Ewald A. W. J. Dumont

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.

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.

Markers of apoptosis in cardiovascular tissues: focus on Annexin V.

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, pros and contras of this new apoptosis detection assay will be discussed.

Annexin A5 Uptake in Ischemic Myocardium: Demonstration of Reversible Phosphatidylserine Externalization and Feasibility of Radionuclide Imaging

Ischemic insult to the myocardium is associated with cardiomyocyte apoptosis. Because apoptotic cell death is characterized by phosphatidylserine externalization on cell membrane and annexin-A5 (AA5) avidly binds to phosphatidylserine, we hypothesized that radiolabeled AA5 should be able to identify the regions of myocardial ischemia. Methods: Models of brief myocardial ischemia by the occlusion of the coronary artery for 10 min (I-10) and reperfusion for 180 min (R-180) for the detection of phosphatidylserine exteriorization using 99mTc-labeled AA5 and γ-imaging were produced in rabbits. 99mTc-AA5 uptake after brief ischemia was compared with an I-40/R-180 infarct model. Histologic characterization of both myocardial necrosis and apoptosis was performed in ischemia and infarct models. Phosphatidylserine exteriorization was also studied in a mouse model, and the dynamics and kinetics of phosphatidylserine exposure were assessed using unlabeled recombinant AA5 and AA5 labeled with biotin, Oregon Green, or Alexa 568. Appropriate controls were established. Results: Phosphatidylserine exposure after ischemia in the rabbit heart could be detected by radionuclide imaging with 99mTc-AA5. Pathologic characterization of the explanted rabbit hearts did not show apoptosis or necrosis. Homogenization and ultracentrifugation of the ischemic myocardial tissue from rabbit hearts recovered two thirds of the radiolabeled AA5 from the cytoplasmic compartment. Murine experiments demonstrated that the cardiomyocytes expressed phosphatidylserine on their cell surface after an ischemic insult of 5 min. Phosphatidylserine exposure occurred continuously for at least 6 h after solitary ischemic insult. AA5 targeted the exposed phosphatidylserine on cardiomyocytes; AA5 was internalized into cytoplasmic vesicles within 10–30 min. Twenty-four hours after ischemia, cardiomyocytes with internalized AA5 had restored phosphatidylserine asymmetry of the sarcolemma, and no detectable phosphatidylserine remained on the cell surface. The preadministration of a pan-caspase inhibitor, zVAD-fmk, prevented phosphatidylserine exposure after ischemia. Conclusions: After a single episode of ischemia, cardiomyocytes express phosphatidylserine, which is amenable to targeting by AA5, for at least 6 h. Phosphatidylserine exposure is transient and internalized in cytoplasmic vesicles after AA5 binding, indicating the reversibility of the apoptotic process.

Bringing cell death alive.

The role of apoptosis in ischemia and reperfusion of the heart has been widely debated. This controversy has been continued because of the lack of an apoptosis detection method that allowed obtaining detailed kinetic and quantitative information on apoptosis. Here we focus on recent findings that look into the detection of apoptosis following ischemia and reperfusion in the heart in animal models and in patients using Annexin-A5 based image technology. Following cardiac cell damage, one major characteristic finding is that apoptotic cells express phosphatidylserines (PS) on the outer leaflet of their cell membrane, serving as a “remove me” signal for the immune system. Annexin-A5, a native plasma protein with a high affinity for PS, can be used to measure this mode of cell death. Several Annexin-A5 based imaging systems have been developed to measure apoptosis from cell culture up to patients. In this review, implications, limitations, and clinical relevance of cell death imaging will be discussed.

Minocycline Inhibits Apoptotic Cell Death in a Murine Model of Partial Flap Loss

For breast reconstruction, the deep inferior epigastric perforator (DIEP) flap has become standard therapy. A feared complication is partial or even total flap loss. In a novel murine model of partial DIEP flap loss, the contribution of apoptotis to flap loss was investigated. The clinically available apoptosis-inhibiting compound minocycline was tested for its ability to reduce cell death. The effect of minocycline on cell proliferation was studied in cell cultures of breast carcinoma. In 12 mice, pedicled DIEP flaps were raised, which were subjected to 15 minutes of ischemia and 4 days of reperfusion. Six mice were treated with minocycline 2 hours before surgery and every 24 hours for 4 days. Apoptosis was revealed by injecting annexin A5 30 minutes before sacrifice. Annexin A5 binds to phosphatidylserines, which are expressed on the cell membrane during apoptotis. Prior to sacrifice, necrosis was measured using planimetry. Minocycline reduced cell death after 4 days from 35.9% (standard deviation = 10.6) to 13.9% (standard deviation = 8.0; P < 0.05). Apoptosis, as shown by annexin A5 binding in nontreated animals, was abundant. Minocycline did not influence tumor growth in cell cultures of human breast cancer. Minocycline treatment leads to increased DIEP flap viability in mice. This study widens the perspective in the improvement of free flap survival in patients.