Am J Physiol Cell Physiol. 2026 Aug 7. doi: 10.1152/ajpcell.00171.2026. Online ahead of print.
ABSTRACT
Tetrahydrobiopterin (BH) is an essential cofactor for endothelial nitric oxide synthase (eNOS), which produces nitric oxide (NO) to maintain vascular homeostasis. When BH is deficient, eNOS becomes uncoupled, generating superoxide (O-) instead of NO, contributing to endothelial dysfunction and cardiovascular disease. The cellular BH concentration is determined by its de novo synthesis via GTP cyclohydrolase I (GTPCH), oxidation of BH to BH, and the regeneration of BH from BH by dihydrofolate reductase (DHFR). A diminished BH/BH ratio, often due to DHFR dysregulation, promotes eNOS uncoupling. This study investigates how hypoxia affects eNOS activity and NO bioavailability in human endothelial cells (ECs) derived from various vascular beds. We show that hypoxia downregulates eNOS and DHFR, impairs BH regeneration, and induces eNOS uncoupling in all human EC types tested. We also demonstrate that human ECs exhibit low basal BH levels, which may result from limited GTPCH expression; consequently, the BH/BH ratio appears to depend substantially on DHFR activity. Importantly, we show for the first time that BH-dependent regulation of eNOS uncoupling varies between ECs derived from distinct vascular beds. This variability is driven by cell-type-specific differences in the relative levels of eNOS and DHFR under hypoxia. In particular, human aortic endothelial cells (HAECs) display high eNOS expression and low DHFR levels, making them especially prone to hypoxic eNOS uncoupling. These findings suggest that certain vascular beds may be intrinsically more susceptible to hypoxia-induced endothelial dysfunction, driven by greater eNOS uncoupling that depends on DHFR activity, highlighting DHFR as a potential therapeutic target.
PMID:42566752 | DOI:10.1152/ajpcell.00171.2026