Angiogenin Induces Nitric Oxide Synthesis in Endothelial Cells through PI-3 and Akt Kinases

Angiogenesis, the formation of new blood vessels, is a critical but complex phenomenon modulated by numerous physicochemical conditions. Nitric oxide (NO) is a very well known biological mediator involved in vascular physiology. This study focuses on relationships between the effect of angiogenin, a major angiogenic factor, and extracellular NO release. NO concentration was sensed electrochemically using a fibronectin-coated multiple microelectrode array. Angiogenin was shown to increase NO levels, thus triggering nitric oxide synthase (NOS) activity. The effect of angiogenin on NOS was demonstrated using l-NAME, a competitive NOS inhibitor. Dose−time dependence was investigated, showing a stimulation threshold in the 250 ng/mL−1 μg/mL range and a maximal NO release after 30 min of exposure to angiogenin. To elucidate the very complex reactive pathway of angiogenin, we have used various selective inhibitors to investigate the mechanism leading to NO production. Neomycin, an antibiotic blocking nuclear translocation, inhibited the angiogenin effect on NOS. This result demonstrates that angiogenin activates NOS by interacting with the cell nucleus. Similarly, NOS activity was stopped by blocking the PI-3/Akt kinase signaling transduction cascade, showing the importance of this pathway.