In order to establish that the Pazopanib-induced radiosensitization is mediated via ASMase/ceramide pathway activation, we assessed ASMase activity and ceramide levels in BAEC after Pazopanib treatment in combination with SDRT. As shown in Figure 6A, 10 Gy radiation rapidly triggered an increase in ASMase activity in BAEC from a baseline of 68.34 ± 2.41 mol/mg/h to peak at 90.42 ± 1.84 mol/mg/h at 2 min after radiation, similar to our previously reported results in these cells. Pre-treatment of Pazopanib further elevated ASMase activity up to 101.78 ± 2.6 mol/mg/h at 2 min, which was significantly higher than radiation alone. Combination of Pazopanib and SDRT also enhanced the total ceramide content compared to Pazopanib or SDRT alone treatment (Table 1). Recent studies revealed that C16-ceramide was preferentially associated with stress-induced apoptosis in a variety of cell types [25–27]. In this study, C16-ceramide, the apoptogenic ceramide, level was immediately enhanced after radiation treatment, and increased from a baseline of 72.71 ± 0.47 pmol/106 cells to 112.25 ± 5.88 pmol/106 cells at 2 min and to 127.36 ± 3.04 pmol/106 cells at 10 min (Figure 6B and Table 1). Pazopanib induced a significant increase in the generation of ceramide (243.03 ± 3.65 pmol/106 cells) at 2 min, which was maintained over 10 min (249.48 ± 1.8 pmol/106 cells, p < 0.05% vs SDRT alone). There were no significant differences in the other ceramide species levels in response to Pazopanib or SDRT alone or in their combination. These results indicate that Pazopanib potentiates radiation-induced apoptosis in endothelial cells via modulation of ASMase/ceramide signaling.