The biochemical characterization of rejecting tissues suggests that apoptosis plays a major role in acute cardiac rejection. We propose to use a novel radiopharmaceutical, radiolabeled (99m Tc) annexin V, to monitor the early and specific biochemical properties of cardiac allografts undergoing acute rejection. Annexin V, a 35kD a human protein, is useful to detect and quantify dying cells in vitro and in situ well in advance of other cell markers of death. Our group has adapted annexin V for use as an imaging agent by radiolabeling this protein with technetium 99m without affecting its bioreactivity. Our work so far has shown that radiolabeled annexin V can detect and monitor dying cells in the liver undergoing fulminant apoptosis in response to Fas antibody, and in normal bone marrow and spleen, as well as lymphomatous tumors following anti-cancer treatment. We also have demonstrated that radiolabeled annexin V can be used to noninvasively detect and monitor acute liver, lung, and heart transplant rejection. In this proposal we will optimize the parameters needed for radiolabeled annexin V detection of acute cardiac rejection imaging by: 1) determining the relationships of administered protein dose, and rate of intravenous infusion with respect to the biodistribution of radiolabeled annexin V; 2) increasing the serum half life radiopharmaceutical by dimerizing annexin V (which retains the same binding capacity as monomeric annexin V) to improve target localization; 3) studying the influence of competing binding sites on tracer distribution using PS containing polymerized liposomes which bind to annexin V in the circulation. The optimized imaging approach will be then applied to study the time course of apoptosis within cardiac allografts during acute rejection. We will then determine the sensitivity and specificity of radiolabeled annexin V to monitor apoptosis during immunosuppressive therapy with cyclosporine of heterotopic cardiac allografts undergoing acute section. Finally we will selectively block apoptosis in rejecting cardiac allografts using the caspase inhibitor, ZVAD.fmk, to determine the contribution of apoptotic relative to necrotic cell death to the uptake of radiolabelled annexin V. The data from our project may allow for direct monitoring of apoptosis in cardiac transplant recipients without the need for endomyocardial biopsy. Using radiolabeled annexin V as a clinical imaging tool may permit more rapid assessment and development of therapies designed to inhibit cardiac apoptotic cell death and decrease immunotherapy related morbidity via more precise adjustment of the intensity and duration of immunosuppression to the severity of rejection.
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