NADPH oxidase and xanthine oxidoreductase as targets and regulators of the nitrate-nitrite-nitric oxide pathway

Overproduction of reactive oxygen species (ROS) by NADPH oxidase (NOX) and xanthine oxidoreductase (XOR) with a concomitant decrease in the bioavailability of nitric oxide (NO) from eNOS contributes to the development of cardiovascular and metabolic disorders. Extensive research has proven the existence of an alternative NOS-independent pathway for NO production. This nitrate-nitrite-NO pathway starts with inorganic nitrate which is derived from the diet and from oxidized NO. Nitrate is reduced to nitrite mainly by oral commensal bacteria and then to NO and other bioactive nitrogen oxides in blood and tissues.

The aim of the current thesis was to investigate the therapeutic role of the nitrate-nitrite-NO pathway in models of hypertension, metabolic dysfunction and inflammation focusing on whether the main ROS-producing enzymes NOX and XOR could be targets or even regulators of the this pathway. We show that NOX in the renal microvasculature is a primary target for the blood pressure lowering effects of dietary nitrate in Angiotensin-II (AngII)-mediated hypertension. In agreement, NOX activity and AngII-induced receptor signaling are downregulated by nitrate in aged and hypertensive rats. Moreover, nitrate targets the elevated liver NOX activity of aged and metabolically dysregulated mice with an improvement of AMPK activity. Finally, we show that nitrite can act on cells of the innate immune response. In particular, NOX mediated superoxide production is strongly attenuated in activated macrophages with a concomitant reduction on iNOS gene expression and peroxynitrite production.

Mechanistically, we observed that symbiotic bacteria and XOR together are responsible for the bioactivation of dietary nitrate to form NO. In addition, the effects of nitrate and nitrite are not only NOS independent but actually potentiated when eNOS activity is pharmacologically, genetically or naturally impaired. Interestingly, the absence of eNOS is associated with a higher XOR activity that partly compensates for the disrupted NO homeostasis and elevated blood pressure in these mice. Also, the blood pressure response to dietary nitrate is augmented in eNOS-/- mice and abolished upon XOR inhibition. Interestingly, nitrate and nitrite were able to switch the function of XOR towards lower ROS and higher NO production which could significantly contribute to the antihypertensive effects.

In conclusion, administration of nitrate or nitrite is associated with a number of therapeutic cardiovascular and metabolic effects in animal models of disease. We propose that NOX and XOR are two main targets and possible regulators of the nitrate-nitrite-NO pathway. This pathway is triggered in situations with higher NOX and XOR activity and acts in parallel with the NOS dependent NO production to uphold NO homeostasis.

List of scientific papers

I. Gao X, Yang T, Liu M, PELELI M, Zollbrecht C, Weitzberg E, Lundberg JO, Persson AE, Carlström M. NADPH oxidase in the renal microvasculature is a primary target for blood pressure-lowering effects by inorganic nitrate and nitrite. Hypertension. 2015 Jan; 65(1):161-70.
https://doi.org/10.1161/HYPERTENSIONAHA.114.04222

II. Yang T, PELELI M, Zollbrecht C, Giulietti A, Terrando N, Lundberg JO, Weitzberg E, Carlström M. Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide dependent mechanism. Free Radic Biol Med. 2015 Jun;83:159-66.
https://doi.org/10.1016/j.freeradbiomed.2015.02.016

III. Peleli M, Hezel M, Zollbrecht C, Persson AE, Lundberg JO, Weitzberg E, Fredholm BB, Carlström M. In adenosine A2B knockouts acute treatment with inorganic nitrate improves glucose disposal, oxidative stress and AMPK signaling in the liver. Front Physiol. 2015 Aug 7;6:222.
https://doi.org/10.3389/fphys.2015.00222

IV. Hezel M, Peleli M, Liu M, Zollebrecht C, Jensen B, Checa A, Giulietti A, Wheelock C, Lundberg JO, Weitzberg E, Carlström M. Dietary nitrate improves age-related hypertension and metabolic abnormalities via modulation of angiotensin II receptor signaling and inhibition of superoxide generation. Free Radic Biol Med. 2016 Jul 26;99:87-98.
https://doi.org/10.1016/j.freeradbiomed.2016.07.025

V. Peleli M, Zollbrecht C, Montenegro MF, Hezel M, Zhong J, Persson E.G, Holmdahl R, Weitzberg E, Lundberg JO and Carlström M. Enhanced XOR activity in eNOS-deficient mice: Effects on the nitrate-nitrite-NO pathway and ROS homeostasis. Free Radic Biol Med. 2016 Sep 5;99:472-484.
https://doi.org/10.1016/j.freeradbiomed.2016.09.004