A Modified Chorioallantoic Membrane Assay Allows for Specific Detection of Endothelial Apoptosis Induced by Antiangiogenic Substances

Current in vivo angiogenesis assays allow for the assessment of vascular growth inhibition induced by a test substance, but they usually do not provide information about the mechanisms underlying such an inhibition. A potential antiangiogenic mechanism is the triggering of endothelial apoptosis in the growing vessels. Apoptogenic substances can be of interest for antiangiogenic therapy specially if they specifically perform their action on the angiogenic endothelium. We have developed a modification of the chorioallantoic membrane (CAM) assay using embryos of quail (Coturnix coturnix japonica). This novel assay allows to elucidate whether an antiangiogenic substance is specifically triggering an apoptotic response in endothelial cells. We have used a quail-specific monoclonal endothelial marker (QH1), a standard TUNEL technique of apoptotic cell labelling together with a general nuclear counterstaining with propidium iodide. Through laser confocal microscopy, paraffin sections of chorioallantoic membranes treated with test substances are stained in three colours: red for normal cell nuclei, yellow—green for apoptotic nuclei and blue for endothelial cells and endothelial progenitors. In a test experience, our assay showed significant differences in the apoptogenic properties of two antiangiogenic substances, camptothecin and aeroplysinin-1.