Imaging of apoptosis (programmed cell death) with Tc-99m annexin V

Apoptosis (programmed cell death) is a critical element in normal physiolog y and in many disease processes. Phosphatidylserine (PS), one component of cell membrane phospholipids, is normally confined to the inner leaflet of t he plasma membrane. Early in the course of apoptosis, this phospholipid is rapidly exposed on the cell's outer surface. Annexin V, an endogenous human protein. has a high affinity for membrane-bound PS. This protein has been labeled with fluorescein and has been used to detect apoptosis in vitro. We describe the use of radiolabeled annexin V to detect apoptosis in vivo. Th e results are compared to histologic and flow cytometric methods to identif y cells and tissues undergoing apoptosis. Methods: Annexin V was coupled to hydrazinonicotinamide (HYNIC) and radiolabeled with Tc-99m. Bioreactivity of Tc-99m-HYNIC annexin V was compared with fluorescein isothiocyanate (FIT C)-labeled annexin V in cultures of Jurkat T-cell lymphoblasts and in ex vi vo thymic cell suspensions undergoing apoptosis in response to different st imuli. In addition, the uptake of FITC annexin V and 99mTc-HYNIC annexin V was studied in heat-treated necrotic Jurkat T-cell cultures. In vivo locali zation of annexin V was studied in Balb/c mice injected with Tc-99m-HYNIC a nnexin V before and after induction of Fas-mediated hepatocyte apoptosis wi th intravenously administered antiFas antibody. Results: Membrane-bound rad iolabeled annexin V activity linearly correlated to total fluorescence as o bserved by FITC annexin V flow cytometry in Jurkat T-cell cultures induced to undergo apoptosis in response to growth factor deprivation (N = 10, r(2) = 0.987), antiFas antibody (N = 8, r(2) = 0.836) and doxorubicin (N = 10, r(2) = 0.804); and in ex vivo experiments on thymic cell suspensions with d examethasone-induced apoptosis from Balb/c mice (N = 6, r(2) = 0.989). Necr otic Jurkat T-cell cultures also demonstrated marked increases in radiophar maceutical (4000-5000-fold) above control values. AntiFas antibody-treated Balb/c mice (N = 6) demonstrated a three-fold rise in hepatic uptake of ann exin V (P < 0.0005) above control (N = 10), identified both by imaging and scintillation well counting. The increase in hepatic uptake in antiFas anti body-treated mice correlated to histologic evidence of fulminant hepatic ap optosis. Conclusion: These data suggest that Tc-99m-HYNIC annexin V can be used to image apoptotic and necrotic cell death in vivo.