Person:

Sips, Patrick

Objective: Soluble guanylate cyclase (sGC), a heterodimer composed of α and β subunits, synthesizes cGMP in response to nitric oxide (NO). NO modulates vascular tone and structure but the relative contributions of cGMP-dependent versus cGMP-independent mechanisms remain uncertain. We studied the response to vascular injury in male (M) and female (F) mice with targeted deletion of exon 6 of the sGCα1 subunit (sGCα1-/-), resulting in a non-functional heterodimer. Methods: We measured aortic cGMP levels and mRNA transcripts encoding sGC α1, α2, and β1 subunits in wild type (WT) and sGCa1-/- mice. To study the response to vascular injury, BrdU-incorporation and neointima formation (maximum intima to media (I/M) ratio) were determined 5 and 28 days after carotid artery ligation, respectively. Results: Aortic cGMP levels were 4-fold higher in F than in M mice in both genotypes, and, within each gender, 4-fold higher in WT than in sGCa1-/-. In contrast, sGCα1, sGCα2, and sGCβ1 mRNA expression did not differ between groups. 3H-thymidine incorporation in cultured sGCa1-/- smooth muscle cells (SMC) was 27%±12% lower than in WT SMC and BrdU-incorporation in carotid arteries 5 days after ligation was significantly less in sGCa1-/- M than in WT M. Neointima area and I/M 28 days after ligation were 65% and 62% lower in sGCa1-/- M than in WT M mice (p<0,05 for both) but were not different in F mice. Conclusion: Functional deletion of sGCa1 resulted in reduced cGMP levels in male sGCa1-/- mice and a gender-specific effect on the adaptive response to vascular injury.

Nitric oxide (NO) plays an essential role in regulating hypertension and blood flow by inducing relaxation of vascular smooth muscle. Male mice deficient in a NO receptor component, the α1 subunit of soluble guanylate cyclase (sGCα1), are prone to hypertension in some, but not all, mouse strains, suggesting that additional genetic factors contribute to the onset of hypertension. Using linkage analyses, we discovered a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) in the context of sGCα1 deficiency. This region is syntenic with previously identified blood pressure–related QTLs in the human and rat genome and contains the genes coding for renin. Hypertension was associated with increased activity of the renin-angiotensin-aldosterone system (RAAS). Further, we found that RAAS inhibition normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-deficient mice. These data identify the RAAS as a blood pressure–modifying mechanism in a setting of impaired NO/cGMP signaling.

Background—Sudden cardiac arrest (CA) is a leading cause of death worldwide. Breathing nitric oxide (NO) reduces ischemia/reperfusion injury in animal models and in patients. The objective of this study was to learn whether inhaled NO improves outcomes after CA and cardiopulmonary resuscitation (CPR). Methods and Results—Adult male mice were subjected to potassium-induced CA for 7.5 minutes whereupon CPR was performed with chest compression and mechanical ventilation. One hour after CPR, mice were extubated and breathed air alone or air supplemented with 40 ppm NO for 23 hours. Mice that were subjected to CA/CPR and breathed air exhibited a poor 10-day survival rate (4 of 13), depressed neurological and left ventricular function, and increased caspase-3 activation and inflammatory cytokine induction in the brain. Magnetic resonance imaging revealed brain regions with marked water diffusion abnormality 24 hours after CA/CPR in mice that breathed air. Breathing air supplemented with NO for 23 hours starting 1 hour after CPR attenuated neurological and left ventricular dysfunction 4 days after CA/CPR and markedly improved 10-day survival rate (11 of 13; P=0.003 versus mice breathing air). The protective effects of inhaled NO on the outcome after CA/CPR were associated with reduced water diffusion abnormality, caspase-3 activation, and cytokine induction in the brain and increased serum nitrate/nitrite levels. Deficiency of the α1 subunit of soluble guanylate cyclase, a primary target of NO, abrogated the ability of inhaled NO to improve outcomes after CA/CPR. Conclusions—These results suggest that NO inhalation after CA and successful CPR improves outcome via soluble guanylate cyclase–dependent mechanisms.

Objectives: Despite advances in resuscitation methods, survival after out-of-hospital cardiac arrest remains low, at least in part, due to postcardiac arrest circulatory and neurologic failure. To elucidate the role of nitric oxide (NO) in the recovery from cardiac arrest and cardiopulmonary resuscitation (CPR), we studied the impact of NO synthase (NOS3)/cGMP signaling on cardiac and neurologic outcomes after cardiac arrest and CPR. Design: Prospective, randomized, controlled study. Setting: Animal research laboratory. Subjects: Mice. Interventions: Female wild-type (WT) mice, NOS3-deficient mice (NOS3−/−), NOS3−/− mice with cardiomyocyte-specific overexpression of NOS3 (NOS3−/−CSTg), and mice deficient for soluble guanylate cyclase α1 (sGCα1−/−) were subjected to potassium-induced cardiac arrest (9 min) followed by CPR. Cardiac and neurologic function and survival were assessed up to 24 hrs post-CPR. Measurements and Main Results: Cardiac arrest and CPR markedly depressed myocardial function in NOS3−/− and sGCα1−/− but not in WT and NOS3−/−CSTg. Neurologic function score and 24 hrs survival rate was lower in NOS3−/− and sGCα1−/− compared with WT and NOS3−/−CSTg. Detrimental effects of deficiency of NOS3 or sGCα1 were associated with enhanced inflammation of heart and liver and increased cell death in heart, liver, and brain that were largely prevented by cardiomyocyte-restricted NOS3 overexpression. Conclusions: These results demonstrate an important salutary impact of NOS3/sGC signaling on the outcome of cardiac arrest. Myocardial NOS3 prevented postcardiac arrest myocardial dysfunction, attenuated end-organ damage, and improved neurologic outcome and survival. Our observations suggest that enhancement of cardiac NOS3 and/or sGC activity may improve outcome after cardiac arrest and CPR.

Anaphylactic shock is a sudden, life-threatening allergic reaction associated with severe hypotension. Platelet-activating factor (PAF) is implicated in the cardiovascular dysfunctions occurring in various shock syndromes, including anaphylaxis. Excessive production of the vasodilator NO causes inflammatory hypotension and shock, and it is generally accepted that transcriptionally regulated inducible iNOS is responsible for this. Nevertheless, the contribution of NO to PAF-induced shock or anaphylactic shock is still ambiguous. We studied PAF and anaphylactic shock in conscious mice. Surprisingly, hyperacute PAF shock depended entirely on NO, produced not by inducible iNOS, but by constitutive eNOS, rapidly activated via the PI3K pathway. Soluble guanylate cyclase (sGC) is generally regarded as the principal vasorelaxing mediator of NO. Nevertheless, although methylene blue partially prevented PAF shock, neither 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) nor sGCα1 deficiency did. Also, in 2 different models of active systemic anaphylaxis, inhibition of NOS, PI3K, or Akt or eNOS deficiency provided complete protection. In contrast to the unsubstantiated paradigm that only excessive iNOS-derived NO underlies cardiovascular collapse in shock, our data strongly support the unexpected concept that eNOS-derived NO is the principal vasodilator in anaphylactic shock and define eNOS and/or PI3K or Akt as new potential targets for treating anaphylaxis.

Oxidative stress, a central mediator of cardiovascular disease, results in loss of the prosthetic haem group of soluble guanylate cyclase (sGC), preventing its activation by nitric oxide (NO). Here we introduce Apo-sGC mice expressing haem-free sGC. Apo-sGC mice are viable and develop hypertension. The haemodynamic effects of NO are abolished, but those of the sGC activator cinaciguat are enhanced in apo-sGC mice, suggesting that the effects of NO on smooth muscle relaxation, blood pressure regulation and inhibition of platelet aggregation require sGC activation by NO. Tumour necrosis factor (TNF)-induced hypotension and mortality are preserved in apo-sGC mice, indicating that pathways other than sGC signalling mediate the cardiovascular collapse in shock. Apo-sGC mice allow for differentiation between sGC-dependent and -independent NO effects and between haem-dependent and -independent sGC effects. Apo-sGC mice represent a unique experimental platform to study the in vivo consequences of sGC oxidation and the therapeutic potential of sGC activators.

BACKGROUND: Nitric oxide (NO) activates soluble guanylate cyclase (sGC), a heterodimer composed of alpha- and beta-subunits, to produce cGMP. NO reduces pulmonary vascular remodeling, but the role of sGC in vascular responses to acute and chronic hypoxia remains incompletely elucidated. We therefore studied pulmonary vascular responses to acute and chronic hypoxia in wild-type (WT) mice and mice with a nonfunctional alpha1-subunit (sGCalpha1-/-). METHODS AND RESULTS: sGCalpha1-/- mice had significantly reduced lung sGC activity and vasodilator-stimulated phosphoprotein phosphorylation. Right ventricular systolic pressure did not differ between genotypes at baseline and increased similarly in WT (22+/-2 to 34+/-2 mm Hg) and sGCalpha1-/- (23+/-2 to 34+/-1 mm Hg) mice in response to acute hypoxia. Inhaled NO (40 ppm) blunted the increase in right ventricular systolic pressure in WT mice (22+/-2 to 24+/-2 mm Hg, P<0.01 versus hypoxia without NO) but not in sGCalpha1-/- mice (22+/-1 to 33+/-1 mm Hg) and was accompanied by a significant rise in lung cGMP content only in WT mice. In contrast, the NO-donor sodium nitroprusside (1.5 mg/kg) decreased systemic blood pressure similarly in awake WT and sGCalpha1-/- mice as measured by telemetry (-37+/-2 versus -42+/-4 mm Hg). After 3 weeks of hypoxia, the increases in right ventricular systolic pressure, right ventricular hypertrophy, and muscularization of intra-acinar pulmonary vessels were 43%, 135%, and 46% greater, respectively, in sGCalpha1-/- than in WT mice (P<0.01). Increased remodeling in sGCalpha1-/- mice was associated with an increased frequency of 5'-bromo-deoxyuridine-positive vessels after 1 and 3 weeks (P<0.01 versus WT). CONCLUSIONS: Deficiency of sGCalpha1 does not alter hypoxic pulmonary vasoconstriction. sGCalpha1 is essential for NO-mediated pulmonary vasodilation and limits chronic hypoxia-induced pulmonary vascular remodeling.

Carbon monoxide (CO) has been shown to cause enteric smooth muscle relaxation by activating soluble guanylyl cyclase (sGC). In gastric fundus, the sGCα1β1 heterodimer is believed to be the most important isoform. The aim of our study was to investigate the role of the sGCα1/α2 subunits in the relaxant effect of CO and CORM-2 in murine gastric fundus using wild-type (WT) and sGCα1 knock-out (KO) mice. In WT mice, CO (bolus)-induced relaxations were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), while CORM-2- and CO (infusion)-induced relaxations were only partially inhibited by ODQ. In sGCα1 KO mice, relaxant responses to CO and CORM-2 were significantly reduced when compared with WT mice, but ODQ still had an inhibitory effect. The sGC sensitizer 1-benzyl-3-(5′-hydroxymethyl-2′-furyl-)-indazol (YC-1) was able to potentiate CO- and CORM-2-induced relaxations in WT mice but lost this potentiating effect in sGCα1 KO mice. Both in WT and sGCα1 KO mice, CO-evoked relaxations were associated with a significant cGMP increase; however, basal and CO-elicited cGMP levels were markedly lower in sGCα1 KO mice. These data indicate that besides the predominant sGCα1β1 isoform, also the less abundantly expressed sGCα2β1 isoform plays an important role in the relaxant effect of CO in murine gastric fundus; however, the sGC stimulator YC-1 loses its potentiating effect towards CO in sGCα1 KO mice. Prolonged administration of CO—either by the addition of CORM-2 or by continuous infusion of CO—mediates gastric fundus relaxation in both a sGC-dependent and sGC-independent manner.

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. Methods and results: Blood pressure was evaluated, using both non-invasive and invasive haemodynamic techniques, in intact and gonadectomized male and female sGCα1−/− and wild-type (WT) mice. Cardiac function was assessed with a conductance catheter inserted in the left ventricle of male and female sGCα1−/− and WT mice. Male sGCα1−/− mice developed hypertension (147 ± 2 mmHg), whereas female sGCα1−/− mice did not (115 ± 2 mmHg). Orchidectomy and treatment with an androgen receptor antagonist prevented hypertension, while ovariectomy did not influence the phenotype. Chronic testosterone treatment increased blood pressure in ovariectomized sGCα1−/− mice but not in WT mice. The NO synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride raised blood pressure similarly in male and female WT and sGCα1−/− mice. The ability of NO donor compounds to reduce blood pressure was slightly attenuated in sGCα1−/− male and female mice as compared to WT mice. The direct sGC stimulator BAY 41-2272 reduced blood pressure only in WT mice. Increased cardiac contractility and arterial elastance as well as impaired ventricular relaxation were observed in both male and female sGCα1−/− mice. Conclusion: These findings demonstrate that sGCα1β1-derived cGMP signalling has gender-specific and testosterone-dependent cardiovascular effects and reveal that the effects of NO on systemic blood pressure do not require sGCα1β1.