Ayelet Reshef

Small-Molecule Biomarkers for Clinical PET Imaging of Apoptosis

Apoptosis is a fundamental biologic process. Molecular imaging of apoptosis in vivo may have important implications for clinical practice, assisting in early detection of disease, monitoring of disease course, assessment of treatment efficacy, or development of new therapies. Although a PET probe for clinical imaging of apoptosis would be highly desirable, this is yet an unachieved goal, mainly because of the required challenging integration of various features, including sensitive and selective detection of the apoptotic cells, clinical aspects such as favorable biodistribution and safety profiles, and compatibility with the radiochemistry and imaging routines of clinical PET centers. Several approaches are being developed to address this challenge, all based on novel small-molecule structures targeting various steps of the apoptotic cascade. This novel concept of small-molecule PET probes for apoptosis is the focus of this review.

Molecular Imaging of Neurovascular Cell Death in Experimental Cerebral Stroke by PET

Clinical molecular imaging of apoptosis is a highly desirable yet unmet challenge. Here we provide the first report on 18F-labeled 5-fluoropentyl-2-methyl-malonic acid (18F-ML-10), a small-molecule, 18F-labeled PET tracer for the imaging of apoptosis in vivo; this report includes descriptions of the synthesis, radiolabeling, and biodistribution of this novel apoptosis marker. We also describe the use of 18F-ML-10 for small-animal PET of neurovascular cell death in experimental cerebral stroke in mice. Methods: 18F-ML-10 was synthesized by nucleophilic substitution from the respective mesylate precursor, and its biodistribution was assessed in healthy rats. Permanent occlusion of the middle cerebral artery (MCA) was induced in mice, and small-animal PET was performed 24 h later. Results: Efficient radiolabeling of ML-10 with 18F was achieved. Biodistribution studies with 18F-ML-10 revealed rapid clearance from blood (half-life of 23 min), a lack of binding to healthy tissues, and rapid elimination through the kidneys. No significant tracer metabolism in vivo was observed. Clear images of distinct regions of increased uptake, selectively in the ischemic MCA territory, were obtained in the in vivo small-animal PET studies. Uptake measurements ex vivo revealed 2-fold-higher uptake in the affected hemisphere and 6- to 10-fold-higher uptake in the region of interest of the infarct. The cerebral uptake of 18F-ML-10 was well correlated with histologic evidence of cell death. The tracer was retained in the stroke area but was cleared from blood and from intact brain areas. Conclusion: 18F-ML-10 is useful for noninvasive PET of neurovascular histopathology in ischemic cerebral stroke in vivo. Such an assessment may assist in characterization of the extent of stroke-related cerebral damage and in the monitoring of disease course and effect of treatment.

Opening of ATP-sensitive potassium channels by cromakalim confers tolerance against chemical ischemia in rat neuronal cultures

The effect of opening and of blocking of ATP-sensitive potassium (K(ATP)) channels on the short-term capacity of neurons to resist ischemia-reperfusion-induced cell injury, was studied in a model of primary rat neuronal cultures, subjected to metabolic poisoning by iodoacetic acid (150 microM, 150 min), followed by reperfusion (1 h). The metabolic poisoning resulted in a marked decrease in cellular ATP content (from 65.3 +/- 13.4 to 21.6 +/- 11.7 nmole/mg protein), simulating an ischemia, or hypoxia-induced condition of energy crisis. The degree of neuronal damage was assessed by the trypan blue exclusion test. Exposure of the neurons to the channel-opener cromakalim (10 microM; 15 min), prior to the insult, induced resistance, which could be abolished by the specific channel blocker glibenclamide (2 microM). Glibenclamide also abolished the protection acquired by preconditioning of the neurons with iodoacetate (IA; 100 microM), the adenosine A1 agonist N6-(R)-phenylisopropyladenosine (R-PIA; 100 microM), or with the protein kinase C (PKC) activator 1,2 dioctanoyl-rac-glycerol (DOG; 1 microM). The results indicate that in the neurons, opening of the K(ATP) channels confers protection against an ATP-depleting crisis, and suggest that the protective effects induced by adenosine and by activation of PKC, are mediated by the opening of these channels.

Opening of KATP channels is mandatory for acquisition of ischemic tolerance by adenosine

Binding of adenosine to neuronal adenosine receptors activates a signal transduction pathway (the adenosine mechanism), leading to a temporary ischemic tolerance. We have demonstrated before that induction of this mechanism in primary rat neuronal cultures, by activation of adenosine receptors, or by activation of protein kinase C (PKC), confers a wide time window of ischemic tolerance, lasting up to 72 h, the early (immediate) part of which depends on opening of KATP channels (glibenclamide sensitive). Here we demonstrate that the entire duration of the ischemic tolerance conferred by activation of the adenosine mechanism depends on opening of the KATP channels. Thus, opening of the KATP channels appears to be a mandatory step in the adenosine mechanism, leading to the creation of the wide time window of ischemic tolerance.

Role of KATP Channels in the Induction of Ischemic Tolerance by the ‘Adenosine Mechanism’ Neuronal Cultures

Activation of the adenosine receptors in the brain and in the heart preconditions these tissues to resist a subsequent ischemic insult . The heart ‘adenosine mechanism’ was demonstrated to produce two windows of protection, an early, rapidly appearing (within minutes) but short lasting (60-120 min) window , and a delayed (reappearing about 24 h later), relatively long lasting (24 to 48 h) ‘second window of protection’ . The short-term heart mechanism was suggested to operate through a signal transduction pathway, including activation of protein kinase C (PKC), and depending on opening of KATP channels (glibenclamide-sensitive) . The adenosine-induced mechanism operating to produce the ‘second window of protection’ was suggested to involve enhanced expression of heat shock proteins (HSP) , and recently to be also dependent on opening of the KATP channels . An ‘adenosine mechanism’, resembling in many aspects that operating in the heart, has also been demonstrated to function in the neurons 1-3,11-14 . We could verify the operation of the neuronal ‘adenosine mechanism’, employing an experimental model of cultured rat neurons, subjected to