Nitric oxide (NO), which is derived from endothelial
NO synthase (eNOS), provides crucial signals for angiogenesis in the tumor microenvironment.
Tetrahydrobiopterin (BH4) is an absolute requirement for eNOS activity. In this study, we investigated whether this activation is both maintained by a wild-type Ras/
phosphatidylinositol 3-kinase (PI3K)/Akt-positive feedback loop in endothelial cells and affects
tumor angiogenesis. We found that supplementation of BH4 (via the
pterin salvage pathway with Sep) increased Akt/eNOS phosphorylation in both human eNOS-transfected COS-7 cells and endothelial cells concomitant with increases in NO production, cell proliferation, migration, and tube formation. This augmentation was abrogated by a PI3K inhibitor.
Sepiapterin (Sep) also increased
GTP-bound wild-type Ras and PI3K/Akt/eNOS activation, which was prevented by the eNOS inhibitor, Nω-Nitro-
L-arginine methyl ester (
L-NAME). Furthermore, expression of
GTP cyclohydrolase I (the rate-limiting
enzyme in de novo BH4 synthesis) under
doxycycline control potentiated in vivo
tumorigenesis,
tumor cell proliferation, as well as angiogenesis. Conversely, both switching off
GTP cyclohydrolase I expression as well as inhibiting its enzymatic activity significantly decreased eNOS expression and
tumor vascularization. This study demonstrates an important role for BH4 synthesis in angiogenesis by the activation of eNOS for NO production, which is maintained by a PI3K/Akt-positive feedback loop through effects on wild-type Ras in endothelial cells. Our findings suggest that BH4 synthesis may be a rational target for antiangiogenesis
therapy for
tumors.