Apoptosis apo  ptosis 1 2 2 2 3 4 5 6 7 8 9 10 11 12 13 14 Biochemistry of apoptotic cell death 15 16 Several checks and balances exist that control burst of the caspase cascade if triggered by minor undesired initiating events. Inhibitors of apoptotic proteins (IAPs) 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. 17 19 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. 20 21 22 Surface-expressed PS provides an attractive target for molecular imaging of apoptosis. Annexin A5 23 26 K a M 27 28 21 22 29 2+ 30 30 34 Detecting apoptosis with Anx A5 35 29 36 23 25 37 41 99m 26 42 46 Detection of apoptosis using alternative methods 1 Fig. 1 Targets 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 47 47 99m 48 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 49 c b r 50 ApoSense 51 52 Role and detection of apoptosis in CVD Apoptosis of cardiomyocytes after myocardial infarction 53 99m 99m 48 99m 54 55 N N S 25 99m 99m 2 3 Fig. 2 a  Arrow  99m b  99m arrow  L  Fig. 3 a  Arrow  99m  L b arrow  Atherosclerotic lesions and plaque instability 56 63 64 65 65 66 67 69 70 73 59 65 74 59 61 59 75 4 Fig. 4 a–c d–f 99m  L  K a, d b, e a b  c  b 99m  d–f e d  f 99m 75  76 v 3 60 62 75 83 99m 18 18 18 84 18 18 18 18 76 82 85 86 18 p 18 18 99m 99m 99m 75 87 99m 99m 43 n n 99m 5 99m Fig. 5 a arrows  b  c  d  ANT  L 43  88 Heart failure 5 5 5 5 89 5 90 99m 201 6 6 99m Fig. 6 a b c d b ANT INF LAT SEPT 90   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 48 19 91 92 93 42 99m 7 99m 99m 8 Fig. 7 99m a  solid circles b c GV  PA  Ao 8  RV  LV 42  Fig. 8 99m 42  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 (patho)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 (inflammation) and aging red blood cells (intra-plaque haemorrhage). This ability of Anx A5 to visualise exposed PS in different biological conditions opens novel opportunities for imaging.