantibodies, when reacted with BD sera. In conclusion, Annexin A1 has a similar immunogenic expression and correlation with its analog Annexin A2 and their association may be a novel immune target of BD in Han Chinese population.

Annexin A1 attenuates microvascular complications through restoration of Akt signalling in a murine model of type 1 diabetes.

AIMS/HYPOTHESIS: Microvascular complications in the heart and kidney are strongly associated with an overall rise in inflammation. Annexin A1 (ANXA1) is an endogenous anti-inflammatory molecule that limits and resolves inflammation. In this study, we have used a bedside to bench approach to investigate: (1) ANXA1 levels in individuals with type 1 diabetes; (2) the role of endogenous ANXA1 in nephropathy and cardiomyopathy in experimental type 1 diabetes; and (3) whether treatment with human recombinant ANXA1 attenuates nephropathy and cardiomyopathy in a murine model of type 1 diabetes. METHODS: ANXA1 was measured in plasma from individuals with type 1 diabetes with or without nephropathy and healthy donors. Experimental type 1 diabetes was induced in mice by injection of streptozotocin (STZ; 45 mg/kg i.v. per day for 5 consecutive days) in C57BL/6 or Anxa1 -/- mice. Diabetic mice were treated with human recombinant (hr)ANXA1 (1 µg, 100 µl, 50 mmol/l HEPES; 140 mmol/l NaCl; pH 7.4, i.p.) or vehicle (100 µl, 50 mmol/l HEPES; 140 mmol/l NaCl; pH 7.4, i.p.). RESULTS: Plasma levels of ANXA1 were elevated in individuals with type 1 diabetes with/without nephropathy compared with healthy individuals (66.0 ± 4.2/64.0 ± 4 ng/ml vs 35.9 ± 2.3 ng/ml; p < 0.05). Compared with diabetic wild-type (WT) mice, diabetic Anxa1 -/- mice exhibited a worse diabetic phenotype and developed more severe cardiac (ejection fraction; 76.1 ± 1.6% vs 49.9 ± 0.9%) and renal dysfunction (proteinuria; 89.3 ± 5.0 µg/mg vs 113.3 ± 5.5 µg/mg). Mechanistically, compared with non-diabetic WT mice, the degree of the phosphorylation of mitogen-activated protein kinases (MAPKs) p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) was significantly higher in non-diabetic Anxa1 -/- mice in both the heart and kidney, and was further enhanced after STZ-induced type 1 diabetes. Prophylactic treatment with hrANXA1 (weeks 1-13) attenuated both cardiac (ejection fraction; 54.0 ± 1.6% vs 72.4 ± 1.0%) and renal (proteinuria; 89.3 ± 5.0 µg/mg vs 53.1 ± 3.4 µg/mg) dysfunction associated with STZ-induced diabetes, while therapeutic administration of hrANXA1 (weeks 8-13), after significant cardiac and renal dysfunction had already developed, halted the further functional decline in cardiac and renal function seen in diabetic mice administered vehicle. In addition, administration of hrANXA1 attenuated the increase in phosphorylation of p38, JNK and ERK, and restored phosphorylation of Akt in diabetic mice. CONCLUSIONS/INTERPRETATION: Overall, these results demonstrate that ANXA1 plasma levels are elevated in individuals with type 1 diabetes independent of a significant impairment in renal function. Furthermore, in mouse models with STZ-induced type 1 diabetes, ANXA1 protects against cardiac and renal dysfunction by returning MAPK signalling to baseline and activating pro-survival pathways (Akt). We propose ANXA1 to be a potential therapeutic option for the control of comorbidities in type 1 diabetes.

The advantageous role of annexin A1 in cardiovascular disease.

The inflammatory response protects the human body against infection and injury. However, uncontrolled and unresolved inflammation can lead to tissue damage and chronic inflammatory diseases. Therefore, active resolution of inflammation is essential to restore tissue homeostasis. This review focuses on the pro-resolving molecule annexin A1 (ANXA1) and its derived peptides. Mechanisms instructed by ANXA1 are multidisciplinary and affect leukocytes as well as endothelial cells and tissue resident cells like macrophages and mast cells. ANXA1 has an outstanding role in limiting leukocyte recruitment and different aspects of ANXA1 as modulator of the leukocyte adhesion cascade are discussed here. Additionally, this review details the therapeutic relevance of ANXA1 and its derived peptides in cardiovascular diseases since atherosclerosis stands out as a chronic inflammatory disease with impaired resolution and continuous leukocyte recruitment.

Identification of two phosphorylation sites essential for annexin A1 in blood-brain barrier protection after experimental intracerebral hemorrhage in rats.

Annexin A1 has been reported to exert a blood-brain barrier protection. This study was designed to examine the role of annexin A1 in intracerebral hemorrhage-induced blood-brain barrier dysfunction. A collagenase intracerebral hemorrhage model was performed in adult male Sprague Dawley rats. First, a possible relationship between annexin A1 and intracerebral hemorrhage pathology was confirmed by a loss of annexin A1 in the cerebrovascular endothelium and serum of intracerebral hemorrhage rats, and the rescue effects of i.v. administration of human recombinant annexin A1 in vivo and annexin A1 overexpression in vitro on the barrier function of brain microvascular endothelial cells exposed to intracerebral hemorrhage stimulus. Second, we found that intracerebral hemorrhage significantly increased the phosphorylation ratio of annexin A1 at the serine/threonine residues. Finally, based on site-specific mutagenesis, we identified two phosphorylation sites (a) annexin A1 phosphorylation at threonine 24 is required for its interaction with actin cytoskeleton, and (b) phosphorylation at serine27 is essential for annexin A1 secretion, both of which were essential for maintaining cytoskeleton integrity and paracellular permeability. In conclusion, annexin A1 prevents intracerebral hemorrhage-induced blood-brain barrier dysfunction in threonine 24 and serine27 phosphorylation-dependent manners. Annexin A1 phosphorylation may be a self-help strategy in brain microvascular endothelial cells after intracerebral hemorrhage; however, that was almost completely abolished by the intracerebral hemorrhage-induced loss of annexin A1.