**1. Introduction**

250 12 Chapters on Nuclear Medicine

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M. & Kanazawa, S. (2011). The local efficacy of I-131 for F-18 FDG PET positive lesions in patients with recurrent or metastatic thyroid carcinomas. *Clincal Nuclear*  In various myocardial disorders including myocardial ischemia, infarction and subsequent cardiac remodelling and heart failure, myocarditis, cardiomyopathy, cardiac allograft rejection, chemotherapy induced cardiotoxicity, both necrosis and apoptosis are considered to play an important role in the underling pathophysioloy. Molecular and cellular dysfunction has been widely investigated in cardiovascular fields using various modalities. Of particular, radionuclide imaging technique has advantage for quantitative assessment of molecular function in vivo in patients. Especially in patients with coronary artery disease, perfusion imaging agents such as 201Tl, 99mTc-MIBI and tetrofosmin with combination of stress testing and ECG-gated data acquisition have been used for the simultaneous assessment of the ventricular function and severity of myocardial perfusion abnormality including its location and size in stress and resting condition. From these data, status of myocardial ischemia or jeopardized myocardium, myocardial viability and reversibility of wall motion abnormality can be diagnosed to some extent but still insufficiently. Molecular imaging may play an important role for assessing the pathophysiology and its severity in these various cardiovascular diseases beyond perfusion imaging. This chapter focuses on the apoptosis imaging that is one of the most possible nuclear molecular imaging in-vivo at this stage, and its clinical application might permit more precise assessment of the pathophysiology in various myocardial abnormalities beyond perfusion imaging.

Four decades ago, the term apoptosis has been introduced by Kerr et. al. as a special form of cell death different from necrosis (Kerr, et al., 1972). Necrosis is passive and unregulated form of cell death, characterized by irreversible loss of plasma membrane integrity with cell swelling and rupture after sudden severe insults which preclude adequate homeostatic energy-dependent cell functions, leading to release of intracellular contents and a subsequent inflammatory response. Apoptosis on the other hand is characterized morphologically by the condensation of nuclear chromatin, cytoplasmic condensation, cell shrinkage, followed by the nuclear and cellular fragmentation and phagocytosis of apoptotic bodies by neighboring cells in the absence of inflammation. Apoptosis is considered to be an active and highly regulated ATP dependent programmed cell daeth process and plays an important roles in embryonic developement and maintenace of postnatal tissues and contributes to both normal physiology and pathology. Dysregulation of apoptosis results in either too littel or too much cell death and implicated in various diseases. For instance, insufficient apoptosis may contribute carcinogenesis, on the otherhand, eccessive apoptosis

Apoptosis Imaging in Diseased Myocardium 253

and designated to be engulfed and phagocytosed by macrophages and neighbouring cells

After the initial description of apoptosis based on the morphological features, several useful biochemical and immunohistochemical detection methods were subsequently introduced based on the understanding of the basic mechanisms of apoptosis. As a histochemical technique for the detection of apoptosis, terminal deoxynucleotidyltransferase-dUTP-nick endlabeling (TUNEL) assays has become a standard technique for in situ labeling and localization of DNA breaks in individual nuclei on tissue section (Gavrieli, et al.,1992). TUNEL is based on the specific binding of terminal deoxynucleotidyltransferase (TdT) to 3'-OH ends of fragmented DNA. As DNA ladder formation is quite a late feature of apoptosis, TUNEL assay appears to be uniquely associated with apoptotic cell death. For the in-vivo imaging initiator caspases and effector caspases can be distinguished and serve as potential targets inside the apoptotic cells. However, for molecular target for in-vivo imaging, it is favorable that the target exists on cell surface rather than in cytoplasm or nucleus. Accordingly, to date, most noninvasive imagings of apoptosis target PS on the cell membrane, which is a membrane aminophospholipid that is normally located on the inner leaflet of cell membrane but is rapidly trnaslocated to the outer leaflet of cell membrane once the cell become apoptotic. Annexin V, a 36-kD physiologic protein, binds with nanomolar affinity to PS in a calcium dependent manner, therefore, 99mTc labeled annexin V permits imaging of apoptosis in vivo in

its early stage (Blankenberg, et al., 1998; Hofstra, et al., 2000; Kemerink, et al., 2003).

production by recombinant DNA technology, and lack of toxicity of the protein.

presents millions of binding sites per cell on the cell surface.

Annexin V (also known as annexin A5) is consisted of 319 amino acids and a 36-kD endogeneous human protein that is mainly distributed intracellularly with very high concentrations in placenta, and lower concentrations in endothelial cells, myocardium, skeletal muscle, kidneys, skin, red blood cells, platelets, and monocytes. Very low concentrations of annexin V (1-6 ng/ml) circulate in blood of healthy humans (Andree et al., 1992). Several advantages for the use of annexin V for apoptotic imaging have been described including very high affinity for PS with low nanomolar to subnanomolar dissociation constant values, ready

In healthy cells, anionic phospholipid PS and phosphatidylethanolamine (PE) confined to the inner leaflet of the lipid bilayer plasma membrane by an ATP dependent enzyme called translocase. On the other hand, ATP dependent enzyme, floppase, pumps cationic phospholipids such as phosphatidylcholine (PC) and sphingomyelin to the cell surface. Therefore, an asymmetric distribution of different phospholipids between the inner and outer leaflet of the plasma membrane is maintained in normal cells. However, at the beginning of the execution phase of apoptosis, rapid redistribution of PS and PC across the cell membrane is facilitated by a calcium ion-dependent deactivation of translocase and floppase and activation of scramblase. Then, PS exposes on the cell surface and annexin V become accessible to PS. This PS exposure is attractive target for imaging apoptosis, since it is a near universal event in apoptosis, it occurs early after the apoptotic stimulus, and it

Annexin V and its derivatives have been labeled with 123I, 124I, 125I, 99mTc, 18F, thereby providing a broad range of imaging applications in apoptosis research from autoradiography

**2. Radipharmaceuticals for molecular imaging of apoptosis** 

without causing inflammation.

**2.1 Radiolabeled annexin V** 

may account for substantial portion of pathogenesis of myocardial infarction and heart failure.

Fig. 1. Schema of two major pathway for apoptosis.

Apoptosis can be mediated through the death receptor pathway, or by mitochondrial pathway that is initiated by the release of cytochrome c into the cytosol. Both pathways result in activation of caspase-3, the final effector enzyme of apoptosis, leading to the cleavage of numerous structural and regulatory cellular proteins, thereby producing the apoptotic phenotype characterized by cell shrinkage, chromatin condensation, nucleus fragmentation, and externalization of phosphatidylserin (PS) on the outside of the cell membrane.

Apoptosis is mediated by 2 central pathway: the extrinsic (or death receptor) pathway that utilizes cell surface receptors (e.g. Fas) and the intrinsic (or mitochondrial) pathway that involves the mitochondria and cytoplasmic reticulum (Fig. 1.). The extrinsic or death receptor pathway relies on the binding of specific cytokines expressed by other cells, including Fas ligand, tumor necrosis factor alfa, or other factors that binds to specific cellular receptors. Ligand binding initiates the activation of caspase-8, which in turn activates downstream effector caspases including caspase-3, the final effector enzyme of apoptosis. In contrast to extrinsic pathway that transduces a specialized set of death stimuli, the intrinsic or mitochondrial pathway integrates a broad spectrum of extracellular and intracellular stresses such as ischemia, reperfusion injury, chemotherapy, oxidative stress etc. These stimuli converges on the mithochondria leading to the release of several factors into cytosol including cytochrome c, which activates the initiator caspase-9 via the apoptosome followed by the activation of effector caspases. The activation of down stream caspases leads to the cleavage of numerous structural and regulatory cellular proteins, thereby producing the apoptotic phenotype characterized by cell shrinkage, chromatin condensation, nucleus fragmentation, and externalization of phosphatidylserin (PS) on the outside of the cell membrane which serves as an "eat me" signal for phagocyte. Intracellular components are packaged by blebbing and the cell is fragmentated into apoptotic bodies

may account for substantial portion of pathogenesis of myocardial infarction and heart

Apoptosis can be mediated through the death receptor pathway, or by mitochondrial pathway

Apoptosis is mediated by 2 central pathway: the extrinsic (or death receptor) pathway that utilizes cell surface receptors (e.g. Fas) and the intrinsic (or mitochondrial) pathway that involves the mitochondria and cytoplasmic reticulum (Fig. 1.). The extrinsic or death receptor pathway relies on the binding of specific cytokines expressed by other cells, including Fas ligand, tumor necrosis factor alfa, or other factors that binds to specific cellular receptors. Ligand binding initiates the activation of caspase-8, which in turn activates downstream effector caspases including caspase-3, the final effector enzyme of apoptosis. In contrast to extrinsic pathway that transduces a specialized set of death stimuli, the intrinsic or mitochondrial pathway integrates a broad spectrum of extracellular and intracellular stresses such as ischemia, reperfusion injury, chemotherapy, oxidative stress etc. These stimuli converges on the mithochondria leading to the release of several factors into cytosol including cytochrome c, which activates the initiator caspase-9 via the apoptosome followed by the activation of effector caspases. The activation of down stream caspases leads to the cleavage of numerous structural and regulatory cellular proteins, thereby producing the apoptotic phenotype characterized by cell shrinkage, chromatin condensation, nucleus fragmentation, and externalization of phosphatidylserin (PS) on the outside of the cell membrane which serves as an "eat me" signal for phagocyte. Intracellular components are packaged by blebbing and the cell is fragmentated into apoptotic bodies

that is initiated by the release of cytochrome c into the cytosol. Both pathways result in activation of caspase-3, the final effector enzyme of apoptosis, leading to the cleavage of numerous structural and regulatory cellular proteins, thereby producing the apoptotic phenotype characterized by cell shrinkage, chromatin condensation, nucleus fragmentation,

and externalization of phosphatidylserin (PS) on the outside of the cell membrane.

Fig. 1. Schema of two major pathway for apoptosis.

failure.

and designated to be engulfed and phagocytosed by macrophages and neighbouring cells without causing inflammation.
