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Nitroxyl anion (HNO) donors are currently being assessed for their therapeutic utility in several cardiovascular disorders including heart failure. Here, we examine their effect on factors that precede atherosclerosis including endothelial cell and monocyte activation, leucocyte adhesion to the endothelium and macrophage polarization. Similar to the NO donor glyceryl trinitrate (GTN), the HNO donors Angeli's salt (AS) and isopropylamine NONOate (IPA/NO) decreased leucocyte adhesion to activated human umbilical vein endothelial cells (HUVECs) and mouse isolated aorta. This reduction in adhesion was accompanied by a reduction in intercellular adhesion molecule-1 (ICAM-1) and the cytokines monocyte chemoattractant protein 1 (MCP-1) and interleukin 6 (IL-6) which was inhibitor of nuclear factor κB (NFκB) α (IκBα)- and subsequently NFκB-dependent. Intriguingly, the effects of AS on leucocyte adhesion, like those on vasodilation, were found to not be susceptible to pharmacological tolerance, unlike those observed with GTN. As well, HNO reduces monocyte activation and promotes polarization of M2 macrophages. Taken together, our data demonstrate that HNO donors can reduce factors that are associated with and which precede atherosclerosis and may thus be useful therapeutically. Furthermore, since the effects of the HNO donors were not subject to tolerance, this confers an additional advantage over NO donors.

Background and Purpose Angiotensin AT2 receptors are upregulated in disease states such as atherosclerosis and blockade of the AT2 receptors exacerbates plaque formation. Direct stimulation of these receptors is anti-atherogenic but the mechanisms and pathways involved remain unknown. We examined the effect of direct AT2 receptor stimulation with Compound 21 (C21) on the leukocyte adhesion cascade in vitro, right through to plaque formation in vivo. Experimental Approach Effects of C21 on TNFα-induced inflammation were assessed in human umbilical vein endothelial cells (HUVECs), activation of monocytes, polarisation of monocyte-derived macrophages and in intact mouse aortae. Key Results C21 attenuated TNFα-induced: monocyte adhesion to cultured HUVECs, adhesion molecule expression and abolished TNFα-induced ROS production. TNFα-induced NFκB translocation from the cytoplasm to the nucleus, essential for cytokine production, was prevented by C21. C21 did not influence monocyte activation or macrophage polarisation but did reduce TNFα and IL-6 mRNA expression in M1 macrophages. The anti-inflammatory effects of C21 were abolished by an AT2 receptor antagonist confirming that the effects of C21 were AT2 receptor-mediated. Also, leukocyte adhesion and cytokine gene expression, induced by high-fat diet (HFD), was attenuated in ApoE-/- mice treated with C21. Plaque size and stability were improved with C21 treatment with increased smooth muscle cell composition and decreased lipid size, compared with HFD-saline treated mice. Conclusion and Implications C21 prevented TNFα-induced and HFD-induced vascular inflammation in vitro and in vivo. Our data provide strong evidence that the anti-atherosclerotic actions of C21 were due to vascular anti-inflammatory effects, mediated by AT2 receptors.

Nitroxyl (HNO) is a redox congener of NO˙. We now directly compare the antihypertrophic efficacy of HNO and NO˙ donors in neonatal rat cardiomyocytes and compare their contributing mechanisms of actions in this setting. Isopropylamine-NONOate (IPA-NO) elicited concentration-dependent inhibition of endothelin-1 (ET1)-induced increases in cardiomyocyte size, with similar suppression of hypertrophic genes. Antihypertrophic IPA-NO actions were significantly attenuated by l-cysteine (HNO scavenger), Rp-8-pCTP-cGMPS (cGMP-dependent protein kinase inhibitor), and 1-H-(1,2,4)-oxodiazolo-quinxaline-1-one [ODQ; to target soluble guanylyl cyclase (sGC)] but were unaffected by carboxy-PTIO (NO˙ scavenger) or CGRP8–37 (calcitonin gene-related peptide antagonist). Furthermore, IPA-NO significantly increased cardiomyocyte cGMP 3.5-fold (an l-cysteine-sensitive effect) and stimulated sGC activity threefold, without detectable NO˙ release. IPA-NO also suppressed ET1-induced cardiomyocyte superoxide generation. The pure NO˙ donor diethylamine-NONOate (DEA-NO) reproduced these IPA-NO actions but was sensitive to carboxy-PTIO rather than l-cysteine. Although IPA-NO stimulation of purified sGC was preserved under pyrogallol oxidant stress (in direct contrast to DEA-NO), cardiomyocyte sGC activity after either donor was attenuated by this stress. Excitingly IPA-NO also exhibited acute antihypertrophic actions in response to pressure overload in the intact heart. Together these data strongly suggest that IPA-NO protection against cardiomyocyte hypertrophy is independent of both NO˙ and CGRP but rather utilizes novel HNO activation of cGMP signaling. Thus HNO acutely limits hypertrophy independently of NO˙, even under conditions of elevated superoxide. Development of longer-acting HNO donors may thus represent an attractive new strategy for the treatment of cardiac hypertrophy, as stand-alone and/or add-on therapy to standard care.

Left ventricular hypertrophy (LVH), an increased left ventricular (LV) mass, is common to many cardiovascular disorders, initially developing as an adaptive response to maintain myocardial function. In the longer term, this LV remodelling becomes maladaptive, with progressive decline in LV contractility and diastolic function. Indeed LVH is recognised as an important blood-pressure independent predictor of cardiovascular morbidity and mortality. The clinical efficacy of current treatments for LVH is reduced, however, by their tendency to slow disease progression rather than induce its reversal, and thus the development of new therapies for LVH is paramount. The signalling molecule cyclic guanosine-3',5'-monophosphate (cGMP), well-recognised for its role in regulating vascular tone, is now being increasingly identified as an important anti-hypertrophic mediator. This review is focused on the various means by which cGMP can be stimulated in the heart, such as via the natriuretic peptides, to exert anti-hypertrophic actions. In particular we address the limitations of traditional nitric oxide (NO*) donors in the face of the potential therapeutic advantages offered by novel alternatives; NO* siblings, ligands of the cGMP-generating enzymes, soluble (sGC) and particulate guanylyl cyclases (pGC), and phosphodiesterase inhibitors. Further impact of cGMP within the cardiovascular system is also discussed with a view to representing cGMP-based therapies as innovative pharmacotherapy, alone or concurrent with standard care, for the management of LVH.

Background and purpose: Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries. Experimental approach: Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording. Key results: Vasorelaxation to the HNO donor, Angeli's salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K+ channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger, l-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (with l-NAME, Nω-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO•) with hydroxocobalamin (HXC) or HNO with l-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC and l-cysteine reduced ACh-mediated relaxation, as did l-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination with l-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO•, as HXC and l-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP. Conclusions and implications: The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO• = HNO > EDHF and EDHF > HNO = NO• respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels. British Journal of Pharmacology (2009) 157, 540–550; doi:10.1111/j.1476-5381.2009.00150.x; published online 26 March 2009 This article is commented on by Martin, pp. 537–539 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009

The nitroxyl anion (HNO) is emerging as a novel regulator of cardiovascular function with therapeutic potential in the treatment of diseases such as heart failure. It remains unknown whether tolerance develops to HNO donors, a limitation of currently used nitrovasodilators. The susceptibility of the HNO donor, Angeli’s salt (AS), to the development of vascular tolerance was compared with the NO donors, glyceryl trinitrate (GTN) and diethylamine/NONOate (DEA/NO) in rat isolated aortae. Vasorelaxation to AS was attenuated ( P l -cysteine, whereas the sensitivity to GTN and DEA/NO was decreased ( P P P P GTN. GTN did not induce cross-tolerance to either AS or DEA/NO. In contrast, pre-exposure to DEA/NO, but not AS, caused a concentration-dependent attenuation ( P

Nitroxyl (HNO), the redox congener of nitric oxide, has numerous vasoprotective actions including an ability to induce vasodilation and inhibit platelet aggregation. Given HNO is resistant to scavenging by superoxide and does not develop tolerance, we hypothesised that HNO would retain its in vivo vasodilatory action in the setting of hypertension. The in vitro and in vivo vasodilator properties of the HNO donors Angeli's salt (AS) and isopropylamine/NONOate (IPA/NO) were compared with the NO˙ donor diethylamine/NONOate (DEA/NO) in spontaneously hypertensive rats (SHR) and normotensive [Wistar-Kyoto (WKY) rats]. AS (10, 50, and 200 μg/kg), IPA/NO (10, 50, and 200 μg/kg), and DEA/NO (1, 5, and 20 μg/kg) caused dose-dependent depressor responses in conscious WKY rats of similar magnitude. Depressor responses to AS and IPA/NO were significantly attenuated ( P < 0.01) after infusion of the HNO scavenger N-acetyl-l-cysteine (NAC), confirming that AS and IPA/NO function as HNO donors in vivo. In contrast, responses to DEA/NO were unchanged following NAC infusion. Depressor responses to AS and IPA/NO in conscious SHR retained their sensitivity to the inhibitory effects of NAC ( P < 0.01), yet those to DEA/NO in SHR were significantly ( P < 0.05) enhanced following NAC infusion. Importantly, depressor responses to AS, IPA/NO, and DEA/NO were preserved in hypertension and vasorelaxation to AS and DEA/NO, in isolated aorta, unchanged in SHR as compared with WKY rats. This study has shown for the first time that HNO donors exert antihypertensive effects in vivo and may, therefore, offer a therapeutic alternative to traditional nitrovasodilators in the treatment of cardiovascular disorders such as hypertension.

Background New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated. Methods Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli's salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. Results We now demonstrate that Angeli's salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli's salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli's salt were mimicked by BNP. We also demonstrate that the effects of Angeli's salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP). Conclusions Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.

Nitroxyl (HNO) displays distinct pharmacology to its redox congener nitric oxide (NO(•)) with therapeutic potential in the treatment of heart failure. It remains unknown if HNO donors are resistant to tolerance development following chronic in vivo administration. Wistar-Kyoto rats received a 3-day subcutaneous infusion of one of the NO(•) donors, glyceryl trinitrate (GTN) or diethylamine/NONOate (DEA/NO), or the HNO donor Angeli's salt (AS). GTN infusion (10 μg/kg/min) resulted in significantly blunted depressor responses to intravenous bolus doses of GTN, demonstrating tolerance development. By contrast, infusion with AS (20 μg/kg/min) or DEA/NO (2 μg/kg/min) did not alter their subsequent depressor responses. Similarly, ex vivo vasorelaxation responses in isolated aortae revealed that GTN infusion elicited a significant 6-fold decrease in the sensitivity to GTN and reduction in the maximum response to acetylcholine (ACh). Chronic infusion of AS or DEA/NO had no effect on subsequent vasorelaxation responses to themselves or to ACh. No functional cross-tolerance between nitrovasodilators was evident, either in vivo or ex vivo, although an impaired ability of a nitrovasodilator to increase tissue cGMP content was not necessarily indicative of a reduced functional response. In conclusion, HNO donors may represent novel therapies for cardiovascular disease with therapeutic potential over clinically used organic nitrates.

Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO(•)), is rapidly emerging as a novel nitrogen oxide with distinct pharmacology and therapeutic advantages over its redox sibling. Whilst the cardioprotective effects of HNO in heart failure have been established, it is apparent that HNO may also confer a number of vasoprotective properties. Like NO(•), HNO induces vasodilatation, inhibits platelet aggregation, and limits vascular smooth muscle cell proliferation. In addition, HNO can be putatively generated within the vasculature, and recent evidence suggests it also serves as an endothelium-derived relaxing factor (EDRF). Significantly, HNO targets signaling pathways distinct from NO(•) with an ability to activate K(V) and K(ATP) channels in resistance arteries, cause coronary vasodilatation in part via release of calcitonin-gene related peptide (CGRP), and exhibits resistance to scavenging by superoxide and vascular tolerance development. As such, HNO synthesis and bioavailability may be preserved and/or enhanced during disease states, in particular those associated with oxidative stress. Moreover, it may compensate, in part, for a loss of NO(•) signaling. Here we explore the vasoprotective actions of HNO and discuss the therapeutic potential of HNO donors in the treatment of vascular dysfunction.

Background Over the last decade, we have shown that cGMP, derived from bradykinin, nitric oxide (NO•, both from endogenous and exogenous sources) or natriuretic peptides, is a potent inhibitor of cardiac hypertrophy, across isolated cardiomyocytes and intact hearts both ex vivo and in vivo. However, NO• bioavailability is reduced due to scavenging by ROS; furthermore, oxidation of sGC may result in sGC dysfunction (including loss of responsiveness to NO•). In the present study, we tested the hypothesis that the NO•-independent sGC stimulator BAY 41-2272 and the NO-independent sGC activator BAY 58-2667 elicit powerful antihypertrophic actions.

Results IPA-NO elicited concentration-dependent inhibition of endothelin-1 (ET1)-induced increases in neonatal rat cardiomyocyte size, with similar suppression of hypertrophic genes. Antihypertrophic IPA-NO actions were significantly attenuated by L-cysteine (HNO scavenger), Rp-8-pCTPcGMPS (cGMP-dependent protein kinase inhibitor), and ODQ (to target soluble guanylyl cyclase, sGC), but were unaffected by carboxy-PTIO (NO• scavenger) or CGRP837 (calcitonin gene-related peptide antagonist). Furthermore, IPA-NO significantly increased cardiomyocyte cGMP 3.5-fold (an L-cysteine-sensitive effect), and stimulated sGC activity 3-fold, without detectable NO• release. IPA-NO also suppressed ET1-induced cardiomyocyte superoxide generation. The pure NO• donor, DEA-NO, reproduced these IPA-NO actions, but were sensitive to carboxy-PTIO rather than L-cysteine. Although IPA-NO stimulation of purified sGC was preserved under pyrogallol oxidant stress (in direct contrast to DEA-NO), cardiomyocyte sGC activity after either donor were attenuated by this stress. Excitingly IPA-NO also exhibited acute antihypertrophic actions in response to acute pressureoverload in the intact heart, at doses that did not affect coronary perfusion pressure or LV function.

We have previously shown that NO•/cGMP signalling is an important antihyper-trophic mechanism in the heart [1-3]. HNO is the one electron reduction of NO•, thought to elicit cardiovascular actions via cGMP and/or calcitonin gene-related peptide (CGRP) [4]; we have recently shown that the HNO donor Angeli's salt inhibits cardiomyocyte hypertrophy and superoxide generation [5]. We now test the hypothesis that isopropylamine/NO (IPA/NO) elicits concentration-dependent anti-hypertrophic and antioxidant actions via HNO/sGC/cGMP-dependent signalling. IPA/NO (0.1–3 μM, replenished 3×/day over 48 h) elicited concentration-dependent inhibition of endothelin-1 (ET1, 60 nM)-stimulated neonatal rat cardiomyocyte (NRCM) hypertrophy (on two dimensional area of live cells). At 3 μM, IPA/NO decreased cell size from 255 ± 28% to 96 ± 27% of paired control (n = 4, p < 0.001). This antihypertrophic action of IPA/NO was significantly attenuated in the presence of the HNO scavenger L-cysteine (3 mM) or the cGMP-dependent protein kinase inhibitor Rp-8 PCTP cGMPS (10 μM, both n = 4, p < 0.05), but was unaffected by the NO scavenger carboxy-PTIO (200 μM) or the CGRP antagonist, CGRP8–37 (1 μM, both n = 4). For comparison, the NO• donor DEA/NO elicited similar concentration-dependent inhibition of ET1-induced cardiomyocyte hypertrophy; this was inhibited by carboxy-PTIO and Rp-8 PCTP cGMPS (10 μM, both n = 4, p < 0.05), but was unaffected by L-cysteine. Both IPA/NO and DEA/NO also blocked ET1-induced cardiomyocyte superoxide generation (both n = 4, p < 0.001, on NADPH-driven lucigenin-enhanced chemiluminescence), a key trigger of hypertrophy [3]. IPA-NO stimulated purified cell-free sGC activity by 3.2 ± 0.6-fold, and elevated NRCM cGMP content by 3.5 ± 0.4-fold (both n = 5, p < 0.05 via cGMP ELISA, as previously described [2,3]. None of these agents alone, or their respective vehicles, elicited any effect on NRCM. Finally, using an NO•-sensing electrode, we demonstrated that IPA/NO (in contrast to DEA/NO), does not release NO• under these conditions, even at supra-pharmacological concentrations. In conclusion, these results provide convincing evidence that IPA/NO prevents cardiomyocyte hypertrophy via HNO activation of sGC. Although the antihypertrophic and antioxidant efficacy of IPA/NO was comparable to NO•, there is no role for extracellular oxidation of HNO to NO• or CGRP-mediated signalling in these IPA/NO actions. These studies may ultimately facilitate the development of HNO donors such as IPA/NO as novel antihypertrophic therapy for patients at risk of heart failure.

Until recently, most of the biological effects of nitric oxide (NO) have been attributed to its uncharged state (NO*), yet NO can also exist in the reduced state as nitroxyl (HNO or NO(-)). Putatively generated from both NO synthase (NOS)-dependent and -independent sources, HNO is rapidly emerging as a novel entity with distinct pharmacology and therapeutic advantages over its redox sibling, NO*. Thus, unlike NO*, HNO can target cardiac sarcoplasmic ryanodine receptors to increase myocardial contractility, can interact directly with thiols and is resistant to both scavenging by superoxide (*O2-) and tolerance development. HNO donors are protective in the setting of heart failure in which NO donors have minimal impact. Here, we discuss the unique pharmacology of HNO versus NO* and highlight the therapeutic potential of HNO donors in the treatment of cardiovascular disease.