C-type natriuretic peptide is a pivotal regulator of metabolic homeostasis.

Thermogenesis and adipogenesis are tightly regulated mechanisms that maintain lipid homeostasis and energy balance; dysfunction of these critical processes underpins obesity and contributes to cardiometabolic disease. C-type natriuretic peptide (CNP) fulfills a multimodal protective role in the cardiovascular system governing local blood flow, angiogenesis, cardiac function, and immune cell reactivity. Herein, we investigated a parallel, preservative function for CNP in coordinating metabolic homeostasis. Global inducible CNP knockout mice exhibited reduced body weight, higher temperature, lower adiposity, and greater energy expenditure in vivo. This thermogenic phenotype was associated with increased expression of uncoupling protein-1 and preferential lipid utilization by mitochondria, a switch corroborated by a corresponding diminution of insulin secretion and glucose clearance. Complementary studies in isolated murine and human adipocytes revealed that CNP exerts these metabolic regulatory actions by inhibiting sympathetic thermogenic programming via Gi-coupled natriuretic peptide receptor (NPR)-C and reducing peroxisome proliferator-activated receptor-γ coactivator-1α expression, while concomitantly driving adipogenesis via NPR-B/protein kinase-G. Finally, we identified an association between CNP/NPR-C expression and obesity in patient samples. These findings establish a pivotal physiological role for CNP as a metabolic switch to balance energy homeostasis. Pharmacological targeting of these receptors may offer therapeutic utility in the metabolic syndrome and related cardiovascular disorders.

Multidrug resistance proteins preferentially regulate natriuretic peptide-driven cGMP signalling in the heart and vasculature.

BACKGROUND AND PURPOSE: cGMP underpins the bioactivity of NO and natriuretic peptides and is key to cardiovascular homeostasis. cGMP-driven responses are terminated primarily by PDEs, but cellular efflux via multidrug resistance proteins (MRPs) might contribute. Herein, the effect of pharmacological blockade of MRPs on cGMP signalling in the heart and vasculature was investigated in vitro and in vivo. EXPERIMENTAL APPROACH: Proliferation of human coronary artery smooth muscle cells (hCASMCs), vasorelaxation of murine aorta and reductions in mean arterial BP (MABP) in response to NO donors or natriuretic peptides were determined in the absence and presence of the MRP inhibitor MK571. The ability of MRP inhibition to reverse morphological and contractile deficits in a murine model of pressure overload-induced heart failure was also explored. KEY RESULTS: MK571 attenuated hCASMC growth and enhanced the anti-proliferative effects of NO and atrial natriuretic peptide (ANP). MRP blockade caused concentration-dependent relaxations of murine aorta and augmented responses to ANP (and to a lesser extent NO). MK571 did not decrease MABP per se but enhanced the hypotensive actions of ANP and improved structural and functional indices of disease severity in experimental heart failure. These beneficial actions of MRP inhibition were associated with a greater intracellular:extracellular cGMP ratio in vitro and in vivo. CONCLUSIONS AND IMPLICATIONS: MRP blockade promotes the cardiovascular functions of natriuretic peptides in vitro and in vivo, with more modest effects on NO. MRP inhibition may have therapeutic utility in cardiovascular diseases triggered by dysfunctional cGMP signalling, particularly those associated with altered natriuretic peptide bioactivity. 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.

Vascular KATP channels protect from cardiac dysfunction and preserve cardiac metabolism during endotoxemia.

KATP channels in the vasculature composed of Kir6.1 regulate vascular tone and may contribute to the pathogenesis of endotoxemia. We used mice with cell-specific deletion of Kir6.1 in smooth muscle (smKO) and endothelium (eKO) to investigate this question. We found that smKO mice had a significant survival disadvantage compared with their littermate controls when treated with a sub-lethal dose of lipopolysaccharide (LPS). All cohorts of mice became hypotensive following bacterial LPS administration; however, mean arterial pressure in WT mice recovered to normal levels, whereas smKO struggled to overcome LPS-induced hypotension. In vivo and ex vivo investigations revealed pronounced cardiac dysfunction in LPS-treated smKO, but not in eKO mice. Similar results were observed in a cecal slurry injection model. Metabolomic profiling of hearts revealed significantly reduced levels of metabolites involved in redox/energetics, TCA cycle, lipid/fatty acid and amino acid metabolism. Vascular smooth muscle-localised KATP channels have a critical role in the response to systemic infection by normalising cardiac function and haemodynamics through metabolic homeostasis. KEY MESSAGES: • Mice lacking vascular KATP channels are more susceptible to death from infection. • Absence of smooth muscle KATP channels depresses cardiac function during infection. • Cardiac dysfunction is accompanied by profound changes in cellular metabolites. • Findings from this study suggest a protective role for vascular KATP channels in response to systemic infection.

C-type natriuretic peptide co-ordinates cardiac structure and function.

AIMS: C-type natriuretic peptide (CNP) is an essential endothelium-derived signalling species that governs vascular homoeostasis; CNP is also expressed in the heart but an intrinsic role for the peptide in cardiac function is not established. Herein, we employ unique transgenic strains with cell-specific deletion of CNP to define a central (patho)physiological capacity of CNP in maintaining heart morphology and contractility. METHODS AND RESULTS: Cardiac structure and function were explored in wild type (WT), cardiomyocyte (cmCNP-/-), endothelium (ecCNP-/-), and fibroblast (fbCNP-/-)-specific CNP knockout mice, and global natriuretic peptide receptor (NPR)-B-/-, and NPR-C-/- animals at baseline and in experimental models of myocardial infarction and heart failure (HF). Endothelium-specific deletion of CNP resulted in impaired coronary responsiveness to endothelium-dependent- and flow-mediated-dilatation; changes mirrored in NPR-C-/- mice. Ex vivo, global ischaemia resulted in larger infarcts and diminished functional recovery in cmCNP-/- and NPR-C-/-, but not ecCNP-/-, vs. WT. The cardiac phenotype of cmCNP-/-, fbCNP-/-, and NPR-C-/- (but not ecCNP-/- or NPR-B-/-) mice was more severe in pressure overload- and sympathetic hyperactivation-induced HF compared with WT; these adverse effects were rescued by pharmacological CNP administration in WT, but not NPR-C-/-, mice. At a molecular level, CNP/NPR-C signalling is impaired in human HF but attenuates activation of well-validated pro-hypertrophic and pro-fibrotic pathways. CONCLUSION: C-type natriuretic peptide of cardiomyocyte, endothelial and fibroblast origins co-ordinates and preserves cardiac structure, function, and coronary vasoreactivity via activation of NPR-C. Targeting NPR-C may prove an innovative approach to treating HF and ischaemic cardiovascular disorders.

C-type Natriuretic Peptide: A Multifaceted Paracrine Regulator in the Heart and Vasculature.

C-type natriuretic peptide (CNP) is an autocrine and paracrine mediator released by endothelial cells, cardiomyocytes and fibroblasts that regulates vital physiological functions in the cardiovascular system. These roles are conveyed via two cognate receptors, natriuretic peptide receptor B (NPR-B) and natriuretic peptide receptor C (NPR-C), which activate different signalling pathways that mediate complementary yet distinct cellular responses. Traditionally, CNP has been deemed the endothelial component of the natriuretic peptide system, while its sibling peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are considered the endocrine guardians of cardiac function and blood volume. However, accumulating evidence indicates that CNP not only modulates vascular tone and blood pressure, but also governs a wide range of cardiovascular effects including the control of inflammation, angiogenesis, smooth muscle and endothelial cell proliferation, atherosclerosis, cardiomyocyte contractility, hypertrophy, fibrosis, and cardiac electrophysiology. This review will focus on the novel physiological functions ascribed to CNP, the receptors/signalling mechanisms involved in mediating its cardioprotective effects, and the development of therapeutics targeting CNP signalling pathways in different disease pathologies.

Neprilysin inhibition for pulmonary arterial hypertension: a randomized, double-blind, placebo-controlled, proof-of-concept trial.

BACKGROUND AND PURPOSE: Pulmonary arterial hypertension (PAH) is an incurable, incapacitating disorder resulting from increased pulmonary vascular resistance, pulmonary arterial remodelling, and right ventricular failure. In preclinical models, the combination of a PDE5 inhibitor (PDE5i) with a neprilysin inhibitor augments natriuretic peptide bioactivity, promotes cGMP signalling, and reverses the structural and haemodynamic deficits that characterize PAH. Herein, we conducted a randomized, double-blind, placebo-controlled trial to assess the efficacy and safety of repurposing the neprilysin inhibitor, racecadotril, in PAH. EXPERIMENTAL APPROACH: Twenty-one PAH patients stable on PDE5i therapy were recruited. Acute haemodynamic and biochemical changes following a single dose of racecadotril or matching placebo were determined; this was followed by a 14-day safety and efficacy evaluation. The primary endpoint in both steps was the maximum change in circulating atrial natriuretic peptide (ANP) concentration (Δmax ), with secondary outcomes including pulmonary and systemic haemodynamics plus mechanistic biomarkers. KEY RESULTS: Acute administration of racecadotril (100 mg) resulted in a 79% increase in the plasma ANP concentration and a 106% increase in plasma cGMP levels, with a concomitant 14% fall in pulmonary vascular resistance. Racecadotril (100 mg; t.i.d.) treatment for 14 days resulted in a 19% rise in plasma ANP concentration. Neither acute nor chronic administration of racecadotril resulted in a significant drop in mean arterial BP or any serious adverse effects. CONCLUSIONS AND IMPLICATIONS: This Phase IIa evaluation provides proof-of-principle evidence that neprilysin inhibitors may have therapeutic utility in PAH and warrants a larger scale prospective trial.

Endothelial C-Type Natriuretic Peptide Is a Critical Regulator of Angiogenesis and Vascular Remodeling.

BACKGROUND: Angiogenesis and vascular remodeling are complementary, innate responses to ischemic cardiovascular events, including peripheral artery disease and myocardial infarction, which restore tissue blood supply and oxygenation; the endothelium plays a critical function in these intrinsic protective processes. C-type natriuretic peptide (CNP) is a fundamental endothelial signaling species that coordinates vascular homeostasis. Herein, we sought to delineate a central role for CNP in angiogenesis and vascular remodeling in response to ischemia. METHODS: The in vitro angiogenic capacity of CNP was examined in pulmonary microvascular endothelial cells and aortic rings isolated from wild-type, endothelium-specific CNP-/-, global natriuretic peptide receptor (NPR)-B-/- and NPR-C-/- animals, and human umbilical vein endothelial cells. These studies were complemented by in vivo investigation of neovascularization and vascular remodeling after ischemia or vessel injury, and CNP/NPR-C expression and localization in tissue from patients with peripheral artery disease. RESULTS: Clinical vascular ischemia is associated with reduced levels of CNP and its cognate NPR-C. Moreover, genetic or pharmacological inhibition of CNP and NPR-C, but not NPR-B, reduces the angiogenic potential of pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, and isolated vessels ex vivo. Angiogenesis and remodeling are impaired in vivo in endothelium-specific CNP-/- and NPR-C-/-, but not NPR-B-/-, mice; the detrimental phenotype caused by genetic deletion of endothelial CNP, but not NPR-C, can be rescued by pharmacological administration of CNP. The proangiogenic effect of CNP/NPR-C is dependent on activation of Gi, ERK1/2, and phosphoinositide 3-kinase γ/Akt at a molecular level. CONCLUSIONS: These data define a central (patho)physiological role for CNP in angiogenesis and vascular remodeling in response to ischemia and provide the rationale for pharmacological activation of NPR-C as an innovative approach to treating peripheral artery disease and ischemic cardiovascular disorders.

Phosphodiesterase 2 inhibition preferentially promotes NO/guanylyl cyclase/cGMP signaling to reverse the development of heart failure.

Heart failure (HF) is a shared manifestation of several cardiovascular pathologies, including hypertension and myocardial infarction, and a limited repertoire of treatment modalities entails that the associated morbidity and mortality remain high. Impaired nitric oxide (NO)/guanylyl cyclase (GC)/cyclic guanosine-3',5'-monophosphate (cGMP) signaling, underpinned, in part, by up-regulation of cyclic nucleotide-hydrolyzing phosphodiesterase (PDE) isozymes, contributes to the pathogenesis of HF, and interventions targeted to enhancing cGMP have proven effective in preclinical models and patients. Numerous PDE isozymes coordinate the regulation of cardiac cGMP in the context of HF; PDE2 expression and activity are up-regulated in experimental and human HF, but a well-defined role for this isoform in pathogenesis has yet to be established, certainly in terms of cGMP signaling. Herein, using a selective pharmacological inhibitor of PDE2, BAY 60-7550, and transgenic mice lacking either NO-sensitive GC-1α (GC-1α-/-) or natriuretic peptide-responsive GC-A (GC-A-/-), we demonstrate that the blockade of PDE2 promotes cGMP signaling to offset the pathogenesis of experimental HF (induced by pressure overload or sympathetic hyperactivation), reversing the development of left ventricular hypertrophy, compromised contractility, and cardiac fibrosis. Moreover, we show that this beneficial pharmacodynamic profile is maintained in GC-A-/- mice but is absent in animals null for GC-1α or treated with a NO synthase inhibitor, revealing that PDE2 inhibition preferentially enhances NO/GC/cGMP signaling in the setting of HF to exert wide-ranging protection to preserve cardiac structure and function. These data substantiate the targeting of PDE2 in HF as a tangible approach to maximize myocardial cGMP signaling and enhancing therapy.

IκB Kinase Inhibitor Attenuates Sepsis-Induced Cardiac Dysfunction in CKD.

Patients with CKD requiring dialysis have a higher risk of sepsis and a 100-fold higher mortality rate than the general population with sepsis. The severity of cardiac dysfunction predicts mortality in patients with sepsis. Here, we investigated the effect of preexisting CKD on cardiac function in mice with sepsis and whether inhibition of IκB kinase (IKK) reduces the cardiac dysfunction in CKD sepsis. Male C57BL/6 mice underwent 5/6 nephrectomy, and 8 weeks later, they were subjected to LPS (2 mg/kg) or sepsis by cecal ligation and puncture (CLP). Compared with sham operation, nephrectomy resulted in significant increases in urea and creatinine levels, a small (P<0.05) reduction in ejection fraction (echocardiography), and increases in the cardiac levels of phosphorylated IκBα, Akt, and extracellular signal-regulated kinase 1/2; nuclear translocation of the NF-κB subunit p65; and inducible nitric oxide synthase (iNOS) expression. When subjected to LPS or CLP, compared with sham-operated controls, CKD mice exhibited exacerbation of cardiac dysfunction and lung inflammation, greater increases in levels of plasma cytokines (TNF-α, IL-1ß, IL-6, and IL-10), and greater increases in the cardiac levels of phosphorylated IKKα/ß and IκBα, nuclear translocation of p65, and iNOS expression. Treatment of CKD mice with an IKK inhibitor (IKK 16; 1 mg/kg) 1 hour after CLP or LPS administration attenuated these effects. Thus, preexisting CKD aggravates the cardiac dysfunction caused by sepsis or endotoxemia in mice; this effect may be caused by increased cardiac NF-κB activation and iNOS expression.

Functional pharmacological characterization of SER100 in cardiovascular health and disease.

BACKGROUND AND PURPOSE: SER100 is a selective nociceptin (NOP) receptor agonist with sodium-potassium-sparing aquaretic and anti-natriuretic activity. This study was designed to characterize the functional cardiovascular pharmacology of SER100 in vitro and in vivo, including experimental models of cardiovascular disease. EXPERIMENTAL APPROACH: Haemodynamic, ECG parameters and heart rate variability (HRV) were determined using radiotelemetry in healthy, conscious mice. The haemodynamic and vascular effects of SER100 were also evaluated in two models of cardiovascular disease, spontaneously hypertensive rats (SHR) and murine hypoxia-induced pulmonary hypertension (PH). To elucidate mechanisms underlying the pharmacology of SER100, acute blood pressure recordings were performed in anaesthetized mice, and the reactivity of rodent aorta and mesenteric arteries in response to electrical- and agonist-stimulation assessed. KEY RESULTS: SER100 caused NOP receptor-dependent reductions in mean arterial blood pressure and heart rate that were independent of NO. The hypotensive and vasorelaxant actions of SER100 were potentiated in SHR compared with Wistar Kyoto. Moreover, SER100 reduced several indices of disease severity in experimental PH. Analysis of HRV indicated that SER100 decreased the low/high frequency ratio, an indicator of sympatho-vagal balance, and in electrically stimulated mouse mesenteric arteries SER100 inhibited sympathetic-induced contractions. CONCLUSIONS AND IMPLICATIONS: SER100 exerts a chronic hypotensive and bradycardic effects in rodents, including models of systemic and pulmonary hypertension. SER100 produces its cardiovascular effects, at least in part, by inhibition of cardiac and vascular sympathetic activity. SER100 may represent a novel therapeutic candidate in systemic and pulmonary hypertension.

Raised arterial blood pressure in neurokinin-1 receptor-deficient mice (NK1R(-/-) ): evidence for a neural rather than a vascular mechanism.

NEW FINDINGS: What is the central question of this study? Does genetic ablation of neurokinin-1 receptors alter arterial blood pressure? What is the main finding and its importance? NK1R(-/-) mice have increased mean arterial blood pressure, but without a concomitant change in vascular reactivity. This finding suggests that neurokinin-1 receptors play a role in the neural regulation of blood pressure. Mice with functional ablation of the neurokinin-1 receptor gene, Tacr1, (NK1R(-/-) ) express behavioural abnormalities equivalent to those seen in attention deficit hyperactivity disorder (ADHD). An established model of ADHD is the spontaneously hypertensive rat, which exhibits high blood pressure owing to increased central sympathetic drive. In light of the evidence that the neurokinin-1 receptor (NK1R) also influences cardiovascular haemodynamics, we have investigated whether NK1R(-/-) mice exhibit raised blood pressure. Cardiovascular parameters were recorded for 24 h in conscious mice using radiotelemetry. Vascular function was assessed in mesenteric resistance arteries by wire myography. The NK1R(-/-) mice exhibited a higher blood pressure than wild-type animals throughout the 24 h period. Heart rate and locomotor activity in NK1R(-/-) mice were higher than in wild-type mice during the night period (active phase), consistent with an ADHD-like phenotype, but not during the day. Mesenteric and renal arteries from NK1R(-/-) mice exhibited normal vascular function; the responses to vasoconstrictors (U46619 and phenylephrine) and the endothelium-dependent vasodilator, acetylcholine, were not altered in these animals, suggesting that the NK1R does not regulate vascular tone. Analysis of heart rate variability revealed a higher low-frequency to high-frequency ratio in NK1R(-/-) mice, indicative of increased cardiac sympathetic activity. We propose that the raised blood pressure in NK1R(-/-) mice could be due to a neural mechanism rather than a change in vascular reactivity. Further studies are required to understand this mechanism and to establish whether a subgroup of ADHD patients with polymorphism of the equivalent (TACR1) gene are affected in a similar way.

Endothelial C-type natriuretic peptide maintains vascular homeostasis.

The endothelium plays a fundamental role in maintaining vascular homeostasis by releasing factors that regulate local blood flow, systemic blood pressure, and the reactivity of leukocytes and platelets. Accordingly, endothelial dysfunction underpins many cardiovascular diseases, including hypertension, myocardial infarction, and stroke. Herein, we evaluated mice with endothelial-specific deletion of Nppc, which encodes C-type natriuretic peptide (CNP), and determined that this mediator is essential for multiple aspects of vascular regulation. Specifically, disruption of CNP leads to endothelial dysfunction, hypertension, atherogenesis, and aneurysm. Moreover, we identified natriuretic peptide receptor-C (NPR-C) as the cognate receptor that primarily underlies CNP-dependent vasoprotective functions and developed small-molecule NPR-C agonists to target this pathway. Administration of NPR-C agonists promotes a vasorelaxation of isolated resistance arteries and a reduction in blood pressure in wild-type animals that is diminished in mice lacking NPR-C. This work provides a mechanistic explanation for genome-wide association studies that have linked the NPR-C (Npr3) locus with hypertension by demonstrating the importance of CNP/NPR-C signaling in preserving vascular homoeostasis. Furthermore, these results suggest that the CNP/NPR-C pathway has potential as a disease-modifying therapeutic target for cardiovascular disorders.

Bradykinin B1 receptor-mediated vasodilation is impaired in myometrial arteries from women with pre-eclampsia.

OBJECTIVE: To investigate the vascular functional activity, localisation and expression of B1 and B2 kinin receptors in normal pregnancy and pre-eclampsia. METHODS: Kinin receptor-mediated relaxation of myometrial arteries was assessed using wire myography. Immunohistochemical staining and gene expression of kinin receptors in the myometrium was determined. RESULTS: B2 receptor-mediated relaxation was reduced in pre-eclampsia. B1 receptor-mediated relaxation was observed in a proportion of healthy women and was impaired in pre-eclampsia. Receptor expression and localisation was unaltered in pre-eclampsia. CONCLUSION: Here, we demonstrate a novel B1 receptor-mediated vasodilatation in healthy myometrial vessels that is absent in pre-eclampsia.

Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model.

INTRODUCTION: The present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs. METHODS: Natriuretic peptide (Npr) 2 and 3 expression were compared in non-diseased (grade 0/1) and diseased (grade IV) human cartilage by immunofluoresence microscopy and western blotting. In separate experiments, constructs were cultured under free-swelling conditions or subjected to dynamic compression with CNP, interleukin-1ß (IL-1ß), the Npr2 antagonist P19 or the Npr3 agonist cANF4⁻²³. Nitric oxide (NO) production, prostaglandin E2 (PGE2) release, glycosaminoglycan (GAG) synthesis and CNP concentration were quantified using biochemical assays. Gene expression of Npr2, Npr3, CNP, aggrecan and collagen type II were assessed by real-time qPCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse the data. RESULTS: The present study demonstrates increased expression of natriuretic peptide receptors in diseased or older cartilage (age 70) when compared to non-diseased tissue (age 60) which showed minimal expression. There was strong parallelism in the actions of CNP on cGMP induction resulting in enhanced GAG synthesis and reduction of NO and PGE2 release induced by IL-1ß. Inhibition of Npr2 with P19 maintained catabolic activities whilst specific agonism of Npr3 with cANF4⁻²³ had the opposite effect and reduced NO and PGE2 release. Co-stimulation with CNP and dynamic compression enhanced anabolic activities and inhibited catabolic effects induced by IL-1ß. The presence of CNP and the Npr2 antagonist abolished the anabolic response to mechanical loading and prevented loading-induced inhibition of NO and PGE2 release. In contrast, the presence of the Npr3 agonist had the opposite effect and increased GAG synthesis and cGMP levels in response to mechanical loading and reduced NO and PGE2 release comparable to control samples. In addition, CNP concentration and natriuretic peptide receptor expression were increased with dynamic compression. CONCLUSIONS: Mechanical loading mediates endogenous CNP release leading to increased natriuretic peptide signalling. The loading-induced CNP/Npr2/cGMP signalling route mediates anabolic events and prevents catabolic activities induced by IL-1ß. The CNP pathway therefore represents a potentially chondroprotective intervention for patients with OA, particularly when combined with physiotherapeutic approaches to stimulate biomechanical signals.

Enhanced angiogenic capacity of human umbilical vein endothelial cells from women with preeclampsia.

Maternal and placental angiogenic abnormalities are a common feature of preeclampsia. The aim of this study was to determine if endothelial cells from women with preeclampsia exhibit different angiogenic responses compared to healthy cells. Using the endothelial tube formation assay, we have shown that primary human umbilical vein endothelial cells (HUVECs) isolated from women with preeclampsia display greater levels of in vitro angiogenic branching compared to cells from healthy women. A comparable increase in tube formation was observed in healthy cells cultured at 0.5% O(2). Vascular endothelial growth factor (VEGF) receptor inhibition resulted in a decrease in angiogenesis in both healthy hypoxic cells and cells from women with preeclampsia. These findings demonstrate that HUVECs from women with preeclampsia exhibit inherent differences in their angiogenic capacity which are apparent in the absence of placental or maternal factors.