Basic Research in Cardiology

, Volume 103, Issue 2, pp 95–104

Annexin A5: an imaging biomarker of cardiovascular risk


  • Edward M. Laufer
    • Dept. of CardiologyUniversity of Maastricht
  • Chris P. M. Reutelingsperger
    • Dept. of CardiologyUniversity of Maastricht
    • Dept. of BiochemistryUniversity of Maastricht
  • Jagat Narula
    • Dept. of CardiologyUniversity of Maastricht
    • Dept. of Cardiology, Irvine Medical CenterUniversity of California
    • Dept. of CardiologyUniversity of Maastricht
    • Dept. of CardiologyThe Netherlands

DOI: 10.1007/s00395-008-0701-8

Cite this article as:
Laufer, E.M., Reutelingsperger, C.P.M., Narula, J. et al. Basic Res Cardiol (2008) 103: 95. doi:10.1007/s00395-008-0701-8


Apoptosis, a form of programmed cell death (PCD), plays an important role in the initiation and progression of a number of cardiovascular disease, such as heart failure, myocardial infarction, and atherosclerosis. One of the most prominent characteristics of apoptosis is the externalisation of phosphatidylserine (PS), a plasma cell membrane phospholipid, which in healthy cells only is present on the inner leaflet of the plasma cell membrane. Annexin A5, a 35 kD plasma protein, has strong affinity for PS in the nano-molar range. Through the coupling of Annexin A5 to contrast agents, visualization of apoptotic cell death in vivo in animal models and in patients has become feasible. These imaging studies have provided novel insight into the extent and kinetics of apoptosis in cardiovascular disease. Furthermore, Annexin A5 imaging has proven to be a suitable imaging biomarker for the evaluation of cell death modifying compounds and plaque stabilizing strategies. Recent insight in PS biology has shown that PS externalisation not only occurs in apoptosis, but is also observed in activated macrophages and stressed cells. In addition, it has been shown that Annexin A5 not only binds to exteriorized PS, but is also internalized through an Annexin A5 specific mechanism. These latter findings indicate that Annexin A5 imaging is not exclusively valuable for apoptosis detection, but can also be used to visualize inflammation and cell stress. This will open novel opportunities for imaging and drug delivery strategies. In this review we will discuss the introduction of Annexin A5 in preclinical and clinical imaging studies and provide an outlook on novel opportunities of Annexin A5 based targeting of PS.

Key words

Annexin A5apoptosisunstable plaquecardiovascular riskimaging

1 Introduction

Cardiovascular disease is the leading cause of death in the Western world [4]. In addition, it is predicted that the incidence of cardiovascular disease will rapidly increase in emerging economies, such as China and India [4]. These developments call for the development of technologies that detect subclinical cardiovascular pathology before catastrophes, such as myocardial infarction, heart failure and/or stroke occur [3].

With the help of risk profiling based on large prospective clinical studies, patients with a high risk of developing myocardial infarction in the next 10 years can be identified, based on relatively simple questionnaires and the assessment of lipid profiles [29]. However, which individual patient with a high vascular risk will develop myocardial infarction, and when exactly this will occur is hard to predict. The lack of diagnostic tools that precisely tell us which of the patients are at imminent risks result in thousands of unnecessary cardiovascular deaths worldwide. This unmet clinical need has triggered a range of initiatives in serum and imaging biomarker research to predict upcoming vascular catastrophe in the individual patient [28].

Similarly, in patients who have suffered from cardiac injury, it is hard to predict which individual patient will develop pump function disorders of the left ventricle of the heart [9, 32]. Although the assessment of left ventricular ejection fraction by echocardiography and/or magnetic resonance imaging has proven to have prognostic relevance, the chances of developing cardiac pump failure in the individual patient are still hard to predict [33]. This precludes optimal therapeutic measures in the patients with a high risk to develop heart failure

Novel clues to predict adverse outcome in patients with a high cardiovascular risk may be discovered in the wealth of data generated by the rapid evolvement of molecular biology in cardiovascular research. One of the challenges we face is how to translate these discoveries in basic science into practical clinical tools that will help to prevent the occurrence of full-blown cardiovascular diseases [2]. Initial successes in this translational process include the introduction of high sensitivity C-reactive protein to better risk stratify patients at risk to develop acute vascular events [32]. This development is based on findings in atherosclerosis research that atherosclerotic plaque rupture is strongly associated with plaque inflammation [5]. However, the usefulness of hs-CRP in the prediction of acute vascular events occurring in the short term in the individual patient is still limited.

Another example of a successful novel serum biomarker in clinical cardiovascular practice is the introduction of NT-pro Brain Natriuretic Peptide (NT pro-BNP)[36]. This introduction is based on basic discoveries that ventricular cardiomyocytes release NT-pro BNP upon left ventricular dysfunction and stretch. Although proven useful in the diagnosis of left ventricular failure, biomarkers that act upstream from NT pro BNP will be required to identify patients before left ventricular pump failure occurs.

Cardiovascular imaging provides a complementary diagnostic approach in the assessment of cardiovascular risk in patients. The rapid development of echocardiography, coronary angiography, MRI, and multi-detector CT have given us superb imaging tools for the assessment of left ventricular function, and the anatomical consequences of atherosclerosis. However, up to now, these imaging modalities lack the ability to identify the underlying biology of cardiovascular disease [15]. The lack of biological detection possibilities of current imaging technologies limits their predictive value. For instance in patients with CHF echocardiography will give us a functional outcome of left ventricular function, but cannot predict whether the underlying biology that drives left ventricular dysfunction will result in worsening of the left ventricle in the short term. Likewise, coronary angiography and multi detector CT (MDCT) are excellent tools to visualize the anatomical consequences of atherosclerosis. However, which of the diagnosed atherosclerotic plaques will undergo plaque rupture and lead to acute vascular events is hard to predict from current imaging technologies [6].

Therefore, imaging technologies that visualize the biology of the underlying cardiovascular disease are urgently needed for early diagnosis and better prediction of cardiovascular risk in the individual patient [14]. Molecular imaging, defined as the non-invasive diagnostic imaging method to visualize biological events in vivo, has emerged rapidly in the recent years.

In this review, we will discuss the possible role of molecular imaging in the early detection of cardiovascular disease and the prediction of cardiovascular catastrophe. The review will focus on the detection of apoptosis [17]. Apoptosis, a form of programmed cell death (PCD), plays a prominent role in the loss of cells during myocardial infarction and heart failure [8, 30]. In addition, apoptosis is strongly linked to the development of atherosclerotic plaque vulnerability [22]. Therefore, apoptosis provides an attractive biological target for molecular imaging studies to better predict cardiovascular risk in the individual patients. For the detection of apoptosis, Annexin A5 has emerged as a valuable imaging biomarker [13]. Annexin A5 has strong affinity for phosphatidylserine (PS), a plasma cell membrane phospholipid that is externalized by apoptotic cells [24]. We will discuss the use of Annexin A5 imaging in atherosclerosis, myocardial ischemia, and heart failure, respectively. In addition, the review will provide novel insights in Annexin A5–PS biology, which form the basis of novel imaging and drug targeting strategies.

2 Apoptosis as a target in atherosclerosis

Coronary artery disease is a major cause of morbidity in the United States afflicting 13.7 million people [4]. Of these, approximately 1.5 million people develop an acute coronary event every year and up to 400,000 die from the acute event [29]. It has been long believed that progressive increase in plaque thickness leading to complete luminal occlusion results in acute infarction [27]. However, emerging biological information has radically altered this concept. It is now evident that although the progressive stenosis of the arterial lumen constitutes the basis of progressive ischemic symptoms, acute coronary events may be associated with less occlusive plaques. Acute vascular events usually occur from plaque rupture or plaque erosion leading to occlusive thrombus [5, 25]. The rupture occurs at the weakest point of the fibrous cap and exposes the thrombogenic lipid core to blood which initiates thrombotic occlusion. However, it is not clear what converts a stable plaque into a vulnerable plaque.

It has been proposed that the risk of plaque rupture depends upon the prevalence of plaques that have large necrotic core, dense inflammation and relatively thin protective fibrous caps [27]. The first characteristic of the vulnerable plaque is a large lipid core. The lipid in the core is likely derived either from necrosis of macrophage foam cells, or gradual insudation of ox-LDL in the plaque. It has recently been proposed that cholesterol in atherosclerotic plaques may also be derived from red blood cell membranes, which may be present in excess during intra-lesional hemorrhages associated with high density of neovascularization of plaque [21]. Thinning of the fibrous cap is a second important component of the vulnerable plaque. The fibrous cap is made up of collagen, which is predominantly derived from proliferating smooth muscle cells (SMC) of the plaque. The extracellular collagen provides stability to the plaque. The conversion of a stable plaque into a vulnerable plaque is associated with thinning of the fibrous cap. The thin-capped fibroatheroma has been described as a lesion with a fibrous cap of 65 µm or less.

The third important component of vulnerable plaques comprises of infiltration of the fibrous caps by macrophages and to some extent, by T lymphocytes [26]. The release of MMP from macrophages, such as collagenases, stromolysin and gelatinases leads to the digestion of extracellular matrix. In addition, the activated T cells release IFN-γ, which suppresses TGF-β induced signaling of production of procollagen type I and III.

Gradual loss of SMC perpetuates attenuation and weakening of the fibrous cap. Further, it has been proposed that extensive apoptosis of macrophages at the site of plaque rupture is associated with the acute coronary event. At the rupture site, other cell populations do not demonstrate significant apoptosis and the prevalence of apoptosis in various cells (including macrophages) is very low at remote site [22]. In conclusion, extensive apoptosis and inflammation appear to be closely associated with higher likelihood of plaque disruption.

3 Feasibility of imaging apoptosis in atherosclerotic lesions

In a pioneering study, Kolodgie and co-workers demonstrated the ability of exogenous radiolabeled Annexin A5 to detect apoptosis and inflammation in experimental atherosclerosis in vivo in a preliminary study of 5 NZW rabbits [23]. Atherosclerotic lesions were induced in three rabbits by de-endothelialization of the infradiaphragmatic aorta followed by 12 weeks of a high fat diet. Two control rabbits were studied without manipulation. Animals were injected intravenously with 0.5–1 mg of Annexin A5 labeled with 7–10 mCi of technetium-99m for in vivo imaging studies. After imaging studies, the abdominal aortas were explanted for ex vivo imaging and macroautoradiography. Histological examination was performed for characterization of the site of localization of the radiotracer. At 2 h, there was clear delineation of the radiolabel within the abdominal aorta by in vivo gamma imaging (Fig. 1). After explantation of the aorta, ex vivo imaging showed a robust uptake of radiotracer in both the arch and infradiaphragmatic aorta corresponding to the in-vivo images and confirming the macroscopic distribution of atherosclerotic lesions.
Fig. 1

Pathologic characterization of Annexin A5 uptake in the experimental atherosclerotic lesions. The lesions were classified as AHA type II (a, fatty streaks), III (b, extracellular lipid pools) and type IV (c, formation of necrotic core). Lesions higher than type IV were not seen. d Type IV lesions constituted 50% of total lesions, type III (30%) and type II (20%). e Significant Annexin A5 uptake was detectable only in type IV lesions. In situ end-labeling demonstrated higher macrophage apoptosis in type IV lesions (h, i) compared to less severe lesions (f, g, i) and hence explained higher annexin uptake in type IV lesions. (Kolodgie, Circulation Dec 2003).

The accumulation of 99mTc-Annexin A5 in atherosclerotic lesions in the balloon-denuded (abdominal) region of the aorta was approximately 9.3-fold greater than in the corresponding control abdominal aortic region. The mean ± SEM percent-injected dose per gram uptake in the specimens with lesions (0.054 ± 0.0095%) was significantly higher than the background activity in the normal specimens (0.0058 ± 0.001, P < 0.000). The Annexin A5 uptake was significantly higher in more advanced lesions (such as AHA type IV lesions, wherein the incidence of apoptosis was maximal.

The uptake correlated with the extent of macrophage infiltration in the plaques; there was no association with SMC burden. Although the rabbit model of atherosclerosis does not develop true unstable lesions, these data indicate that Annexin A5 targets to macrophages in the plaque. Since human unstable lesions exhibit an abundance of activated and/or apoptotic macrophages, the data suggest that Annexin A5 may be a suitable marker for plaque instability in patients with atherosclerotic disease.

4 Detection of Annexin A5 in the coronary tree in a swine model

A study in a swine model of atherosclerosis showed that the detection of uptake in the coronary tree is feasible using non-invasive nuclear imaging technology [16]. The hypothesis was tested that apoptosis in coronary arteries of swine with high cholesterol diet and vascular injury could be detected in vivo using Tc-99m Annexin A5 and SPECT imaging. In juvenile male swine atherosclerosis was induced by high fat diet combined with injury to the coronary vessels. After average of 51 ± 9 days the animals underwent coronary angiography, injection with 8–10 mCi of Tc-99m Annexin A5 and SPECT imaging. The animals were sacrificed, the hearts removed, vessels imaged by autoradiography and counted in the well counter. Immunohistopathology was performed for α-actin and caspase 3.

Atherosclerotic lesions were early and mild and characterized by smooth muscle cells. Thirteen of the 22 vessels showed focal uptake of Tc-99m Annexin A5 in vivo and corresponded to uptake on autoradiography (Fig. 2). The count ratio of the injured over control vessel was 2.7 ± 0.8 for positive scans and was 1.3 ± 0.4 for negative scans. The percent injected dose per gram uptake for the positive and negative scans were 1.54 ± 1.30 × 10−3 and 0.38 ± 0.15 × 10−3, respectively. Scan positive for focal annexin uptake correlated with caspase-positive staining by quantitative morphometry. These data indicate that detection of the uptake of Annexin A5 in atherosclerotic lesions of the coronary tree is feasible.
Fig. 2

Left upper panel shows SPECT reconstructions from swine with high cholesterol diet and injury to the RCA. White arrows point to focal uptake in the region of the injured RCA. Right upper panel shows autorads of RCA with a long segment of uptake of radioactivity in the proximal vessel. The lower panel shows histology from two vessels—scan positive on left and scan negative on right. The sections were stained with anti-caspase 3 (brown) and counter stained with methyl green which is a nuclear stain. The number of caspase positive cells as percentage of total cells was 55% for the scan positive experiment and 15% for the scan negative experiment.

In a pilot attempt to evaluate apoptosis in clinical scenario, evaluation of Annexin A5 imaging was performed in four patients with recent or remote history of transient ischemic attack (TIA), respectively, 1–3 days prior to carotid endarterectomy [19]. Six hours after infusion of 600–800 MBq 99mTc-Annexin-V, gamma images were obtained with a multi-SPECT camera.

Two patients had suffered from a TIA 3–4 days prior to imaging, and showed distinct Annexin-V uptake (Fig. 3) in the area of the ultrasonically verified carotid artery lesion. Histopathological characterization of the endarterectomy specimen revealed unstable plaque morphology, including significant macrophage infiltration and intraplaque hemorrhage. Immunohistochemical analysis demonstrated binding of Annexin A5 predominantly to the macrophages. The remaining two patients, who had suffered from TIA 3–4 months prior to imaging, showed no Annexin-V uptake in the carotid artery. These patients had severe carotid lesion and were being treated with statins and anti-platelet agents after the acute event. Endarterectomy specimens revealed stable plaque characteristics with insignificant macrophage infiltration and no intra-plaque hemorrhage; negligible Annexin binding was observed on immuno-histochemical analysis. These data indicate that 99mTc-Annexin-V targets to macrophage rich lesions of plaques with unstable plaque morphology. In addition the data show that Annexin A5 imaging of unstable lesions is clinically feasible and may identify patients at risk for acute vascular events, such as stroke and/or acute myocardial infarction (AMI).
Fig. 3

Imaging of unstable carotid artery atherosclerotic lesion with radiolabeled Annexin-V. a Transverse and coronal SPECT images from a patient, who suffered from left sided TIA 3 days prior to imaging. Although this patient demonstrated significant stenosis of both carotid arteries, Annexin-V uptake was only seen in the culprit lesion. b Histopathological analysis of endarterectomy specimen from the same patient shows significant macrophage infiltration in the neointima with extensive Annexin-V binding. c SPECT images from the another patient, who had a right sided TIA 3 months prior to imaging, did not show Annexin A5 uptake in the carotid region on either side; however, Doppler ultrasound revealed significant obstructive lesion on the affected side. d Histopathological analysis of endarterectomy specimen from this patient showed smooth muscle cell (SMC) rich lesion with no Annexin-V binding. (Kietselaer 2004).

5 The use of Annexin A5 as an endpoint for plaque stabilizing compounds

In a first attempt to evaluate experimental compounds for acute manipulation of apoptosis the potential role of statins was investigated using imaging with 99mTc-labeled annexin A5 [11]. In NZW rabbits experimental atherosclerosis was induced by abdominal aorta balloon de-endothelialization and high cholesterol diet (0.5%) and 6 unmanipulated rabbits, fed normal chow, were used as controls. The hypercholesterolemic animals were randomized to receive diet withdrawal or statin therapy. These data show that after 1 month of treatment substantial decrease (40–50%) in the uptake of Annexin A5 is be observed, which is in concordance with clinical data, showing treatment benefit of statins after 1 month (Fig. 4).
Fig. 4

Annexin A5 as an imaging biomarker for plaque stabilizing compounds. In contrast to the control animals (lane 1) extensive uptake of Annexin A5 is observed in the high fat diet group (lane 2). Diet withdrawal for 3 months results in substantial less uptake of Annexin A5 (lane 3). However, statin therapy for 1 month results in an as extensive reduction of Annexin A5 uptake (lane 4) as compared to diet withdrawal.

Histopathologic characterization revealed that there was a substantial decrease in TUNEL-staining-verified prevalence of apoptosis in macrophages in the statin treated group. These data suggest that acute changes in plaque biology can be induced through the use of statin therapy, which is reflected by a decrease in the uptake of Annexin A5. Whether this will result in acute plaque stabilization remains to be investigated. These data suggest that Annexin A5 may serve as a surrogate imaging biomarker for the efficacy of plaque stabilizing compounds, such as statins.

6 Annexin A4 as an imaging biomarker in heart failure

Heart failure has emerged as one of the most important cardiovascular health problems. It has become the number one reason for hospital admission in the USA in patients of 65 years of age and older [34].

Apoptosis or PCD of cardiomyocytes has been projected as an important biological process that results in ongoing loss of heart muscle cells and ventricular dysfunction [20]. Preclinical studies have shown that anti-apoptotic intervention results in delay in the development of left ventricular dilatation and heart failure [12]. Furthermore, clinical studies in explanted hearts of patients have demonstrated increased apoptosis of cardiomyocytes as compared to controls, suggesting that apoptosis in the failing hearts of patients may play a role in the slowly deteriorating pump function of the left ventricle [30].

Activation of caspase 3, one of the hallmarks of apoptosis, has been observed both in preclinical models of heart failure as in patients with heart failure [10]. It has been demonstrated that cytokinemia and ischemic/oxidative stress results in the release of cytochrome c from the mitochondria into the cytoplasmic compartment, which in turn and leads to activation of caspase 3. Active caspase 3 cleaves contractile proteins, within cardiomyocytes and activates DNA fragmentation enzymes in most cell types. However, it has been demonstrated that in cardiomyocytes the activation of DNA-es is counterbalanced by anti-apoptotic factors. The result is that cardiomyocytes may become dysfunctional upon activation of the apoptotic program through the caspase induced cleavage of contractile proteins, but survive due to appropriate inhibition of DNA cleaving enzymes.

Activation of caspase 3 leads to a change in phospholipid distribution in the sarcolemmal lipid bilayer, resulting in exposure of PS to the outer surface of the plasma cell membrane. In theory, the extent of PS exposure is a reflection of the activation of the apoptotic program. Therefore, PS may be an attractive target to use as a biological marker for the detection of cell death activation in the failing heart, which could be imaged by Annexin A5. Identification of apoptosis activation in the failing heart may become of future use to identify patients suitable for novel therapeutic interventions [31].

To evaluate the feasibility of Annexin A5 imaging for the detection of PCD, a small group of patients with recently diagnosed idiopatic dilated cardiomyopathy (DCMP) heart failure were evaluated using Annexin A5 [18]. SPECT imaging of technetium labeled Annexin was performed in nine consecutive heart failure patients with advanced non-ischemic cardiomyopathy (dilated, N = 8 and hypertrophic, N = 1), and also in two relatives of the hypertrophic cardiomyopathy patient having a similar genetic background, but no heart failure. In four patients with dilated cardiomyopathy and one with hypertrophic cardiomyopathy and heart failure showed focal, multifocal or global left ventricular uptake of Annexin A5 (Fig. 5).
Fig. 5

Uptake of technetium labeled Annexin A5 in dilated cardiomyopathy. a Patient with a recent history of idiopatic DCMP focal uptake is found on SPECT imaging in the anterolateral region of the heart. b Patient with a recent history of DCMP global uptake of Annexin A was found. c A negative case of Annexin A5 uptake in DCMP is shown, and in d a negative control (adapted from Kietselaer 2007)

No uptake was visualized in the remaining four patients, and two controls. All cases showing Annexin A5 uptake within the left ventricle suffered significant reduction in left ventricular function and/or functional class, as assessed by echocardiography after one year follow up. In cases with no Annexin A5 uptake, left ventricular function and clinical status remained stable or even improved (Fig. 6). These data indicate the feasibility of non-invasive PCD detection with Annexin A5 imaging in heart failure patients. Annexin A5 uptake is associated with deterioration in left ventricular function and may lend itself to development of novel management strategies.
Fig. 6

Graph depicting the change in left ventricular ejection fraction 1 year after follow up in patients with a postive Annexin A5 scan (red) and a negative Annexin A5 (adapted from Kietselaer 2007)

The explanation for the uptake of Annexin A5 in the context of activation of the apoptotic program in cardiomyocytes in given in Fig. 7. The activation of caspase 3 results in the externalisation of PS. However, since activation of DNA-es is inhibited in cardiomyocytes, these cells do not necessarily die from activation of the cell death program. Therefore, the concept put forward is, is that the extent of PS externalisation is a reflection of the degree of caspase 3 activation within cardiomyocytes. This concept indicates that Annexin A5 uptake is not necessarily equivalent to cells dying from apoptosis, but merely reflects PS exposure, which may be a consequence of activation of essential parts of the apoptotic machinery.
Fig. 7

The concept of PS externalization in the failing heart. In the failing heart, cytokinaemia, ischemia, and the generator of reactive oxygen species (ROS) result in either the activation of caspase-8 or the release of cytochrome c from mitochondria. These events in turn activate caspase 3, one of the executore caspases. In the cardiac myocyte, caspase 3 mediated activation of DNA-es is counter balanced by inhibitors. Substrates of caspase 3, such as contractile proteins, are cleaved by caspase 3, which hamper the function of the cardiomyocytes. PS exposure in the failing cardiomyocyte reflects the balance between caspase 3 activation and inhibition through XIAP

7 Annexin A5 as an imaging biomarker in myocardial ischemia

One of the strongest triggers for apoptotic cell death in the heart is ischemia, followed by reperfusion [1]. In animal models of ischaemia/reperfusion, it has been shown that mitochondria release pro-apoptotic factors such as cytochrome c and reactive oxygen species (ROS), which may in turn activate downstream caspases. In addition proteolytically cleaved caspases 2, 3 and 7 have also been detected in ischaemia/reperfusion models, suggesting a role for receptor-mediated cell death. Thirdly, calcium overload plays an important role in cardiomyocyte apoptosis following ischaemia/reperfusion. Cardiomyocyte apoptosis usually occurs within the first 2–4 h after the acute insult, and is subsequently followed by myocyte necrosis.

Since the activation of caspase 3 mediates externalization of PS, as was discussed previously, Annexin A5 may provide an efficient means to detect the kinetics of apoptosis following ischemia and reperfusion of the heart. In an ischemia reperfusion model of the mouse heart, we have started to use optical imaging technology and fluorescently labeled Annexin A5 to detect PS exposure [7]. These data showed that Annexin A5 binding occurs rapidly after reperfusion and progresses in a wave front over the area at risk in the left ventricle. The kinetics of the progression show that PS exposure starts within minutes after the onset of reperfusion, and seems completed after about 30 min of reperfusion (Fig. 8).
Fig. 8

Planar imaging of fluorescently labeled Annexin A5 following ischemia and reperfusion of the mouse heart in vivo (I/R 30/1 means 30 min of ischemia and 1 min of reperfusion). Binding of Annexin A5 occurs early after reperfusion (upper panel), and proceed rapidly within the first 30 min after the onset of reperfusion (adapted from Dumont 2001)

To follow up on the work in animal models, clinical studies were undertaken to evaluate the feasibility of technetium labeled Annexin A5 to detect cell death in the heart of patients suffering from AMI [13]. These data show that uptake of Annexin A5 occurred in exactly the same localization as the perfusion defect observed in on perfusion scintigraphic imaging (Fig. 9).
Fig. 9

Cell death imaging using Annexin A5 in a patients with acute anteroseptal myocardial infarction. The arrow in the upper panel shows uptake of technetium labelled Annexin A5 on the day of admission (L liver uptake). Perfusion scintigraphy taken at rest using sestamibi in the lower panel shows a defect at exactly the same localisation (adapted from Hofstra 2000)

In a follow up study in patients with AMI it was found that the uptake of Annexin A5 in the acute stage extended beyond the perfusion defect as shown on the perfusion scintigram 1 week after the cardiac insult. These data may suggest that not all PS positive cardiomyocytes will eventually undergo apoptosis [35]. This is in line with the concept proposed for patients with CHF, where activation of caspase 3 result in PS externalization, but not necessarily the entire execution of PCD.

8 Outlook

Based on the preclinical and clinical evidence obtained over the years, PS exposure in the cardiovascular system provides an attractive biological target in atherosclerosis, heart failure, and cardiac ischemia. The high affinity of Annexin A5 for PS, combined with the cross-modality imaging options of the molecule have given Annexin A5 an appealing status as an imaging biomarker for cardiovascular disease.

However, before Annexin A5 could be adopted as an imaging biomarker for routine clinical use, extensive clinical studies need to be conducted. For instance, it is still unknown whether uptake of Annexin A5 in carotid artery lesions of patients is associated with increased event rate. To evaluate this clinical question, large prospective studies need to be performed in patients not undergoing surgery, for instance in patients with 50–70% lesions of the carotid artery. Likewise, prospective cohort studies in patients with CHF are needed to assess the predictive value of Annexin A5 imaging in these patients.

Other prospects of the use of Annexin A5 may be given by the need of the pharmaceutical industry to develop imaging biomarkers as read out for the evaluation of novel cardiovascular drugs. For instance, the strong association of Annexin A5 with unstable atherosclerotic lesions in patients, may open novel opportunities for early clinical evaluation of novel plaque stabilizing compounds.

For ischemic heart disease, the use of Annexin A5 is probably of limited value. The existence of alternative cheap biomarkers for the detection of acute ischemia, such as the ECG, and the need for acute revascularization precludes the use of a relatively time consuming Annexin A5 imaging study.

Conflict of Interest


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