Novel bifunctional natriuretic peptides as potential therapeutics

Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics.     

Poly(lactic-co-glycolic acid) nanospheres and microspheres for short- and long-term delivery of bioactive ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) has been shown to be neuroprotective in the central nervous system (CNS). However, systemic administration and bolus injections have shown significant side effects and limited efficacy. Sustained, local delivery may lead to effective neuroprotection and avoid or limit adverse side effects, but sustained CNTF delivery has proven difficult to achieve and control. For controlled, sustained delivery, we investigated several processing variables in making poly(DL-lactic-co-glycolic acid) (PLGA) nano- and microspheres to optimize CNTF encapsulation and release. Nano- and microspheres were 314.9 +/- 24.9 nm and 11.69 +/- 8.16 microm in diameter, respectively. CNTF delivery from nanospheres was sustained over 14 days, and delivery from microspheres continued over more than 70 days. To assess protein bioactivity after encapsulation, neural stem cells (NSCs) were treated with CNTF released from nanospheres and compared to those treated with unencapsulated CNTF as a control. NSCs treated with CNTF expressed markers specific to mature cells, notably astrocytes; some increase in oligodendrocytic and neuronal marker expression was also observed. Significantly, cells treated with CNTF released by nanospheres exhibited a similar degree of differentiation when compared to those treated with control CNTF of equivalent concentration, showing that the process of protein encapsulation did not reduce its potency.     

Evaluation of Sustained BMP-2 Release Profiles Using a Novel Fluorescence-Based Retention Assay

The purpose of this study was to develop and characterize a novel fluorescence-based retention assay for the evaluation of the release profile of bone morphogenetic protein-2 (BMP-2) released from bone graft carrier. In this study, we evaluated the binding, release kinetics, and delivery efficacies of BMP-2 incorporated into hydroxyapatite (HA) bone grafts. The evaluation of the release profile of BMP-2 from HA bone grafts using a fluorescence-based retention assay revealed initial burst releases from the HA bone grafts followed by long sustained releases up to 14 weeks. The sustained biological activity of the released BMP-2 from HA bone grafts over the full 14-week period supports a long sustained mechanism via fluorescence-based retention assay. Thus, the results from this study show that BMP-2 could be incorporated into HA bone grafts for sustained release over a prolonged period of time with retention of bioactivity and our fluorescence-based retention assay, which is principally detecting the retention profile of BMP-2 in HA bone grafts, is more accurate than conventionally collecting the released BMP-2 for evaluation of BMP-2 release profiles.     

Human cardiac plasma concentrations of atrial natriuretic peptide quantified by radioreceptor assay

The presence of high affinity receptors for atrial natriuretic peptide in bovine adrenal cortex has enabled the development of a sensitive, specific and rapid radioreceptor assay for this peptide in human plasma. In 18 normal subjects, venous plasma atrial natriuretic peptide concentration ranged from 6 to 65 pM. This plasma concentration was two-fold higher in right atrium as compared to venous blood in 12 patients investigated by cardiac catheterisation, confirming that the right atrium is the site of release of atrial natriuretic peptide into circulation. There was a further step up in plasma atrial natriuretic peptide concentration between pulmonary arterial and aortic plasma. This finding indicates that released hormone in man may undergo further activation in the lungs, or that there may be direct release from the left atrium.     

Natriuretic peptides in cardiovascular diseases

The natriuretic peptide family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin. The activities of natriuretic peptides and endothelins are strictly associated with each other. ANP and BNP inhibit endothelin-1 (ET-1) production. ET-1 stimulates natriuretic peptide synthesis. All natriuretic peptides are synthesized from polypeptide precursors. Changes in natriuretic peptides and endothelin release were observed in many cardiovascular diseases: e.g. chronic heart failure, left ventricular dysfunction and coronary artery disease.     

Expression of constitutively active guanylate cyclase in cardiomyocytes inhibits the hypertrophic effects of isoproterenol and aortic constriction on mouse hearts

Evidence from several rodent models has suggested that a reduction of either atrial natriuretic peptide or its receptor in the heart affects cardiac remodeling by promoting the onset of cardiac hypertrophy. The atrial natriuretic peptide receptor mediates signaling at least in part via the generation of intracellular cyclic GMP. To directly test whether accumulation of intracellular cyclic GMP conveys protection against cardiac hypertrophy, we engineered transgenic mice that overexpress a catalytic fragment of constitutively active guanylate cyclase domain of the atrial natriuretic peptide receptor in a cardiomyocyte-specific manner. Expression of the transgene increased the intracellular concentration of cyclic GMP specifically within cardiomyocytes and had no detectable effect on cardiac performance under basal conditions. However, expression of the transgene attenuated the effects of the pharmacologic hypertrophic agent isoproterenol on cardiac wall thickness and prevented the onset of the fetal gene expression program normally associated with cardiac hypertrophy. Likewise, expression of the transgene inhibited the hypertrophic effects of abdominal aortic constriction, since it abolished its effects on ventricular wall thickness and greatly attenuated its effects on cardiomyocyte size. Altogether, our results suggest that cyclic GMP is a cardioprotective agent against hypertrophy that acts via a direct local effect on cardiomyocytes.     

CD-NP: A Novel Engineered Dual Guanylyl Cyclase Activator with Anti-Fibrotic Actions in the Heart

Natriuretic peptides (NPs) are cardioprotective through the activation of guanylyl cyclase (GC) receptors A and B. CD-NP, also known as cenderitide, is a novel engineered NP that was designed to uniquely serve as a first-in-class dual GC receptor agonist. Recognizing the aldosterone suppressing actions of GC-A activation and the potent inhibitory actions on collagen synthesis and fibroblast proliferation through GC-B activation, the current study was designed to establish the anti-fibrotic actions of CD-NP, administered subcutaneously, in an experimental rat model of early cardiac fibrosis induced by unilateral nephrectomy (UNX). Our results demonstrate that a two week subcutaneous infusion of CD-NP significantly suppresses left ventricular fibrosis and circulating aldosterone, while preserving both systolic and diastolic function, in UNX rats compared to vehicle treated UNX rats. Additionally we also confirmed, in vitro, that CD-NP significantly generates the second messenger, cGMP, through both the GC-A and GC-B receptors. Taken together, this novel dual GC receptor activator may represent an innovative anti-fibrotic therapeutic agent.     

Tissue scaffolds functionalized with therapeutic elastin-like biopolymer particles

Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin-like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF–ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF–ELPs.     

Designer natriuretic peptides: a vision for the future of heart failure therapeutics

Despite recent pharmacological advances in heart failure therapy, mortality from acute decompensated heart failure remains high. Conventional therapies are often insufficient to address the complex interplay between structural, functional, neurohumoral, and renal mechanisms involved in the heart failure syndrome. The natriuretic peptide system, however, offers a unique pleiotropic strategy which could bridge this gap in heart failure therapy. Exogenous administration of native A-type and B-type natriuretic peptides has been met with both success and limitations, and despite the limitations, remains a worthwhile endeavor. Alternatively, synthetic modification to create "designer" chimeric peptides holds the possibility to extend both the application and therapeutic benefits possible with a natriuretic peptide based approach. Herein we describe the development of natriuretic peptide based heart failure therapies, including the design, rationale, and preliminary studies of the novel chimeric peptides CD-NP and CU-NP.     

Visfatin aggravates transverse aortic constriction-induced cardiac remodelling by enhancing macrophage-mediated oxidative stress in mice

Previous studies have reported that visfatin can regulate macrophage polarisation, which has been demonstrated to participate in cardiac remodelling. The aims of this study were to investigate whether visfatin participates in transverse aortic constriction (TAC)-induced cardiac remodelling by regulating macrophage polarisation. First, TAC surgery and angiotensin II (Ang II) infusion were used to establish a mouse cardiac remodelling model, visfatin expression was measured, and the results showed that TAC surgery or Ang II infusion increased visfatin expression in the serum and heart in mice, and phenylephrine or hydrogen peroxide promoted the release of visfatin from macrophages in vitro. All these effects were dose-dependently reduced by superoxide dismutase. Second, visfatin was administered to TAC mice to observe the effects of visfatin on cardiac remodelling. We found that visfatin increased the cross-sectional area of cardiomyocytes, aggravated cardiac fibrosis, exacerbated cardiac dysfunction, further regulated macrophage polarisation and aggravated oxidative stress in TAC mice. Finally, macrophages were depleted in TAC mice to investigate whether macrophages mediate the regulatory effect of visfatin on cardiac remodelling, and the results showed that the aggravating effects of visfatin on oxidative stress and cardiac remodelling were abrogated. Our study suggests that visfatin enhances cardiac remodelling by promoting macrophage polarisation and enhancing oxidative stress. Visfatin may be a potential target for the prevention and treatment of clinical cardiac remodelling.     

Histamine release induced by dendroaspis natriuretic peptide from rat mast cells

Dendroaspis natriuretic peptide (DNP), recently isolated from the venom of the green Mamba snake Dendroaspis angusticeps, is a 38 amino acid peptide containing a 17 amino acid disulfide ring structure similar to that of the natriuretic peptide family. The natriuretic peptide family is known to induce histamine release from human and rat mast cells, but there are no published data concerning the effects of DNP on histamine release from mast cells. The purpose of this study is to investigate whether DNP induces the histamine release from rat peritoneal mast cells (RMPCs) and to determine the mechanism of DNP-induced histamine release from RPMCs. After treatment of RPMC with DNP, mast cell degranulation was observed, and calcium uptake and histamine release were measured. DNP released the histamine, induced the mast cell degranulation, and increased the calcium uptake of RPMCs, in a dose-dependent manner. The results indicate that DNP can increase Ca-uptake and induce histamine release.     

Alteration of gene expression during progression of hypertension-induced cardiac dysfunction in rats

Hypertension induced by high-salt diet in Dahl salt-sensitive rats leads to compensatory cardiac hypertrophy by approximately 11 wk, cardiac dysfunction at approximately 17 wk, and death from cardiac dysfunction at approximately 21 wk. It is unclear what molecular hallmarks distinguish the compensatory hypertrophy from the decompensated cardiac dysfunction phase. Here we compared the gene expression in rat cardiac tissue from the compensatory hypertrophic phase (11 wk, n = 6) with the cardiac dysfunction phase (17 wk, n = 6) and with age-matched normotensive controls. Messenger RNA levels of 93 genes, selected based on predicted association with cardiac dysfunction, were measured by quantitative real-time PCR. In the hypertrophic phase, the expression of three genes, atrial natriuretic peptide (ANP; P = 0.0089), brain natriuretic peptide (P = 0.0012), and endothelin-1 precursor (P = 0.028), significantly increased, whereas there was decreased expression of 24 other genes including SOD2 (P = 0.0148), sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (P = 0.0002), and ryanodine receptor 2 (P = 0.0319). In the subsequent heart cardiac dysfunction phase, the expression of an additional 20 genes including inducible nitric oxide synthase (NOS; P = 0.0135), angiotensin I-converting enzyme (P = 0.0082), and IL-1beta (P < 0.0001) increased, whereas the expression of seven genes decreased compared with those of age-matched controls. Furthermore, the expression of 22 genes, including prepro-endothelin-1, ANP, angiotensin I-converting enzyme, beta(1)-adrenergic receptor, SOD2, and endothelial NOS, significantly changed in the cardiac dysfunction phase compared with the compensatory hypertrophic phase. Finally, principal component analysis successfully segregated animals with decompensatory cardiac dysfunction from controls, as well as from animals at the compensated hypertrophy phase, suggesting that we have identified molecular markers for each stage of the disease.     

Atrial natriuretic peptide inhibits cardiomyocyte hypertrophy through mitogen-activated protein kinase phosphatase-1

Cardiac hypertrophy is formed in response to hemodynamic overload. Although a variety of factors such as catecholamines, angiotensin II (AngII), and endothelin-1 (ET-1) have been reported to induce cardiac hypertrophy, little is known regarding the factors that inhibit the development of cardiac hypertrophy. Production of atrial natriuretic peptide (ANP) is increased in the hypertrophied heart and ANP has recently been reported to inhibit the growth of various cell types. We therefore examined whether ANP inhibits the development of cardiac hypertrophy. Pretreatment of cultured cardiomyocytes with ANP inhibited the AngII- or ET-1-induced increase in the cell size and the protein synthesis. ANP also inhibited the AngII- or ET-1-induced hypertrophic responses such as activation of mitogen-activated protein kinase (MAPK) and induction of immediate early response genes and fetal type genes. To determine how ANP inhibits cardiomyocyte hypertrophy, we examined the mechanism of ANP-induced suppression of the MAPK activation. ANP strongly induced expression of MAPK phosphatase-1 (MKP-1) and overexpression of MKP-1 inhibited AngII- or ET-1-induced hypertrophic responses. These growth-inhibitory actions of ANP were mimicked by a cyclic GMP analog 8-bromo-cyclic GMP. Taken together, ANP directly inhibits the growth factor-induced cardiomyocyte hypertrophy at least partly via induction of MKP-1. Our present study suggests that the formation of cardiac hypertrophy is regulated not only by positive but by negative factors in response to hemodynamic load.     

Effect of a chronic infusion of atrial natriuretic peptide on vascular reactivity in normotensive and renal hypertensive rats

In a previous study, we found that a long-term infusion of atrial natriuretic peptide (ANP) produced a sustained reduction of mean arterial pressure and peripheral vascular resistance in two-kidney, one-clip (2K-1C) hypertensive rats, whereas in control rats it had only a transient effect on cardiac output. However, plasma levels of ANP were actually 3-fold higher in normotensive than in hypertensive rats. Previous studies suggested that plasma ANP levels might modulate the vascular reactivity to the peptide. The present study examined whether the lack of chronic hemodynamic effects of ANP in control rats was due to changes in vascular reactivity to the peptide. In control rats, vascular reactivity to ANP was reduced 50% by a chronic infusion of ANP. However, in 2K-1C hypertensive rats, a long-term infusion of ANP had no effect on the vascular reactivity to ANP. The results of the present study indicate that the lack of persistent hemodynamic effects of a chronic infusion of ANP in control rats may be due to a decrease in the vascular reactivity to the peptide. The sustained hypotensive and vasodilatory effects of a long-term infusion of ANP in 2K-1C hypertensive rats are associated with no changes in the vascular reactivity to ANP.     

Modulating Drug Release from Gastric-Floating Microcapsules through Spray-Coating Layers

Floating dosage forms with prolonged gastric residence time have garnered much interest in the field of oral delivery. However, studies had shown that slow and incomplete release of hydrophobic drugs during gastric residence period would reduce drug absorption and cause drug wastage. Herein, a spray-coated floating microcapsule system was developed to encapsulate fenofibrate and piroxicam, as model hydrophobic drugs, into the coating layers with the aim of enhancing and tuning drug release rates. Incorporating fenofibrate into rubbery poly(caprolactone) (PCL) coating layer resulted in a complete and sustained release for up to 8 h, with outermost non-drug-holding PCL coating layer serving as a rate-controlling membrane. To realize a multidrug-loaded system, both hydrophilic metformin HCl and hydrophobic fenofibrate were simultaneously incorporated into these spray-coated microcapsules, with metformin HCl and fenofibrate localized within the hollow cavity of the capsule and coating layer, respectively. Both drugs were observed to be completely released from these coated microcapsules in a sustained manner. Through specific tailoring of coating polymers and their configurations, piroxicam loaded in both the outer polyethylene glycol and inner PCL coating layers was released in a double-profile manner (i.e. an immediate burst release as the loading dose, followed by a sustained release as the maintenance dose). The fabricated microcapsules exhibited excellent buoyancy in simulated gastric fluid, and provided controlled and sustained release, thus revealing its potential as a rate-controlled oral drug delivery system.     

Ventricular and atrial myocytes of newborn rats synthesise and secrete atrial natriuretic peptide in culture: Light-and electron-microscopical localisation and chromatographic examination of stored and secreted molecular forms

Summary We have demonstrated that atrial natriuretic peptide-like immunoreactivity is stored and secreted by ventricular and atrial myocytes in dissociated cell culture preparations from the heart of newborn rat. Culture preparations were maintained in either foetal calf serum-supplemented medium 199 or in hormone-supplemented, serum-free medium 199. The presence of atrial natriuretic peptidelike immunoreactivity in the cultured myocytes was demonstrated at both light-and electron-microscopical levels. Release of atrial natriuretic peptide-like immunoreactivity into the culture medium was measured by radioimmunoassay; molecular forms of the stored and secreted peptide were determined by gel column chromatography. The atrial natriuretic peptide-like immunoreactivity of cultured atrial and ventricular myocytes was concentrated in the perinuclear cytoplasm and was localised to electron-dense secretory granules. The number of immunoreactive ventricular myocytes and the intensity of their immunofluorescence changed with time in culture and was higher in cultures in foetal calf serum-supplemented medium than in serum-free medium. Gamma-atrial natriuretic peptide was stored and released by cultured atrial and ventricular myocytes, but was broken down to alpha-atrial natriuretic peptide in the growth medium. This process was foetal calf serum-independent, since it occurred in both the media used, indicating that cardiac myocytes in culture may release a factor that cleaves gamma-atrial natriuretic peptide to form alphaatrial natriuretic peptide.