Amie J. Moyes

Manganese carbonyls bearing tripodal polypyridine ligands as photoactive carbon monoxide-releasing molecules.

The recently discovered cytoprotective action of CO has raised interest in exogenous CO-releasing materials (CORMs) such as metal carbonyls (CO complexes of transition metals). To achieve control on CO delivery with metal carbonyls, we synthesized and characterized three Mn(I) carbonyls, namely, [Mn(tpa)(CO)(3)]ClO(4) [1, where tpa = tris(2-pyridyl)amine], [Mn(dpa)(CO)(3)]Br [2, where dpa = N,N-bis(2-pyridylmethyl)amine], and [Mn(pqa)(CO)(3)]ClO(4) [3, where pqa = (2-pyridylmethyl)(2-quinolylmethyl)amine], by crystallography and various spectroscopic techniques. All three carbonyls are sensitive to light and release CO when illuminated with low-power UV (5-10 mW) and visible (λ > 350 nm, ~100 mW) light. The sensitivity of 1-3 to light has been assessed with respect to the number of pyridine groups in their ligand frames. When a pyridine ring is replaced with quinoline, extended conjugation in the ligand frame increases the absorptivity and makes the resulting carbonyl 3 more sensitive to visible light. These photosensitive CORMs (photoCORMs) have been employed to deliver CO to myoglobin under the control of light. The superior stability of 3 in aqueous media makes it a photoCORM suitable for inducing vasorelaxation in mouse aortic muscle rings.

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.

C2238 Atrial Natriuretic Peptide Molecular Variant Is Associated With Endothelial Damage and Dysfunction Through Natriuretic Peptide Receptor C Signaling

Rationale: C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events. Objective: We investigated the mechanisms underlying the vascular effects of C2238-&agr;ANP. Methods and Results: In vitro, human umbilical vein endothelial cell were exposed to either wild-type (T2238)- or mutant (C2238)-&agr;ANP. Cell survival and apoptosis were tested by Trypan blue, annexin V, and cleaved caspase-3 assays. C2238-&agr;ANP significantly reduced human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-&agr;ANP reduced endothelial tube formation, as assessed by matrigel. C2238-&agr;ANP did not differentially modulate natriuretic peptide receptor (NPR)-A/B activity with respect to T2238-&agr;ANP, as evaluated by intracellular cGMP levels. In contrast, C2238-&agr;ANP, but not T2238-&agr;ANP, markedly reduced intracellular cAMP levels in an NPR-C–dependent manner. Accordingly, C2238-&agr;ANP showed higher affinity binding to NPR-C, than T2238-&agr;ANP. Either NPR-C inhibition by antisense oligonucleotide or NPR-C gene silencing by small interfering RNA rescued survival and tube formation of human umbilical vein endothelial cell exposed to C2238-&agr;ANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-&agr;ANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. Adenovirus-mediated Akt1 reactivation rescued the detrimental effects of C2238-&agr;ANP. Overall, these data indicate that C2238-&agr;ANP affects endothelial cell integrity through NPR-C–dependent inhibition of the cAMP/protein kinase A/Akt1 pathway and increased reactive oxygen species production. Accordingly, C2238-&agr;ANP caused impairment of acetylcholine-dependent vasorelaxation ex vivo, which was rescued by NPR-C pharmacological inhibition. Finally, subjects carrying C2238 minor allele showed early endothelial dysfunction, which highlights the clinical relevance of our results. Conclusions: C2238-&agr;ANP reduces endothelial cell survival and impairs endothelial function through NPR-C signaling. NPR-C targeting represents a potential strategy to reduce cardiovascular risk in C2238 minor-allele carriers.

Light-triggered carbon monoxide delivery with Al-MCM-41-based nanoparticles bearing a designed manganese carbonyl complex

A photoactive manganese carbonyl complex namely, fac-[Mn(pqa)(CO)3]ClO4 (abbreviated as {Mn-CO}, pqa = (2-pyridylmethyl)(2-quinolylmethyl)amine) has been incorporated in the pores of 60 nm mesoporous Al-MCM-41 nanoparticles. Strong electrostatic interactions with the negatively charged walls of the aluminosilicate host entrap the cationic carbonyl complex in the resulting material {Mn-CO}@Al-MCM-41 which has been characterized by various physical techniques and chemical analysis. The sample morphology and microstructure of the material have been established by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results of powder X-ray diffraction (PXRD) data and the SEM-EDX elemental maps of Si, O, Al and Mn confirm that the carbonyl complex is within the pores of the nanoparticles. The 2% manganese content of {Mn-CO}@Al-MCM-41, determined by acid digestion followed by inductively coupled plasma-optical emission spectroscopy (ICP-OES), indicates efficient loading of the carbonyl complex in the nanoparticles. In biological buffer solutions, less than 2% leaching of {Mn-CO} from the nanoparticles was noted within a period of 24 h. When exposed to a broadband visible light source (λ > 350 nm), {Mn-CO}@Al-MCM-41 rapidly releases CO, as confirmed by the reduced myoglobin assay. The utility of light-induced CO delivery by {Mn-CO}@Al-MCM-41 has been established by its capacity to relax rat aorta muscle rings in tissue bath experiments.

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-1β, IL-6, and IL-10), and greater increases in the cardiac levels of phosphorylated IKKα/β 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.

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

IntroductionThe present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs.MethodsNatriuretic 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-1β (IL-1β), 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.ResultsThe 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-1β. 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-1β. 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.ConclusionsMechanical 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-1β. The CNP pathway therefore represents a potentially chondroprotective intervention for patients with OA, particularly when combined with physiotherapeutic approaches to stimulate biomechanical signals.

Functional pharmacological characterization of SER100 in cardiovascular health and disease

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.

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

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.

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

Significance The morbidity and mortality associated with heart failure (HF) are unacceptably high. Cyclic guanosine-3′,5′-monophosphate (cGMP) plays a key role in preserving cardiac structure and function, and therapeutically targeting cGMP in HF has shown promise in experimental models and patients. Phosphodiesterases (PDEs) metabolize and curtail the actions of cGMP (and cAMP), and increased PDE activity is thought to contribute to HF pathogenesis. Herein, we show that inhibition of one specific isoform, PDE2, enhances the salutary effects of cGMP in the context of HF, and that this beneficial action facilitates a distinct pathway, driven by nitric oxide, that is impaired in this disorder. These observations validate PDE2 inhibitors as a demonstrable means of boosting cardiac cGMP and advancing HF therapy. 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.

Investigation of the role of multidrug resistance proteins (MRPs) in vascular homeostasis

Cellular levels of cyclic GMP (cGMP) are tightly controlled by synthetic and degradative mechanisms. Pharmacological manipulation of these processes (e.g. soluble guanylate cyclase stimulators and phosphodiesterase 5 inhibitors) augments cGMP-dependent signalling and is beneficial in treating cardiovascular disease (eg. pulmonary hypertension). An additional mechanism potentially important in the inactivation of cGMP is cellular extrusion, driven by a family of multidrug resistance proteins (MRPs). Herein, we investigated if MRPs modulate vascular reactivity, smooth muscle cell proliferation, and systemic hemodynamics. The functional reactivity of murine aortic rings and proliferation of human pulmonary artery smooth muscle cells (PASMC) were determined in the absence and presence of the MRP inhibitor MK571. Hemodynamic changes in vivo in response to MK571 were analysed acutely by bolus dosing and chronically by radiotelemetry. MK571 (1nM-50µM) caused a concentration-dependent relaxation of mouse aortic r...