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Does the Mediterranean G6PD S188F Polymorphism Confer Vascular Protection?: A Novel Rat Model Offering CRISPR Insight Into High Fat-Induced Vascular Dysfunction and Hypertension.

Glucose-6-phosphate dehydrogenase (G6PD), the first enzyme and rate-limiting step in the pentose phosphate pathway, oxidizes glucose-6-phosphate to 6-phosphogluconolactone, while also serving as a major cell generator of NADPH. As such, G6PD predictably subserves both antiand pro-oxidant effects in cells. Via the generation of NADPH, G6PD replenishes reduced glutathione (GSH) from oxidized glutathione (GSSG). It stands to reason then that a polymorphism in G6PD leading to G6PD insufficiency would be expected to increase the GSSG-to-GSH ratio and decrease GSH reductase activity and H 2 O 2 levels. eNOS (endothelial NO synthase) and other NOS (NO synthase) isozymes rely on NADPH to convert arginine to citrulline while liberating NO. Further, NADPH is the substrate for the Nox (NADPH oxidase) family of enzymes, major sources of reactive oxygen species/reactive nitrogen species in the cardiovascular system and myriad other organ systems. Modulation in NADPH levels is expected to cause aberrations in liver metabolic cytochrome P450 and other oxidoreductases that require NADPH as a cofactor. All told, the contribution of G6PD and NADPH is likely to be much more complex than its effects on GSH peroxidase, NOS, and oxidoreductases since an increased oxidation state of a cell or cellular compartment is known to have profound effects on proteins ranging from tyrosine kinases to calcium channels. In this issue, Kitagawa et al shed light on our understanding of the impact of G6PD deficiency (one of the most common enzyme deficiencies worldwide) on vascular function and blood pressure. As the authors reveal, the most common complications from G6PD deficiency are neonatal jaundice and hemolytic anemia. However, relevant to the current spotlighted study, the deficiency has been implicated in a higher incidence of hypertension in some cases although the data are not conclusive in that regard. On the contrary, studies have suggested that the Mediterranean polymorphism is vasoprotective. Employing CRISPR-Cas9 gene editing, the authors generate a novel rat model replicating a specific human Mediterranean single-nucleotide polymorphism (S188F) that is epidemiologically associated with a lower incidence of vascular disease in humans. With this model, the authors set out to interrogate a potential mechanism afforded by this polymorphism as to whether or not it can be vasculoprotective. Compared with WT (wild type) rats, livers from G6PD mutants display lower G6PD activity by 80%, increased GSSG-to-GSH ratio, and decreased nucleotide levels, all of which rather faithfully recapitulate the human phenotype. To date, in vitro and in vivo manipulation of G6PD gene expression has yielded mixed results for vascular reactivity. Indeed, G6PD-deficient cells and animals can be more susceptible to oxidative damage and decreased NO levels than WT cells/mice; and animals overexpressing G6PD may be protected against oxidation and generate greater NO. On the other hand, studies have suggested that hyperlipidemia and hyperglycemia potentiate G6PD activity and NADPH levels leading to augmented superoxide anion production from Nox2 and rat myocardial and vascular dysfunction. Intriguingly in the current study, the authors’ findings are supportive of a potential reduced propensity for cardiovascular disease in the Mediterranean single-nucleotide polymorphism cohort consistent with clinical data in humans. Indeed, the high-fat diet representative of metabolic syndrome and applied in the current study is expected to induce vascular abnormalities including vascular dysfunction, inflammation, and vessel stiffening, which all combine to cause greater vasoconstriction and increased blood pressure. However, a lesser increase in systolic and diastolic blood pressure and arterial stiffness was observed in G6PD mutant rats versus WT rats, suggesting that these G6PD-deficient rats are less prone to vascular pathologies elicited by high fat. Moreover, the authors show that the same changes (observed in response to NOS inhibition) in WT rats are markedly attenuated or abrogated in G6PD-deficient rats. As G6PD-derived NADPH is required for NOS-mediated NO production, it stands to reason that with lower G6PD activity, mutant rats would have decreased NO synthase activity, and thus application of the NO synthase inhibitor L-NAME (N[ω]-Nitro-L-arginine methyl ester hydrochloride) would yield a lesser response in the G6PD-deficient rats. While the authors conclude that these The opinions expressed in this article are not necessarily those of the American Heart Association. From the Heart, Lung, Blood and Vascular Medicine Institute and Department of Pharmacology and Chemical Biology, University of Pittsburgh, PA. Correspondence to Patrick J. Pagano, Department of Pharmacology and Chemical Biology, University of Pittsburgh, 200 Lothrop St, BST-E1247, Pittsburgh, PA 15261. Email pagano@pitt.edu Does the Mediterranean G6PD S188F Polymorphism Confer Vascular Protection? A Novel Rat Model Offering CRISPR Insight Into High Fat–Induced Vascular Dysfunction and Hypertension