Characterization and Proteomic Analysis of Endometrial Stromal Cell-Derived Small Extracellular Vesicles.

CONTEXT: Small extracellular vesicles (sEVs) have emerged as modulators of the disease microenvironment, thereby supporting disease progression. However, the potential role of EVs and their content to the pathophysiology of endometriosis remain unclear. OBJECTIVE: This work aimed to investigate whether the EVs from eutopic (Eu) and ectopic (Ec) endometrial stromal cells (ESCs) differ with respect to protein composition and role in endometriosis. METHODS: Human Eu and Ec endometrium-derived ESCs were isolated from samples of the same patients (n = 3). sEVs were isolated from ESCs via ultracentrifugation; these sEVs were characterized by Western blotting, transmission electron microscopy, and nanoparticle tracking analysis and analyzed using mass spectrometry. The potential role of EcESCs-derived sEVs (EcESCs-sEVs) in endometriosis was explored by assaying their effects on cell viability/proliferation, migration, and angiogenesis. RESULTS: In total, 105 ESCs-sEV-associated proteins were identified from EcESCs-sEVs and EuESCs-sEVs by mass spectrometry analysis. The protein content differed between EcESCs-sEVs and EuESCs-sEVs, with annexin A2 (ANXA2) being the most prominent difference-present in EcESCs-sEVs but not EuESCs-sEVs. We also found that sEVs-ANXA2 regulates the motility, proliferation, and angiogenesis of ESCs via the extracellularly regulated kinase (ERK)/STAT3 pathway. Notably, treatment of ESCs with sEVs-ANXA2 resulted in increased proliferation and motility, suggesting that sEVs-ANXA2 may be involved in regulating endometriosis. Our data suggest that EcESCs-sEVs-ANXA2 regulates the motility and the angiogenic potential of ESCs, implying a role for sEVs-ANXA2 in the pathogenesis of endometriosis. CONCLUSION: The study of sEVs-ANXA2 from Ec endometriotic cells uncovers a new mechanism of endometriosis progression and will inform the development of novel therapeutic strategies.

Annexin A2 acts as an adherent molecule under the regulation of steroids during embryo implantation.

We previously showed that annexin A2 (Axna2) was transiently expressed at the embryo-uterine luminal epithelium interface during the window of implantation and was involved in mouse embryo implantation. At the same time, Axna2 was reported to be upregulated in human receptive endometrium, which was critical for embryo attachment as an intracellular molecule. Here, we identified Axna2 as a membrane-bound molecule on human endometrial epithelial cells and trophoblast cells, and the outer surface membrane-bound Axna2 was involved in human embryo attachment. In addition, physiological levels of estrogen and progesterone increased the expression of overall Axna2 as well as that in the extracellular surface membrane protein fraction in human endometrial cells. Furthermore, p11 (or S100A10, a member of the S100 EF-hand family protein, molecular weight 11 kDa) was involved in the translocation of Axna2 to the outer surface membrane of endometrial epithelial cells without affecting its overall expression. Finally, the surface relocation of Axna2 was also dependent on cell-cell contact and calcium binding. A better understanding of the function and regulation of Axna2 in human endometrium may help us to identify a potential therapeutic target for subfertile and infertile patients.

ANXA2P2/miR-9/LDHA axis regulates Warburg effect and affects glioblastoma proliferation and apoptosis.

BACKGROUND: Aerobic glycolysis is a unique tumor cell phenotype considered as one of the hallmarks of cancer. Aerobic glycolysis can accelerate tumor development by increasing glucose uptake and lactate production. In the present study, lactate dehydrogenase A (LDHA) is significantly increased within glioma tissue samples and cells, further confirming the oncogenic role of LDHA within glioma. METHODS: Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were applied for histopathological examination. The protein levels of LDHA, transporter isoform 1 (GLUT1), hexokinase 2 (HK2), phosphofructokinase (PFK) in target cells were detected by Immunoblotting. The predicted miR-9 binding to lncRNA Annexin A2 Pseudogene 2 (ANXA2P2) or the 3' untranslated region (UTR) of LDHA was verified using Luciferase reporter assay. Cell viability or apoptosis were examined by MTT assay or Flow cytometry. Intracellular glucose and Lactate levels were measured using glucose assay kit and lactate colorimetric assay kit. RESULTS: The expression of ANXA2P2 showed to be dramatically upregulated within glioma tissue samples and cells. Knocking down ANXA2P2 within glioma cells significantly inhibited cell proliferation and aerobic glycolysis, as manifested as decreased lactate and increased glucose in culture medium, and downregulated protein levels of glycolysis markers, GLUT1, HK2, PFK, as well as LDHA. miR-9 was predicted to target both lncRNA ANXA2P2 and LDHA. The overexpression of miR-9 suppressed the cell proliferation and aerobic glycolysis of glioma cells. Notably, miR-9 could directly bind to LDHA 3'UTR to inhibit LDHA expression and decrease the protein levels of LDHA. ANXA2P2 competitively targeted miR-9, therefore counteracting miR-9-mediated repression on LDHA. Within tissues, miR-9 exhibited a negative correlation with ANXA2P2 and LDHA, respectively, whereas ANXA2P2 and LDHA exhibited a positive correlation with each other. CONCLUSIONS: In conclusion, ANXA2P2/miR-9/LDHA axis modulates the aerobic glycolysis progression in glioma cells, therefore affecting glioma cell proliferation.

Epicardial adipose tissue volume and annexin A2/fetuin-A signalling are linked to coronary calcification in advanced coronary artery disease: Computed tomography and proteomic biomarkers from the EPICHEART study.

BACKGROUND & AIMS: The role of epicardial adipose tissue (EAT) in the pathophysiology of late stage-coronary artery disease (CAD) has not been investigated. We explored the association of EAT volume and its proteome with advanced coronary atherosclerosis. METHODS: The EPICHEART Study prospectively enrolled 574 severe aortic stenosis patients referred to cardiac surgery. Before surgery, EAT volume was quantified by computed tomography (CT). During surgery, epicardial, mediastinal (MAT) and subcutaneous (SAT) adipose tissue samples were collected to explore fat phenotype by analyzing the proteomic profile using SWATH-mass spectrometry; pericardial fluid and peripheral venous blood were also collected. CAD presence was defined as coronary artery stenosis ≥50% in invasive angiography and by CT-derived Agatston coronary calcium score (CCS). RESULTS: EAT volume adjusted for body fat was associated with higher CCS, but not with the presence of coronary stenosis. In comparison with mediastinal and subcutaneous fat depots, EAT exhibited a pro-calcifying proteomic profile in patients with CAD characterized by upregulation of annexin-A2 and downregulation of fetuin-A; annexin-A2 protein levels in EAT samples were also positively correlated with CCS. We confirmed that the annexin-A2 gene was overexpressed in EAT samples of CAD patients and positively correlated with CCS. Fetuin-A gene was not detected in EAT samples, but systemic fetuin-A was higher in CAD than in non-CAD patients, suggesting that fetuin-A was locally downregulated. CONCLUSIONS: In an elderly cohort of stable patients, CCS was associated with EAT volume and annexin-A2/fetuin-A signaling, suggesting that EAT might orchestrate pro-calcifying conditions in the late phases of CAD.

GroEL Protein (Heat Shock Protein 60) of Mycoplasma gallisepticum Induces Apoptosis in Host Cells by Interacting with Annexin A2.

Mycoplasma gallisepticum is an avian respiratory and reproductive tract pathogen that has a significant economic impact on the poultry industry worldwide. Although membrane proteins of Mycoplasma spp. are thought to play crucial roles in host interactions, very few have had their biochemical function defined. In this study, we found that the GroEL protein (heat shock protein 60) of Mycoplasma gallisepticum could induce apoptosis in peripheral blood mononuclear cells, and the underlying molecular mechanism was further determined. The GroEL gene from Mycoplasma gallisepticum was cloned and expressed in Escherichia coli to facilitate the functional analysis of recombinant protein. The purified GroEL protein was shown to adhere to peripheral blood mononuclear cells (PBMCs) and DF-1 cells and cause apoptosis in PBMCs. A protein pulldown assay coupled with mass spectrometry identified that annexin A2 possibly interacted with GroEL protein. Coimmunoprecipitation assays confirmed that GroEL proteins could bind to annexin A2, and confocal analysis further demonstrated that GroEL colocolized with annexin A2 in HEK293T cells and PBMCs. Moreover, annexin A2 expression was significantly induced by a recombinant GroEL protein in PBMCs, and knocking down annexin A2 expression resulted in significantly reduced apoptosis. Taken together, these data suggest that GroEL induces apoptosis in host cells by interacting with annexin A2, a novel virulence mechanism in Mycoplasma gallisepticum Our findings lead to a better understanding of molecular pathogenesis in Mycoplasma gallisepticum.

A new role for host annexin A2 in establishing bacterial adhesion to vascular endothelial cells: lines of evidence from atomic force microscopy and an in vivo study.

Understanding bacterial adhesion is challenging and critical to our understanding of the initial stages of the pathogenesis of endovascular bacterial infections. The vascular endothelial cell (EC) is the main target of Rickettsia, an obligately intracellular bacterium that causes serious systemic disease in humans and animals. But the mechanism(s) underlying bacterial adherence to ECs under shear stress from flowing blood prior to activation are unknown for any bacteria. Although host surface annexin a2 (ANXA2) has been identified to participate in efficient bacterial invasion of epithelial cells, direct evidence is lacking in the field of bacterial infections of ECs. In the present study, we employ a novel, anatomically based, in vivo quantitative bacterial-adhesion-to-vascular-EC system, combined with atomic force microscopy (AFM), to examine the role of endothelial luminal surface ANXA2 during rickettsial adherence to ECs. We also examined whether ANXA2 antibody affected binding of Staphylococcus aureus to ECs. We found that deletion of ANXA2 impeded rickettsial attachment to the ECs in vitro and blocked rickettsial adherence to the blood vessel luminal surface in vivo. The AFM studies established that EC surface ANXA2 acts as an adherence receptor for rickettsiae, and that rickettsial adhesin OmpB is the associated bacterial ligand. Furthermore, pretreatment of ECs with anti-ANXA2 antibody reduced EC surface-associated S. aureus. We conclude that the endothelial surface ANXA2 plays an important role in initiating pathogen-host interactions, ultimately leading to bacterial anchoring on the vascular luminal surface.

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.

Role of the long non­coding RNA­Annexin A2 pseudogene 3/Annexin A2 signaling pathway in biliary atresia­associated hepatic injury.

Biliary atresia (BA) is the most common cause of chronic cholestasis in children. The long non­coding RNA (lncRNA) Annexin A2 pseudogene 3 (ANXA2P3) and Annexin A2 (ANXA2) have been suggested to serve pivotal roles in BA; however, the clinical significance and biological roles of ANXA2P3 and ANXA2 in BA remain to be elucidated. The present study aimed to elucidate the function of ANAX2P3 and ANXA2 in BA­induced liver injury using a human liver cell line and liver tissues from patients with BA. Reverse transcription­quantitative polymerase chain reaction, western blotting and immunohistochemistry were conducted to determine the expression levels of ANXA2 and ANXA2P3 in liver tissues from patients with BA. Classification of fibrosis was analyzed by Masson staining. The functional roles of ANXA2 and ANXA2P3 in liver cells were determined by Cell Counting kit­8 assay, and flow cytometric and cell cycle analyses. Activation of the ANXA2/ANXA2P3 signaling pathway in liver cells was evaluated by western blot analysis. According to the present results, the expression levels of ANXA2 and ANXA2P3 were significantly increased in liver tissues from patients with BA. In addition, knocking down the expression of ANXA2P3 and ANXA2 may result in reduced liver cell proliferation, cell cycle arrest in G1 phase and increased apoptosis of liver cells in vitro. Furthermore, in cells in which ANXA2 and ANXA2P3 were overexpressed, cell apoptosis was reduced and cell cycle arrest in G2 phase. Taken together, these results indicated that ANXA2P3 and ANXA2 may have protective effects against liver injury progression and may be considered biomarkers in patients with BA.

ANXA2 could act as a moderator of EGFR-directed therapy resistance in triple negative breast cancer.

Triple negative breast cancer (TNBC) patients cannot benefit from EGFR-targeted therapy even though the EGFR is highly expressed, because patients exhibit resistance to these drugs. Unfortunately, the molecular mechanisms remain relatively unknown. ANXA2, highly expressed in invasive breast cancer cells, is closely related with poor prognosis, and acts as a molecular switch to EGFR activation. In this study, MDA-MB-231 cells and MCF7 cells were used. Our results showed that ANXA2 expression is inversely correlated with cell sensitivity to gefitinib. Knockdown of ANXA2 expression in MDA-MB-231 cells increased the gefitinib induced cell death. When ANXA2 was overexpressed in MCF7 cells, the gefitinib induced cell death was decreased. Furthermore, we demonstrated that phosphorylation of ANXA2 at Tyr23 is negatively correlated with the sensitivity of TNBC to gefitinib. Altogether, our results suggest a new role of ANXA2 in regulating sensitivity of TNBC MDA-MB-231 cells to the EGFR inhibitor gefitinib.

Annexin A2 supports pulmonary microvascular integrity by linking vascular endothelial cadherin and protein tyrosine phosphatases.

Relative or absolute hypoxia activates signaling pathways that alter gene expression and stabilize the pulmonary microvasculature. Alveolar hypoxia occurs in disorders ranging from altitude sickness to airway obstruction, apnea, and atelectasis. Here, we report that the phospholipid-binding protein, annexin A2 (ANXA2) functions to maintain vascular integrity in the face of alveolar hypoxia. We demonstrate that microvascular endothelial cells (ECs) from Anxa2-/- mice display reduced barrier function and excessive Src-related tyrosine phosphorylation of the adherens junction protein vascular endothelial cadherin (VEC). Moreover, unlike Anxa2+/+ controls, Anxa2-/- mice develop pulmonary edema and neutrophil infiltration in the lung parenchyma in response to subacute alveolar hypoxia. Mice deficient in the ANXA2-binding partner, S100A10, failed to demonstrate hypoxia-induced pulmonary edema under the same conditions. Further analyses reveal that ANXA2 forms a complex with VEC and its phosphatases, EC-specific protein tyrosine phosphatase (VE-PTP) and Src homology phosphatase 2 (SHP2), both of which are implicated in vascular integrity. In the absence of ANXA2, VEC is hyperphosphorylated at tyrosine 731 in response to vascular endothelial growth factor, which likely contributes to hypoxia-induced extravasation of fluid and leukocytes. We conclude that ANXA2 contributes to pulmonary microvascular integrity by enabling VEC-related phosphatase activity, thereby preventing vascular leak during alveolar hypoxia.

[Effect of annexin A2 on EGFR/NF-κB signal transduction and mucin expression in human airway epithelial cells treated with Mycoplasma pneumoniae].

OBJECTIVE: To investigate the effect of annexin A2 (AnxA2) on epithelial growth factor receptor (EGFR)/nuclear factor-κB (NF-κB) signal transduction and mucin expression in human airway epithelial H292 cells treated with Mycoplasma pneumoniae (MP). METHODS: H292 cells were divided into control group, MP group, NC-siRNA+MP group, and AnxA2 siRNA+MP group. The cells in the MP group were incubated with 5 µg/mL MP antigen for 2 hours. The cells in the NC-siRNA+MP and AnxA2 siRNA+MP groups were transfected with NC-siRNA and AnxA2 siRNA for 24 hours, followed by MP antigen stimulation for 2 hours. The MTT method was used to measure cell viability; quantitative real-time PCR was used to measure the mRNA expression of AnxA2; Western blot was used to measure the protein expression of AnxA2, phosphorylated EGFR (p-EGFR), and phosphorylated p65 NF-κB (p-p65 NF-κB); ELISA was used to measure the secretion of mucin 5AC (MUC5AC) and mucin 5B (MUC5B). RESULTS: The MP and NC-siRNA+MP groups had lower cell viability than the control group (P<0.05). The AnxA2 siRNA+MP group had higher cell viability than the MP and NC-siRNA+MP groups and lower cell viability than the control group (P<0.05). The MP and NC-siRNA+MP groups had significantly higher mRNA and protein expression of AnxA2 than the AnxA2 siRNA+MP group (P<0.05). Compared with the control group, the MP and NC-siRNA+MP groups had significant increases in the protein expression of p-EGFR, p-p65 NF-κB, MUC5AC, and MUC5B (P<0.05); the AnxA2 siRNA+MP group had lower protein expression than the MP and NC-siRNA+MP groups, but higher protein expression than the control group (P<0.05). CONCLUSIONS: AnxA2 is involved in the airway lesion induced by MP antigen via mediating EGFR/NF-κB signaling activation and mucin expression in human airway epithelial cells.

S100A10 identified in a genome-wide gene × cannabis dependence interaction analysis of risky sexual behaviours.

BACKGROUND: We conducted a genome-wide gene × environment interaction analysis to identify genetic variants that interact with cannabis dependence (CaD) in influencing risky sexual behaviours (RSB). METHODS: Our sample included cannabis-exposed and sexually experienced African-American and European-American participants. A DSM-IV CaD diagnosis and RSB were evaluated using the Semi-Structured Assessment for Drug Dependence and Alcoholism. We analyzed RSBs as a score that takes into account experiences of unprotected sex and multiple sexual partners. RESULTS: A total of 3350 people participated in our study; 43% had a CaD diagnosis, 56% were African-American and 33% were women. We identified a genome-wide significant locus in African-American participants (S100A10 rs72993629, p = 2.73 × 10-8) and a potential transpopulation signal in women (CLTC rs12944716, p = 5.27 × 10-8). A resting-state fMRI follow-up analysis of S100A10 rs72993629 conducted in an independent cohort showed 2 significant associations: reduced power of the left paracentral lobule in amplitude of low frequency fluctuations (ALFF) analysis (p = 7.8 × 10-3) and reduced power of the right pallidum in fractional ALFF analysis (p = 4.6 × 10-3). The activity of these brain regions is known to be involved in sexual functions and behaviours. The S100A10 result functionally recapitulated our S100B finding observed in our previous genome-wide association study of CaD. The probability of identifying 2 S100 genes in 2 independent genome-wide investigations by chance is approximately 1 in 1.1 million. LIMITATIONS: We were not able to identify any African-American cohort with appropriate sample size, and phenotypic assessment is available to replicate our findings. CONCLUSION: The S100A10 and S100B genes, which are located on different chromosomes, encode specialized calcium-binding proteins. These data support a role for calcium homeostasis in individuals with CaD and its induced behaviours.

Annexin A2 links poor myofiber repair with inflammation and adipogenic replacement of the injured muscle.

Repair of skeletal muscle after sarcolemmal damage involves dysferlin and dysferlin-interacting proteins such as annexins. Mice and patient lacking dysferlin exhibit chronic muscle inflammation and adipogenic replacement of the myofibers. Here, we show that similar to dysferlin, lack of annexin A2 (AnxA2) also results in poor myofiber repair and progressive muscle weakening with age. By longitudinal analysis of AnxA2-deficient muscle we find that poor myofiber repair due to the lack of AnxA2 does not result in chronic inflammation or adipogenic replacement of the myofibers. Further, deletion of AnxA2 in dysferlin deficient mice reduced muscle inflammation, adipogenic replacement of myofibers, and improved muscle function. These results identify multiple roles of AnxA2 in muscle repair, which includes facilitating myofiber repair, chronic muscle inflammation and adipogenic replacement of dysferlinopathic muscle. It also identifies inhibition of AnxA2-mediated inflammation as a novel therapeutic avenue for treating muscle loss in dysferlinopathy.

Cellular and molecular basis for stress-induced depression.

Chronic stress has a crucial role in the development of psychiatric diseases, such as anxiety and depression. Dysfunction of the medial prefrontal cortex (mPFC) has been linked to the cognitive and emotional deficits induced by stress. However, little is known about the molecular and cellular determinants in mPFC for stress-associated mental disorders. Here we show that chronic restraint stress induces the selective loss of p11 (also known as annexin II light chain, S100A10), a multifunctional protein binding to 5-HT receptors, in layer II/III neurons of the prelimbic cortex (PrL), as well as depression-like behaviors, both of which are reversed by selective serotonin reuptake inhibitors (SSRIs) and the tricyclic class of antidepressant (TCA) agents. In layer II/III of the PrL, p11 is highly concentrated in dopamine D2 receptor-expressing (D2+) glutamatergic neurons. Viral expression of p11 in D2+ PrL neurons alleviates the depression-like behaviors exhibited by genetically manipulated mice with D2+ neuron-specific or global deletion of p11. In stressed animals, overexpression of p11 in D2+ PrL neurons rescues depression-like behaviors by restoring glutamatergic transmission. Our results have identified p11 as a key molecule in a specific cell type that regulates stress-induced depression, which provides a framework for the development of new strategies to treat stress-associated mental illnesses.

Annexin A2 is involved in Ca2+-dependent plasma membrane repair in primary human endothelial cells.

Many cells in an organism are exposed to constant and acute mechanical stress that can induce plasma membrane injuries. These plasma membrane wounds have to be resealed rapidly to guarantee cell survival. Plasma membrane resealing in response to mechanical strain has been studied in some detail in muscle, where it is required for efficient recovery after insult. However, less is known about the capacity of other cell types and tissues to perform membrane repair and the underlying molecular mechanisms. Here we show that vascular endothelial cells, which are subject to profound mechanical burden, can reseal plasma membrane holes inflicted by laser ablation. Resealing in endothelial cells is a Ca2+-dependent process, as it is inhibited when cells are wounded in Ca2+-free medium. We also show that annexin A1 (AnxA1), AnxA2 and AnxA6, Ca2+-regulated membrane binding proteins previously implicated in membrane resealing in other cell types, are rapidly recruited to the site of plasma membrane injury. S100A11, a known protein ligand of AnxA1, is also recruited to endothelial plasma membrane wounds, albeit with a different kinetic. Mutant expression experiments reveal that Ca2+ binding to AnxA2, the most abundant endothelial annexin, is required for translocation of the protein to the wound site. Furthermore, we show by knock-down and rescue experiments that AnxA2 is a positive regulator of plasma membrane resealing. Thus, vascular endothelial cells are capable of active, Ca2+-dependent plasma membrane resealing and this process requires the activity of AnxA2.

Annexin A2, an essential partner of the exocytotic process in chromaffin cells.

Annexin A2 is a calcium-, actin-, and lipid-binding protein implicated in exocytosis in different cell types, such as neuroendocrine cells. In chromaffin cells, cytosolic annexin A2 is recruited to the plasma membrane upon cell stimulation. Here, we review the latest evidence detailing the functional importance of annexin A2 in different stages of exocytosis. These include the recruitment of annexin A2 to the plasma membrane near soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, the role of annexin A2 in the formation of lipid domains at exocytotic sites, and finally the annexin A2 bundling of actin microfilaments associated with chromaffin granules. These structures induce first the coalescence of lipid domains required for the formation of the exocytotic site, and in the second time, exert mechanical force on the granule to favor fusion pore expansion and squeeze the granule to facilitate catecholamine release. Annexin A2 is a calcium-, actin-, and lipid-binding protein implicated in exocytosis in different cell types, including neuroendocrine cells. Upon cell stimulation, annexin A2 translocates from the cytosol to the plasma membrane of chromaffin cells and bundles actin filaments associated with chromaffin granules. This promotes the formation of lipid domains required for granule docking, and facilitates catecholamine release by compressing the granule. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

p11 modulates L-DOPA therapeutic effects and dyskinesia via distinct cell types in experimental Parkinsonism.

The reduced movement repertoire of Parkinson's disease (PD) is mainly due to degeneration of nigrostriatal dopamine neurons. Restoration of dopamine transmission by levodopa (L-DOPA) relieves motor symptoms of PD but often causes disabling dyskinesias. Subchronic L-DOPA increases levels of adaptor protein p11 (S100A10) in dopaminoceptive neurons of the striatum. Using experimental mouse models of Parkinsonism, we report here that global p11 knockout (KO) mice develop fewer jaw tremors in response to tacrine. Following L-DOPA, global p11KO mice show reduced therapeutic responses on rotational motor sensitization, but also develop less dyskinetic side effects. Studies using conditional p11KO mice reveal that distinct cell populations mediate these therapeutic and side effects. Selective deletion of p11 in cholinergic acetyltransferase (ChAT) neurons reduces tacrine-induced tremor. Mice lacking p11 in dopamine D2R-containing neurons have a reduced response to L-DOPA on the therapeutic parameters, but develop dyskinetic side effects. In contrast, mice lacking p11 in dopamine D1R-containing neurons exhibit tremor and rotational responses toward L-DOPA, but develop less dyskinesia. Moreover, coadministration of rapamycin with L-DOPA counteracts L-DOPA-induced dyskinesias in wild-type mice, but not in mice lacking p11 in D1R-containing neurons. 6-OHDA lesioning causes an increase of evoked striatal glutamate release in wild type, but not in global p11KO mice, indicating that altered glutamate neurotransmission could contribute to the reduced L-DOPA responsivity. These data demonstrate that p11 located in ChAT or D2R-containing neurons is involved in regulating therapeutic actions in experimental PD, whereas p11 in D1R-containing neurons underlies the development of L-DOPA-induced dyskinesias.

Atorvastatin protects endothelial colony­forming cells against H2O2­induced oxidative damage by regulating the expression of annexin A2.

Endothelial dysfunction and injury are central events in the pathogenesis of ischemic vascular disorders. Endothelial progenitor cells (EPCs) are mobilized from the bone marrow into the peripheral circulation, where they locate to sites of injured endothelium and are involved in endothelial repair and vascular regeneration. During these processes, EPCs are exposed to oxidative stress, a crucial pathological condition, which occurs during vascular injury and limits the efficacy of EPCs in the repair of injured endothelium. Statins are effective inhibitors of 3­hydroxy­3­methylglutaryl coenzyme A reductase, and are commonly used to manage and prevent ischemic vascular disease by reducing plasma cholesterol levels. In addition to lowering cholesterol, statins have also been reported to exert pleiotropic actions, including anti­inflammatory and anti­oxidative activities. The present study aimed to investigate the ability of atorvastatin to protect endothelial colony­forming cells (ECFCs), a homogeneous subtype of EPCs, from hydrogen peroxide (H2O2)­induced oxidative damage, and to determine the mechanism underlying this protective action. MTT assay, acridine orange/ethidium bromide staining, reactive oxygen species assay, western blot analysis and tube formation assay were employed. The results demonstrated that H2O2 induced cell death and decreased the tube­forming ability of the ECFCs, in a concentration­dependent manner; however, these effects were partially attenuated following administration of atorvastatin. The reversion of the quantitative and qualitative impairment of the H2O2­treated ECFCs appeared to be mediated by the regulation of annexin A2, as the expression levels of annexin A2 were decreased following treatment with H2O2 and increased following treatment with atorvastatin. These results indicated that annexin A2 may be involved in the H2O2­induced damage of ECFCs, and in the protective activities of atorvastatin in response to oxidative stress.

Characterization of annexin A2 in chicken follicle development: Evidence for its involvement in angiogenesis.

Annexin A2 (ANXA2) is a calcium-dependent, phospholipid-binding protein found in various cells and tissues. ANXA2 plays multiple roles in regulating cellular functions and is often over-expressed in different types of tumors including ovarian cancer. Others and we previously found that ANXA2 was up-regulated in the ovaries of hens with higher laying rate, indicated that ANXA2 is involved in avian follicle development. In this study, we found that ANXA2 mRNA expression increased during chicken ovary maturation and follicle development. In the pre-ovulatory follicles, ANXA2 expression level was significantly higher in theca cells than granulosa cells. In theca cells, ANXA2 expression could be stimulated by follicle-stimulating hormone (FSH) and estrogen but not luteinizing hormone (LH) or progesterone. The core promoter regions control the basal and FSH-induced ANXA2 gene expression were identified. Forced expression of ANXA2 could induce the expression of angiogenic factors and receptors in theca cells. Furthermore, ANXA2 overexpression resulted increased vascular endothelial growth factor A (VEGFA) secretion and theca cell proliferation. Current study not only provides the first evidence of expression and regulation of ANXA2 in chicken ovary, but also suggests that ANXA2 is involved in follicular angiogenesis and contributes to successful follicle development and ovulation.