RESUMO
BACKGROUND: Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level. RESULTS: Here, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis. CONCLUSIONS: Our findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and seed development.
Assuntos
Anexina A5/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Proteínas de Cloroplastos/farmacologia , Plastídeos/fisiologia , Pólen/crescimento & desenvolvimento , Proteínas rab1 de Ligação ao GTP/farmacologia , Anexina A5/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/farmacologia , Clorofila/metabolismo , Proteínas de Cloroplastos/genética , Técnicas de Silenciamento de Genes , Genes de Plantas , Homeostase , Pólen/anatomia & histologia , Pólen/genética , Tubo Polínico/crescimento & desenvolvimento , Plântula/metabolismo , Nicotiana/genética , Nicotiana/fisiologia , Transcriptoma , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
Background: Although domain IV of annexin A5 (anxA5) may be less effective in binding phosphatidylserine (PS), the four domains together may guarantee the maximum binding of anxA5 to the PS membrane. Additionally, previous research has shown that annexin mutants lacking one or more domain(s) have different biological activities compared to the wild-type. The present research mainly aims to study the role of domain IV in the crucial PS-binding function of anxA5. Methods: The domain IV-truncated anxA5 protein was constructed and purified. Isothermal titration calorimetry, flow cytometry and activated partial thromboplastin time were adopted to examine the function of domain IV in anxA5-PS binding directly or indirectly. Results: The domain IV-truncated form of anxA5 is impaired in binding PS liposome and apoptotic cells, and anticoagulation activity. The mutant cannot bind calcium, but binds PS only in the presence of calcium. Conclusions: Truncation of domain IV of anxA5 destroys its calcium-binding ability and impairs its PS-binding activity. Truncation of domain IV may induce conformation change of anxA5 or reduce the hydrophobic interactions between protein and membrane, which may explain the decrease of PS-binding affinity of the mutant.
Assuntos
Anexina A5/química , Cálcio/química , Fosfatidilserinas/química , Anexina A5/fisiologia , Apoptose , Sítios de Ligação , Membrana Celular , Humanos , Células Jurkat , Fosfatidilserinas/metabolismo , Ligação ProteicaRESUMO
Eryptosis, the suicidal erythrocyte death, leads to cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to the vascular wall by binding of phosphatidylserine to the CXC chemokine ligand 16 (CXCL16). Stimulators of eryptosis include increased cytosolic Ca(2+) activity, energy depletion, and activation of ceramide-producing sphingomyelinase. The present study explored whether sphingomyelinase triggers erythrocyte adhesion to endothelial cells. To this end, human erythrocytes were exposed for 6 h to bacterial sphingomyelinase (1-10 mU/ml) and phosphatidylserine exposure was estimated from fluorescent annexin-V-binding, cell volume from forward scatter in FACS-analysis, erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber under flow conditions at arterial shear rates, and CXCL16 protein abundance utilizing Western blotting and FACS analysis of fluorescent antibody binding. As a result, sphingomyelinase (≥1 mU/ml) triggered cell shrinkage, phosphatidylserine exposure and erythrocyte adhesion to HUVEC, effects blunted by Ca(2+) removal. Adhesion was significantly blunted by phosphatidylserine-coating annexin-V (5 µl/ml), following addition of neutralizing antibodies against endothelial CXCL16 (4 µg/ml) and following silencing of the CXCL16 gene with small interfering RNA. Pretreatment of HUVEC with sphingomyelinase upregulated CXCL16 protein abundance. Six hours pretreatment of HUVEC with sphingomyelinase (10 mU/ml) or C6-ceramide (50 µM) augmented erythrocyte adhesion following a 30-min treatment with Ca(2+) ionophore ionomycin (1 µM) or following energy depletion by 48-h glucose removal. Thus exposure to sphingomyelinase or C6-ceramide triggers eryptosis followed by phosphatidylserine- and CXCL16-sensitive adhesion of eryptotic erythrocytes to HUVEC.
Assuntos
Apoptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Esfingomielina Fosfodiesterase/farmacologia , Anexina A5/fisiologia , Anticorpos Neutralizantes/farmacologia , Apoptose/fisiologia , Cálcio/farmacologia , Ionóforos de Cálcio/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Tamanho Celular , Células Cultivadas , Ceramidas/farmacologia , Quimiocina CXCL16 , Quimiocinas CXC/antagonistas & inibidores , Quimiocinas CXC/genética , Quimiocinas CXC/fisiologia , Eritrócitos/fisiologia , Inativação Gênica , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Ionomicina/farmacologia , Fosfatidilserinas/fisiologia , Receptores Depuradores/antagonistas & inibidores , Receptores Depuradores/genética , Receptores Depuradores/fisiologiaRESUMO
Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In CF, the most common mutant DeltaF508-CFTR is misfolded, is retained in the ER and is rapidly degraded. If conditions could allow DeltaF508-CFTR to reach and to stabilize in the plasma membrane, it could partially correct the CF defect. We have previously shown that annexin V (anxA5) binds to both the normal CFTR and the DeltaF508-CFTR in a Ca(2+)-dependent manner and that it regulates the chloride channel function of Wt-CFTR through its membrane integration. Our aim was to extend this finding to the DeltaF508-CFTR. Because some studies show that thapsigargin (Tg) increases the DeltaF508-CFTR apical expression and induces an increased [Ca(2+)](i) and because anxA5 relocates and binds to the plasma membrane in the presence of Ca(2+), we hypothesized that the Tg effect upon DeltaF508-CFTR function could involve anxA5. Our results show that raised anxA5 expression induces an augmented function of DeltaF508-CFTR due to its increased membrane localization. Furthermore, we show that the Tg effect involves anxA5. Therefore, we suggest that anxA5 is a potential therapeutic target in CF.
Assuntos
Anexina A5/fisiologia , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Mutação , Anexina A5/genética , Anexina A5/metabolismo , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Humanos , Imunoprecipitação , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Tapsigargina/farmacologia , TransfecçãoRESUMO
Several clinical studies have suggested that blood transfusions are immunosuppressive. Whereas there have been reports describing immunosuppression induced by leukocytes or fragments thereof, the possibility that microparticles, released by erythrocytes during storage, are also involved was not investigated. We present evidence here that such microparticles have all the properties of ectosomes including size, the presence of a lipid membrane, and the specific sorting of proteins. These erythrocyte-derived ectosomes (E-ecto) fixed C1q, which was followed by activation of the classical pathway of complement with binding of C3 fragments. Similarly to ectosomes released by PMN, they express phosphatidylserine on their surface membrane, suggesting that they may react with and down-regulate cells of the immune system. In vitro, they were taken up by macrophages, and they significantly inhibited the activation of these macrophages by zymosan A and LPS, as shown by a significant drop in TNF-alpha and IL-8 release (respectively, 80% and 76% inhibitions). In addition, the effect of E-ecto was not transient but lasted for at least 24 h. In sum, E-ecto may interfere with the innate immune system/inflammatory reaction. Therefore, E-ecto transfused with erythrocytes may account for some of the immunosuppressive properties attributed to blood transfusions.
Assuntos
Eritrócitos/fisiologia , Frações Subcelulares/fisiologia , Animais , Anexina A5/fisiologia , Anticorpos Monoclonais , Transfusão de Sangue , Ativação do Complemento , Complemento C1q/fisiologia , Eritrócitos/citologia , Eritrócitos/imunologia , Eritrócitos/ultraestrutura , Citometria de Fluxo , Humanos , Macrófagos/fisiologia , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Neutrófilos/imunologia , Neutrófilos/fisiologia , Ligação Proteica , Frações Subcelulares/imunologia , Frações Subcelulares/ultraestruturaRESUMO
OBJECTIVE: The expression of tissue factor (TF) in vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of artherosclerosis (AS) and thrombosis. Hyperhomocysteinemia is a risk factor for AS. Annexin A5, a calcium-dependent anionic-phospholipid-binding protein has anticoagulant effect mediated by its interaction with phosphatidylserine. We investigated the effects of Annexin A5 on homocysteine (Hcy)-induced TF expression and activity in VSMCs. METHODS: Human umbilical artery VSMCs were cultured by tissue explanting method, incubated with Hcy at various concentrations in the absence and presence of Annexin A5 (50 microg/ml)/mono-TFAb (10 microg/ml). Flow Cytometry (FCM) was used to detect the expression of TF on the surface of VSMCs. Protein expression of TF was detected by Western blot. Determination of TF activity by factor Xa generation. RESULTS: The expression level of TF protein on the surface of the resting VSMCs was low [the positive rate was (4.01 +/- 2.11)%] and could be upregulated by Hcy [peaked at 1000 micromol/L, the positive rate was (37.67 +/- 4.96)%]. Annexin A5, as well as mono-TFAb could significantly inhibit Hey-induced TF membrane expression, release activity and protein expression. CONCLUSIONS: These results suggest that Annexin A5 could inhibit Hcy-induced expression and activity of TF in VSMCs as well as TF release of VSMCs. Annexin A5 might play an active role on attenuating AS and reducing coronary thrombosis by inhibiting TF pathway.
Assuntos
Anexina A5/fisiologia , Homocisteína/antagonistas & inibidores , Músculo Liso Vascular/metabolismo , Tromboplastina/metabolismo , Western Blotting , Células Cultivadas , Citometria de Fluxo , Humanos , Artérias UmbilicaisRESUMO
Annexin V is a Ca(2+)-dependent membrane-binding protein that forms voltage-dependent Ca2+ channels in phospholipid bilayers and is the first ion channel to be structurally and functionally characterized. Data outlined here indicate that key amino acid residues act as selectivity filters and voltage sensors, thereby regulating the permeability of the channel pore to ions.
Assuntos
Anexina A5/fisiologia , Canais Iônicos/fisiologia , Anexina A5/química , Canais de Cálcio/química , Canais de Cálcio/fisiologia , Membrana Celular/fisiologia , Análise Mutacional de DNA , Humanos , Ativação do Canal Iônico , Mutação , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Death receptors are a subgroup of the tumor necrosis factor receptor superfamily (TNFRSF) and mediate activation of what is widely known as the "extrinsic" apoptosis pathway. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is one of the most recent death receptor ligands identified. The TRAIL receptor family consists of four distinct membrane-bound receptors, named TRAIL-R1 to -R4. TRAIL-R1 and TRAIL-R2 belong to the "death receptor" subfamily of the TNFRSF. Unlike other death receptor ligands, such as FasL/CD95L and TNF, TRAIL seems to display selective toxicity by killing tumor and transformed, but not normal, cells. Importantly, there also seems to be a complete lack of apparent toxicity, specifically hepatotoxicity, when TRAIL is used in vivo. Taken together, these observations led to TRAIL being proposed as a potential anti-tumor therapeutic, thus explaining the intense activity surrounding TRAIL and TRAIL receptor research over the past few years. This chapter describes a number of methods for the production of recombinant TRAIL in E. coli followed by labeling of recombinant TRAIL with either biotin or fluorochromes. These recombinant TRAIL preparations are then used to study various aspects of TRAIL signaling from cell surface receptor levels to the composition of death receptor complexes. This combination of direct binding and functional analysis provides a very powerful approach to aid in further characterization of TRAIL/TRAIL-Receptor regulation and signaling.
Assuntos
Receptores do Ligante Indutor de Apoptose Relacionado a TNF/análise , Ligante Indutor de Apoptose Relacionado a TNF/análise , Anexina A5/fisiologia , Anticorpos/metabolismo , Apoptose/fisiologia , Biotinilação/métodos , Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/biossínteseRESUMO
After several decades of debate, it is now widely acknowledged that apoptosis, also known as programmed cell death, is central to homoeostasis and normal development and physiology in all multicellular organisms, including humans. The dysregulation of apoptosis can lead to the destruction of normal tissues in a variety of disorders, including autoimmune and neurodegenerative diseases (too much apoptosis) or the growth of tumors (too little apoptosis). In addition, effective therapy of tumors requires the iatrogenic induction of programmed cell death by radiation, chemotherapy, or both. Given the central role of apoptosis, it would be desirable to have a noninvasive imaging method to serially detect and monitor this process in cancer patients undergoing conventional radiation and chemotherapy treatments as well as for the development and testing of new drugs. In this article, the latest modalities and contrast agents described in the literature for the imaging of apoptosis in vivo are reviewed. First, the most recent developments in the biochemical characterization of the many intracellular pathways involved in this complex process are discussed. Next, a variety of new radionuclide tracers, including radiolabeled annexin V and caspase inhibitors for PET and SPECT, are described. Finally, the use of MRI, MR spectroscopy, and ultrasound as possible alternative imaging modalities for the imaging of apoptosis is addressed.
Assuntos
Anexina A5 , Apoptose/fisiologia , Neoplasias/metabolismo , Compostos Radiofarmacêuticos , Animais , Anexina A5/fisiologia , Inibidores de Caspase , Caspases/metabolismo , Morte Celular/fisiologia , Meios de Contraste , Humanos , Metabolismo dos Lipídeos , Imageamento por Ressonância Magnética , Camundongos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Tomografia por Emissão de Pósitrons , UltrassonografiaRESUMO
OBJECTIVES: Basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbapatite (CA) and hydroxyapatite (HA)) are associated with severe forms of osteoarthritis. In advanced osteoarthritis, cartilage shows chondrocyte apoptosis, overexpression of annexin 5 (A5) and BCP crystal deposition within matrix vesicles. We assessed in vitro whether BCP crystals and overexpression of A5 increased chondrocyte apoptosis. METHODS: Apoptosis was induced by BCP crystals, tumour necrosis factor (TNF)-alpha (20 ng/ml) and Fas ligand (20 ng/ml) in normal articular chondrocytes (control) and in A5 overexpressed chondrocytes, performed by adenovirus infection. Apoptosis was assessed by caspase 3 (Cas3) activity, and DNA fragmentation. RESULTS: All BCP crystals, TNF-alpha and Fas ligand induced chondrocyte apoptosis as demonstrated by decreased cell viability and increased Cas3 activity and DNA fragmentation. TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end-labelling)-positive staining chondrocytes were increased by OCP (12.4 (5.2)%), CA (9.6 (2.6)%) and HA (9.2 (3.0)%) crystals and TNF-alpha (9.6 (2.4)%) stimulation compared with control (3.1 (1.9)%). BCP crystals increased Cas3 activity in a dose-dependent fashion. BCP-crystal-induced chondrocyte apoptosis was independent from TNF-alpha and interleukin-1beta pathways but required cell-crystal contact and intralysosomal crystal dissolution. Indeed, preincubation with ammonium chloride, a lysosomal inhibitor of BCP crystal dissolution, significantly decreased BCP-crystal-induced Cas3 activity. Finally, overexpression of A5 enhanced BCP crystal- and TNF-alpha-induced chondrocyte apoptosis. CONCLUSIONS: Overexpression of A5 and the presence of BCP crystals observed in advanced osteoarthritis contributed to chondrocyte apoptosis. Our results suggest a new pathophysiological mechanism for calcium-containing crystal arthropathies.
Assuntos
Anexina A5/fisiologia , Apoptose/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Animais , Anexina A5/metabolismo , Apoptose/fisiologia , Fosfatos de Cálcio/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Caspase 3/metabolismo , Bovinos , Células Cultivadas , Condrócitos/metabolismo , Cristalização , Fragmentação do DNA , Fator de Necrose Tumoral alfa/fisiologia , Ácido Úrico/metabolismoRESUMO
OBJECTIVES: High-dose chemotherapy with the alkylating agent busulfan has been widely used in the treatment of patients with high-risk Ewing's sarcoma. Because of risks for toxicity, busulfan and radiotherapy can not be applied together, leading to the omission of one effective therapy component. Treosulfan is a derivative of busulfan which has a lower side effect profile than busulfan and which can be used together with radiotherapy. We investigated the effect of treosulfan in a panel of Ewing's sarcoma cell lines on cell survival, cell cycle and apoptosis in vitro and compared it to busulfan. Furthermore, the anti-tumor effect of treosulfan was studied in an orthotopic Ewing's sarcoma mouse xenograft model. METHODS: Cell survival was measured by MTT assay and cell cycle analysis by flow cytometry. Apoptosis was analyzed via detection of DNA fragmentation, Hoechst 33258 staining, Annexin V, and cleavage of caspases-3 and 9. The effect of treosulfan and busulfan on primary tumor growth was assessed in Ewing's sarcoma xenografts in NOD/SCID mice (10 mice per group), pharmacokinetics of treosulfan were analyzed in nude mice. RESULTS: Treosulfan inhibited cell growth to at least 70% in all cell lines at concentrations achievable in vivo. Treosulfan had a greater effect on the inhibition of cell growth at equivalent concentrations compared to busulfan. The growth inhibitory effect of treosulfan at low concentrations was mainly due to a G2 cell cycle arrest, whereas at higher concentrations it was due to apoptosis. Apoptosis was induced at lower concentrations compared to busulfan. In contrast to busulfan, treosulfan induced cell death in an apoptosis-deficient cell line at concentrations achievable in vivo. In mice, treosulfan suppressed tumor growth at dosages of 2,500 and 3,000 mg/kg. Pharmacokinetic exposures of treosulfan in mice were similar to previous reports in human patients. At maximal tolerated dosages treosulfan had a higher anti-tumor activity than busulfan. CONCLUSIONS: Our results suggest that treosulfan has efficacy against Ewing's sarcoma cells in vitro and in mice. Therefore, controlled trials examining the role of treosulfan in patients with Ewing's sarcoma are warranted.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Bussulfano/análogos & derivados , Sarcoma de Ewing/tratamento farmacológico , Animais , Anexina A5/fisiologia , Antineoplásicos Alquilantes/farmacocinética , Apoptose/efeitos dos fármacos , Bisbenzimidazol , Western Blotting , Bussulfano/farmacocinética , Bussulfano/uso terapêutico , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes , Fase G1/efeitos dos fármacos , Indicadores e Reagentes , Injeções Intraperitoneais , Camundongos , Camundongos Nus , Sais de Tetrazólio , Tiazóis , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Apoptotic and necrotic cells expose phosphatidylserine (PS). This membrane modification ensures a swift recognition and uptake by phagocytes of the dying and dead cells. Annexin V (AxV) preferentially binds to anionic phospholipids and thereby, modulates the clearance process. First, we analyzed the influence of AxV on the immunogenicity of apoptotic cells. The addition to apoptotic cells of AxV prior to their injection into mice increased their immunogenicity significantly. Next, we studied the influence of endogenous AxV on the allogeneic reaction against apoptotic and necrotic cells. To preserve heat-labile, short-lived "danger signals," we induced necrosis by mechanical stress. Wild-type mice showed a strong, allogeneic delayed-type hypersensitivity (DTH) reaction. In contrast, AxV-deficient animals showed almost no allogeneic DTH reaction, indicating that endogenous AxV increases the immune response against dead cells. Furthermore, AxV-deficient macrophages had a higher immunosuppressive potential in vitro. Next, we analyzed the influence of AxV on chronic macrophage infection with HIV-1, known to expose PS on its surface. The infectivity in human macrophages of HIV-1 was reduced significantly in the presence of AxV. Finally, we show that AxV also blocked the in vitro uptake by macrophages of primary necrotic cells. Similar to apoptotic cells, necrotic cells generated by heat treatment displayed an anti-inflammatory activity. In contrast, mechanical stress-induced necrotic cells led to a decreased secretion of IL-10, indicating a more inflammatory potential. From the experiments presented above, we conclude that AxV influences the clearance of several PS-exposing particles such as viruses, dying, and dead cells.
Assuntos
Anexina A5/farmacologia , Anti-Inflamatórios/farmacologia , Imunidade Celular , Fatores Imunológicos/farmacologia , Animais , Anexina A5/genética , Anexina A5/fisiologia , Apoptose , Células Cultivadas , Citocinas/metabolismo , Feminino , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Humanos , Imunização , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Imunológicos , Necrose , Fagocitose/imunologia , Fosfatidilserinas/metabolismoRESUMO
Antiphospholipid syndrome is defined by the presence of thrombosis and/or obstetrical adverse events (≥3 recurrent early miscarriage or fetal death or a prematurity<34 weeks of gestation) associated with persistent antiphospholipid antibodies. The pregnancy outcome has been improved by the conventional treatment (aspirin 100mg/day with low molecular weight heparin [LMWH] from 30 to 75% of uncomplicated pregnancies. In PROMISSE study, 19% of pregnancies had at least one obstetrical adverse event despite treatment (maternal, fetal or neonatal complications) in relation with APS. In the European registry of babies born from APS mothers, maternal and foetal adverse events were observed in 13% of cases, with prematurity in 14% despite treatment. The presence of lupus erythematosus, a history of thrombosis, presence of lupus anticoagulant and APL triple positivity are considered as factors associated with unfavorable obstetrical outcome. Hydroxychloroquine (HCQ) has anti-inflammatory and anti-thrombotic properties. Studies in vitro have shown that HCQ is able to restore the placental expression of Annexin V, which has an anticoagulant effect and to prevent the placental injury induced by APL. HCQ used for lupus erythematosus decrease the thrombotic risk and its value for thrombotic APS has been raised in an open labelled French study. In European retrospective study, the addition of HCQ to conventional treatment improved refractory obstetrical APS. Its use during the pregnancy of patients with lupus erythematosus, the evidence of good safety during the pregnancy and follow-up of children born to mothers exposed to HCQ demonstrate an overall good safety profile for mothers and the fetus. This clinical trial is designed to assess the interest of the addition of hydroxychloroquine to conventional treatment in APS during the pregnancy.
Assuntos
Síndrome Antifosfolipídica/complicações , Síndrome Antifosfolipídica/tratamento farmacológico , Hidroxicloroquina/uso terapêutico , Complicações na Gravidez/tratamento farmacológico , Complicações na Gravidez/imunologia , Resultado da Gravidez , Aborto Habitual/imunologia , Aborto Habitual/prevenção & controle , Anexina A5/fisiologia , Aspirina/administração & dosagem , Quimioterapia Combinada , Feminino , Morte Fetal/etiologia , Morte Fetal/prevenção & controle , França , Heparina de Baixo Peso Molecular/administração & dosagem , Humanos , Hidroxicloroquina/administração & dosagem , Recém-Nascido , Placebos , Doenças Placentárias/tratamento farmacológico , Doenças Placentárias/imunologia , GravidezRESUMO
Annexin 5 is a Ca(2+)-binding protein, the function of which is poorly understood. Structural and electrophysiological studies have shown that annexin 5 can mediate Ca(2+) fluxes across phospholipid membranes in vitro [1]. There is, however, no direct evidence for the existence of annexin 5 Ca(2+) channels in living cells. Here, we show that annexin 5 inserts into phospholipid vesicle membranes at neutral pH in the presence of peroxide. We then used targeted gene disruption to explore the role of annexin 5 in peroxide-induced Ca(2+) signaling in DT40 pre-B cells. DT40 clones lacking annexin 5 exhibited normal Ca(2+) responses to both thapsigargin and B-cell receptor stimulation, but lacked the sustained phase of the response to peroxide. This late phase was due to Ca(2+) influx from the extracellular space, demonstrating that annexin 5 mediates a peroxide-induced Ca(2+) influx. Thus, peroxide induces annexin 5 membrane insertion in vitro, and peroxide-induced Ca(2+) entry in vivo in DT40 cells requires annexin 5. Our results are consistent with a role for annexin 5 either as a Ca(2+) channel, or as a signaling intermediate in the peroxide-induced Ca(2+)-influx pathway.
Assuntos
Anexina A5/fisiologia , Linfócitos B/metabolismo , Cálcio/metabolismo , Animais , Anexina A2/genética , Anexina A2/metabolismo , Anexina A5/genética , Anexina A5/metabolismo , Linfócitos B/efeitos dos fármacos , Cálcio/agonistas , Linhagem Celular , Galinhas , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Ácido Clorídrico/farmacologia , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Imunoglobulina M/farmacologia , Tapsigargina/farmacologia , Fatores de TempoRESUMO
Annexins are characterized by the ability to bind phospholipids of membranes in the presence of Ca2+. Annexin A5 represents a typical member of this protein family and is a natural occurring highly specific ligand for phosphatidylserine (PS). The exposure of PS is one major "eat me" signal for phagocytes of apoptotic and necrotic cells. Apoptotic cells are normally cleared via an anti-inflammatory pathway. In contrast, the uptake and removal of necrotic cells normally involves inflammation and an immune response. Interestingly, the lack of endogenous annexin A5 also leads to a reduced inflammatory potential of necrotic cells. Annexin A5 may interfere in vivo with the immunosuppressive effects of apoptotic cells since it preferentially binds PS with high affinity and inhibits apoptotic cell uptake by macrophages. In this review we focus on how defects in the clearance process can lead to chronic autoimmunity. Furthermore, the role of annexin A5 as important adjuvant for apoptotic cell-based tumour vaccines is discussed. The mechanism of how the immunogenicity of apoptotic cells can be restored by blocking their PS-dependent clearance is outlined in detail. Taken together, annexin A5 is an important modulator of the immune response against PS-exposing particles like apoptotic cells, necrotic cells, and certain viruses.
Assuntos
Anexina A5/fisiologia , Morte Celular/imunologia , Fagocitose/imunologia , Animais , Apoptose/imunologia , Humanos , Inflamação/fisiopatologia , Macrófagos/fisiologia , Necrose/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Fosfatidilserinas/fisiologiaRESUMO
Annexin A5 (AnxA5) exerts anti-inflammatory, anticoagulant and anti-apoptotic effects through its binding to cell surface expressed phosphatidylserine. We previously showed that AnxA5 can stabilize advanced atherosclerotic plaques by reducing macrophage infiltration. We now investigated the effects of AnxA5 administration on the onset of atherosclerosis development. Eight-week-old ApoE-/-mice were fed a western diet while being administered AnxA5 or control (M1234) for a total of 6 weeks. AnxA5 administration reduced plaque size in the aortic root as well as the aortic arch by 36% and 55% respectively. As determined by immunohistochemistry, administration of AnxA5 further stabilized plaque by reducing macrophage content and increasing smooth muscle cell content. Furthermore, the pre-treatment of HUVEC's with AnxA5 reduced monocyte adhesion under flow-conditions. Finally, AnxA5 administration results in a trend to reduced cell death more pronounced in the aortic arch than the aortic root. In conclusion, treatment with AnxA5 before the onset of atherosclerosis reduces plaque formation in a murine model of atherosclerosis in part by reducing apoptotic rates further to its beneficial effect on macrophage infiltration and activation.
Assuntos
Anexina A5/fisiologia , Apolipoproteínas E/genética , Placa Aterosclerótica/metabolismo , Animais , Anexina A5/genética , Apoptose , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: Annexin V, an abundant anticoagulant protein, has been proposed to exert its effects by self-assembling into highly ordered arrays on phospholipid membranes to form a protective anti-thrombotic shield at the cell surface. The protein exhibits very high-affinity calcium-dependent interactions with acidic phospholipid membranes, as well as specific binding to glycosaminoglycans (GAGs) such as heparin and heparan sulfate, a major component of cell surface proteoglycans. At present, there is no structural information to elucidate this interaction or the role it may play in annexin V function at the cell surface. RESULTS: We report the 1.9 A crystal structure of annexin V in complex with heparin-derived tetrasaccharides. This structure represents the first of a heparin oligosaccharide binding to a protein where calcium ions are essential for the interaction. Two distinct GAG binding sites are situated on opposite protein surfaces. Basic residues at each site were identified from the structure and site-directed mutants were prepared. The heparin binding properties of these mutants were measured by surface plasmon resonance. The results confirm the roles of these mutated residues in heparin binding, and the kinetic and thermodynamic data define the functionally distinct character of each distal binding surface. CONCLUSION: The annexin V molecule, as it self-assembles into an organized array on the membrane surface, can bind the heparan sulfate components of cell surface proteoglycans. A novel model is presented in which proteoglycan heparan sulfate could assist in the localization of annexin V to the cell surface membrane and/or stabilization of the entire molecular assembly to promote anticoagulation.
Assuntos
Anexina A5/química , Anexina A5/fisiologia , Membrana Celular/metabolismo , Heparina/química , Oligossacarídeos/química , Animais , Sítios de Ligação , Biotinilação , Cálcio/metabolismo , Membrana Celular/química , Cristalografia por Raios X , Elétrons , Cinética , Lipossomos/química , Modelos Químicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fosfolipídeos/metabolismo , Ratos , Proteínas Recombinantes/química , Estreptavidina/química , Ressonância de Plasmônio de Superfície , Suínos , Termodinâmica , Fatores de TempoRESUMO
Annexin V binds to phospholipids in a calcium-dependent manner and exhibits calcium channel activity in vitro. We prepared a variety of mutants yielding information about the structure-function relationship of the ion channel activity. All mutants were characterized by X-ray crystallography, electron microscopy and electrophysiological measurements. Their structures are insignificantly changed whereas their electrophysiological properties are drastically different. Glu95, located in the central hydrophilic pore of the molecule, is crucial for the ion selectivity filter as its exchange leads to reduced calcium and increased sodium conductance. The removal of Glu17, located on the protein surface and far from the ion conduction pathway, leads to the appearance of a second conductance level of 9 pS in addition to the conductance level of about 30 pS in the wild-type molecule. This was also the case for Glu78, which is part of a weak calcium binding site. The exchange of Glu17 and Glu78 produced a mutant retaining only the smaller conductance level. We conclude that these two residues influence the angle between the two halves of the molecule, which determines the diameter of the ion conduction pathway, thereby leading to the occurrence of a second conductance level.
Assuntos
Anexina A5/química , Anexina A5/fisiologia , Canais de Cálcio/química , Canais de Cálcio/fisiologia , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Anexina A5/ultraestrutura , Sequência de Bases , Cálcio/metabolismo , Canais de Cálcio/biossíntese , Clonagem Molecular , Cristalografia por Raios X/métodos , Eletrofisiologia/métodos , Humanos , Bicamadas Lipídicas , Potenciais da Membrana , Microscopia Eletrônica , Modelos Moleculares , Modelos Estruturais , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Mapeamento por Restrição , Sódio/metabolismoRESUMO
Antiphospholipid syndrome (APS) has been defined as a clinical and laboratory entity. Laboratory criteria include the presence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA), collectively termed as antiphospholipid antibodies (aPL). However, there has been a rising interest in antibodies against so-called protein cofactors, particularly in beta(2)-glycoprotein I. In the early 90s, annexins were considered as target antigens for aPL, but at present the exact role of antibodies against annexins (aANX) remains puzzling. This review is concerned with annexin V or annexin A5 (ANXA5), a widespread member of the annexin family, and antibodies directed towards it. We have endeavoured to summarise essential information about the detection of anti-annexin V antibodies (aANXA5) and their clinical relevance. This review has also brought together some relevant published data concerning the structure, physiological role and therapeutic potential of ANXA5.
Assuntos
Anexina A5/análise , Anexina A5/imunologia , Autoanticorpos/análise , Autoanticorpos/biossíntese , Aborto Espontâneo/imunologia , Anexina A5/fisiologia , Anexina A5/uso terapêutico , Síndrome Antifosfolipídica/imunologia , Feminino , Humanos , Gravidez , Trombose/imunologiaRESUMO
Annexins are soluble proteins that bind to biological membranes containing negatively charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. Annexin-A5 (AnxA5), the smallest member of the annexin family, presents unique properties of membrane binding and self-assembly into ordered two-dimensional (2D) arrays on membrane surfaces. We have previously reported that AnxA5 plays a central role in the machinery of membrane repair by enabling rapid resealing of plasma membrane disruption in murine perivascular cells. AnxA5 promotes membrane repair via the formation of a protective 2D bandage at membrane damaged site. Here, we review current knowledge on cell membrane repair and present recent findings on the role of AnxA5 in membrane resealing of human trophoblasts.