RESUMEN
Click here to view the article Letter to the Editor by S. Shetty et al.
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Anexina A5/sangre , Micropartículas Derivadas de Células/metabolismo , Insuficiencia Cardíaca/mortalidad , Biomarcadores/sangre , Insuficiencia Cardíaca/sangre , HumanosRESUMEN
BACKGROUND: Natriuretic peptides are currently used to predict mortality in patients with heart failure (HF). However, novel independent biomarkers are needed to improve risk stratification in these patients. We hypothesized that annexin A5 (anxA5) would be highly expressed by organs which are generally affected by HF and that circulating anxA5 levels would predict mortality in HF patients. METHODS: We prospectively determined the diagnostic value of anxA5, N-terminal pro-B-type natriuretic peptide (NT-proBNP), C-reactive protein (CRP) and estimated glomerular filtration rate (eGFR) to predict mortality in 180 HF patients during a median follow-up of 3.6 years. Studies were conducted with anxA5(-/-) mice to investigate the underlying mechanisms. RESULTS: AnxA5 levels were significantly elevated in HF patients compared to healthy control subjects. Cox regression analysis demonstrated that anxA5, NT-proBNP and eGFR all predict mortality independently. AnxA5 significantly improved the diagnostic efficiency of NT-proBNP alone (improvement of c-statistic from 0.662 to 0.705, P < 0.001) and also combined with eGFR and CRP (improvement of c-statistic from 0.675 to 0.738, P < 0.001) to predict mortality in the Cox regression model. Receiver operating characteristic curve analysis showed that anxA5 predicted 3-year survival (area under curve 0.708) with an optimal cut-off value of 2.24 ng mL(-1) . Using anxA5(-/-) mice, we demonstrated that anxA5 is highly expressed in organs that are often affected by HF including lung, kidney, liver and spleen. Lysis of these organs in vitro resulted in a marked and significant increase in anxA5 concentrations. CONCLUSION: AnxA5 improves the diagnostic efficiency of conventional biomarkers to predict mortality in HF patients. Whereas natriuretic peptides originate from the myocardium, high circulating anxA5 levels in patients with HF are likely to reflect peripheral organ damage secondary to HF.
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Anexina A5/sangre , Insuficiencia Cardíaca/mortalidad , Animales , Biomarcadores/sangre , Proteína C-Reactiva/análisis , Femenino , Predicción , Tasa de Filtración Glomerular , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Estudios Prospectivos , Análisis de RegresiónRESUMEN
Sex differences have been widely reported in neuroinflammatory disorders, focusing on the contributory role of estrogen. The microvascular endothelium of the brain is a critical component of the blood-brain barrier (BBB) and it is recognized as a major interface for communication between the periphery and the brain. As such, the cerebral capillary endothelium represents an important target for the peripheral estrogen neuroprotective functions, leading us to hypothesize that estrogen can limit BBB breakdown following the onset of peripheral inflammation. Comparison of male and female murine responses to peripheral LPS challenge revealed a short-term inflammation-induced deficit in BBB integrity in males that was not apparent in young females, but was notable in older, reproductively senescent females. Importantly, ovariectomy and hence estrogen loss recapitulated an aged phenotype in young females, which was reversible upon estradiol replacement. Using a well-established model of human cerebrovascular endothelial cells we investigated the effects of estradiol upon key barrier features, namely paracellular permeability, transendothelial electrical resistance, tight junction integrity and lymphocyte transmigration under basal and inflammatory conditions, modeled by treatment with TNFα and IFNγ. In all cases estradiol prevented inflammation-induced defects in barrier function, action mediated in large part through up-regulation of the central coordinator of tight junction integrity, annexin A1. The key role of this protein was then further confirmed in studies of human or murine annexin A1 genetic ablation models. Together, our data provide novel mechanisms for the protective effects of estrogen, and enhance our understanding of the beneficial role it plays in neurovascular/neuroimmune disease.
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Anexina A1/fisiología , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/metabolismo , Estradiol/fisiología , Inflamación/inmunología , Inflamación/fisiopatología , Linfocitos/fisiología , Animales , Anexina A1/administración & dosificación , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Estradiol/administración & dosificación , Femenino , Humanos , Inflamación/inducido químicamente , Lipopolisacáridos , Linfocitos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Endothelial cell (EC) apoptosis seems to play an important role in the pathophysiology of pulmonary arterial hypertension (PAH). We aimed to test the hypothesis that circulating anti-endothelial cell antibodies (AECA) of PAH patients induce EC apoptosis. Immunoglobulin (Ig)G was purified from sera of PAH patients (n = 26), patients with systemic lupus erythematosus (SLE) nephritis without PAH (n = 16), patients with systemic sclerosis (SSc) without PAH (n = 58) and healthy controls (n = 14). Human umbilical vein endothelial cells (HUVECs) were incubated with patient or healthy control IgG for 24 h. Thereafter, apoptosis was quantified by annexin A5 binding and hypoploid cell enumeration by flow cytometry. Furthermore, real-time cell electronic sensing (RT-CES™) technology was used to monitor the effects of purified IgG from patient and healthy control IgG on HUVECs. As demonstrated previously, IgG of AECA-positive SLE nephritis patients (n = 7) induced a higher percentage of apoptosis of HUVECs compared to IgG of AECA-negative SLE nephritis patients and healthy controls. Furthermore, IgG of AECA-positive SLE nephritis patients induced a marked decrease in cell index as assessed by RT-CES™ technology. IgG of AECA-positive PAH patients (n = 12) and SSc patients (n = 13) did not alter the percentage of HUVEC apoptosis or cell index compared to IgG of AECA-negative PAH and SSc patients and healthy controls. AECA-positive PAH patients, in contrast to SLE nephritis patients, do not have circulating IgG AECA that enhances apoptosis of HUVECsâ in vitro. Further studies should focus on other mechanisms by which AECA may enhance EC apoptosis in PAH, such as antibody-dependent cell-mediated cytotoxicity.
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Apoptosis/inmunología , Autoanticuerpos/inmunología , Células Endoteliales/metabolismo , Hipertensión Pulmonar/metabolismo , Inmunoglobulina G/inmunología , Adolescente , Adulto , Anciano , Autoanticuerpos/sangre , Células Endoteliales/inmunología , Hipertensión Pulmonar Primaria Familiar , Femenino , Humanos , Hipertensión Pulmonar/inmunología , Inmunoglobulina G/sangre , Nefritis Lúpica/sangre , Nefritis Lúpica/inmunología , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/inmunología , Adulto JovenRESUMEN
We report a novel real-time imaging model to visualize apoptotic membrane changes of single cardiomyocytes in the injured heart of the living mouse, using fluorescent labeled annexin-V. Annexin-V binds to externalized phosphatidylserine (PS) of cells undergoing programmed cell death. With high-magnification (x100-160) real-time imaging, we visualized the binding of annexin-V to single cardiomyocytes. Kinetic studies at the single-cell level revealed that cardiomyocytes started to bind annexin-V within minutes after reperfusion, following an ischemic period of 30 minutes. The amount of bound annexin-V increased rapidly and reached a maximum within 20-25 minutes. Caspase inhibitors decreased the number of annexin-V-positive cardiomyocytes and slowed down the rate of PS exposure of cardiomyocytes that still bound annexin-V. This technology to study cell biology in the natural environment will enhance knowledge of intracellular signaling pathways relevant for cell-death regulation and strategies to manipulate these pathways for therapeutic effect.
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Anexina A5/metabolismo , Apoptosis , Membrana Celular/patología , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Animales , Colorantes Fluorescentes/metabolismo , Procesamiento de Imagen Asistido por Computador/instrumentación , Cinética , Ratones , Microscopía Fluorescente/instrumentación , Unión ProteicaRESUMEN
Vitamin K was discovered early last century at the same time as the vitamin K-antagonists. For many years the role of vitamin K was solely ascribed to coagulation and coagulation was thought to be involved only at the venous blood side. This view has dramatically changed with the discovery of vitamin K-dependent proteins outside the coagulation cascade and the role of coagulation factors at the arterial side. Vitamin K-dependent proteins are involved in the regulation of vascular smooth muscle cell migration, apoptosis, and calcification. Vascular calcification has become an important independent predictor of cardiovascular disease. Vitamin K-antagonists induce inactivity of inhibitors of vascular calcification, leading to accelerated calcification. The involvement of vitamin K-dependent proteins such as MGP in vascular calcification make that calcification is amendable for intervention with high intake of vitamin K. This review focuses on the effect of vitamin K-dependent proteins in vascular disease.
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Anticoagulantes/metabolismo , Arteriopatías Oclusivas/metabolismo , Arterias/metabolismo , Factores de Coagulación Sanguínea/metabolismo , Proteínas Sanguíneas/metabolismo , Calcinosis/metabolismo , Vitamina K/metabolismo , Animales , Humanos , Modelos CardiovascularesRESUMEN
Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Transmembrane protein carbonic anhydrase IX (CAIX) is hypoxia-induced and can be shed into the circulation as soluble CAIX (sCAIX). As plaque macrophages are hypoxic, we hypothesized a role for CAIX in macrophage function, and as biomarker of hypoxic plaque burden and CVD. As tumor patients with probable CVD are treated with CAIX inhibitors, this study will shed light on their safety profile. CAIX co-localized with macrophages (CD68) and hypoxia (pimonidazole), and correlated with lipid core size and pro-inflammatory iNOS+ macrophages in unstable human carotid artery plaques. Although elevated pH and reduced lactate levels in culture medium of CAIX knock-out (CAIXko) macrophages confirmed its role as pH-regulator, only spare respiratory capacity of CAIXko macrophages was reduced. Proliferation, apoptosis, lipid uptake and expression of pro- and anti-inflammatory genes were not altered. Plasma sCAIX levels and plaque-resident CAIX were below the detection threshold in 50 and 90% of asymptomatic and symptomatic cases, respectively, while detectable levels did not associate with primary or secondary events, or intraplaque hemorrhage. Initial findings show that CAIX deficiency interferes with macrophage metabolism. Despite a correlation with inflammatory macrophages, plaque-resident and sCAIX expression levels are too low to serve as biomarkers of future CVD.
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Antígenos de Neoplasias/fisiología , Anhidrasa Carbónica IX/fisiología , Enfermedades Cardiovasculares , Macrófagos/metabolismo , Anciano , Animales , Antígenos de Neoplasias/genética , Aterosclerosis/diagnóstico , Aterosclerosis/genética , Aterosclerosis/metabolismo , Biomarcadores/metabolismo , Anhidrasa Carbónica IX/genética , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Células Cultivadas , Estudios de Cohortes , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos , Proteínas Proto-Oncogénicas c-abl/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Anexina A5/metabolismo , Biomarcadores , Ciclo Celular , Proteína Ligando Fas , Humanos , Leucemia-Linfoma de Células T del Adulto/patología , Glicoproteínas de Membrana/fisiología , Ratones , Modelos Biológicos , Neutrófilos/metabolismo , Fagocitosis , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Recombinantes de Fusión/biosíntesis , Timo/citología , Transfección , Células Tumorales Cultivadas , Receptor fas/fisiologíaRESUMEN
Efferocytosis, the clearing of dead or dying cells from living tissues, is a highly programmed, vital process to maintain the healthy functioning of every organism. Disorders of efferocytosis have been linked to several chronic diseases including atherosclerosis and auto-immune diseases. To date several different assays to determine phagocytosis, using microscopy or FACS analysis with labelled targets, have been developed. However, many of these are unable to differentiate between cells that have truly been phagocytosed and those still present on the surface of the macrophages hindering exact assessment of efferocytotic capacity. We herein describe AnxA5-pHrodo and its negative control M1234-pHrodo as new molecular probes to measure in vitro as well as ex-vivo efferocytotic capacity.
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Anexina A5/metabolismo , Fagocitosis/fisiología , Animales , Apoptosis/fisiología , Aterosclerosis/metabolismo , Línea Celular , Humanos , Células Jurkat , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Sondas Moleculares/metabolismoRESUMEN
BACKGROUND: Blockade of the thrombin receptors protease-activated receptor (PAR)1 and PAR4 with pepducins, cell-penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen-induced externalization of phosphatidylserine (PS) at the platelet plasma membrane. OBJECTIVE: In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)-initiated thrombin generation in platelet-rich plasma (PRP) and the interaction between PAR4 pepducin-loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data. RESULTS: Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF-initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin-induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus. CONCLUSIONS: Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet-platelet interactions.
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Anticoagulantes/farmacología , Plaquetas/efectos de los fármacos , Fibrinolíticos/farmacología , Lipoproteínas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Proteinasa-Activados/antagonistas & inhibidores , Animales , Anticoagulantes/metabolismo , Anticoagulantes/uso terapéutico , Plaquetas/metabolismo , Arteria Carótida Común/cirugía , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fibrinolíticos/metabolismo , Fibrinolíticos/uso terapéutico , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Técnicas In Vitro , Lipoproteínas/metabolismo , Lipoproteínas/uso terapéutico , Masculino , Ratones , Microscopía por Video , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/metabolismo , Inhibidores de Agregación Plaquetaria/uso terapéutico , Receptor PAR-1/antagonistas & inhibidores , Receptores Proteinasa-Activados/metabolismo , Receptores de Trombina/antagonistas & inhibidores , Trombina/metabolismo , Tromboplastina/metabolismo , Trombosis/sangre , Trombosis/metabolismo , Trombosis/prevención & control , Factores de TiempoRESUMEN
BACKGROUND: Apoptosis induces cellular membrane changes that are thought to be linked to thrombotic processes, for example, surface exposure of procoagulant phosphatidylserine (PtdSer), upregulation of tissue factor (TF), and microvesicle formation. The latter, though, could downregulate this cellular response by shedding prothrombotic membrane elements, for example, integrins and TF. To test this hypothesis, etoposide-treated, apoptotic, monocytic cells (human monocytic leukemia cell line [THP-1]) were examined for rolling and adhesion on adherent platelets and for TF expression. METHODS AND RESULTS: Etoposide treatment did not result in a significant change in TF antigen expression. However, TF activity, measured in a continuous factor Xa generation assay, was increased fivefold concomitantly with increased exposure of PtdSer. Laminar flow adhesion assays specific for interaction between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) revealed that in contrast to non-treated cells, apoptotic cells did not roll or firmly attach on adherent platelets. Lack of apoptotic THP-1 platelet interaction could be attributed to both a loss of cell surface-expressed PSGL-1 and loss of functional PSGL-1 as a result of disruption of the binding of PSGL-1 with the cytoskeleton. CONCLUSION: Etoposide-induced apoptosis in THP-1 cells evokes a procoagulant response by increasing TF activity associated with an increased PtdSer exposure. However, in contrast to TF, PSGL-1 shedding and loss of function, makes that apoptotic monocytes are unlikely involved in a thrombotic action because of their inability to adhere to an injured vessel wall or developing thrombus.
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Apoptosis/fisiología , Glicoproteínas de Membrana/deficiencia , Monocitos/fisiología , Apoptosis/efectos de los fármacos , Adhesión Celular , Línea Celular Tumoral , Etopósido/farmacología , Humanos , Rodamiento de Leucocito , Glicoproteínas de Membrana/fisiología , Monocitos/citología , Fosfatidilserinas/metabolismo , Tromboplastina/análisis , Trombosis/etiologíaRESUMEN
Lysophospholipase-transacylase (lysolecithin acylhydrolase, EC 3.1.1.5) from rat lung catalyzes the transfer of acyl groups from lysophosphatidylcholine to either water or another molecule of lysophosphatidylcholine. Studies on the substrate specificity of the purified enzyme showed that a phosphate group in the substrate is essential for enzymatic activity; monoacylglycerol is not hydrolyzed, nor does it serve as an acceptor of acyl groups. The influence of the acyl chain in lysophosphatidylcholine was investigated by using mixtures of differently labelled lysophosphatidylcholine species, or by studying the transfer of [1-14C]Palmitate from [1-14C]palmitoylpropane (1,3)diol-phosphocholine to various 1-acyl-sn-glycero-3-phosphocholines. Lysophosphatidylcholines with acyl chains comprised of ten or more C-atoms were found to serve as acyl acceptors. This finding was used to determine the action of the enzyme on 1-[1-14C]auroyl- and 1[1-14C]myristoyl-sn-glycero-3-phosphocholine both below and above the critical micelle concentration of the substrate. Monomeric substrate was effectively hydrolyzed, but the transacylase activity of the enzyme was only expressed when substrate micelles were present. Likewise, no transacylase activity was found when lysophosphatidylcholine was embedded in liposomal membranes prepared from lung total lipids. These findings, which persist with crude enzyme preparations (100 000 x g supernatant), are discussed in relation to the putative function of the lysophospholipase-transacylase in the synthesis of disaturated phosphatidylcholine in lung.
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Aciltransferasas/metabolismo , Pulmón/enzimología , Lisofosfolipasa/metabolismo , Fosfolipasas/metabolismo , Animales , Radioisótopos de Carbono , Cinética , Liposomas , Lisofosfatidilcolinas , Ácidos Palmíticos , Técnica de Dilución de Radioisótopos , Ratas , Especificidad por SustratoRESUMEN
Interactions between human platelets and human umbilical vein endothelial cells (HUVEC) were studied by monitoring changes in cytosolic [Ca2+]i in both cell types. Confluent monolayers of Fura-2-loaded HUVEC, grown on gelatin-coated coverslips, responded to repeated addition of a suspension of unstimulated platelets by transient increases in cytosolic [Ca2+]i. These platelet-evoked Ca2+ responses were not caused by products secreted from the platelets and were insensitive to inhibitors of platelet activation and/or platelet aggregation. The platelet-evoked rises in [Ca2+]i in endothelial cells, similarly as the thrombin-evoked rises, were blocked by preincubation of HUVEC with the phospholipase C inhibitor U73122 or the Ca2+ influx blocker Ni2+. In contrast, treatment with the protein tyrosine kinase inhibitor genistein was without effect. Video imaging experiments, in which the fluorescence signal was analysed from the individual cells of an endothelial monolayer, showed that only 2-20% of the cells, scattered over the monolayer, responded to the addition of platelets by a transient increase in [Ca2+]i, whereas most of the cells responded to thrombin. This leads to the conclusion that unstimulated platelets can activate HUVEC putatively by mechanical interaction with individual endothelial cells in the monolayer.
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Plaquetas/fisiología , Calcio/metabolismo , Comunicación Celular/fisiología , Endotelio Vascular/metabolismo , Plaquetas/citología , Células Cultivadas , Quelantes , Citosol/metabolismo , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Fura-2 , Humanos , Ionomicina/farmacología , Ionóforos/farmacología , Níquel/farmacología , Pirrolidinonas/farmacología , Trombina/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/fisiología , Venas Umbilicales/citologíaRESUMEN
INTRODUCTION: Phosphatidylserine (PS) externalization is regarded as one of the earliest hallmarks of cells undergoing programmed cell death. We studied the use of labeled human recombinant annexin-V, a protein selectively binding to PS, to detect cardiomyocyte death in an in vivo mouse model of cardiac ischemia and reperfusion (I/R). METHODS AND RESULTS: I/R was induced in mouse hearts by ligation and subsequent release of a suture around the left anterior descending coronary artery. Annexin-V (25 mg/kg) fused to a marker molecule was injected intra-arterially 30 minutes before euthanasia. After 15 minutes of ischemia followed by 30 minutes of reperfusion, 1.4+/-1. 2% (mean+/-SD) of the cardiomyocytes in the area at risk were annexin-V positive (n=6). This increased to 11.4+/-1.9% after 15 minutes of ischemia followed by 90 minutes of reperfusion (n=7) and to 20.2+/-3.3% after 30 minutes of ischemia followed by 90 minutes of reperfusion (n=7). In control mice, including those injected with annexin-V at the binding site of PS, no annexin-V-positive cells were observed. DNA gel electrophoresis showed typical laddering starting after 15 minutes of ischemia followed by 30 minutes of reperfusion, suggesting activation of the cell death program. Intervention in the cell death program by pretreatment with a novel Na(+)-H(+) exchange inhibitor substantially decreased annexin-V-positive cardiomyocytes from 20.2% to 2.2% in mice after 30 minutes of ischemia followed by 90 minutes of reperfusion. CONCLUSIONS: These data suggest that labeled annexin-V is useful for in situ detection of cell death in an in vivo model of I/R in the heart and for the evaluation of cell death-blocking strategies.
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Anexina A5/análisis , Apoptosis/fisiología , Corazón/fisiopatología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Animales , Circulación Cerebrovascular/fisiología , Modelos Animales de Enfermedad , Ratones , Factores de TiempoRESUMEN
The distribution of phospholipids across the two leaflets of the plasma membrane is important for many cellular processes including phagocytosis and hemostasis. In the present study we investigated the in vivo plasma membrane distribution of the aminophospholipid phosphatidylserine in mouse embryos with a novel technique employing Annexin V, a Ca2+ dependent phosphatidylserine binding protein, conjugated to fluorescein isothiocyanate and biotin. Annexin V directly applied to cryostat sections labeled the plasma membrane of all cells at the interface. In contrast, Annexin V injected intracardially into viable mouse embryos labeled almost exclusively apoptotic cells. These apoptotic cells were visible in all tissues and derived from all germ layers. Our experiments demonstrate that phosphatidylserine is asymmetrically distributed between the two leaflets of the plasma membrane in virtually all cell types in vivo and that this asymmetry is lost early during apoptosis.
RESUMEN
In the last decade, apoptosis (or programmed cell death) has become appreciated as an important process in the development of the cardiovascular system. Moreover, apoptosis contributes to the adaptation of the system to the environment. We are at the beginning of understanding its relevance to cardiovascular physiology and pathology. This understanding forms the key to implement apoptosis in diagnosis and therapy of cardiovascular diseases. New avenues for pharmacological intervention are expected to arise from the synergy of our knowledge about the molecular mechanisms of apoptosis, and how apoptosis integrates in the complex environment of the cardiovascular tissue. The latter strongly depends on techniques to measure apoptosis. Currently, we are facing a relative paucity in available techniques, covering both specificity and sensitivity, and furthermore allowing quantitative analysis, preferably in combination with morphology. This field, however, is rapidly evolving and is fed by the expanding knowledge about the molecular mechanisms of apoptosis. In this paper we will briefly review the available techniques to detect and/or quantify apoptosis. These methods are based on the analysis of cellular morphology, either by light- or electron microscopy, DNA fragmentation (TdT-mediated X-dUTP nick end labeling or in situ nick end labeling), or cytoplasmic and membrane changes. Furthermore, the advantages and limitations of these techniques for their use in cardiovascular research will be outlined. In the text we will refer to available reviews and protocols which discuss the techniques in more detail. The main part of this article will, however, focus on a recently introduced technique, the Annexin V-based apoptosis detection assay. The principle, characteristics, pro's and contra's of this new apoptosis detection assay will be discussed.
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Anexina A5/análisis , Apoptosis , Enfermedades Cardiovasculares/fisiopatología , Miocardio/metabolismo , Animales , Biomarcadores/análisis , Calcio/análisis , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Caspasas/análisis , Membrana Celular/metabolismo , Citoplasma/metabolismo , Fragmentación del ADN , Citometría de Flujo , Humanos , Etiquetado Corte-Fin in Situ , Microscopía Electrónica , Mitocondrias Cardíacas/metabolismo , Miocardio/patología , Miocardio/ultraestructura , Fosfatidilserinas/metabolismoRESUMEN
Cell surface exposure of phosphatidylserine (PS) during apoptosis serves recognition and removal of the dying cell by phagocytes. Loss of phospholipid asymmetry and PS exposure is investigated by immunocytochemistry and related to morphological changes. Loss of membrane asymmetry was determined on dexamethasone-treated rat thymocytes using the PS specific probe annexin V. Thymocytes incubated in the presence of dexamethasone were studied in time series during the execution of the apoptotic program. Thymocytes first start to expose PS at their cell surface. At this initial stage the barrier function of the plasma membrane remains intact. At a later stage the plasma membrane becomes leaky for compounds like propidium iodide and subsequently the cell disintegrates into apoptotic bodies. Microscopical evaluation of dexamethasone-treated thymocytes showed that the cells with an apoptotic morphology all bound annexin V. The cells with a normal viable morphology lacked annexin V binding except for those cells that started to shed small vesicles. These vesicles were positive for annexin V, indicating a local disturbance of the phospholipid asymmetry. The local exposure of PS is considered to be a very early event of apoptosis, preceding the full sequence of morphological changes at the ultrastructural level.
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Apoptosis , Núcleo Celular/ultraestructura , Fosfatidilserinas/metabolismo , Animales , Anexina A5/metabolismo , Femenino , Citometría de Flujo , Fluoresceína-5-Isotiocianato/metabolismo , Cinética , Microscopía Inmunoelectrónica , Propidio , Ratas , Ratas Endogámicas Lew , TimoRESUMEN
The potency of annexin V to transport Ca2+ ions across phospholipid membranes was investigated, using large unilamellar phospholipid vesicles loaded with the Ca2+ indicator fura-2. It was demonstrated that annexin V leaves the vesicle membranes intact when added in the presence of 1 mM Ca2+. However, if the vesicles were first incubated with annexin V in the absence of Ca2+, subsequent addition of Ca2+ produced a fluorescence signal due to binding of Ca2+ to fura-2. Centrifugation of the vesicle suspension immediately thereafter showed that this signal originated from the supernatant and not from the sedimented vesicles. Our results show that annexin V causes loss of vesicle integrity in the absence of Ca2+, and leakage of trapped fura-2, rather than inward Ca2+ transport. Bovine serum albumin or Ca2+ concentrations higher than 2.5 mM also caused such fura-2 leakage. Apparently, calcium-dependent binding of annexin V to the membrane prevents aspecific membrane damage caused by this protein.
Asunto(s)
Anexina A5/fisiología , Calcio/metabolismo , Membrana Celular/metabolismo , Fosfolípidos/metabolismo , Canales de Calcio/metabolismo , Humanos , Técnicas In Vitro , Lípidos de la Membrana/metabolismoRESUMEN
The expression of Annexins V and VIII by human lung, liver, kidney, skin, heart, uterus, spleen and skeletal muscle was investigated by ELISA. All investigated tissues contained Annexin V. Its level varied with the tissue from around 5 microgram (skin) to approximately 120 micrograms (spleen) per g of wet tissue. Contradistinctionally Annexin VIII expression was less ubiquitous and less abundant. Only lung, skin, liver, and kidney expressed Annexin VIII. Its levels were approximately 100-fold less then the Annexin V levels. Immunohistochemical analysis of lung sections revealed Annexin VIII presence exclusively in the endothelia. Annexin V and VIII levels of cultured human umbilical vein endothelial cells, human arterial smooth muscle cells, human lung fibroblasts and HeLa cells were measured by ELISA. All cell types expressed Annexin V whereas only HeLa cells had detectable levels of Annexin VIII. The results indicate a tissue specific expression of Annexin VIII by lung endothelium, suggesting a highly specialised function.
Asunto(s)
Anexina A5/metabolismo , Anexinas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunohistoquímica , Pulmón/metabolismo , Distribución TisularRESUMEN
Phospholipase A2 (PLA2)-mediated hydrolysis of membrane phospholipids was measured by ellipsometry, and the inhibition of this process by annexin V was studied. Planar membranes, consisting of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (PC/PE/PS; 54:33:13, on molar basis), were degraded by pancreatic PLA2, and the rate of hydrolysis was limited to about 0.7%/min. The influence of graded coverage of the membrane with annexin V was studied. The degree of PLA2 inhibition was nonlinearly related to the amount of membrane-bound annexin V, and binding of only 12% and 54% of full membrane coverage resulted in, respectively, 50% and 93% inhibition. These findings indicate that the inhibition of PLA2-mediated hydrolysis by annexin V cannot be simply explained by shielding of phospholipid substrates from the enzyme. Moreover, the present results leave room for a role of endogenous annexin V in regulating phospholipid turnover in the plasma membrane of parenchymal cells such as cardiomyocytes.