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Endothelial dysfunction due to eNOS uncoupling: molecular mechanisms as potential therapeutic targets - PubMed

  • ️Sun Jan 01 2023

Endothelial dysfunction due to eNOS uncoupling: molecular mechanisms as potential therapeutic targets

Anna Janaszak-Jasiecka et al. Cell Mol Biol Lett. 2023.

Abstract

Nitric oxide (NO) is one of the most important molecules released by endothelial cells, and its antiatherogenic properties support cardiovascular homeostasis. Diminished NO bioavailability is a common hallmark of endothelial dysfunction underlying the pathogenesis of the cardiovascular disease. Vascular NO is synthesized by endothelial nitric oxide synthase (eNOS) from the substrate L-arginine (L-Arg), with tetrahydrobiopterin (BH4) as an essential cofactor. Cardiovascular risk factors such as diabetes, dyslipidemia, hypertension, aging, or smoking increase vascular oxidative stress that strongly affects eNOS activity and leads to eNOS uncoupling. Uncoupled eNOS produces superoxide anion (O2-) instead of NO, thus becoming a source of harmful free radicals exacerbating the oxidative stress further. eNOS uncoupling is thought to be one of the major underlying causes of endothelial dysfunction observed in the pathogenesis of vascular diseases. Here, we discuss the main mechanisms of eNOS uncoupling, including oxidative depletion of the critical eNOS cofactor BH4, deficiency of eNOS substrate L-Arg, or accumulation of its analog asymmetrical dimethylarginine (ADMA), and eNOS S-glutathionylation. Moreover, potential therapeutic approaches that prevent eNOS uncoupling by improving cofactor availability, restoration of L-Arg/ADMA ratio, or modulation of eNOS S-glutathionylation are briefly outlined.

Keywords: ADMA; BH4; Cardiovascular disease; Endothelial dysfunction; Nitric oxide; Oxidative/nitroxidative stress; Peroxynitrite; Tetrahydrobiopterin; eNOS uncoupling.

© 2023. The Author(s).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1

Schematic eNOS homodimer structure with two monomers orientated "head-to-tail". Each monomer consists of a C-terminal reductase domain that binds NADPH, FAD, and FMN, a central calmodulin-binding region, and an N-terminal oxygenase domain that binds substrate L-Arg, oxygen, heme, and BH4. The formation of a homodimer enables the transfer of electrons from the reductase domain of one monomer to the oxygenase domain of the second monomer. The dimeric structure is stabilized by heme binding and by zinc ion in the zinc-thiolate cluster at the dimer interface. During catalysis, electrons from NADPH flow through the flavins FAD and FMN to the heme of the opposite monomer and CaM increases the rate of interdomain electron transfer. Heme reduction enables O2 binding, and BH4 can donate an electron to reduce and activate O2. When cellular redox balance is maintained, and the substrate L-Arg and the essential cofactor BH4 availabilities are optimal, O2 reduction is coupled to L-Arg oxidation and NO synthesis. L-Cit is formed as a byproduct

Fig. 2
Fig. 2

eNOS uncoupling due to BH4 deficiency. Under conditions of oxidative stress, O2− can combine with NO yielding ONOO−, which strongly oxidizes BH4 to BH2. Decreased DHFR expression or activity prevents effective regeneration of the cofactor. BH2 competes with BH4 at the heme oxygenase domain but is not catalitically active, thus disturbing the normal electron flow and promoting superoxide formation

Fig. 3
Fig. 3

eNOS uncoupling due to diminished L-Arg/ADMA ratio. Under reduced L-Arg availability (resulting from excessive arginase activity) and/or accumulation of ADMA (due to decreased DDAH activity), the substrate concentration may not be sufficient to saturate eNOS and/or L-Arg is outcompeted by ADMA. As a result, molecular oxygen is a final electron acceptor, leading to superoxide formation

Fig. 4
Fig. 4

eNOS uncoupling due to eNOS S-glutathionylation. Oxidative stress decreases the cellular GSH/GSSG ratio, leading to protein S-glutathionylation. Glutathionylated cysteine residues (Cys689 and Cys908) of eNOS are located at the interface of the FAD and FMN binding sites, thus disrupting FAD-FMN alignment and electron transfer between flavins, which causes the transfer of an electron to molecular oxygen and the production of a superoxide radical instead of NO. Prolonged retention of S-glutathionylated eNOS (SG-eNOS) in the cytoplasm can result in its degradation via chaperone-mediated autophagy (CMA), leading to irreversible loss of eNOS

Fig. 5
Fig. 5

Major causes of eNOS uncoupling as targets of potential therapeutic interventions. Oxidative stress associated with cardiovascular risk factors leads to eNOS uncoupling by: A decreased BH4/BH2 ratio due to oxidation of BH4 and impairment of DHFR expression/activity; B decreased L-Arg/ADMA ratio due to excessive arginase expression/activity and diminished DDAH expression/activity; C eNOS S-glutathionylation at Cys689 and Cys908

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