Novel Therapeutic Directions for the Natriuretic Peptides in Cardiovascular Diseases: What’s on the Horizon

Introduction

The natriuretic peptide system (NPS) consists of three known peptides which are distinct gene products and include atrial natriuretic peptide (ANP} and brain natriuretic peptide (BNP), primarily from cardiomyocytes, and C-type natriuretic peptide (CNP), chiefly from endothelial cells. These three peptides following binding to their respective receptors elevate intracellular cGMP leading to activation of cGMP dependent protein kinase (cGMP-PK). Specifically, ANP and BNP bind to NPR-A while CNP binds to NPR-B.¹⁾ These receptors are membrane bound and represent well-characterized particulate guanylyl cyclases that serve similarly to soluble guanylyl cyclase that is activated by nitric oxide (NO). All three peptides are cleared by a clearance receptor, which is a non-particulate guanylyl cyclase receptor, termed NPR-C. All are degraded enzymatically by widely distributed neutral endopeptidase 24.11(NEP). Following studies in cell systems, novel mouse models of altered natriuretic peptide production or receptor function, integrative studies in pathophysiological models and in human subjects, a unifying understanding of the biology of these peptides has emerged as playing a fundamental role in cardiovascular biology and medicine.

The conventional view of the natriuretic peptide system is one in which the natriuretic peptides form an important cardiorenal axis. Specifically, the heart synthesizes and releases ANP and BNP to optimize intravascular volume and arterial pressure. This homeostatic function is mediated by cardiorenal and endocrine actions that adapt to the volume status of the individual. An emerging paradigm is one of regulator of cardiac structure and function leading to the use of the natriuretic peptides as novel therapy to maintain optimal myocardial structure and function. Most importantly, we will review new knowledge that underscores that the natriuretic peptide system functions to maintain myocardial structure and function through actions on both cardiomyocytes and non-myocytes. Such cardiac properties are important in cardiac development as well as having therapeutic relevance in cardiovascular disease. Indeed, as will be discussed below, evidence is emerging in heart failure and hypertension that there may be a deficiency of biologically active natriuretic peptides which argues for the use of the natriuretic peptides as therapy to maintain optimal myocardial structure and function.

Natriuretic Peptide and Cardiomyocyte Function

A key observation has been that in mice lacking the natriuretic peptide receptor A (NPR-A) gene (Nprl^−/−) survival is reduced with hearts enlarged at birth and with possible cardiac developmental abnormalities.^2,3) The adult phenotype of such mice is elevated blood pressure and marked cardiac hypertrophy and fibrosis, indicating that the natriuretic peptide system plays an important role in cardiomyocyte growth and development. The increase in myocardial load which occurs with hypertension in NPR-A or ANP gene disruptions makes interpretation of the findings in such genetically altered mice difficult from the perspective of understanding the direct actions of the natriuretic peptides on cardiomyocyte growth and function. Mounting evidences underscore the important role of this receptor in regulating the anti-growth and anti-fibrotic actions of the cardiac peptides ANP and BNP. Holtwick and co-workers⁴⁾ reported that mice with cardiac specific disruption of the NPR-A demonstrated impaired relaxation and exaggerated hypertrophic responses to pressure overload supporting a key role for the natriuretic peptide system as a regulator of myocardial structure and function.

We developed a mouse model of cardiac specific expression of the DN-NPR-A to further investigate the autocrine and paracrine actions of the natriuretic peptide system in the heart.⁵⁾ Our hypothesis was that DN-NPR-A mice would demonstrate alterations in cardiac structure and function in the absence of alterations in blood pressure when exposed to pressure overload produced by aortic banding. In the absence of pressure overload, basal and BNP stimulated guanylyl cyclase activity was reduced. In contrast, blood pressure, myocardial cGMP, ANP and myocardial structure and function were normal in the DN-NPR-A when compared to wild-type mice. With pressure overload, myocardial cGMP was reduced, and ventricular filling pressures were increased in association with greater ventricular hypertrophy, fibrosis and mortality in the DN-NPR-A mice compared to wild-types. These studies support the conclusion that the endogenous natriuretic peptide systems exert physiologically relevant autocrine and paracrine effects via cardiomyocyte NPR-A receptors to modulate cardiac hypertrophy and fibrosis in response to pressure overload.

The molecular mechanism of natriuretic peptide regulation of cardiomyocyte growth is only now emerging. To address this issue, Tokudome et al.⁶⁾ investigated the role of calcineurin, a calcium-dependent phosphatase, in cardiac remodeling in NPR-A knockout mice. They reported that in young NPR-A knockout mice, calcineurin activity, nuclear translocation of nuclear factor of activated T cells c3(NFATc3)and modulatory calcineurin-interacting protein 1 (MCIPI) gene expressions were increased in hearts of the NPR-A knockout mice compared to wild-type mice. Inhibition of calcineurin activity by FK506 decreased the heart to body weight ratio, cardiomyocyte size and collages volume fraction but such inhibition had no impact in wild-type mice. These studies concluded that their findings supported the concept that activation of NPR-A by locally secreted natriuretic peptides protects the heart from excessive cardiomyotye hypertrophy by inhibition of the calcineurin-NFATc3 pathway.

Natriuretic Peptides and Cardiac Fibroblasts

We recently investigated whether cardiac fibroblasts (CFs) produce BNP and whether BNP and its signaling system contributes to the regulation of collagen synthesis and to the activation of MMPs.⁷⁾ In these studies BNP mRNA was detected in CFs, and a specific radioimmunoassay demonstrated that BNP 1–32 was secreted from these cells. The amount of BNP secretion was significantly augmented by tumor necrosis factor. BNP inhibited de-novo collagen synthesis whereas zymographic MMP-2 (gelatinase) abundance was stimulated by BNP. In addition, protein expression of MMP-1, -2, and -3 and membranous type-1 MMP was increased by BNP. The cGMP analogue 8-bromo-cGMP mimicked the BNP effect, whereas inhibition of protein kinase G by KT5823 attenuated BNP-induced zymographic MMP-2 abundance. These in vitro findings support a role for BNP as a regulator of myocardial structure via control of cardiac fibroblast function. More recently, Calderone et al.⁸⁾ extended our studies to localize both ANP and BNP in CFs associated with scar formation following acute myocardial infarction. They observed that both ANP and BNP mRNA levels were significantly increased in the non-infarcted left ventricle and scar of 1-week post-myocardial infarction male rats, as compared to the left ventricle of normal rats. Following 4–7 days in culture, myofibroblasts expressed organized alpha-smooth muscle actin filaments. However, natriuretic peptides were predominantly detected in the nucleus and cytoplasm, and thin filaments occupying the perinuclear region were positive for pre pro-ANP and BNP. The authors concluded that natriuretic peptide synthesis by CFs may in part influence reparative fibrosis.

Building the case for a functionally important anti-fibrotic property of the natriuretic peptides has been its interaction with the profibrotic cytokine transforming growth factor-beta (TGF-beta). Here Kapoun and co-workers⁹⁾ reported that BNP inhibited TGF-beta-induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. Elegant cDNA microarray analysis was performed on CFs incubated in the presence or absence of TGF-beta and BNP. BNP treatment reduced the TGF-beta induced effects. Specifically, 88% and 85% of all TGF-beta-regulated mRNAs were affected at 24 and 48 hr, respectively. Further, BNP inhibited TGF-beta-regulated genes related to fibrosis (collagen I, fibronectin, CTGF PAI-1, and TIMP3), myofibroblast conversion (alpha-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA. IGF1. FGF18, and IGFBP10), and inflammation (COX2, IL6, TNF alpha-induced protein 6, and TNF superfamily, member 4), These studies strongly support the conclusion that BNP has a direct effect on CFs to inhibit fibrotic responses, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.

Cardiac remodeling involves the accumulation of extracellular matrix (ECM) proteins including fibronectin. It has been recognized that fibronectin contains RGD motifs that bind integrins at DDX sequences allowing signaling from the ECM to the nucleus. We noted that the NPR-A sequence also contains both RGD and DDX Sequences. We therefore pursued studies to determine if there could be cross talk between the ECM and BNP in CFs by investigating potential interactions between fibronectin and NPR-A on BNP induction of cGMP in CFs. We further sought to determine if we could augment this interaction with a Mayo designed NPR-A specific RGD peptide that could have therapeutic potential in the prevention of cardiac fibrosis as a small molecule.¹⁰⁾ We noted that CFs placed on fibronectin coated plates demonstrated a pronounced increase in cGMP production to BNP compared to noncoated plates. This cGMP production was also enhanced by the NPR-A specific RGD peptide. Further, we defined a possible role for the NPR-C receptor through a non-cGMP mechanism in mediating the antiproliferative actions of BNP in CFs where the NPR-C receptor antagonist blocked BNP inhibition of proliferation in these cells. Thus, evidence is strong that BNP and most likely ANP and CNP are potent anti-fibrotic peptides with therapeutic potential in the inhibition of cardiac fibrosis warranting further studies.

While the studies discussed above are largely in vitro investigations, one seminal in vivo study strongly supports this interaction between the natriuretic peptides and modulation of the ECM of the heart, Tamura and co-workers¹¹⁾ developed mice with targeted disruption of BNP. They observed multifocal fibrotic lesions in the ventricles from BNP knockout mice. Interestingly, unlike ANP knockout mice, no systemic hypertension nor ventricular hypertrophy were noted. However, in response to ventricular pressure overload, focal fibrotic lesions were markedly increased to a greater extent than to what observed in wild-type mice. One may conclude-that BNP is an antifibrotic factor in vivo and there is evidence for its role as a local regulator of ventricular structure and function independently of cardiac load and pressure.

Deficiencies of Natriuretic Peptides in Cardiovascular Disease

While the conventional view of circulating natriuretic peptides in cardiovascular disease is that they are elevated and serve as exceptional biomarkers especially in heart failure, a new concept is beginning to evolve as mounting evidence suggests that BNP circulates in different structural forms. We recently developed and used an immunoaffinity purification assay to isolate endogenous BNP 1–32 from New York Heart Association (NYHA) class IV patient plasma for subsequent analysis by nano-liquid chromatography (LC) electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR)-MS.¹²⁾ Here we introduced stable isotope-labeled BNP 1–32 to the assayed plasma to enable quantification of endogenous levels of BNP 1–32. Unlike the chemically nonspecific point-of-care tests (POCTs) and RIAs used worldwide to quantify BNP 1–32 from plasma, FT-ICR-MS (unprecedented mass measurement accuracy) coupled with LC (retention time) affords extraordinary molecular specificity, and when combined with the use of internal standards is able to confidently identify and quantify BNP 1–32. We observed that, despite exceedingly high circulating levels of BNP 1–32 in the NYHA class IV patients as determined by POCTs, nano-LC-electrospray ionization-FT-ICR-MS data did not reveal any endogenous BNP 1–32. We concluded that these results provide molecularly specific evidence for the absence of circulating BNP 1–32 in advanced-stage heart failure patients and suggest the existence of altered forms of BNP that are contributing to the POCT values.

Evidence also suggests a lack of or genetically altered activation of the natriuretic peptide system in human hypertension contributing to a relative deficiency. Such a deficiency could have major implications and contribute to adverse cardiac remodeling as well as further elevation of arterial pressure due to the lack of cardiovascular protection provided by the endogenous natriuretic peptides. Recently, Belluardo et al.¹³⁾ evaluated the relationship between two circulating molecular forms of BNP (BNP 1–32 and NT-pro-BNP), the severity of hypertension and cardiac hypertrophy in subjects with mild, moderate and severe hypertension. The important finding was that in grade 1 hypertension BNP 1–32 was not elevated and NT-pro-BNP was reduced when compared to controls, thus suggesting that early stages of hypertension are characterized by a lack of activation of the cardiac protective system of BNP.

Work by Dries et al.¹⁴⁾ has reported the existence of a corin gene allele defined by two missense mutations that is associated with hypertension. This observation is important as corin cleaves ANP and BNP into smaller biologically active molecules and its genetic alteration or maladaptive modulation by hypertension itself could suppress activation and processing of ANP and BNP with pathophysiological consequences. This was clearly shown recently in corin-deficient (Cor−/−) mice. Cor−/− mice have elevated levels of pro-ANP but no detectable levels of mature ANP.¹⁵⁾ Using radiotelemetry to assess blood pressure, Cor−/− mice had spontaneous hypertension as compared with wild-type mice, which was enhanced after dietary salt loading. This data and that of Dries establish corin as the physiological pro-natriuretic peptide convertase and indicate that a corin deficiency may contribute to hypertensive heart disease.

Rubattu et al.¹⁶⁾ has reported a link between the natriuretic peptides and cardiac structure. These investigators addressed the relationship between the natriuretic peptides and cardiac mass in human hypertension exploring the possibility of an ANP deficiency based upon ANP polymorphisms. They specifically focused on hypertensives carrying the ANP gene promoter allelic variant and found that they demonstrated increased left ventricular mass index and relative wall thickening compared with the wild-type genotype. These carriers also demonstrated significantly lower plasma pro-ANP levels also compared to heterozygote subjects. These studies also strongly support the existence of genetic derangements in human hypertension for the natriuretic peptide system that may have important functional relevance to the development and treatment cardiac hypertrophy and fibrosis.

Therapeutic Implications for Natriuretic Peptide Therapy in Cardiovascular Disease: Next Generation Peptides, Delivery Strategies and Novel Indications

Dendroaspis Natriuretic Peptide

A new member of the natriuretic peptide family, dendroaspis natriuretic peptide (DNP), has been reported, DNP, originally isolated from the venom of the Dendroaspis angusticeps (green mamba snake), is a 38-amino-acid peptide that contains a 17-amino-acid disulfide ring structure with 15-residue C-terminal extension.¹⁷⁾ This peptide, which shares structural similarity to ANP, BNP, and CNP, potently vasorelaxes isolated precontracted rodent aorta and canine coronary arteries and augments the formation of cGMP in aortic endothelial and smooth muscle cells.¹⁸⁾

DNP as a novel therapy is supported by recent studies in normal animals in which intravenous administration of synthetic DNP had potent natriuretic and diuretic properties, which were associated with marked increases in plasma and urinary cGMP.¹⁹⁾ Studies also have reported that DNP has greater affinity for the NPR-A than ANP or BNP that may explain its potency.^20,21) In addition, Chen et al.²²⁾ reported that DNP is highly resistant to renal degradation by neutral endopeptidase that may also explain the potency of DNP in mediating cardiorenal actions. Thus, in contrast to the other known natriuretic peptides, DNP may have unique characteristics that support its development as a new intravenous agent for acutely decompensated severe congestive heart failure (CHF) but also may have a role as a modulator of cardiac structure and function in the setting of acute myocardial infarction and as a potential oral agent.

Supporting this therapeutic potential are studies which have demonstrated in a model of severe heart failure that DNP markedly enhances renal function including both glomerular filtration rate and sodium excretion together with myocardial unloading and suppression of plasma renin activity.²³⁾ Clinical trials are being planned at the Mayo Clinic to characterize the cardiorenal actions of a DNP-like chimeric peptide as a first step in the clinical development of this new member of the natriuretic peptide family.

Oral Brain Natriuretic Peptide

A therapeutic challenge has been oral administration of intact and biologically active peptides. Most recently, new technologies have been developed that may be instrumental in achieving this objective. We and others recently applied these new technologies to BNP. First in experimental hypertension, administration of long-acting BNP synthesized as a fusion peptide with albumin resulted in sustained blood pressure-lowering actions, supporting a strategy for longer-term BNP therapy in cardiovascular diseases, especially hypertension in which BNP or ANP may be deficient and the cardiac phenotype is hypertrophy and fibrosis with diastolic dysfunction.²⁴⁾

We employed proprietary technology (Nobex) that had been developed in which short, amphiphilic oligomers are covalently attached to peptides. In contrast to standard PEGylation technology, this technique employs comparatively small, amphiphilic oligomers that are monodispersed and comprise both a hydrophobic (alkyl) moiety and a hydrophilic polyethylene glycol (PEG) moiety. The oligomers are intended to improve the pharmacokinetic and pharmacodynamic profiles of the peptide and enable oral administration.

We addressed the feasibility and the biological activity of acute orally administered conjugated BNP (CONJ-BNP).²⁵⁾ In these recent studies we evaluated for the first time a novel form of CONJ-hBNP (hBNP-021) through oral administration. In a randomized crossover-designed study we tested the biological activity of oral CONJ-BNP compared with oral native BNP in normal conscious dogs. Measurements of MAP, plasma BNP, and cGMP were made at baseline and repeated at 10, 30, 60, 120, 180, and 240 min after oral administration. As expected, plasma human BNP was not detectable in dogs at baseline while plasma human BNP that was used for oral BNP was detected after CONJ-hBNP administration. Importantly, plasma human BNP concentration was significantly higher after CONJ-BNP administration than after native BNP. Plasma cGMP increased after CONJ-hBNP for 60 min whereas it did not change after native hBNP. MAP decreased at 10 min and remained decreased for 60 min after CONJ-hBNP while remaining unchanged after native hBNP. Thus, this study clearly demonstrated that BNP is absorbed intact and promotes sustained biological actions when administered orally as conjugated form. These data suggest the importance of pursuing further studies with oral administration of conjugated forms of human BNP for the long-term treatment of cardiovascular diseases.

Acute Myocardial Infarction

Acute myocardial infarction remains a major cardiovascular disease entity and is a complication of coronary artery disease and may lead to loss of ventricular myocardium with cardiac enlargement and fibrosis, as the heart cannot regenerate itself. Evidence is strong for the concept that the cardiac natriuretic peptides ANP and BNP are small endogenous hormones possessing cardioprotective properties that may protect the heart from injury thus preserving cardiac structure and function. Data are emerging which support the therapeutic application of ANP and BNP in human acute myocardial infarction as an approach in cardioprotection,²⁶⁾ To complement the direct actions on the cardiomyocyte and cardiac fibroblast by ANP and BNP via cGMP are their ability to mediate coronary vasodilatation and reduce myocardial oxygen consumption, enhance myocardial relaxation, suppress aldosterone release including aldosterone synthesis in cultured cardiomyocytes, retard adrenergic activation and induce vascular regeneration^27–31). Based in part on the elegant human studies of Hayashi et al.,³²⁾ two human clinical trials are now ongoing in the US and Japan to test the hypothesis that BNP and ANP respectively infused at the time of acute myocardial infarction without heart failure for three days can preserve myocardial structure and function and reduce the ultimate development of human heart failure.

Summary

Remarkable advances have occurred since the discovery of ANP. The field of the natriuretic peptides has moved beyond their role as regulators of renal function; Growing evidence has now established the natriuretic peptides as regulators of myocardial structure and function as well as in cardiovascular regulation. Further, they emerge as being products not only of the cardiomyocyte but also of the cardiac fibroblast. Studies in vitro and in vivo have clearly established these cardiac hormones as antihypertrophic and antifibrotic. Further, indications such as cardioprotection for acute myocardial infarction, chronic therapy with oral BNP for hypertension and next generation peptides such as DNP-like peptides for acute heart failure may be in the near future. Novel therapeutic strategies to be tested in clinical trials in humans with cardiovascular disease are clearly on the horizon.