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

Authors

Anna Janaszak-Jasiecka, Adrianna Radulska, Anna Siekierzycka, Agata Płoska, Tomasz Borkowski, Iwona T Dobrucki, Rafał Bartoszewski, Leszek Kalinowski

Published in

American journal of physiology. Cell physiology. Aug 07, 2026. Epub Aug 07, 2026.

Abstract

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 (O₂^-) 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.

PMID:
42566752
Bibliographic data and abstract were imported from PubMed on 08 Aug 2026.

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