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Journal of Investigative Medicine:
doi: 10.231/JIM.0b013e3181948b37
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Cardiac Natriuretic Peptides Gene Expression and Secretion in Inflammation

Vesely, David L. MD, PhD; de Bold, Adolfo J. PhD

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Author Information

From the Cardiovascular Endocrinology Laboratory, University of Ottawa Heart Institute, Ottawa, Ontario, Canada.

Received September 3, 2008, and in revised form October 3, 2008.

Accepted for publication October 3, 2008.

Reprints: Adolfo J. de Bold, PhD, Cardiovascular Endocrinology Laboratory,University of Ottawa Herat Institute, 40 Ruskin St., Ottawa,Ontario K1Y 4W7, Canada. E-mail: adebold@ottawaheart.ca.

Cardiac natriuretic peptides gene expression and secretion in inflammation: Erratum

Dr. Vesely should not have been included as an author. The authorship was only Dr. de Bold's.

Reference:

Vesely DL, de Bold AJ: Cardiac natriuretic peptides gene expression and secretion in inflammation. J Investig Med. 2009;57(1):29-32

Should have read:

de Bold AJ: Cardiac natriuretic peptides gene expression and secretion in inflammation. J Investig Med. 2009;57(1):29-32

This erratum is published in the August 2009 issue of the journal.

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Abstract

The genetic expression and secretion of the cardiac polypeptide hormones atrial natriuretic factor (ANF or ANP) and brain natriuretic peptide (BNP) have been studied mainly in the context of cardiac diseases associated with neuroendocrine and hemodynamic changes arising from cardiac dysfunction such as in chronic congestive heart failure. In this type of pathology, both ANF and BNP plasma levels change in an approximate coordinated fashion so that the use of these hormones as biomarkers of cardiac disease is, in principle, indistinctive. However, we reported that during an acute cardiac allograft rejection episode, BNP plasma levels can significantly increase in the absence of a similar increase in ANF plasma levels. We tested the hypothesis that these changes were related to cytokines and found that some pro-inflammatory cytokines, including TNFα and IL-1β, selectively promote BNP synthesis and secretion in cultures of neonatal rat ventricular cardiocytes. This effect was found related to increased BNP promoter activity and sensitive to p38 mitogen-activated protein kinase inhibition.

In order to determine in vivo if the selective up-regulation of BNP would be observed in inflammatory processes other than acute cardiac allograft rejection, we carried out investigation using the experimental autoimmune myocarditis rat model, which histologically resembles human giant cell myocarditis. It was found that this model is also accompanied by a specific increase in BNP-circulating levels although the cytokines involved seem to differ from those characterized earlier through in vitro studies.

Recent studies in humans have found that in sepsis, plasma BNP levels increase in the absence of hemodynamic changes.

In conclusion, BNP appears to be regulated uniquely in the setting of an inflammatory process. This sets it apart from ANF in terms of potential roles in the pathogenesis of disease and in its use as a biomarker of cardiac disease.

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CARDIAC NATRIURETIC PEPTIDE GENE EXPRESSION AND SECRETION

Cardiac muscle cells of the heart atria in mammals produce and secrete in a regulated manner the polypeptide hormones atrial natriuretic factor (ANF or ANP) and brain natriuretic peptide (BNP). These hormones are referred to as cardiac natriuretic peptides (NP). The ventricular cardiocytes do not produce appreciable amounts of these hormones except during fetal life, when ventricular cardiocytes display a secretory phenotype. After birth, this phenotype is rapidly down-regulated at both the morphological and functional levels. As a result, the amount of ANF and BNP found in the adult ventricles is several orders of magnitude smaller that in the atria. The statement that is often repeated in the clinical literature asserting that BNP is a ventricular hormone, whereas ANF is of atrial origin is baseless (for reviews, see1,2).

The pathological expression and secretion of cardiac NP was first studied with respect to ANF in situations of chronic hemodynamic overload.3,4 These early studies led to many others that established that ANF might be used as a biomarker. The realization that BNP behaved similarly to ANF-a fact to be expected because both ANF and BNP are costored and secreted from the same storage granules-established both cardiac NP as biomarkers for clinical entities involving cardiac dysfunction. The relationship of NP with ventricular dysfunction should not obscure the fact that both ANF and BNP of atrial origin are by far the most up-regulated NP during chronic hemodynamic overload. Ventricular NP expression, although increased as part of the reexpression of fetal genes observed during the ventricular hypertrophic process, remain modest compared with atrial expression.5

The fact that BNP is the peptide most widely used as a cardiac biomarker, although demonstrated to be marginally superior to ANF, perhaps has to do more with commercialization of tests than with superiority over the measurement of plasma ANF with the same purposes. Nevertheless, the biggest significance of NP plasma values as an inexpensive screening method was demonstrated by BNP measurements and their ability to discriminate between symptoms arising from heart failure and those arising from other sources like lung pathologies with high sensitivity and specificity.6 Many other applications have since been developed or suggested for either BNP or ANF as biomarkers or as therapeutic agents.

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NP IN ACUTE CARDIAC ALLOGRAFT REJECTION

Conceptually central in the previous notions is the fact that the increases in NP gene expression and secretion observed in physiological and pathophysiological situations affect both ANF and BNP in a similar manner. That is, cardiac NP gene expression and secretion change in a coordinated manner. This concept suffered its first exception after the studies of NP plasma levels during cardiac allograft acute rejection in humans.

During studies carried out 15 years ago concerning elevated NP plasma levels observed in heart transplant recipients, we found that ANF did not normalize after transplantation from the elevated levels found in advanced heart failure before transplantation. The elevated circulating levels of ANF persisted even after intracardiac pressures and the renin-angiotensin-aldosterone system normalized. Altogether, these findings suggested that factors other than hemodynamic ones or activation of renin-angiotensin-aldosterone system or sympathetic nerve activity were at play in the observed elevation of NP circulating levels in transplant recipients.7

Later, we found that not only ANF but also BNP continued to be highly elevated.8,9 Furthermore circulating levels of BNP but, importantly, not ANF, increased in most patients that were about to or were suffering from an acute rejection episode. Successful treatment of the rejection episodes with various immunosuppressive agents resulted in a decrease of BNP plasma levels together with an improvement of the grade of endomyocardial biopsies suggesting that activated T-lymphocyte secretion products during a rejection episode modulate cardiocyte BNP secretion.8 This effect was not merely due to an improvement in hemodynamic parameters because ANF plasma levels did not show the same changes.

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MODULATION OF NP GENE EXPRESSION AND SECRETION BY CYTOKINES

Given that the changes in BNP plasma levels occurred in a context of an acute inflammatory process, we tested whether pro-inflammatory cytokines were capable of selectively up-regulating BNP gene expression and secretion in vitro. We found that specific cytokines (TNFα and IL-1β) up-regulate BNP at the transcriptional and translational levels through a pathway using p38 signaling.10 These findings made utilizing neonatal ventricular cardiocytes in culture provided evidence that the changes specifically affecting BNP secretion can occur independently of hemodynamic influences.

Although we were able to reproduce the specific up-regulation of BNP gene expression during acute cardiac rejection by exposing cardiocyte cultures to TNFα and IL-1β,10 and elucidated the signaling pathway of this phenomenon, our clinical studies9 did not show an increase of TNFα and IL-1β in plasma during periods of acute rejection in conjunction with the elevation of BNP plasma levels. However, by reverse transcriptase polymerase chain reaction as well as by immunocytochemistry, several observations indicate that there is an enhanced gene expression of both the proinflammatory cytokines IL-1β, IL-6, and TNF-β and the immunoregulatory cytokines IL-2, IL-4, and IFN-γ in the graft during the acute phase of cardiac allograft rejection.11 Hence, there are 2 possibilities that may help explain our findings. First, cytokines are local mediators and plasma levels do not accurately reflect in situ levels at the graft (ie, lack of sufficient cardiac spillover to be reflected peripherally).12,13 Second, cytokines other than or in addition to TNFα and IL-1β participate in the observed up-regulation of BNP during allograft rejection.

In order to address the possibility that cytokines other than or in addition to TNFα and IL-1β participate in the observed up-regulation of BNP during allograft rejection, we used a proteomic approach using cytokine arrays and plasma samples from rejecting and nonrejecting patients. Regulated on activation, normal T expressed and secreted (RANTES), neutrophil-activating protein-2 (NAP-2), and insulin growth factor binding protein-1 (IGFBP-1) had significant correlations with BNP plasma levels during acute allograft rejection as diagnosed by endomyocardial biopsy. Furthermore, in rat neonatal ventricular cardiocyte cultures, IGFBP-1 and RANTES were capable of inducing BNP, but not ANF secretion. In a similar manner to findings made with TNFα and IL-1β, the effect of IGFBP-1 and RANTES was abolished by the specific p38 mitogen-activated protein kinase inhibitor SB203580.

Altogether, these findings suggest that cytokines other than proinflammatory can specifically promote BNP secretion and that they share a common p39 mitogen-activated protein kinase signaling pathway.

We previously showed that conditioned medium from mixed lymphocyte reaction (MLR) cultures significantly stimulated BNP secretion from neonatal rat ventricular cardiocyte cultures.10 We used this approach to replicate the conditions seen during transplant rejection using an established in vitro model of transplantation immunity. The increase was specific for BNP. Atrial natriuretic factor secretion was not altered. Cytokines that have been found in MLR conditioned medium include IL-1β, TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, and LIF.14-23 The nature of cytokines that are secreted in an MLR, however, seems to vary according to species. Indeed, in our MLR experiments using Brown Norway and Wistar rat lymphocytes, we did not detect IL-1β or TNF-α.

The presence of the BNP cognate receptor has been demonstrated in cells of the immune system including lymphocytes and ligands to the NPR-A receptor induce production of cGMP as expected.24,25 Our unpublished observations confirm the expression of all 3 NP receptors in lymphocytes and clearly indicate that all 3 NP are potentially able to interact with the proliferative response in MLRs. Indeed, we have observed that all 3 NP can modulate proliferation in the MLR.

Our studies on BNP gene regulation suggest that the differential regulation of ANF and BNP and, by extension, their participation in some biological processes, is determined at the transcriptional level.10 This finding is based on the fact that the increase in steady-state BNP messenger RNA caused by TNF-α or IL-1β was prevented by actinomycin D, suggesting an effect of cytokines at the transcriptional level. We also demonstrated that IL-1β and TNF-α can stimulate a -2.2 kbp rat BNP proximal promoter and that pharmacological inhibition of p38 MAP kinase can completely suppress this effect. The precise 5' cis-acting regulatory regions on the BNP promoter responsible for this phenomenon have not been identified, but it has been demonstrated that IL-1β can target a muscle-specific cytidine-adenosine-thymidine sequence element (-97 bp) in the proximal human BNP promoter.26 Although ablation of the muscle-specific cytidine-adenosine-thymidine sequence element reduces IL-1β-mediated BNP expression, the large proportion of activity still remaining implies that other uncharacterized factors are involved in mediating the increased BNP transcription. Other proximal enhancer elements such as the GATA elements do not appear to be involved in IL-1β-induced increase in BNP transcription.26

Another mediator of inflammation, lipopolysaccharide (LPS) can also regulate the expression of the BNP gene. The release of LPS from gram-negative bacteria results in an acute inflammatory response that can cause an increase in myocardial depression accompanied by profound changes in cardiac gene expression.27 Investigations have shown that LPS significantly up-regulates BNP messenger RNA in rat heart. Transfection experiments with a -1000-bp rat BNP promoter have shown that LPS targets the GATA elements located in the proximal rat BNP promoter to increase activity by 2.1-fold.28 However, when the promoter is truncated to -112-bp, the LPS-induced activity increased to 4.1-fold, hinting at the existence of important uncharacterized negative regulatory elements between -112 bp and -1000 bp.

Altogether, these findings suggest that different cis-acting elements in the BNP promoter can participate in the up-regulation of BNP in inflammation and that 1 common pathway appears to be signaling through p38.

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NP IN AN ANIMAL MODEL OF MYOCARDITIS

The question that arises from the previously mentioned study relates to whether the increase in BNP expression and secretion observed can be reproduced in models of myocarditis other than that observed during acute cardiac allograft rejection. We have used experimental autoimmune myocarditis in rats to determine if the discoordinate regulation of ANF and BNP is observed in such pathology that histologically resembles giant cell myocarditis.29 Experimental autoimmune myocarditis specifically increased BNP, but not ANF circulating levels, thus resembling findings made in acute cardiac allograft rejection and the effect of some proinflammatory cytokines on cardiocyte cultures, suggesting that inflammatory processes are generally associated with the disregulation of cardiac ANF and BNP synthesis and secretion.

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THE EFFECT OF INFLAMMATION ON THE USE OF NP AS BIOMARKERS OF DISEASE

Given the previously mentioned findings, it is to be expected that BNP, when used as a biomarker, might be modulated not only by hemodynamic parameters but also by inflammatory processes. Hence, the elevation of BNP plasma values in clinical entities such as chronic congestive heart failure, which is known to be associated with inflammation,30 may partly reflect this process. Conceivably related to this possibility, recent literature point to elevation in BNP plasma values in sepsis in the absence of hemodynamic changes.31-39 There is a scarcity of data regarding the values for ANF plasma levels in these circumstances. Perhaps the concomitant determination of these levels may provide a measure of volume load independently of the effect of cytokines on BNP plasma levels.

In conclusion, the inflammatory process affects uniquely NP gene expression and secretion. Changes induced by cytokines appear specific to BNP and can be independent of hemodynamic status. This concept is one to take into account when using NP as biomarkers.

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REFERENCES

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2. de Bold AJ. Determinants of brain natriuretic peptide gene expression and secretion in acute cardiac allograft rejection. Curr Opin Cardiol. 2007;22:146-150.

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5. Yokota N, Bruneau BG, Fernandez BE, et al. Dissociation of cardiac hypertrophy, myosin heavy chain isoform expression, and natriuretic peptide production in DOCA-salt rats. Am J Hypertens. 1995;8:301-310.

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7. Masters RG, Davies RA, Keon WJ, et al. Neuroendocrine response to cardiac transplantation. Can J Cardiol. 1993;9:609-617.

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9. Ogawa T, Veinot JP, Davies RA, et al. Neuroendocrine profiling of humans receiving cardiac allografts. J Heart Lung Transplant. 2005;24:1046-1054.

10. Ma KK, Ogawa T, de Bold AJ. Selective upregulation of cardiac brain natriuretic peptide at the transcriptional and translational levels by pro-inflammatory cytokines and by conditioned medium derived from mixed lymphocyte reactions via p38 MAP kinase. J Mol Cell Cardiol. 2004;36:505-513.

11. Duquesnoy RJ, Demetris AJ. Immunopathology of cardiac transplant rejection. Curr Opin Cardiol. 1995;10:193-206.

12. Azzawi M, Grant SD, Hasleton PS, et al. TNF alpha mRNA and protein in cardiac transplant biopsies: comparison with serum TNF alpha levels. Cardiovasc Res. 1996;32:551-556.

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15. Leenaerts PL, Ceuppens JL, Van Damme J, et al. Evidence that stimulator cell-derived IL-6 and IL-1 are released in the mixed lymphocyte culture but are not requisite for responder T cell proliferation. Transplantation. 1992;54:1071-1078.

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18. Baan CC, van Emmerik NE, Balk AH, et al. Cytokine mRNA expression in endomyocardial biopsies during acute rejection from human heart transplants. Clin Exp Immunol. 1994;97:293-298.

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20. van Emmerik N, Baan C, Vaessen L, et al. Cytokine gene expression profiles in human endomyocardial biopsy (EMB) derived lymphocyte cultures and in EMB tissue. Transpl Int. 1994;7 Suppl 1:S623-S626.

21. Bishara A, Malka R, Brautbar C, et al. Cytokine production in human mixed leukocyte reactions performed in serum-free media. J Immunol Methods. 1998;215:187-190.

22. Kohka H, Iwagaki H, Yoshino T, et al. Involvement of interleukin-18 (IL-18) in mixed lymphocyte reactions (MLR). J Interferon Cytokine Res. 1999;19:1053-1057.

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24. Vollmar AM. The role of atrial natriuretic peptide in the immune system. Peptides. 2005;26:1086-1094.

25. Kurihara M, Katamine S, Saavedra JM. Atrial natriuretic peptide, ANP(99-126), receptors in rat thymocytes and spleen cells. Biochem Biophys Res Commun. 1987;145:789-796.

26. He Q, LaPointe MC. Interleukin-1beta regulation of the human brain natriuretic peptide promoter involves Ras-, Rac-, and p38 kinase-dependent pathways in cardiac myocytes. Hypertension. 1999;33:283-289.

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29. Ogawa T, Veinot JP, Kuroski de Bold ML, et al. Angiotensin II Receptor Antagonism Reverts the Selective Cardiac BNP Upregulation and Secretion Observed in Myocarditis. Submitted for publication, 2008.

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32. Brueckmann M, Huhle G, Lang S, et al. Prognostic value of plasma N-terminal pro-brain natriuretic peptide in patients with severe sepsis. Circulation. 2005;112:527-534.

33. Burjonroppa SC, Tong AT, Xiao LC, et al. Cancer patients with markedly elevated B-type natriuretic peptide may not have volume overload. Am J Clin Oncol. 2007;30:287-293.

34. Castillo JR, Zagler A, Carrillo-Jimenez R, et al. Brain natriuretic peptide: a potential marker for mortality in septic shock. Int J Infect Dis. 2004;8:271-274.

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36. Nikolaou NI, Goritsas C, Dede M, et al. Brain natriuretic peptide increases in septic patients without severe sepsis or shock. Eur J Intern Med. 2007;18:535-541.

37. Roch A. What does high NT-proBNP mean in septic shock patients? A part of the puzzle. Crit Care. 2007;11:122.

38. Rudiger A, Gasser S, Fischler M, et al. Comparable increase of B-type natriuretic peptide and amino-terminal pro-B-type natriuretic peptide levels in patients with severe sepsis, septic shock, and acute heart failure. Crit Care Med. 2006;34:2140-2144.

39. Shor R, Rozenman Y, Bolshinsky A, et al. BNP in septic patients without systolic myocardial dysfunction. Eur J Intern Med. 2006;17:536-540.

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Keywords:

cardiac natriuretic peptides; gene expression; inflammation; cytokines

© 2009 American Federation for Medical Research

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