Disruption of the balance between nitric oxide (NO) and superoxide has been linked to the development of pulmonary hypertension (PH). PH, associated with right ventricular hypertrophy (RVH) and pulmonary vascular remodelling (PVR) develops also in response to chronic hypoxia. We thus hypothetized that hypoxia affects the balance between NO and superoxide and investigated a possible link to PH.

Exposure to 24 h hypoxia lowered NO levels in human pulmonary artery endothelial cells (HPAEC) or murine pulmonary arteries (PA) compared to normoxic controls as measured by electron paramagnetic resonance (EPR). Concomitantly, the ratio between tetrahydrobiopterin (BH4), a cofactor for endothelial NO synthase (eNOS) activity, and dihydrobiopterin (BH2) was reduced, while expression of dihydrofolate reductase (DHFR) which controls BH4 recycling from BH2, was decreased under hypoxia. Treatment with folic acid (FA) which can induce and activate DHFR, restored not only hypoxic DHFR levels but also the hypoxic BH4/BH2 ratio to normoxic levels.

Since reduced BH4 levels have been associated with uncoupling of eNOS we measured superoxide levels in hypoxic HPAEC or PA by EPR. Compared to normoxia, superoxide levels were decreased. Inhibition of eNOS further decreased hypoxic superoxide levels, and this response was restored by folic acid further indicating that eNOS was uncoupled under hypoxia generating superoxide rather than NO. Subsequently, blood NO levels and BH₄/BH₂ ratio were decreased in mice exposed to chronic hypoxia while treatment with FA restored NO and BH4 levels to normoxic values. In addition, the development of PVR and RVH in chronic hypoxic mice was diminished upon treatment with FA.

Collectively, these findings indicate that hypoxia uncouples eNOS by reducing BH2 to BH4 conversion due to decreased DHFR expression, thus lowering NO and increasing superoxide levels. Restoration of DHFR levels by FA not only improves the disturbed balance between NO and superoxide but also reduces PVR and RVH due to chronic hypoxia. Thus, FA treatment may be a novel therapeutic option in PH.