Annexin A5: an imaging biomarker of cardiovascular risk
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- Laufer, E.M., Reutelingsperger, C.P.M., Narula, J. et al. Basic Res Cardiol (2008) 103: 95. doi:10.1007/s00395-008-0701-8
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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 wordsAnnexin A5apoptosisunstable plaquecardiovascular riskimaging
Cardiovascular disease is the leading cause of death in the Western world . In addition, it is predicted that the incidence of cardiovascular disease will rapidly increase in emerging economies, such as China and India . 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 .
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 . 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 .
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 . 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 . 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 . This development is based on findings in atherosclerosis research that atherosclerotic plaque rupture is strongly associated with plaque inflammation . 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). 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 . 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 .
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 . 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 . 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 . 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 . Annexin A5 has strong affinity for phosphatidylserine (PS), a plasma cell membrane phospholipid that is externalized by apoptotic cells . 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 . Of these, approximately 1.5 million people develop an acute coronary event every year and up to 400,000 die from the acute event . It has been long believed that progressive increase in plaque thickness leading to complete luminal occlusion results in acute infarction . 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 . 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 . 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 . 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 . 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
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 . 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.
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 . Six hours after infusion of 600–800 MBq 99mTc-Annexin-V, gamma images were obtained with a multi-SPECT camera.
5 The use of Annexin A5 as an endpoint for plaque stabilizing compounds
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 .
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 . Preclinical studies have shown that anti-apoptotic intervention results in delay in the development of left ventricular dilatation and heart failure . 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 .
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 . 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 .
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 . 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.
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 . 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.
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