Vasoprotectiven actions of nitroxyl (HNO) in vascular disease

Nitroxyl (HNO), the one electron reduced and protonated congener of nitric oxide (NO) has recently gained therapeutic interest as HNO exhibits similar vasoprotective actions to that of NO, but does not share its limitations. Additionally, HNO displays unique pharmacological actions that may enable its effects to be preserved and/or enhanced in disease states where NO function is compromised. This thesis examined whether the vasoprotective actions of endogenous and exogenous HNO were maintain in the setting of hypercholesterolemia and advanced atherosclerosis (apolipoprotein E-deficient mice maintained on a high fat diet for 7 and 21 weeks, respectively; ApoE-/--HFD) and these effects were directly compared to NO. Given the role that oxidative stress plays in the pathogenesis of these diseases, this thesis also determined whether the absence of the superoxide (.O2-) generating enzyme Nox2-NADPH oxidase, conferred vasoprotection in advanced atherosclerosis. In Chapter 2, we confirmed that the vasorelaxant and anti-aggregatory effects of the HNO donors, Angeli’s salt and isopropylamine NONOate (IPA/NO) were mediate via HNO and were dependent upon sGC/cGMP signalling. Furthermore, in hypercholesterolemic mice (ApoE-/--HFD mice) we observed that vasorelaxation responses to IPA/NO and the clinically used NO donor, glyceryl trinitrate (GTN) were conserved. However, GTN, but not IPA/NO, was susceptible to vascular tolerance development and its anti-aggregatory response was abolished in hypercholesterolemia. Given the vasoprotective actions of HNO were preserved in hypercholesterolemia, Chapter 3 sought to compare the vasoprotective actions of HNO and NO in advanced atherosclerosis (ApoE-/--HFD mice). In these mice, IPA/NO and the NO donor, diethylamine NONOate (DEA/NO) maintained their ability to induce relaxation in carotid arteries, inhibit platelet aggregation, supress . O2- generation and did not develop vascular tolerance. Whilst relaxation responses to GTN were sustained, GTN developed vascular tolerance and it’s anti-aggregatory and . O2- limiting actions were compromised in atherosclerotic mice. Additionally, the contribution of HNO to endothelium-dependent relaxation was enhanced in advanced atherosclerosis and may compensate for a reduction in endogenous NO. In Chapter 4 we found that the ability of the NO donor sodium nitroprusside (SNP) to inhibit platelet aggregation and elevate cGMP levels was impaired in hypercholesterolemic (ApoE-/--HFD) mice, indicating a model of platelet NO resistance. In this model, dysfunction at the level of sGC and/or downstream mediators of cGMP appeared to contribute to resistance. Our main finding was that the anti-platelet effects of NO-independent sGC stimulators IPA/NO and BAY 41-2272 were maintained in hypercholesterolemic mice, indicating that IPA/NO and BAY 41-2272 can circumvent platelet NO resistance. In Chapter 5 we examined the effect of genetic deletion of the . O2- generating enzyme Nox2-NAD oxidase upon vascular function and plaque morphology in advanced atherosclerosis (ApoE-/- mice on a HFD for 21 weeks). We observed that Nox2 oxidase deletion had no effect upon endogenous NO generation or endothelium-dependent and –independent relaxation responses but reduced vascular . O2- generation in advanced atherosclerosis. Moreover, in aortae and innominate arteries Nox2 deletion did not reduce lesion size but did lead to an increase in plaque stability and appeared to promote an anti-inflammatory (M2) macrophage phenotype. These findings suggest that, Nox2 oxidase plays an important role in plaque instability in atherosclerosis. In conclusion, the findings of this thesis indicate that the vasoprotective actions of endogenous and exogenously generated HNO are sustained or augmented, in the setting of hypercholesterolemia and advanced atherosclerosis. These properties, coupled with the lack of tolerance development, resistance to scavenging by . O2- and an ability of HNO to circumvent platelet NO resistance and target sGC/cGMP-independent signalling pathways confers advantages over traditional nitrovasodilators. Thus, HNO donors represent an innovative pharmacotherapy for the treatment of cardiovascular diseases.