Praliciguat Promotes Ischemic Leg Reperfusion in Leptin Receptor-Deficient Mice.

BACKGROUND: Lower-limb peripheral artery disease is one of the major complications of diabetes. Peripheral artery disease is associated with poor limb and cardiovascular prognoses, along with a dramatic decrease in life expectancy. Despite major medical advances in the treatment of diabetes, a substantial therapeutic gap remains in the peripheral artery disease population. Praliciguat is an orally available sGC (soluble guanylate cyclase) stimulator that has been reported both preclinically and in early stage clinical trials to have favorable effects in metabolic and hemodynamic outcomes, suggesting that it may have a potential beneficial effect in peripheral artery disease. METHODS: We evaluated the effect of praliciguat on hind limb ischemia recovery in a mouse model of type 2 diabetes. Hind limb ischemia was induced in leptin receptor-deficient (Leprdb/db) mice by ligation and excision of the left femoral artery. Praliciguat (10 mg/kg/day) was administered in the diet starting 3 days before surgery. RESULTS: Twenty-eight days after surgery, ischemic foot perfusion and function parameters were better in praliciguat-treated mice than in vehicle controls. Improved ischemic foot perfusion was not associated with either improved traditional cardiovascular risk factors (ie, weight, glycemia) or increased angiogenesis. However, treatment with praliciguat significantly increased arteriole diameter, decreased ICAM1 (intercellular adhesion molecule 1) expression, and prevented the accumulation of oxidative proangiogenic and proinflammatory muscle fibers. While investigating the mechanism underlying the beneficial effects of praliciguat therapy, we found that praliciguat significantly downregulated Myh2 and Cxcl12 mRNA expression in cultured myoblasts and that conditioned medium form praliciguat-treated myoblast decreased ICAM1 mRNA expression in endothelial cells. These results suggest that praliciguat therapy may decrease ICAM1 expression in endothelial cells by downregulating Cxcl12 in myocytes. CONCLUSIONS: Our results demonstrated that praliciguat promotes blood flow recovery in the ischemic muscle of mice with type 2 diabetes, at least in part by increasing arteriole diameter and by downregulating ICAM1 expression.

Brown Adipose Tissue and BMP3b Decrease Injury in Cardiac Ischemia-Reperfusion.

BACKGROUND: Despite advances in treatment, myocardial infarction (MI) is a leading cause of heart failure and death worldwide, with both ischemia and reperfusion (I/R) causing cardiac injury. A previous study using a mouse model of nonreperfused MI showed activation of brown adipose tissue (BAT). Recent studies showed that molecules secreted by BAT target the heart. We investigated whether BAT attenuates cardiac injury in I/R and sought to identify potential cardioprotective proteins secreted by BAT. METHODS: Myocardial I/R surgery with or without BAT transplantation was performed in wild-type (WT) mice and in mice with impaired BAT function (uncoupling protein 1 [Ucp1]-deficient mice). To identify potential cardioprotective factors produced by BAT, RNA-seq (RNA sequencing) was performed in BAT from WT and Ucp1-/- mice. Subsequently, myocardial I/R surgery with or without BAT transplantation was performed in Bmp3b (bone morphogenetic protein 3b)-deficient mice, and WT mice subjected to myocardial I/R were treated using BMP3b. RESULTS: Dysfunction of BAT in mice was associated with larger MI size after I/R; conversely, augmenting BAT by transplantation decreased MI size. We identified Bmp3b as a protein secreted by BAT after I/R. Compared with WT mice, Bmp3b-deficient mice developed larger MIs. Increasing functional BAT by transplanting BAT from WT mice to Bmp3b-deficient mice reduced I/R injury whereas transplanting BAT from Bmp3b-deficient mice did not. Treatment of WT mice with BMP3b before reperfusion decreased MI size. The cardioprotective effect of BMP3b was mediated through SMAD1/5/8. In humans, the plasma level of BMP3b increased after MI and was positively correlated with the extent of cardiac injury. CONCLUSIONS: The results of this study suggest a cardioprotective role of BAT and BMP3b, a protein secreted by BAT, in a model of I/R injury. Interventions increasing BMP3b levels or targeting Smad 1/5 may represent novel therapeutic approaches to decrease myocardial damage in I/R injury.

Pharmacologically increasing cGMP improves proteostasis and reduces neuropathy in mouse models of CMT1.

Increasing cyclic GMP activates 26S proteasomes via phosphorylation by Protein Kinase G and stimulates the intracellular degradation of misfolded proteins. Therefore, agents that raise cGMP may be useful therapeutics against neurodegenerative diseases and other diseases in which protein degradation is reduced and misfolded proteins accumulate, including Charcot Marie Tooth 1A and 1B peripheral neuropathies, for which there are no treatments. Here we increased cGMP in the S63del mouse model of CMT1B by treating for three weeks with either the phosphodiesterase 5 inhibitor tadalafil, or the brain-penetrant soluble guanylyl cyclase stimulator CYR119. Both molecules activated proteasomes in the affected peripheral nerves, reduced polyubiquitinated proteins, and improved myelin thickness and nerve conduction. CYR119 increased cGMP more than tadalafil in the peripheral nerves of S63del mice and elicited greater biochemical and functional improvements. To determine whether raising cGMP could be beneficial in other neuropathies, we first showed that polyubiquitinated proteins and the disease-causing protein accumulate in the sciatic nerves of the C3 mouse model of CMT1A. Treatment of these mice with CYR119 reduced the levels of polyubiquitinated proteins and the disease-causing protein, presumably by increasing their degradation, and improved myelination, nerve conduction, and motor coordination. Thus, pharmacological agents that increase cGMP are promising treatments for CMT1 neuropathies and may be useful against other proteotoxic and neurodegenerative diseases.

SGLT2 inhibition potentiates the cardiovascular, renal, and metabolic effects of sGC stimulation in hypertensive rats with prolonged exposure to high-fat diet.

Prolonged high-fat diet (HFD) accelerates the cardiovascular, renal, and metabolic dysfunction in hypertensive rats with altered renal development (ARDev). Soluble guanylate cyclase (sGC) stimulation or sodium-glucose cotransporter 2 (SGLT2) inhibition may improve cardiovascular, renal, and metabolic function in settings of hypertension and obesity. This study examined whether 6 wk treatment with an SGLT2 inhibitor (empagliflozin, 7 mg/kg/day) enhances the cardiovascular, renal, and metabolic effects of a sGC stimulator (praliciguat, 10 mg/kg/day) in hypertensive rats with ARDev and prolonged exposure to HFD. Arterial pressure (AP), renal vascular resistance (RVR), fat abdominal volume (FAV), insulin resistance, leptin and triglycerides levels, and intrarenal infiltration of inflammatory cells were higher, but cardiac output and creatinine clearance were lower in hypertensive rats (n = 15) than in normotensive rats (n = 7). Praliciguat administration (n = 10) to hypertensive rats reduced (P < 0.05) AP, FAV, plasma concentrations of leptin and triglycerides, and increased (P < 0.05) cardiac output and creatinine clearance. Empagliflozin administration (n = 8) only increased (P < 0.05) glucosuria and creatinine clearance and decreased (P < 0.05) plasma leptin and triglycerides concentrations in hypertensive rats. Simultaneous administration of praliciguat and empagliflozin (n = 10) accelerated the decrease in AP, improved glucose tolerance, reduced (P < 0.05) incremental body weight gain, and decreased (P < 0.05) insulin resistance index, RVR, and the infiltration of T-CD3 lymphocytes in renal cortex and renal medulla. In summary, the combined administration of praliciguat and empagliflozin leads to a greater improvement of the cardiovascular, renal, and metabolic dysfunction secondary to prolonged exposure to HFD in hypertensive rats with ARDev than the treatment with either praliciguat or empagliflozin alone.NEW & NOTEWORTHY This is the first study, to our knowledge, showing that SGLT2 inhibition potentiates the beneficial cardiovascular, renal, and metabolic effects elicited by sGC stimulation in hypertensive rats with prolonged high-fat diet. The effects of the simultaneous administration of praliciguat and empagliflozin are greater than those elicited by either one alone. The effects of the simultaneous treatment may be related to a greater reduction in the inflammatory status.

Inhaled nitric oxide improves post-cardiac arrest outcomes via guanylate cyclase-1 in bone marrow-derived cells.

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 GC-1 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.

Soluble guanylate cyclase stimulator praliciguat attenuates inflammation, fibrosis, and end-organ damage in the Dahl model of cardiorenal failure.

Reduced nitric oxide (NO) and a decrease in cGMP signaling mediated by soluble guanylate cyclase (sGC) has been linked to the development of several cardiorenal diseases. Stimulation of sGC is a potential means for enhancing cGMP production in conditions of reduced NO bioavailability. The purpose of our studies was to determine the effects of praliciguat, a clinical-stage sGC stimulator, in a model of cardiorenal failure. Dahl salt-sensitive rats fed a high-salt diet to induce hypertension and organ damage were treated with the sGC stimulator praliciguat to determine its effects on hemodynamics, biomarkers of inflammation, fibrosis, tissue function, and organ damage. Praliciguat treatment reduced blood pressure, improved cardiorenal damage, and attenuated the increase in circulating markers of inflammation and fibrosis. Notably, praliciguat affected markers of renal damage at a dose that had minimal effect on blood pressure. In addition, liver fibrosis and circulating markers of tissue damage were attenuated in praliciguat-treated rats. Stimulation of the NO-sGC-cGMP pathway by praliciguat attenuated or normalized indicators of chronic inflammation, fibrosis, and tissue dysfunction in the Dahl salt-sensitive rat model. Stimulation of sGC by praliciguat may present an effective mechanism for treating diseases linked to NO deficiency, particularly those associated with cardiac and renal failure. Praliciguat is currently being evaluated in patients with diabetic nephropathy and heart failure with preserved ejection fraction.

Deficiency of bone morphogenetic protein-3b induces metabolic syndrome and increases adipogenesis.

Bone morphogenetic protein (BMP) receptor signaling is critical for the regulation of the endocrine system and cardiovascular structure and function. The objective of this study was to investigate whether Bmp3b, a glycoprotein synthetized and secreted by adipose tissue, is necessary to regulate glucose and lipid metabolism, adipogenesis, and cardiovascular remodeling. Over the course of 4 mo, Bmp3b-knockout (Bmp3b-/-) mice gained more weight than wild-type (WT) mice. The plasma levels of cholesterol and triglycerides were higher in Bmp3b-/- mice than in WT mice. Bmp3b-/- mice developed insulin resistance and glucose intolerance. The basal heart rate was higher in Bmp3b-/- mice than in WT mice, and echocardiography revealed eccentric remodeling in Bmp3b-/- mice. The expression of adipogenesis-related genes in white adipose tissue was higher in Bmp3b-/- mice than in WT control mice. In vitro studies showed that Bmp3b modulates the activity of the C/ebpα promoter, an effect mediated by Smad2/3. The results of this study suggest that Bmp3b is necessary for the maintenance of homeostasis in terms of age-related weight gain, glucose metabolism, and left ventricular (LV) remodeling and function. Interventions that increase the level or function of BMP3b may decrease cardiovascular risk and pathological cardiac remodeling.

Increased bioavailability of cyclic guanylate monophosphate prevents retinal ganglion cell degeneration.

The nitric oxide - guanylyl cyclase-1 - cyclic guanylate monophosphate (NO-GC-1-cGMP) pathway has emerged as a potential pathogenic mechanism for glaucoma, a common intraocular pressure (IOP)-related optic neuropathy characterized by the degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. NO activates GC-1 to increase cGMP levels, which are lowered by cGMP-specific phosphodiesterase (PDE) activity. This pathway appears to play a role in both the regulation of IOP, where reduced cGMP levels in mice leads to elevated IOP and subsequent RGC degeneration. Here, we investigated whether potentiation of cGMP signaling could protect RGCs from glaucomatous degeneration. We administered the PDE5 inhibitor tadalafil orally (10 mg/kg/day) in murine models of two forms of glaucoma - primary open angle glaucoma (POAG; GC-1-/- mice) and primary angle-closure glaucoma (PACG; Microbead Occlusion Model) - and measured RGC viability at both the soma and axon level. To determine the direct effect of increased cGMP on RGCs in vitro, we treated axotomized whole retina and primary RGC cultures with the cGMP analogue 8-Br-cGMP. Tadalafil treatment increased plasma cGMP levels in both models, but did not alter IOP or mean arterial pressure. Nonetheless, tadalafil treatment prevented degeneration of RGC soma and axons in both disease models. Treatment of whole, axotomized retina and primary RGC cultures with 8-Br-cGMP markedly attenuated both necrotic and apoptotic cell death pathways in RGCs. Our findings suggest that enhancement of the NO-GC-1-cGMP pathway protects the RGC body and axon in murine models of POAG and PACG, and that enhanced signaling through this pathway may serve as a novel glaucoma treatment, acting independently of IOP.

Low guanylyl cyclase activity in Weddell seals: implications for peripheral vasoconstriction and perfusion of the brain during diving.

Nitric oxide (NO) is a potent vasodilator, which improves perfusion and oxygen delivery during tissue hypoxia in terrestrial animals. The vertebrate dive response involves vasoconstriction in select tissues, which persists despite profound hypoxia. Using tissues collected from Weddell seals at necropsy, we investigated whether vasoconstriction is aided by downregulation of local hypoxia signaling mechanisms. We focused on NO-soluble guanylyl cyclase (GC)-cGMP signaling, a well-known vasodilatory transduction pathway. Seals have a lower GC protein abundance, activity, and capacity to respond to NO stimulation than do terrestrial mammals. In seal lung homogenates, GC produced less cGMP (20.1 ± 3.7 pmol·mg protein-1·min-1) than the lungs of dogs (-80 ± 144 pmol·mg protein-1·min-1 less than seals), sheep (-472 ± 96), rats (-664 ± 104) or mice (-1,160 ± 104, P < 0.0001). Amino acid sequences of the GC enzyme α-subunits differed between seals and terrestrial mammals, potentially affecting their structure and function. Vasoconstriction in diving Weddell seals is not consistent across tissues; perfusion is maintained in the brain and heart but decreased in other organs such as the kidney. A NO donor increased median GC activity 49.5-fold in the seal brain but only 27.4-fold in the kidney, consistent with the priority of cerebral perfusion during diving. Nos3 expression was high in the seal brain, which could improve NO production and vasodilatory potential. Conversely, Pde5a expression was high in the seal renal artery, which may increase cGMP breakdown and vasoconstriction in the kidney. Taken together, the results of this study suggest that alterations in the NO-cGMP pathway facilitate the diving response.

The nitric oxide-guanylate cyclase pathway and glaucoma.

Glaucoma is a prevalent optic neuropathy characterized by the progressive dysfunction and loss of retinal ganglion cells (RGCs) and their optic nerve axons, which leads to irreversible visual field loss. Multiple risk factors for the disease have been identified, but elevated intraocular pressure (IOP) remains the primary risk factor amenable to treatment. Reducing IOP however does not always prevent glaucomatous neurodegeneration, and many patients progress with the disease despite having IOP in the normal range. There is increasing evidence that nitric oxide (NO) is a direct regulator of IOP and that dysfunction of the NO-Guanylate Cyclase (GC) pathway is associated with glaucoma incidence. NO has shown promise as a novel therapeutic with targeted effects that: 1) lower IOP; 2) increase ocular blood flow; and 3) confer neuroprotection. The various effects of NO in the eye appear to be mediated through the activation of the GC- guanosine 3:5'-cyclic monophosphate (cGMP) pathway and its effect on downstream targets, such as protein kinases and Ca2+ channels. Although NO-donor compounds are promising as therapeutics for IOP regulation, they may not be ideal to harness the neuroprotective potential of NO signaling. Here we review evidence that supports direct targeting of GC as a novel pleiotrophic treatment for the disease, without the need for direct NO application. The identification and targeting of other factors that contribute to glaucoma would be beneficial to patients, particularly those that do not respond well to IOP-dependent interventions.

Intrinsic anti-inflammatory properties in the serum of two species of deep-diving seal.

Weddell and elephant seals are deep-diving mammals, which rely on lung collapse to limit nitrogen absorption and prevent decompression injury. Repeated collapse and re-expansion exposes the lungs to multiple stressors, including ischemia-reperfusion, alveolar shear stress and inflammation. There is no evidence, however, that diving damages pulmonary function in these species. To investigate potential protective strategies in deep-diving seals, we examined the inflammatory response of seal whole blood exposed to lipopolysaccharide (LPS), a potent endotoxin. Interleukin-6 (IL6) cytokine production elicited by LPS exposure was 50 to 500 times lower in blood of healthy northern elephant seals and Weddell seals compared with that of healthy human blood. In contrast to the ∼6× increased production of IL6 protein from LPS-exposed Weddell seal whole blood, isolated Weddell seal peripheral blood mononuclear cells, under standard cell culture conditions using medium supplemented with fetal bovine serum (FBS), produced a robust LPS response (∼300×). Induction of Il6 mRNA expression as well as production of IL6, IL8, IL10, KC-like and TNFα were reduced by substituting FBS with an equivalent amount of autologous seal serum. Weddell seal serum also attenuated the inflammatory response of RAW 267.4 mouse macrophage cells exposed to LPS. Cortisol level and the addition of serum lipids did not impact the cytokine response in cultured cells. These data suggest that seal serum possesses anti-inflammatory properties, which may protect deep divers from naturally occurring inflammatory challenges such as dive-induced hypoxia-reoxygenation and lung collapse.

Endothelial dysfunction inhibits the ability of haptoglobin to prevent hemoglobin-induced hypertension.

Intravascular hemolysis produces injury in a variety of human diseases including hemoglobinopathies, malaria, and sepsis. The adverse effects of increased plasma hemoglobin are partly mediated by depletion of nitric oxide (NO) and result in vasoconstriction. Circulating plasma proteins haptoglobin and hemopexin scavenge extracellular hemoglobin and cell-free heme, respectively. The ability of human haptoglobin or hemopexin to inhibit the adverse effects of NO scavenging by circulating murine hemoglobin was tested in C57Bl/6 mice. In healthy awake mice, the systemic hemodynamic effects of intravenous coinfusion of cell-free hemoglobin and exogenous haptoglobin or of cell-free hemoglobin and hemopexin were compared with the hemodynamic effects of infusion of cell-free hemoglobin or control protein (albumin) alone. We also studied the hemodynamic effects of infusing hemoglobin and haptoglobin as well as injecting either hemoglobin or albumin alone in mice fed a high-fat diet (HFD) and in diabetic (db/db) mice. Coinfusion of a 1:1 weight ratio of haptoglobin but not hemopexin with cell-free hemoglobin prevented hemoglobin-induced systemic hypertension in healthy awake mice. In mice fed a HFD and in diabetic mice, coinfusion of haptoglobin mixed with an equal mass of cell-free hemoglobin did not reverse hemoglobin-induced hypertension. Haptoglobin retained cell-free hemoglobin in plasma, but neither haptoglobin nor hemopexin affected the ability of hemoglobin to scavenge NO ex vivo. In conclusion, in healthy C57Bl/6 mice with normal endothelium, coadministration of haptoglobin but not hemopexin with cell-free hemoglobin prevents acute hemoglobin-induced systemic hypertension by compartmentalizing cell-free hemoglobin in plasma. In murine diseases associated with endothelial dysfunction, haptoglobin therapy appears to be insufficient to prevent hemoglobin-induced vasoconstriction.NEW & NOTEWORTHY Coadministraton of haptoglobin but not hemopexin with cell-free hemoglobin prevents hemoglobin-induced systemic hypertension in mice with a normal endothelium. In contrast, treatment with the same amount of haptoglobin is unable to prevent hemoglobin-induced vasoconstriction in mice with hyperlipidemia or diabetes mellitus, disorders that are associated with endothelial dysfunction.

Erectile Dysfunction in Heme-Deficient Nitric Oxide-Unresponsive Soluble Guanylate Cyclase Knock-In Mice.

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 (sGCß1ki/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 sGCß1ki/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 sGCß1ki/ki and wild-type mice after stimulation with sGC-dependent and -independent vasorelaxant agents. RESULTS: NO-induced responses were abolished in sGCß1ki/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 sGCß1ki/ki mice. In contrast, the relaxation response to the heme- and NO-independent sGC activator BAY 58-2667 was significantly enhanced in sGCß1ki/ki mice. The relaxing effect of sGC-independent vasorelaxant agents was similar in wild-type and sGCß1ki/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 sGCα1ß1, sGCα2ß1 is important for erectile function. In addition, the significant relaxation observed in sGCß1ki/ki mice with the cumulative addition of the sGC activator BAY 58-2667 indicates that sGC activators might offer value in treating erectile dysfunction.

Sensitivity to Sevoflurane anesthesia is decreased in mice with a congenital deletion of Guanylyl Cyclase-1 alpha.

BACKGROUND: Volatile anesthetics increase levels of the neurotransmitter nitric oxide (NO) and the secondary messenger molecule cyclic guanosine monophosphate (cGMP) in the brain. NO activates the enzyme guanylyl cyclase (GC) to produce cGMP. We hypothesized that the NO-GC-cGMP pathway contributes to anesthesia-induced unconsciousness. METHODS: Sevoflurane-induced loss and return of righting reflex (LORR and RORR, respectively) were studied in wild-type mice (WT) and in mice congenitally deficient in the GC-1α subunit (GC-1-/- mice). Spatial distributions of GC-1α and the GC-2α subunit in the brain were visualized by in situ hybridization. Brain cGMP levels were measured in WT and GC-1-/- mice after inhaling oxygen with or without 1.2% sevoflurane for 20 min. RESULTS: Higher concentrations of sevoflurane were required to induce LORR in GC-1-/- mice than in WT mice (1.5 ± 0.1 vs. 1.1 ± 0.2%, respectively, n = 14 and 14, P < 0.0001). Similarly, RORR occurred at higher concentrations of sevoflurane in GC-1-/- mice than in WT mice (1.0 ± 0.1 vs. 0.8 ± 0.1%, respectively, n = 14 and 14, P < 0.0001). Abundant GC-1α and GC-2α mRNA expression was detected in the cerebral cortex, medial habenula, hippocampus, and cerebellum. Inhaling 1.2% sevoflurane for 20 min increased cGMP levels in the brains of WT mice from 2.6 ± 2.0 to 5.5 ± 3.7 pmol/mg protein (n = 13 and 10, respectively, P = 0.0355) but not in GC-1-/- mice. CONCLUSION: Congenital deficiency of GC-1α abolished the ability of sevoflurane anesthesia to increase cGMP levels in the whole brain, and increased the concentration of sevoflurane required to induce LORR. Impaired NO-cGMP signaling raises the threshold for producing sevoflurane-induced unconsciousness in mice.

Soluble epoxide hydrolase deficiency or inhibition enhances murine hypoxic pulmonary vasoconstriction after lipopolysaccharide challenge.

Hypoxic pulmonary vasoconstriction (HPV) is the response of the pulmonary vasculature to low levels of alveolar oxygen. HPV improves systemic arterial oxygenation by matching pulmonary perfusion to ventilation during alveolar hypoxia and is impaired in lung diseases such as the acute respiratory distress syndrome (ARDS) and in experimental models of endotoxemia. Epoxyeicosatrienoic acids (EETs) are pulmonary vasoconstrictors, which are metabolized to less vasoactive dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). We hypothesized that pharmacological inhibition or a congenital deficiency of sEH in mice would reduce the metabolism of EETs and enhance HPV in mice after challenge with lipopolysaccharide (LPS). HPV was assessed 22 h after intravenous injection of LPS by measuring the percentage increase in the pulmonary vascular resistance of the left lung induced by left mainstem bronchial occlusion (LMBO). After LPS challenge, HPV was impaired in sEH+/+, but not in sEH-/- mice or in sEH+/+ mice treated acutely with a sEH inhibitor. Deficiency or pharmacological inhibition of sEH protected mice from the LPS-induced decrease in systemic arterial oxygen concentration (PaO2 ) during LMBO. In the lungs of sEH-/- mice, the LPS-induced increase in DHETs and cytokines was attenuated. Deficiency or pharmacological inhibition of sEH protects mice from LPS-induced impairment of HPV and improves the PaO2 after LMBO. After LPS challenge, lung EET degradation and cytokine expression were reduced in sEH-/- mice. Inhibition of sEH might prove to be an effective treatment for ventilation-perfusion mismatch in lung diseases such as ARDS.

BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy.

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.

Androgen-sensitive hypertension associated with soluble guanylate cyclase-α1 deficiency is mediated by 20-HETE.

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.

An open-source computational tool to automatically quantify immunolabeled retinal ganglion cells.

A fully automated and robust method was developed to quantify ß-III-tubulin-stained retinal ganglion cells, combining computational recognition of individual cells by CellProfiler and a machine-learning tool to teach phenotypic classification of the retinal ganglion cells by CellProfiler Analyst. In animal models of glaucoma, quantification of immunolabeled retinal ganglion cells is currently performed manually and remains time-consuming. Using this automated method, quantifications of retinal ganglion cell images were accelerated tenfold: 1800 images were counted in 3 h using our automated method, while manual counting of the same images took 72 h. This new method was validated in an established murine model of microbead-induced optic neuropathy. The use of the publicly available software and the method's user-friendly design allows this technique to be easily implemented in any laboratory.

Regulation of B-type natriuretic peptide synthesis by insulin in obesity in male mice.

Human studies suggest that insulin resistance and obesity are associated with a decrease in B-type natriuretic peptide (BNP) plasma concentrations. The objective of the study was to gain insights into the mechanisms involved in the association between insulin resistance and decreased BNP plasma concentrations. Mice fed a high-fat, high-fructose (HFHF) diet for 4 weeks developed mild obesity and systemic insulin resistance. Elevated plasma concentrations of insulin, glucose and triglycerides were noted. The HFHF diet was also associated with myocardial insulin resistance, characterized by an impaired response of the phosphoinositide 3-kinase-AKT (PI3K-AKT) pathway to insulin in the left ventricle. Myocardial BNP expression and protein were decreased in HFHF-fed mice compared with control animals. Exposure of cardiomyocytes to 100 nm insulin activated PI3K-AKT signalling (15 min) and induced a 1.9 ± 0.3-fold increase in BNP gene expression (6 h). Prolonged exposure of cardiomyocytes to a high insulin concentration (100 nm) for 48 h induced insulin resistance, characterized by an impaired response of the PI3K-AKT signalling pathway and a decreased response of the BNP gene expression to insulin. The decreased response in BNP gene expression was reproduced by treating cardiomyocytes for 7 h with a PI3-kinase inhibitor (wortmannin). In conclusion, HFHF diet in vivo, prolonged exposure to an elevated concentration of insulin or inhibition of the PI3K-AKT pathway in vitro all decrease BNP mRNA levels; this decrease may in turn contribute to the decreased BNP peptide concentrations in plasma observed in insulin-resistant individuals.

Functional brown adipose tissue limits cardiomyocyte injury and adverse remodeling in catecholamine-induced cardiomyopathy.

Brown adipose tissue (BAT) has well recognized thermogenic properties mediated by uncoupling protein 1 (UCP1); more recently, BAT has been demonstrated to modulate cardiovascular risk factors. To investigate whether BAT also affects myocardial injury and remodeling, UCP1-deficient (UCP1(-/-)) mice, which have dysfunctional BAT, were subjected to catecholamine-induced cardiomyopathy. At baseline, there were no differences in echocardiographic parameters, plasma cardiac troponin I (cTnI) or myocardial fibrosis between wild-type (WT) and UCP1(-/-) mice. Isoproterenol infusion increased cTnI and myocardial fibrosis and induced left ventricular (LV) hypertrophy in both WT and UCP1(-/-) mice. UCP1(-/-) mice also demonstrated exaggerated myocardial injury, fibrosis, and adverse remodeling, as well as decreased survival. Transplantation of WT BAT to UCP1(-/-) mice prevented the isoproterenol-induced cTnI increase and improved survival, whereas UCP1(-/-) BAT transplanted to either UCP1(-/-) or WT mice had no effect on cTnI release. After 3 days of isoproterenol treatment, phosphorylated AKT and ERK were lower in the LV's of UCP1(-/-) mice than in those of WT mice. Activation of BAT was also noted in a model of chronic ischemic cardiomyopathy, and was correlated to LV dysfunction. Deficiency in UCP1, and accompanying BAT dysfunction, increases cardiomyocyte injury and adverse LV remodeling, and decreases survival in a mouse model of catecholamine-induced cardiomyopathy. Myocardial injury and decreased survival are rescued by transplantation of functional BAT to UCP1(-/-) mice, suggesting a systemic cardioprotective role of functional BAT. BAT is also activated in chronic ischemic cardiomyopathy.