Abstract
The process of controlled cellular death known as apoptosis has an important central role not only in normal homeostatic maintenance of tissues, but also in numerous diseases such as cancer, neurodegenerative, autoimmune, and cardiovascular diseases. As a result, new technologies with the capability to selectively detect apoptotic cells represent a central focus of research for the study of these conditions. We have developed a new biosensor for the detection of apoptotic cells, incorporating the targeted selectivity for apoptotic cells from Annexin V with the sensitivity of bioluminescence signal generation from a serum-stable mutant of Renilla luciferase (RLuc8). Our data presents a complete characterization of the structural and biochemical properties of this new Annexin-Renilla fusion protein (ArFP) construct, as well as a validation of its ability to detect apoptosis in vitro. Moreover, this work represents the first report of a bioluminescent Annexin V apoptosis sensor utilized in vivo. With this new construct, we examine apoptosis within disease-relevant animal models of surgery-induced ischemia/reperfusion, corneal injury, and retinal cell death as a model of age-related macular degeneration. In each of these experiments, we demonstrate successful application of the ArFP construct for detection and bioluminescence imaging of apoptosis within each disease or treatment model. ArFP represents an important new tool in the continuously growing kit of technologies for apoptosis detection, and our results from both in vitro and in vivo experiments suggest a diverse range of potential clinically relevant applications including cancer therapeutic screening and efficacy analysis, atherosclerosis and cardiovascular disease detection, and the monitoring of any number of other conditions in which apoptosis has a central role.