RESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
PURPOSE: The purpose of this paper is to study molecular imaging of apoptosis and necrosis, two key players in atherosclerosis instability, using a multimodal imaging approach combining single photon emission computed tomography (SPECT), positron emission tomography (PET), and computed tomography (CT). PROCEDURES: Collar-induced carotid atherosclerosis ApoE knockout mice were imaged with (99m)Tc-AnxAF568 SPECT-CT to study apoptosis and sequentially with PET-CT following (124)I-Hypericin ((124)I-Hyp) injection to visualize necrosis. RESULTS: SPECT depicted increased (99m)Tc-AnxAF568 uptake in both atherosclerotic carotid arteries, whereas our data suggest that this uptake is not merely apoptosis related. Although PET of (124)I-Hyp was hampered by the slow blood clearance in atherosclerotic mice, (124)I-Hyp was able to target necrosis in the atherosclerotic plaque. CONCLUSION: Both (99m)Tc-AnxAF568 and (124)I-Hyp uptake are increased in atherosclerotic carotid vasculature compared to control arteries. While apoptosis imaging remains challenging, necrosis imaging can be feasible after improving the biodistribution characteristics of the probe.
Asunto(s)
Apoptosis , Imagen Molecular/métodos , Imagen Multimodal , Placa Aterosclerótica/diagnóstico por imagen , Tomografía de Emisión de Positrones , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada por Rayos X , Animales , Anexina A5/metabolismo , Antracenos , Humanos , Ratones , Necrosis , Perileno/análogos & derivados , Placa Aterosclerótica/patología , Tecnecio , Distribución TisularRESUMEN
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.
Asunto(s)
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
Impaired efferocytosis has been shown to be associated with, and even to contribute to progression of, chronic inflammatory diseases such as atherosclerosis. Enhancing efferocytosis has been proposed as strategy to treat diseases involving inflammation. Here we present the strategy to increase 'eat me' signals on the surface of apoptotic cells by targeting cell surface-expressed phosphatidylserine (PS) with a variant of annexin A5 (Arg-Gly-Asp-annexin A5, RGD-anxA5) that has gained the function to interact with α(v)ß(3) receptors of the phagocyte. We describe design and characterization of RGD-anxA5 and show that introduction of RGD transforms anxA5 from an inhibitor into a stimulator of efferocytosis. RGD-anxA5 enhances engulfment of apoptotic cells by phorbol-12-myristate-13-acetate-stimulated THP-1 (human acute monocytic leukemia cell line) cells in vitro and resident peritoneal mouse macrophages in vivo. In addition, RGD-anxA5 augments secretion of interleukin-10 during efferocytosis in vivo, thereby possibly adding to an anti-inflammatory environment. We conclude that targeting cell surface-expressed PS is an attractive strategy for treatment of inflammatory diseases and that the rationally designed RGD-anxA5 is a promising therapeutic agent.
Asunto(s)
Anexina A5/farmacología , Oligopéptidos/farmacología , Fosfatidilserinas/metabolismo , Animales , Anexina A5/farmacocinética , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Adhesión Celular/fisiología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Células Jurkat , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Terapia Molecular Dirigida , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Oligopéptidos/farmacocinética , Fagocitosis/efectos de los fármacos , Fagocitosis/fisiología , Fosfatidilserinas/biosíntesisRESUMEN
Atherosclerosis still represents killer number one in industrialized nations, and is starting to have increased impact in developing countries. Atherosclerotic plaques are the net result of a complex interplay between vascular cholesterol deposition, inflammatory activity and extracellular matrix formation. The result is luminal narrowing of arteries, which may ultimately lead to compromised blood flow to essential body organs, most notoriously to the heart. Most of the cardiovascular events that are caused by atherosclerosis, such as acute myocardial infarction or stroke, are the result of a transition of so-called stable atherosclerotic plaques to vulnerable plaques, that are prone to rupture. The direct consequence of atherosclerotic plaque rupture is exposure of thrombogenic plaque constituents to the blood, leading to instant local thrombus formation. The formation of this localized thrombus may ultimately result in sudden obstruction of blood flow and consequent infarction of distal tissue. Clinical risk profiling methods, such as the Framingham and Procam risk scores, are reasonable predictors of myocardial infarction over a 10-year time-span. However, the challenge remains to identify those patients with a very high risk of suffering from myocardial infarction in the coming months. Imaging may provide the necessary diagnostic information to identify such individuals. The transition of stable atherosclerotic plaques to vulnerable plaques is typically heralded by inflammation, thinning of the overlying fibrous cap, and the presence of a large necrotic core. Apoptosis is linked to all of these features of plaque vulnerability, and may, therefore, provide uniquely useful targets for the identification of plaque vulnerability. In recent years, a number of molecular imaging technologies have been developed to image apoptosis, which will be discussed in this review. Further development of apoptosis imaging technologies may aid us in the years to come in the quest to identify patients with critical cardiovascular risks, to treat myocardial infarction in its imminent, instead of its evident phase.
Asunto(s)
Anexina A5/análisis , Apoptosis , Aterosclerosis/diagnóstico por imagen , Aterosclerosis/patología , Animales , Anexina A5/metabolismo , Aterosclerosis/metabolismo , Humanos , Tomografía de Emisión de Positrones , Coloración y Etiquetado , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
The surge in apoptosis research and the discovery of the phosphatidylserine binding properties of annexin A5 have propelled a tremendous interest in cell death detection technologies. In the past years, annexin A5 has evolved from an efficient assay for detection of apoptotic cells in vitro to an in vivo molecular imaging technology with potential clinical use. A second key discovery, the specific internalization properties of annexin A5, has opened the opportunity to use annexin A5 for therapeutic applications. Annexin A5-mediated internalization creates a novel therapeutic platform for targeted drug delivery and cell entry to treat various diseases, including cancer and cardiovascular disease.
Asunto(s)
Anexina A5/uso terapéutico , Animales , Anexina A5/metabolismo , Apoptosis , Enfermedades Cardiovasculares/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Humanos , Neoplasias/tratamiento farmacológico , Fosfatidilserinas/metabolismoRESUMEN
INTRODUCTION: Molecular imaging strives to visualise processes at the molecular and cellular level in vivo. Understanding these processes supports diagnosis and evaluation of therapeutic efficacy on an individual basis and thereby makes personalised medicine possible. APOPTOSIS AND MOLECULAR IMAGING: Apoptosis is a well-organised mode of cell suicide that plays a role in cardiovascular diseases (CVD). Apoptosis is associated with loss of cardiomyocytes following myocardial infarction, atherosclerotic plaque instability, congestive heart failure and allograft rejection of the transplanted heart. Thus, apoptosis constitutes an attractive target for molecular imaging of CVD. Our current knowledge about the molecular players and mechanisms underlying apoptosis offers a rich palette of potential molecular targets for molecular imaging. However, only a few have been successfully developed so far. AIMS: This review highlights aspects of the molecular machinery and biochemistry of apoptosis relevant to the development of molecular imaging probes. It surveys the role of apoptosis in four major areas of CVD and portrays the importance and future perspectives of apoptosis imaging. The annexin A5 imaging protocol is emphasised since it is the most advanced protocol to measure apoptosis in both preclinical and clinical studies.
Asunto(s)
Apoptosis , Enfermedades Cardiovasculares/diagnóstico , Diagnóstico por Imagen/métodos , Corazón/diagnóstico por imagen , Técnicas de Sonda Molecular , Miocardio/patología , Humanos , CintigrafíaRESUMEN
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.
Asunto(s)
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.
Asunto(s)
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
BACKGROUND: Increased systemic levels of endotoxin have been detected in human alcoholics and are thought to be derived from the gut. Although a 'leaky gut' is considered to be a necessary factor for alcohol-induced endotoxemia followed by chronic liver injury, the effects of low concentrations of ethanol on intestinal epithelial cells have not been fully understood. The aim of this study was to evaluate intestinal epithelial cell death induced by acute, low concentrations of ethanol in an in vitro system. METHODS: The human intestinal Caco-2 cell line was incubated with 0%, 5%, 10% ethanol for up to 3 h. Phosphatidylserine (PS) externalization, caspase-mediated cytokeratin 18 (CK18) cleavage, and DNA fragmentation were evaluated using flow cytometry. The caspase inhibitor zVAD-fmk was used to test the role of caspases in ethanol-induced cell death. RESULTS: Treatment with 5% and 10% ethanol for 3 h led to a gradual increase in PS externalization. Caspase-mediated CK18 was significantly enhanced as early as 1 h after 10% ethanol incubation, while DNA fragmentation was detected from 2 h onwards. Not only caspase activation but also both PS externalization and DNA fragmentation were completely prevented by pretreatment with the caspase inhibitor. CONCLUSIONS: Apoptotic cell death in confluent Caco-2 cells was induced by acute and low concentrations of ethanol. These results suggest that clinically achievable doses of ethanol impair intestinal barrier function by induction of apoptosis in intestinal epithelial cells. This impairment of the barrier function would allow endotoxin to enter the circulation and evoke hepatic inflammation.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Etanol/administración & dosificación , Intestinos/efectos de los fármacos , Solventes/administración & dosificación , Adenocarcinoma/metabolismo , Células CACO-2/efectos de los fármacos , Células CACO-2/metabolismo , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Neoplasias del Colon/metabolismo , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Células Epiteliales/enzimología , Humanos , Intestinos/citología , Intestinos/enzimología , Fosfatidilserinas/metabolismo , Células Tumorales CultivadasRESUMEN
Annexin A5 (AnxA5) is a protein with high affinity for phosphatidyl serine, a phospholipid exposed on the cell surface during apoptosis. This phenomenon has been used for determination of cell death after myocardial infarction. To evaluate the potential of (99m)Tc-AnxA5 for in vivo scintigraphy of apoptotic cells, the pharmacokinetics and imaging properties of two radiopharmaceuticals, (99m)Tc-(n-1-imino-4-mercaptobutyl)-AnxA5 (I-AnxA5) and (99m)Tc-(4,5-bis(thioacetamido)pentanoyl)-AnxA5 (B-AnxA5), were studied. I-AnxA5 was administered intravenously to seven patients and one healthy volunteer, and B-AnxA5 was administered to 12 patients. All patients in the pharmacokinetic study had myocardial disease. Additionally, imaging was performed in a patient with acute myocardial infarction, as well as in three patients with different malignancies. The plasma concentration, excretion and biodistribution of (99m)Tc-AnxA5 were measured, as well as levels of AnxA5 antigen. The kinetic data of both radiopharmaceuticals in plasma fitted a two-compartment model. Both preparations had similar half-lives, but a different distribution over the two compartments. Plasma levels of AnxA5 antigen showed a broad variation. Both radiopharmaceuticals accumulated in the kidney, liver and gut. B-AnxA5 was excreted significantly faster than I-AnxA5. Both compounds can be used for imaging of the head/neck region, the thorax and the extremities. B-AnxA5 has a faster clearance and a lower radiation dose. Imaging of apoptosis in the abdomen will be difficult with both radiopharmaceuticals, and especially with B-AnxA5 because of its faster appearance in the gut.
Asunto(s)
Anexina A5/farmacocinética , Cardiomiopatías/diagnóstico por imagen , Compuestos de Organotecnecio/farmacocinética , Radiofármacos/farmacocinética , Adulto , Anciano , Anciano de 80 o más Años , Anexina A5/sangre , Apoptosis , Disponibilidad Biológica , Neoplasias de la Mama/diagnóstico por imagen , Semivida , Humanos , Linfoma no Hodgkin/diagnóstico por imagen , Persona de Mediana Edad , Infarto del Miocardio/diagnóstico por imagen , Compuestos de Organotecnecio/sangre , Radiofármacos/sangre , Sarcoma/diagnóstico por imagen , Tomografía Computarizada de Emisión de Fotón Único/métodosRESUMEN
Annexin A5 has been proposed to be important for shielding of negatively charged phospholipids from blood, thereby preventing the binding of clotting factors. It has been suggested that antiphospholipid antibodies can disrupt the binding of annexin A5 from negatively phospholipid-containing surfaces, resulting in uncontrolled coagulation. If this hypothesis is correct, than the plasma levels of annexin A5 will be increased in patients with antiphospholipid antibodies. Therefore, we have measured plasma levels of annexin A5 of 175 patients with systemic lupus erythematosus (SLE), of which 104 had antiphospholipid antibodies and 23 patients had primary antiphospholipid syndrome. The annexin A5 levels were compared with the annexin A5 plasma levels measured in 23 patients with diabetes mellitus type 2 and 35 healthy volunteers. We found a significant increase of annexin A5 plasma levels in patients with SLE (median 6.7 ng mL(-1)) and primary antiphospholipid syndrome (median 7.1 ng mL(-1)) as compared to patients with diabetes mellitus type 2 (median 3.3 ng mL(-1)) and healthy volunteers (median 3.9 ng mL(-1)). However, no correlation was found with the presence of antiphospholipid antibodies or with a history of thromboembolic complications. Based on these observations, we conclude that displacement of annexin A5 from cellular surfaces by antiphospholipid antibodies is not a common mechanism in patients with antiphospholipid antibodies.
Asunto(s)
Anexinas/sangre , Anticuerpos Antifosfolípidos/sangre , Anexina A5 , Síndrome Antifosfolípido/sangre , Estudios de Casos y Controles , Reacciones Cruzadas , Diabetes Mellitus Tipo 2/sangre , Ensayo de Inmunoadsorción Enzimática/normas , Humanos , Lupus Eritematoso Sistémico/sangre , Estadísticas no Paramétricas , TromboemboliaRESUMEN
Programmed cell death plays a critical role in embryology, homeostasis and disease. However, until recently no non-invasive imaging modality has been able to visualize this process directly. Annexin A5 binds to cells undergoing programmed cell death. When labeling this protein, Annexin A5 becomes a tool for the detection of programmed cell death in vitro and in vivo. Labeled Annexin A5 has enabled our group and others to detect programmed cell death non-invasively in animals and patients. This review will highlight the development of this imaging modality in cellular and animal models. Furthermore, we will discuss Annexin A5 imaging in human disease. We will focus on the clinical applications and their relevance, limitations and future perspectives of non-invasive imaging of programmed cell death using labeled Annexin A5.
Asunto(s)
Anexina A5/farmacocinética , Apoptosis , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Radiofármacos/farmacocinética , Animales , Humanos , Ratones , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/metabolismo , Compuestos de Organotecnecio/farmacocinética , CintigrafíaRESUMEN
Cardiopulmonary bypass (CPB) is associated with a generalized hemostatic defect, in which platelet dysfunction seems to play a central role. The present study was designed to elucidate whether the potential procoagulant activity of platelets, detected as annexin V binding, was altered during coronary bypass surgery, using non-coated and heparin-coated extracorporeal circuits. Thirty patients undergoing elective coronary artery bypass grafting were prospectively randomized using either a standard untreated extracorporeal circuit (n = 15) or a heparin-treated extracorporeal circuit (n=15). Besides measurement of the procoagulant phospholipid activity, the mediastinal blood loss after surgery, and the blood transfusion requirements were also monitored. CPB induced a decrease in the percentage of activated platelets in whole blood, manifest directly after start of CPB, which was significantly attenuated using a non-treated system. Postoperatively, the percentage of activated platelets recovered in both systems, reaching a point of significance 24 hours after the operation, compared to the values 2 hours after the operation. The differences among the groups for mediastinal blood loss during the first 2 and 24 postoperative hours coincided with the differences in procoagulant phospholipid activity. Furthermore, there was no statistical difference among the groups for blood transfusion requirements. The platelets in both groups showed a significantly lower ability to generate ionomycin-induced procoagulant activity after blood-material interaction when compared to the baseline values. These observations are compatible with the notion that during CPB, irrespective of the heparin coating, platelets become modestly activated.
Asunto(s)
Anexina A5/metabolismo , Puente de Arteria Coronaria , Circulación Extracorporea/métodos , Heparina/administración & dosificación , Ionomicina/farmacología , Ionóforos/farmacología , Activación Plaquetaria/efectos de los fármacos , Anciano , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Circulación Extracorporea/instrumentación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Complicaciones PosoperatoriasRESUMEN
Maturation arrest and interference with selection are two well-documented effects of cyclosporin-A (CsA) on the thymus. We recently hypothesized that these effects are related and owing to the reduced T-cell receptor (TCR)-CD3 complex-mediated signal transduction in thymocytes upon CsA treatment. In this hypothesis, the maturation arrest is the result of the additional depletion of thymocytes that normally survive by positive selection, whereas the impaired self-tolerance induction is caused by an increased survival of thymocytes that normally undergo negative selection. In this view, it is anticipated that CsA differentially affects thymocyte apoptosis during in vivo thymocyte maturation. Indeed, we report in this study a strong increase in apoptotic cells in the thymic cortex on in situ analysis. Simultaneously, the number of apoptotic cells had decreased at the cortico-medullary zone which is held to be the site for negative selection. Rapamycin (Rapa) also interferes with thymocyte maturation by inhibiting cytokine-driven proliferation. Hence, Rapa preferentially affects the early maturational stages of thymocyte development and is considered not to alter thymocyte selection and subsequent apoptotic events. Indeed, the number of apoptotic events appears not to be altered. However, possibly owing to the decrease in cortical macrophages, the apoptotic cells revealed an atypical enumeration around blood vessels. Taken together, our results favour the hypothesis that the dominant effect of CsA on the thymus is the reduction of the TCR-CD3 complex-mediated signal transduction in thymocytes upon interaction with stromal cells. Furthermore, the preferential localization of apoptotic cells next to blood vessels upon Rapa administration may indicate that endothelial cells are a back-up system for the removal of apoptotic cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclosporina/farmacología , Inmunosupresores/farmacología , Linfocitos T/efectos de los fármacos , Animales , Atrofia , Diferenciación Celular/efectos de los fármacos , Supresión Clonal/efectos de los fármacos , Ciclosporina/antagonistas & inhibidores , Endotelio Vascular/fisiología , Femenino , Inmunosupresores/antagonistas & inhibidores , Modelos Inmunológicos , Ratas , Ratas Endogámicas Lew , Complejo Receptor-CD3 del Antígeno de Linfocito T/efectos de los fármacos , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Autotolerancia/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Organismos Libres de Patógenos Específicos , Linfocitos T/citología , Timo/efectos de los fármacos , Timo/patologíaRESUMEN
BACKGROUND AND OBJECTIVES: In this study we examined whether prestorage leucocyte reduction prevents the accumulation of bioreactive substances in red cell units. MATERIALS AND METHODS: Measurements were performed in the supernatants of buffy-coat-depleted (standard red cells) and leucocyte-reduced (filtered red cells) red cell units. The effect of storage was evaluated by taking repetitive samples up to 35 days after donation. We determined the concentrations of polymorphonuclear neutrophil (PMN)-derived bactericidal permeability increasing protein (BPI), defensins and annexin A5. In addition, leucocyte counts (using nageotte chamber) were performed on days 0 and 35. RESULTS: During storage, the concentrations of BPI, defensins and annexin A5 in standard red cells gradually increased. However, in the filtered red cells BPI and defensins were found in only a few samples, whereas the annexin A5 concentration in these units did not change during storage. Haemolysis data in both types of red cell components were similar at all time-points, except prestorage. Significant correlations were found between the release of BPI, defensins and annexin A5 into red cell units and the loss of leucocytes during storage. CONCLUSIONS: PMNs lose their membrane integrity during cold storage and release their contents into red cell components. Prestorage leucocyte reduction of red cell components prevents the accumulation of BPI, defensins and annexin A5.
Asunto(s)
Conservación de la Sangre , Proteínas Sanguíneas/metabolismo , Defensinas/metabolismo , Transfusión de Eritrocitos/normas , Leucocitos , Proteínas de la Membrana , Anexina A5 , Anexinas/análisis , Péptidos Catiónicos Antimicrobianos , Proteínas Sanguíneas/análisis , Separación Celular , Defensinas/análisis , Filtración , Humanos , Recuento de Leucocitos , Neutrófilos/química , Factores de TiempoRESUMEN
BACKGROUND: Primary endocardial tumours are rare, but may impose a difficult clinical problem. The definite diagnosis regarding the nature of the tumour is often made after surgery. This is due to the fact that current non-invasive imaging techniques are unable to inform us about the nature of the tumour. In addition, invasive techniques can not be used to obtain biological information of the tumour in these cases, because they carry a high risk of embolic complications. OBJECTIVE: To assess the possibility of a novel modality of imaging, molecular imaging, in the diagnosis of primary intracardiac tumours. METHODS: We evaluated two patients with a primary cardiac tumour. Prior to therapy, we infused human recombinant annexin-V Tc99-m and thallium 201. We used a dual isotope single photon emission computed tomography technique. This allowed us to obtain information about the relation between the anatomical position of the left ventricle and the uptake of the labelled annexin-V within the thoracic cavity. RESULTS: The patient with a malignant primary cardiac tumour showed uptake of labelled annexin-V within the area of the tumour. After surgery, the malignant nature was confirmed by histological analysis. The patient with a benignant intracardiac tumour showed no uptake of annexin-V within the area of the tumour. CONCLUSION: This novel imaging technique, molecular imaging, may be of help to differentiate non-invasively between a malignant and benignant primary intracardiac tumour.