The intense inflammatory reaction following myocardial infarction and reperfusion has been implicated as a crucial factor, which ultimately leads to ventricular remodeling, heart failure, and cardiovascular death. Our long-term goal in our research is to develop highly effective SPECT imaging techniques to screen and diagnose patients with cardiovascular diseases and to guide their therapy. The objective of the present proposal toward this goal is to validate 99mTc-labeled bispecific cytokine ligands for SPECT imaging of inflammation induced by myocardial ischemia and reperfusion. Tumor necrosis factor (TNF-1), Interleukin-1 (IL-12), and Interleukin-18 (IL-18) are the most potent proinflammatory cytokines involved in the progress of ischemia- reperfusion and ventricular remodeling;their actions are regulated by soluble Type I/Type II TNF receptor (TNFR1/TNFR2), IL-1 receptor antagonist (IL-1ra), and IL-18 binding protein (IL-18bp), respectively. Two 99mTc-labeled recombinant bispecific proteins, [99mTc]IL-18bp-Fc-IL-1ra (99mTc-IL181) and [99mTc]TNFR2-Fc- IL-1ra (99mTc-TR21), each containing an amino-terminal segment that specifically binds to TNFR2 or IL-18, the Fc portion of human IgG1, and a carboxy-terminal segment with the sequence of IL-1ra, have been recently developed in our laboratory for high-affinity targeting inflammatory sites via dual-cytokine pathways. In this proposal, we aim to: (1) determine the inflammation-targeting properties and radiopharmaceutical kinetics of these two bispecific radioligands in comparison with 99mTc-labeled individual cytokine ligands, 99mTc-TNFR2- Fc, 99mTc-IL-1ra-Fc and 99mTc-IL-18bp-Fc, in vitro and in vivo;(2) correlate the uptake of 99mTc-IL181 and 99mTc-TR21 in acute infarct and non-infarct regions with inflammatory intensity and progression of myocardial injury;and (3) evaluate 99mTc-IL181 and 99mTc-ILTR21 uptake associated with progressive left ventricular dilatation and pathophysiological alterations during the chronic remodeling phase and anti-inflammation therapy. To ascertain the abilities of 99mTc-IL181 and 99mTc-TR21 for specific cytokine targeting, cell-based binding assay will be carried out in IL-1-dependent murine D10.G4.1 (D10) cells. We will explore the interactions of the cytokine radioligands and cardiac cells (myocytes, endothelium and fibroblasts). Furthermore, we propose using a state-of-the-art small-animal SPECT imager, FastSPECT II, to collect dynamic cardiac imaging data in ischemic-reperfused rat hearts. Kinetic data of inflammatory reactions will be correlated with the extent of myocardial infarct and changes in cardiac function. The proposed research will apply SPECT imaging techniques toward the development of an innovative specific tool for fundamentally understanding inflammatory response in ischemic-reperfused hearts, providing insights into the intergradations of the proinflammatory cytokine pathway at different phases, and timing therapeutic modalities to limit the intense inflammatory reaction before severe cardiac function failure.
Development of highly specific imaging agents for noninvasive detection of inflammation induced by heart attacks.
