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Aims Nitroglycerine (NTG) given prior to an ischaemic insult exerts cardioprotective effects. However, whether administration of an acute low dose of NTG in a clinically relevant manner following an ischaemic episode limits infarct size, has not yet been explored. Methods and results Adult mice were subjected to acute myocardial infarction in vivo and then treated with vehicle or low-dose NTG prior to reperfusion. This treatment regimen minimized myocardial infarct size without affecting haemodynamic parameters but the protective effect was absent in mice rendered tolerant to the drug. Mechanistically, NTG was shown to nitrosate and inhibit cyclophilin D (CypD), and NTG administration failed to limit infarct size in CypD knockout mice. Additional experiments revealed lack of the NTG protective effect following genetic (knockout mice) or pharmacological inhibition (L-NAME treatment) of the endothelial nitric oxide synthase (eNOS). The protective effect of NTG was attributed to preservation of the eNOS dimer. Moreover, NTG retained its cardioprotective effects in a model of endothelial dysfunction (ApoE knockout) by preserving CypD nitrosation. Human ischaemic heart biopsies revealed reduced eNOS activity and exhibited reduced CypD nitrosation. Conclusion Low-dose NTG given prior to reperfusion reduces myocardial infarct size by preserving eNOS function, and the subsequent eNOS-dependent S-nitrosation of CypD, inhibiting cardiomyocyte necrosis. This novel pharmacological action of NTG warrants confirmation in clinical studies, although our data in human biopsies provide promising preliminary results. [ABSTRACT FROM AUTHOR]

Aims Endothelial nitric oxide (NO) synthase (eNOS) is known to play a cardioprotective protective. However, the molecular mechanisms regulating eNOS activity during ischaemia/reperfusion (I/R) injury are incompletely understood. eNOS is a substrate for several kinases that positively or negatively affect its enzymatic activity. Herein, we sought to correlate eNOS phosphorylation status with cardiomyocyte survival and we investigated the contribution of the proline-rich tyrosine kinase 2 (PYK2)/eNOS axis to the regulation of myocardial infarct size in vivo. Methods and results Exposure of H9c2 cardiomyocytes to H2O2 lead to PYK2 phosphorylation on its activator site (Y402) and eNOS phosphorylation on the inhibitor site Y656 and the activator site S1176. Both H2O2-induced eNOS phosphorylation events were abolished by PYK2 pharmacological inhibition or gene knockdown. Activity assays demonstrated that phosphorylation of the tyrosine inhibitory site exerts a dominant effect over S1176. In cardiomyocytes subjected to oxidative stress or oxygen-glucose deprivation, inhibition of PYK2 limited cell injury; this effect was prevented by inhibition of NO production. In vivo, ischaemia-reperfusion induced an early activation of PYK2, leading to eNOS phosphorylation on Y656, which, in turn, reduced NO output, as judged by the low tissue levels of its downstream effector cGMP. Moreover, pharmacological blockade of PYK2 alleviated eNOS inhibition and prevented cardiac damage following I/R injury in wild-type, but not in eNOS KO mice. Conclusion The current studies demonstrate that PYK2 is a pivotal regulator of eNOS function in myocardial infarction and identify PYK2 as a novel therapeutic target for cardioprotection. [ABSTRACT FROM AUTHOR]

Aims Cardiac progenitor cells (CPC) from adult hearts can differentiate to several cell types composing the myocardium but the underlying molecular pathways are poorly characterized. We examined the role of paracrine nitric oxide (NO) in the specification of CPC to the cardiac lineage, particularly through its inhibition of the canonical Wnt/β-catenin pathway, a critical step preceding cardiac differentiation. Methods and Results Sca1+CPC from adult mouse hearts were isolated by MACS and clonally expanded. Pharmacologic NO donors increased their expression of cardiac myocyte-specific sarcomeric proteins in a concentration and time-dependent manner. The optimal time window for NO efficacy coincided with upregulation of CPC expression of GUCY1A3 (coding the alpha1 subunit of guanylyl cyclase). The effect of paracrine NO was reproduced in vitro upon co-culture of CPC with cardiac myocytes expressing a transgenic NOS3 (endothelial nitric oxide synthase) and in vivo upon injection of CPC in infarcted hearts from cardiac-specific NOS3 transgenic mice. In mono- and co-cultures, this effect was abrogated upon inhibition of soluble guanylyl cyclase or nitric oxide synthase, and was lost in CPC genetically deficient in GUCY1A3. Mechanistically, NO inhibits the constitutive activity of the canonical Wnt/β-catenin in CPC and in cell reporter assays in a guanylyl cyclase-dependent fashion. This was paralleled with decreased expression of β-catenin and downregulation of Wnt target genes in CPC and abrogated in CPC with a stabilized, non-inhibitable β-catenin. Conclusions Exogenous or paracrine sources of NO promote the specification towards the myocyte lineage and expression of cardiac sarcomeric proteins of adult CPC. This is contingent upon the expression and activity of the alpha1 subunit of guanylyl cyclase in CPC that is necessary for NO-mediated inhibition of the canonical Wnt/β-catenin pathway. [ABSTRACT FROM AUTHOR]

Aims H2S is known to confer cardioprotection; however, the pathways mediating its effects in vivo remain incompletely understood. The purpose of the present study is to evaluate the contribution of cGMP-regulated pathways in the infarct-limiting effect of H2S in vivo. Methods and results Anaesthetized rabbits were subjected to myocardial ischaemia (I)/reperfusion (R), and infarct size was determined in control or H2S-exposed groups. The H2S donor sodium hydrosulfide (NaHS, an agent that generates H2S) increased cardiac cGMP and reduced the infarct size. The cGMP-dependent protein kinase (PKG)-I inhibitor DT2 abrogated the protective effect of NaHS, whereas the control peptide TAT or L-nitroarginine methyl ester (L-NAME) did not alter the effect of NaHS. Moreover, the KATP channel inhibitor, glibenclamide, partially reversed the effects of NaHS, whereas inhibition of mitochondrial KATP did not modify the NaHS response. NaHS enhanced phosphorylation of phospholamban (PLN), in a PKG-dependent manner. To further investigate the role of PLN in H2S-mediated cardioprotection, wild-type and PLN KOmice underwent I/R. NaHS did not exert cardioprotection in PLN KOmice. Unlike whatwas observed in rabbits, genetic or pharmacological inhibition of eNOS abolished the infarct-limiting effect of NaHS in mice. Conclusions Our findings demonstrate (i) that administration of NaHS induces cardioprotection via a cGMP/PKG/PLN pathway and (ii) contribution of nitric oxide to the H2S response is species-specific. [ABSTRACT FROM AUTHOR]

Aims Septic shock is the leading cause of death in intensive care units worldwide, resulting from a progressive systemic inflammatory reaction causing cardiovascular and organ failure. Nitric oxide (NO) is a potent vasodilator and inhibition of NO synthases (NOS) can increase blood pressure in septic shock. However, NOS inhibition does not improve outcome, on the contrary, and certain NO donors may even provide protection. In addition, NOS produce superoxide in case of substrate or cofactor deficiency or oxidation. We hypothesized that excessive systemic iNOS-derived NO production is insufficient to trigger cardiovascular failure and shock. Methods and results We found that the systemic injection with various synthetic Toll-like receptor-2 (TLR2), TLR3, or TLR9 agonists triggered systemic NO production identical to that of lipopolysaccharide (LPS) or tumour necrosis factor. In contrast to the latter, however, these agonists did not cause hypothermia or any other signs of discomfort or morbidity, and inflammatory cytokine production was low. TLR2 stimulation with the triacylated lipopeptide Pam3CSK4 not only caused identical NO levels in circulation, but also identical iNOS expression patterns as LPS. Nevertheless, Pam3CSK4 did not cause hypotension, bradycardia, reduced blood flow, or inadequate tissue perfusion in the kidney or the liver. Conclusion We demonstrate that excessive iNOS-derived NO in circulation is not necessarily linked to concomitant cardiovascular collapse, morbidity, or mortality. As such, our data indicate that the central role of iNOS-derived NO in inflammation-associated cardiovascular failure may be overestimated. [ABSTRACT FROM AUTHOR]

Severe sepsis and septic shock, which are among the most common causes of death in intensive care units worldwide, cause high morbidity, mortality, and social and economic costs. Therefore, developing successful therapies against sepsis is one of the most important challenges in critical care medicine. Death from septic shock is caused by refractory hypotension and multiple organ failure (MOF). Although excessive systemic vasodilation triggered by nitric oxide (NO) is believed to mediate the hypotension, several endogenous factors and phenomena are responsible for MOF, including tissue hypoperfusion and ischaemia, mitochondrial dysfunction, and other cytotoxic effects, all of which might be directly or indirectly antagonized by local NO. Hence, selective inhibition of the production of hypotension-causing NO in the macrocirculation and/or selective treatment with microvasculature-specific NO donors could theoretically constitute a successful therapy. Recently, the NO metabolite nitrite was recognized as an NO donor specifically in hypoxic/acidic conditions, which can be expected in the septic microvasculature. We recently showed that treatment with nitrite can protect mice against progressive hypothermia, mitochondrial dysfunction, organ damage, and even death induced by tumour necrosis factor or lipopolysaccharide. In this review, we discuss the rationale for using nitrite for the treatment of shock, the possible mechanisms of nitrite-mediated protection, and the lessons that can be drawn for possible future translation of the results from mouse models to the clinic. [ABSTRACT FROM AUTHOR]

Background. Acute peritonitis is the most frequent complication of peritoneal dialysis (PD). Increased nitric oxide (NO) release by NO synthase (NOS) isoforms has been implicated in acute peritonitis, but the role played by the NOS isoforms expressed in the peritoneum is unknown. [ABSTRACT FROM PUBLISHER]

Aim: The effects of nitric oxide (NO) in the cardiovascular system are attributed in part to cGMP synthesis by the α1β1 isoform of soluble guanylate cyclase (sGC). Because available sGC inhibitors are neither enzyme- nor isoform-specific, we generated knockout mice for the α1 subunit (sGCα1−/− mice) in order to investigate the function of sGCα1β1 in the regulation of blood pressure and cardiac function. [ABSTRACT FROM PUBLISHER]

Abstract: Objective: Soluble guanylyl cyclase (sGC), the predominant receptor for nitric oxide (NO), exists in 2 active isoforms (α2β1 and α1β1). In vascular tissue sGCα1β1 is believed to be the most important. The aim of our study was to investigate the functional importance of the sGCα1-subunit in vasorelaxation. Methods: Aortic and femoral artery segments from male and/or female sGCα1 −/− mice and wild-type littermates were mounted in a small-vessel myograph for isometric tension recording. This was supplemented with biochemical measurements of the cGMP concentration and sGC enzyme activity. Results: The functional importance of sGCα1β1 was demonstrated by the significantly decreased relaxing effects of acetylcholine (ACh), sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), NO gas, YC-1, BAY 41-2272 and T-1032 in the sGCα1 −/− mice of both genders. Moreover, the basal and SNP-stimulated cGMP levels and basal sGC activity were significantly lower in the sGCα1 −/− mice. However, the relaxing effects of NO, BAY 41-2272 and YC-1 seen in blood vessels from sGCα1 −/− mice indicate a role for an sGCα1β1-independent mechanism. The increase in sGC activity after addition of BAY 41-2272 and the inhibition of the ACh-, SNP-, SNAP- and NO gas-induced response by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the sGCα1 −/− mice are observations suggesting that the sGCα2β1 isoform is also functionally active. However, the insignificant increase in cGMP in response to SNP and the non-upregulated sGCα2 expression level in the sGCα1 −/− mice suggest rather the involvement of (an) sGC-independent mechanism(s). Conclusions: We conclude that sGCα1β1 is involved in the vasorelaxation induced by NO-dependent and NO-independent sGC activators in both genders. However, the remaining relaxation seen in the sGCα1 −/− mice suggests that besides sGCα1β1 also the minor isoform sGCα2β1 and/or (an) sGC-independent mechanism(s) play(s) a substantial role. [Copyright &y& Elsevier]

In experimental murine trypanosomiasis, resistance is often scored as the capacity to control peak parasitemia levels, which results in prolonged survival. Infection-induced pathology has not systematically been used as a resistance criterion. Because this parameter could be the most relevant for comparative analysis of natural and experimental infections, as well as for understanding of pathology-associated immune alterations, we analyzed Trypanosoma brucei infections in 4 different established conventional mouse models, as well as in tumor necrosis factor (TNF)­deficient and TNF-receptor­deficient mice. Results indicate the following: (1) there is no correlation between peak parasitemia control or survival and the induction of infection-associated anemia, loss of body weight, liver pathology, reduced locomotor activity, and general morbidity; (2) serum levels of TNF, interferon-g, and interleukin-10, which are known to affect survival, do not correlate with induction of pathology; and (3) infection-induced occurrence of lipopolysaccharide hypersensitivity does not correlate with survival. However, one parameter that was found to correlate with the inhibition of trypanosomiasis-associated pathology in all models was the shedding of soluble p75 TNF-receptor during peak parasitemia stages. These results are important for future cytokine and trypanosomiasis pathology studies,because the interplay between TNF and the soluble receptors it sheds has not been considered in either human clinical sleeping sickness studies or in veterinary trypanosomiasis research. [ABSTRACT FROM AUTHOR]

Introduction Because of their nitric oxide ( NO)-donating capacities, oxime derivatives have shown to offer some therapeutic perspective for the treatment of erectile dysfunction ( ED) as well as cardiovascular diseases. However, to date the in vivo effect of these oximes on erectile function remains unknown. In many disease states oxidative stress occurs, impairing NO-mediated relaxations. Hence the influence of oxidative stress on oxime-induced effects is also of interest. Aims This study aimed to evaluate the in vivo effect of formaldoxime ( FAL) and formamidoxime ( FAM) on blood pressure and intracavernosal pressure (ICP); and to examine the role of soluble guanylyl cyclase ( sGC) and the influence of oxidative stress on the FAL and FAM responses. Methods Blood pressure and ICP were monitored in vivo after resp. intravenous or intracavernosal injection of FAL and FAM. Moreover isometric tension was measured in vitro on isolated mice corpora cavernosa ( CC), thoracic aorta, and femoral artery in organ baths. The role of sGC was investigated using transgenic mice lacking the alpha 1 subunit of sGC. Main Outcome Measures Mean arterial pressure ( MAP) and ICP were measured after FAL/ FAM injection. In vitro relaxation of CC strips was evaluated in response to addition of FAL/ FAM. Results In vivo both FAL and FAM elicit a dose-dependent lowering of blood pressure (maximal Δ MAP: 33.66 ± 4.07 mm Hg [ FAL] and 20.43 ± 2.06 mm Hg [ FAM] ) as well as an increase of ICP (maximal increase of ICP/ MAP: 70.29 ± 2.88% [ FAL] and 52.91 ± 8.61% [ FAM] ). The FAL/ FAM effect is significantly lower in knockout vs. wild-type mice. Oxidative stress has an inhibitory effect on corporal NO-mediated relaxations induced by electrical field stimulation, acetylcholine, and sodium nitroprusside whereas the responses to 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate sodium salt, FAL and FAM were not influenced. Conclusions Oximes induce erection which is mediated by sGC. The oxime-induced relaxations are resistant to oxidative stress, which increases their therapeutic potential for the treatment of ED. Pauwels B, Boydens C, Brouckaert P, and Van de Voorde, J. Oximes Induce Erection and Are Resistant to Oxidative Stress. J Sex Med 2015;12:906-915. [ABSTRACT FROM AUTHOR]

Introduction The nitric oxide (NO), soluble guanylate cyclase (sGC), and cyclic guanosine monophosphate (cGMP) pathway is the leading pathway in penile erection. Aim To assess erectile function in a mouse model in which sGC is deficient in heme (apo-sGC) and unresponsive to NO. Methods Mutant mice (sGCβ 1 ki/ki ) that express an sGC enzyme that retains basal activity but fails to respond to NO because of heme deficiency (apo-sGC) were used. Isolated corpora cavernosa from sGCβ 1 ki/ki and wild-type mice were mounted in vitro for isometric tension recordings in response to sGC-dependent and -independent vasorelaxant agents. In addition, the erectile effects of some of these agents were tested in vivo at intracavernosal injection. Main Outcome Measures In vitro and in vivo recordings of erectile responses in sGCβ 1 ki/ki and wild-type mice after stimulation with sGC-dependent and -independent vasorelaxant agents. Results NO-induced responses were abolished in sGCβ 1 ki/ki mice in vitro and in vivo. The ability of the heme-dependent, NO-independent sGC stimulator BAY 41-2272 to relax the corpora cavernosa was markedly attenuated in sGCβ 1 ki/ki mice. In contrast, the relaxation response to the heme- and NO-independent sGC activator BAY 58-2667 was significantly enhanced in sGCβ 1 ki/ki mice. The relaxing effect of sGC-independent vasorelaxant agents was similar in wild-type and sGCβ 1 ki/ki mice, illustrating that the observed alterations in vasorelaxation are limited to NO-sGC-cGMP–mediated processes. Conclusion Our results suggest that sGC is the sole target of NO in erectile physiology. Furthermore, this study provides indirect evidence that, in addition to sGCα 1 β 1 , sGCα 2 β 1 is important for erectile function. In addition, the significant relaxation observed in sGCβ 1 ki/ki mice with the cumulative addition of the sGC activator BAY 58-2667 indicates that sGC activators might offer value in treating erectile dysfunction. [ABSTRACT FROM AUTHOR]

Introduction The red wine polyphenols resveratrol and quercetin are known for their vasorelaxant and antioxidant capacity, which is assumed to rely on the activation of the nitric oxide (NO)/soluble guanylyl cyclase (sGC) pathway. Vasodilators as well as antioxidants can regulate penile erection and be beneficial for the treatment of erectile dysfunction ( ED). Aims The goal of this study was to evaluate the NO/ sGC dependency of the relaxant effect of resveratrol and quercetin on mice aorta and corpora cavernosa ( CC), as well as to explore their influence on oxidative stress-induced ED. Methods Isolated mice aorta and CC were mounted for isometric tension recordings into organ baths. Cumulative concentration-response curves were constructed for resveratrol and quercetin in the absence/presence of inhibitors of the NO/ sGC pathway. In addition, in CC the effect of resveratrol and quercetin was studied on NO-mediated relaxations using acetylcholine ( Ach), sodium nitroprusside ( SNP), and electrical field stimulation ( EFS). In certain experiments, corporal tissues were exposed to oxidative stress using palmitic acid ( PA, 0.5 mM). Main Outcome Measures Corporal responses to resveratrol and quercetin were measured in the presence/absence of inhibitors of different molecular pathways. The effect of resveratrol and quercetin incubation on Ach-, SNP-, or EFS-mediated responses was explored in the presence/absence of PA. Results While both polyphenols are potent vasodilators of mice aorta, only resveratrol relaxes mice CC. The relaxation response to resveratrol on aorta was diminished in sGCα1−/− mice, but not on CC. The polyphenols did not influence Ach-, SNP-, or EFS-mediated relaxations as such. Resveratrol, but not quercetin, was able to significantly reverse PA-induced decrease of EFS relaxations. Conclusion The red wine compound resveratrol, but not quercetin, relaxes isolated mice CC concentration-dependently through mechanisms independent of the NO/ sGC pathway. Resveratrol is a more potent antioxidant than quercetin, being able to restore decreased neuronal NO responses in mice CC. Boydens C, Pauwels B, Decaluwé K, Brouckaert P, and Van de Voorde J. Relaxant and antioxidant capacity of the red wine polyphenols, resveratrol and quercetin, on isolated mice corpora cavernosa. J Sex Med 2015;12:303-312. [ABSTRACT FROM AUTHOR]