Redox sensor and effector functions of tetrahydrobiopterin and the mitochondria in molecular models of cardiovascular disease

Tetrahydrobiopterin (BH4) is a redox active molecule that is essential for the production of nitric oxide (NO) by the NO synthase (NOS) enzymes. Loss of endothelial BH4 leads to decreased NO and increased superoxide generation by endothelial NOS (eNOS) uncoupling, and the resulting endothelial dysfunction is involved in the pathogenesis of vascular diseases. Although genetic augmentation of endothelial BH4 synthesis has proven successful at normalising dysfunction in murine studies, clinical trials of BH4 supplementation have been limited by systemic oxidation. This highlights the need to explore the wider roles of BH4 signalling to allow alternate therapeutic approaches. Thus the aims of this thesis were to identify novel pathways modulated by BH4, both independent from and via NO signalling.
Our laboratory has previously reported a major NOS-independent but unidentified source of superoxide in BH4-deficient endothelial cells. In this thesis, RNA interference targeting Gch1 was used to knock down synthesis of the BH4 synthetic enzyme, GTPCH, to deplete murine endothelial cells of BH4. This model was used to reveal that mitochondria were the main site of superoxide generation in BH4-deficient cells, and that this occurred independent from the NOS cofactor function of BH4. Due to the localised nature of ROS signalling, the effects of BH4 depletion on mitochondrial structure and function were subsequently investigated. BH4-deficient endothelial cells contained larger mitochondria, elevated succinate and decreased isocitrate accumulations indicating altered tricarboxylic acid (TCA) cycle metabolism. Overall, this study shows that targeting mitochondrial superoxide generated by BH4-deficient endothelium may present a potential therapeutic target.
In addition, inflammatory macrophages synthesise BH4 and NO (by iNOS) to fight pathogens, however these cells are also implicated in the development of atherosclerosis. Inflammatory polarisation of macrophages involves dramatic metabolic remodelling, including NO-dependent repression of respiration, up-regulation of glycolysis and accumulation of specific TCA cycle metabolites. These changes support cytokine production and the pro-inflammatory phenotype, but mechanisms regulating this metabolic remodelling are still being elucidated and the roles of BH4 and NO beyond modulation of respiration are unknown. In this thesis, whole cell proteomic and metabolomic analyses were carried out on inflammatory Gch1 knockout (KO) bone marrow derived macrophages from Gchfl/flTie2cre mice. This revealed that BH4 modulated aspects of mitochondrial and metabolic pathways in inflammatory macrophages, and Nos2-/- (iNOS KO) macrophages were used to investigate whether changes occurred due to loss of NO signalling. BH4 was shown to modulate the abundance of subunits making up the catalytic N-module of Complex I, HIF1a-mediated glycolytic remodelling and levels of key inflammatory mediators and TCA cycle metabolites: succinate, citrate and itaconate, via NO signalling. This study shows that NO-signalling directs critical aspects of immuno-metabolism in macrophages.

Keywords: Mitochondria
Metabolism
Macrophages
Redox
Tetrahydrobiopterin
Immunometabolism
Endotheliall cells
Nitric oxide
Advisor: Channon, K
Crabtree, M