Relation between dysfunction of nitric oxide synthase and angiogenesis

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Reactive oxygen species (ROS) have been known to induce tissue injury in ischemialreperfusion and inflammation. Recently, however, it has beenTeported that-ROS regulates cel-lular-function such as proliferation. Interestingly, nitric oxide synthase (NOS) from brain releases ROS instead of nitric oxide (NO) when tetrahydrobiopterin (BH4), a cofactor of NOS, is decreased. The purpose of this study was to determine whether endothelial isoform of NOS also produces ROS with decreasing BH4, and the dysfunction of NOS affects angiogenesis. Addition of calcium ionophore to endothelial cells (ECs) released 0_2 which was measured by using MCLA, a Cypridina luciferin analogue. The calcium ionophore-induced 0_2 release was further stimulated by the treatment with 2,4-diamino-6-hydroxyprimidine (DAHP), an inhibitor of BH4 synthesis. Moreover, he calcium ionophore-induced O_2 release in the DAHP treated cells was strongly inhibited by NOS inhibitor. These findings suggest that endothelial isoform of NOS also produces ROS with decreasing BH4 content. We next examined the effect of H_20_2, one of the ROS, on in vitro angiogenesis. The low concentrations of H_20 stimulated angiogenesis. Ets-1 is a member of the ets gene family of transcription factors, which regulates the expression of urokinase plasminogen activator and matrix metalloprotease-1. Interestingly, H_20_2, increased the ets-l mRNA in ECs. The H_2_2-stimulated angiogenesis was completely blocked by an ets-1 antisense oligonucleotide. These results indicate that low concentrations of H_2O_2 stimulate angiogenesis, and the H_20_2-induced angiogenesis is likely to be mediated by the transcription factor ets-l. In the present study, we were not able to determine whether ROS from NOS affect angiogenesis, since DAHP itself has an inhibiting effect of angiogenesis. Future studies will be needed to find more selective inhibitor for BH4 synthesis.