Induction of apoptosis following proton irradiation of endothelial cells

In addition to the treatment of some cancers, proton therapy is currently being used to treat arteriovenous malformations and neovascular eye diseases such as senile macular degeneration. However little is known of the radiobiological responses of endothelial cells to proton radiation. In this study, we have investigated the effects of 62MeV protons from the Clatterbridge Cyclotron on the clonogenic survival, growth and apoptosis of bovine aortic endothelial cells grown in vitro. Analysis of clonogenic survival showed endothelial cells to be relatively radiosensitive to protons, with an a value of 0.75 ±0.004 and a βvalue of 0.0008 ±0.004, indicating little repair of radiation damage. Following 2Gy of proton irradiation, the clonogenic surviving fraction was 0.22 and growth of subconfluent cultures was retarded by 22%. A wave of apoptotic cells, identified morphologically, occurred in monolayers of confluent endothelial cells, peaking around 6 hours post-irradiation and returning to insignificant levels by 24 hours post-irradiation. The number of apoptotic cells in these monolayers was greatly increased in the absence of serum post-irradiation and was dependent on dose. In addition, radiation induced a small but significant number of endothelial cells to detach from the monolayer; some of these cells were apoptotic. DMA extracted from monolayers of cells following 10Gy proton irradiation showed the DMA laddering pattern characteristic of apoptosis, confirming that the monolayer did indeed contain apoptotic cells. These studies indicate that the cell death by apoptosis following proton irradiation of endothelial cells is influenced by environmental factors, and may contribute to the biological responses of endothelial cells following proton irradiation.