Peptides 58 (2014) 108–116

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Peptides journal homepage: www.elsevier.com/locate/peptides

Review

Atrial natriuretic peptide: An old hormone or a new cytokine? Paolo De Vito ∗ Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy

a r t i c l e

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Article history: Received 8 May 2014 Received in revised form 19 June 2014 Accepted 19 June 2014 Available online 26 June 2014 Keywords: Atrial natriuretic peptide Cytokines Reactive oxygen species Cell proliferation Cytoprotective effects

a b s t r a c t Atrial natriuretic peptide (ANP) a cardiovascular hormone mainly secreted by heart atria in response to stretching forces induces potent diuretic, natriuretic and vasorelaxant effects and plays a major role in the homeostasis of blood pressure as well as of water and salt balance. The hormone can also act as autocrine/paracrine factor and modulate several immune functions as well as cytoprotective effects. ANP contributes to innate immunity being able to: (i) stimulate the host defense against extracellular microbes by phagocytosis and Reactive Oxygen Species (ROS) release; (ii) inhibit the synthesis and release of proinflammatory markers such as TNF-␣, IL-1, MCP-1, nitric oxide (NO), cyclooxygenase-2 (COX-2); (iii) inhibit the expression of adhesion molecules such as ICAM-1 and E-selectin. ANP can also affect the adaptive immunity being able to: (i) reduce the number of CD4+ CD8+ lymphocytes as well as to increase the CD4− CD8− cells; (ii) stimulate the differentiation of naïve CD4+ cells toward the Th2 and/or Th17 phenotype. The hormone shows protective effects during: (i) ventricular hypertrophy and myocardial injury; (ii) atherosclerosis and hypertension by the induction of antiproliferative effects; (iii) oxidative stress counteracting the dangerous effects of ROS; (iv) growth of tumors cells by the induction of apoptosis or necrosis. Since not much is known about of the role of ANP locally produced and released by noncardiac cells, this review outlines the contribution of ANP in different aspect of innate as well as adaptive immunity also with respect to the excessive cell growth in physiological and/or pathological conditions. © 2014 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunomodulatory function of ANP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytoprotective effects of ANP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ANP protective effects on cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ANP cytoprotective effects on Vascular Smooth Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ANP role on endothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ANP-induced protection in liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ANP effects on tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Atrial Natriuretic Petide (ANP) belongs to a family of polypeptide hormones, reported as natriuretic peptides, also including Brain Natriuretic Peptide (BNP) and the type-C natriuretic

∗ Tel.: +39 06 72594357; fax: +39 06 2023500. E-mail address: [email protected] http://dx.doi.org/10.1016/j.peptides.2014.06.011 0196-9781/© 2014 Elsevier Inc. All rights reserved.

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peptide (CNP) (Fig. 1). ANP mainly produced by the atrium, derives from a precursor peptide reported as preproANP which in humans consists of 151 amino acids; the subsequent cleavage of preproANP produces a 126-amino acid peptide called proANP1126 that is stored in the atrium granules [15]. Specific signals such as mechanical stretching of atrium cardiomyocytes and/or several messenger molecules such as angiotensin II, catecholamines and vasopressin, can promote proANP1-126 splitting into an NH2terminal fragment, reported as proANP1-98, and a COOH-terminal

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Fig. 1. Amino acid sequence and structures of the natriuretic peptide family. ANP = atrial natriuretic peptide; BNP = brain natriuretic peptide; CNP C-type natriuretic peptide.

peptide ANP99-126, usually called ANP, which is considered to be the biologically active hormone [89]. The main ANP targets are kidney, vessels, and adrenal glands: the hormone, by different mechanisms, produces potent diuretic, natriuretic and vasorelaxant effects and plays a major role in the homeostasis of blood pressure as well as of salt and water balance [15]. ANP is able to induce an increase of the glomerular filtration rate (GFR) by the stimulation of afferent arterioles vasodilation together with vasoconstriction of efferent arterioles, the induction of natriuresis depends on the inhibition of the: (i) sodium/hydrogen exchanger (NHE) in the proximal tubule; (ii) sodium/chloride co-transporter in the distal tubule; (iii) and sodium channel in the collecting duct. Finally, the hormone-induced diuresis with not completely defined effects on aquaporin-2 incorporation into the collecting duct of apical membrane of distal tubuli. Several biological effects of ANP are elicited by the suppression of the release of different messenger molecules such as renin, angiotensin, aldosterone, endothelin and vasopressin. ANP can also stimulate the extravasation of fluids from the intravascular to the extravascular compartments through an increase of endothelial permeability [1]. ANP ring structure is disrupted by metalloendopeptidases including various neutral endopeptidases (NEP) and its half-life after i.v. injection is 2–4 min in humans [76]. ANP degrading enzymes have been maximally found in the proximal renal tubule, but are also located in the lung, intestine, seminal vesicles and neutrophils [76]. Moreover, ANP is also removed from plasma by the binding to its clearance receptor [109]. Outside the heart ANP-specific mRNA has been detected in lung, brain, liver, gastrointestinal tract, thymus, and in several cell types such as Vascular Smooth Muscle (VSM) cells, endothelial cells and immune cells [88]. Several findings suggest that the biological functions of ANP are not only restricted to blood pressure homeostasis, but the hormone can also play an important role to modulate cell growth, counteract the oxidant-induced cell damage and contribute to antiinflammatory processes [22,112]. ANP exerts its biological effects by binding to specific plasma membrane receptors called Natriuretic Peptide Receptor (NPR)-A and/or NPR-C [70]. The NPR-A receptors are transmembrane receptors with a molecular weight of about 120–140 kDa showing an intrinsic intracellular guanylate cyclase (GC) domain [81]. NPR-A receptor activation requires ANP binding to the extracellular domain of the receptor and the phosphorylation of six residues within the intracellular kinase

homology domain (KHD). The subsequent hormone-induced receptor activation stimulates the GC domain which, in turn, induces an intracellular production of guanylate cyclase able to stimulate a specific cGMP-dependent protein kinase, reported as PKG; the latter can stimulate several biological events such as ion transport mechanisms, transcription of specific factors, cell growth and proliferation, apoptosis and inflammation [81]. NPR-C receptors or clearance receptors (molecular weight 60–70 KDa) show a short intracellular domain (37-amino acids) and are coupled to adenylyl cyclase inhibition with a subsequent decrease of intracellular levels of cAMP and/or to phospholipase C (PLC) activation: the enzyme able to catalyze the transformation of phosphatydilinositolbisphosphate (PIP2) to inositol trisphoshate (IP3) and diacylglicerol (DAG), an important activator of protein kinase C (PKC) (Fig. 2). ANP

Fig. 2. Structure of the different types of natriuretic peptide receptors. NPR-A and B receptors are transmembrane receptors with a molecular weight of about 120–140 kDa and show: (i) extracellular domain (ECD); (ii) intramembrane domain (ICD); (iii) intracellular kinase homology domain (KHD); (iv) an intrinsic intracellular guanylate cyclase (GC) domain by which a hormone-induced cGMP intracellular signaling cascade can operate. NPR-C receptors or clearance receptors (molecular weight 60–70 KDa) show a short intracellular domain (37-amino acids) and are coupled to adenylyl cyclase inhibition through a subsequent decrease of intracellular levels of cAMP and/or to phospholipase C (PLC) activation.

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receptors are reported in several tissues such as lung, brain cardiovascular system and in different cell types [3,82].

Immunomodulatory function of ANP Immune cells involved in innate immunity, such as macrophages and neutrophils, provide the first line of defence of the immune system by phagocytosis, cytokine and chemokine synthesis and release, as well as by Reactive Oxygen Species (ROS) generation. Several evidences suggest that ANP is involved in innate immunity being able to stimulate superoxide anion production, leukotriene B4 synthesis and the upregulation of CD11 expression in polymorphonuclear neutrophils (PMN) [14,114]. PMNs represent the main source of soluble neutral endopeptidase (NEP) and by their ability to adhere to hypoxic endothelial cells and release NEP could elicit dangerous effects on vascular cells [38]. NEP through its ability to promote ANP degradation seems involved in ANP clearance [10], and its inhibition represents an important mechanism to stimulate ANP effects on PMNs-vascular cells in vitro in terms of matrix metalloproteinase (MMP)-9 and elastase release, fibronectin degradation and vascular cell apoptosis [74]. Moreover, there is interesting evidence that ANP locally produced can act as a pleiotropic modulator in the regulation of immune response [17]. The ability of several immune cells to produce ANP and – at same time – present specific natriuretic receptors supports this hypothesis [22,68,101,108,111]. ANP, can also contribute to innate immunity by stimulation of macrophage phagocytosis and killing activity by ROS production [12,69,105]. During the inflammation process macrophages, through the activation of the inducible nitric oxide synthase (iNOS), produce and release high amounts of Nitric Oxide (NO) [55]. ANP inhibits Lipopolysaccharide (LPS)-induced NO release in macrophage cells; after ANP binding to NPR-A receptors, the subsequent increase of intracellular levels of cGMP represents an upstream event for the inactivation of Nuclear Factor-kappa B (NF-␬B), an important transcriptional factor involved in the iNOS synthesis; moreover, ANP-induced cGMP production increases intracellular calcium levels in murine macrophages and this seems to be involved in the decrease of iNOS mRNA stability [48,49]. ANP effects on NO release are partly mediated by the involvement on l-arginine transport operated by the cationic amino acid transport (CAT-2B) [50]. It has been reported in macrophages that ANP can counteract the LPS-induced CAT activation and l-arginine transport [77]. On such a basis it is possible to hypothesize that ANP locally released by macrophages can act in an autocrine manner to reduce the NO macrophage synthesis and release thus counteracting the NO-induced dangerous effects. ANP, in several macrophages cell lines can inhibit LPS-induced Tumor Necrosis Factor-alpha (TNF-␣) and Interleukin-1(IL-1) synthesis through transcriptional as well as post-transcriptional mechanisms [44,102]. However, in human THP-1 monocytes stimulated with hydrogen peroxide (H2 O2 ) it has been reported [21] that H2 O2 alone is able to induce a marked release of TNF-␣ and IL-9, whereas ANP can inhibit same pathway. Interestigly, a similar experimental model, ANP was able to stimulate H2 O2 -induced cell death and decrease cell migration. Recently, Zhu et al. [121] have shown that the treatment of ANP on oleic acid-dependent acute lung injury rats induces a marked decrease of different pro-inflammatory markers such as IL-1beta, IL-6, IL-10 and TNF-␣; but histological examination showed an attenuation of the inflammation effects in tissues. Moreover, the hormone can induce important anti-inflammatory effects also in LPS-induced acute lung injury in C57/B6 mice; ANP was able to counteract the LPS-induced E-selectin expression in human pulmonary artery endothelial cells as well as TNF-␣ and IL-6 levels in the bronchoalveolar lavage fluid [99].

ANP inhibits the activation of NF-␬B as well as of Activating Protein-1 (AP-1), two important pro-inflammatory transcriptional factors [102]; the hormone can contribute to inhibit TNF-␣ synthesis by the suppression of the downstream regulatory p38 MAPK [62]. The ANP effects on TNF-␣ synthesis seem mediated by a signaling involving the hormone interaction with NPR-A receptors and the subsequent intracellular increase of cGMP levels [44,102]. Several reports indicate a complicate interaction between vascular endothelial cells and macrophages during the inflammatory processes [100]. ANP, through the NPR-A/cGMP signaling, is able to counteract the TNF-␣-induced expression of adhesion molecules such as E-selectin, Intracellular Adhesion Molecules (ICAM) and Vascular Cell Adhesion Molecules (VCAM) as well as of chemokines such as Chemoattractant Protein-1 (MCP-1) and IL-8; moreover, ANP plays an important role also in the polymerization of Gactin which represents an important step in the TNF-␣-induced cytoskeletal changes [112]. Taken together these observations suggest that following the interaction between endothelial cells and macrophages, ANP can operate in autocrine and/or paracrine manner promoting anti-inflammatory effects. The expression of the enzyme cyclooxygenase-2 (COX-2) in macrophages is an important event during pro-inflammatory processes and Kiemer et al. [46] have shown that ANP, through the interaction with NPR-C receptors and the subsequent decrease of intracellular levels of cAMP inhibits in murine macrophages, LPS-induced COX-2 production. Such effects seem to be dependent on the ANP ability to reduce both the levels of mRNA specific for the inducible form of COX-2 (transcriptional effects) and the quantitative expression of the enzyme (post-transcriptional effects) [46]. The Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase plays a crucial role in host defense and through this multienzyme complex, macrophages can produce high levels of ROS to counteract and kill invading microbes [92]. We have shown that ANP, in human macrophages interacting with its NPR-C receptor, can inhibit the sodium/hydrogen exchanger (NHE) activity, an important modulator of intracellular pH (pHi) [7]. The subsequent ANP-induced pHi decrease is an upstream signal able to induce phosphatidylcholinephospholipase C (PC-PLC) and phospholipase D (PLD) activation. The second messengers produced, DAG and phospatidic acid, can activate NADPH oxidase and promote ROS generation. The hormone effects on pHi, by the involvement of NHE, can be relevant because NHE activity plays a key role in innate as well as adaptative immunity [23]. Natural Killer (NK) cells elicit several activities including the immunoregulation of both B and T cells, natural resistance to tumors, bacterial and viral infection, and transplantation rejection [79]. Moss and Golightly [73] have shown that ANP significantly increases NK cytotoxicity in vitro without increasing the NK cell number. The localization of ANP in lymphoid organs such as thymus, lymphoid follicles of the intestine and spleen strengthens the concept of a possible link between ANP and the lymphoid system [60,61]. Vollmar et al. [104] have shown an increased production of ANP in rat thymus after irradiation. Since it is well known that macrophages play a relevant role in the production of several humoral factors that contribute to proliferation, maturation and differentiation of thymocytes ANP released from macrophages may act as a cytokine affecting the development of thymocytes. ANP is able to inhibit the proliferation of concanavalin A-stimulated thymocytes suggesting that the hormone, in a cytokine-like manner, can contribute to thymus physiology [107]. Thymus is a major site of T-lymphocytes differentiation, a very complicate process involving several events such as receptor gene rearrangement, positive selection for major histocompatibility complex (MHC) restriction and deletion of self reactive cells. T cells are an important component of adaptive immunity, and it has been reported that ANP reduces the number of lymphocytes CD4+ CD8+ as well as increases the CD4− CD8− cells [110]. Interestingly, thymic stromal cells play

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Cytoprotective effects of ANP In several diseases such as renal failure, liver cirrhosis and congestive heart failure plasmatic ANP concentration appears higher with respect to the physiological range, and although the pathophysiological mechanisms causing such increment have not been completely understood, the hormone levels can provide useful information for the diagnosis, prognosis and risk of individual patients, and may be of great help when choosing the most appropriate therapeutic strategy [91]. ANP protective effects on cardiomyocytes

Fig. 3. Immunomodulatory effects of ANP. ANP affects the innate immunity by the inhibition of the expression of TNF-␣, NO and COX-2 as well as by the stimulation of NK activity, neutrophil migration and ROS release. ANP affects the adaptive immunity by the stimulation of: (i) Th2 and or Th17 phenotype in naïve DC; (ii) the release of IL-10 by DCs; (iii) the decrease of the number of lymphocytes CD4+ CD8+ as well as the increase of the CD4− CD8− cells. ANP interferes in the cross talk between immune and endothelial cells by the inhibition of: (i) the expression of adhesion molecules such as E-selectin, V CAM as well as ICAM-1; (ii) the expression of MCP-1 chemokine; (iii) the release of IL-8 cytokine.

a key role during the lymphocyte maturation, and the observation that these cells express ANP receptors [2] supports the hypothesis that locally produced ANP can contribute to lymphocyte maturation. An important cellular component in the modulation of adaptive immunity is given by the Dendritic Cells (DCs), which play an important role as antigen presenting cells and in the induction of primary immune response: it seems to be remarkably important that these cells express ANP receptors [72]. Several cytokines and soluble mediators have been shown to induce differentiation of immature DCs into mature DCs and polarized phenotype toward Th1 or Th2 responses [40] Morita et al. [72] have shown that DCs respond to ANP with an increase of intracellular cGMP in a dosedependent manner, indicating that NPR-A receptors are functional in DCs. Moreover, DCs stimulated with LPS in the presence of ANP decrease the production of IL-12 and TNF-␣ and increase the synthesis of the antinflammatory cytokine IL-10. In accordance with this shift of cytokine production, DCs treated with ANP plus LPS promoted differentiation of naïve CD4+ T cells into Th2 phenotype. Naïve CD4+ T cells besides the classic phenotypes Th1 and Th2 can also differentiate in Th17 (able to produce IL-17) and Treg phenotypes playing a key role in several pathological as well as inflammatory conditions [24]. A role for ANP/NPR-A signaling in DCs has been reported by Zhang et al. [120]; NPR-A activation leads to immune tolerance by the involvement of several transcription factors such as Toll-Like Receptor-2 (TLR-2), Signal Transducer and Activator of Transcription-3 (STAT-3) and Suppressor of Cytokine Signaling 3 (SOCS3) and through a modified expression of IL-6, IL10 and TGF-beta but not IL-12. Morita et al. [71] have shown that plasmacytoid DCs express NPR-A receptors, being the NPR-A/cGMP system involved in the immune regulation also in cells present in secondary lymphoid organs. Libing et al. [63] have shown that ANP through a NRP-A/cGMP signaling that in turn activate a downstream pathway such as PKG/PI3K-Akt, could inhibit the development of murine naive CD4+ T cells maturation in Th17 phenotype. The major ANP effects in innate and adaptive immunity are reported in Fig. 3.

ANP, by the interaction with its NPR-A receptor, elicits protective effects against ventricular hypertrophy; knockout mice, lacking NPR-A receptors, show a severe hypertrophic cardiomyopathy, whereas in genetic models characterized by NPR-A receptor overexpression the hormone can inhibit hypertrophy by the regulation of cardiac size [53]. ANP by a signaling which includes the hormone interaction with NPR-A receptors and the subsequent cGMP production can inhibit Mitogen-Activated Protein Kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways and contribute to the reduction of cardiac hypertrophy through inhibitory effects on cardiac cells growth [78]. Finally, ANP ability to induce apoptosis in neonatal cardiomyocytes by the involvement of NPR-A/cGMP signaling suggests a possible role in the modulation of cardiomyocytes growth and survival [41,116]. It is conceivable that ANP can act as an endogenous regulator of cardiac hypertrophy because mice lacking natriuretic receptors in the heart show markedly elevated ventricular mass and, in cultured neonatal rat ventricular myocytes, ANP through an involvement of cGMP production is able to reduce the phenylephrine-induced increase of cells size [34]. Hayashi et al. [31] have shown that ANP can inhibit the Angiotensin II and Endothelin-induced cardiomyocyte hypertrophy by the activation of mitogen-activated protein kinase phosphatase-1 (MKP-1). An antiproliferative effect of ANP coupled to the decrease of hypertrophyc effects has also been reported in cardiac fibroblasts where the hormone effects seem to operate by the inhibition of endothelin-induced factor transcription GATA4 phosphorylation [27]. Several genes are expressed during the hypoxia, and in particular the activation of ANP gene and the release of the hormone plays an important role in heart adaptation to hypoxia [19]. Generation of ROS, in particular, superoxide anion has been reported for Angiotensin II-induced hypertrophic changes in rat cardiomyocytes, and the ANP ability to reduce the Angiotensin II-induced superoxide release, suggests that the antioxidant property of ANP can contribute to elicit an antihypertrophic effect [87]. However, while in vascular cells and cardiomyocytes ANP stimulates the antioxidant defense, in other systems such as hepatoblastoma and macrophages ANP produces either antioxidant or pro-oxidant effects, depending on experimental conditions and cell context [22]. The hormone can contribute to reduce the cardiomyocyte growth also by inhibition of NHE-1 activity; Kilic et al. [52] have shown that the ANP/cGMP signaling can counteract the excessive activation of NHE-1 and the subsequent increase of pHi, [Ca2+ ]I and Ca2+ /calmodulin-dependent kinase II as well as the increase of Akt activity in response to pressure overload suggesting how to understand the mechanisms by which the hormone can promote antihypertrophic effects in adult cardiomyocytes. A relationship between NHE-1 inhibition and the antihypertrophic effects in cardiomyocytes seems also to be induced by the AMP-activated protein kinase (AMPK)/glycogen syntase kinase 3beta (GSK-3beta), which in turn can reduce several mytochondrial dysfunctions [39]. The transport systems involved in pHi and [Ca2+ ]I homeostasis are essential for cellular viability and their alteration may

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represent an early event after ischemic conditions [30]. NHE may contribute to myocardial injury during ischemia: in fact, the steep H+ gradient across the sarcolemma during the reperfusion which follows ischemic conditions causes a maximal activation of NHE striving to compensate cytosolic acidosis and returning the pHi at physiological values. As a consequence, cardiomyocytes show a massive increase of intracellular Na+ which causes the activation of the Na+ /Ca2+ exchanger with an increase of cytosolic calcium. However, high intracellular Ca2+ levels can cause cellular damage in many different ways: e.g., through the uncoupling of the mitochondrial function, through local cellular hypercontraction, or through arrhythmia that can aggravate the ischemic conditions [36]. Interestingly, ANP-induced NHE inhibition contribute to counteract many of these dangerous effects and, therefore, locally produced the hormone represents a protective mechanism against this serious disease, also supporting the hypothesis that ANP acts as an emergency hormone [25]. Studies concerning ANP effects on the gene promoters involved in cardiomyogenesis has shown that the hormone plays a key role in the expression of several transcription factors involved in cardiac development such as GATA-4, T-box 5 (TBX5) and Homeobox protein NKX2-5 [32]. Lyu et al. [64] has shown that the serum infusion of ANP acts as an adjunctive treatment to counteract the acute myocardial infarction. Similar protective effects have been documented in human trials where the infusion of synthetic forms of ANP usually reported as Anartide elicits defensive effects with respect to heart and renal failure as well as to hypertension [54]. Zhang et al. [119], in a rabbit atrial model, have shown that the hypoxic condition induces a marked increase of ANP secretion and the subsequent expression of the Hypoxia-Inducible Factor 1 (HIF-1) alpha by a mechanism which involves MAPK/ERK and PI3K pathway activation; taken together such events play a key role for the reduction of dangerous effects due to hypoxic conditions. ANP cytoprotective effects on Vascular Smooth Muscle In VSM cell changes of pHi affect several VSM cellular functions such as cell contraction, myosin ATPase activity, the sensitivity of contractile machinery to calcium ions and the transmitter release [115]. Since lactic acid in VSM cells is produced under normal conditions, the acidosis is a common phenomenon in these cells, and one of the main recovery mechanisms is given by the activation of NHE [65]. Previous data from our laboratory have shown that in Rat Aortic Smooth Muscle (RASM) cells ANP can induce a dual modulation of NHE: the hormone at physiological concentrations can activate NHE through a mechanism involving PLC activation and DAG production as well as the decrease of cAMP intracellular levels, whereas at pharmacological concentrations it inhibits NHE by a mechanism involving the increase of cGMP intracellular levels [86]. Since VSM cells can locally produce ANP and contribute to the formation of local microgradients caracterized by high hormone concentrations, it is possible that the hormone, at pharmacological concentrations may plays a physiological role for the recovery from acidosis [11]. During the inflammatory process, the interplay between inflammatory immune cells such as neutrophils, monocytes, macrophages as well as lymphocytes and VSM cells determines an excessive proliferation of VSM cells, a common phenomenon in several diseases such as atherosclerosis and hypertension [96]. We have shown that in RASM cells physiological concentration of ANP can counteract cell growth induced by mitogens such as Platelet-Derived Growth Factor (PDGF) and insulin through a mechanism involving the hormone-induced decrease of phosphatidic acid production and MAP kinases activation [6]. The ANP role in the modulation of VSM cells growth induced by growth factors appears particularly interesting since locally produced ANP could counterbalance the potentially dangerous effect of an excess of cell growth (i.e.,

atherosclerosis). Lysophosphatidic acid (LPA), a lipid mediator with several biological functions is able to induce ROS production and cell growth in RASM cells [5]. Moreover, it was reported that ANP, by the inhibition of LPA-induced PI3K/Akt activation can counteract the excessive RASM cells proliferation and ROS production, indicating this PI3/Akt pathway as a mediator of the antiproliferative effects of the hormone [5]. Oxidative stress, an altered balance between the production of free radicals and antioxidant defenses, plays a role in different pathological processes modifying the activity of several plasma membrane ion transport systems as well as of different enzymes, such as phospholipases [58,59]. ANP has been identified as a protective factor in early stages of myocardial ischemia and its exogenous administration has been proposed in order to counteract diseases characterized by an excessive oxidative stress such as atherosclerosis, hypertension and congestive heart failure [112]. We have shown that the free radical generating system 2-2 Azobis(2-amidinopropane) dihydrochloride (AAPH) in RASM cells is able to induce oxidative effects by the increase of phopholipase D (PLD) activity that, in turn, is responsible for the increase of [Ca2+ ]I and decrease of pHi [20]. However, ANP through the induction of the NPR-A/cGMP pathway, is able to abolish all AAPH-induced effects. Since the shift in [Ca2+ ]I and/or pHi may be considered an early event linked to oxidative stress, these observations suggest a possible role for the hormone as a defensive modulator against oxidative stress. Moreover, ANP effects on PLD activity suggest that the inhibition of PLD could represent a suitable target for the protection of vascular cells against inflammatory cell injury. ANP role on endothelial cells ANP plays a key role also in the modulation of endothelial cell proliferation, since the vascular endothelial growth factor (VEGF)-induced signals linked to endothelial proliferation can be counteracted by cell exposure to ANP [80]. Lysophospatidylcholine (LPC) a major atherogenic lipid reported within the low-density lipoprotein (ox-LDL) fraction induces several endothelial dysfunction; however, pharmacological concentrations of ANP are able to interfere with LPC-induced endothelial cytotoxicity, and this is in good agreement with observations reported for VSM cells suggesting that the hormone may be considered as a local modulator of protective cellular effects [75]. Vascular endothelium regeneration is a complex process involving different steps including vascular endothelial cell activation, proliferation, migration; it represents an important event to counteract the dangerous effects coupled to the development of atherogenic lesions [83]. Physiological concentrations of ANP can induce an increase of human endothelial cell proliferation and migration by the stimulation of a signal pathway including NPR-A/cGMP/PKG activation and downstream modulation of both Akt and p42/44 MAPK, suggesting a possible role for the hormone during the regeneration of endothelial cells after injury [57]. TNF-␣ is a crucial mediator in the pathogenesis of atherosclerosis and, thereby, is an important factor in hypertension [90]. ANP can elicit anti-inflammatory effects in TNF-␣ endothelial cells by the activation of a complex pathway involving NPR-A induced cGMP production, the downstream p38 MAPK inhibition and the over-expression of MKP-1 [51]. Moreover, ANP through a signaling involving a reduced p38 MAPK activity as well as the phosphorylation of the chaperone Heat Shock Protein 27 (HSP27) and G-actin polymerization can operate protective actions with respect to TNF-␣ induced effects on endothelial permeability and cytoskeleton changes [51]. Furst et al. [26] have shown that, in endothelial cells, the ANP-induced Rac1 and NADPH oxidase signaling represents an upstream event coupled to Mitogen-Activated Protein Kinase Phosphatase-1 (MKP-1) over-expression and cytoprotective effects. An inhibitory effect of

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ANP on LPS and TNF-␣ induced endothelial cell permeability, by the attenuation of Rho inflammatory pathway, has been reported in lung endothelial cells suggesting that the hormone plays a relevant role in the preservation of acute lung injury and sepsis [117]. ANP, by increasing intracellular levels of cGMP, stimulates the expression of heme oxygenase-1: a cytoprotective enzyme able to degrade heme which plays a protective role against TNF-␣ induced endothelial cell injury [42]. The monocyte MCP-1 is critically involved in the TNF-␣ induced pro-inflammatory cell recruitment [85]. Weber et al. [113] have shown that ANP by the inhibition of the p38MAPK inhibits the TNF-␣ induced endothelial MCP-1 expression and thus the hormone can operate as an endogenous antiatherosclerotic and anti-inflammatory regulator. ANP induces apoptosis in rat endothelial cells, and such effect can be blocked by a HS-142-1, an inhibitor which selectively antagonizes NPR-A and NPR-B, as well as by KT-5823, a selective inhibitor of PKG [98]. Since the hormone effects are evident only at pharmacological concentrations, the physiological relevance of such observation remains uncertain; however, it is possible that pharmacological concentrations of ANP can be of help during the vascular endothelium exposure to pathological conditions such as heart failure and myocardial infarction. ANP-induced protection in liver ANP-induced cytoprotective effects have also been reported in liver where, during ischemia/reperfusion several factors including ROS and inflammatory cytokines produced by activated Kupffer cells are responsible for the liver damage [29]. Thus, ANP signaling during reperfusion, should be considered as a new pharmacological approach for liver survival against the inflammatory response activated by Kupffer cells [13]. Kiemer et al. [47] have shown that ANP prevents liver damage caused by ischemia/reperfusion through the activation of NPR-A/cGMP signaling and the inhibition of downstream pro-inflammatory effectors such as NF-kB and AP-1 with subsequent decrease of TNF-␣ synthesis and release. ANP, through the induction of the NPR-A/cGMP signaling, can also enhance the resistance of liver during ischemia/reperfusion by the activation of the heat shock transcription factor (HSF), an important factor for the regulation of Heat Shock Protein (HSP) synthesis, able to suppress the induction of proinflammatory mediators such as TNF-␣ and/or NO [43]. In rat hepatocytes ANP prevents calcium-dependent cell injury through the activation of NPR-A/cGMP pathway: the hormone can modulate the intracellular levels of Ca2+ stimulating Ca2+ efflux and partially inhibiting Ca2+ influx, and these mechanisms seem to be able to promote hepatocyte resistance [97]. ANP stimulates hepatocyte tolerance to hypoxia reducing intracellular Na+ accumulation: a consequence of intracellular acidosis [16]. ANP, by the activation of two independent signal pathways mediated by cGMP/PKG and/or Gi protein/PLC/PKC-delta, can activate the downstream p38 MAPK that, in turn, can downregulate NHE and reduce intracellular Na+ accumulation during hypoxia and stimulate the hepatocyte tolerance to hypoxia. The differential role of p38 MAPK and c-Jun N-Terminal Kinases (JNK) activities, during rat liver ischemia/reperfusion, has been investigated by Kiemer et al. [45]: p38MAPK activity decreases through the whole course of ischemia and reperfusion, whereas JNK appear only activated during reperfusion. In particular, during liver preincubation with ANP, the hormone exerts a tremendous effect on p38MAPK activity with an increase up to 30-fold, thus counteracting the liver disease. Therefore, ANP contributes to liver survival during ischemia/reperfusion by the induction of anti-apoptotic effects following the decrease of caspase-3 activity during reperfusion, due to the induction of NPR-A/cGMP/PKA and/or ANP/NPR-A/PI3K pathways [28,33]. Finally, the continuous intravenous infusion of ANP decreases the expression of several factors including type 1 procollagen, metalloproteinase such as Tissue Inhibitor of

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Metalloproteinase (TIMP-1-2) and the alpha-smooth muscle actin reported as alpha-SMA, thus eliciting a useful capacity to suppress liver fibrosis [37]. ANP effects on tumor cells Among different biological effects, in hepatoblastoma (HepG2) cells, ANP inhibits cell proliferation through the selective upregulation of the expression of the NPR-C receptors associated with decreased intracellular concentration of cAMP [84]. Vollmar et al. [106] have shown that patients with hepatic tumors express about 5.5 fold higher mRNA concentrations of ANP, CNP and their receptors in tumor with respect to normal tissues. The presence of natriuretic peptide hormones and their receptors in hepatic tissue suggests that they may have autocrine or paracrine functions. However, it is uncertain whether the ANP expression in tumor tissues can represent the ability of these cells to produce the hormone or whether it derives from endothelial cells which are highly presented in tumor areas. The speculation about a local involvement of Natriuretic Peptides hormones in tumor development clearly needs further investigation. In cancerous tissues, extracellular microenvironment is more acidic than in normal tissues and NHE-1 concurs to minimize pHi and to maintain tumor cell viability [35]. In hepatic tumors, the presence of high levels of ANP, as well as of its receptors, could be relevant to understand the possible link between ANP and NHE activity in liver cells survival. Our laboratory has shown a significant pHi decrease in ANP-treated HepG2 cells with an enhancement of PLD activity and ROS production [8]. The ANP effect was coupled with: (i) an increase of p38 MAPK phosphorylation; (ii) a down regulation NHE-1 expression (in terms of both specific mRNA and protein). Because such effects were mimicked by cANF, the NPR-C specific ring-deleted analog of ANP, it is possible to suppose that NPR-C receptors play a role in ANP-induced ROS production. Moreover, in HepG2 cells, the hormone activates the NADPH oxidase and the increasing ROS levels can inhibit the caspase 3-enzyme and switch cell death from apoptosis to necrosis [4]. This effect is not in contrast with data showing an ANP cytoprotective action in liver cells following ischemia-reperfusion injury [43], because the spectrum of biological actions of ANP appears to follow different pathways depending on cell phenotype [6,18]. For example, cells which are known to undergo phenotypic modulation, such as those observed in “vivo” atheromatous lesions and cancer cell such as HepG2, are highly sensitive to ANP [18,84] because they express a high number of NPR-C receptors. Therefore, it is reasonable to hypothesize that the different viability of hepatoblastoma cells (HepG2) and hepatocytes could be dependent on the presence of a threshold signal, linked to the higher expression of NPR-C receptors and increased intracellular ROS level. This consideration may be relevant in different pathological conditions, such as atherosclerosis and tumors, in which a strong resistance to apoptosis is observed and necrotic death represents the only pathway to eliminate pathological cells. An involvement of NPRC receptors in the antiproliferative effects observed in tumors cells has also been reported in metastatic murine (B16-F10) and human (SK-MEL 110) cells lines where the hormone, at physiological concentrations, seems to act by the induction of a decrease of polyamine levels as well as by the enhancement of the transglutaminase enzyme activity [9]. These findings are particularly interesting in light of a possible use of ANP as a potential selective antineoplastic agent. The hormone, in fact, is able to eliminate up to 86% of cells in human small-cell lung carcinomas, two thirds of cell in human breast cancer, and up to 80% of cells in human pancreatic adenocarcinoma cells [103]. ANP, after the interaction with specific receptors on cancer cells can inhibit several pathway such as RAS/MAPK, cJun terminal kinase, Wnt/␤-catenin, thus

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can be considered an important hormone for the development of new therapeutic strategies. The major ANP–induced cytoprotective effects are reported in Fig. 4.

Concluding remarks

Fig. 4. ANP-induced cytoprotective effects. ANP elicits cytoprotective effects in: (i) cardiac cells, by the inhibition of ventricular hypertrophy as well as myocardial injury; (ii) Vascular Smooth Muscle cells (VSMc), by the inhibition of cell growth during atherosclerotic disease; (iii) endothelial cells, by the inhibition of the expression of adhesion molecules, cytoskeletal and permeability changes, and cell proliferation; (iv) liver counteracting the dangerous effects of the ischemia/reperfusion; (v) tumor cells by the induction of apotosis or necrosis.

decreasing the gene expression leading to malignancy [103]. Interestingly, whereas the hormone can induce cell death in pancreatic adenocarcinoma cells and prostate cancer cells, ANP cannot induce cell death in normal prostate, kidney as well as in lung cells [95]. Recently, Zhang et al. [118] have shows that ANP is able to modulate the proliferation of human gastric cancer cells via potassium voltage-gated channel member 1 (KCNQS) expression. In several tumor diseases specific glycoproteins called frizzeled-related proteins (sFRPs), are released in the tumor microenvirement and elicit an important role in the tumor growth; Skelton et al. [95] have shown that ANP is a strong inhibitor of sFRP-3 in different human cancer cells, suggesting that the hormone, at least partially elicits antitumor activity by such mechanism. ANP and NPR-A are highly expressed in inflammatory and tumor tissues [56], and interestingly, the NPR-A deficiency markedly decreases the inflammation as well as tumors growth but the mechanisms involved are not completely understood [56]. Malella et al. [66] have shown that in NPR-A–KO mice at variance with the wild type C57BL/6 mice after the infusion of mesenchymal stem cells (MSCs) these are not able to recruit in the tumor microenviroment and at same time also the angiogenesis and tumorogenesis appear decreased. On the basis of these data it is possible to suppose that the NPR-A signaling manipulation provides an important step in the crosstalk between inflammation and tumor development and could represent a key target for tumor therapy. Moreover, it have been shown that ANP and other cardiac hormones such as long-acting natriuretic peptide (LANP), vessel dilators and kaliuretic peptide (KP) inhibit the growth of several tumor cell lines such as human hepatocellular cancer, small-cell lung cancer and renal adenocarcinoma, through a signaling pathway which include the inhibition of RAS-MEK 1/2ERK1/2 kinase cascade that, in turn, through the involvement of STAT-3 induces the inactivaction of oncogenes such as c-FOS and c-Jun [66,67]. Recently, Serafino et al. [93] have shown that ANP can induce antiproliferative effects on colorectal cancer cells by an Aktmediated cross talk between NHE-1 and Wnt signaling, suggesting that Akt activity may be an important target in a new strategy for the development of novel antitumor therapy. The ANP ability to inhibit cancer cell growth by a mechanism that include Wnt/Betacatenin pathway and pHi regulation was also been reported by Serafino and Pierimarchi [94] and such evidences suggest that ANP

Studies carried out in the last 20 years support the hypothesis that ANP plays a relevant role in the complex network linking cardiovascular and immune system. Although fascinating, the hypothesis that ANP released by immune cells can act as autocrine/paracrine effector able to counteract the excessive proliferation of vascular cells as well as the growth of cancer cells needs further evidences. However, the ANP ability to decrease synthesis and release of proinflammatory cytokines and, at the same time, to reduce the expression of several adhesion molecules with a pivotal role during the inflammation process, suggest that the hormone can act as an anti-inflammatory agent. Moreover, the ANP-induced antimitogenic effects and death in cancerous cells, without any interference with normal cells, stimulates research about new strategy for the development of novel anticancer therapy. At the same time, ANP-dependent NHE-1 inhibition can represent an important upstream target to modulate immune response and cell growth and may help to clarify the role of NHE-1 in the complex interrelationship between cardiovascular system and immune system. In conclusion it is possible speculate that locally released ANP can act in a cytokine-like manner in order to operate antinflammatory and antiproliferative effects thus representing an important effector to preserve different cell types in pathological condition.

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