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Soluble guanylyl cyclase (sGC) is a well-established pharmacological target for the treatment of acute angina pectoris, pulmonary hypertension and heart failure. Histidine 105 in the heme binding pocket of sGC is a crucial residue for heme binding and natural enzyme activation by NO. It was assumed that the heme-free sGC mutants α 1 /β 1 H105F and α 1 /β 1 H105A were valuable research tools for studying NO independent sGC activators. These mutants have been used in drug screening and animal models. We confirm that the first generation of sGC activators cinaciguat and BAY 60-2770 activate the α 1 /β 1 H105F and α 1 /β 1 H105A mutants. In contrast, we show that the second generation sGC activators runcaciguat and BAY 543 only activate heme-free sGC when the β 1 H105 residue is present. By testing runcaciguat in β 1 H105F knock-in mice, we confirm this histidine-dependency in vivo. We propose a novel classification of sGC activators, distinguishing between the histidine-dependent activators runcaciguat and BAY 543 and the histidine-independent activators cinaciguat, BAY 60-2770 and BI703704. The histidine-dependency of some of the sGC activators provides a compelling rationale for a re-evaluation of previous research and drug development programs based on sGC histidine mutants. Whether the classification of sGC activators based on the activation mechanism also makes a therapeutic difference needs to be clarified in the future., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ilka Mathar and Peter Sandner are employees of Bayer AG., (Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nitric oxide (NO)-stimulated cyclic guanosine monophosphate (cGMP) is a key regulator of cardiovascular health, as NO-cGMP signalling is impaired in diseases like pulmonary hypertension, heart failure and chronic kidney disease. The development of NO-independent sGC stimulators and activators provide a novel therapeutic option to restore altered NO signalling. sGC stimulators have been already approved for the treatment of pulmonary arterial hypertension (PAH), chronic thromboembolic pulmonary hypertension (CTEPH), and chronic heart failure (HFrEF), while sGC activators are currently in phase-2 clinical trials for CKD. The best characterized effect of increased cGMP via the NO-sGC-cGMP pathway is vasodilation. However, to date, none of the sGC agonists are in development for hypertension (HTN). According to WHO, the global prevalence of uncontrolled HTN continues to rise, contributing significantly to cardiovascular mortality. While there are effective antihypertensive treatments, many patients require multiple drugs, and some remain resistant to all therapies. Thus, in addition to improved diagnosis and lifestyle changes, new pharmacological strategies remain in high demand. In this review we explore the potential of sGC stimulators and activators as novel antihypertensive agents, starting with the overview of NO-sGC-cGMP signalling, followed by potential mechanisms by which the increase in cGMP may regulate vascular tone and BP. These effects may encompass not only acute vasodilation, but also mid-term and chronic effects, such as the regulation of salt and water balance, as well as mitigation of vascular ageing and remodelling. The main section summarizes the preclinical and clinical evidence supporting the BP-lowering efficacy of sGC agonists., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Can direct activation of soluble guanylyl cyclase (sGC) provide kidney-protection? To answer this, we tested the kidney-protective effects of a sGC activator, which functions independent of nitric oxide and with oxidized sGC, in an acute kidney injury (AKI) model with transition to chronic kidney disease (CKD). We hypothesize this treatment would provide protection of kidney microvasculature, kidney blood flow, fibrosis, inflammation, and kidney damage. Assessment took place on days three, seven, 14 (acute phase) and 84 (late phase) after unilateral ischemia reperfusion injury (IRI) in rats. Post-ischemia, animals received vehicle or the sGC activator BAY 60-2770 orally. In the vehicle group, medullary microvessels narrowed and cortical microvessels showed hypertrophic inward remodeling. The mRNA levels of acute injury markers (Kim-1, Ngal) were high in the acute phase but declined in the late phase. Kidney weight decreased after the acute phase, while fibrosis started after day seven. Abundance of fibrotic (Col1a, Tgf-β1) and inflammatory markers (Il-6, Tnf-α) remained elevated throughout, along with mononuclear cell invasion, with elevated plasma cystatin C and creatinine. BAY 60-2770 treatment increased tissue cGMP concentration, dilated kidney microvasculature, and enhanced blood flow and oxygenation. This intervention significantly attenuated kidney weight loss, cell damage, fibrosis, and inflammation. Plasma cystatin C and creatinine improved significantly with sGC activator treatment indicating functional recovery, though possible GFR increase above kidney reserve in uninjured kidneys could not be excluded. In cultured human tubular cells (HK-2 cells) exposed to hypoxia or profibrotic TGF-β, BAY 60-2770 improved abundance patterns of pathologically relevant genes. Overall, our results show that sGC activation may provide effective kidney-protection and attenuate the AKI-to-CKD transition., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objective: To examine disease and target engagement biomarkers in the RISE-SSc trial of riociguat in early diffuse cutaneous systemic sclerosis and their potential to predict the response to treatment., Methods: Patients were randomized to riociguat (n = 60) or placebo (n = 61) for 52 weeks. Skin biopsies and plasma/serum samples were obtained at baseline and week 14. Plasma cyclic guanosine monophosphate (cGMP) was assessed using radio-immunoassay. α-Smooth muscle actin (αSMA) and skin thickness were determined by immunohistochemistry, mRNA markers of fibrosis by qRT-PCR in skin biopsies, and serum CXC motif chemokine ligand 4 (CXCL-4) and soluble platelet endothelial cell adhesion molecule-1 (sPECAM-1) by enzyme-linked immunosorbent assay., Results: By week 14, cGMP increased by 94 (78)% with riociguat and 10 (39)% with placebo (P < 0.001, riociguat vs placebo). Serum sPECAM-1 and CXCL-4 decreased with riociguat vs placebo (P = 0.004 and P = 0.008, respectively). There were no differences in skin collagen markers between the two groups. Higher baseline serum sPECAM-1 or the detection of αSMA-positive cells in baseline skin biopsies was associated with a larger reduction of modified Rodnan skin score from baseline at week 52 with riociguat vs placebo (interaction P-values 0.004 and 0.02, respectively)., Conclusion: Plasma cGMP increased with riociguat, suggesting engagement with the nitric oxide-soluble guanylate cyclase-cGMP pathway. Riociguat was associated with a significant reduction in sPECAM-1 (an angiogenic biomarker) vs placebo. Elevated sPECAM-1 and the presence of αSMA-positive skin cells may help to identify patients who could benefit from riociguat in terms of skin fibrosis., Trial Registration: Clinicaltrials.gov, NCT02283762., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

The discovery of soluble GC (sGC) stimulators and sGC activators provided valuable tools to elucidate NO-sGC signalling and opened novel pharmacological opportunities for cardiovascular indications and beyond. The first-in-class sGC stimulator riociguat was approved for pulmonary hypertension in 2013 and vericiguat very recently for heart failure. sGC stimulators enhance sGC activity independent of NO and also act synergistically with endogenous NO. The sGC activators specifically bind to, and activate, the oxidised haem-free form of sGC. Substantial research efforts improved on the first-generation sGC activators such as cinaciguat, culminating in the discovery of runcaciguat, currently in clinical Phase II trials for chronic kidney disease and diabetic retinopathy. Here, we highlight the discovery and development of sGC stimulators and sGC activators, their unique modes of action, their preclinical characteristics and the clinical studies. In the future, we expect to see more sGC agonists in new indications, reflecting their unique therapeutic potential., (© 2021 Bayer AG. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway plays a key role in the pathogenesis of pulmonary hypertension (PH). Targeted treatments include phosphodiesterase type 5 inhibitors (PDE5i) and sGC stimulators. The sGC stimulator riociguat is approved for the treatment of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). sGC stimulators have a dual mechanism of action, enhancing the sGC response to endogenous NO and directly stimulating sGC, independent of NO. This increase in cGMP production via a dual mechanism differs from PDE5i, which protects cGMP from degradation by PDE5, rather than increasing its production. sGC stimulators may therefore have the potential to increase cGMP levels under conditions of NO depletion that could limit the effectiveness of PDE5i. Such differences in mode of action between sGC stimulators and PDE5i could lead to differences in treatment efficacy between the classes. In addition to vascular effects, sGC stimulators have the potential to reduce inflammation, angiogenesis, fibrosis and right ventricular hypertrophy and remodelling. In this review we describe the evolution of treatments targeting the NO-sGC-cGMP pathway, with a focus on PH., Competing Interests: Conflict of interest: R.L. Benza reports receiving grants from Actelion, Bayer AG, Bellerophon Therapeutics and Eiger Biopharmaceuticals. E. Grünig reports fees for lectures and/or consultations from Actelion, Bayer AG, GlaxoSmithKline, Merck Sharp & Dohme Corp., Pfizer and United Therapeutics outside the submitted work. P. Sandner and J.-P. Stasch are employees of Bayer AG, Wuppertal, Germany. G. Simonneau reports personal fees and nonfinancial support from Actelion, Bayer AG and Merck Sharp & Dohme outside the submitted work., (Copyright ©The authors 2024.)

Vascular cognitive impairment (VCI) describes neurodegenerative disorders characterized by a vascular component. Pathologically, it involves decreased cerebral blood flow (CBF), white matter lesions, endothelial dysfunction, and blood-brain barrier (BBB) impairments. Molecularly, oxidative stress and inflammation are two of the major underlying mechanisms. Nitric oxide (NO) physiologically stimulates soluble guanylate cyclase (sGC) to induce cGMP production. However, under pathological conditions, NO seems to be at the basis of oxidative stress and inflammation, leading to a decrease in sGC activity and expression. The native form of sGC needs a ferrous heme group bound in order to be sensitive to NO (Fe(II)sGC). Oxidation of sGC leads to the conversion of ferrous to ferric heme (Fe(III)sGC) and even heme-loss (apo-sGC). Both Fe(III)sGC and apo-sGC are insensitive to NO, and the enzyme is therefore inactive. sGC activity can be enhanced either by targeting the NO-sensitive native sGC (Fe(II)sGC), or the inactive, oxidized sGC (Fe(III)sGC) and the heme-free apo-sGC. For this purpose, sGC stimulators acting on Fe(II)sGC and sGC activators acting on Fe(III)sGC/apo-sGC have been developed. These sGC agonists have shown their efficacy in cardiovascular diseases by restoring the physiological and protective functions of the NO-sGC-cGMP pathway, including the reduction of oxidative stress and inflammation, and improvement of vascular functioning. Yet, only very little research has been performed within the cerebrovascular system and VCI pathology when focusing on sGC modulation and its potential protective mechanisms on vascular and neural function. Therefore, within this review, the potential of sGC as a target for treating VCI is highlighted., Competing Interests: Declaration of Competing Interest JP has a proprietary interest in sGC agonists in the treatment of cognitive impairments. PS is a full time employee of Bayer AG, Pharmaceuticals. All other authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background and Purpose: First-generation soluble guanylate cyclase (sGC) stimulators have shown clinical benefit in pulmonary hypertension (riociguat) and chronic heart failure (vericiguat). However, given the broad therapeutic opportunities for sGC stimulators, tailored molecules for distinct indications are required., Experimental Approach: We report the high-throughput screening (HTS)-based discovery of a second generation of sGC stimulators from a novel imidazo[1,2-a]pyridine lead series. An intense medicinal chemistry programme resulted in the discovery of the sGC stimulator BAY 1165747 (BAY-747). The pharmacokinetic profile of BAY-747 was determined in different species, and it was broadly characterized in pharmacological model systems relevant for vasodilatation and hypertension., Key Results: BAY-747 is a highly potent sGC stimulator in vitro. In addition, BAY-747 showed an excellent pharmacokinetic profile with long half-life and low peak-to-trough ratio. BAY-747 was investigated in experimental in vivo models of malignant and resistant hypertension (rHT). In spontaneously hypertensive (SH) rats, BAY-747 caused a dose-related and long-lasting decrease in mean arterial blood pressure (MAP). Oral treatment over 12 days resulted in a persistent decrease. BAY-747 provided additional benefit when dosed on top of losartan, amlodipine or spironolactone and even on top of triple combinations of frequently used antihypertensive drugs. In a new canine model of rHT, BAY-747 caused a dose-related and long-lasting (>6 h) MAP decrease., Conclusion and Implications: BAY-747 is a potent, orally available sGC stimulator. BAY-747 shows long-acting pharmacodynamic effects with a very low peak-to-trough ratio. BAY-747 could be a treatment alternative for patients with hypertension, especially those not responding to standard-of-care therapy., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Chronic kidney disease (CKD) progression is associated with persisting oxidative stress, which impairs the NO-sGC-cGMP signaling cascade through the formation of oxidized and heme-free apo-sGC that cannot be activated by NO. Runcaciguat (BAY 1101042) is a novel, potent, and selective sGC activator that binds and activates oxidized and heme-free sGC and thereby restores NO-sGC-cGMP signaling under oxidative stress. Therefore, runcaciguat might represent a very effective treatment option for CKD/DKD. The potential kidney-protective effects of runcaciguat were investigated in ZSF1 rats as a model of CKD/DKD, characterized by hypertension, hyperglycemia, obesity, and insulin resistance. ZSF1 rats were treated daily orally for up to 12 weeks with runcaciguat (1, 3, 10 mg/kg/bid) or placebo. The study endpoints were proteinuria, kidney histopathology, plasma, urinary biomarkers of kidney damage, and gene expression profiling to gain information about relevant pathways affected by runcaciguat. Furthermore, oxidative stress was compared in the ZSF1 rat kidney with kidney samples from DKD patients. Within the duration of the 12-week treatment study, kidney function was significantly decreased in obese ZSF1 rats, indicated by a 20-fold increase in proteinuria, compared to lean ZSF1 rats. Runcaciguat dose-dependently and significantly attenuated the development of proteinuria in ZSF1 rats with reduced uPCR at the end of the study by -19%, -54%, and -70% at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo treatment. Additionally, average blood glucose levels measured as HbA1C, triglycerides, and cholesterol were increased by five times, twenty times, and four times, respectively, in obese ZSF1 compared to lean rats. In obese ZSF1 rats, runcaciguat reduced HbA1c levels by -8%, -34%, and -76%, triglycerides by -42%, -55%, and -71%, and cholesterol by -16%, -17%, and -34%, at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo. Concomitantly, runcaciguat also reduced kidney weights, morphological kidney damage, and urinary and plasma biomarkers of kidney damage. Beneficial effects were accompanied by changes in gene expression that indicate reduced fibrosis and inflammation and suggest improved endothelial stabilization. In summary, the sGC activator runcaciguat significantly prevented a decline in kidney function in a DKD rat model that mimics common comorbidities and conditions of oxidative stress of CKD patients. Thus, runcaciguat represents a promising treatment option for CKD patients, which is in line with recent phase 2 clinical study data, where runcaciguat showed promising efficacy in CKD patients (NCT04507061).

Increasing cGMP is a unique therapeutic principle, and drugs inhibiting cGMP-degrading enzymes or stimulating cGMP production are approved for the treatment of various diseases such as erectile dysfunction, coronary artery disease, pulmonary hypertension, chronic heart failure, irritable bowel syndrome, or achondroplasia. In addition, cGMP-increasing therapies are preclinically profiled or in clinical development for quite a broad set of additional indications, e.g., neurodegenerative diseases or different forms of dementias, bone formation disorders, underlining the pivotal role of cGMP signaling pathways. The fundamental understanding of the signaling mediated by nitric oxide-sensitive (soluble) guanylyl cyclase and membrane-associated receptor (particulate) guanylyl cyclase at the molecular and cellular levels, as well as in vivo, especially in disease models, is a key prerequisite to fully exploit treatment opportunities and potential risks that could be associated with an excessive increase in cGMP. Furthermore, human genetic data and the clinical effects of cGMP-increasing drugs allow back-translation into basic research to further learn about signaling and treatment opportunities. The biannual international cGMP conference, launched nearly 20 years ago, brings all these aspects together as an established and important forum for all topics from basic science to clinical research and pivotal clinical trials. This review summarizes the contributions to the "10th cGMP Conference on cGMP Generators, Effectors and Therapeutic Implications," which was held in Augsburg in 2022 but will also provide an overview of recent key achievements and activities in the field of cGMP research., (© 2023. The Author(s).)

Diabetes mellitus (DM) is an independent risk factor for micro- and macrovascular complications such as nephropathy and atherosclerosis respectively, which are the major causes of premature morbidity and mortality in Type 1 and Type 2 diabetic patients. Endothelial dysfunction is the critical first step of vascular disease and is characterized by reduced bioavailability of the essential endothelial vasodilator, nitric oxide (NO), coupled with an elevation in inflammation and oxidative stress. A novel pathway to bolster NO activity is to upregulate soluble guanylate cyclase (sGC), an enzyme responsible for mediating the protective actions of NO. Two classes of sGC modulators exist, activators and stimulators, with differing sensitivity to oxidative stress. In this study, we investigated the therapeutic effects of the sGC stimulator BAY 41-2272 (Bay 41) and the sGC activator BAY 60-2770 (Bay 60) on endpoints of atherosclerosis and renal disease as well as inflammation and oxidative stress in diabetic Apolipoprotein E knockout (ApoE-/-) mice. We hypothesized that under oxidative conditions known to accompany diabetes, sGC activation might be more efficacious than sGC stimulation in limiting diabetic vascular complications. We demonstrate that Bay 60 not only significantly decreased nitrotyrosine staining ( P  < 0.01) and F4/80 positive cells by 75% ( P  < 0.05), but it also significantly reduced total plaque area ( P  < 0.05) and improved endothelial function ( P  < 0.01). Our data suggest an important anti-atherogenic role for Bay 60 accompanied by reduced oxidative stress and inflammation under diabetic settings. Treatment with the stimulator Bay 41, on the other hand, had minimal effects or caused no changes with respect to cardiovascular or renal pathology. In the kidneys, treatment with Bay 60 significantly lessened urinary albuminuria, mesangial expansion and nitrotyrosine staining under diabetic conditions. In summary, our head-to-head comparator is the first preclinical study to show that a sGC activator is more efficacious than a sGC stimulator for the treatment of diabetes-associated vascular and renal complications., Competing Interests: This paper is funded by Bayer Pharmaceuticals. PS and LD who are both co-authors on the paper are employees of Bayer Pharmaceuticals and have assisted with the studies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Sharma, Choi, Sim, Dey, Mohan, Kantharidis, Dietz, Sandner and de Haan.)

Herein, we describe the identification, chemical optimization, and preclinical characterization of novel soluble guanylate cyclase (sGC) stimulators. Given the very broad therapeutic opportunities for sGC stimulators, new tailored molecules for distinct indications with specific pharmacokinetics, tissue distribution, and physicochemical properties will be required in the future. Here, we report the ultrahigh-throughput (uHTS)-based discovery of a new class of sGC stimulators from an imidazo[1,2- a ]pyridine lead series. Through the extensive and staggered optimization of the initial screening hit, liabilities such as potency, metabolic stability, permeation, and solubility could be substantially improved in parallel. These efforts resulted ultimately in the discovery of the new sGC stimulators 22 and 28 . It turned out that BAY 1165747 (BAY-747, 28 ) could be an ideal treatment alternative for patients with hypertension, especially those not responding to standard anti-hypertensive therapy (resistant hypertension). BAY-747 ( 28 ) demonstrated sustained hemodynamic effects up to 24 h in phase 1 studies.

Diabetic kidney disease (DKD) is the most common cause of renal failure. Therapeutics development is hampered by our incomplete understanding of animal models on a cellular level. We show that ZSF1 rats recapitulate human DKD on a phenotypic and transcriptomic level. Tensor decomposition prioritizes proximal tubule (PT) and stroma as phenotype-relevant cell types exhibiting a continuous lineage relationship. As DKD features endothelial dysfunction, oxidative stress, and nitric oxide depletion, soluble guanylate cyclase (sGC) is a promising DKD drug target. sGC expression is specifically enriched in PT and stroma. In ZSF1 rats, pharmacological sGC activation confers considerable benefits over stimulation and is mechanistically related to improved oxidative stress regulation, resulting in enhanced downstream cGMP effects. Finally, we define sGC gene co-expression modules, which allow stratification of human kidney samples by DKD prevalence and disease-relevant measures such as kidney function, proteinuria, and fibrosis, underscoring the relevance of the sGC pathway to patients., Competing Interests: Declaration of interests A.F., J.V., I.M., K. Siudak, F.E., P.S., M.G., and M.P. are employees of Bayer AG. M.S.B. reports consultancy: Boehringer Ingelheim; editorial board membership: Journal of the American Society of Nephrology. K. Susztak reports research support: AstraZeneca, Bayer, Boehringer Ingelheim, Calico, Genentech, Gilead, GSK, Jnana, Lilly, Maze, Merck, Novartis, Novo Nordisk, Regeneron, Variant Bio, and Ventus; advisory board membership: Jnana Therapeutics and Pfizer; consultancy: AstraZeneca, Bayer, GSK, Jnana Therapeutics, Maze, Novo Nordisk, Pfizer, and Ventus; patents: Jag1- and Notch-based targeting of chronic kidney disease; editorial board membership: Cell Metabolism, eBioMedicine, Journal of the American Society of Nephrology, Journal of Clinical Investigation, Kidney International, and Med., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a deadly disease with limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECIIs. Deficiency of CYB5R3 in AECIIs led to sustained activation of the pro-fibrotic factor TGF-β1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and the sGC/cGMP/protein kinase G axis that modulates activation of the TGF-β1 signaling pathway. We demonstrate that sGC agonists (BAY 41-8543 and BAY 54-6544) are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECIIs. Taken together, these results show that CYB5R3 in AECIIs is required to maintain resilience after lung injury and fibrosis and that therapeutic manipulation of the sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.

The complex mechanism by which stress can affect sensory processes such as hearing is still poorly understood. In a previous study, the mineralocorticoid (MR) and/or glucocorticoid receptor (GR) were deleted in frontal brain regions but not cochlear regions using a CaMKIIα-based tamoxifen-inducible Cre ERT2 /loxP approach. These mice exhibit either a diminished (MR TMX cKO) or disinhibited (GR TMX cKO) auditory nerve activity. In the present study, we observed that mice differentially were (MR TMX cKO) or were not (GR TMX cKO) able to compensate for altered auditory nerve activity in the central auditory pathway. As previous findings demonstrated a link between central auditory compensation and memory-dependent adaptation processes, we analyzed hippocampal paired-pulse facilitation (PPF) and long-term potentiation (LTP). To determine which molecular mechanisms may impact differences in synaptic plasticity, we analyzed Arc/Arg3.1, known to control AMPA receptor trafficking, as well as regulators of tissue perfusion and energy consumption (NO-GC and GC-A). We observed that the changes in PPF of MR TMX cKOs mirrored the changes in their auditory nerve activity, whereas changes in the LTP of MR TMX cKOs and GR TMX cKOs mirrored instead the changes in their central compensation capacity. Enhanced GR expression levels in MR TMX cKOs suggest that MRs typically suppress GR expression. We observed that hippocampal LTP, GC-A mRNA expression levels, and ABR wave IV/I ratio were all enhanced in animals with elevated GR (MR TMX cKOs) but were all lower or not mobilized in animals with impaired GR expression levels (GR TMX cKOs and MRGR TMX cKOs). This suggests that GC-A may link LTP and auditory neural gain through GR-dependent processes. In addition, enhanced NO-GC expression levels in MR, GR, and MRGR TMX cKOs suggest that both receptors suppress NO-GC; on the other hand, elevated Arc/Arg3.1 levels in MR TMX cKOs and MRGR TMX cKOs but not GR TMX cKOs suggest that MR suppresses Arc/Arg3.1 expression levels. Conclusively, MR through GR inhibition may define the threshold for hemodynamic responses for LTP and auditory neural gain associated with GC-A., Competing Interests: PS was employed by the company BAYER. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Calis, Hess, Marchetta, Singer, Modro, Nelissen, Prickaerts, Sandner, Lukowski, Ruth, Knipper and Rüttiger.)

Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr -/- mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. I mp or ta ntly, p ha rm ac ol ogical sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr -/- mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD., Competing Interests: Competing interests H.S. has received personal fees from MSD Sharp & Dohme, Amgen, Bayer Vital GmbH, Boehringer Ingelheim, Daiichi Sankyo, Novartis, Servier, Brahms, Bristol Myers Squibb, Medtronic, Sanofi Aventis, Synlab, Pfizer and Vifor T as well as grants and personal fees from AstraZeneca that are unrelated to the submitted work. H.S. and T.K. are named inventors on a patent application for the prevention of restenosis after angioplasty and stent implantation (patent applicants: Klinikum rechts der Isar, German Heart Centre Munich; inventors: T. Kessler, A. Kastrati, H. Schunkert; application no. PCT/EP2021/053116; status: pending), which is unrelated to the submitted work. T.K. received lecture fees from Bayer HealthCare Pharmaceuticals. L.D., F.W. and P.S. are full-time employees of Bayer HealthCare Pharmaceuticals. The other authors declare no competing interests.

Background: Oxidative stress associated with severe cardiopulmonary diseases leads to impairment in the nitric oxide/soluble guanylate cyclase signaling pathway, shifting native soluble guanylate cyclase toward heme-free apo-soluble guanylate cyclase. Here we describe a new inhaled soluble guanylate cyclase activator to target apo-soluble guanylate cyclase and outline its therapeutic potential., Methods: We aimed to generate a novel soluble guanylate cyclase activator, specifically designed for local inhaled application in the lung. We report the discovery and in vitro and in vivo characterization of the soluble guanylate cyclase activator mosliciguat (BAY 1237592)., Results: Mosliciguat specifically activates apo-soluble guanylate cyclase leading to improved cardiopulmonary circulation. Lung-selective effects, e.g., reduced pulmonary artery pressure without reduced systemic artery pressure, were seen after inhaled but not after intravenous administration in a thromboxane-induced pulmonary hypertension minipig model. These effects were observed over a broad dose range with a long duration of action and were further enhanced under experimental oxidative stress conditions. In a unilateral broncho-occlusion minipig model, inhaled mosliciguat decreased pulmonary arterial pressure without ventilation/perfusion mismatch. With respect to airway resistance, mosliciguat showed additional beneficial bronchodilatory effects in an acetylcholine-induced rat model., Conclusion: Inhaled mosliciguat may overcome treatment limitations in patients with pulmonary hypertension by improving pulmonary circulation and airway resistance without systemic exposure or ventilation/perfusion mismatch. Mosliciguat has the potential to become a new therapeutic paradigm, exhibiting a unique mode of action and route of application, and is currently under clinical development in phase Ib for pulmonary hypertension., (© 2022. The Author(s).)

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.)

Background and Purpose: Chronic pelvic pain syndrome (CPPS) is a common and bothersome condition for which no pharmacological treatment options with acceptable efficacy exist. The aim of this study was to investigate the effects of the soluble guanylate cyclase (sGC) activator BAY 60-2770 and the COX-2 inhibitor celecoxib on bladder function in a rat model of CPPS., Experimental Approach: Forty-eight male Sprague-Dawley rats were intraprostatically injected with either saline, serving as control, or zymosan, to induce prostatitis. On days 8-20, the rats were treated with either dimethylsulphoxide (DMSO; vehicle), celecoxib, BAY 60-2770 or a combination of celecoxib and BAY 60-2770. Thereafter, micturition parameters were assessed in a metabolic cage and urine samples were collected. The following day, cystometry was performed. Subsequently, the urinary bladder and prostate were removed and examined histopathologically., Key Results: Induction of prostatitis led to a significant increase of micturition frequency and corresponding decrease of volume per micturition. These alterations were ameliorated by celecoxib, and completely normalized by BAY 60-2770. Induction of prostatitis led to a significantly increased number of non-voiding contractions, decreased bladder compliance and increased voiding time. These parameters were normalized by treatment with BAY 60-2770, either alone or in combination with celecoxib. The immunohistochemical analysis showed signs of prostate inflammation, but not bladder inflammation., Conclusion and Implications: Induction of prostatitis led to significant impairment in bladder function. These alterations could be prevented by BAY 60-2770, alone or in combination with celecoxib. This is the first study to show that sGC activators could be a promising option for the treatment of CPPS., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Purpose: Generation of cGMP via NO-sensitive soluble guanylyl cyclase (sGC) has been implicated in the regulation of renal functions. Chronic kidney disease (CKD) is associated with decreased NO bioavailability, increased oxidative stress and oxidation of sGC to its haem-free form, apo-sGC. Apo-sGC cannot be activated by NO, resulting in impaired cGMP signalling that is associated with chronic kidney disease progression. We hypothesised that sGC activators, which activate apo-sGC independently of NO, increase renal cGMP production under conditions of oxidative stress, thereby improving renal blood flow (RBF) and kidney function., Experimental Approach: Two novel sGC activators, runcaciguat and BAY-543, were tested on murine kidney. We measured cGMP levels in real time in kidney slices of cGMP sensor mice, vasodilation of pre-constricted glomerular arterioles and RBF in isolated perfused kidneys. Experiments were performed at baseline conditions, under L-NAME-induced NO deficiency, and in the presence of oxidative stress induced by ODQ., Key Results: Mouse glomeruli showed NO-induced cGMP increases. Under baseline conditions, sGC activator did not alter glomerular cGMP concentration or NO-induced cGMP generation. In the presence of ODQ, NO-induced glomerular cGMP signals were markedly reduced, whereas sGC activator induced strong cGMP increases. L-NAME and ODQ pretreated isolated glomerular arterioles were strongly dilated by sGC activator. sGC activator also increased cGMP and RBF in ODQ-perfused kidneys., Conclusion and Implication: sGC activators increase glomerular cGMP, dilate glomerular arterioles and improve RBF under disease-relevant oxidative stress conditions. Therefore, sGC activators represent a promising class of drugs for chronic kidney disease treatment., Linked Articles: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Micro-RNAs (miRNAs) are regulators of gene expression and play an important role in physiological homeostasis and disease. In biofluids, miRNAs can be found in protein complexes or in extracellular vesicles (EVs). Altered urinary miRNAs are reported as potential biomarkers for chronic kidney disease (CKD). In this context, we compared established urinary protein biomarkers for kidney injury with urinary miRNA profiles in obese ZSF1 and hypertensive renin transgenic rats. Additionally, the benefit of urinary EV enrichment was investigated in vivo and the potential association of urinary miRNAs with renal fibrosis in vitro. Kidney damage in both rat models was confirmed by histopathology, proteinuria, and increased levels of urinary protein biomarkers. In total, 290 miRNAs were elevated in obese ZSF1 rats compared with lean controls, whereas 38 miRNAs were altered in obese ZSF1 rats during 14-26 weeks of age. These 38 miRNAs correlated better with disease progression than established urinary protein biomarkers. MiRNAs increased in obese ZSF1 rats were associated with renal inflammation, fibrosis, and glomerular injury. Eight miRNAs were also changed in urinary EVs of renin transgenic rats, including one which might play a role in endothelial dysfunction. EV enrichment increased the number and detection level of several miRNAs implicated in renal fibrosis in vitro and in vivo. Our results show the benefit of EV enrichment for miRNA detection and the potential of total urine and urinary EV-associated miRNAs as biomarkers of altered kidney physiology, renal fibrosis and glomerular injury, and disease progression in hypertension and obesity-induced CKD., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Benign prostatic hyperplasia (BPH) is a feature of ageing males. Up to half demonstrate bladder outlet obstruction (BOO) with associated lower urinary tract symptoms (LUTS) including bladder overactivity. Current therapies to reduce obstruction, such as α1-adrenoceptor antagonists and 5α-reductase inhibitors, are not effective in all patients. The phosphodiesterase-5 inhibitor (PDE5I) tadalafil is also approved to treat BPH and LUTS, suggesting a role for nitric oxide (NO • ), soluble guanylate cyclase (sGC), and cGMP signalling pathways. However, PDE5I refractoriness can develop for reasons including nitrergic nerve damage and decreased NO • production, or inflammation-related oxidation of the sGC haem group, normally maintained in a reduced state by the cofactor cytochrome-b5-reductase 3 (CYB5R3). sGC activators, such as cinaciguat (BAY 58-2667), have been developed to enhance sGC activity in the absence of NO • or when sGC is oxidised. Accordingly, their effects on the prostate and LUT function of aged mice were evaluated. Aged mice (≥24 months) demonstrated a functional BPH/BOO phenotype, compared with adult animals (2-12 months), with low, delayed voiding responses and elevated intravesical pressures as measured by telemetric cystometry. This was consistent with outflow tract histological and molecular data that showed urethral constriction, increased prostate weight, greater collagen deposition, and cellular hyperplasia. All changes in aged animals were attenuated by daily oral treatment with cinaciguat for 2 weeks, without effect on serum testosterone levels. Cinaciguat had only transient (1 h) cardiovascular effects with oral gavage, suggesting a positive safety profile. The benefit of cinaciguat was suggested by its reversal of an overactive cystometric profile in CYB5R3 smooth muscle knockout mice that mirrors a profile of oxidative dysfunction where PDE5I may not be effective. Thus, the aged male mouse is a suitable model for BPH-induced BOO and cinaciguat has a demonstrated ability to reduce prostate-induced obstruction and consequent effects on bladder function. © 2021 The Pathological Society of Great Britain and Ireland., (© 2021 The Pathological Society of Great Britain and Ireland.)

Soluble guanylate cyclase (sGC) requires a heme-group bound in order to produce cGMP, a second messenger involved in memory formation, while heme-free sGC is inactive. Two compound classes can increase sGC activity: sGC stimulators acting on heme-bound sGC, and sGC activators acting on heme-free sGC. In this rodent study, we investigated the potential of the novel brain-penetrant sGC stimulator BAY-747 and sGC activator runcaciguat to enhance long-term memory and attenuate short-term memory deficits induced by the NOS-inhibitor L-NAME. Furthermore, hippocampal plasticity mechanisms were investigated. In vivo, oral administration of BAY-747 and runcaciguat to male Wistar rats enhanced memory acquisition in the object location task (OLT), while only BAY-747 reversed L-NAME induced memory impairments in the OLT. Ex vivo, both BAY-747 and runcaciguat enhanced hippocampal GluA1-containing AMPA receptor (AMPAR) trafficking in a chemical LTP model for memory acquisition using acute mouse hippocampal slices. In vivo only runcaciguat acted on the glutamatergic AMPAR system in hippocampal memory acquisition processes, while for BAY-747 the effects on the neurotrophic system were more pronounced as measured in male mice using western blot. Altogether this study shows that sGC stimulators and activators have potential as cognition enhancers, while the underlying plasticity mechanisms may determine disease-specific effectiveness., (© 2022. The Author(s).)

In heart failure with reduced ejection fraction (HFrEF), nitric oxide-soluble guanylyl cyclase (sGC) pathway dysfunction impairs skeletal muscle arteriolar vasodilation and thus capillary hemodynamics, contributing to impaired oxygen uptake (V̇O 2 ) kinetics. Targeting this pathway with sGC activators offers a new treatment approach to HFrEF. We tested the hypotheses that sGC activator administration would increase the O 2 delivery (Q̇O 2 )-to-V̇O 2 ratio in the skeletal muscle interstitial space (PO 2 is) of HFrEF rats during twitch contractions due, in part, to increases in red blood cell (RBC) flux (f RBC ), velocity (V RBC ), and capillary hematocrit (Hct cap ). HFrEF was induced in male Sprague-Dawley rats via myocardial infarction. After 3 weeks, rats were treated with 0.3 mg/kg of the sGC activator BAY 60-2770 (HFrEF + BAY; n = 11) or solvent (HFrEF; n = 9) via gavage b.i.d for 5 days prior to phosphorescence quenching (PO 2 is, in contracting muscle) and intravital microscopy (resting) measurements in the spinotrapezius muscle. Intravital microscopy revealed higher f RBC (70 ± 9 vs 25 ± 8 RBC/s), V RBC (490 ± 43 vs 226 ± 35 μm/s), Hct cap (16 ± 1 vs 10 ± 1%) and a greater number of capillaries supporting flow (91 ± 3 vs 82 ± 3%) in HFrEF + BAY vs HFrEF (all P < 0.05). Additionally, PO 2 is was especially higher during 12-34s of contractions in HFrEF + BAY vs HFrEF (P < 0.05). Our findings suggest that sGC activators improved resting Q̇O 2 via increased f RBC , V RBC , and Hct cap allowing for better Q̇O 2 -to-V̇O 2 matching during the rest-contraction transient, supporting sGC activators as a potential therapeutic to target skeletal muscle vasomotor dysfunction in HFrEF., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Vitamin D contributes to blood pressure (BP) regulation. We compared the association of BP in diabetic patients with either total vitamin D - the standard way of analyzing the vitamin D status - or free vitamin D, because only free vitamin D passes the cell membrane and interacts with the nuclear vitamin D receptor (VDR). An analytical cross-sectional study was conducted with 178 diabetic patients with impaired kidney function. Free and total vitamin D concentrations were measured in all patients. Multiple linear regression analysis considering patient age, sex, body mass index, height, smoking and drinking situation, the use of antihypertensive drugs, cholecalciferol treatment, C-reactive protein and estimated glomerular filtration rate as confounding factors were conducted to compare the association of free and total vitamin D with systolic and diastolic blood pressure (SBP and DBP). Multiple linear regression analysis revealed that neither SBP nor DBP was correlated with total vitamin D (SBP, 95% CI -0.405 ~ 0.159, p = 0.390; DBP, 95% CI -0.131 ~ 0.142, p = 0.933) (Table 2). However, the concentration of free vitamin D was independently associated with SBP (95% CI -2.691 ~ -0.210; p = 0.022) (Table 3), but not with DBP (95% CI -0.934 ~ 0.285; p = 0.293). In conclusion, free - but not total - vitamin D serum concentrations in patients with diabetes and impaired kidney function are inversely correlated with SBP. This study suggests that free vitamin D measurements might be more clinically relevant - as compared to measurements of total vitamin D - to adjust vitamin D therapy in diabetic patients with impaired kidney function.

Therapy-resistant hypertension is a serious medical problem, causing end-organ damage, stroke, and heart failure if untreated. Since the standard of care fails in resistant hypertension patients, there is still a substantial unmet medical need for effective therapies. Active stimulation of soluble guanylyl cyclase via novel soluble guanylyl cyclase stimulators might provide an effective treatment option. To test this hypothesis, we established a new experimental dog model and investigated the effects of the soluble guanylyl cyclase-stimulator BAY 41-2272. In beagle dogs, a resistant hypertension phenotype was established by combining unilateral renal wrapping with the occlusion of the renal artery in the contralateral kidney. The most frequently used antihypertensive drugs were administered orally, either alone or in combination, and their acute effect on telemetric measured blood pressure was assessed and compared with that of BAY 41-2272. The chosen disease stimulus led to a moderate and stable increase in blood pressure. Even high doses of standard-of-care antihypertensives only slightly decreased blood pressure. In contrast, the administration of the soluble guanylyl cyclase stimulator BAY 41-2272 as standalone therapy led to a dose-dependent reduction in blood pressure (-14.1 ± 1.8 mmHg). Moreover, BAY 41-2272 could also further decrease blood pressure in addition to a triple combination of standard-of-care antihypertensives (-28.6 ± 13.2 mmHg). BAY 41-2272 was highly efficient as a standalone treatment in resistant hypertension but was also effective in addition to standard-of-care treatment. These data strongly suggest that soluble guanylyl cyclase stimulators might provide an effective pharmacologic therapy for patients with resistant hypertension., (© 2021. The Author(s).)

Chronic kidney diseaQueryse (CKD) is associated with oxidative stress which can interrupt the nitric oxide (NO)/soluble guanylyl cyclase (sGC) signaling and decrease cyclic guanosine monophosphate (cGMP) production. Low cGMP concentrations can cause kidney damage and progression of CKD. The novel sGC activator runcaciguat targets the oxidized and heme-free form of sGC, restoring cGMP production under oxidative stress. The purpose of this study is to investigate if runcaciguat could provide an effective treatment for CKD. Runcaciguat was used for the treatment not only in rat CKD models with different etiologies and comorbidities, namely of hypertensive rats, the renin transgenic (RenTG) rat, and angiotensin-supplemented (ANG-SD) rat, but also in rats with diabetic and metabolic CKD, the Zucker diabetic fatty (ZDF) rat. The treatment duration was 2 to 42 weeks and runcaciguat was applied orally in doses from 1 to 10 mg/kg/bid. In these different rat CKD models, runcaciguat significantly reduced proteinuria (urinary protein to creatinine ratio; uPCR). These effects were also significant at doses which did not or only moderately decrease systemic blood pressure. Moreover, runcaciguat significantly decreased kidney injury biomarkers and attenuated morphological kidney damages. In RenTG rats, runcaciguat improved survival rates and markers of heart injury. These data demonstrate that the sGC activator runcaciguat exhibits cardio-renal protection at doses which did not reduce blood pressure and was effective in hypertensive as well as diabetic and metabolic CKD models. These data, therefore, suggest that runcaciguat, with its specific mode of action, represents an efficient treatment approach for CKD and associated CV diseases., (© 2021. The Author(s).)

Background: Riociguat is a first-in-class soluble guanylate cyclase stimulator for which preclinical data suggested improvements in cystic fibrosis transmembrane conductance regulator (CFTR) function., Methods: This international, multicenter, two-part, Phase II study of riociguat enrolled adults with cystic fibrosis (CF) homozygous for Phe508del CFTR. Part 1 was a 28-day, randomized, double-blind, placebo-controlled study in participants not receiving CFTR modulator therapy. Twenty-one participants were randomized 1:2 to placebo or oral riociguat (0.5 mg three times daily [tid] for 14 days, increased to 1.0 mg tid for the subsequent 14 days). The primary and secondary efficacy endpoints were change in sweat chloride concentration and percent predicted forced expiratory volume in 1 second (ppFEV 1 ), respectively, from baseline to Day 14 and Day 28 with riociguat compared with placebo., Results: Riociguat did not alter CFTR activity (change in sweat chloride) or lung function (change in ppFEV 1 ) at doses up to 1.0 mg tid after 28 days. The most common drug-related adverse event (AE) was headache occurring in three participants (21%); serious AEs occurred in one participant receiving riociguat (7%) and one participant receiving placebo (14%). This safety profile was consistent with the underlying disease and the known safety of riociguat for its approved indications., Conclusions: The Rio-CF study was terminated due to lack of efficacy and the changing landscape of CF therapeutic development. The current study⁠, within its limits of a small sample size, did not provide evidence that riociguat could be a valid treatment option for CF., Clinical Trial Registration Number: NCT02170025., Competing Interests: Declaration of Competing Interest N. Derichs received a speaker honorarium from Vertex Pharmaceuticals, Inc. for participation in a symposium, and served as a consultant for Vertex Pharmaceuticals, Inc. at educational activities and advisory boards. J.L. Taylor-Cousar reports grants paid to her institution from the Cystic Fibrosis Foundation; grants paid to her institution and consulting and speaking fees from Vertex Pharmaceuticals, Inc.; grants paid to her institution from Bayer; grants, consulting, and speaking fees from Celtaxys; grants paid to her institution and consulting fees from Proteostasis Therapeutics, Inc.; consulting fees from Santhera, 4DMT, AbbVie, and Polarean; and grants paid to her institution from Eloxx Pharmaceuticals. J.C. Davies reports grants from the CF Trust and other funding from Algipharma AS, Bayer AG, Boehringer Ingelheim Pharma GmbH & Co. KG, Galapagos NV, ImevaX GmbH, Nivalis Therapeutics, Inc., ProQR Therapeutics III B.V., Proteostasis Therapeutics, Inc., Raptor Pharmaceuticals, Inc., Vertex Pharmaceuticals, Inc., Enterprise, Novartis, Pulmocide, Flatley, Nivalis Therapeutics, Inc., and Teva. I. Fajac received speaker honoraria from Vertex Pharmaceuticals, Inc. for participation in symposia and served as a consultant for Novartis, Proteostasis Therapeutics, Inc., and Vertex Pharmaceuticals, Inc. E. Tullis reports grants paid to her institution from Vertex Pharmaceuticals, Inc., Proteostasis Therapeutics, Bayer AG, Boehringer Ingelheim, AbbVie, Celtaxis, Corbus, and Spyryx, and consultancy fees and honoraria from Vertex Pharmaceuticals, Proteostasis Therapeutics, Astra Zeneca and Horizon. D. Nazareth has nothing to disclose. D.G. Downey reports grants from Vertex Pharmaceuticals, Inc., Proteostasis Therapeutics, Inc., Gilead, and Chiesi; and honoraria or speaker fees from Vertex Pharmaceuticals, Inc., Proteostasis Therapeutics, Inc., and Chiesi. D. Rosenbluth has nothing to disclose. A. Malfroot has nothing to disclose. C. Saunders has nothing to disclose. R. Jensen has nothing to disclose. G.M. Solomon reports work on advisory boards for Bayer and Electromed. F. Vermeulen has nothing to disclose. A. Kaiser is an employee of Bayer AG. S. Willmann is an employee of Bayer AG. S. Saleh is an employee of Bayer AG. K. Droebner is an employee of Bayer AG. P. Sandner is an employee of Bayer AG. C.E. Bear has nothing to disclose. A. Hoffmann is an employee of Bayer AG. F. Ratjen reports other consultancy fees from Vertex Pharmaceuticals, Inc., Bayer, Talecris, CSL Behring, Roche, and Gilead; grants and consultancy from Novartis; and developed speaker bureau for Genentech outside the submitted work. S.M. Rowe reports grants and consulting fees from Arrowhead, Bayer, Celtaxsys, Galapagos/AbbVie, Novartis, Renovion, Synedgen/Synspira, andVertex Pharmaceuticals Inc.; consulting fees from Arcturus, and Cysteic Medicines; and grants from AstraZeneca, Eloxx, N30 Pharmaceuticals, PTC Therapeutics, Translate Bio, and Proteostasis Therapeutics, Inc., (Copyright © 2021. Published by Elsevier B.V.)

Vascular cognitive impairment (VCI) is characterized by impairments in cerebral blood flow (CBF), endothelial function and blood-brain barrier (BBB) integrity. These processes are all physiologically regulated by the nitric oxide (NO)-soluble guanylate cyclase (sGC)-cGMP signaling pathway. Additionally, cGMP signaling plays an important role in long-term potentiation (LTP) underlying memory formation. Therefore, targeting the NO-sGC-cGMP pathway may be a therapeutic strategy for treating VCI. Hence, in this study we investigated whether sGC stimulator vericiguat has potential as a cognitive enhancer. The effects of vericiguat on long-term memory were measured in rats using an object location task. Due to the low brain-penetrance of vericiguat found in this study, it was investigated whether in the absence of BBB limitations, vericiguat enhanced hippocampal plasticity using an ex vivo memory acquisition-like chemical LTP model. Finally, peripheral effects were measured by means of blood pressure and cerebral blood volume. Vericiguat successfully enhanced long-term memory and increased hippocampal plasticity via enhanced translocation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors to the cell membrane, while blood pressure and cerebral blood volume were unaltered. Although the memory enhancing effects in this study are likely due to peripheral effects on the cerebral microvasculature, sGC stimulation may provide a new therapeutic strategy for treating VCI, especially when BBB integrity is reduced.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder, affecting one in 3500 to 5000 boys worldwide. The NO-sGC-cGMP pathway plays an important role in skeletal muscle function, primarily by improving blood flow and oxygen supply to the muscles during exercise. In fact, PDE5 inhibitors have previously been investigated as a potential therapy for DMD, however, a large-scale Phase III clinical trial did not meet its primary endpoint. Since the efficacy of PDE5i is dependent on sufficient endogenous NO production, which might be impaired in DMD, we investigated if NO-independent sGC stimulators, could have therapeutic benefits in a mouse model of DMD. Male mdx/mTR G2 mice aged six weeks were given food supplemented with the sGC stimulator, BAY-747 (150 mg/kg of food) or food alone (untreated) ad libitum for 16 weeks. Untreated C57BL6/J mice were used as wild type (WT) controls. Assessments of the four-limb hang, grip strength, running wheel and serum creatine kinase (CK) levels showed that mdx/mTR G2 mice had significantly reduced skeletal muscle function and severe muscle damage compared to WT mice. Treatment with BAY-747 improved grip strength and running speed, and these mice also had reduced CK levels compared to untreated mdx/mTR G2 mice. We also observed increased inflammation and fibrosis in the skeletal muscle of mdx/mTR G2 mice compared to WT. While gene expression of pro-inflammatory cytokines and some pro-fibrotic markers in the skeletal muscle was reduced following BAY-747 treatment, there was no reduction in infiltration of myeloid immune cells nor collagen deposition. In conclusion, treatment with BAY-747 significantly improves several functional and pathological parameters of the skeletal muscle in mdx/mTR G2 mice. However, the effect size was moderate and therefore, more studies are needed to fully understand the potential treatment benefit of sGC stimulators in DMD.

Herein we describe the discovery, mode of action, and preclinical characterization of the soluble guanylate cyclase (sGC) activator runcaciguat. The sGC enzyme, via the formation of cyclic guanosine monophoshphate, is a key regulator of body and tissue homeostasis. sGC activators with their unique mode of action are activating the oxidized and heme-free and therefore NO-unresponsive form of sGC, which is formed under oxidative stress. The first generation of sGC activators like cinaciguat or ataciguat exhibited limitations and were discontinued. We overcame limitations of first-generation sGC activators and identified a new chemical class via high-throughput screening. The investigation of the structure-activity relationship allowed to improve potency and multiple solubility, permeability, metabolism, and drug-drug interactions parameters. This program resulted in the discovery of the oral sGC activator runcaciguat (compound 45 , BAY 1101042). Runcaciguat is currently investigated in clinical phase 2 studies for the treatment of patients with chronic kidney disease and nonproliferative diabetic retinopathy.

Introduction : In 2013, riociguat a potent and specific stimulator of the soluble guanylyl cyclase (sGC) was approved as first in class sGC stimulator which reflected a first culmination of intense research and development efforts starting in the mid 1990ies. In the meantime, it turned out that triggering cGMP production by sGC stimulators could have a broad treatment potential. In consequence, various pharmaceutical companies are still very active in identifying novel chemistry for sGC stimulators. After the first generation of sGC stimulators like riociguat or lificiguat, new compound classes with different physicochemical and kinetic profiles were identified, like the sGC stimulators vericiguat or praliciguat. Area covered : Patent literature on sGC stimulators with a focus on recent compounds of the years 2014-2019 as on claimed use and formulations of these compounds. The information was collected from publicly available data sources only (MedLine, EmBase, Chemical Abstracts, Orbit, Dolphin). Expert Opinion : With the recent advancements reported in the patent literature, sGC stimulators might be differentiated due to tissue selectivity or route of application although exhibiting the same molecular mode of action. The indication space of these compounds is potentially very broad and multiple indications in cardiovascular diseases and beyond are under investigation.

When Furchgott, Murad, and Ignarro were honored with the Nobel prize for the identification of nitric oxide (NO) in 1998, the therapeutic implications of this discovery could not be fully anticipated. This was due to the fact that available therapeutics like NO donors did not allow a constant and long-lasting cyclic guanylyl monophosphate (cGMP) stimulation and had a narrow therapeutic window. Now, 20 years later, the stimulator of soluble guanylate cyclase (sGC), riociguat, is on the market and is the only drug approved for the treatment of two forms of pulmonary hypertension (PAH/CTEPH), and a variety of other sGC stimulators and sGC activators are in preclinical and clinical development for additional indications. The discovery of sGC stimulators and sGC activators is a milestone in the field of NO/sGC/cGMP pharmacology. The sGC stimulators and sGC activators bind directly to reduced, heme-containing and oxidized, heme-free sGC, respectively, which results in an increase in cGMP production. The action of sGC stimulators at the heme-containing enzyme is independent of NO but is enhanced in the presence of NO whereas the sGC activators interact with the heme-free form of sGC. These highly innovative pharmacological principles of sGC stimulation and activation seem to have a very broad therapeutic potential. Therefore, in both academia and industry, intensive research and development efforts have been undertaken to fully exploit the therapeutic benefit of these new compound classes. Here we summarize the discovery of sGC stimulators and sGC activators and the current developments in both compound classes, including the mode of action, the chemical structures, and the genesis of the terminology and nomenclature. In addition, preclinical studies exploring multiple aspects of their in vitro, ex vivo, and in vivo pharmacology are reviewed, providing an overview of multiple potential applications. Finally, the clinical developments, investigating the treatment potential of these compounds in various diseases like heart failure, diabetic kidney disease, fibrotic diseases, and hypertension, are reported. In summary, sGC stimulators and sGC activators have a unique mode of action with a broad treatment potential in cardiovascular diseases and beyond.

Introduction: The nonsteroidal mineralocorticoid receptor (MR) antagonist finerenone and sodium-glucose cotransporter-2 (SGLT2) inhibitors have demonstrated clinical benefits in CKD patients with type 2 diabetes. Clinical data analyzing the potential value of a combination therapy are currently limited. We therefore investigated cardiorenal protection of respective mono- and combination therapy in a preclinical model of hypertension-induced end-organ damage., Methods: Cardiovascular (CV) morbidity and mortality were studied in hypertensive, N(ω)-nitro-L-arginine methyl ester-treated, renin-transgenic (mRen2)27 rats. Rats (10- to 11-week-old females, n = 13-17/group) were treated once daily orally for up to 7 weeks with placebo, finerenone (1 and 3 mg/kg), empagliflozin (3 and 10 mg/kg), or a combination of the respective low doses. Key outcome parameters included mortality, proteinuria, plasma creatinine and uric acid, blood pressure, and cardiac and renal histology., Results: Placebo-treated rats demonstrated a 50% survival rate over the course of 7 weeks. Drug treatment resulted in variable degrees of survival benefit, most prominently in the low-dose combination group with a survival benefit of 93%. Monotherapies of finerenone or empagliflozin dose-dependently reduced proteinuria, while low-dose combination revealed an early, sustained, and over-additive reduction in proteinuria. Empagliflozin induced a strong and dose-dependent increase in urinary glucose excretion which was not influenced by finerenone coadministration in the combination arm. Low-dose combination but not respective low-dose monotherapies significantly reduced plasma creatinine and plasma uric acid after 6 weeks. Treatment with finerenone and the low-dose combination significantly decreased systolic blood pressure after 5 weeks. There was a dose-dependent protection from cardiac and kidney fibrosis and vasculopathy with both agents, while low-dose combination therapy was more efficient than the respective monotherapy dosages on most cardiorenal histology parameters., Discussion/conclusions: Nonsteroidal MR antagonism by finerenone and SGLT2 inhibition by empagliflozin confer CV protection in preclinical hypertension-induced cardiorenal disease. Combination of these 2 independent modes of action at low dosages revealed efficacious reduction in important functional parameters such as proteinuria and blood pressure, plasma markers including creatinine and uric acid, cardiac and renal lesions as determined by histopathology, and mortality indicating a strong potential for combined clinical use in cardiorenal patient populations., (The Author(s). Published by S. Karger AG, Basel.)

Aims: Duchenne muscular dystrophy (DMD) is an X-linked inherited disease due to dystrophin deficiency causing skeletal and cardiac muscle dysfunction. Affected patients lose ambulation by age 12 and usually die in the second to third decades of life from cardiac and respiratory failure. Symptomatic treatment includes the use of anti-inflammatory corticosteroids, which are associated with side effects including weight gain, osteoporosis, and increased risk of cardiovascular disease. Novel treatment options include blockade of the renin-angiotensin-aldosterone system, because angiotensin as well as aldosterone contribute to persistent inflammation and fibrosis, and aldosterone blockade represents an efficacious anti-fibrotic approach in cardiac failure. Recent preclinical findings enabled successful clinical testing of a combination of steroidal mineralocorticoid receptor antagonists (MRAs) and angiotensin converting enzyme inhibitors in DMD boys. The efficacy of MRAs alone on dystrophic skeletal muscle and heart has not been investigated. Here, we tested efficacy of the novel non-steroidal MRA finerenone as a monotherapy in a preclinical DMD model., Methods and Results: The dystrophin-deficient, utrophin haploinsufficient mouse model of DMD was treated with finerenone and compared with untreated dystrophic and wild-type controls. Grip strength, electrocardiography, cardiac magnetic resonance imaging, muscle force measurements, histological quantification, and gene expression studies were performed. Finerenone treatment alone resulted in significant improvements in clinically relevant functional parameters in both skeletal muscle and heart. Normalized grip strength in rested dystrophic mice treated with finerenone (40.3 ± 1.0 mN/g) was significantly higher (P = 0.0182) compared with untreated dystrophic mice (35.2 ± 1.5 mN/g). Fatigued finerenone-treated dystrophic mice showed an even greater relative improvement (P = 0.0003) in normalized grip strength (37.5 ± 1.1 mN/g) compared with untreated mice (29.7 ± 1.1 mN/g). Finerenone treatment also led to significantly lower (P = 0.0075) susceptibility to limb muscle damage characteristic of DMD measured during a contraction-induced injury protocol. Normalized limb muscle force after five lengthening contractions resulted in retention of 71 ± 7% of baseline force in finerenone-treated compared with only 51 ± 4% in untreated dystrophic mice. Finerenone treatment also prevented significant reductions in myocardial strain rate (P = 0.0409), the earliest sign of DMD cardiomyopathy. Moreover, treatment with finerenone led to very specific cardiac gene expression changes in clock genes that might modify cardiac pathophysiology in this DMD model., Conclusions: Finerenone administered as a monotherapy is disease modifying for both skeletal muscle and heart in a preclinical DMD model. These findings support further evaluation of finerenone in DMD clinical trials., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

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.)

Aims: Our aim was to investigate the effect of nitric oxide (NO)-independent activation of soluble guanylyl cyclase (sGC) on cardiomyocyte function in a hypertensive animal model with diastolic dysfunction and in biopsies from human heart failure with preserved ejection fraction (HFpEF)., Methods: Dahl salt-sensitive (DSS) rats and control rats were fed a high-salt diet for 10 weeks and then acutely treated in vivo with the sGC activator BAY 58-2667 (cinaciguat) for 30 min. Single skinned cardiomyocyte passive stiffness (F passive ) was determined in rats and human myocardium biopsies before and after acute treatment. Titin phosphorylation, activation of the NO/sGC/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade, as well as hypertrophic pathways including NO/sGC/cGMP/PKG, PKA, calcium-calmodulin kinase II (CaMKII), extracellular signal-regulated kinase 2 (ERK2), and PKC were assessed. In addition, we explored the contribution of pro-inflammatory cytokines and oxidative stress levels to the modulation of cardiomyocyte function. Immunohistochemistry and electron microscopy were used to assess the translocation of sGC and connexin 43 proteins in the rat model before and after treatment., Results: High cardiomyocyte F passive was found in rats and human myocardial biopsies compared to control groups, which was attributed to hypophosphorylation of total titin and to deranged site-specific phosphorylation of elastic titin regions. This was accompanied by lower levels of PKG and PKA activity, along with dysregulation of hypertrophic pathway markers such as CaMKII, PKC, and ERK2. Furthermore, DSS rats and human myocardium biopsies showed higher pro-inflammatory cytokines and oxidative stress compared to controls. DSS animals benefited from treatment with the sGC activator, as F passive , titin phosphorylation, PKG and the hypertrophic pathway kinases, pro-inflammatory cytokines, and oxidative stress markers all significantly improved to the level observed in controls. Immunohistochemistry and electron microscopy revealed a translocation of sGC protein toward the intercalated disc and t-tubuli following treatment in both control and DSS samples. This translocation was confirmed by staining for the gap junction protein connexin 43 at the intercalated disk. DSS rats showed a disrupted connexin 43 pattern, and sGC activator was able to partially reduce disruption and increase expression of connexin 43. In human HFpEF biopsies, the high F passive , reduced titin phosphorylation, dysregulation of the NO-sGC-cGMP-PKG pathway and PKA activity level, and activity of kinases involved in hypertrophic pathways CaMKII, PKC, and ERK2 were all significantly improved by sGC treatment and accompanied by a reduction in pro-inflammatory cytokines and oxidative stress markers., Conclusion: Our data show that sGC activator improves cardiomyocyte function, reduces inflammation and oxidative stress, improves sGC-PKG signaling, and normalizes hypertrophic kinases, indicating that it is a potential treatment option for HFpEF patients and perhaps also for cases with increased hypertrophic signaling., (Copyright © 2020 Kolijn, Kovács, Herwig, Lódi, Sieme, Alhaj, Sandner, Papp, Reusch, Haldenwang, Falcão-Pires, Linke, Jaquet, Van Linthout, Mügge, Tschöpe and Hamdani.)

Thermal shift assay is a fluorescence dye based biochemical method to determine the melting point of a protein. It can be used to investigate the ligand-induced stabilization of proteins and helps to increase the likelihood of crystallization in biological samples. Dimeric proteins like soluble guanylyl cyclase (sGC) have specific structural and functional properties which may pose a challenge in thermal shift measurements. In this paper, thermal shift assay was used to examine ligand-induced thermostabilization of the dimeric heme-containing protein soluble guanylyl cyclase. Adjustment of the parameters buffer solution, pH, protein / dye ratio and protein amount per well yielded a one-phase melting curve of sGC with a sharp transition and high reproducibility. We found that thermal shift measurement is not affected by heme state or heme content of the enzyme preparation. We used the method to investigate the thermostabilization of sGC induced by the heme-mimetic activator drugs cinaciguat, BAY 60-2770 and BR 11257 in combination with non-hydrolyzable nucleotides. Measurements with the dicarboxylic drugs cinaciguat and BAY 60-2770 yielded steep melting curves with high amplitudes. In contrast, in the presence of the monocarboxylic sGC activator BR 11257, melting curves appear flattened in the dye-based measurements. In the present paper, we show that activity-based thermostability measurements are superior to dye-based measurements in detecting the thermostabilizing influence of sGC activator drugs., Competing Interests: Declaration of Competing Interest PS is a full-time employee of Bayer AG., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Previous research in the field of cardiovascular diseases suggests a relaxing effect of testosterone (T) on smooth muscle cells. Therefore, it was hypothesized that T could play a significant role in erection development., Aim: To investigate the relaxing effect of T and other molecules of the T signaling pathway on human corpus cavernosum (HCC) tissue., Methods: Samples of the HCC tissue were obtained from men who underwent penile prosthesis implantation (n = 33) for erectile dysfunction. Samples were used for isometric tension measurement in Ex Vivo experiments. Following standardized precontraction with phenylephrine, increasing doses of T or dihydrotestosterone were administered and blocked by NO/H 2 S synthesis inhibitors, a K ATP blocker, and flutamide (androgen receptor inhibitor)., Main Outcome Measure: The outcome was relaxation of the HCC tissue, normalized to a maximum precontraction achieved by phenylephrine., Results: A dose-dependent relaxing effect of dihydrotestosterone and T was observed with a relaxation of, respectively, 24.9% ± 23.4% (P < .0001) and 41.7% ± 19.1% (P = .01) compared with 6.8% ± 15.9% for vehicle (dimethylsulfoxide) at 300 μM. The relaxing effect of T was not countered by blocking NO synthesis, H 2 S synthesis, K ATP channels, or the androgen receptor., Clinical Implications: By understanding the underlying mechanisms of T-induced HCC relaxation, potential new therapeutic targets can be identified., Strengths & Limitations: The strength of the study is the use of fresh HCC tissues with reproducible results. The limitation is the need for supraphysiological T levels to induce the observed effect., Conclusion: Rapid androgen-induced relaxation of HCC is likely to occur via nongenomic mechanisms. Previously suggested mechanisms of action by which T modulates HCC relaxation have been excluded. Van den Broeck T, Soebadi MA, Falter A, et al. Testosterone Induces Relaxation of Human Corpus Cavernosum Tissue of Patients With Erectile Dysfunction. J Sex Med 2019; 8:114-119., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Cyclic guanosine monophosphate (cGMP) is a unique second messenger molecule formed in different cell types and tissues. cGMP targets a variety of downstream effector molecules and, thus, elicits a very broad variety of cellular effects. Its production is triggered by stimulation of either soluble guanylyl cyclase (sGC) or particulate guanylyl cyclase (pGC); both enzymes exist in different isoforms. cGMP-induced effects are regulated by endogenous receptor ligands such as nitric oxide (NO) and natriuretic peptides (NPs). Depending on the distribution of sGC and pGC and the formation of ligands, this pathway regulates not only the cardiovascular system but also the kidney, lung, liver, and brain function; in addition, the cGMP pathway is involved in the pathogenesis of fibrosis, inflammation, or neurodegeneration and may also play a role in infectious diseases such as malaria. Moreover, new pharmacological approaches are being developed which target sGC- and pGC-dependent pathways for the treatment of various diseases. Therefore, it is of key interest to understand this pathway from scratch, beginning with the molecular basis of cGMP generation, the structure and function of both guanylyl cyclases and cGMP downstream targets; research efforts also focus on the subsequent signaling cascades, their potential crosstalk, and also the translational and, ultimately, the clinical implications of cGMP modulation. This review tries to summarize the contributions to the "9th International cGMP Conference on cGMP Generators, Effectors and Therapeutic Implications" held in Mainz in 2019. Presented data will be discussed and extended also in light of recent landmark findings and ongoing activities in the field of preclinical and clinical cGMP research.

The soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate signaling pathway evokes vascular smooth muscle relaxation; whether this pathway mediates airway smooth muscle relaxation remains controversial. We posit that sGC activators are equi-effective as β-agonists in reversing contractile agonist-induced airway smooth muscle shortening. To provide clarity, we tested the efficacy of sGC stimulator and activator drugs, BAY 41-2272 and BAY 60-2270, respectively, in reversing bronchoconstriction of human small airways using human precision-cut lung slices (hPCLS). Both BAY drugs reversed carbachol-induced bronchoconstriction to a maximal degree comparable to that of formoterol. Moreover, the sGC drugs remained effective bronchodilators despite formoterol-induced desensitization of the airways. Analysis of the hPCLS after their activation by sGC or β 2 -adrenergic receptor agonist showed distinct cyclic nucleotide accumulation in the hPCLS. Collectively, these data suggest that cAMP and cyclic guanosine monophosphate pathways are equi-effective for reversing carbachol-induced bronchoconstriction in the human airway via separate and distinct second messenger pathways. This should open the door for future studies to test whether sGC-targeted drugs alone or in combination can serve as effective bronchodilators in asthma and chronic obstructive pulmonary disease.

Background and Purpose: The intracellular signalling molecule cGMP, formed by NO-sensitive GC (NO-GC), has an established function in the vascular system. Despite numerous reports about NO-induced cGMP effects in the heart, the underlying cGMP signals are poorly characterized., Experimental Approach: Therefore, we analysed cGMP signals in cardiac myocytes and fibroblasts isolated from knock-in mice expressing a FRET-based cGMP indicator., Key Results: Whereas in cardiac myocytes, none of the known NO-GC-activating substances (NO, GC activators, and GC stimulators) increased cGMP even in the presence of PDE inhibitors, they induced substantial cGMP increases in cardiac fibroblasts. As cardiac myocytes and fibroblasts are electrically connected via gap junctions, we asked whether cGMP can take the same route. Indeed, in cardiomyocytes co-cultured on cardiac fibroblasts, NO-induced cGMP signals were detectable, and two groups of unrelated gap junction inhibitors abolished these signals., Conclusion and Implication: We conclude that NO-induced cGMP formed in cardiac fibroblasts enters cardiac myocytes via gap junctions thereby turning cGMP into an intercellular signalling molecule. The findings shed new light on NO/cGMP signalling in the heart and will potentially broaden therapeutic opportunities for cardiac disease., (© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Nitric oxide regulates BP by binding the reduced heme iron (Fe2+) in soluble guanylyl cyclase (sGC) and relaxing vascular smooth muscle cells (SMCs). We previously showed that sGC heme iron reduction (Fe3+ → Fe2+) is modulated by cytochrome b5 reductase 3 (CYB5R3). However, the in vivo role of SMC CYB5R3 in BP regulation remains elusive. Here, we generated conditional smooth muscle cell-specific Cyb5r3 KO mice (SMC CYB5R3-KO) to test if SMC CYB5R3 loss affects systemic BP in normotension and hypertension via regulation of the sGC redox state. SMC CYB5R3-KO mice exhibited a 5.84-mmHg increase in BP and impaired acetylcholine-induced vasodilation in mesenteric arteries compared with controls. To drive sGC oxidation and elevate BP, we infused mice with angiotensin II. We found that SMC CYB5R3-KO mice exhibited a 14.75-mmHg BP increase, and mesenteric arteries had diminished nitric oxide-dependent vasodilation but increased responsiveness to sGC heme-independent activator BAY 58-2667 over controls. Furthermore, acute injection of BAY 58-2667 in angiotensin II-treated SMC CYB5R3-KO mice showed greater BP reduction compared with controls. Together, these data provide the first in vivo evidence to our knowledge that SMC CYB5R3 is an sGC heme reductase in resistance arteries and provides resilience against systemic hypertension development.

Pharmacological inhibition of the renin-angiotensin-aldosterone system (RAAS) is, in combination with diuretics, the first-choice treatment for hypertension, although 10-20% of patients do not respond adequately. Next to the RAAS, the nitric oxide/cGMP/protein kinase G (PKG) system is the second fundamental blood pressure regulator. Whether both systems influence each other is not well-studied. It has been shown that nitric oxide (NO) supports renin recruitment via activation of soluble guanylate cyclase (sGC) and subsequent generation of cGMP. Whether this leads to an ensuing activation of PKGs in this context is not known. PKGIα, as well as PKGII, is expressed in renin-producing cells. Hence, we analyzed whether these enzymes play a role regarding renin synthesis, secretion, or recruitment. We generated renin-cell-specific PKGI-knockout mice and either stimulated or inhibited the renin system in these mice by salt diets. To exclude the possibility that one kinase isoform can compensate the lack of the other, we also studied double-knockout animals with a conditional knockout of PKGI in juxtaglomerular cells (JG cells) and a ubiquitous knockout of PKGII. We analyzed blood pressure, renin mRNA and renal renin protein content as well as plasma renin concentration. Furthermore, we stimulated the cGMP system in these mice using BAY 41-8543, an sGC stimulator, and examined renin regulation either after acute administration or after 7 days (application once daily). We did not reveal any striking differences regarding long-term renin regulation in the studied mouse models. Yet, when we studied the acute effect of BAY 41-8543 on renin secretion in isolated perfused kidneys as well as in living animals, we found that the administration of the substance led to a significant increase in plasma renin concentration in control animals. This effect was completely abolished in double-knockout animals. However, after 7 days of once daily application, we did not detect a persistent increase in renin mRNA or protein in any studied genotype. Therefore, we conclude that in mice, cGMP and PKG are involved in the acute regulation of renin release but have no influence on long-term renin adjustment.

The soluble guanylyl cyclase (sGC) plays a key role in NO/cGMP signalling and is widely recognised to be important in different disease pathomechanisms. The discovery of sGC agonists provides a new opportunity to stimulate the NO/cGMP pathway. One class of compounds are the heme-independent sGC activators, which are thought to bind to oxidised or heme-free sGC. This enzyme is preferentially formed under disease situations accompanied by oxidative stress. Accordingly, this binding mode of sGC activators has quite some appeal for the clinical use of sGC activator drugs in diseases with high oxidative stress burden. However, none of the previous sGC activators, most of them dicarboxylic acid derivatives, has passed clinical trials to date, also because of the potent blood pressure lowering effects. In the current study, we investigate the effects of a new monocarboxylic drug BR 11257 in vitro and in vivo. Activity measurements with purified enzyme indicated gentle sGC activation for BR 11257 resembling a partial agonistic behaviour. In thermal shift measurements, we observed an unexpected difference between BR 11257 and the sGC activators from the dicarboxylic acid type. While activators from the dicarboxylic acid type had a highly thermostabilising influence on sGC, this effect was absent with BR 11257. We hypothesize that the key interaction partner for thermostabilisation is the second carboxylic acid in BAY 60-2770 which is missing in BR 11257. The absence of this thermodynamic receptor stabilisation and the partial agonism may be advantageous to overcome limitations of this class of drugs by avoiding excessive hypotension., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The NO/sGC/cGMP signaling cascade plays a pivotal role in regulation of cardiovascular, cardiopulmonary and cardiorenal diseases and impairment of this cascade results in severe pathologies. Therefore, pharmacological interventions, targeting this pathway are promising strategies for treating a variety of diseases. Nitrates, supplementing NO and, PDE5 inhibitors preventing cGMP degradation, are used for angina pectoris treatment and the treatment of pulmonary arterial hypertension (PAH), respectively. More recently, a new class of drugs which directly stimulate the sGC enzyme and trigger NO-independent cGMP production was introduced and termed sGC stimulators. In 2013, the first sGC stimulator, riociguat, was approved for the treatment of PAH and chronic thromboembolic pulmonary hypertension (CTEPH). Since cGMP targets multiple intracellular downstream targets, sGC stimulators have shown - beyond the well characterized vasodilatation - anti-fibrotic, anti-inflammatory and anti-proliferative effects. These additional modes of action might extend the therapeutic potential of this drug class substantially. This review summarizes the NO/sGC/cGMP signaling cascades, the discovery and the mode of action of sGC stimulators. Furthermore, the preclinical evidence and development of riociguat for the treatment of PAH and CTEPH is reviewed. Finally, a summary of the antifibrotic effects of sGC stimulators, especially the most recent finding for skin fibrosis are included which may indicate efficacy in fibrotic diseases like Systemic Sclerosis (SSc)., (Copyright © 2018. Published by Elsevier Inc.)

Nitric oxide (NO) signaling represents one of the major regulatory pathways for cardiovascular function. After the discovery of NO, awarded with the Nobel Prize in 1998, this signaling cascade was stepwise clarified. We now have a good understanding of NO production and NO downstream targets such as the soluble guanylyl cyclases (sGCs) which catalyze cGMP production. Based on the important role of NO-signaling in the cardiovascular system, intense research and development efforts are currently ongoing to fully exploit the therapeutic potential of cGMP increase. Recently, NO-independent stimulators of sGC (sGC stimulators) were discovered and characterized. This new compound class has a unique mode of action, directly binding to sGC and triggering cGMP production. The first sGC stimulator made available to patients is riociguat, which was approved in 2013 for the treatment of different forms of pulmonary hypertension (PH). Besides riociguat, other sGC stimulators are in clinical development, with vericiguat in phase 3 clinical development for the treatment of chronic heart failure (HF). Based on the broad impact of NO/cGMP signaling, sGC stimulators could have an even broader therapeutic potential beyond PH and HF. Within this review, the NO/sGC/cGMP/PKG/PDE-signaling cascade and the major pharmacological intervention sites are described. In addition, the discovery and mode of action of sGC stimulators and the clinical development in PH and HF is covered. Finally, the preclinical and clinical evidence and treatment approaches for sGC stimulators beyond these indications and the cardiovascular disease space, like in fibrotic diseases as in systemic sclerosis (SSc), are reviewed.

Chronic Kidney Disease (CKD) is a highly prevalent disease with a substantial medical need for new and more efficacious treatments. The Nitric Oxide (NO), soluble guanylyl cyclase (sGC), cyclic guanosine monophosphate (cGMP) signaling cascade regulates various kidney functions. cGMP directly influences renal blood flow, renin secretion, glomerular function, and tubular exchange processes. Downregulation of NO/sGC/cGMP signaling results in severe kidney pathologies such as CKD. Therefore, treatment strategies aiming to maintain or increase cGMP might have beneficial effects for the treatment of progressive kidney diseases. Within this article, we review the NO/sGC/cGMP signaling cascade and its major pharmacological intervention sites. We specifically focus on the currently known effects of cGMP on kidney function parameters. Finally, we summarize the preclinical evidence for kidney protective effects of NO-donors, PDE inhibitors, sGC stimulators, and sGC activators.