Irradiation-induced angiogenesis through the up-regulation of the nitric oxide pathway: implications for tumor radiotherapy.

The combination of radiotherapy and antiangiogenic strategies has been shown to increase the tumor response in various experimental models. The rationale for this cotherapy was initially related to the expected gain in efficacy by acting on two different targets, e.g., tumor cells and endothelial cells (ECs). However, recent studies have documented more than additive effects due to apparent mutual potentiation of these approaches. In this study, we tested the hypothesis that these synergistic effects could stem from the stimulatory effects of ionizing radiations on angiogenesis, which would then need to be restrained to avoid tumor regrowth after irradiation. We found that irradiation dose-dependently induced the activation of the proangiogenic NO pathway in ECs through increases in endothelial nitric oxide synthase abundance and phosphorylation. Using 2- and 3-dimensional cultures of ECs and isolated mouse tumor arterioles, we documented that the irradiation-induced enhanced production of NO accounted for EC migration and sprouting. Irradiation was also shown to stimulate the colonization of Matrigel plugs implanted in mouse by ECs, where they formed capillary-like structures in a NO-dependent manner. These findings were confirmed by documenting the NO-mediated infiltration of CD31-positive ECs after local irradiation of Lewis lung carcinoma tumor-bearing mice. Finally, we measured a consistent increase in endothelial nitric oxide synthase mRNA by real-time PCR experiments in human biopsies of head and neck squamous cell carcinoma after low-dose irradiation. In conclusion, we have demonstrated that the potentiation of the NO signaling pathway after irradiation induces profound alterations in the EC phenotype leading to tumor angiogenesis. Moreover, our demonstration that the inhibition of NO production suppresses these provascular effects of irradiation highlights new potentials for the coordinated use of antiangiogenic strategies and radiotherapy in clinical practice.