Annexin A1 Research Articles

STARD3NL inhibits the osteogenic differentiation by inactivating the Wnt/β-catenin pathway via binding to Annexin A2 in osteoporosis.

Osteoporosis is one of the leading forms of systemic diseases related to bone metabolism in the world. STARD3 N‐terminal like (STARD3NL) showed robust association with osteoporosis‐related traits. Yet, the molecular functional mechanisms of STARD3NL in osteoblasts is still obscure. In this study, we demonstrated a high level of STARD3NL expression in the bone tissues from the patients with low bone mass and ovariectomized (OVX)‐induced osteoporotic mice. We identified Stard3nl as a potent factor that negatively and positively regulates osteoblast differentiation and cell proliferation, respectively. Furthermore, inhibition of Stard3nl induced β‐catenin gene expression and the nuclear translocation of β‐catenin, as well as Wnt signalling activities, contributing to the activation of Wnt/β‐catenin signalling. Mechanistic studies revealed that Stard3nl bound with Annexin A2 (Anxa2) to suppress β‐catenin expression, resulting into the suppression of Wnt signalling and downstream osteogenic differentiation. Moreover, adeno‐associated virus 9 (AAV9)‐mediated silencing of Stard3nl reversed bone loss in OVX‐induced osteoporotic mice by the injection into the knee joints. Collectively, our study revealed that Stard3nl suppressed osteogenesis via binding with Anxa2, resulting into the inactivation of Wnt signalling. It also highlights the preventive and therapeutic potential of STARD3NL as a specific and novel target for osteoporotic patients.

The In Vitro Anti-Cancer Activities and Mechanisms of Action of 9-Methoxycanthin-6-one from Eurycoma longifolia in Selected Cancer Cell Lines.

An alkaloid compound from the hairy root culture of Eurycoma longifolia has been isolated and characterised as 9-methoxycanthin-6-one. The aims of these studies were to investigate the in vitro anti-cancer activities of 9-methoxycanthin-6-one against ovarian cancer (A2780, SKOV-3), breast cancer (MCF-7), colorectal cancer (HT29), skin cancer (A375) and cervical cancer (HeLa) cell lines by using a Sulphorhodamine B assay, and to evaluate the mechanisms of action of 9-methoxycanthin-6-one via the Hoechst 33342 assay and proteomics approach. The results had shown that 9-methoxycanthin-6-one gave IC50 values of 4.04 ± 0.36 µM, 5.80 ± 0.40 µM, 15.09 ± 0.99 µM, 3.79 ± 0.069 µM, 5.71 ± 0.20 µM and 4.30 ± 0.27 µM when tested in A2780, SKOV-3, MCF-7, HT-29, A375 and HeLa cell lines, respectively. It was found that 9-methoxycanthin-6-one induced apoptosis in a concentration dependent manner when analysed via the Hoechst 33342 assay. 9-methoxycanthine-6-one were found to affect the expressions of apoptotic-related proteins, that were proteins pyruvate kinase (PKM), annexin A2 (ANXA2), galectin 3 (LGAL3), heterogeneous nuclear ribonucleoprotein A1 (HNRNP1A1), peroxiredoxin 3 (PRDX3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the differential analysis of 2-DE profiles between treated and non-treated 9-methoxycanthine-6-one. Proteins such as acetyl-CoA acyltransferase 2 (ACAA2), aldehyde dehydrogenase 1 (ALDH1A1), capping protein (CAPG), eukaryotic translation elongation factor 1 (EEF1A1), malate dehydrogenase 2 (MDH2), purine nucleoside phosphorylase (PNP), and triosephosphate isomerase 1 (TPI1) were also identified to be associated with A2780 cell death induced by 9-methoxycanthine-6-one. These findings may provide a new insight on the mechanisms of action of 9-methoxycanthin-6-one in exerting its anti-cancer effects in vitro.

Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions

Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

Endothelial Retargeting of AAV9 In Vivo.

Adeno‐associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV‐transduction. AAVs are retargeted here toward endothelial cells by coating with second‐generation polyamidoamine dendrimers (G2) linked to endothelial‐affine peptides (CNN). G2CNN AAV9‐Cre (encoding Cre recombinase) are injected into mTmG‐mice or mTmG‐pigs, cell‐specifically converting red to green fluorescence upon Cre‐activity. Three endothelial‐specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of ‐ G2CNN AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti‐inflammatory Annexin A1 (Anxa1) in ApoE−/− mice (day 28). Moreover, 3) in Cas9‐transgenic mice, blood pressure is monitored till day 56 after systemic application of G2CNN AAV9‐gRNAs, targeting exons 6–10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2CNN AAV9‐Cre transduces microvascular endothelial cells in mTmG‐mice or mTmG‐pigs. Functionally, G2CNN AAV9‐S1FG mediates S1FG‐leukocyte adhesion, whereas G2CNN AAV9‐Anxa1‐application reduces long‐term leukocyte recruitment. Moreover, blood pressure increases in Cas9‐expressing mice subjected to G2CNN AAV9‐gRNAeNOS. Therefore, G2CNN AAV9 may enable gene transfer in vascular and atherosclerosis models.

Annexin A1 promotes the progression of bladder cancer via regulating EGFR signaling pathway

BackgroundBladder cancer (BLCA) is one of the most common malignancies worldwide. One of the main reasons for the unsatisfactory management of BLCA is the complex molecular biological mechanism. Annexin A1 (ANXA1), a Ca2+-regulated phospholipid-binding protein, has been demonstrated to be implicated in the progression and prognosis of many cancers. However, the expression pattern, biological function and mechanism of ANXA1 in BLCA remain unclear.MethodsThe clinical relevance of ANXA1 in BLCA was investigated by bioinformatics analysis based on TCGA and GEO datasets. Immunohistochemical (IHC) analysis was performed to detect the expression of ANXA1 in BLCA tissues, and the relationships between ANXA1 and clinical parameters were analyzed. In vitro and in vivo experiments were conducted to study the biological functions of ANXA1 in BLCA. Finally, the potential mechanism of ANXA1 in BLCA was explored by bioinformatics analysis and verified by in vitro and in vivo experiments.ResultsBioinformatics and IHC analyses indicated that a high expression level of ANXA1 was strongly associated with the progression and poor prognosis of patients with BLCA. Functional studies demonstrated that ANXA1 silencing inhibited the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of BLCA cells in vitro, and suppressed the growth of xenografted bladder tumors in vivo. Mechanistically, loss of ANXA1 decreased the expression and phosphorylation level of EGFR and the activation of downstream signaling pathways. In addition, knockdown of ANXA1 accelerated ubiquitination and degradation of P-EGFR to downregulate the activation of EGFR signaling.ConclusionsThese findings indicate that ANXA1 is a reliable clinical predictor for the prognosis of BLCA and promotes proliferation and migration by activating EGFR signaling in BLCA. Therefore, ANXA1 may be a promising biomarker for the prognosis of patients with BLCA, thus shedding light on precise and personalized therapy for BLCA in the future.

The expression of Annexin A1 and A5 mRNA by gonadotropin-releasing hormone in LβT2 gonadotrope cells.

Gonadotropin-releasing hormone (GnRH) stimulation of annexin A1 (ANXA1) and A5 (ANXA5) mRNA expression was analyzed in LβT2 gonadotrope cells. Quantitative polymerase chain reaction results showed that a GnRH analog (GnRHa) stimulated the expression of both ANXA1 and A5 mRNA with a peak at 12 h of incubation; however, ANXA1 mRNA was extremely stimulated (60 folds). Immunocytochemical analysis confirmed these findings. A GnRH antagonist inhibited the effect of GnRHa. ANXA1 and A5 mRNA levels were significantly increased by protein kinase C (PKC) activator (12-O-Tetradecanoylphorbol-13-acetate; TPA), but not by dibutyryl cAMP. GnRHa-stimulated induction of ANXA1 and A5 mRNA was inhibited by PKC (GF109203) and MEK inhibitors (PD98059). TPA increased ANXA1 and A5 mRNA expression in a dose-dependent manner (1 nM to 10 μM), while the extent of the increase was much greater in ANXA1. After stimulation with 10 nM or 1 μM TPA, ANXA1 and A5 mRNA levels were increased at 6 h. ANXA1 mRNA levels were higher in the 1 μM TPA than in the 10 nM TPA treatment, whereas 1 μM TPA did not show further stimulation of ANXA5 mRNA compared to 10 nM TPA. These results clearly show that ANXA1 mRNA expression is stimulated by GnRH through PKC like ANXA5, and the response of ANXA1 is much larger than that of ANXA5. A close relationship between these annexins and a significant role for ANXA1 in GnRH action at gonadotropes is suggested.

Changes in the expression of annexin A1 in the anterior pituitary gland after ovariectomy in rats.

The expression of annexin A1 (ANXA1) is augmented by gonadotrophin releasing hormone (GnRH) in LβT2 gonadotroph. We examined the distribution of ANXA1 in the pituitary tissues and the effect of ovariectomy. ANXA1 was mainly stained on folliculostellate cell-like irregular shaped cells with extended process of adult female rats. Large gonadotroph, so called castration cells, appeared two weeks after the ovariectomy. ANXA1 in castration cells exists around cells although another GnRH responsive annexin, ANXA5, was apparent also in the cytoplasm. The pituitary expression of ANXA1 after ovariectomy was significantly higher than intact rats. These difference in tissue distribution of two annexins suggest ANXA1 and ANXA5 bear different physiological function in the gonadotroph under GnRH regulation.

MiR-206 is involved in neuroprotective effects of Dexmedetomidine in H2O2-induced SK-N-SH cells by targeting ANXA1

This study focused on exploring the neuroprotective role of Dexmedetomidine (DEX) and miR-206 in H2O2-induced SK-N-SH cells. First, we dectected the cell viability, apotosis, oxidative stress and miR-206 expression in H2O2-treated SK-N-SH cells.Next, we examined above content in H2O2-induced SK-N-SH cells though DEX treatment. Besides, the level of cell viability, apotosis, oxidative stress were evaluated in H2O2-treated SK-N-SH cells transfected with miR-206 mimics and miR-206 inhibitor. Moreover, the target gene of miR-206 were predicted and verifed by binformatics tools and luciferase reporter assay. These data indicated that H2O2 evoked the apotosis, oxidative stress and inhibition of miR-206 expression in SK-N-SH cells in a dose manner. Besides, DEX attenuated H2O2-induced oxidative damage, apotosis and promoted miR-206 expression in SK-N-SH cells. Moreover, overexpression of miR-206 augmented the cell viablity as well as suppressed apotosis and oxidative stress in H2O2-induced SK-N-SH cells, while down-expression of miR-206 showed opppsite effects. Further, Annexin A1 (ANXA1) was verified as a directly target of miR-206, and over-expression of ANXA1 could slack the neuroprotective effect of miR-206 in H2O2-induced SK-N-SH cells. DEX exerted neuroprotective effects on H2O2-treated SK-N-SH cells in vitro by negatively reguating ANXA1 expression via activation of miR-206.

Gonadotropin-releasing hormone stimulation of annexin A5 expression in the thymus of male rats.

As gonadotropin-releasing hormone (GnRH) is expressed in the thymus, its direct action on thymic cells, including thymic involution, has been suggested. Annexin A5 (ANXA5), a biomarker of GnRH, was used to determine whether GnRH affects the thymus of male rats. Immunohistochemistry showed positive reactions for ANXA5 in large medullary epithelial cells at 30 days of age, and the expression continued until 180 days of age. Organ culture of thymus pieces was performed to examine the direct action of a GnRH agonist (GnRHa) on the expression of Anxa5 and Gnrh mRNA. Thymus tissues obtained from male rats (40–60 days old) were cut into small pieces (2–3 mm3) and incubated for 3 hr with the GnRHa. The expression levels of Anxa5 and Gnrh mRNA were augmented by the GnRHa. Immunohistochemistry of these tissue fragments showed that ANXA5 expression was enhanced, especially in medullary epithelial cells. These results revealed that GnRH synthesis in the thymus could affect thymic epithelial cells after puberty.

Polymorphisms and avascular necrosis in patients with sickle cell disease - A systematic review.

Objective:To systematically establish whether there is an association between polymorphisms and avascular necrosis in patients with sickle cell disease.Data source:The review, conducted according to PRISMA guidelines and registered with PROSPERO, was based on research of studies in PubMed, SciELO, LILACS, BVS databases and in the gray literature (Google Scholar and Open Gray) published until June 2020. The STROBE initiative was used to analyze the articles’ quality.Data synthesis:Ten articles were selected from the databases and two were included through manual search, totaling 12 studies. All samples gathered 2,362 patients. According to STROBE, seven studies fully and/or partially covered more than 70% of the essential items and two studies reached less than 60%, with an overall variation of 86.4–54.5%. The results indicate that polymorphisms in the genes of the bone morphogenetic protein 6 (BMP6), Klotho (KL) and Annexin A2 (ANXA2) may be associated with osteonecrosis in the context of sickle cell disease. Six articles addressed the polymorphism in the MTHFR enzyme gene, but only one found a positive association. Polymorphisms associated with the DARC receptor, the ITGA4 gene, CD36 and thrombophilia protein genes were not associated in any of the studies.Conclusions:The results indicate that the polymorphisms in BMP6, Klotho and ANXA2 genes may be associated with avascular necrosis in patients with sickle cell disease. However, in order to confirm these genetic changes as risk factors, further studies with greater statistical power and methodological rigor are needed.

Abstract P041: Simultaneous checkpoint inhibition and immune cell activation that is safely localized to solid tumors

Abstract Unlike other checkpoint inhibitors, our targeted immunotherapeutic localizes to any solid tumor and simultaneously shields an agent of immunosuppression while presenting a signal for immunostimulation. Phosphatidylserine (PS) exposure on the extracellular surface of living tumor cells and their vasculatures provides one avenue by which the tumor microenvironment promotes immunosuppression. Extracellular surface PS is inherent to a tumor and its vasculature, even for inoperable tumors, and its expression cannot be mutated nor affected by acquired drug resistance. Annexin A5 (AnxA5) is a direct, high-affinity PS-binding protein that localizes to cells with PS exposed on the outer plasma membrane. In our studies, we conjugated a proprietary modified AnxA5, lacking cellular internalization, to TNFα (AnxA5MOD-TNFα) to convert the immunosuppresive environs of a murine 4T1 triple negative breast cancer (TNBC) into an immunostimulated one. This strategy localized the immune response to the tumor and minimized side effects, as evidenced by a lack of toxicity for up to 7 days in non-tumor bearing Balb/c female mice given up to 1 mg/kg. Proper assembly and functionality of AnxA5MOD-TNFα was verified simultaneously by ellipsometry, an optical technique similar to plasmon resonance. Fully assembled constructs were tested for binding to PS coated slides. The degree of light polarization is proportional to the amount of PS bound by the AnxA5 complex. Samples could be further incubated with TNF receptors to verify TNFα activity. Based on dose escalation studies in 4T1 tumor-bearing mice where the TNBC tumors were grown in the mammary fat pads, optimal dosages were determined for AnxA5MOD-TNFα (18 µg) and AnxA5MOD alone as a control (180 µg). These doses were further tested in a 4T1 growth inhibition study. Tumor size was tracked by caliper in two groups of mice (n=5/group) receiving drug treatment on days 12, 14 and 16 and a repeated measures ANOVA was conducted on measurements taken before, during and post-treatment. While median tumor size did not differ between control and drug treatment groups during the pre-treatment interval (p=0.84), there was a significant difference post-treatment (p<0.001) with mice receiving AnxA5MOD-TNFα having much smaller TNBC tumors. Tumors from the study were embedded in paraffin, sectioned (5 µm) and the overall immune cell content determined by H&E staining. Once it was evident there was a greater quantity of immune cells in AnxA5MOD-TNFα treated tumors vs. controls, sections were stained with validated antibodies to identify and count the immunoactivated T-cells, NK-cells and macrophages. There was a 3X greater mean percentage of CD8 and CD4 T-cells in mice receiving drug vs. control (p=0.03) along with 2.5X and 5X increases in NK-cells and M1 immunoactive macrophages, respectively. Conclusion: Our AnxA5MOD-TNFα inhibits the PS inhibitor while simultaneously activating TNF activators! Citation Format: Richard A. Richieri, Navneet Narula, Cynthia A. Loomis, Valeria Mezzano, John Billimek, Glenn T. Reynolds, Chris Reutelingsperger, Andries Zijlstra, Missag H. Parseghian. Simultaneous checkpoint inhibition and immune cell activation that is safely localized to solid tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P041.

Activin A specifically suppresses the expression of annexin A5 mRNA and augments gonadotropin-releasing hormone stimulation of A1 expression in LβT2 gonadotrope cells.

Recently, we reported that gonadotropin-releasing hormone (GnRH) stimulates annexin A1 (Anxa1) and A5 (Anxa5) mRNA expression through the GnRH-receptor-mitogen-activated protein kinase cascade in LβT2 cells. As LβT2 cells respond to activin, we examined the effect of activin A on Anxa1 and a5 expression in LβT2 cells. Activin A (0.4 and 4 ng/mL) treatment decreased Anxa5 mRNA levels in a dose-dependent manner, but did not affect Anxa1 mRNA levels at concentrations up to 40 ng/mL. After activin A treatment (4 ng/mL), Anxa5 mRNA levels significantly decreased at 6 h, gradually declined until 24 h, and remained low until 48 h, whereas Anxa1 mRNA levels did not significantly change following treatment. Pretreatment with activin A for 24 h increased GnRH agonist (GnRHa)-induced Anxa1 increase by approximately 7-fold compared with GnRHa stimulation alone, but Anxa5 was not affected. As previously reported, these activin A treatments increased gonadotropin β subunit and GnRH receptor mRNA levels and slightly decreased common α-glycoprotein subunit mRNA levels. Furthermore, we examined the effect of activin A on Nr4a3, which is repressed by ANXA5 and which reduces Fshb expression, and found that activin A augmented Nr4a3 expression and slightly decreased the GnRHa-induced increase in Nr4a3. These results suggest that in gonadotrope cells, the mechanism regulating Anxa1 and a5 expression is differentially coupled with activin A signal transduction. Activin A suppresses Anxa5 expression under increased Nr4a3 expression, whereas activin A and GnRH synergistically stimulate Anxa1 expression. These GnRH-inducible annexins may have different specific functions in gonadotropes.

Changes in the expressions of annexin A1, annexin A5, inhibin/activin subunits, and vitamin D receptor mRNAs in pituitary glands of female rats during the estrous cycle: correlation analyses among these factors.

Pituitary gonadotropin secretion is regulated by several pituitary factors as well as GnRH and ovarian hormones. To elucidate the regulatory mechanisms of pituitary gonadotropin secretions, we observed changes in the mRNA levels of pituitary factors, namely annexin A1 (Anxa1) and Anxa5, inhibin/activin subunits, follistatin (Fst), and vitamin D receptor (Vdr), in female rat pituitary glands during the estrous cycle. Additionally, levels of LHβ subunit (Lhb), FSHβ subunit (Fshb), and GnRH receptor (Gnrh-r) mRNA were examined. During proestrus, Anxa1, Anxa5, Vdr, and inhibin α-subunit (Inha) exhibited the lowest levels, while during estrus, activin βB-subunit (Actbb), Lhb, and Gnrh-r were the lowest. Moreover, Fshb exhibited the highest value during metestrus, whereas Fst did not differ significantly. Correlation analyses revealed 16 statistically significant gene combinations. In particular, four combinations, namely Anxa5 and Inha, Anxa5 and Actbb, Inha and Vdr, and Inha and Actbb, were highly significant (P<0.0001), while four combinations, Anxa1 and Anxa5, Anxa1 and Vdr, Anxa5 and Vdr, and Lhb and Gnrh-r, were moderately significant (P<0.001). The remaining eight combinations that exhibited statistical significance were Anxa1 and Inha, Anxa1 and Actbb, Vdr and Actbb, Anxa1 and Fshb, Inha and Lhb, Actbb and Fshb, Actbb and Lhb, and Fst and Fshb (P<0.05). These results highlight strong correlations among Anxa1, Anxa5, Vdr, Inha, and Actbb, thereby suggesting that an interaction among ANXA1, ANXA5, and VDR may lead to further communications with inhibin and/or activin in the pituitary gland.

The role of annexin A1 peptide in regulating PI3K/Akt signaling pathway to reduce lung injury after cardiopulmonary bypass in rats.

Cardiopulmonary bypass (CPB) -induced lung ischemia-reperfusion (I/R) injury remains a large challenge in cardiac surgery; up to date, no effective treatment has been found. Annexin A1 (AnxA1) has an anti-inflammatory effect, and it has been proven to have a protective effect on CPB-induced lung injury. However, the specific mechanism of AnxA1 in CPB-induced lung injury is not well studied. Therefore, we established a CPB-induced lung injury model to explore the relevant mechanism of AnxA1 and try to find an effective treatment for lung protection. Male rats were randomized into five groups (n = 6, each): sham (S group), I/R exposure (I/R group), I/R + dimethyl sulfoxide (D group), I/R + Ac2-26 (AnxA1 peptide) (A group), and I/R + LY294002 (a PI3K specific inhibitor) (AL group). Arterial blood gas analysis and calculation of the oxygenation index, and respiratory index were performed. The morphological changes in lung tissues were observed under light and electron microscopes. TNF-α and IL-6 and total protein in lung bronchoalveolar lavage fluid were detected via enzyme-linked immunosorbent assay. The expressions of PI3K, Akt, and NF-κB (p65) as well as p-PI3K, p-Akt, p-NF-κB (p65), and AnxA1 were detected via western blotting. Compared with the I/R group, the A group showed the following: lower lung pathological damage score; decreased expression of IL-6 and total protein in the bronchoalveolar lavage fluid, and TNF-α in the lung; increased lung oxygenation index; and improved lung function. These imply the protective role of Ac2-26, and show that LY294002 inhibited the ameliorative preconditioning effect of Ac2-26. This finding suggested that the AnxA1 peptide Ac2-26 decreased the inflammation reaction and CPB-induced lung injury in rats, the lung protective effects of AnxA1may be correlated with the activation of PI3K/Akt signaling pathway.

Annexin A2 Promotes Development of Retinal Neovascularization through PI3K/ AKT Signaling Pathway

Purpose Retinal Neovascularization (RNV) is a pathological characteristic of ocular diseases. Annexin A2 (ANXA2) plays important roles in RNV while the mechanism remains unclear. The study aimed to explore relationship between ANXA2 and PI3K/AKT signaling pathway in RNV. Methods We used human retinal vascular endothelial cells (HRECs) and oxygen-induced retinopathy (OIR) mice model to show ANXA2 can promote the development of RNV through PI3K/AKT signaling pathway. We divided HRECs into 6 groups by infecting lentivirus containing appropriate plasmid and adding corresponding solution. Assays showing ability of HRECs were performed in vitro. Mice were randomly divided into 3 groups and treated accordingly. Results Expression of ANXA2 and activity of PI3K/AKT signaling pathway in HRECs were detected. RNV and expression of ANXA2 in mice retinas were detected. Results showed that ANXA2 expression is positively related with RNV-forming ability of HRECs in vitro and development of RNV in vivo while low activity of PI3K/AKT signaling pathway could attenuate the role of ANXA2. Conclusions We can make ANXA2 and PI3K/ AKT signaling pathway as a promising target for the regulation of pathological neovascularization of the retina, which also provides a novel idea for effective prevention and treatment of diseases related to RNV in future.

From NSAIDs to Glucocorticoids and Beyond.

Our interest in inflammation and its treatment stems from ancient times. Hippocrates used willow bark to treat inflammation, and many centuries later, salicylic acid and its derivative aspirin’s ability to inhibit cyclooxygenase enzymes was discovered. Glucocorticoids (GC) ushered in a new era of treatment for both chronic and acute inflammatory disease, but their potentially dangerous side effects led the pharmaceutical industry to seek other, safer, synthetic GC drugs. The discovery of the GC-inducible endogenous anti-inflammatory protein annexin A1 (AnxA1) and other endogenous proresolving mediators has opened a new era of anti-inflammatory therapy. This review aims to recapitulate the last four decades of research on NSAIDs, GCs, and AnxA1 and their anti-inflammatory effects.

Electroacupuncture promotes microglial M2 polarization in ischemic stroke via annexin A1.

Neuroinflammation is the leading cause of neurological sequelae in ischemic stroke. Recently, we reported that the anti-inflammatory mediator annexin A1 (ANXA1) favored microglial M2 polarization in brain injury. The purpose of this study was to investigate electroacupuncture (EA) treatment and its potentially ANXA1-mediated anti-inflammatory effects in the middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model of stroke. Treatment with EA consisted of dense-sparse frequencies (alternating 4 Hz sparse waves for 1.5 s and 16 Hz dense waves for 1.5 s) at CV24 and GV26. Intracerebroventricular (ICV) injection of Boc-2 (5 µM) or short hairpin RNA (sh)ANXA1 (2 µL) 3 days before EA was performed to block the effects of ANXA1. EA pretreatment enhanced expression of ANXA1 and its receptor, formyl peptide receptor (FPR), when compared to MCAO/R alone. EA treatment also rescued MCAO/R-induced deficits in neurological performance, and learning and memory, and reduced infarct volume. Double immunofluorescent labeling showed that EA prevented MCAO/R-induced changes in microglial activation and morphology. EA also reduced the release of pro-inflammatory cytokines, such as interleukin (IL)-1β, inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α, while increasing the release of anti-inflammatory cytokines, such as arginase-1 (Arg-1) and brain-derived neurotrophic factor (BDNF). All EA-induced effects were either partially or completely prevented by prior administration of FPR antagonist Boc-2 or shANXA1. The current study provides strong evidence that EA treatment has protective effects against ischemic stroke in the MCAO/R mouse model and that the mechanism likely involves the promotion of M2 polarization in microglia via ANXA1.

Genetic analysis of ANXA5 haplotype and its effect on recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is often associated with dysregulated AnnexinA5 (ANXA5) expression. Moreover, the variants of Anxa5, a protein that is enriched in the placenta to prevent coagulation, have been reported to affect RPL risks. The haplotypes M1 [including single nucleotide polymorphisms (SNPs) 1A/C and 27T/C] and M2 (including SNPs 19G/A, 1A/C, 27T/C and 76G/A) of ANXA5 were also reported to affect RPL risks. The present study aimed to investigate the association between the haplotype located in the promoter region of ANXA5 and the risk of RPL. Patients with RPL (n=235) or intrauterine fetus death (IUFD; n=154), as well as healthy control subjects (n=375) were enrolled in the current research. Their haplotypes of ANXA5 were determined using genotyping, and the association between ANXA5 haplotypes and the risk of RPL was accordingly analyzed. A luciferase assay was conducted to investigate the haplotype responsible for ANXA5 activity. Reverse transcription‑quantitative PCR, western blot analysis, immunohistochemistry and ELISA were performed to assess the expression level and activity of ANXA5 in patients with RPL. Consequently, the majority (n=214) of patients with RPL had a history of early RPL, whereas 31patients with RPL had a history of both early and late RPL episodes. A significant difference was found between cases and controls in terms of gravidity and parity, whereas no significant differences were found in terms of age. The percentage of patients with RPL carrying the M2 haplotype of ANXA5 was significantly higher compared with that in control subjects, indicating that the M2 haplotype of ANXA5 was an independent risk of RPL as it influenced the transcription efficiency of ANXA5 promoter. In patients with RPL, ANXA5 activity was suppressed and the mRNA and protein expression levels of Anxa5 were decreased. Thus, the ANXA5 M2 haplotype may be an independent risk factor of RPL by affecting Anxa5 activity.

Relationships between values of myocardial remodeling parameters and level of apoptosis biomarkers in patients with ischemic cardiomyopathy

The aim of the research. To study levels of the apoptosis biomarkers annexin A5 (AnxA5) and Bcl‑2 and to identify the presence of correlations of structural and functional parameters of the myocardium with the level of the studied biomarkers in patients with ischemic cardiomyopathy (ICMP).Materials and methods. Patients with ICMP (n = 47) were examined as the main group. The control group included 30 somatically healthy individuals. Bcl‑2 and APA5 levels were determined in the blood serum by the enzyme immunoassay.Results. It was found that in the group of patients with ICMP. The level of AnxA5 was statistically significantly higher (p &lt; 0.001), and the level of Bcl‑2 was statistically significantly lower (p &lt; 0.001) than in the control group. Based on the results of the correlation analysis a noticeable close relationship on the Cheddock scale was revealed between levels of the studied apoptosis biomarkers and the values of the sphericity index of in diastole and systole of left ventricle (LV), final diastolic and systolic volume of LV and ejection fraction of LV. Between the other parameters of myocardial remodeling and levels of the studied biomarkers of apoptosis, the closeness of relationships on the Cheddock scale were weak and moderate.

Quantitative proteomic screen identifies annexin A2 as a host target for Salmonella pathogenicity island-2 effectors SopD2 and PipB2

Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein–protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.