Chris Reutelingsperger

A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.

In the early stages of apoptosis changes occur at the cell surface, which until now have remained difficult to recognize. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding protein with high affinity for PS. Hence this protein can be used as a sensitive probe for PS exposure upon the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, but occurs also during cell necrosis. The difference between these two forms of cell death is that during the initial stages of apoptosis the cell membrane remains intact, while at the very moment that necrosis occurs the cell membrane looses its integrity and becomes leaky. Therefore the measurement of Annexin V binding to the cell surface as indicative for apoptosis has to be performed in conjunction with a dye exclusion test to establish integrity of the cell membrane. This paper describes the results of such an assay, as obtained in cultured HSB-2 cells, rendered apoptotic by irradiation and in human lymphocytes, following dexamethasone treatment. Untreated and treated cells were evaluated for apoptosis by light microscopy, by measuring the amount of hypo-diploid cells using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish whether or not DNA fragmentation had occurred. Annexin V binding was assessed using bivariate FCM, and cell staining was evaluated with fluorescein isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The test described, discriminates intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing traditional tests the Annexin V assay is sensitive and easy to perform. The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity and permits measurements of the kinetics of apoptotic death in relation to the cell cycle. More extensive FCM will allow discrimination between different cell subpopulations, that may or may not be involved in the apoptotic process.

Annexin V‐Affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure

Apoptosis is a programmed, physiological mode of cell death that plays an important role in tissue homeostasis. Understanding of the basic mechanisms that underlie apoptosis will point to potentially new targets of therapeutic treatment of diseases that show an imbalance between cell proliferation and cell loss. In order to conduct such research, techniques and tools to reliably identify and enumerate death by apoptosis are essential. This review focuses on a novel technique to detect apoptosis by targeting for the loss of phospholipid asymmetry of the plasma membrane. It was recently shown that loss of plasma membrane asymmetry is an early event in apoptosis, independent of the cell type, resulting in the exposure of phosphatidylserine (PS) residues at the outer plasma membrane leaflet. Annexin V was shown to interact strongly and specifically with PS and can be used to detect apoptosis by targeting for the loss of plasma membrane asymmetry. Labeled annexin V can be applied both in flow cytometry and in light microscopy in both vital and fixed material by using appropriate protocols. The annexin V method is an extension to the current available methods. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses the novel annexin V-binding assay.

Flow cytometry of apoptotic cell death

The term apoptosis or programmed cell death defines a genetically encoded cell death program, which is morphologically and biochemically distinct from necrosis or accidental cell death. The characteristic morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events which is an integral part of physiological homeostasis. Techniques designed to identify, quantitate and characterize apoptosis are numerous, but flow cytometry (FCM) remains the methodology of choice to study the apoptotic cascade in relation to cell type, trigger and time. This review outlines the main stages of the apoptotic cascade together with current FCM methods. All FCM apoptosis assays described have a solid experimental basis and have been used successfully in basic research on molecular and biochemical mechanisms of apoptosis. In various clinical settings the ability to follow the apoptotic process in patient samples may offer the rationale for optimal treatment schedules.

A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture.

Early during the process of apoptosis, cells lose their phospholipid membrane asymmetry and expose phosphatidylserine (PS) at the cell surface while maintaining their plasma membrane integrity intact. This process can be monitored for suspended cell types by using annexin V-FITC, which is a Ca(2+)-dependent, phospholipid-binding protein with high affinity for PS, and flow cytometry. If adherent cell types are to be studied for this apoptosis-associated phenomenon, then a problem is encountered, in that specific membrane damage occurs during harvesting. In this paper, a flow cytometric-based method is described that allows the measurement of loss of phospholipid asymmetry during apoptosis of adherent cells in culture. The method relies on the phospholipid binding property of biotinylated annexin V. Furthermore, the use of this conjugate allows tricolor flow cytometric analysis of apoptosis. Employing the method to MR65 cells, which were initiated by olomoucine to enter apoptosis, it is shown that PS exposure occurs early after the onset of apoptosis and, at the prevalent time-resolution, that PS exposure is accompanied by loss of both cytokeratin and DNA. The annexin V+ cells appear as a characteristic sub-G1 peak in the DNA histogram.

Visualisation of cell death in vivo in patients with acute myocardial infarction

BACKGROUND In-vivo visualisation and quantification of the extent and time-frame of cell death after acute myocardial infarction would be of great interest. We studied in-vivo cell death in the hearts of patients with an acute myocardial infarction using imaging with technetium-99m-labelled annexin-V-a protein that binds to cells undergoing apoptosis. METHODS Seven patients with an acute myocardial infarction and one control were studied. All patients were treated by percutaneous transluminal coronary angioplasty (six primary and one rescue), resulting in thrombolysis in myocardial infarction (TIMI) III flow of the infarct-related artery. 2 h after reperfusion, 1 mg annexin-V labelled with 584 MBq Tc-99m was injected intravenously. Early (mean 3.4 h) and late (mean 20.5 h) single-photon-emission computed tomographic (SPECT) images of the heart were obtained. Routine myocardial resting-perfusion imaging was also done to verify infarct localisation. FINDINGS In six of the seven patients, increased uptake of Tc-99m-labelled annexin-V was seen in the infarct area of the heart on early and late SPECT images. No increased uptake was seen in the heart outside the infarct area. All patients with increased Tc-99m-labelled annexin-V uptake in the infarct area showed a matching perfusion defect. In a control individual, no increased uptake in the heart was seen. INTERPRETATION Increased uptake of Tc-99m-labelled annexin-V is present in the infarct area of patients with an acute myocardial infarction, suggesting that programmed cell death occurs in that area. The annexin-V imaging protocol might allow us to study the dynamics of reperfusion-induced cell death in the area at risk and may help to assess interventions that inhibit cell death in patients with an acute myocardial infarction.

Noninvasive detection of plaque instability with use of radiolabeled annexin A5 in patients with carotid-artery atherosclerosis.

To the Editor: Although progressive stenosis of the arterial lumen constitutes the basis for ischemic symptoms in atherosclerotic vascular disease, acute vascular events are for the most part assoc...

Targeting of apoptotic macrophages and experimental atheroma with radiolabeled annexin V: a technique with potential for noninvasive imaging of vulnerable plaque.

Background—Apoptosis is common in advanced human atheroma and contributes to plaque instability. Because annexin V has a high affinity for exposed phosphatidylserine on apoptotic cells, radiolabeled annexin V may be used for noninvasive detection of apoptosis in atherosclerotic lesions. Methods and Results—Atherosclerotic plaques were produced in 5 rabbits by deendothelialization of the infradiaphragmatic aorta followed by 12 weeks of cholesterol diet; 5 controls were studied without manipulation. Animals were injected with human recombinant annexin V labeled with technetium-99m before imaging. Aortas were explanted for ex vivo imaging, macroautoradiography, and histological characterization of plaque. Radiolabeled annexin V cleared rapidly from the circulation (T1/2, &agr; 9 and &bgr; 46 minutes). There was intense uptake of radiolabel within lesions by 2 hours; no uptake was seen in controls. The results were confirmed in the ex vivo imaging of the explanted aorta. Quantitative annexin uptake was 9.3-fold higher in lesion versus nonlesion areas; the lesion-to-blood ratio was 3.0±0.37. Annexin uptake paralleled lesion severity and macrophage burden; no correlation was observed with smooth muscle cells. DNA fragmentation staining of apoptotic nuclei was increased in advanced lesions with evolving necrotic cores, predominantly in macrophages; the uptake of radiolabel correlated with the apoptotic index. Conclusions—Because annexin V clears rapidly from blood and targets apoptotic macrophage population, it should constitute an attractive imaging agent for the noninvasive detection of unstable atherosclerotic plaques.

Novel Conformation-Specific Antibodies Against Matrix γ-Carboxyglutamic Acid (Gla) Protein: Undercarboxylated Matrix Gla Protein as Marker for Vascular Calcification

Objective—Matrix γ-carboxyglutamic acid (Gla) protein (MGP), a vitamin K–dependent protein, is a potent in vivo inhibitor of arterial calcification. We hypothesized that low endogenous production of MGP and impaired carboxylation of MGP may contribute to the development or the progression of vascular disease. Methods and Results—Novel conformation-specific antibodies against MGP were used for immunohistochemistry of healthy and sclerotic arteries. In healthy arteries, MGP was mainly displayed around the elastin fibers in the tunica media. The staining colocalized with that for carboxylated MGP, whereas undercarboxylated MGP (ucMGP) was not detected. In atherosclerotic arteries, ucMGP was found in the intima, where it was associated with vesicular structures. In Mönckeberg’s sclerosis of the media, ucMGP was localized around all areas of calcification. The results indicate that ucMGP is strongly associated with vascular calcification of different etiologies. In a separate study, serum MGP concentrations in a cohort of 172 subjects who had undergone percutaneous coronary intervention were significantly reduced compared with an apparently healthy population. Conclusions—These data show that impaired carboxylation of MGP is associated with intimal and medial vascular calcification and suggest the essentiality of the vitamin K modification to the function of MGP as an inhibitor of ectopic calcification.

Annexin V, the regulator of phosphatidylserine-catalyzed inflammation and coagulation during apoptosis

Abstract. Annexin V belongs to a family of phospholipid binding proteins, the Annexins. It binds in the presence of Ca2+-ions with high affinity to negatively charged phospholipids like phosphatidylserine (PS). On the basis of its protein structure and biological activity Annexin V is considered as a protein exhibiting its hitherto unknown function within the intracellular environment. One argument comes from the understanding that PS is predominantly located in membrane leaflets, which face the cytosol. However, recent findings show that each cell type has the molecular machinery to expose PS at its cell surface. This machinery is activated during the execution of apoptosis. Once PS is exposed at the cell surface it exhibits procoagulant and proinflammatory activities. Annexin V will bind to the PS-exposing apoptotic cell and can inhibit thereby the procoagulant and pro-inflammatory activities of the dying cell. These findings together with the presence of Annexin V in the extracellular space depict a novel (patho)physiological significance for Annexin V in vivo.

Cardiomyocyte Death Induced by Myocardial Ischemia and Reperfusion Measurement With Recombinant Human Annexin-V in a Mouse Model

IntroductionPhosphatidylserine (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 ResultsI/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. ConclusionsThese 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.