Nitroxyl: A Novel Strategy to Circumvent Diabetes Associated Impairments in Nitric Oxide Signaling

Abstract

Diabetes is associated with an increased mortality risk due to cardiovascular complications. Hyperglycemia-induced oxidative stress underlies these complications, leading to an impairment in endogenous nitric oxide (NO•) generation, together with reductions in NO• bioavailability and NO• responsiveness in the vasculature, platelets and myocardium. The latter impairment of responsiveness to NO•, termed NO• resistance, compromises the ability of traditional NO•-based therapeutics to improve hemodynamic status during diabetes-associated cardiovascular emergencies, such as acute myocardial infarction. Whilst a number of agents can ameliorate (e.g. angiotensin converting enzyme [ACE] inhibitors, perhexiline, statins and insulin) or circumvent (e.g. nitrite and sGC activators) NO• resistance, nitroxyl (HNO) donors offer a novel opportunity to circumvent NO• resistance in diabetes. With a suite of vasoprotective properties and an ability to enhance cardiac inotropic and lusitropic responses, coupled with preserved efficacy in the setting of oxidative stress, HNO donors have intact therapeutic potential in the face of diminished NO• signaling. This review explores the major mechanisms by which hyperglycemia-induced oxidative stress drives NO• resistance, and the therapeutic potential of HNO donors to circumvent this to treat cardiovascular complications in type 2 diabetes mellitus.

Keywords: HNO; cardiovascular disease; diabetes; nitric oxide; nitric oxide resistance; nitroxyl; type 2 diabetes.

Figure 1

Nitric oxide signaling in the vasculature and myocardium. In endothelial cells and cardiomyocytes, nitric oxide (NO•) is produced by endothelial nitric oxide synthase (eNOS) following stimulation by shear stress (blood flow) or the presence of agonists such as bradykinin. Upon stimulation, the cofactor tetrahydrobiopterin (BH₄) is recruited, resulting in the conversion of L-arginine to NO• and L-citrulline. In the vascular lumen, NO• inhibits platelet aggregation and leukocyte adhesion and migration. NO• produced by endothelial cells diffuses into underlying vascular smooth muscle cells (VSMCs) where it suppresses proliferation and binds to the ferrous (Fe²⁺) heme group on its biological target, soluble guanylyl cyclase (sGC). Activation of sGC leads to the production of 3',5'-cyclic guanosine monophosphate (cGMP) resulting in vasodilation. NO• produced by endothelial cells from coronary vessels, diffuses into cardiomyocytes, where in combination with NO• produced intracellularly by eNOS and neuronal nitric oxide synthase (nNOS), induces myocardial relaxation, and has anti-hypertrophic effects. NO• also suppresses thioredoxin-interacting protein (TXNIP) formation in cardiomyocytes and the vasculature.

Figure 2

Hyperglycemia-induced oxidative stress impairs nitric oxide signaling. Hyperglycemia induces oxidative stress via increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes including Nox2, resulting in superoxide (O₂ ⁻) generation. In mitochondria, hyperglycemia increases reactive oxygen species (ROS) production including O₂ ⁻, which stimulates thioredoxin-interacting protein (TXNIP) expression. TXNIP promotes activation of the NLRP3 inflammasome, which activates caspase-1, resulting in the maturation and secretion of the pro-inflammatory cytokines, interleukin-1β (IL-1β) and interleukin-18 (IL-18). O₂ ⁻ also causes strand breaks in DNA, leading to activation of poly (ADP-ribose) polymerase (PARP), which reduces activity of glyceraldehyde-3 phosphate dehydrogenase (GAPDH). Decreased GAPDH activity leads to overactivation of the hexosamine pathway, upregulation of protein kinase C (PKC), elevated glucose flux through the polyol pathway and increased formation of advanced glycation end-products (AGEs). This leads to activation of Nox and NF-κB signaling, resulting in increased expression of pro-inflammatory and pro-atherogenic mediators including vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and interleukin-6 (IL-6). Upregulation of these pathways results in impaired flow-mediated vasodilation, reflecting both endothelial dysfunction and nitric oxide (NO•) resistance.

Figure 3

Nitric oxide resistance and its circumvention by nitroxyl. Under oxidative stress, activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes, such as Nox2, is elevated, resulting in increased superoxide (O₂ ⁻) generation. O₂ ⁻ reacts with nitric oxide (NO•), generating the powerful oxidant, peroxynitrite (ONOO^-), which oxidizes the ferrous (Fe²⁺) heme group of sGC to the ferric (Fe³⁺) or heme-free state, desensitizing the enzyme to NO•. Consequently, tissue responsiveness to NO• is impaired, resulting in NO• resistance. Nitroxyl (HNO) is resistant to scavenging by O₂ ⁻ and HNO donors offer an opportunity to circumvent NO• resistance. In the vasculature, HNO causes vasorelaxation, inhibits platelet aggregation and reduces monocyte activation. In vascular smooth muscle cells (VSMCs), HNO signals predominantly via activation of sGC and the subsequent increase in 3',5'-cyclic guanosine monophosphate (cGMP) and may activate the oxidized (Fe³⁺) form of sGC. HNO also targets vascular voltage-dependent and ATP-sensitive K⁺ channels through a cGMP-dependent mechanism. In the vasculature and myocardium, HNO interacts directly with Nox2 to suppresses O₂ ^- generation. In cardiomyocytes, HNO has anti-hypertrophic effects, and reacts directly with thiols and thiol-containing proteins including the sarcoplasmic reticulum Ca²⁺-ATPase pump (SERCA2a) and ryanodine receptors (RyR2) to enhance Ca²⁺ cycling, together with increasing myofilament Ca²⁺ sensitivity, resulting in enhanced myocardial contractility and relaxation. The vaso- and cardio-protective actions of HNO are preserved in the setting of oxidative stress and HNO donors offer a new therapeutic approach to treat diabetes-associated cardiovascular complications.