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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.

Original publication

DOI

10.2353/ajpath.2010.100025

Type

Journal article

Journal

The American journal of pathology

Publication Date

11/2010

Volume

177

Pages

2671 - 2680

Addresses

Department of Clinical Pharmacology, University of Oxford, John Radcliffe Hospital, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ, UK.

Keywords

COS Cells, NIH 3T3 Cells, Animals, Cercopithecus aethiops, Humans, Mice, Neovascularization, Pathologic, Nitric Oxide, Pterins, Biopterin, ras Proteins, Signal Transduction, Cell Proliferation, Cell Movement, Enzyme Activation, Proto-Oncogene Proteins c-akt, Nitric Oxide Synthase Type III, Phosphatidylinositol 3-Kinases, Tumor Microenvironment