The role of labeled Annexin A5 in imaging of programmed cell death. From animal to clinical imaging.

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.

Molecular imaging of cell death in intracardiac tumours: A new approach to differential diagnosis in cardiac tumours.

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.

Real-time imaging of apoptotic cell-membrane changes at the single-cell level in the beating murine heart.

We report a novel real-time imaging model to visualize apoptotic membrane changes of single cardiomyocytes in the injured heart of the living mouse, using fluorescent labeled annexin-V. Annexin-V binds to externalized phosphatidylserine (PS) of cells undergoing programmed cell death. With high-magnification (x100-160) real-time imaging, we visualized the binding of annexin-V to single cardiomyocytes. Kinetic studies at the single-cell level revealed that cardiomyocytes started to bind annexin-V within minutes after reperfusion, following an ischemic period of 30 minutes. The amount of bound annexin-V increased rapidly and reached a maximum within 20-25 minutes. Caspase inhibitors decreased the number of annexin-V-positive cardiomyocytes and slowed down the rate of PS exposure of cardiomyocytes that still bound annexin-V. This technology to study cell biology in the natural environment will enhance knowledge of intracellular signaling pathways relevant for cell-death regulation and strategies to manipulate these pathways for therapeutic effect.

Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model.

INTRODUCTION: Phosphatidylserine (PS) externalization is regarded as one of the earliest hallmarks of cells undergoing programmed cell death. We studied the use of labeled human recombinant annexin-V, a protein selectively binding to PS, to detect cardiomyocyte death in an in vivo mouse model of cardiac ischemia and reperfusion (I/R). METHODS AND RESULTS: I/R was induced in mouse hearts by ligation and subsequent release of a suture around the left anterior descending coronary artery. Annexin-V (25 mg/kg) fused to a marker molecule was injected intra-arterially 30 minutes before euthanasia. After 15 minutes of ischemia followed by 30 minutes of reperfusion, 1.4+/-1. 2% (mean+/-SD) of the cardiomyocytes in the area at risk were annexin-V positive (n=6). This increased to 11.4+/-1.9% after 15 minutes of ischemia followed by 90 minutes of reperfusion (n=7) and to 20.2+/-3.3% after 30 minutes of ischemia followed by 90 minutes of reperfusion (n=7). In control mice, including those injected with annexin-V at the binding site of PS, no annexin-V-positive cells were observed. DNA gel electrophoresis showed typical laddering starting after 15 minutes of ischemia followed by 30 minutes of reperfusion, suggesting activation of the cell death program. Intervention in the cell death program by pretreatment with a novel Na(+)-H(+) exchange inhibitor substantially decreased annexin-V-positive cardiomyocytes from 20.2% to 2.2% in mice after 30 minutes of ischemia followed by 90 minutes of reperfusion. CONCLUSIONS: These data suggest that labeled annexin-V is useful for in situ detection of cell death in an in vivo model of I/R in the heart and for the evaluation of cell death-blocking strategies.

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.

Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling.

Endochondral ossification in the epiphyseal growth plate of long bones is associated with programmed cell death (PCD) of a major portion of the chondrocytes. Here we tested the hypothesis that at the ossification front of the epiphyseal growth plate osteoclasts preferentially phagocytose chondrocytes that are undergoing PCD. We injected biotin-labelled annexin-V (anx-V-biotin, an early marker of PCD) intravenously in young adult mice. After 30 min of labelling, long bones were recovered and the tissue distribution examined of anx-V-biotin-labelled cells in the growth plate using ABC-peroxidase histochemistry. Positive staining for anx-V-biotin was detected in hypertrophic chondrocytes still present in closed lacunae at some distance from the ossification front. At the ossification front, chondrocyte lacunae were opened and close contacts were seen between tartrate-resistant acid phosphatase-positive osteoclasts and hypertrophic cartilage cells. Osteoclasts were significantly more frequently in contact with anx-V-biotin-labelled chondrocytes than with unlabelled chondrocytes. Osteoclasts also contained labelled and unlabelled phagocytic fragments within their cytoplasm. We conclude that in the growth plate osteoclasts preferentially phagocytose hypertrophic chondrocytes that are dying, suggesting these dying cells may signal osteoclasts for their removal.