Rationale: Nitric oxide (NO) exerts its biological effects primarily via activation of guanylate cyclase (GC) and production of cyclic guanosine monophosphate. Inhaled NO improves outcomes after cardiac arrest and cardiopulmonary resuscitation (CPR). However, mechanisms of the protective effects of breathing NO after cardiac arrest are incompletely understood.Objective: To elucidate the mechanisms of beneficial effects of inhaled NO on outcomes after cardiac arrest. Methods: Adult male C57BL/6J wild-type (WT) mice, GC-1 knockout mice, and chimeric WT mice with WT or GC1 knockout bone marrow were subjected to 8 min of potassium-induced cardiac arrest to determine the role of GC-1 in bone marrow-derived cells. Mice breathed air or 40 parts per million NO for 23 h starting at 1 h after CPR.Results: Breathing NO after CPR prevented hypercoagulability, cerebral microvascular occlusion, an increase in circulating polymorphonuclear neutrophils and neutrophil-to-lymphocyte ratio, and right ventricular dysfunction in WT mice, but not in GC-1 knockout mice, after cardiac arrest. The lack of GC-1 in bone marrow-derived cells diminished the beneficial effects of NO breathing after CPR. Conclusions: GC-dependent signaling in bone marrow-derived cells is essential for the beneficial effects of inhaled NO after cardiac arrest and CPR.

BACKGROUND:Epidemiological, laboratory and clinical studies have established an association between elevated urate and high blood pressure (BP). However, the inference of causality remains controversial. A naturally occurring antioxidant, urate may also be neuroprotective, and urate-elevating treatment with its precursor inosine is currently under clinical development as a potential disease-modifying strategy for Parkinson's disease (PD). METHODS:Our study takes advantage of a recently completed phase II trial evaluating oral inosine in de novo non-disabling early PD with no major cardiovascular and nephrological conditions, and of three lines of genetically engineered mice: urate oxidase (UOx) global knockout (gKO), conditional KO (cKO), and transgenic (Tg) mice with markedly elevated, mildly elevated, and substantially reduced serum urate, respectively, to systematically investigate effects of urate-modifying manipulation on BP. FINDINGS:Among clinical trial participants, change in serum urate but not changes in systolic, diastolic and orthostatic BP differed by treatment group. There was no positive correlation between urate elevations and changes in systolic, diastolic and orthostatic BP ((p = .05 (in inverse direction), 0.30 and 0.63, respectively)). Between UOx gKO, cKO, or Tg mice and their respective wildtype littermates there were no significant differences in systolic or diastolic BP or in their responses to BP-regulating interventions. INTERPRETATION:Our complementary preclinical and human studies of urate modulation in animal models and in generally healthy early PD do not support a hypertensive effect of urate elevation or an association between urate and BP. FUND: U.S. Department of Defense, RJG Foundation, Michael J. Fox Foundation LEAPS program, National Institutes of Health, American Federation for Aging Research, Parkinson's Disease Foundation Advancing Parkinson's Therapies initiative.

PURPOSE. While nitric oxide (NO) donors are emerging as treatments for glaucoma, the mechanism by which NO lowers intraocular pressure (IOP) is unclear. NO activates the enzyme guanylyl cyclase (GC) to produce cyclic guanosine monophosphate. We studied the ocular effects of inhaled and topically applied NO gas in mice and lambs, respectively.METHODS. IOP and aqueous humor (AqH) outflow were measured in WT and GC-1 alpha subunit null (GC-1-/-) mice. Mice breathed 40 parts per million (ppm) NO in O-2 or control gas (N-2/O-2). We also studied the effect of ocular NO gas exposure (80, 250, 500, and 1000 ppm) on IOP in anesthetized lambs. NO metabolites were measured in AqH and plasma.RESULTS. In awake WT mice, breathing NO for 40 minutes lowered IOP from 14.4 1.9 mm Hg to 10.9 1.0 mm Hg (n = 11, P < 0.001). Comparable results were obtained in anesthetized WT mice (n = 10, P < 0.001). In awake or anesthetized GC-1(-/-)mice, IOP did not change under similar experimental conditions (P >= 0.08, n = 20). Breathing NO increased in vivo outflow facility in WT but not GC-1(-/-)mice (+13.7 14.% vs. -12.1 9.4%, n = 4 each, P < 0.05). In lambs, ocular exposure to NO lowered IOP in a dose-dependent manner (-0.43 mm Hg/ppm NO; n = 5 with 40 total measurements; P = 0.04) without producing corneal pathology or altering pulmonary and systemic hemodynamics. After ocular NO exposure, NO metabolites were increased in AqH (n = 8, P < 0.001) but not in plasma.CONCLUSIONS. Breathing NO reduced IOP and increased outflow facility in a GC-dependent manner in mice. Exposure of ovine eyes to NO lowers IOP.

Objective: Inhaled nitric oxide (iNO) improved outcomes after cardiac arrest (CA) and CPR via a soluble guanylate cyclase α1 (sGCα1)-dependent mechanism in mice. However, mechanisms responsible for...

Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis.

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1 (-/-) mice) display sex- and strain-specific hypertension: male but not female sGCα1 (-/-) mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex- and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGCα1 (-/-)S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGCα1 (-/-)S6 than of male sGCα1 (-/-)B6 mice. Furthermore, treating male sGCα1 (-/-)S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background- and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling.

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.

Since the discovery of functional brown adipose tissue (BAT) in adult humans, an inverse correlation between aging, type 2 diabetes and the amount and activity of the BAT‐depots have been reported. Upon activation, BAT converts the energy of free fatty acids and glucose oxidation into heat through the mitochondrial carrier protein,uncoupling protein 1 (UCP1). BAT has recently been demonstrated to increase triglyceride uptake and insulin sensitivity. Although the thermogenic responses of UCP1 KO mice have been well studied, little is known about the short and long term effects of UCP1 deficiency and how it affects BAT function and metabolism in general.We used RNAseq to analyze and compare the BAT transcriptome of WT and UCP1 KO mice, under thermoneutral conditions at baseline and after catecholamine induced activation. The effects of aging on glucose and insulin tolerance and molecular markers of type 2 diabetes were investigated in WT and UCP1 KO mice in thermoneutrality for up to 1 year.Whole transcriptome analysis, followed by qPCR and immunoblotting, revealed marked dysfunction of key metabolic pathways in activated BAT from UCP1KO mice compared to WT mice, while at baseline thermoneutral conditions the BAT transcriptome from UCP1KO and WT mice were similar. Further, UCP1KO mice age‐dependently develop hallmarks of type 2 diabetes, such as impaired glucose tolerance and increased insulin resistance, without developing an obesity phenotype.We conclude that UCP1 KO mice are a model of severe BAT dysfunction. Long‐term BAT dysfunction is associated with age‐dependent development of hallmarks of type 2 diabetes.

Background The nitric oxide (NO)-soluble guanylate cyclase (sGC)cyclic guanosine 3’5’-monophosphate (cGMP) pathway regulates intraocular pressure (IOP). Preclinical and clincial studies have demonstrated the ability of NO-donor compounds to lower IOP (e.g. VESNEO). The use of inhaled NO gas (iNO), a specific pulmonary but not systemic vasodilator, is an approved therapy for pulmonary hypertension and is under development as a treatment for other cardiovascular diseases (e.g. for myocardial ischemia, the NOMI trial). We hypothesized that breathing NO lowers IOP in an sGC-dependent manner.

Cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids contribute to the regulation of pulmonary vascular tone and hypoxic pulmonary vasoconstriction. We investigated whether the attenuated acute vasoconstrictor response to hypoxic exposure of Cyp2j(-/-) mice would protect these mice against the pulmonary vascular remodeling and hypertension associated with prolonged exposure to hypoxia. Cyp2j(-/-) and Cyp2j(+/+) male and female mice continuously breathed an inspired oxygen fraction of 0.21 (normoxia) or 0.10 (hypoxia) in a normobaric chamber for 6 weeks. We assessed hemoglobin (Hb) concentrations, right ventricular (RV) systolic pressure (RVSP), and transthoracic echocardiographic parameters (pulmonary acceleration time [PAT] and RV wall thickness). Pulmonary Cyp2c29, Cyp2c38, and sEH mRNA levels were measured in Cyp2j(-/-) and Cyp2j(+/+) male mice. At baseline, Cyp2j(-/-) and Cyp2j(+/+) mice had similar Hb levels and RVSP while breathing air. After 6 weeks of hypoxia, circulating Hb concentrations increased but did not differ between Cyp2j(-/-) and Cyp2j(+/+) mice. Chronic hypoxia increased RVSP in Cyp2j(-/-) and Cyp2j(+/+) mice of either gender. Exposure to chronic hypoxia decreased PAT and increased RV wall thickness in both genotypes and genders to a similar extent. Prolonged exposure to hypoxia produced similar levels of RV hypertrophy in both genotypes of either gender. Pulmonary Cyp2c29, Cyp2c38, and sEH mRNA levels did not differ between Cyp2j(-/-) and Cyp2j(+/+) male mice after breathing at normoxia or hypoxia for 6 weeks. These results suggest that murine Cyp2j deficiency does not attenuate the development of murine pulmonary vascular remodeling and hypertension associated with prolonged exposure to hypoxia in mice of both genders.

Introduction: Hypertension is a multifactorial disease affecting one in three adults in the US. Evidence suggests that dysregulated signaling of the vasodilator nitric oxide (NO) is involved in its pathogenesis. Previously, we reported that mice deficient in the α1 subunit of the NO receptor guanylate cyclase (sGCα1-/- mice), display gender- and strain-specific hypertension: male mice on an Sv129/J (S6) but not a C57BL6/J (B6) background are hypertensive. Methods and Results: Via linkage analysis, we identified a quantitative trait locus (QTL) associated with elevated blood pressure in male sGCα1-/-S6 mice. This QTL encompasses the gene encoding CYP4a12a, a predominant synthase of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) in murine kidneys. Renal CYP4a12a gene expression, assessed using qRT-PCR, was higher in male WT and sGCα1-/- S6 mice than in female S6 mice or male and female, WT and sGCα1-/- B6 mice. In addition, 20-HETE levels, measured via liquid chromatography-tandem mass spectrometry, were higher in renal preglomerular microvessels of male sGCα1-/-S6 than of male sGCα1-/-B6 mice (2.3±0.4 vs 1.6±0.4 ng/mg protein, respectively, n=5 and 3, P <0.05). Furthermore, treating male sGCα1-/-S6 mice with the 20-HETE synthesis inhibitor HET0016 (10 mg/kg/day IP for 14 days) reduced the mean arterial pressure (MAP; 130±13 mmHg in 6 HET0016-treated mice vs. 159±2 mmHg in 2 vehicle-treated mice). Similarly, administration of the 20-HETE antagonist, 20-6,15-HEDGE (20-HEDGE, 10 mg/kg IV) to male sGCα1-/-S6 mice lowered MAP in 20-HEDGE-treated but not in vehicle treated mice (delta MAP = -10±3 vs 1±2 mmHg, n=8 for both, P <0.05). Finally, the more significant impairment of acetylcholine-induced relaxation of renal interlobar arteries in male sGCα1-/-S6 than in sGCα1-/-B6 mice, was rescued by adding 20-HEDGE (10 μM). Conclusions: Gender- and strain-specific hypertension in sGCα1-/-S6 mice is associated with elevated CYP4a12a gene expression and higher 20-HETE levels. Hypertension and vascular dysfunction in sGCα1-/-S6 mice is abrogated by 20-HETE inhibition. Our data identify CYP4a12a as a candidate blood pressure modifying gene in the context of deficient NO-sGC signaling.

AIMS:The use of doxorubicin, a potent chemotherapeutic agent, is limited by cardiotoxicity. We tested the hypothesis that decreased soluble guanylate cyclase (sGC) enzyme activity contributes to the development of doxorubicin-induced cardiotoxicity.RESULTS:Doxorubicin administration (20 mg/kg, intraperitoneally [IP]) reduced cardiac sGC activity in wild-type (WT) mice. To investigate whether decreased sGC activity contributes to doxorubicin-induced cardiotoxicity, we studied mice with cardiomyocyte-specific deficiency of the sGC α1-subunit (mice with cardiomyocyte-specific deletion of exon 6 of the sGCα1 allele [sGCα1-/-CM]). After 12 weeks of doxorubicin administration (2 mg/kg/week IP), left ventricular (LV) systolic dysfunction was greater in sGCα1-/-CM than WT mice. To further assess whether reduced sGC activity plays a pathogenic role in doxorubicin-induced cardiotoxicity, we studied a mouse model in which decreased cardiac sGC activity was induced by cardiomyocyte-specific expression of a dominant negative sGCα1 mutant (DNsGCα1) upon doxycycline removal (Tet-off). After 8 weeks of doxorubicin administration, DNsGCα1tg/+, but not WT, mice displayed LV systolic dysfunction and dilatation. The difference in cardiac function and remodeling between DNsGCα1tg/+ and WT mice was even more pronounced after 12 weeks of treatment. Further impairment of cardiac function was attenuated when DNsGCα1 gene expression was inhibited (beginning at 8 weeks of doxorubicin treatment) by administering doxycycline. Furthermore, doxorubicin-associated reactive oxygen species generation was higher in sGCα1-deficient than WT hearts. Innovation and Conclusion: These data demonstrate that a reduction in cardiac sGC activity worsens doxorubicin-induced cardiotoxicity in mice and identify sGC as a potential therapeutic target. Various pharmacological sGC agonists are in clinical development or use and may represent a promising approach to limit doxorubicin-associated cardiotoxicity. Antioxid. Redox Signal. 26, 153-164.

Primary open angle glaucoma (POAG) is a leading cause of blindness, due to irreversible retinal ganglion cell (RGC) loss with optic nerve (ON) degeneration. Currently, there is no cure for POAG and available therapies offer incomplete protection, highlighting the need for novel drug targets. Although several POAG risk factors have been identified, including elevated intraocular pressure (IOP) and genetic factors, the molecular signaling pathways involved remain largely unknown. Impaired nitric oxide (NO) signaling has been implicated in the pathogenesis of POAG. Here, we report that mice lacking the α1 subunit of the NO receptor soluble guanylate cyclase (sGCα1-/- mice) represent a novel animal model of POAG. sGCα1-/- mice develop moderate increases in IOP, a decrease in aqueous humor outflow rate, thinning of the retinal nerve fiber layer (RNFL), and loss of ON axons in the context of an open iridocorneal angle, all features of POAG. Importantly, a targeted gene association study in POAG patients with incident paracentral vision loss, a subtype of POAG thought to be associated with vascular dysregulation, identified a locus containing the genes encoding the α1 and β1 subunits of sGC. Our findings provide new insights into the genetics and biology of POAG, demonstrating that a well-characterized signaling pathway (NO-cGMP) is involved in the pathogenesis of POAG. In addition, identifying sGCα1-/- mice as a unique model for POAG, provides a tool for investigators to test new strategies for disease prevention, potentially informing the clinical development of existing cGMP-elevating therapeutic compounds for treatment of POAG.

Male mice deficient in the α1 subunit of soluble guanylate cyclase, a nitric oxide (NO) receptor, are hypertensive when on a 129S6 (S6) background (sGCα1-/-S6) but not when on a C57BL/6 (B6) background (sGCα1-/-B6), suggesting that hypertension associated with sGCα1-deficiency is modulated by genetic factors. Genetic linkage analyses in 284 male F2 offspring from an sGCα1-/-S6 X sGCα1-/-B6 intercross (sGCα1-/-F2) revealed a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) with a maximal logarithm of the odds (LOD) score of 6.3. 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): plasma angiotensin II levels were higher in S6 mice than in B6 mice (of either genotype), and plasma aldosterone levels were greater in sGCα1-/-S6 than in WTS6 mice. Renal function, as assessed by glomerular filtration rate, urinary volume and blood urea nitrogen, was similar in sGCα1-/-S6 mice and in WTS6 mice. Inhibiting the RAAS normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-/-S6 mice. These data identify the RAAS as a blood pressure-modifying signaling pathway in a setting of impaired NO-cGMP signaling. The finding that NO-cGMP and RAAS signaling (established pathways in the regulation of blood pressure) interact to regulate blood pressure, has great potential to advance our understanding of the etiology of human hypertension, and may help define genetically distinct subgroups of men and women with essential hypertension.