Cardiovascular Research

Cardiovascular Research 2000 45(3):549-559; doi:10.1016/S0008-6363(99)00396-X
© 2000 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Articles by van Heerde, W. L Articles by Reutelingsperger, C. P.M Search for Related Content PubMed PubMed Citation Articles by van Heerde, W. L Articles by Reutelingsperger, C. P.M Social Bookmarking          What's this?

Copyright © 2000, European Society of Cardiology

Markers of apoptosis in cardiovascular tissues

focus on Annexin V

Waander L van Heerde^*, Saskia Robert-Offerman, Ewald Dumont, Leo Hofstra, Pieter A Doevendans, Jos F.M Smits, Mat J.A.P Daemen and Chris P.M Reutelingsperger

Departments of Biochemistry, Pathology, Pharmacology and Cardiology, Cardiovascular Research Institute Maastricht, University of Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands

^* Corresponding author. Tel.: +31-43-388-1674; fax: +31-43-367-0988 WL.vanHeerde{at}bioch.unimaas.nl

Received 23 July 1999; accepted 15 October 1999

	Abstract

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
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.

KEYWORDS Apoptosis; Atherosclerosis; Cardiomyopathy; Heart failure; Ischemia

	1 Introduction

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
Over the last 10 years, there has been a steep increase in research into apoptosis, mainly because it represents a new concept of how multicellular organisms, ranging from worms to mammals, regulate their cell numbers. In contrast to this recent interest, this concept depicts a phylogenetic conserved form of cell death, which turned out to be crucial for life in many ways. It became clear that apoptosis is a process, which is accurately orchestrated and organized inside the cell by gene products. To date, it is accepted that every cell type harbors the machinery to commit suicide by apoptosis. Subsequently, it is acknowledged that apoptosis plays a crucial role in homeostasis and pathology. As such, apoptosis has also been recognized as a key process in the cardiovascular system. It is an important contributor to the intact and complete development of the cardiovascular system [1] and to the adaptation of the cardiovascular system to its continuously changing demands (e.g. stress, sports, and illness). Moreover, apoptosis not only turned out to be an important feature in (sub)acute cardiovascular incidence like myocardial infarction, hypertension and heart failure, but also in a broad spectrum of other cardiovascular diseases (Table 1). Increasing knowledge of the apoptotic pathway will help to understand its regulation and importance for the cardiovascular system. It may also lead to new therapeutic interventions in cardiovascular diseases.

View this table: [in this window] [in a new window]   Table 1 Examples of cardiovascular entities in which apoptosis have been reported

 
The molecular biology and biochemistry of the apoptotic death machinery are far from being completely resolved. The apoptotic process shows great diversity in the signaling pathways by which it is induced in various cell types. Beyond this diversity, three functionally distinct phases of apoptosis, common to all cell types, can be distinguished [44,45]. First, the initiation phase can be induced by death inducing signals, like Fas ligand and tumor necrosis factor (TNF) [46] a lack of growth and/or survival signals, or DNA damage [47], which may devise the cell to prepare for suicide. This preparation may proceed in a manner, which involves a subclass of proteases, the so-called upstream or decision caspases [48]. The initiation phase will result in the activation of the second more general decision phase, in which the cell is still able to make the decision to live. This phase is characterized, in most cases, by the involvement of the mitochondrion [49,50]. This organelle provides the molecular links between the upstream initiation phase and the downstream execution phase, by releasing apoptosis inducing factor [51], cytochrome c [52], and procaspases 2, 3 and 9 [53,54]. When the cell is committed to die, and thus the point of no return has been passed, the third phase the execution phase is activated. This phase is characterized by the activation of the downstream or effector caspases [48], which subsequently orchestrate a sequence of events by their hierarchical activation [55]. These events include loss of cell junctions, cell shrinkage, chromatin condensation and margination, nuclear pyknosis and fragmentation, membrane blebbing, and disassembly of the cell into membrane-enclosed vesicles (apoptotic bodies) [56]. Several events have been identified on the biochemical level, including the degradation of DNA into fragments of multiples of 200 base pairs, the proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and cytoskeleton components, and the cell surface exposure of phosphatidylserine (PS).

	2 Techniques to detect apoptosis

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
The original techniques to measure apoptosis employed the early definition of apoptosis based on morphology. During the last decade, techniques have been extended to biochemistry, molecular biology and immunology due to our expanding knowledge about the molecular mechanisms of apoptosis (see also Table 1). Despite our increasing insight in the initiation and decision phases of apoptosis, most of these techniques are based on what is happening during the execution phase (Table 2). In the next sections the different techniques will be briefly outlined. For more details and protocols the reader is referred to the user's guide: Techniques in apoptosis [57].

View this table: [in this window] [in a new window]   Table 2 Overview of techniques used to detect apoptosis

 

	3 Morphological changes

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
3.1 Light microscopy (LM)
The earliest technique to detect apoptosis, and so far still the golden standard, is to study the morphology of the cell by LM. Membrane blebbing, nuclear pyknosis and fragmentation can thus be visualized on haematoxylin and eosin stained histological sections. A disadvantage of LM, however, is its low sensitivity especially, since apoptotic cells are rapidly removed from the tissues by phagocytosis, which can be completed within 30–60 min following the onset of apoptosis. In myocardium, cells with apoptotic morphology have only been observed incidentally. Hence, it is very well possible that LM reveals only the tip of the iceberg [56,58].

3.2 Electron microscopy (EM)
EM has the great advantage of its high specificity. Morphological changes can be detected on a subcellular level, thereby increasing sensitivity and specificity as compared to LM. The cumbersome workup of the material and the limited number of cells that can be studied, make it unsuitable for routine use. Therefore, EM is considered to be a valuable, specific and sensitive, but merely a qualitative methodology, for the detection of apoptosis [59].

3.3 Flow cytometry (FC)
FC enables the analysis of a relatively large number of cells on a per cell basis in a short period of time. Assay-required manipulation of cells is in most cases restricted to a minimum. As the apoptotic cell shrinks (in contrast to the necrotic cell which shows swelling) and subsequently condenses, the forward scatter decreases and sideward scatter slightly increases [60]. FC can be performed on cultured nonadherent as well as adherent cells. In general, cells have to be in suspension for analysis. This feature limits its applicability as a routine technique. FC is therefore an excellent tool for in vitro studies, in cardiovascular research, but not suitable for the detection of apoptosis in tissues, since it lacks the histological context.

	4 Cytoplasmic changes

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
The apoptotic process causes a dramatic change in the biochemistry of the cytoplasm. Some cytoplasmic parameters have been found to be useful markers for the detection of the apoptotic process. These include activated enzymes like caspases, "de novo" antigens, which arise from proteolytic action of caspases, Ca²⁺-ions, and mitochondrial proteins, which are translocated from the mitochondrion into the cytosolic compartment.

4.1 Caspase activity
Caspases constitute a family of cysteine proteases that cleave target proteins at an aspartate residue in a recognition sequence [48,61]. They reside in the cytoplasm and in organelles as inactive precursors and are activated by cleavage after specific aspartate residues. Currently various fluorogenic and chromogenic substrates are available for different active caspases. Most of these substrates are cell impermeable and require cell and tissue homogenization to measure the caspase activity [62]. These substrates are in general not specific for individual caspases. So, in most cases it is difficult to attribute fluorogenic and chromogenic activity to one specific caspase.

Caspase activity can also be measured by immunohistochemical techniques using antibodies against neoepitopes, which arise from their proteolytic activation [63–65]. Caspase activity can also be detected immunologically by using antibodies, which recognize only the cleaved substrates of caspases. Recently, antibodies were described which recognize caspase cleaved cytokeratin 18 [63], actin [64] and PARP [65]. These immunological approaches require fixation of the specimen.

4.2 Calcium flux
Activation of the apoptotic pathways is in most cases associated with an increase in cytosolic Ca²⁺ concentration. Such increase can be measured by using Ca²⁺ indicators like fura-2 [66]. A drawback of this technique is that the rise in cytosolic calcium is not exclusive for apoptosis and can also be associated with the activation of a variety of signalling pathways, which do not lead to the execution of apoptosis [67]. Furthermore, a lack of sensitivity has been observed, because, in some cases, apoptosis proceeds in the absence of Ca²⁺ changes [68]. Finally, it has to be addressed that the currently available indicators are only useful for in vitro assays.

4.3 Mitochondrial dysfunction
A decrease in mitochondrial membrane potential is an early feature of apoptotic cell death and is considered to mark the point of no return. The mitochondrial potential can be measured by a variety of fluorescent probes, which accumulate into the mitochondrion, as a function of the membrane potential. Collapse of the mitochondrial membrane potential, due to apoptosis, will result in a diminished ability of these fluorochromes to accumulate in the mitochondria [69]. Next, the translocation of several mitochondrial proapoptotic proteins from the intermembrane space into the cytosol can be measured. These proteins include apoptosis inducing factor [51], cytochrome c [52], and procaspases 2, 3 and 9 [53,54]. Translocation of these proteins can be visualized immunologically, using specific antibodies [70,71]. The drawback of these approaches may be their low sensitivity since certain death pathways that have been recently described circumvent the mitochondrion and activate the downstream executioner directly [70].

	5 DNA fragmentation

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
5.1 DNA laddering
Degradation of nuclear DNA is one of the key features of apoptosis. During apoptosis activated endonucleases cleave the DNA into fragments of multiples of 180–200 base pairs. These fragments appear as a DNA ladder on agarose gels [72]. One limitation of this technique is the large quantities of degraded DNA required to visualize a ladder pattern. Selective amplification of DNA fragments by ligation-mediated polymerase chain reaction (PCR) reduces this need [73]. A major disadvantage, however, is the loss of tissue morphology, which render unable the localization and identification of apoptotic cells. In some cases apoptosis generates only high-molecular-weight DNA fragments, thus lowering the sensitivity of this technique [51].

5.2 DNA content by FC
The DNA content reflects grossly its phase in the cell cycle (G0/G1, S/G2 and M). Apoptotic cells show a decreased DNA content below the G0/G1 level. This can be measured by FC and DNA probes like propidium iodide. This technique also discriminates between apoptosis and necrosis. The applicability of this technique for cardiovascular research is, however, limited (see Section FC) [60,74].

5.3 DNA strand break labeling
The presence of 3' hydroxyl-termini at the DNA strand breaks, characteristic of endonuclease-cleaved DNA, can be detected by a labeling reaction with modified nucleotides, like biotin, digoxigenin — or fluorescein-labeled dUTP. This reaction requires enzymes like terminal deoxynucleotidyltransferase (TdT) or DNA polymerase. The commonly used techniques are the in situ nick end labeling (ISEL) technique using DNA polymerase [75] and the TdT-mediated X-dUTP nick end labeling (TUNEL) technique (Fig. 1) [76,77]. Both techniques stain the nuclei of the cells and vesicles in the tissue [78]. Recent improvements of the protocol, to exclude the nonspecific staining of Ca²⁺ filled vesicles and RNA splicing factors, provide more consistent staining results [4,79].

View larger version (67K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 TUNEL staining of cardiomyocytes in human myocardial infarction. The apoptotic cardiomyocyte is detected by the brown nuclear staining in contrast to the haematoxylin stained normal cardiomyocyte nucleus.

 

	6 Plasma membrane alterations

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
6.1 Membrane permeability
Apoptosis is marked by altered cell morphology while the plasma membrane excludes the uptake of dyes such as trypan blue and propidium iodide. This phenomenon has great advantages with respect to the specificity, as it allows discrimination between apoptotic and necrotic cells. Especially the combined analyses of morphological changes and nonpermanent dye uptake, due to an altered membrane permeability, makes flow cytometric analysis very useful [74]. However, because of the toxicity of these dyes, this approach is less suitable for in vivo detection of apoptosis.

6.2 Membrane changes
Apoptotic cells express PS on their outer leaflet, which can be measured by derivatisation with fluorescamine and subsequent analysis of the derivatized, extracted membrane lipids by two-dimensional thin layer chromatography [80]. Furthermore, incorporation of the hydrophobic dye merocyanine 540 into the cellular membrane shows a loosely packed membrane, characteristic of apoptotic cells [80]. Both features indicate that the plasma membrane is altered due to the apoptotic process, but obviously, the above mentioned techniques are only suitable for in vitro analyses.

	7 Limitations of the currently available techniques

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
The most important limitation of the techniques described above is that these methods measure parameters of the late execution phase of apoptosis (Table 2). It is expected that cells in this phase are removed from the tissue by phagocytosis. Hence, measuring apoptosis in cardiovascular tissue by these techniques harbors the risk of underestimating this process. It will be of great advantage if techniques are available which measure parameters active at the transition from the decision to the execution phase. Also, analyzing only one parameter will in most cases not result in sufficient sensitivity and specificity. This can be improved by measuring multiple parameters. A general disadvantage of the current methods is that they are single endpoint measurements, which do not provide information of the dynamics of the apoptotic process in the tissues.

Most techniques can only be performed after fixation of the material or after lysis of the cells. With respect to cardiovascular research only the TUNEL/ISEL gives information on the localization of apoptosis in situ. LM and EM are time consuming because, in myocardium, morphological changes are rare. FC requires cells in suspension whereas assessment of DNA laddering requires cell/tissue homogenates. Therefore, there is a need for new methods to gain more understanding of the role of apoptosis in physiology and pathology of the cardiovascular system.

	8 Annexin V, a new marker for the detection of apoptosis

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
In 1992, Fadok et al. established the fundamentals for a novel apoptosis detection methodology by showing that apoptotic cells expose PS at their cell surface [80]. This observation triggered us to develop a novel technique to measure apoptosis using Annexin V. The basis of this technique is simple, yet elegant. Annexin V is able to bind to PS-exposing membranes in a calcium dependent manner [81]. Conjugation of Annexin V to detectable prosthetic groups, like fluorescein and biotin, thus allows the detection of cells, which expose PS [82–85]. The sensitivity and specificity of the Annexin V-based assay depends on the biological properties of PS, and the physicochemical property of Annexin V to bind to PS. Below, we will address these two aspects in more detail.

8.1 PS localization and (patho)physiological significance
The aminophospholipid PS localizes predominantly in membrane leaflets facing the cytosol as was firstly shown for erythrocytes and platelets and later for nucleated cells [86–88]. This PS asymmetry of the plasma membrane is generated and maintained by an aminophospholipid translocase, which transports selectively aminophospholipids from the outer to the inner leaflet, thereby creating a situation in which PS is exclusively localized to the leaflets facing the cytosol [86–88].

Blood platelets were the first cells for which it was demonstrated that a change of PS asymmetry could be caused by the action of agonists like thrombin and collagen [89,90]. Stimulation of platelets results in a rise of cytosolic Ca²⁺, which on the one hand, inhibits the aminophospholipid translocase, and on the other hand, activates a so-called scramblase, which scrambles the phospholipid species symmetrically over the two leaflets [91]. Within minutes the architecture of the plasma membrane is changed such that the platelet exposes significant amounts of PS at its outer plasma membrane leaflet. Comparable mechanisms appear to operate during apoptosis giving rise to the cell surface exposure of PS [80,92].

8.2 Functions of PS in apoptosis
Cell surface exposure of PS has a functional significance in the removal of senescent and dying cells from the tissue. The reticuloendothelial system recognizes PS by receptors and removes PS-exposing cells, like aged erythrocytes, from the circulation [93]. A similar scavenging system appears to be operational in tissues, where phagocytes recognize and engulf PS-exposing cells through receptor mediated processes [80]. Hence, surface-exposed PS appears to be one of the signals to communicate termination of existence to the environment, and it fulfills a distinct role in the physiological process to remove unwanted and superfluous cells from the tissues. It is therefore not surprising, that surface exposure of PS is an event that occurs while the plasma membrane integrity is uncompromised. If this would not be the case, the PS-exposing cell would leak its proinflammatory content into the tissue, eliciting undesirable inflammatory reactions.

8.3 Annexin V and its binding to PS
Annexin V was originally isolated from the human umbilical cord artery by virtue of its anticoagulant activity [94], which, in retrospect, can be explained by its binding to and shielding of negatively charged phospholipids [81,95]. In model systems, Annexin V hardly associates with phosphatidylcholine and sphingomyelin (at <5 mM Ca²⁺) [81], but it binds avidly to PS at 1 mM Ca²⁺, likely because Annexin V bears a putative binding pocket for the phosphoserine headgroup [96]. Once bound to the phospholipid surface, Annexin V forms two-dimensional lattices, which are stabilized by protein–protein interactions [97,98]. Binding of Annexin V to phospholipid membranes is reversible when calcium ions are chelated. The rates of association and dissociation suggest that Annexin V does not penetrate the membrane and behaves as an extrinsic membrane protein [81,99]. Altogether, these binding features make Annexin V an excellent tool to detect cell surface-exposed PS in vitro as well as in vivo.

8.4 Annexin V and cell surface-exposed PS, a revealing pas de deux of apoptosis
Using leukocytes, it was demonstrated for the first time that Annexin V discriminates between viable and apoptotic cells [82–84]. Competition experiments, using PS containing phospholipid vesicles, indeed demonstrated that the binding site for Annexin V on the apoptotic cell comprises PS [85]. In combination with the vital dye propidium iodide, it was shown that apoptotic cells expose PS while maintaining their plasma membrane integrity [82–84]. The Annexin V-based assay was first developed for cells in suspension (Fig. 2). Later it was shown that this assay could also be applied to adherent cell types [100] and in tissues [101].

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Course of the apoptotic process of antiFas stimulated Jurkat cells as visualized by Annexin V–Oregon Green (green) and propidium iodide (red). Jurkat cells were incubated with antiFas. At various time points, cells were sampled and stained with Annexin V–Oregon Green and propidium iodide. The cells were analyzed by confocal scanning laser microscopy.

 
The Annexin V-based assay [102] rapidly increased our knowledge about the embedding of PS exposure in the apoptotic process. The accumulated experimental data point towards a position at the transition from the decision to the execution phase, hence early in the execution phase [103] before morphological changes of the nucleus can be detected [104]. The exposure of PS does not depend on the involvement of the nucleus, but requires the activation of caspase 3 and a Ca²⁺ flux over the plasma membrane [105]. Next, it turned out that the exposure of PS is under the control of the Bcl-2 checkpoint, in case the mitochondrion participates in the death process [85]. These insights were generated by in vitro experiments using cultured or isolated cells. Recently, it was demonstrated that the Annexin V assay could be used in vivo by injecting Annexin V–biotin into the bloodstream of living mouse embryos [101]. In vivo Annexin V–biotin stains cells in various phases of apoptosis ranging from the early phase in which no morphological changes of the nucleus are detected on the EM level, until the late phase with pyknotic nucleus and condensed cytoplasm [101]. The combination of the Annexin V-based assay and the TUNEL method revealed the presence of three subpopulations of apoptotic cells in tissues. Firstly, Annexin V positive/TUNEL negative cells which are in the early execution phase. Secondly, both TUNEL and Annexin V positive cells, which are in the late stage of the execution phase. And thirdly, Annexin V negative/TUNEL positive cells, which are located in phagolysosomes [106]. The latter subpopulation reflects cells, which started to execute apoptosis well before the time point of the experiment. This could range from hours to days. These data indicate that the Annexin V-based assay is so far the most sensitive technique to detect ongoing apoptosis.

	9 Use of Annexin V in detection of apoptosis in the heart

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
To date, the TUNEL assay or DNA gel electrophoresis are the most utilized techniques to detect apoptosis in the heart [107–110]. Both techniques identify events occurring late in the apoptotic process, thus detecting apoptosis approximately 3 h after the onset of ischemia [110]. It has been argued that TUNEL and/or DNA laddering are suitable to assess neither the full extent of apoptosis nor the early kinetics of apoptosis (see above section). An insight into these is, however, indispensable to judge whether intervention with agents interfering with the apoptotic pathway is an option and whether such option is therapeutically of value. The Annexin V-based assay could provide answers to these questions. Therefore we injected labeled Annexin V intraarterially in an established ischemia and reperfusion model of the mouse heart in vivo [111].

After 15 min of ischemia followed by 30 min of reperfusion, some Annexin V positive cardiomyocytes were already observed in the area at risk. Specifically, stained cardiomyocytes could be observed, which also showed shrinkage and membrane blebbing. As PS expression and subsequent binding of Annexin V are, at least in vitro, downstream from the activation of executioner caspases, our data suggest that activation of the cell death program beyond the point of no return already may have occurred. Extending the time of ischemia to 30 min and the reperfusion time to 90 min leads to a massive increase in Annexin V positive cardiomyocytes in the area at risk (Fig. 3A). In most cardiomyocytes only plasma membrane staining was observed (Fig. 3B), and different stages of apoptosis were observed in the cardiomyocytes, similar to the findings reported from in vitro assays.

View larger version (82K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Cross section of a murine heart after 30 min ischemia and 90 min reperfusion. Annexin V–biotin was injected during the reperfusion phase. After reperfusion, the murine heart was dissected and cross sections were incubated with peroxidase-conjugated streptavidin. Peroxidase activity is represented by the dark brown color. The sections were counterstained with haematoxylin (A). Detailed picture of a mouse heart subjected to 30 min of ischemia followed by 90 min of reperfusion, which shows specific staining of the cell membrane of the cardiomyocyte (B).

 
In the contralateral areas of the heart and in hearts of sham-operated controls, no Annexin V positive cardiomyocytes were visualized. Moreover, in mouse hearts subjected to 30 min of ischemia and 90 min of reperfusion, no positive cardiomyocytes were observed after application of an Annexin V mutant, which lacks the ability to bind PS. Altogether, we conclude that the Annexin V-based assay is a specific, sensitive, and reliable method to detect apoptosis in the heart in vivo. Furthermore, we demonstrated that ischemia/reperfusion rapidly elicits programmed cell death in a substantial region of the area at risk in the murine heart. These findings bear important consequences for future intervention strategies since the observed kinetics suggest that the intervention window is narrow. Thus, only agents should be considered which interfere before the point of no return.

Annexin V also offers the possibility to monitor apoptosis in vivo by noninvasive imaging techniques. Such an approach has been described previously in a cardiac transplant model in rats [22]. We explored this technique in our murine ischemia/reperfusion model by using fluorescent-labeled Annexin V, and detected the apoptotic cells using a surgical microscope equipped with a fluorescence detection system. Ischemia and reperfusion resulted in a time dependent increase of Annexin V labeling in the area at risk, suggesting that continuous in vivo imaging is possible. Next, we evaluated the use of technetium labeled Annexin V and nuclear single photon emission computed tomography (SPECT) imaging for detection of apoptosis under similar conditions. Again an enhanced uptake of Annexin V–technetium was observed in the area at risk in contrast to sham-operated controls where no uptake was detected. These data suggest that in vivo imaging of apoptosis in the heart is possible with Annexin V conjugates. Whether such a technique is also suitable for patients with myocardial infarction remains to be established.

	10 Sensitivity, specificity and applicability of the Annexin V assay

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 
As already discussed, detection of apoptosis with Annexin V is a rather simple and easily applicable technique. The assay is one of the most sensitive methods because it detects apoptosis in the early execution phase (Table 2). Moreover, the assay is versatile since it can be applied in vitro, ex vivo and even in vivo. Theoretically, the sensitivity of the Annexin V-based assay could be impaired by endogenous Annexin V, which may compete with administered Annexin V. However, so far such a competition was not observed. This may be explained by the physicochemical characteristics of Annexin V to form a dynamic two-dimensional lattice on the cell surface in which exogenous Annexin V is easily incorporated.

The specificity of the assay is determined by the cell surface exposure of PS independent of an apoptotic process. So far, three examples have been reported. Firstly, myoblasts in the embryo express PS [24]. Secondly, embryonic megakaryoblasts expose PS before they fragment into multiple platelets [101]. Thirdly, murine syncytiotrophoblasts in the placenta possess Annexin V binding sites suggesting the existence of viable cells with surface-exposed PS [112]. Another potential drawback on the specificity of the Annexin V-based assay is related to the potential binding of Annexin V to necrotic cells. For reasons that remain to be elucidated, Annexin V preferably binds to apoptotic cells even in conditions of excess necrosis. Until the underlying mechanism is resolved a combination of the Annexin V-based assay and a DNA marker such as propidium iodine, which will stain DNA if the plasma membrane is perturbed, will increase the specificity of the assay. Until now, such a combination can only be performed in vitro due to the toxicity of the DNA dye. In vivo, a combination with assays for substances, which accumulate in necrotic cells, like fibronectin [113] or IgG [114], may increase the specificity of the assay.

Another possible pitfall of the administration of Annexin V in mammals results from our relative lack of knowledge of the physiological function of Annexin V. Studies concentrated around endogenous Annexin V, do suggest that the PS recognition of Annexin V also forms the basis for its physiological function. Since Annexin V is widely distributed in mammals, it is conceivable that administration of exogenous Annexin V may interfere with physiological processes in which Annexin V is involved, such as the blood coagulation, inflammation and phagocytosis, although such interference has never been reported.

The Annexin V-based assay allows also the performance of kinetic experiments. Preliminary experiments have shown that the Annexin V-based assay allows real time imaging of apoptosis since no fixation or other artifactual handling is required. In the ischemia and reperfusion model administration of Annexin V–fluorescence isothiocyanate (FITC) to the circulation results in the visualization of the appearance of apoptotic cells in the area at risk.

In conclusion, the Annexin V-based assay seems to be a valuable technique to detect apoptosis in cardiovascular research. So far, it is a very sensitive technique to detect apoptosis and applicable in a variety of settings, including in vivo. Its applicability to detect apoptosis in patients with cardiovascular diseases remains to be established.

Time for primary review 32 days

	Acknowledgements

 
Waander L. van Heerde is a research fellow of the Netherlands Heart Foundation and supported by grant D96-025.

	References

Top Abstract 1 Introduction 2 Techniques to detect... 3 Morphological changes 4 Cytoplasmic changes 5 DNA fragmentation 6 Plasma membrane alterations 7 Limitations of the... 8 Annexin V, a... 9 Use of Annexin... 10 Sensitivity, specificity and... References

 

1. Watanabe M, Choudhry A, Berlan M, et al. Developmental remodeling and shortening of the cardiac outflow tract involves myocyte programmed cell death. Development (1998) 125:3809–3820.[Abstract]
2. Mallat Z, Hugel B, Ohan J, et al. Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity. Circulation (1999) 99:348–353.[Abstract/Free Full Text]
3. Kockx M.M, De Meyer G.R, Buyssens N, et al. Cell composition, replication, and apoptosis in atherosclerotic plaques after 6 months of cholesterol withdrawal. Circ Res (1998) 83:378–387.[Abstract/Free Full Text]
4. Lutgens E, Daemen M.J.A.P, Kockxs M, et al. Atherosclerosis in APOE*3-Leiden transgenic mice. From proliferative to atheromatous stage. Circulation (1999) 99:276–283.[Abstract/Free Full Text]
5. Shindo J, Ishibashi T, Yokoyama K, et al. Granulocyte–macrophage colony-stimulating factor prevents the progression of atherosclerosis via changes in the cellular and extracellular composition of atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbits. Circulation (1999) 99:2150–2156.[Abstract/Free Full Text]
6. Fukumoto H, Naito Z, Asano G, Aramaki T. Immunohistochemical and morphometric evaluations of coronary atherosclerotic plaques associated with myocardial infarction and diabetes mellitus. J Atheroscler Thromb (1998) 5:29–35.[Medline]
7. Hamet P, Moreau P, Dam T.V, et al. The time window of apoptosis: a new component in the therapeutic strategy for cardiovascular remodeling. J Hypertens Suppl (1996) 14:S65–S70.[Medline]
8. DeBlois D, Tea B.S, Than V.D, Tremblay J, Hamet P. Smooth muscle apoptosis during vascular regression in spontaneously hypertensive rats. Hypertension (1997) 29:340–349.[Abstract/Free Full Text]
9. Akyurek L.M, Johnsson C, Lange D, et al. Tolerance induction ameliorates allograft vasculopathy in rat aortic transplants. Influence of Fas-mediated apoptosis. J Clin Invest (1998) 101:2889–2899.[ISI][Medline]
10. Hirsch G.M, Kearsey J, Burt T, Karnovsky M.J, Lee T. Medial smooth muscle cell loss in arterial allografts occurs by cytolytic cell induced apoptosis. Eur J Cardiothorac Surg (1998) 14:89–96.[Medline]
11. Slomp J, Gittenberger-de Groot A.C, Glukhova M.A, et al. Differentiation, dedifferentiation, and apoptosis of smooth muscle cells during the development of the human ductus arteriosus. Arterioscler Thromb Vasc Biol (1997) 17:1003–1009.[Abstract/Free Full Text]
12. Lizard G, Monier S, Cordelet C, et al. Characterization and comparison of the mode of cell death, apoptosis versus necrosis, induced by 7beta-hydroxycholesterol and 7-ketocholesterol in the cells of the vascular wall. Arterioscler Thromb Vasc Biol (1999) 19:1190–1200.[Abstract/Free Full Text]
13. Langille B.L. Arterial remodeling: relation to hemodynamics. Can J Physiol Pharmacol (1996) 74:834–841.[CrossRef][ISI][Medline]
14. Cho A, Courtman D.W, Langille B.L. Apoptosis (programmed cell death) in arteries of the neonatal lamb. Circ Res (1995) 76:168–175.[Abstract/Free Full Text]
15. Bauriedel G, Schluckebier S, Hutter R, et al. Apoptosis in restenosis versus stable-angina atherosclerosis: implications for the pathogenesis of restenosis. Arterioscler Thromb Vasc Biol (1998) 18:1132–1139.[Abstract/Free Full Text]
16. Frustaci A, Chimenti C, Setoguchi M, et al. Cell death in acromegalic cardiomyopathy. Circulation (1999) 99:1426–1434.[Abstract/Free Full Text]
17. Valente M, Calabrese F, Thiene G, et al. In vivo evidence of apoptosis in arrhythmogenic right ventricular cardiomyopathy. Am J Pathol (1998) 152:479–484.[Abstract]
18. Fontaliran F, Arkwright S, Vilde F, Fontaine G. Arrhythmogenic right ventricular dysplasia and cardiomyopathy. Clinical and anatomic–pathologic aspects, nosologic approach. Arch Anat Cytol Pathol (1998) 46:171–177.[Medline]
19. Kageyama Y, Li X.K, Suzuki S, et al. Apoptosis is involved in acute cardiac allograft rejection in rats. Ann Thorac Surg (1998) 65:1604–1609.[Abstract/Free Full Text]
20. Koglin J, Russell M.E. Alloimmune-mediated apoptosis: comparison in mouse models of acute and chronic cardiac rejection. Transplantation (1999) 67:904–909.[CrossRef][ISI][Medline]
21. Vriens P.W, Blankenberg F.G, Stoot J.H, et al. The use of technetium Tc 99m Annexin V for in vivo imaging of apoptosis during cardiac allograft rejection. J Thorac Cardiovasc Surg (1998) 116:844–853.[Abstract/Free Full Text]
22. Blankenberg F.G, Katsikis P.D, Tait J.F, et al. In vivo detection and imaging of phosphatidylserine expression during programmed cell death. Proc Natl Acad Sci USA (1998) 95:6349–6354.[Abstract/Free Full Text]
23. Takeda K, Yu ZX, Nishikawa T, et al. Apoptosis and DNA fragmentation in the bulbus cordis of the developing rat heart. J Mol Cell Cardiol (1996) 28:209–215.[CrossRef][ISI][Medline]
24. Poelmann R.E, Mikawa T, Gittenberger-De Groot A.C. Neural crest cells in outflow tract septation of the embryonic chicken heart: differentiation and apoptosis. Dev Dyn (1998) 212:373–384.[CrossRef][ISI][Medline]
25. Saphier C.J, Yeh J. Altered apoptosis levels in hearts of human fetuses with Down syndrome. Am J Obstet Gynecol (1998) 179:962–965.[CrossRef][ISI][Medline]
26. Elsässer A, Schlepper M, Klövekorn W.P, et al. Hibernating myocardium: an incomplete adaptation to ischemia. Circulation (1997) 96:2920–2931.[Abstract/Free Full Text]
27. Chen C, Ma L, Linfert D.R, et al. Myocardial cell death and apoptosis in hibernating myocardium. J Am Coll Cardiol (1997) 30:1407–1412.[Abstract]
28. Baghelai K, Graham L.J, Wechsler A.S, Jakoi E.R. Delayed myocardial preconditioning by alpha₁-adrenoceptors involves inhibition of apoptosis. J Thorac Cardiovasc Surg (1999) 117:980–986.[Abstract/Free Full Text]
29. Narula J, Hajjar R.J, Dec G.W. Apoptosis in the failing heart. Cardiol Clin (1998) 16:691–710.[CrossRef][Medline]
30. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev (1999) 79:215–262.[Abstract/Free Full Text]
31. Freude B, Masters T.N, Kostin S, Robicsek F, Schaper J. Cardiomyocyte apoptosis in acute and chronic conditions. Basic Res Cardiol (1998) 93:85–89.[CrossRef][ISI][Medline]
32. Zhang J, Andrade Z.A, Yu Z.X, et al. Apoptosis in a canine model of acute Chagasic myocarditis. J Mol Cell Cardiol (1999) 31:581–596.[CrossRef][ISI][Medline]
33. Rossi M.A, Souza A.C. Is apoptosis a mechanism of cell death of cardiomyocytes in chronic Chagasic myocarditis? Int J Cardiol (1999) 68:325–331.[CrossRef][ISI][Medline]
34. James T.N, St. Martin E, Willis P.W III, Lohr T.O. Apoptosis as a possible cause of gradual development of complete heart block and fetal arrhythmias associated with absence of the AV node, sinus node, and internodal pathways. Circulation (1996) 93:1424–1438.[Abstract/Free Full Text]
35. Seki Y, Kai H, Kai M, et al. Myocardial DNA strand breaks are detected in biopsy tissues from patients with dilated cardiomyopathy. Clin Cardiol (1998) 21:591–596.[ISI][Medline]
36. Saraste A, Pulkki K, Kallajoki M, et al. Cardiomyocyte apoptosis and progression of heart failure to transplantation. Eur J Clin Invest (1999) 29:380–386.[CrossRef][ISI][Medline]
37. Feuerstein G, Yue T.L, Ma X, Ruffolo R.R. Novel mechanisms in the treatment of heart failure: inhibition of oxygen radicals and apoptosis by carvedilol. Prog Cardiovasc Dis (1998) 48(Suppl_1):17–24.
38. Diez J, Panizo A, Hernandez M, et al. Cardiomyocyte apoptosis and cardiac angiotensin-converting enzyme in spontaneously hypertensive rats. Hypertension (1997) 30:1029–1034.[Abstract/Free Full Text]
39. Ohno M, Takemura G, Ohno A, et al. "Apoptotic" myocytes in infarct area in rabbit hearts may be oncotic myocytes with DNA fragmentation: analysis by immunogold electron microscopy combined with in situ nick end-labeling. Circulation (1998) 98:1422–1430.[Abstract/Free Full Text]
40. Anversa P, Cheng W, Liu Y, et al. Apoptosis and myocardial infarction. Basic Res Cardiol (1998) 93(Suppl 3):8–12.[CrossRef][ISI][Medline]
41. Dong C, Wilson J.E, Winters G.L, McManus B.M. Human transplant coronary artery disease: pathological evidence for Fas-mediated apoptotic cytotoxicity in allograft arteriopathy. Lab Invest (1996) 74:921–931.[ISI][Medline]
42. Colston J.T, Chandrasekar B, Freeman G.L. Expression of apoptosis-related proteins in experimental coxsackievirus myocarditis. Cardiovasc Res (1998) 38:158–168.[Abstract/Free Full Text]
43. Gebhard J.R, Perry C.M, Harkins S, et al. Coxsackievirus B3-induced myocarditis: perforin exacerbates disease, but plays no detectable role in virus clearance. Am J Pathol (1998) 153:417–428.[Abstract/Free Full Text]
44. Thompson C.B. Apoptosis in the pathogenesis and treatment of disease. Science (1995) 267:1456–1462.[Abstract/Free Full Text]
45. Kroemer G, Petit P.X, Zamzami N, Vayssiere J.L, Mignotte B. The biochemistry of apoptosis. FASEB J (1995) 9:1277–1287.[Abstract]
46. Ashkenazi A, Dixit V.M. Death receptors: signaling and modulation. Science (1998) 281:1305–1308.[Abstract/Free Full Text]
47. Evan G, Littlewood T. A matter of life and cell death. Science (1998) 281:1317–1322.[Abstract/Free Full Text]
48. Thornberry N.A, Lazebnik Y. Caspases: enemies within. Science (1998) 281:1312–1316.[Abstract/Free Full Text]
49. Susin S.A, Zamzami N, Castedo M, et al. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J Exp Med (1997) 186:25–37.[Abstract/Free Full Text]
50. Green D.R, Reed J.C. Mitochondria and apoptosis. Science (1998) 281:1309–1312.[Abstract/Free Full Text]
51. Susin S.A, Lorenzo H.K, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature (1999) 397:441–446.[CrossRef][Medline]
52. Kluck R.M, Bossy Wetzel E, Green D.R, Newmeyer D.D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science (1997) 275:1132–1136.[Abstract/Free Full Text]
53. Mancini M, Nicholson D.W, Roy S, et al. The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling. J Cell Biol (1998) 140:1485–1495.[Abstract/Free Full Text]
54. Susin S.A, Lorenzo H.K, Zamzami N, et al. Mitochondrial release of caspase-2 and -9 during the apoptotic process. J Exp Med (1999) 189:381–394.[Abstract/Free Full Text]
55. Slee E.A, Harte M.T, Kluck R.M, et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol (1999) 144:281–292.[Abstract/Free Full Text]
56. Kerr J.F.R, Wyllie A.H, Currie A.R. Apoptosis: a basis biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer (1972) 26:239–257.[ISI][Medline]
57. Cotter T.G, Martin S.J. Techniques in apoptosis. A user's guide. (1996) London: Portland Press.
58. Majno G, Joris I. Apoptosis, oncosis and necrosis. Am J Pathol (1995) 146:3–15.[Abstract]
59. Gorman A, McCarthy J, Finucane D, Reville W, Cotter T. Techniques in apoptosis. A user's guide. Cotter T.G, Martin S.J, eds. (1996) London: Portland Press. 1–20.
60. Darzynkiewicz Z, Bruno S, DelBino G, et al. Features of apoptotic cells measured by flow cytometry. Cytometry (1992) 13:795–808.[CrossRef][ISI][Medline]
61. Nagata S. Apoptosis by death factor. Cell (1997) 88:355–365.[CrossRef][ISI][Medline]
62. Gurtu V, Kain S.R, Zhang G. Fluorometric and colorometric detection of caspase activity associated with apoptosis. Anal Biochem (1997) 251:98–102.[CrossRef][ISI][Medline]
63. Leers M.P.G, Kolgen W, Bjorkland V, et al. Immunocytochemical detection and mapping of a cytokeratin 18 neoepitope exposed during early apoptosis. J Pathol (1999) 187:567–572.[CrossRef][ISI][Medline]
64. Yang F, Sun X, Beech W, et al. Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and microglia in Alzheimer's disease. Am J Pathol (1998) 152:379–389.[Abstract]
65. Sallmann F.R, Bourassa S, Saint-Cyr J, Poirier G.G. Characterization of antibodies specific for the caspase cleavage site on poly(ADP-ribose) polymerase: specific detection of apoptotic fragments and mapping of the necrotic fragments of poly(ADP-ribose) polymerase. Biochem Cell Biol (1997) 75:451–456.[CrossRef][ISI][Medline]
66. McConkey D.J. Techniques in apoptosis. A user's guide. Cotter T.G, Martin S.J, eds. (1996) London: Portland Press. 133–148.
67. Berridge M, Lipp P, Bootman M. Calcium signalling. Curr Biol (1999) 9:R157–159.[CrossRef][ISI][Medline]
68. Whyte M.K, Hardwick S.J, Meagher L.C, Savill J.S, Haslett C. Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro. J Clin Invest (1993) 92:446–453.[ISI][Medline]
69. Metivier D, Dallaporta B, Zamzami N, et al. Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells. Comparison of seven mitochondrion-specific fluorochromes. Immunol Lett (1998) 61:157–163.[CrossRef][ISI][Medline]
70. Bossy-Wetzel E, Newmeyer D.D, Green D.R. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J (1998) 17:37–49.[CrossRef][ISI][Medline]
71. Krajewski S, Krajewska M, Ellerby L.M, et al. Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia. Proc Natl Acad Sci USA (1999) 96:5752–5757.[Abstract/Free Full Text]
72. Itoh G, Tamura J, Suzuki M, et al. DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis. Am J Pathol (1995) 146:1325–1331.[Abstract]
73. Staley K, Blaschke A.J, Chun J. Apoptotic DNA fragmentation is detected by a semiquantitative ligation mediated PCR of blunt DNA ends. Cell Death Diff (1997) 4:66–75.[CrossRef][ISI][Medline]
74. Darzynkiewicz Z, Li X. Techniques in apoptosis. A user's guide. Cotter T.G, Martin S.J, eds. (1996) London: Portland Press. 71–106.
75. Meyaard L, Otto S.A, Jonker R.R, et al. Programmed death of T cells in HIV-1 infection. Science (1992) 257:217–219.[Abstract/Free Full Text]
76. Gorczyca W, Bruno S, Darzynkiewicz R.J, Gong J, Darzynkiewicz Z. DNA strand breaks occurring during apoptosis: their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int J Oncol (1992) 1:639–648.[ISI]
77. Gavrieli Y, Sherman Y, Ben-Sasson S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol (1992) 119:493–501.[Abstract/Free Full Text]
78. Kockx M.M, Muhring J, Bortier H, DeMeyer G.R.Y, Jacob W. Biotin- or digoxigenin-conjugated nucleotides bind to matrix vesicles in atherosclerotic plaques. Am J Pathol (1996) 148:1771–1777.[Abstract]
79. Kockx M.M, Muhring J, Knaapen M.W.M, de Meyer G.R. RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis. Am J Pathol (1998) 152:885–888.[Abstract]
80. Fadok V.A, Voelker D.R, Campbell P.A, et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol (1992) 148:2207–2216.[Abstract]
81. Andree H.A, Reutelingsperger C.P.M, Hauptmann R, et al. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J Biol Chem (1990) 265:4923–4928.[Abstract/Free Full Text]
82. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C.P.M. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods (1995) 184:39–52.[CrossRef][ISI][Medline]
83. Koopman G, Reutelingsperger C.P.M, Kuijten G.A.M, et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood (1994) 84:1415–1420.[Abstract/Free Full Text]
84. Homburg C.H.E, De Haas M, Von Dem Borne A.E.G.K, et al. Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro. Blood (1995) 85:532–540.[Abstract/Free Full Text]
85. Martin S.J, Reutelingsperger C.P.M, McGahon A.J, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med (1995) 182:1545–1557.[Abstract/Free Full Text]
86. Seigneuret M, Devaux P.F. ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythr