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Soluble guanylate cyclase (sGC) serves as a receptor of nitric oxide (NO) and is the core metalloenzyme in the NO signal transduction pathway. sGC plays a key role in the NO-cGMP signal transduction pathway and participates in various physiological processes, including cell differentiation, neuron transmission, and internal environment homeostasis. sGC consists of two subunits, α and β, each subunit containing multiple isoforms. In this study, we cloned and analyzed the sGC-α1 gene in the silkworm Bombyx mori (BmsGC-α1). The BmsGC-α1 gene was expressed highest at the pupal stages. The highest BmsGC-α1 mRNA expression was observed in the head of fifth instar larvae and in fat body during the wandering stage of B. mori. Furthermore, we observed that feeding fifth instar larvae with thyroid hormone and nitroglycerin induced the expression of the BmsGC-α1 gene. Injection of BmsGC-α1 siRNA into silkworms at the prepupal stage resulted in a significant decrease in BmsGC-α1 expression levels at 48 and 72 h postinjection. After silencing BmsGC-α1, both the egg-laying amount and hatching rate of silkworm eggs were significantly reduced compared to the control group. These results suggest that BmsGC-α1 plays an important role in regulating the reproductive system of silkworms. This finding enhances our understanding of the functional diversity of sGC in insects., (© 2024 Wiley Periodicals LLC.)

Nitric oxide (NO) regulates large swaths of animal physiology including wound healing, vasodilation, memory formation, odor detection, sexual function, and response to infectious disease. The primary NO receptor is soluble guanyly/guanylate cyclase (sGC), a dimeric protein of ∼150 kDa that detects NO through a ferrous heme, leading to a large change in conformation and enhanced production of cGMP from GTP. In humans, loss of sGC function contributes to multiple disease states, including cardiovascular disease and cancer, and is the target of a new class of drugs, sGC stimulators, now in clinical use. sGC evolved through the fusion of four ancient domains, a heme nitric oxide / oxygen (H-NOX) domain, a Per-ARNT-Sim (PAS) domain, a coiled coil, and a cyclase domain, with catalysis occurring at the interface of the two cyclase domains. In animals, the predominant dimer is the α1β1 heterodimer, with the α1 subunit formed through gene duplication of the β1 subunit. The PAS domain provides an extensive dimer interface that remains unchanged during sGC activation, acting as a core anchor. A large cleft formed at the PAS-PAS dimer interface tightly binds the N-terminal end of the coiled coil, keeping this region intact and unchanged while the rest of the coiled coil repacks, and the other domains reposition. This interface buries ∼3000 Å 2 of monomer surface and includes highly conserved apolar and hydrogen bonding residues. Herein, we discuss the evolutionary history of sGC, describe the role of PAS domains in sGC function, and explore the regulatory factors affecting sGC function., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cytoglobin is a heme protein with unresolved physiological function. Genetic deletion of zebrafish cytoglobin (cygb2) causes developmental defects in left-right cardiac determination, which in humans is associated with defects in ciliary function and low airway epithelial nitric oxide production. Here we show that Cygb2 co-localizes with cilia and with the nitric oxide synthase Nos2b in the zebrafish Kupffer's vesicle, and that cilia structure and function are disrupted in cygb2 mutants. Abnormal ciliary function and organ laterality defects are phenocopied by depletion of nos2b and of gucy1a, the soluble guanylate cyclase homolog in fish. The defects are rescued by exposing cygb2 mutant embryos to a nitric oxide donor or a soluble guanylate cyclase stimulator, or with over-expression of nos2b. Cytoglobin knockout mice also show impaired airway epithelial cilia structure and reduced nitric oxide levels. Altogether, our data suggest that cytoglobin is a positive regulator of a signaling axis composed of nitric oxide synthase-soluble guanylate cyclase-cyclic GMP that is necessary for normal cilia motility and left-right patterning., (© 2023. The Author(s).)

Hemoglobin (Hb) present in the lung epithelium is of unknown significance. However Hb being an nitric oxide (NO) scavenger can bind to NO and reduce its deleterious effects. Hence we postulated an NO scavenging role for this lung Hb. Doing transwell co-culture with bronchial epithelial cells, A549/16-HBE (apical) and human airway smooth muscle cells (HASMCs as basal), we found that Hb can protect the smooth muscle soluble guanylyl cyclase (sGC) from excess NO. Inducing the apical A549/16-HBE cells with cytokines to trigger iNOS expression and NO generation caused a time dependent increase in SNO-sGC and this was accompanied with a concomitant drop in sGC-α1β1 heterodimerization. Silencing Hbαβ in the apical cells further increased the SNO on sGC with a faster drop in the sGC heterodimer and these effects were additive along with further silencing of thioredoxin 1 (Trx1). Since heme of Hb is critical for NO scavenging we determined the Hb heme in a mouse model of allergic asthma (OVA) and found that Hb in the inflammed OVA lungs was low in heme or heme-free relative to those of naïve lungs. Further we established a direct correlation between the status of the sGC heterodimer and the Hb heme from lung samples of human asthma, iPAH, COPD and cystic fibrosis. These findings present a new mechanism of protection of lung sGC by the epithelial Hb, and suggests that this protection maybe lost in asthma or COPD where lung Hb is unable to scavenge the NO due to it being heme-deprived., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Moyamoya angiopathy (MMA) is a rare cerebrovascular condition leading to stroke. Mutations in 15 genes have been identified in Mendelian forms of MMA, but they explain only a very small proportion of cases. Our aim was to investigate the genetic basis of MMA in consanguineous patients having unaffected parents in order to identify genes involved in autosomal recessive MMA., Methods: Exome sequencing (ES) was performed in 6 consecutive consanguineous probands having MMA of unknown etiology. Functional consequences of variants were assessed using western blot and protein 3D structure analyses., Results: Causative homozygous variants of NOS3, the gene encoding the endothelial nitric oxide synthase (eNOS), and GUCY1A3, the gene encoding the alpha1 subunit of the soluble guanylate cyclase (sGC) which is the major nitric oxide (NO) receptor in the vascular wall, were identified in 3 of the 6 probands. One NOS3 variant (c.1502 + 1G > C) involves a splice donor site causing a premature termination codon and leads to a total lack of eNOS in endothelial progenitor cells of the affected proband. The other NOS3 variant (c.1942 T > C) is a missense variant located into the flavodoxine reductase domain; it is predicted to be destabilizing and shown to be associated with a reduction of eNOS expression. The GUCY1A3 missense variant (c.1778G > A), located in the catalytic domain of the sGC, is predicted to disrupt the tridimensional structure of this domain and to lead to a loss of function of the enzyme. Both NOS3 mutated probands suffered from an infant-onset and severe MMA associated with posterior cerebral artery steno-occlusive lesions. The GUCY1A3 mutated proband presented an adult-onset MMA associated with an early-onset arterial hypertension and a stenosis of the superior mesenteric artery. None of the 3 probands had achalasia., Conclusions: We show for the first time that biallelic loss of function variants in NOS3 is responsible for MMA and that mutations in NOS3 and GUCY1A3 are causing fifty per cent of MMA in consanguineous patients. These data pinpoint the essential role of the NO pathway in MMA pathophysiology., (© 2023. The Author(s).)

Disruption of endothelial cell (ECs) and pericytes interactions results in vascular leakage in acute lung injury (ALI). However, molecular signals mediating EC-pericyte crosstalk have not been systemically investigated, and whether targeting such crosstalk could be adopted to combat ALI remains elusive. Using comparative genome-wide EC-pericyte crosstalk analysis of healthy and LPS-challenged lungs, we discovered that crosstalk between endothelial nitric oxide and pericyte soluble guanylate cyclase (NO-sGC) is impaired in ALI. Indeed, stimulating the NO-sGC pathway promotes vascular integrity and reduces lung edema and inflammation-induced lung injury, while pericyte-specific sGC knockout abolishes this protective effect. Mechanistically, sGC activation suppresses cytoskeleton rearrangement in pericytes through inhibiting VASP-dependent F-actin formation and MRTFA/SRF-dependent de novo synthesis of genes associated with cytoskeleton rearrangement, thereby leading to the stabilization of EC-pericyte interactions. Collectively, our data demonstrate that impaired NO-sGC crosstalk in the vascular niche results in elevated vascular permeability, and pharmacological activation of this crosstalk represents a promising translational therapy for ALI., (© 2022 He et al.)

The binding of nitric oxide (NO) to heme in the β 1 subunit of soluble guanylyl cyclase (sGC) activates both the heterodimeric α 1 β 1 and α 2 β 1 isoforms of the enzyme, leading to the increased production of cGMP from GTP. In cultured human mast cells, exogenous NO is able to inhibit mast cell degranulation via NO-cGMP signaling. However, under inflammatory oxidative or nitrosative stress, sGC becomes insensitive to NO. The occurrence of mast cells in healthy and inflamed human tissues and the in vivo expression of the α 1 and β 1 subunits of sGC in human mast cells during inflammation remain largely unresolved and were investigated here. Using peroxidase and double immunohistochemical incubations, no mast cells were found in healthy dental pulp, whereas the inflammation of dental pulp initiated the occurrence of several mast cells expressing the α 1 and β 1 subunits of sGC. Since inflammation-induced oxidative and nitrosative stress oxidizes Fe 2+ to Fe 3+ in the β 1 subunit of sGC, leading to the desensitization of sGC to NO, we hypothesize that the NO- and heme-independent pharmacological activation of sGC in mast cells may be considered as a regulatory strategy for mast cell functions in inflamed human dental pulp.

Background: Genetic manipulation on the NO-sGC-cGMP pathway has been rarely achieved, partially due to complexity of the soluble guanylyl cyclase (sGC) enzyme., Objective: We aim to develop gene therapy directly targeting the pathway to circumvent cytotoxicity and tolerance after prolonged use of NO-donors and the insufficiency of PDE inhibitors., Methods: In this study, we constructed lentivirus vectors expressing GUCY1A3 and GUCY1B3 genes, which encoded the α1 and β1 subunits of soluble guanylyl cyclase (sGC), respectively, to enhance cGMP synthesis. We also constructed lentiviral vector harboring PDE5A shRNA to alleviate phosphodiesterase activity and cGMP degradation., Results: Transductions of human HEK293 cells with the constructs were successful, as indicated by the fluorescent signal and altered gene expression produced by each vector. Overexpression of GUCY1A3 and GUCY1B3 resulted in increased sGC enzyme activity and elevated cGMP level in the cells. Expression of PDE5A shRNA resulted in decreased PDE5A expression and elevated cGMP level. Co-transduction of the three lentiviral vectors resulted in a more significant elevation of cGMP in HEK293 cells without obvious cytotoxicity., Conclusion: To the best of our knowledge, this is the first study to show that co-expression of exogenous subunits of the soluble guanylyl cyclase could form functional enzyme and increase cellular cGMP level in mammalian cells. Simultaneous expression of PDE5A shRNA could alleviate feedback up-regulation on PDE5A caused by cGMP elevation. Further studies are required to evaluate the effects of these constructs in vivo.

Chronic obstructive pulmonary disease (COPD), whose main risk factor is cigarette smoking (CS), is one of the most common diseases globally. Some COPD patients also develop pulmonary hypertension (PH), a severe complication that leads to premature death. Evidence suggests reactive oxygen species (ROS) involvement in COPD and PH, especially regarding pulmonary artery smooth muscle cells (PASMC) dysfunction. However, the effects of CS-driven oxidative stress on the pulmonary vasculature are not completely understood. Herein we provide evidence on the effects of CS extract (CSE) exposure on PASMC regarding ROS production, antioxidant response and its consequences on vascular tone dysregulation. Our results indicate that CSE exposure promotes mitochondrial fission, mitochondrial membrane depolarization and increased mitochondrial superoxide levels. However, this superoxide increase did not parallel a counterbalancing antioxidant response in human pulmonary artery (PA) cells. Interestingly, the mitochondrial superoxide scavenger mitoTEMPO reduced mitochondrial fission and membrane potential depolarization caused by CSE. As we have previously shown, CSE reduces PA vasoconstriction and vasodilation. In this respect, mitoTEMPO prevented the impaired nitric oxide-mediated vasodilation, while vasoconstriction remained reduced. Finally, we observed a CSE-driven downregulation of the Cyb5R3 enzyme, which prevents soluble guanylyl cyclase oxidation in PASMC. This might explain the CSE-mediated decrease in PA vasodilation. These results provide evidence that there might be a connection between mitochondrial ROS and altered vasodilation responses in PH secondary to COPD, and strongly support the potential of antioxidant strategies specifically targeting mitochondria as a new therapy for these diseases., Competing Interests: Declaration of competing interest We declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Nitric oxide (NO) is a signal molecule and plays a critical role in the regulation of vascular tone, displays anti-platelet and anti-inflammatory properties. While our earlier and current studies found that low NO doses trigger a rapid heme insertion into immature heme-free soluble guanylyl cyclase β subunit (apo-sGCβ), resulting in a mature sGC-αβ heterodimer, more recent evidence suggests that low NO doses can also trigger heme-maturation of hemoglobin and myoglobin. This low NO phenomena was not only limited to sGC and the globins, but was also found to occur in all three nitric oxide synthases (iNOS, nNOS and eNOS) and Myeloperoxidase (MPO). Interestingly high NO doses were inhibitory to heme-insertion for these hemeproteins, suggesting that NO has a dose-dependent dual effect as it can act both ways to induce or inhibit heme-maturation of key hemeproteins. While low NO stimulated heme-insertion of globins required the presence of the NO-sGC-cGMP signal pathway, iNOS heme-maturation also required the presence of an active sGC. These effects of low NO were significantly diminished in the tissues of double (n/eNOS -/- ) and triple (n/i/eNOS -/- ) NOS knock out mice where lung sGC was found be heme-free and the myoglobin or hemoglobin from the heart/lungs were found be low in heme, suggesting that loss of endogenous NO globally impacts the whole animal and that this impact of low NO is both essential and physiologically relevant for hemeprotein maturation. Effects of low NO were also found to be protective against ischemia reperfusion injury on an ex vivo lung perfusion (EVLP) system prior to lung transplant, which further suggests that low NO levels are also therapeutic., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Age-related changes in the transcriptome lead to memory impairment. Several genes have been identified to cause age-dependent memory impairment (AMI) by changes in their expression, but genetic screens to identify genes critical for AMI have not been performed. The fruit fly is a useful model for studying AMI due to its short lifespan and the availability of consistent techniques and environments to assess its memory ability. We generated a list of candidate genes that act as AMI regulators by performing a comprehensive analysis of RNAsequencing data from young and aged fly heads and genome-wide RNAi screening data to identify memory-regulating genes. A candidate screen using temporal and panneuronal RNAi expression was performed to identify genes critical for AMI. We identified the guanylyl cyclase β-subunit at 100B (gycβ) gene, which encodes a subunit of soluble guanylyl cyclase (sGC), the only intracellular nitric oxide (NO) receptor in fruit flies, as a negative regulator of AMI. RNAi knockdown of gycβ in neurons and NO synthase (NOS) in glia or neurons enhanced the performance of intermediate-term memory (ITM) without apparent effects on memory acquisition. We also showed that pharmacological inhibition of sGC and NOS enhanced ITM in aged individuals, suggesting the possibility that age-related enhancement of the NO-sGC pathway causes memory impairment., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

DNA damage is a causative factor in ageing of the vasculature and other organs. One of the most important vascular ageing features is reduced nitric oxide (NO)soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signaling. We hypothesized that the restoration of NO-sGC-cGMP signaling with an sGC activator (BAY 54-6544) may have beneficial effects on vascular ageing and premature death in DNA repair-defective mice undergoing accelerated ageing. Eight weeks of treatment with a non-pressor dosage of BAY 54-6544 restored the decreased in vivo microvascular cutaneous perfusion in progeroid Ercc1 ∆/- mice to the level of wild-type mice. In addition, BAY 54-6544 increased survival of Ercc1 ∆/- mice. In isolated Ercc1 ∆/- aorta, the decreased endothelium-independent vasodilation was restored after chronic BAY 54-6544 treatment. Senescence markers p16 and p21, and markers of inflammation, including Ccl2, Il6 in aorta and liver, and circulating IL-6 and TNF-α were increased in Ercc1 ∆/- , which was lowered by the treatment. Expression of antioxidant genes, including Cyb5r3 and Nqo1, was favorably changed by chronic BAY 54-6544 treatment. In summary, BAY 54-6544 treatment improved the vascular function and survival rates in mice with accelerated ageing, which may have implication in prolonging health span in progeria and normal ageing., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Human soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. In this work, fluorescent proteins were employed to study the NO-induced sGC molecular mechanism via mutagenesis at the catalytic domain. The conformational change of sGC by mutant α 1 C595 was investigated in living cells through fluorescence lifetime imaging microscopy (FLIM). The results indicated that the NO-induced conformational change of the catalytic domain of sGC from "open to "closed" upon GTP-binding was regulated by the hydrogen (H)-bonding network of the catalytic domain. The mutation of C595 caused a big conformational change of catalytic domain with H-bond variation, which not only demonstrates the key role of the C595 site in the process of conformational change of the catalytic domain, but also reveals the regulatory mechanism of sGC at the catalytic domain. This finding would guide the design of small-molecule drugs targeting the catalytic domain to modulate sGC activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) binds soluble guanylyl cyclase β (sGCβ) to produce cGMP and relax vascular smooth muscle cells (SMCs) needed for vasodilation. Although the regulation of NO-stimulated sGC activity has been well characterized at the posttranslational level, the mechanisms that govern sGC transcription remain incompletely understood. Recently, we identified Forkhead box subclass O (FoxO) transcription factors as essential for expression of sGC; however, the specific FoxO family member responsible for the expression of sGCβ in SMC remains unknown. Using FoxO shRNA knockdown adenovirus treatment in rat aortic SMCs, we show that FoxO1 or FoxO3 knockdown causes greater than twofold increases in Gucy1a3 and Gucy1b3 mRNA expression, without changes in NO-dependent cGMP production or cGMP-dependent phosphorylation. FoxO4 knockdown produced a 50% decrease in Gucy1a3 and Gucy1b3 mRNA with 70% loss of sGCα and 50% loss of sGCβ protein expression. Knockdown of FoxO4 expression decreased cGMP production and downstream protein kinase G-dependent phosphorylation more than 50%. Triple FoxO knockdown exacerbated loss of sGC-dependent function, phenocopying previous FoxO inhibition studies. Using promoter luciferase and chromatin immunoprecipitation assays, we find that FoxO4 acts as a transcriptional activator by directly binding several FoxO DNA motifs in the promoter regions of GUCY1B3 in human aortic SMCs. Collectively, our data show FoxO4 is a critical transcriptional regulator of sGCβ expression in SMC. NEW & NOTEWORTHY One of the key mechanisms of vascular smooth muscle cell (SMC) dilation occurs through nitric oxide (NO)-dependent induction of soluble guanylyl cyclase (sGC) by means of its β-subunit. Herein, we are the first to identify Forkhead box subclass O protein 4 (FoxO4) as a key transcriptional regulator of GUCY1B3 expression, which codes for sGCβ protein in human and animal SMCs. This discovery will likely have important implications for the future usage of antihypertensive and vasodilatory therapies which target NO production, sGC, or FoxO transcription factors.

Chronic hypoxia is a major driver of cardiovascular complications, including heart failure. The nitric oxide (NO) - soluble guanylyl cyclase (sGC) - cyclic guanosine monophosphate (cGMP) pathway is integral to vascular tone maintenance. Specifically, NO binds its receptor sGC within vascular smooth muscle cells (SMC) in its reduced heme (Fe 2+ ) form to increase intracellular cGMP production, activate protein kinase G (PKG) signaling, and induce vessel relaxation. Under chronic hypoxia, oxidative stress drives oxidation of sGC heme (Fe 2+ →Fe 3+ ), rendering it NO-insensitive. We previously showed that cytochrome b5 reductase 3 (CYB5R3) in SMC is a sGC reductase important for maintaining NO-dependent vasodilation and conferring resilience to systemic hypertension and sickle cell disease-associated pulmonary hypertension. To test whether CYB5R3 may be protective in the context of chronic hypoxia, we subjected SMC-specific CYB5R3 knockout mice (SMC CYB5R3 KO) to 3 weeks hypoxia and assessed vascular and cardiac function using echocardiography, pressure volume loops and wire myography. Hypoxic stress caused 1) biventricular hypertrophy in both WT and SMC CYB5R3 KO, but to a larger degree in KO mice, 2) blunted vasodilation to NO-dependent activation of sGC in coronary and pulmonary arteries of KO mice, and 3) decreased, albeit still normal, cardiac function in KO mice. Overall, these data indicate that SMC CYB5R3 deficiency potentiates bilateral ventricular hypertrophy and blunts NO-dependent vasodilation under chronic hypoxia conditions. This implicates that SMC CYB5R3 KO mice post 3-week hypoxia have early stages of cardiac remodeling and functional changes that could foretell significantly impaired cardiac function with longer exposure to hypoxia., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Small cell lung cancer (SCLC) has a 5-year survival rate of <7%. Rapid emergence of acquired resistance to standard platinum-etoposide chemotherapy is common and improved therapies are required for this recalcitrant tumour. We exploit six paired pre-treatment and post-chemotherapy circulating tumour cell patient-derived explant (CDX) models from donors with extensive stage SCLC to investigate changes at disease progression after chemotherapy. Soluble guanylate cyclase (sGC) is recurrently upregulated in post-chemotherapy progression CDX models, which correlates with acquired chemoresistance. Expression and activation of sGC is regulated by Notch and nitric oxide (NO) signalling with downstream activation of protein kinase G. Genetic targeting of sGC or pharmacological inhibition of NO synthase re-sensitizes a chemoresistant CDX progression model in vivo, revealing this pathway as a mediator of chemoresistance and potential vulnerability of relapsed SCLC., (© 2021. The Author(s).)

Background: Many patients with heart failure with preserved ejection fraction have metabolic syndrome and develop exercise-induced pulmonary hypertension (EIPH). Increases in pulmonary vascular resistance in patients with heart failure with preserved ejection fraction portend a poor prognosis; this phenotype is referred to as combined precapillary and postcapillary pulmonary hypertension (CpcPH). Therapeutic trials for EIPH and CpcPH have been disappointing, suggesting the need for strategies that target upstream mechanisms of disease. This work reports novel rat EIPH models and mechanisms of pulmonary vascular dysfunction centered around the transcriptional repression of the soluble guanylate cyclase (sGC) enzyme in pulmonary artery (PA) smooth muscle cells., Methods: We used obese ZSF-1 leptin-receptor knockout rats (heart failure with preserved ejection fraction model), obese ZSF-1 rats treated with SU5416 to stimulate resting pulmonary hypertension (obese+sugen, CpcPH model), and lean ZSF-1 rats (controls). Right and left ventricular hemodynamics were evaluated using implanted catheters during treadmill exercise. PA function was evaluated with magnetic resonance imaging and myography. Overexpression of nuclear factor Y α subunit (NFYA), a transcriptional enhancer of sGC β1 subunit (sGCβ1), was performed by PA delivery of adeno-associated virus 6. Treatment groups received the SGLT2 inhibitor empagliflozin in drinking water. PA smooth muscle cells from rats and humans were cultured with palmitic acid, glucose, and insulin to induce metabolic stress., Results: Obese rats showed normal resting right ventricular systolic pressures, which significantly increased during exercise, modeling EIPH. Obese+sugen rats showed anatomic PA remodeling and developed elevated right ventricular systolic pressure at rest, which was exacerbated with exercise, modeling CpcPH. Myography and magnetic resonance imaging during dobutamine challenge revealed PA functional impairment of both obese groups. PAs of obese rats produced reactive oxygen species and decreased sGCβ1 expression. Mechanistically, cultured PA smooth muscle cells from obese rats and humans with diabetes or treated with palmitic acid, glucose, and insulin showed increased mitochondrial reactive oxygen species, which enhanced miR-193b-dependent RNA degradation of nuclear factor Y α subunit (NFYA), resulting in decreased sGCβ1-cGMP signaling. Forced NYFA expression by adeno-associated virus 6 delivery increased sGCβ1 levels and improved exercise pulmonary hypertension in obese+sugen rats. Treatment of obese+sugen rats with empagliflozin improved metabolic syndrome, reduced mitochondrial reactive oxygen species and miR-193b levels, restored NFYA/sGC activity, and prevented EIPH., Conclusions: In heart failure with preserved ejection fraction and CpcPH models, metabolic syndrome contributes to pulmonary vascular dysfunction and EIPH through enhanced reactive oxygen species and miR-193b expression, which downregulates NFYA-dependent sGCβ1 expression. Adeno-associated virus-mediated NFYA overexpression and SGLT2 inhibition restore NFYA-sGCβ1-cGMP signaling and ameliorate EIPH.

Nitric oxide (NO) is a ubiquitous cell signaling molecule which mediates widespread and diverse processes in the cell. These NO dependent effects often involve activation (e.g. NO binding to the heme group of soluble guanylyl cyclase for cGMP production) or inactivation (e.g. S-nitrosation) of protein targets. We studied the effect of NO and heme-NO on the transmembrane signaling enzyme NADPH oxidase 5 (NOX5), a heme protein which produces superoxide in response to increases in intracellular calcium. We found that treatment with NO donors increases NOX5 activity through heme-dependent effects, and that this effect could be recapitulated by the addition of heme-NO. This work adds to our understanding of NOX5 regulation in the cell but also provides a framework for understanding how NO could cause widespread changes in hemeprotein activity based on different affinities for heme v. heme-NO, and helps explain the opposing roles NO plays in activation and inactivation of hemeprotein targets., (Copyright © 2021 Elsevier Inc. All rights reserved.)

NO sensitive soluble guanylyl cyclase (sGC) plays a key role in mediating physiological functions of NO. Genetic alterations of the GUCY1A3 gene, coding for the α1 subunit of sGC, are associated with several cardiovascular dysfunctions. A rare sGC variant with Cys517 → Tyr substitution in the α1subunit, has been associated with moyamoya disease and achalasia. In this report we characterize the properties of this rare sGC variant. Purified α 1 C517Yβ 1 sGC preserved only ~25% of its cGMP-forming activity and showed an elevated K m for GTP substrate. However, the mutant enzyme retained a high affinity for and robust activation by NO, similar to wild type sGC. Purified α 1 C517Yβ 1 enzyme was more sensitive to specific sGC heme oxidizers and less responsive to heme reducing agents. When expressed in COS7 cells, α 1 C517Yβ 1 sGC showed a much stronger response to cinaciguat or gemfibrozil, which targets apo-sGC or sGC with ferric heme, as compared to its NO response or the relative response of the wild type sGC. A stronger response to cinaciguat was also observed for purified α 1 C517Yβ 1 in the absence of reducing agents. In COS7 cells, αCys517β sGC was less stable than the wild type enzyme under normal conditions and exhibited accelerated degradation upon induction of cellular oxidative stress. We conclude that diminished cGMP-forming activity of this sGC variant is aggravated by its high susceptibility to oxidative stress and diminished protein stability. The combination of these deficiencies contributes to the severity of observed moyamoya and achalasia symptoms in human carriers of this rare α 1 C517Yβ 1 sGC variant., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Renal arteriolar tone depends considerably on the dilatory action of nitric oxide (NO) via activation of soluble guanylyl cyclase (sGC) and cGMP action. NO deficiency and hypoxia/reoxygenation are important pathophysiological factors in the development of acute kidney injury. It was hypothesized that the NO-sGC-cGMP system functions differently in renal afferent arterioles (AA) compared with efferent arterioles (EA) and that the sGC activator cinaciguat differentially dilates these arterioles. Experiments were performed in isolated, perfused mouse glomerular arterioles. Hypoxia (0.1% oxygen) was achieved by using a hypoxia chamber. Phosphodiesterase 5 (PDE5) and sGC subunits were considerably expressed on the mRNA level in AA. PDE5 inhibition with sildenafil, which blocks cGMP degradation, diminished the responses to ANG II bolus application in AA, but not significantly in EA. Vasodilation induced by sildenafil in ANG II-preconstricted vessels was stronger in EA than AA. Cinaciguat, an NO- and heme-independent sGC activator, dilated EA more strongly than AA after N G -nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) treatment and preconstriction with ANG II. Cinaciguat-induced dilatation of l-NAME-pretreated and ANG II-preconstricted arterioles was similar to controls without l-NAME treatment. Cinaciguat also induced dilatation in iodinated contrast medium treated AA. Furthermore, it dilated EA, but not AA, after hypoxia/reoxygenation. The results reveal an important role of the NO-sGC-cGMP system for renal dilatation and that EA have a more potent sGC activated dilatory system. Furthermore, AA seem to be more sensitive to hypoxia/reoxygenation than EA under these experimental conditions.

A subset of asthmatics develop a severe form of the disease whose etiology involves airway inflammation along with inherent drivers that remain ill-defined. To address this, we studied human airway smooth muscle cells (HASMC), whose relaxation drives airway bronchodilation and whose dysfunction contributes to airway obstruction and hypersensitivity in severe asthma. Because HASMC relaxation can be driven by the NO-soluble guanylyl cyclase (sGC)-cGMP signaling pathway, we questioned if HASMC from severe asthma donors might possess inherent defects in their sGC or in redox enzymes that support sGC function. We analyzed HASMC primary lines derived from 17 severe asthma and 16 normal donors and corresponding lung tissue samples regarding sGC activation by NO or by pharmacologic agonists, and also determined expression levels of sGC α1 and β1 subunits, supporting redox enzymes, and related proteins. We found a majority of the severe asthma donor HASMC (12/17) and lung samples primarily expressed a dysfunctional sGC that was NO-unresponsive and had low heterodimer content and high Hsp90 association. This sGC phenotype correlated with lower expression levels of the supporting redox enzymes cytochrome b5 reductase, catalase, and thioredoxin-1, and higher expression of heme oxygenases 1 and 2. Together, our work reveals that severe asthmatics are predisposed toward defective NO-sGC-cGMP signaling in their airway smooth muscle due to an inherent sGC dysfunction, which in turn is associated with inherent changes in the cell redox enzymes that impact sGC maturation and function., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α 1 - and β 1 -subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α 1 - and β 1 -subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α 1 - and β 1 -subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.

Nitric oxide (NO) binds to soluble guanylyl cyclase (GC1) and stimulates its catalytic activity to produce cGMP. Despite the key role of the NO-cGMP signaling in cardiovascular physiology, the mechanisms of GC1 activation remain ill-defined. It is believed that conserved cysteines (Cys) in GC1 modulate the enzyme's activity through thiol-redox modifications. We previously showed that GC1 activity is modulated via mixed-disulfide bond by protein disulfide isomerase and thioredoxin 1. Herein we investigated the novel concept that NO-stimulated GC1 activity is mediated by thiol/disulfide switches and aimed to map the specific Cys that are involved. First, we showed that the dithiol reducing agent Tris (2-carboxyethyl)-phosphine reduces GC1 response to NO, indicating the significance of Cys oxidation in NO activation. Second, using dibromobimane, which fluoresces when crosslinking two vicinal Cys thiols, we demonstrated decreased fluorescence in NO-stimulated GC1 compared to unstimulated conditions. This suggested that NO-stimulated GC1 contained more bound Cys, potentially disulfide bonds. Third, to identify NO-regulated Cys oxidation using mass spectrometry, we compared the redox status of all Cys identified in tryptic peptides, among which, ten were oxidized and two were reduced in NO-stimulated GC1. Fourth, we resorted to computational modeling to narrow down the Cys candidates potentially involved in disulfide bond and identified Cys489 and Cys571. Fifth, our mutational studies showed that Cys489 and Cys571 were involved in GC1'response to NO, potentially as a thiol/disulfide switch. These findings imply that specific GC1 Cys sensitivity to redox environment is critical for NO signaling in cardiovascular physiology., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: To observe the effect of electroacupuncture (EA) on the expression of soluble guanylate cyclase (sGC), cyclic guanosine phosphate (cGMP) and protein kinase G (PKG) of cerebral vascular smooth muscle in cerebral infarction (CI) rats, so as to study its dynamic regulation mechanism., Methods: Male Wistar rats were randomly divided into normal control ( n =10), sham operation ( n =40), model ( n =40), and EA ( n =40) groups, and the latter three groups were further di-vided into 3, 6, 12 and 24 h subgroups ( n =10 in each subgroup). The CI model was established by occlusion of the middle cerebral artery (MCAO). EA(15 Hz, 2 mA)was applied to "Shuigou" (GV26) for 20 min. The cGMP, sGC and PKG activity and expression levels in the vascular smooth muscle of cerebral artery were detected using ELISA and Western blot, respectively., Results: After modeling, the immunoactivity and activities of sGC at 3 h, PKG at 3 and 6 h and cGMP from 3 h to 24 h were ob-viously decreased in the model group relevant to the normal control and sham-operation groups ( P <0.05, P <0.01). After the intervention, the expression levels and activities of sGC at 3 h, PKG at 3 and 6 h and cGMP at 3 and 6 h were apparently up-regulated in the EA group relevant to the model group ( P <0.05)., Conclusion: EA intervention can significantly inhibit the down-regulation of sGC, PKG and cGMP expression of cerebral artery smooth muscle in MCAO model rats, which plays an important role in inhibiting cerebral artery smooth muscle spasm after ischemia, maintaining normal vascular function and state, and thus increasing blood perfusion around cerebral infarction area. However, acupuncture effect has a certain time-effectiveness.

Soluble guanylyl cyclase (sGC), the major receptor for nitric oxide (NO), is a heterodimer consisting of two subunits, the α and the β subunit. The NO/sGC/cGMP signaling pathway is protective in different disease pathomechanisms including angina pectoris, pulmonary hypertension and fibrotic diseases. The natural ligand heme has two carboxylic acids which interact in the β 1 heme nitric oxide oxygen binding (HNOX) domain with the amino acids of the highly conserved Y-x-S-x-R motif. The Y-x-S-x-R motif is also involved in binding of the dicarboxylic activators cinaciguat and BAY 60-2770 as indicated by crystallization studies of sGC activator and bacterial HNOX homologs. To what extent the Y-x-S-x-R motif hydrogen bond network contributes to binding of monocarboxylic acids has not been examined so far. In the current paper, the chemical structural formula of the novel monocarboxylic drug BAY-543 is described for the first time. Using this novel drug, we evaluate the importance of the amino acids Y135 and R139 for thermostabilization and activation in comparison to the dicarboxylic acid BAY 60-2770. Measurements with point mutated sGC variants demonstrate tyrosine 135 as exclusive binding site of the monocarboxylic acid BAY-543 but not the dicarboxylic BAY 60-2770., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Previous genome-wide association studies have found two single nucleotide polymorphisms (SNP) rs7692387 and rs1842896 located on or near the GUCY1A3 gene were associated with coronary artery disease (CAD). GUCY1A3 was considered to be involved in the process of atherosclerosis, but there was little information about the association between genotypic polymorphisms of the GUCY1A3 and large artery atherosclerotic (LAA) stroke. This study aimed to investigate the associations between the GUCY1A3 rs7692387, rs1842896 polymorphisms and LAA stroke susceptibility., Methods: A total of 298 LAA stroke patients and 300 control subjects from a southern Chinese Han population were included. SNaPshot technique was used for genotype analysis. Associations between genotypes and LAA stroke susceptibility were analyzed with logistic regression model., Results: Our study found that under the recessive model (TT vs. GT + GG), the GUCY1A3 rs1842896 polymorphism was significantly correlated with LAA stroke (OR = 1.48, 95%CI: 1.07-2.04, P = 0.018). After adjustment for its effects on age, gender, cigarette smoking, total cholesterol, low-density lipoprotein cholesterol, HbA1c, hypertension, diabetes mellitus, and CAD, the rs1842896 TT genotype retained association with increased susceptibility to LAA stroke (recessive model: adjusted OR = 1.96, 95%CI: 1.22-3.17, P = 0.006). However, association between rs7692387 polymorphism with LAA stroke was not observed., Conclusion: Our results indicate that the GUCY1A3 rs1842896 polymorphism is an LAA stroke risk factor in Southern Han Chinese.

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) and a central component of the NO-cGMP pathway, critical to cardiovascular function. NO binding to the N-terminal sensor domain in sGC enhances the cyclase activity of the C-terminal catalytic domain. Our understanding of the structural elements regulating this signaling cascade is limited, hindering structure-based drug design efforts that target sGC to improve the management of cardiovascular diseases. Conformational changes are thought to propagate the NO-binding signal throughout the entire sGC heterodimer, via its coiled-coil domain, to reorient the catalytic domain into an active conformation. To identify the structural elements involved in this signal transduction cascade, here we optimized a cGMP-based luciferase assay that reports on heterologous sGC activity in Escherichia coli and identified several mutations that activate sGC. These mutations resided in the dorsal flaps, dimer interface, and GTP-binding regions of the catalytic domain. Combinations of mutations from these different elements synergized, resulting in even greater activity and indicating a complex cross-talk among these regions. Molecular dynamics simulations further revealed conformational changes underlying the functional impact of these mutations. We propose that the interfacial residues play a central role in the sGC activation mechanism by coupling the coiled-coil domain to the active site via a series of hot spots. Our results provide new mechanistic insights not only into the molecular pathway for sGC activation but also for other members of the larger nucleotidyl cyclase family., (© 2019 Childers et al.)

Aims: Efficacy of aspirin in primary prevention of cardiovascular disease (CVD) may be influenced by a common allele in guanylate cyclase GUCY1A3, which has been shown to modify platelet function and increase CVD risk., Methods and Results: We investigated whether homozygotes of the GUCY1A3 rs7692387 risk (G) allele benefited from aspirin in two long-term, randomized placebo-controlled trials of aspirin in primary CVD prevention: the Women's Genome Health Study (WGHS, N = 23 294) and a myocardial infarction (MI, N = 550) and stroke (N = 382) case-control set from the Physician's Health Study (PHS, N = 22 071). Bleeding risk was evaluated in the WGHS. In the placebo group of the WGHS, the GUCY1A3 risk (G) allele was confirmed to increase CVD risk [hazard ratio 1.38; 95% confidence interval (CI) 1.08-1.78; P = 0.01]. Random-effects meta-analysis of the WGHS and PHS revealed that aspirin reduced CVD events among risk allele homozygotes [G/G: odds ratio (OR) 0.79; 95% CI 0.65-0.97; P = 0.03] but increased CVD events among non-risk allele carriers (e.g. G/A: OR 1.39; 95% CI 1.03-1.87; P = 0.03) thus implying an interaction between genotype stratum and aspirin intake (Pinteraction = 0.01). Bleeding associated with aspirin increased in all genotype groups, with higher risks in heterozygotes., Conclusion: In two randomized placebo-controlled trials in the setting of primary prevention, aspirin reduced the incidence of CVD events in individuals homozygous for the GUCY1A3 risk (G) allele, whereas heterozygote individuals had more events when taking aspirin., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme constituted by two subunits, α1 and β1. Previously we have shown that 17β-estradiol (E2) exerts opposite effects on these subunits by increasing α1 and decreasing both β1 expression and enzymatic activity. To date, the physiological relevance of E2-induced sGC subunits' imbalance has not been addressed. Also, increased levels strongly correlate with E2-induced proliferation in E2-dependent tissues. The aim of the present study was to investigate the role of sGCα1 in proliferation, survival, and migration in two E2-responsive and non-responsive tumour cell lines. Here we showed that E2 stimulated sGCα1 expression in ECC-1 endometrial cancer cells. sGCα1 knock-down significantly reduced E2-dependent cell proliferation. Moreover, sGCα1 silencing caused G1 arrest together with an increase in cell death and dramatically inhibited cell migration. Surprisingly, disruption of sGCα1 expression caused a similar effect even in absence of E2. Confirming this effect, sGCα1 knock-down also augmented cell death and decreased proliferation and migration in E2-unresponsive HeLa cervical cancer cells. Our results show that sGCα1 mediated cell proliferation, survival, and migration in ECC-1 and HeLa cells and suggest that sGCα1 can not only mediate E2-tumour promoting effects but can also be involved in hormone-independent tumour progression.

Soluble guanylate cyclase (sGC) is the primary sensor of nitric oxide. It has a central role in nitric oxide signalling and has been implicated in many essential physiological processes and disease conditions. The binding of nitric oxide boosts the enzymatic activity of sGC. However, the mechanism by which nitric oxide activates the enzyme is unclear. Here we report the cryo-electron microscopy structures of the human sGCα1β1 heterodimer in different functional states. These structures revealed that the transducer module bridges the nitric oxide sensor module and the catalytic module. Binding of nitric oxide to the β1 haem-nitric oxide and oxygen binding (H-NOX) domain triggers the structural rearrangement of the sensor module and a conformational switch of the transducer module from bending to straightening. The resulting movement of the N termini of the catalytic domains drives structural changes within the catalytic module, which in turn boost the enzymatic activity of sGC.

Cardiovascular complications contribute to the major mortality and morbidity in type 2 diabetes. Diabetic cardiomyopathy (DCM) is increasingly recognized as an important cause of heart failure. EMPA-REG OUTCOME trial has reported that empagliflozin, the sodium-glucose cotransporter 2 inhibitor, exerts cardiovascular benefits on diabetic population. However, the mechanism by which empagliflozin alleviates DCM still remains unclear. In the current study, we investigated the cardiac protective effects of empagliflozin on spontaneous type 2 diabetic db/db mice and its potential mechanism. Eight weeks of empagliflozin treatment (10 mg/kg/day) decreased body weight and blood glucose level, and increased urinary glucose excretion (UGE) in diabetic mice. Echocardiography revealed that both systolic and diastolic functions of db/db mice were also obviously improved by empagliflozin. Furthermore, empagliflozin-treated diabetic mice presented with amelioration of cardiac hypertrophy and fibrosis. In addition, diabetic hearts exhibited the deterioration of oxidative stress, apoptosis and pyroptosis, while these effects were significantly counteracted after empagliflozin treatment. Moreover, empagliflozin rescued diabetes-induced suppression of sGC (soluble guanylate cyclase enzyme)-cGMP (cyclic guanosine monophosphate)-PKG (cGMP-dependent protein kinase) pathway. However, when sGC-β expression of hearts was inhibited by transvascular delivery of small interfering RNA, cardiac dysfunction was aggravated and the advantages of empagliflozin were reversed through inhibiting sGC-cGMP-PKG pathway. Collectively, these findings indicate that empagliflozin improves cardiac function involving the inhibition of oxidative stress-induced injury via sGC-cGMP-PKG pathway and may be a promising therapeutic option for DCM., (© 2019 The Author(s).)

Aim: A common genetic variant at the GUCY1A3 coronary artery disease locus has been shown to influence platelet aggregation. The risk of ischaemic events including stent thrombosis varies with the efficacy of aspirin to inhibit platelet reactivity. This study sought to investigate whether homozygous GUCY1A3 (rs7692387) risk allele carriers display higher on-aspirin platelet reactivity and risk of ischaemic events early after coronary intervention., Methods and Results: The association of GUCY1A3 genotype and on-aspirin platelet reactivity was analysed in the genetics substudy of the ISAR-ASPI registry (n = 1678) using impedance aggregometry. The clinical outcome cardiovascular death or stent thrombosis within 30 days after stenting was investigated in a meta-analysis of substudies of the ISAR-ASPI registry, the PLATO trial (n = 3236), and the Utrecht Coronary Biobank (n = 1003) comprising a total 5917 patients. Homozygous GUCY1A3 risk allele carriers (GG) displayed increased on-aspirin platelet reactivity compared with non-risk allele (AA/AG) carriers [150 (interquartile range 91-209) vs. 134 (85-194) AU⋅min, P < 0.01]. More homozygous risk allele carriers, compared with non-risk allele carriers, were assigned to the high-risk group for ischaemic events (>203 AU⋅min; 29.5 vs. 24.2%, P = 0.02). Homozygous risk allele carriers were also at higher risk for cardiovascular death or stent thrombosis (hazard ratio 1.70, 95% confidence interval 1.08-2.68; P = 0.02). Bleeding risk was not altered., Conclusion: We conclude that homozygous GUCY1A3 risk allele carriers are at increased risk of cardiovascular death or stent thrombosis within 30 days after coronary stenting, likely due to higher on-aspirin platelet reactivity. Whether GUCY1A3 genotype helps to tailor antiplatelet treatment remains to be investigated., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

In this study the nitric oxide (NO)-soluble guanylate cyclase (sGC) and phosphodiesterase-5 (PDE-5) pathway was characterized in tortoise Chelonoidis carbonaria aorta. Concentration response curves (CCR) to ATP, ADP, AMP, adenosine and histamine were performed in the presence and absence of L-NAME in aorta pre-contracted with ACh (3 μM). CCR to SNP, BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator) and tadalafil (PDE-5 inhibitor) were constructed in the presence and absence of ODQ (10 μM). ATP (pEC 50 6.1 ± 0.1), ADP (pEC 50 6.0 ± 0.2), AMP (pEC 50 6.8 ± 0.1) and histamine (pEC 50 6.8 ± 0.12) relaxed Chelonoidis aorta and the addition of L-NAME reduced their efficacy (p < .05). Adenosine effects (pEC 50 6.6 ± 0.1) were not changed in the presence of L-NAME. SNP (pEC 50 7.5 ± 0.7; Emax 102.2 ± 2.5%), BAY 41-2272 (pEC 50 7.3 ± 0.2; Emax 130.3 ± 10.2%), BAY 60-2770 (pEC 50 11.4 ± 0.1; Emax 130.3 ± 6.5%) and tadalafil (pEC 50 6.7 ± 0.3; Emax 121.3 ± 15.3%) relaxed Chelonoidis aorta. The addition of ODQ reduced the SNP and tadalafil maximum response (p < .05) and promoted 63 fold right shift on BAY 41-2272 curve. In contrast, no alteration was observed on BAY 60-2770 response. Transcriptomic analysis for eNOS and sGC were found in aorta and brain libraries with high homology when compared with human transcripts. The NO-sGC-PDE-5 is functionally present in Chelonoidis aorta with a functional and genomic similarity to mammalian vessels. Unlike most of mammalian vessels, ACh did not cause endothelium-dependent relaxation in Chelonoidis carbonaria aortic rings., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) stimulates soluble guanylyl cyclase (sGC) activity, leading to elevated intracellular cyclic guanosine 3',5'-monophosphate (cGMP) and subsequent vascular smooth muscle relaxation. It is known that downregulation of sGC expression attenuates vascular dilation and contributes to the pathogenesis of cardiovascular disease. However, it is not well understood how sGC transcription is regulated. Here, we demonstrate that pharmacological inhibition of Forkhead box subclass O (FoxO) transcription factors using the small-molecule inhibitor AS1842856 significantly blunts sGC α and β mRNA expression by more than 90%. These effects are concentration-dependent and concomitant with greater than 90% reduced expression of the known FoxO transcriptional targets, glucose-6-phosphatase and growth arrest and DNA damage protein 45 α (Gadd45 α ). Similarly, sGC α and sGC β protein expression showed a concentration-dependent downregulation. Consistent with the loss of sGC α and β mRNA and protein expression, pretreatment of vascular smooth muscle cells with the FoxO inhibitor decreased sGC activity measured by cGMP production following stimulation with an NO donor. To determine if FoxO inhibition resulted in a functional impairment in vascular relaxation, we cultured mouse thoracic aortas with the FoxO inhibitor and conducted ex vivo two-pin myography studies. Results showed that aortas have significantly blunted sodium nitroprusside-induced (NO-dependent) vasorelaxation and a 42% decrease in sGC expression after 48-hour FoxO inhibitor treatment. Taken together, these data are the first to identify that FoxO transcription factor activity is necessary for sGC expression and NO-dependent relaxation., (Copyright © 2019 by The Author(s).)

The aberrant activation of the ERG oncogenic pathway due to the TMPRSS2-ERG gene fusion is the major event that contributes to prostate cancer (PCa) development. However, the critical downstream effectors that can be therapeutically targeted remain to be identified. In this study, we have found that the expression of the α1 and β1 subunits of soluble guanylyl cyclase (sGC) was directly and specifically regulated by ERG in vitro and in vivo and was significantly associated with TMPRSS2-ERG fusion in clinical PCa cohorts. sGC is the major mediator of nitric oxide (NO)-cGMP signaling in cells that, upon NO binding, catalyzes the synthesis of cGMP and subsequently activates protein kinase G (PKG). We showed that cGMP synthesis was significantly elevated by ERG in PCa cells, leading to increased PKG activity and cell proliferation. Importantly, we also demonstrated that sGC inhibitor treatment repressed tumor growth in TMPRSS2-ERG-positive PCa xenograft models and can act in synergy with a potent AR antagonist, enzalutamide. This study strongly suggests that targeting NO-cGMP signaling pathways may be a novel therapeutic strategy to treat PCa with TMPRSS2-ERG gene fusion.

Signaling pathways that involve diatomic gases in photosynthetic organisms are not well understood. Exposure to nitric oxide or carbon monoxide is known to elicit certain responses in some photosynthetic organisms. For example, Chlamydomonas reinhardtii grown in low-iron media responds to exogenous carbon monoxide by increasing cell growth and intracellular chlorophyll levels. Here, we characterize Cyg11, a gas-responsive soluble guanylate cyclase from the eukaryotic green alga C. reinhardtii that converts GTP to cGMP. Cyg11 transcription is upregulated when C. reinhardtii is grown in iron-limited media, suggesting its importance in nutrient-limited environments. Cyg11 is purified as a homodimer and is activated by nitric oxide (2.5-fold over basal activity) and carbon monoxide (6.3-fold). The heme binding stoichiometry of Cyg11 was found to be one heme per homodimer, an unexpected result based on the sequence and oligomerization state of the enzyme. Gas binding properties, the kinetics of gas binding, and the ligand-modulated activity of Cyg11 are consistent with CO as the relevant physiological ligand.

The soluble form of guanylyl cyclase (sGC) is the main receptor for the signaling agent nitric oxide (NO), which regulates cardiomyocyte contractile function and attenuates cardiomyocyte hypertrophy. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), a regulator of vascular tone, and cardiac NO-sGC-cGMP signaling modulates cardiac stress responses, including ischemia and reperfusion (IR) injury. Here, we investigated the role of GUCY1B3 (the β subunit of sGC) in cardiomyocyte IR injury and myocardial infarction (MI) in vitro and in vivo. GUCY1B3 was upregulated in neonatal rat ventricular myocytes in response to IR injury, and GUCY1B3 overexpression restored IR-induced cell death and apoptosis. Treatment with specific inhibitors of PKCδ, PKCε, and Akt suggested that the protective effects of GUCY1B3 were mediated by PKCε/Akt signaling. In a mouse model of coronary artery ligation-induced MI, GUCY1B3 silencing aggravated MI-induced cardiac dysfunction and increased infarct size and exacerbated cardiomyocyte apoptosis in association with the inactivation of PKCε and Akt. Our results suggest that GUCY1B3 exerts cardioprotective effects through the modulation of the PKCε/Akt activity and identify a potential mechanism involved in NO-sGC-cGMP signaling in the heart., (© 2018 Wiley Periodicals, Inc.)

A large body of evidence indicates that nitric oxide (NO)/cGMP signaling essentially contributes to the processing of chronic pain. In general, NO-induced cGMP formation is catalyzed by 2 isoforms of guanylyl cyclase, NO-sensitive guanylyl cyclase 1 (NO-GC1) and 2 (NO-GC2). However, the specific functions of the 2 isoforms in pain processing remain elusive. Here, we investigated the distribution of NO-GC1 and NO-GC2 in the spinal cord and dorsal root ganglia, and we characterized the behavior of mice lacking either isoform in animal models of pain. Using immunohistochemistry and in situ hybridization, we demonstrate that both isoforms are localized to interneurons in the spinal dorsal horn with NO-GC1 being enriched in inhibitory interneurons. In dorsal root ganglia, the distribution of NO-GC1 and NO-GC2 is restricted to non-neuronal cells with NO-GC2 being the major isoform in satellite glial cells. Mice lacking NO-GC1 demonstrated reduced hypersensitivity in models of neuropathic pain, whereas their behavior in models of inflammatory pain was normal. By contrast, mice lacking NO-GC2 exhibited increased hypersensitivity in models of inflammatory pain, but their neuropathic pain behavior was unaltered. Cre-mediated deletion of NO-GC1 or NO-GC2 in spinal dorsal horn neurons recapitulated the behavioral phenotypes observed in the global knockout. Together, these results indicate that cGMP produced by NO-GC1 or NO-GC2 in spinal dorsal horn neurons exert distinct, and partly opposing, functions in chronic pain processing.

Vascular smooth muscle cells (VSMCs) play an important role in maintaining vascular function. Inflammation-mediated VSMC dysfunction leads to atherosclerotic intimal hyperplasia and preeclamptic hypertension; however, the underlying mechanisms are not clearly understood. We analyzed the expression levels of microRNA-155 (miR-155) in cultured VSMCs, mouse vessels, and clinical specimens and then assessed its role in VSMC function. Treatment with tumor necrosis factor-α (TNF-α) elevated miR-155 biogenesis in cultured VSMCs and vessel segments, which was prevented by NF-κB inhibition. MiR-155 expression was also increased in high-fat diet-fed ApoE -/- mice and in patients with atherosclerosis and preeclampsia. The miR-155 levels were inversely correlated with soluble guanylyl cyclase β1 (sGCβ1) expression and nitric oxide (NO)-dependent cGMP production through targeting the sGCβ1 transcript. TNF-α-induced miR-155 caused VSMC phenotypic switching, which was confirmed by the downregulation of VSMC-specific marker genes, suppression of cell proliferation and migration, alterations in cell morphology, and NO-induced vasorelaxation. These events were mitigated by miR-155 inhibition. Moreover, TNF-α did not cause VSMC phenotypic modulation and limit NO-induced vasodilation in aortic vessels of miR-155 -/- mice. These findings suggest that NF-κB-induced miR-155 impairs the VSMC contractile phenotype and NO-mediated vasorelaxation by downregulating sGCβ1 expression. These data suggest that NF-κB-responsive miR-155 is a novel negative regulator of VSMC functions by impairing the sGC/cGMP pathway, which is essential for maintaining the VSMC contractile phenotype and vasorelaxation, offering a new therapeutic target for the treatment of atherosclerosis and preeclampsia.

Extensive alterations in splicing is one of the molecular indicator for human cancers. Soluble guanylyl cyclase (sGC), an obligatory heterodimer, is composed of α1 and β1 subunits. Each subunit is encoded by a separate gene, GUCY1a3 and GUCY1b3, correspondingly. sGC activity has been regulated by an alternative splicing and it has an important effect on the breast cancer. sGC alternative splicing has been evaluated in the 55 malignant, 25 benign and 30 normal breast tissues using qRT-PCR and RT-PCR. The differences between groups were analyzed by Mann-Whitney U. The expression of six different splice forms have been detected, three for α1 and three for β1 sGC. Expressions of Tr1, Tr2 β1 sGC and Tr7, Tr6 α1 sGC mRNA in the malignant breast tumors were significantly lower than those of benign and normal breast tissues. However, the expression of Tr3 α1 sGC mRNA was significantly higher than that of benign and normal tissues. Present data have provided some evidences for an alteration in the expression of α1 and β1 sGC alternative splicing forms which may contribute to the loss of sGC functions in the breast cancer. The observed information might be discussed by the cGMP status., (Copyright © 2018. Published by Elsevier Inc.)

Objectives: Hypertension can impair structure and function of blood vessels. Experimental data describing the reverse remodeling process after a mechanical pressure unloading therapy in the vasculature is limited. We studied the influence of pressure unloading on both the structural and functional alterations of the aorta in a hypertensive rat model., Methods: Using isolated thoracic aortic rings in an in-vitro organ bath system, endothelium-dependent and endothelium-independent vasorelaxation were studied 6-weeks or 12-weeks after abdominal aortic banding (aortic banding-6-week or aortic banding-12-week), and 6-weeks after an aortic debanding procedure performed after the sixth experimental week of aortic banding (aortic banding + debanding-12-week). Age-matched rats were sham-operated (sham-6-week or sham-12-week). The aortic morphometry and histological fibrosis were studied, and the mRNA-expression of metalloproteinase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-2, and soluble guanylate cyclase subunits GUCY1a3 and GUCY1b3 were determined., Results: Aortic banding significantly increased systolic, diastolic, and pulse pressures. Structural changes (increased intima-media thickness and area normalized to body weight, aortic collagen content, higher MMP-2 and TIMP-2, and lower GUCY1a3 and GUCY1b3 mRNA-levels) and functional alterations (impaired endothelium-dependent and endothelium-independent vasorelaxation) have already taken place after 6 weeks of aortic banding. Pressure unloading, after established vascular changes, improved vascular function, resulted in reduced collagen content, and decreased both MMP-2 and TIMP-2 mRNA-expression., Conclusion: Pressure-overload-induced vascular changes regressed due to mechanical unloading. Furthermore, debanding leads to a reductive tendency in fibrosis-associated gene expression and collagen accumulation. Collectively, the addition of drugs that target fibrosis to existing hypertensive treatment may present an attractive therapy against vascular remodeling.

Soluble guanylyl cyclase (sGC, EC 4.6.1.2) is a key enzyme in the regulation of vascular tone. In view of the therapeutic interest of the NO/cGMP pathway, drugs were developed that either increase the NO sensitivity of the enzyme or activate heme-free apo-sGC. However, modulation of sGC activity by endogenous agents is poorly understood. In the present study we show that the maximal activity of NO-stimulated purified sGC is significantly increased by cytosolic preparations of porcine coronary arteries. Purification of the active principle by several chromatographic steps resulted in a protein mixture consisting of 100, 70, and 40 kDa bands on SDS polyacrylamide gel electrophoresis. The respective proteins were identified by LC-MS/MS as gelsolin, annexin A6, and actin, respectively. Further purification resulted in loss of activity, indicating an interaction of sGC with a protein complex rather than a single protein. The partially purified preparation had no effect on basal sGC activity or enzyme activation by the heme mimetic BAY 60-2770, suggesting a specific effect on the conformation of the NO-bound heterodimeric holoenzyme. Since the three proteins identified are all related to contractile elements of smooth muscle, our data suggest that regulation of vascular tone involves a modulatory interaction of sGC with the cytoskeleton., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Foods rich in polyphenols such as procyanidins (PC) have been proposed to have anti-inflammatory properties, and we have previously reported inhibition of lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in human dendritic cells (DCs) by PC derived from cocoa. To explore the mechanistic basis of this inhibition, here we conducted transcriptomic analysis on DCs cultured with either LPS or LPS combined with oligomeric cocoa PC. Procyanidins suppressed a number of genes encoding cytokines and chemokines such as CXCL1, but also genes involved in the cGMP pathway such as GUCY1A3 (encoding guanylate cyclase soluble subunit alpha-3). Upregulated genes were involved in diverse metabolic pathways, but notably two of the four most upregulated genes (NMB, encoding neuromedin B and ADCY3, encoding adenyl cyclase type 3) were involved in the cAMP signalling pathway. Gene-set enrichment analysis demonstrated that upregulated gene pathways were primarily involved in nutrient transport, carbohydrate metabolism and lysosome function, whereas down-regulated gene pathways involved cell cycle, signal transduction and gene transcription, as well as immune function. qPCR analysis verified differential expression of GUCY1A3, ADCY3, NMB as well as a number of other genes, and marked suppression of LPS-induced CXCL1 and IL-23 protein secretion was also observed. Thus, our results confirm a marked anti-inflammatory effect of PC in human DCs, which may be related mechanistically to second-messenger function and metabolic activity. Our results provide a foundation to further investigate metabolic pathways altered by PC during intestinal inflammation, and further encourage investigation of the health-promoting potential of PC-rich functional foods.

Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of one α and one β subunit. Each subunit consists of four domains: the N-terminal heme-nitric oxide oxygen binding (HNOX) domain, a PAS domain, a coiled-coil domain and the C-terminal catalytic domain. Upon activation by the endogenous ligand NO or activating drugs, NOsGC catalyses the conversion of GTP to cGMP. Although several crystal structures of the isolated domains are known, the structure of the full-length enzyme and the interdomain conformational changes during activation remain unsolved to date. In the current study, we performed protein thermal shift assays of purified NOsGC to identify discrete conformational states amenable to further analysis e.g. by crystallisation. A non-hydrolysable substrate analogue binding to the catalytic domain led to a subtle change in melting temperature. An activator drug binding to the HNOX domain led to a small increase. However, the combination of substrate analogue and activator drug led to a marked synergistic increase from 51 °C to 60 °C. This suggests reciprocal communication between HNOX domain and catalytic domain and formation of a stable activated conformation amenable to further biophysical characterization., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims: It has been suggested that the nitric oxide-sensitive guanylyl cyclase (NO-GC)/cyclic guanosine monophosphate (cGMP)-dependent signalling pathway affords protection against cardiac damage during acute myocardial infarction (AMI). It is, however, not clear whether the NO-GC/cGMP system confers its favourable effects through a mechanism located in cardiomyocytes (CMs). The aim of this study was to evaluate the infarct-limiting effects of the endogenous NO-GC in CMs in vivo., Methods and Results: Ischemia/reperfusion (I/R) injury was evaluated in mice with a CM-specific deletion of NO-GC (CM NO-GC KO) and in control siblings (CM NO-GC CTR) subjected to an in vivo model of AMI. Lack of CM NO-GC resulted in a mild increase in blood pressure but did not affect basal infarct sizes after I/R. Ischemic postconditioning (iPost), administration of the phosphodiesterase-5 inhibitors sildenafil and tadalafil as well as the NO-GC activator cinaciguat significantly reduced the amount of infarction in control mice but not in CM NO-GC KO littermates. Interestingly, NS11021, an opener of the large-conductance and Ca2+-activated potassium channel (BK), an important downstream effector of cGMP/cGKI in the cardiovascular system, protects I/R-exposed hearts of CM NO-GC proficient and deficient mice., Conclusions: These findings demonstrate an important role of CM NO-GC for the cardioprotective signalling following AMI in vivo. CM NO-GC function is essential for the beneficial effects on infarct size elicited by iPost and pharmacological elevation of cGMP; however, lack of CM NO-GC does not seem to disrupt the cardioprotection mediated by the BK opener NS11021.

Rain, Manjari, Himanshi Chaudhary, Ritushree Kukreti, Tashi Thinlas, Ghulam Mohammad, and Qadar Pasha. Elevated vasodilatory cyclases and shorter telomere length contribute to high-altitude pulmonary edema. High Alt Med Biol. 19:60-68, 2018., Aim: High-altitude (HA) genetics is complex with respect to health and disease (HA pulmonary edema i.e., HAPE). Based on the widely recognized fact that oxidative stress is a major trigger of several physiological processes, this study was designed to establish the significance of vasodilatory cyclases and telomere length in HA physiology. The study was performed in three groups, namely HAPE-free sojourners (HAPE-f, n = 150), HAPE patients (HAPE-p, n = 150), and healthy highland natives or highlanders (HLs, n = 150). Variations in soluble guanylyl cyclase β1-subunit (GUCY1B3) and adenylyl cyclase type 6 (ADCY6) were genotyped by the SNaPshot method and/or Fluidigm SNP type genotyping. Plasma GUCY1B3 and ADCY6 levels were estimated using ELISA, and relative telomere length was estimated by qRT-PCR., Results: The rs7638AA genotype was over-represented in HLs compared with HAPE-f and HAPE-p (p = 0.035 and p = 0.012, respectively). Similarly, the rs7638A allele was prevalent in HLs compared with both groups, but significance was attained against HAPE-p (p = 0.012). Significantly elevated plasma levels of GUCY1B3 and ADCY6 were obtained in HAPE-p compared with HAPE-f (p = 0.001 and p = 0.006, respectively) and HLs (p = 3.31E-05 and p = 0.05, respectively). Shorter telomere length was observed in HAPE-p compared with HAPE-f (p > 0.05) and HLs (p = 0.017)., Conclusion: Elevated cyclases and shorter telomere length associate with HAPE pathophysiology.

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the β1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis , Shewanella woodyi , and Clostridium botulinum , indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Nitric oxide signaling plays a key role in the regulation of vascular tone and platelet activation. Here, we seek to understand the impact of a genetic predisposition to enhanced nitric oxide signaling on risk for cardiovascular diseases, thus informing the potential utility of pharmacological stimulation of the nitric oxide pathway as a therapeutic strategy., Methods: We analyzed the association of common and rare genetic variants in 2 genes that mediate nitric oxide signaling (Nitric Oxide Synthase 3 [ NOS3 ] and Guanylate Cyclase 1, Soluble, Alpha 3 [ GUCY1A3 ]) with a range of human phenotypes. We selected 2 common variants (rs3918226 in NOS3 and rs7692387 in GUCY1A3 ) known to associate with increased NOS3 and GUCY1A3 expression and reduced mean arterial pressure, combined them into a genetic score, and standardized this exposure to a 5 mm Hg reduction in mean arterial pressure. Using individual-level data from 335 464 participants in the UK Biobank and summary association results from 7 large-scale genome-wide association studies, we examined the effect of this nitric oxide signaling score on cardiometabolic and other diseases. We also examined whether rare loss-of-function mutations in NOS3 and GUCY1A3 were associated with coronary heart disease using gene sequencing data from the Myocardial Infarction Genetics Consortium (n=27 815)., Results: A genetic predisposition to enhanced nitric oxide signaling was associated with reduced risks of coronary heart disease (odds ratio, 0.37; 95% confidence interval [CI], 0.31-0.45; P =5.5*10 -26 ], peripheral arterial disease (odds ratio 0.42; 95% CI, 0.26-0.68; P =0.0005), and stroke (odds ratio, 0.53; 95% CI, 0.37-0.76; P =0.0006). In a mediation analysis, the effect of the genetic score on decreased coronary heart disease risk extended beyond its effect on blood pressure. Conversely, rare variants that inactivate the NOS3 or GUCY1A3 genes were associated with a 23 mm Hg higher systolic blood pressure (95% CI, 12-34; P =5.6*10 -5 ) and a 3-fold higher risk of coronary heart disease (odds ratio, 3.03; 95% CI, 1.29-7.12; P =0.01)., Conclusions: A genetic predisposition to enhanced nitric oxide signaling is associated with reduced risks of coronary heart disease, peripheral arterial disease, and stroke. Pharmacological stimulation of nitric oxide signaling may prove useful in the prevention or treatment of cardiovascular disease., (© 2017 American Heart Association, Inc.)

Oxygenic phototrophs frequently encounter environmental conditions that result in intracellular energy crises. Growth of the unicellular green alga Chlamydomonas reinhardtii in hypoxia in the light depends on acclimatory responses of which the induction of photosynthetic cyclic electron flow is essential. The microalga cannot grow in the absence of molecular oxygen (O 2 ) in the dark, although it possesses an elaborate fermentation metabolism. Not much is known about how the microalga senses and signals the lack of O 2 or about its survival strategies during energy crises. Recently, nitric oxide (NO) has emerged to be required for the acclimation of C. reinhardtii to hypoxia. In this study, we show that the soluble guanylate cyclase (sGC) CYG12, a homologue of animal NO sensors, is also involved in this response. CYG12 is an active sGC, and post-transcriptional down-regulation of the CYG12 gene impairs hypoxic growth and gene expression in C. reinhardtii. However, it also results in a disturbed photosynthetic apparatus under standard growth conditions and the inability to grow heterotrophically. Transcriptome profiles indicate that the mis-expression of CYG12 results in a perturbation of responses that, in the wild-type, maintain the cellular energy budget. We suggest that CYG12 is required for the proper operation of the photosynthetic apparatus which, in turn, is essential for survival in hypoxia and darkness., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)