Hypoxic eNOS uncoupling as a consequence of DHFR downregulation in various human endothelial cell lines: the crucial role of the BH4 regeneration pathway.

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS), which produces nitric oxide (NO) to maintain vascular homeostasis. When BH4 is deficient, eNOS becomes uncoupled, generating superoxide ([Formula: see text]) instead of NO, contributing to endothelial dysfunction and cardiovascular disease. The cellular BH4 concentration is determined by its de novo synthesis via GTP cyclohydrolase I (GTPCH), oxidation of BH4 to BH2, and the regeneration of BH4 from BH2 by dihydrofolate reductase (DHFR). A diminished BH4/BH2 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 BH4 regeneration, and induces eNOS uncoupling in all human EC types tested. We also demonstrate that human ECs exhibit low basal BH4 levels, which may result from limited GTPCH expression; consequently, the BH4/BH2 ratio appears to depend substantially on DHFR activity. Importantly, we show for the first time that BH4-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.NEW & NOTEWORTHY This study identifies DHFR-dependent BH4 regeneration as the key determinant of eNOS coupling in human endothelial cells with intrinsically low BH4 levels. We show that hypoxia disrupts this pathway, leading to eNOS uncoupling in a vascular bed-specific manner. Notably, human aortic endothelial cells are particularly susceptible due to high eNOS and low DHFR expression, highlighting DHFR as a potential therapeutic target in hypoxia-driven endothelial dysfunction.