Cardiovascular molecular imaging of apoptosis Introduction Molecular imaging strives to visualise processes at the molecular and cellular level in vivo . Understanding these processes supports diagnosis and evaluation of therapeutic efficacy on an individual basis and thereby makes personalised medicine possible . Apoptosis The term ` apoptosis ' is derived from the Greek -LRB- apo = from and ptosis = falling , commonly pronounced ap-a-tow ' - sis -RRB- and denotes a regulated process of cell suicide resulting in a cell corpse that is distinct from the necrotic cell -LSB- 1 , 2 -RSB- . Whereas necrosis is manifested by cellular swelling , plasma membrane rupture and rapid release of intracellular constituents into the environment , causing an inflammatory response -LSB- 2 -RSB- , apoptosis is characterised by cell shrinkage , long-lasting maintenance of plasma membrane integrity and lack of inflammatory responses in the vicinity of the dying cell -LSB- 2 -RSB- . It has long been thought that apoptosis and necrosis are the only two modes of cell death counterbalancing mitosis -LSB- 3 -RSB- . However , recent insights have taught us that cells can follow more roads to death than only apoptosis and necrosis . In 2001 , Leist and Jaättelä suggested a model in which various forms of cell death could be positioned on a gliding scale between two extremes , apoptosis and necrosis -LSB- 4 -RSB- . The mode of execution depends on the cell type and the cell death-inducing trigger . Most knowledge about various modes of cell death has been generated in the field of oncology , where cell death plays an important role in the progression and treatment of cancer -LSB- 5 -RSB- . Several intermediate forms of cell death have been identified and characterised -LSB- 6 , 7 -RSB- , including mitotic catastrophe and autophagic cell death . Mitotic catastrophe can be the result of unsuccessful chromosome segregation , an event that requires malfunctioning of multiple cell cycle checkpoints . Catastrophic cells are large and non-viable cells with a compromised nuclear blueprint leading eventually to cell death -LSB- 8 -RSB- . Mitotic catastrophe appears to be a rare event in cardiovascular diseases . Autophagic cell death is characterised by the total destruction of the cell through autophagy . It is a process in which cytoplasmic constituents are degraded through the lysosomal machinery -LSB- 9 -RSB- . Autophagic cell death occurs both in oncology -LSB- 10 , 11 -RSB- and in cardiovascular diseases -LSB- 12 -RSB- . The various described modes of cell death differ in morphology and biochemistry -LSB- 13 -RSB- . The caspase cascade is the most remarkable biochemical distinction -LSB- 14 -RSB- . It is activated during apoptosis but hardly , or not at all , during other modes of cell death . Biochemistry of apoptotic cell death Two major biochemical routes dominate apoptotic cell death . They involve the activation of the caspase cascade through either death-receptor mediated signal transduction or stress-induced release of cytochrome C from mitochondria -LSB- 15 -RSB- . Caspases involved in apoptosis can be grouped into initiator -LRB- caspases 8 and 9 -RRB- and effector caspases -LRB- caspases 3 and 7 -RRB- . The principal event of the caspase cascade is the activation of an inactive procaspase into a proteolytically active caspase . Activation of initiator caspases occurs through auto-activation , catalysed by the interaction with ligated death receptors -LRB- procaspase 8 -RRB- or cytochrome C-containing apoptosome -LRB- procaspase 9 -RRB- . Effector caspases are activated through intra-chain cleavage by active initiator caspases -LSB- 16 -RSB- . In both cases , activation results in the exposure of active sites that selectively cleave peptide bonds after an aspartate residue in caspase substrates . Several checks and balances exist that control burst of the caspase cascade if triggered by minor undesired initiating events . Inhibitors of apoptotic proteins -LRB- IAPs -RRB- suppress proteolytic activity by binding to activated caspases . Inhibition of IAPs is in turn required to allow execution of apoptosis . Smac/DIABLO residing in the mitochondria and co-released with cytochrome C inhibits IAPs and permits propagation and amplification of the proteolytic signal through the caspase cascade . Anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-X prevent the release of cytochrome C and Smac/DIABLO from mitochondria . Pro-apoptotic members such as Bax and tBid , which is the result of Bid cleavage by activated caspase 8 , neutralise the protective effects of the anti-apoptotic members and provoke mitochondria to release their pro-apoptotic cargo . The intricate scheme of the apoptotic machinery offers several potential targets for molecular imaging . For example , knowledge about the mechanism of caspase activation has triggered the design of several strategies to measure apoptosis -LSB- 17 -- 19 -RSB- . The hurdle these methods have to take is entrance of the reporter of caspase activity into the cell . Consequently , in vivo applications of such strategies face unfavourable biodistribution profiles and high background levels . Targets embedded in the plasma membrane encompass a more promising set of molecules for molecular imaging . Phosphatidylserine An essential part of the apoptotic program consists of the appearance of ` eat me ' flags at the cell surface . Phagocytes recognise these flags and respond by engulfing the dying cell before it leaks pro-inflammatory components into the surrounding tissue . The ` eat me ' flags are , alone or in combination , specific for the dying cell , allowing phagocytes to make the right choice in an environment filled with living cells . In 1992 , Valerie Fadok and co-workers reported that phosphatidylserine -LRB- PS -RRB- becomes exposed on the surface of apoptotic lymphocytes , where it functions as an ` eat me ' signal for phagocytes -LSB- 20 -RSB- . PS , a negatively charged aminophospholipid , is predominantly found in the cytofacial membrane leaflets of living cells . The exofacial plasma membrane leaflet contains predominantly phosphatidylcholine and sphingomyelin while PS is almost completely lacking . The PS asymmetry results from the ATP-dependent action of the aminophospholipid translocase that transports PS from the outer to the inner leaflet -LSB- 21 , 22 -RSB- . During apoptosis the aminophospholipid translocase is inhibited and concomitantly a scramblase is activated . The combined action results in the surface expression of PS whilst the plasma membrane integrity remains intact . Surface-expressed PS provides an attractive target for molecular imaging of apoptosis . Annexin A5 In the past decade , a molecular imaging protocol was established to detect and measure apoptosis in vitro and in vivo in animal models and patients -LSB- 23 -- 26 -RSB- . This molecular imaging protocol is based upon the protein annexin A5 -LRB- Anx A5 -RRB- , which binds with high selectivity and with great affinity -LRB- Ka = 7 nM -RRB- -LSB- 27 , 28 -RSB- to the PS abnormally expressed on the cell membrane of apoptotic cells -LSB- 21 , 22 , 29 -RSB- . Anx A5 is a member of a multiprotein family of more than 160 proteins that share the property of Ca2 + - dependent binding to negatively charged phospholipid surfaces -LSB- 30 -RSB- . The biophysical , biochemical and biological properties of Anx A5 and other members of the annexin family have been extensively described in a number of excellent reviews -LSB- 30 -- 34 -RSB- . Detecting apoptosis with Anx A5 The development of annexin-based detection of apoptosis started in 1992 when Fadok et al. revealed that PS is expressed on the cell surface of apoptotic cells -LSB- 35 -RSB- . This revelation led Koopman et al. to the design of an apoptosis detection assay on the basis of fluorescence -LRB- fluorescein isothiocyanate -RRB- - labelled Anx A5 -LSB- 29 -RSB- . This Anx A5 affinity assay was further developed by labelling Anx A5 with biotin or with several radionuclides to facilitate various protocols for measuring apoptosis both in vitro -LSB- 36 -RSB- and in vivo -LSB- 23 -- 25 , 37 -- 41 -RSB- in animal models . The availability of 99mTc-labelled Anx A5 produced under GMP regulations led to the first studies of non-invasive detection of apoptosis in patients -LSB- 26 , 42 -- 46 -RSB- . Detection of apoptosis using alternative methods In addition to Anx A5 , several other proteins may have a high specificity and tight binding to PS . If labelled with a fluorescent or nuclear probe , these proteins could possibly be used for in vivo detection of apoptotic cells in the field of cardiovascular disease and in other disciplines . In addition , other steps in the apoptotic signalling cascade , such as the activation of caspase-3 , can be utilised to visualise activation of the apoptotic machinery -LRB- Fig. 1 -RRB- . Fig. 1Targets for apoptosis detection . During apoptosis , initiator caspases are activated , via either cell death receptor-mediated or mitochondrial signalling . These initiator caspases , in turn , trigger the activation of effector caspases , such as caspase-3 . The activation of caspase-3 results in the typical characteristics of apoptosis , such as DNA fragmentation , substrate cleavage of cytoplasmic proteins and cell membrane alterations . These apoptotic characteristics and the activation of caspase-3 offer targets for molecular imaging of apoptosis Synaptotagmin I One such PS binding protein is the C2 domain of synaptotagmin I , which binds to anionic phospholipids in cell membranes -LSB- 47 -RSB- . By conjugation of the C2 domain , synaptotagmin can be used for in vivo molecular imaging of apoptosis . Zhao and co-workers conjugated the C2 domain with superparamagnetic iron oxide -LRB- SPIO -RRB- particles , a very effective T2 relaxing MRI contrast agent , and injected the product in tumour-bearing mice . They revealed that a murine lymphoma -LRB- EL4 -RRB- tumour model treated with cyclophosphamide and etoposide showed an increase in apoptotic cells from a basal value of 4 % to 32 % during chemotherapy . This was readily detected with C2-labelled SPIO particles and T2W MRI . Tumour regions with the greatest MRI changes correlated with regions having the highest proportion of apoptotic and necrotic cells -LSB- 47 -RSB- . More recently , the same group labelled the C2A domain of synaptotagmin I radioactively -LRB- 99mTc -RRB- or fluorescently -LRB- FITC -RRB- and used the labelled synaptotagmin in a reperfused acute myocardial infarction model -LRB- AMI -RRB- rat model . They found both ex vivo and in vivo accumulation of the radiotracer in the area at risk . Although some of the uptake was caused by passive leakage due to elevated vascular permeability in the area at risk , the majority was caused by specific binding to PS -LSB- 48 -RSB- . Both Anx A5 and synaptotagmin I detect apoptotic cells by selective binding to externalised PS . An alternative method to detect apoptosis may be to use a target more upstream in the apoptotic cascade , the effector caspases . 5-Pyrrolidinylsulphonyl isatins In one study the peptide-based irreversible pan-caspase inhibitor Z-VAD-fmk was radio-iodinated and suggested as a caspase imaging agent . However , due to poor cellular permeability , intracellular targeting of activated caspases was limited , preventing in vivo application -LSB- 49 -RSB- . In more recent work , non-peptidyl caspase inhibitors of the 5-pyrrolidinylsulphonyl isatin-type were proposed instead of a peptide-based caspase , such as Z-VAD-fmk . These caspase binding radioligands -LRB- CBRs -RRB- may be capable of directly targeting apoptosis in vivo . CBRs are expected to form intracellular enzyme inhibitor complexes by means of binding to the activated caspases . Kopka and co-workers succeeded in labelling several 5-pyrrolidinylsulphonyl isatins with iodine-125 -LSB- 50 -RSB- . However , none of these compounds have been used for non-invasive imaging in patients yet . ApoSense The ApoSense family is a group of low molecular weight amphipathic apoptosis markers targeting the cell membrane of apoptotic cells . ApoSense was developed by the Israel-based company NST . ApoSense has been used in vitro and in vivo in several disease models associated with cell death , such as radiation-induced lymphoma , renal ischaemia/reperfusion and cerebral stroke -LSB- 51 , 52 -RSB- . However , it is still not known to which cell membrane target ApoSense exactly binds . Therefore , it remains a challenge to link ApoSense binding to the signalling cascades in apoptosis signalling . Role and detection of apoptosis in CVD Apoptosis of cardiomyocytes after myocardial infarction Several forms of cell death have been observed in the infarcted myocardium . The central ischaemic zone is dominated by necrotic cell death , whereas in the periphery of the area at risk mainly apoptotic cell death is observed . Apoptotic cell death in the infarct periphery is not the exclusive domain of the cardiomyocyte . Also non-myocytes , including endothelial cells , macrophages and blood cells , contribute to a comparable degree to apoptotic cell death -LSB- 53 -RSB- . Since the process of cell death can be pharmaceutically manipulated , thereby limiting the extent of myocardial cell loss , detection and quantification of apoptotic cells are of importance for determination of the full extent of reversible myocardial damage . Once the apoptotic program is activated , several targets are present both for imaging apoptosis and for therapeutic interventions . As described earlier , the C2A domain of synaptotagmin I binds to the exposed PS on apoptotic cells . Zhao and co-workers developed a radiotracer for PS expression by labelling synaptotagmin via 2-iminothiolane thiolation with 99mTc , forming a technetium C2A glutathione S-transferase complex -LRB- 99mTc-C2A-GST -RRB- -LSB- 48 -RSB- . In vivo planar imaging of AMI in rats was performed on a gamma camera using a parallel-hole collimator . The uptake of 99mTc-C2A-GST within the area at risk was quantified by direct gamma counting . The foremost finding was that in seven of seven rats the infarct was clearly identifiable as focal uptake in planar images . This and other findings led the authors to conclude that the C2A domain of synaptotagmin I labelled with a radioisotope binds to both apoptotic and necrotic cells . Ex vivo and in vivo data indicate that , because of elevated vascular permeability , both specific binding and passive leakage contribute to the accumulation of the radiotracer in the area at risk . However , the latter component alone is insufficient to achieve detectable target-to-background ratios using in vivo planar imaging . Annexin A5 has been more widely used to target PS exposure . Hiller and co-workers -LSB- 54 -RSB- recently reported on a new Anx A5-based T1 contrast agent . They linked Anx A5 to gadolinium diethylenetriamine penta-acetate -LRB- Gd-DTPA -RRB- - coated liposomes . In their model of perfused isolated rat heart , ligation of the left coronary artery for 30 min was followed by reperfusion . T1 and T2 * images were acquired using an 11.7-T magnet before and after intracoronary injection of the contrast agent . A significant increase in signal intensity , visible in those regions containing cardiomyocytes in the early stage of apoptosis , was found . The group of Weissleder also reported on Anx A5-based MRI contrast agents -LSB- 55 -RSB- , demonstrating the construction of a magneto-optical nanoparticle AnxCLIO-Cy5.5 , which was tested in a murine model of transient coronary artery -LRB- LAD -RRB- occlusion . The synthesis of the probe was achieved in three steps . First , the amino-CLIO nanoparticle was labelled with Cy5 .5 and activated with SPDP -LSB- N-succinimidyl 3 - -LRB- 2-pyridyldithio -RRB- propionate -RSB- , to yield a compound termed 2PySS-CLIO-Cy5.5 . Second , Anx A5 was reacted with SATA -LRB- N-succinimidyl S-acetylthioacetate -RRB- . In the final step of the process , SATAylated Anx A5 was linked with 2PySS-CLIO-Cy5.5 to yield the multimodal AnxCLIO-Cy5.5 nanoparticle . It was demonstrated that it is feasible to obtain high-resolution MR images of cardiomyocyte apoptosis in vivo with the use of the nanoparticle AnxCLIO-Cy5.5 . In early work preformed by our group a fluorescent invasive Anx A5-based imaging protocol for myocardial apoptosis was developed in mice -LSB- 25 -RSB- . The promising findings of this study encouraged use of modified human recombinant Anx A5 to construct -LRB- Anx A5 -RRB- - n-1-imono-4-mercaptobutyl , labelled with pertechnetate for clinical studies . One milligram of 99mTc-Anx A5 -LRB- 584 MBq -RRB- was injected intravenously 2 h after reperfusion in seven AMI patients . In six of the seven patients , increased uptake of 99mTc-Anx A5 was seen in the infarcted region of the heart on single-photon emission computed tomography -LRB- SPECT -RRB- images . No uptake was seen in the heart outside the infarcted area . In all individuals with increased uptake , a matching perfusion defect -LRB- on MIBI SPECT -RRB- was observed -LRB- Figs. 2 , 3 -RRB- . These results demonstrated the feasibility of non-invasive monitoring of apoptosis in patients . These techniques may allow measurement not only of the extent of reversible damage but also of the efficacy of novel therapies targeting apoptosis . Fig. 2Transverse tomographic images in a patient with acute anteroseptal infarction . a Arrow shows increased 99mTc-labelled Anx A5 uptake in the anteroseptal region 22 h after reperfusion . b Perfusion scintigraphy with sestamibi 6 -- 8 weeks after discharge shows an irreversible perfusion defect which coincides with the area of increased 99mTc-labelled Anx A5 uptake -LRB- arrow -RRB- . L liver . Reprinted with permission from Elsevier -LRB- The Lancet , 2000 , 356 , 211 -RRB- Fig. 3Transverse tomographic images in a patient with acute anterior wall myocardial infarction . a Arrow shows increased uptake of 99mTc-labelled Anx A5 in the infarct area 17.5 h after reperfusion . L liver . b Perfusion defect on sestamibi perfusion scintigraphy 6 -- 8 weeks after discharge matches the uptake of Anx A5 -LRB- arrow -RRB- . Reprinted with permission from Elsevier -LRB- The Lancet , 2000 , 356 , 211 -RRB- Atherosclerotic lesions and plaque instability Extensive histopathological studies and investigations on the initiation of atherosclerosis in transgenic mice have generated a quite detailed picture of the development of atherosclerosis in the various stages of disease -LSB- 56 -- 63 -RSB- . The onset of plaque formation is characterised by circulating levels of oxidised low-density lipoproteins , which are capable of penetrating the endothelial border while inflicting oxidative damage on the endothelial cells in the process . Subsequently , the reactionary expression of inflammatory markers on the endothelial cell surface attracts circulating monocytes to the site of injury , which , upon arrival at the scene , differentiate into macrophages and start ingesting the available lipid and other oxidised particles . Uninhibited intracellular lipid accumulation compromises the function of macrophages and converts them into foam cells -LSB- 64 -RSB- . The accompanying secretion of pro-inflammatory cytokines and matrix-metalloproteinases -LRB- MMP -RRB- likely contributes to the hostility of the plaque environment and weakening of collagen matrix structures , both indirectly through loss of smooth muscle cells and directly through MMP activity -LSB- 65 -RSB- . At a certain point , excess damage to macrophages and smooth muscle cells induces apoptosis , causing remnants of lipid-laden dead cells to form small extracellular lipid droplets . A luxating moment in atherogenesis is thought to occur when the remaining population of functional phagocytes becomes unable to engulf all apoptotic remnants of dying cells in the environment -LSB- 65 -RSB- . Once the lesion contains a large , necrotic , lipid pool that is covered by a fibrous cap , primarily consisting of SMCs and extracellular matrix , it is named an atheroma . A critical finding for vascular risk prediction is that although plaque rupture generally requires progression of lesion size , acute events resulting from rupture may just as well originate from only mildly stenotic lesions -LSB- 66 -RSB- . Coronary catheterisation studies in patients admitted with acute coronary syndromes have shown that most of the culprit lesions are below the 50 % stenosis threshold -LSB- 67 -- 69 -RSB- . In reality , the extent of the lesion may be underestimated , since conventional coronary catheterisation does not take the extent of outward remodelling of the coronary artery at the site of atherosclerotic lesions into account . The emerging cardiac multislice CT technology may be better equipped to take outward remodelling into account and to assess the total coronary plaque burden in patients . Nevertheless , these data imply that though plaque dimensions will in general be sufficient for imaging-target visualisation , focussing on mere plaque size entails unsatisfactory prognostic information for clinical use . From a biological perspective , several inflammatory parameters are associated with vulnerable lesions , such as elevated extracellular levels of MMP and pro-inflammatory cytokines , including a variety of interleukins and chemotactic proteins , as has been shown in both experimental and clinical studies -LSB- 70 -- 73 -RSB- . Moreover , high-risk lesions are notorious for displaying high levels of inflammation and apoptotic cells ; in particular , large amounts of apoptotic macrophages have been found to reside in fragile and ruptured fibrous caps -LSB- 59 , 65 , 74 -RSB- . In 2000 , Kolodgie et al. -LSB- 59 -RSB- examined 40 culprit lesions from cases of sudden coronary death and reported apoptotic macrophages to constitute up to 50 % of the total macrophage population , preferentially localising at the site of rupture . Also , once atheromas have formed , plaques have been found to be far more sensitive to SMC death than regular arterial walls , as recently demonstrated by the group of Bennett -LSB- 61 -RSB- . Since all these features share their relation to inflammation and more specifically apoptosis , detection of apoptosis in vivo seems a sensible approach to vulnerable plaque identification . This approach builds on the pivotal role that apoptosis plays in promoting plaque progression and disruption ; however , the underlying mechanism is a complex interplay in which apoptosis affects endothelial cells , smooth muscle cells and macrophages in a different manner and with different consequences . Given the firmly established direct causal relationship between apoptosis and lesion instability -LSB- 59 , 75 -RSB- , in vivo visualisation of the apoptotic plaque status could represent a significant leap forward in the field of therapeutic management for individual patients -LRB- Fig. 4 -RRB- . Fig. 4Feasibility of non-invasive imaging of apoptosis by radiolabelled Anx A5 . Left lateral oblique gamma images of experimental atherosclerotic -LRB- a -- c -RRB- and control -LRB- d -- f -RRB- rabbits injected with 99mTc-Anx A5 ; L and K indicate liver and kidney activities , respectively . Images at the time of injection -LRB- a , d -RRB- and at 2 h after injection -LRB- b , e -RRB- are shown . Although blood pool activity is seen at the time of injection -LRB- a -RRB- in the atherosclerotic animal , tracer uptake is clearly visible in the abdominal aorta -LRB- with lesions -RRB- at 2 h -LRB- b -RRB- . c Ex vivo image of b shows intense 99mTc-Anx A5 uptake in the arch and abdominal region . Annexin-positive areas were confirmed to contain macrophage - and apoptosis-rich regions in the atherosclerotic plaque by histology . d -- f show the corresponding images in the control animal . Note that the aorta is indistinguishable from background at 2 h after injection -LRB- e -RRB- . The blood pool at the time of injection in the control animal -LRB- d -RRB- is comparable to that in the atherosclerotic animal . f Ex vivo aortic image of the control animal demonstrates the absence of 99mTc-labelled Anx A5 uptake . From Kolodgie et al. -LSB- 75 -RSB- The expanding knowledge about plaque biology has led to the identification of a variety of potentially suitable molecular markers for vulnerable plaque imaging -LSB- 76 -RSB- . Studies have been reported on the targeting of several inflammation-related molecular targets , including vascular adhesion molecules -LRB- ICAM1 , VCAM1 , E-selectin -RRB- , neoangiogenesis molecules -LRB- αvβ3 integrin -RRB- , scavenger receptors on macrophages , MMP proteolytic activity , macrophage metabolic activity and apoptosis -LRB- PS -RRB- -LSB- 60 , 62 , 75 -- 83 -RSB- . Although some of these approaches have yielded promising experimental data , there is still a paucity of clinical studies confirming the feasibility of these targeted approaches in humans . Of the clinical results available , the most impressive have been achieved using 99mTc-Anx A5 SPECT imaging and -LSB- 18F -RSB- fluorodeoxyglucose -LRB- 18FDG -RRB- positron emission tomography -LRB- PET -RRB- imaging . The latter has been used for many years to detect metabolically hyperactive processes in nuclear oncology . Since the glucose analogue 18FDG is a marker for foci of increased metabolic activity -LSB- 84 -RSB- and it is known that macrophages involved in atherosclerotic plaques have a high metabolic rate , there is sound logic behind this strategy . In addition , 18FDG imaging capitalises on the merits of oncological medicine since 18FDG is a clinically approved tracer that can be detected with high sensitivity and accuracy by PET technology , whereas PET conjugates for most alternative plaque imaging targets are still in the process of development . 18FDG imaging of arterial inflammation has been performed with rather promising results in retrospective studies of oncological 18FDG scans , in experimental settings and in in vivo clinical studies by Rudd and colleagues -LSB- 76 , 82 , 85 , 86 -RSB- . In an eight-patient pilot study , they were able to correlate 18FDG uptake in carotid lesions of patients scheduled for endarterectomy with histologically assessed macrophage density -LRB- p < 0.005 -RRB- . However , a practical difficulty of performing 18FDG imaging in the search for coronary lesion instability lies in the physical activity of the cardiac muscle , which results in high glucose requirements and thus severe background signal . In addition to detection of the vulnerable plaque with 18FDG , imaging of the vulnerable plaque with 99mTc-Anx A5 has yielded some exciting results as well . The first demonstration of the feasibility of in vivo imaging of plaque instability with 99mTc-Anx A5 , in animals , was given in 2003 by Kolodgie et al. . It was demonstrated in a rabbit model of aortic de-endothelialisation followed by a high-fat diet that 99mTc-Anx A5 showed a ninefold higher binding to the atherosclerotic vessels than to the control vessels -LSB- 75 -RSB- . Further validation of the Anx A5 imaging concept in the vulnerable plaque was done by Johnson and colleagues in 2005 -LSB- 87 -RSB- . In a porcine model of atherosclerosis the feasibility of SPECT imaging with 99mTc-Anx A5 to assess atheroma progression in coronary arteries was demonstrated . This was ultimately challenging owing to the low mass of the imaging target and cardiopulmonary motion . As swine and human hearts have similar cardiac dimensions , these results indicate that non-invasive functional imaging of coronary plaque instability could be realistic in the near future . The first clinical demonstration of the feasibility of nuclear vulnerable plaque imaging with 99mTc-Anx A5 was given by Kietselaer et al. in 2004 -LSB- 43 -RSB- . In a pilot study , four patients were included who were scheduled for carotid endarterectomy and had a history of recent -LRB- n = 2 -RRB- or remote -LRB- n = 2 -RRB- transient ischaemic attack -LRB- TIA -RRB- . Before surgery 99mTc-Anx A5 was administered and SPECT imaging was performed . In the two patients who had suffered a TIA more than 3 months before imaging and had since been treated with statins and antiplatelet agents , no tracer uptake of Anx A5 was observed -LRB- Fig. 5 -RRB- . Histological analysis confirmed stable lesions with negligible macrophage infiltration and intra-plaque haemorrhage . In contrast , the two patients who had experienced transient ischaemia shortly before imaging -LRB- 3 and 4 days , respectively -RRB- demonstrated marked uptake of Anx A5 in the culprit carotid vessel , while the contralateral arteries remained clear of tracer binding . Anx A5 binding was histologically localised to apoptotic macrophage membranes . An interesting finding during this study was that echo-Doppler examination in one of the patients with a recent TIA identified a severe stenotic lesion in the contralateral carotid artery prior to imaging . On the 99mTc-Anx A5 scan , however , the lesion was negative , though it might have been suspected as the culprit lesion by conventional techniques . This finding underlines the potential added value of functional versus anatomical imaging in predicting the likelihood of future thrombotic events . Fig. 5Images of unstable atherosclerotic carotid artery lesions obtained with radiolabelled Anx A5 . a Transverse and coronal views obtained by SPECT in patient 1 , who had a left-sided TIA 3 days before imaging . Although this patient had clinically significant stenosis of both carotid arteries , uptake of radiolabelled Anx A5 is evident only in the culprit lesion -LRB- arrows -RRB- . b Histopathological analysis of an endarterectomy specimen from patient 1 -LRB- polyclonal rabbit anti-Anx A5 antibody , × 400 -RRB- shows substantial infiltration of macrophages into the neointima , with extensive binding of Anx A5 -LRB- brown -RRB- . c In contrast , SPECT images of patient 3 , who had had a right-sided TIA 3 months before imaging , do not show evidence of Anx A5 uptake in the carotid artery region on either side . Doppler ultrasonography revealed a clinically significant obstructive lesion on the affected side . d Histopathological analysis of an endarterectomy specimen from patient 3 -LRB- polyclonal rabbit anti-Anx A5 antibody , × 400 -RRB- shows a lesion rich in smooth muscle cells , with negligible binding of Anx A5 . ANT anterior , L left . Copyright © 2004 Massachusetts Medical Society . All rights reserved . -LSB- 43 -RSB- Among the discussed imaging modalities for vulnerable plaque detection , nuclear imaging -LRB- PET in particular -RRB- is far superior to MRI , being capable of detecting probes at the picomolar range -LSB- 88 -RSB- . PET further outperforms SPECT imaging regarding spatial resolution , with 4 - to 6-mm versus 10 - to 15-mm resolution in the centre of the field . The major difficulty in evaluating PET or SPECT usually originates from orientation and localisation of the measured signal in the body . To overcome this , nuclear imaging modalities can be co-registered and overlaid with conventional CT or MRI imaging , hence providing anatomical landmarks in relation to molecular information . Improvements in the availability and costs of PET/CT or PET/MRI imaging devices will therefore imply great advantages . Given the high costs of imaging , diagnostic algorithms in the future most likely will combine relatively cheap serum biomarkers that indicate plaque instability with the subsequent use of expensive imaging technology in selected cases . This indicates that the specific niche for both serum and imaging biomarkers needs to be defined in large prospective studies . In addition , much is to be gained from refinements of the radioligand with regard to blood clearance and biodistribution , since molecular imaging typically requires fast binding of the probe to the target , rapid subsequent clearance from the blood and not too short radioactive half-lives . Heart failure Heart failure -LRB- HF -RRB- is growing to epidemic proportions , partially due to the better treatment of AMI . The pathogenesis of heart failure is still poorly understood . There may be an important role for cell death in the progression of congestive HF . A number of papers have reported on the differences in the amount of cell death in healthy and failing hearts . The baseline rate of apoptosis in healthy human hearts is around one to ten cardiac myocytes per 105 nuclei . On the other hand , 80 -- 250 heart muscle cells per 105 cardiac nuclei undergo apoptosis at any given time in late-stage dilated cardiomyopathy . However , it remains unclear whether this cell death is a coincidental finding , a protective process or a causal component in the pathogenesis . In work done by Wencker and co-workers , the uncertainty of the role of cell death was addressed . In a transgenic mouse model with a conditionally active caspase expressed exclusively in the myocardium , they demonstrated that even very low levels of cardiomyocyte apoptosis -LRB- 23 per 105 nuclei , compared with 1.5 per 105 nuclei in controls -RRB- caused a lethal dilated cardiomyopathy . Inhibition of this active caspase largely prevented the development of cardiac dilation and contractile dysfunction . These and other data suggest that cardiomyocyte apoptosis and necrosis may be a causal mechanism of heart failure , contributing to adverse left ventricle -LRB- LV -RRB- remodelling . Inhibition of this cell death process may constitute the basis for novel therapies . Consequently , anti-apoptotic agents have been developed and tested in animal models of HF with promising results -LSB- 89 -RSB- . Although , as discussed earlier , it is possible to detect cell death in MI leading to HF , the small amount of cell death present in heart failure -LRB- 80 -- 250 cardiomyocytes per 105 cardiac nuclei -RRB- makes detection with the current techniques very challenging . However , the feasibility of detecting apoptosis in patients with progressive idiopathic dilated cardiomyopathy was demonstrated in a proof of principle study done by Kietselaer -LSB- 90 -RSB- . Nine patients with idiopathic dilated cardiomyopathy -LRB- IDCM -RRB- were evaluated , as well as two family members with hypertrophic cardiomyopathy in different stages of disease . Patients were imaged with 99mTc-labelled Anx A5 . 201Tl images were used for orientation . Other causes of IDCM were excluded using laboratory testing , echocardiography and coronary angiography . It was found that five patients with IDCM showed focal uptake of radiolabelled Anx A5 and that one patient showed global uptake in the left ventricle -LRB- Fig. 6 , left panel -RRB- . No enhanced uptake could be visualised in the other six patients -LRB- Fig. 6 -RRB- and controls . All cases that showed Anx A5 uptake in the heart displayed recent onset or recent worsening of left ventricular function and functional class . In cases that did not show Anx A5 uptake , LV function and clinical status remained stable . Kietselaer concluded that non-invasive imaging using 99mTc-Anx A5 detects apoptosis in patients suffering from IDCM , providing further evidence for the role of apoptosis in the pathogenesis of IDCM . In addition , Anx A5 imaging identifies high-risk patients who might benefit from cell death blocking strategies . Fig. 6Dual-isotope imaging using 201Tl for left ventricular contour detection and , simultaneously , radiolabeled annexin A5 in patients with dilated cardiomyopathy . a Dilated cardiomyopathy patient with rapid deterioration of left ventricular function . Note focal uptake in apex and lateral wall , and slight septal uptake . b Dilated cardiomyopathy patient in acute heart failure . Note global uptake of radiolabeled annexin A5 . c Dilated cardiomyopathy patient in stable clinical condition . Uptake is absent even when image is enhanced to the extent that background radioactivity can be observed . d Family member of patient in panel b. No clinical evidence is seen of dilated cardiomyopathy . Note absence of uptake of radiolabeled annexin A5 . ANT , anterior ; INF , inferior ; LAT , lateral ; SEPT , septal . Reprinted by permission of the Society of Nuclear Medicine from -LSB- 90 -RSB- In addition to imaging of apoptosis , the detection of the molecular substrates of early-stage alterations leading to HF , such as vascular remodelling in infarcted tissue , is an emerging technique . Vascular remodelling is associated with non-contractile scar tissue formation that may contribute to adverse LV remodelling . However , none of these molecular techniques has reached the clinical stage yet . Cardiac allograft rejection Until 1997 , almost 46,300 heart transplantations had been carried out worldwide -LSB- 48 -RSB- . Despite this encouraging number , only 50 % of cardiac transplant recipients will survive 10 years and almost all will die within 20 years unless re-transplantation is preformed -LSB- 19 -RSB- . One of the possible complications for patients who have received a donor heart is transplant rejection . Under the current medical guidelines , endomyocardial biopsies -LRB- EMBs -RRB- are recommended 15 -- 20 times in the first year after transplantation to monitor potential allograft rejection . EMB is an invasive diagnostic procedure and not without risk of complications . Allograft rejection is pathologically characterised by perivascular and interstitial mononuclear inflammatory cell infiltration associated with myocyte necrosis and apoptosis -LSB- 91 , 92 -RSB- . Because the process of apoptosis can be pharmaceutically manipulated , identifying apoptosis non-invasively is of critical importance -LSB- 93 -RSB- . In a study done by Narula and co-workers -LSB- 42 -RSB- , 18 patients who had undergone heart transplantation within the past year were subjected to non-invasive imaging with Anx A5 . Patients received an injected dose of 555 -- 1,110 MBq -LRB- 15 -- 30 mCi -RRB- of 99mTc-Anx A5 and underwent SPECT imaging 1 h after injection -LRB- Fig. 7 -RRB- . In 13 out of these 18 patients , no myocardial uptake of 99mTc-Anx A5 was observed . In the remaining five patients , uptake of 99mTc-Anx A5 in the myocardial region was observed , which was associated with at least moderate transplant rejection -LRB- Fig. 8 -RRB- , of International Society of Heart and Lung Transplantation -LRB- ISHLT -RRB- grade 2/4 . Histological analysis revealed active caspase-3 in EMB specimens . This study demonstrated the clinical feasibility of detecting cardiac allograft rejection induced apoptosis , which , after confirming these findings in larger clinical studies , might lead to a reduction for the need of EMBs . Fig. 7SPECT images of 99mTc-labelled Anx A5 in three patients -LRB- 10 , 3 and 4 -RRB- are demonstrated . a In patient 10 , myocardial uptake -LRB- as outlined by solid circles -RRB- is clearly seen in all tomographic orientations and can be differentiated from the left ventricular cavity , especially in the transaxial slice . b In patient 3 , by contrast , no Anx A5 uptake is observed , and only background activity is seen . c Patient 4 demonstrates Anx A5 activity in the blood pool originating from the great vessels -LRB- GV great cardiac vein , PA pulmonary artery , Ao aorta -RRB- and ventricular contours , but no activity is observed in the myocardium . The scan of patient 10 , with myocardial uptake , was further processed -LRB- Fig. 8 -RRB- . RV right ventricle , LV left ventricle . Adapted by permission from Macmillan Publishers Ltd : Nature Medicine -LSB- 42 -RSB- , copyright 2001Fig . 8Diffuse myocardial uptake of 99mTc-labelled Anx A5 in cardiac allograft . Patient 10 underwent Anx A5 imaging 9 months after orthotopic heart transplantation . SPECT imaging 3 h after intravenous injection of radiolabelled Anx A5 demonstrated multifocal myocardial uptake of radiotracer . Smoothing of the images and cardiac SPECT processing in the short axis , vertical long axis and horizontal long axis revealed myocardial perfusion scan-like images with diffuse uptake in the whole myocardium , suggesting apoptosis in the myocardium . Adapted by permission from Macmillan Publishers Ltd : Nature Medicine -LSB- 42 -RSB- , copyright 2001 Future perspectives Of all the apoptotic imaging agents , Anx A5 has made the most successful transition from the test tube to the clinical arena . Novel imaging modalities like PET/CT and SPECT/CT are making a significant contribution to the growing role of molecular imaging . The combination of anatomical imaging with CT and biological imaging using SPECT or PET yields a new synergistic imaging modality that provides detailed information on the molecular -LRB- patho -RRB- physiological processes in relation to exact anatomical orientation . The next challenge is to assess the incremental clinical value of molecular imaging and its ability to change patient management decisions . For this purpose , prospective cohort studies need to be designed to evaluate the clinical meaning of molecular imaging scan results in relation to the progression of the underlying disease . For instance , data on the association between Anx A5 uptake in plaques in the carotid arteries and clinical event rate are still lacking . The availability of such large clinical datasets may allow for better stratification of patients and therefore more optimal treatment decisions . Finally , we and others have found that not only apoptotic cells but also viable cells are detectable with Anx A5 . Our work on the vulnerable plaque showed that several processes leading to plaque instability are associated with PS expression , such as activated macrophages -LRB- inflammation -RRB- and aging red blood cells -LRB- intra-plaque haemorrhage -RRB- . This ability of Anx A5 to visualise exposed PS in different biological conditions opens novel opportunities for imaging .

O B I O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O B I O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

apoptosis
molecular imaging
cardiovascular disease
annexin a5


A 15-year-old girl with a large pericardial effusion Pericarditis is a rare manifestation of tuberculosis and can be fatal . We describe a 15-year-old girl admitted for a large pericardial effusion . Subxiphoid pericardial biopsy was performed . Biopsy samples were positive for M. tuberculosis DNA by PCR , which confirmed the diagnosis of tuberculous pericarditis . A 15-year-old girl was admitted with chest pain , progressive shortness of breath , fever , nonproductive cough , decreased appetite , weight loss , and fatigue . The patient was born in Angola , but had lived in the Netherlands for 14 years . Seven months before admission she had traveled to the Democratic Republic of Congo in Western Africa . Her temperature was 39.7 °C , her pulse 90 beats/min , respiratory rate 28 breaths/min , and blood pressure 96/58 mmHg . No enlarged lymph nodes were found . Soft cardiac tones and a friction rub were noticed . The liver was enlarged . Laboratory results showed normal white blood cells and platelets with a decreased hemoglobin -LRB- 4.3 mmol/l -RRB- . The erythrocyte sedimentation rate was 60 mm/h . A chest radiograph showed marked enlargement of the cardiac silhouette with no other abnormalities . Echocardiography revealed a large pericardial effusion containing exudative debris and fibrin strands -LRB- Fig. 1 -RRB- . Extensive laboratory studies for auto-immune diseases and infections , including HIV , did not reveal the cause . Fig. 1Echocardiography showing a large pericardial effusion with exudative debris and fibrin strands . RV right ventricle , LV left ventricle , LA left atrium , PE pericardial effusion Under general anesthesia the pericardial fluid was drained and a subxiphoid pericardial biopsy was performed . The PCR for M. tuberculosis DNA was positive on the pericardium , though not in the pericardial fluid . The patient was treated with four anti-tuberculous drugs -LRB- isoniazide , ethambutol , pyrazinamide , rifampicin -RRB- for 4 months and two anti-tuberculous drugs for an additional 4 months . Adjunctive treatment with predinisolone was tapered over a 12-week period . The patient completely recovered and did not develop any signs of constrictive pericarditis . She was followed up for more than 1 year and then discharged because of uneventful recovery . Discussion Pericarditis is a rare manifestation of tuberculosis that can be fatal and accounts for approximately 1 -- 2 % of all cases of tuberculosis -LSB- 6 -RSB- . Children in particular are at high risk of the development of extra-pulmonary disease -LSB- 5 -RSB- . Clinical manifestations of tuberculous pericarditis are nonspecific and cardiopulmonary complaints usually develop later . Accumulation of pericardial fluid may compromise venous return , leading to cardiac failure . Echocardiography plays a major role in confirming the presence of a pericardial effusion . In addition , several echocardiographic abnormalities including pericardial thickening , exudative coating and strands crossing the pericardial space are suggestive of tuberculous pericarditis . Thickened pericardium and fibrin strands are highly specific -LRB- 94 % and 88 % respectively -RRB- , and exudative coating has a high sensitivity -LRB- 100 % -RRB- in tuberculous pericarditis -LSB- 4 -RSB- . The diagnosis is confirmed with detection of Mycobacterium tuberculosis in either pericardial fluid or pericardial tissue . Pericardial fluid aspiration , however , has low diagnostic yield , both for PCR and culture , whereas pericardial biopsy provides the best chance of definitive diagnosis -LSB- 2 , 6 -RSB- . A positive tuberculin skin test may increase the suspicion of tuberculous pericarditis , but a negative skin test does not exclude the diagnosis . A tuberculin skin test was not performed in this case because the girl had been immunized with BCG . There was no history of any family members being diagnosed with TBC . The spread of tuberculosis to the pericardium , resulting in an effusion , occurs most often from the mediastinal lymph glands . However , pericarditis may also represent reactivation of disease in the absence of an apparent primary focus of infection -LSB- 3 -RSB- . Current recommendations for treatment of tuberculous pericarditis include an initial four-drug regimen for a period of 8 weeks with a subsequent continuation phase of treatment for 4 months when the isolate is drug-susceptible -LSB- 1 -RSB- . Controversy exists with respect to the use of corticosteroids for tuberculous pericarditis . The American Thoracic Society 's consensus statement on tuberculosis recommends adjunctive therapy with corticosteroids -LRB- 1 mg/kg of prednisolone with tapering over 11 weeks -RRB- for tuberculous pericarditis -LSB- 1 -RSB- .

O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O