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Circulating nucleosomes in chronic heart failure
International Journal of Cardiology 203 (2016) 742–743
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Correspondence
Circulating nucleosomes in chronic heart failure S.H. Nymo a,⁎, T. Ueland a, E. Askevold a, C.P. Dahl a, L. Gullestad b, P. Aukrust a, A. Yndestad a a b
Research Institute of Internal Medicine, Oslo University Hospital, Norway Department of Cardiology, Oslo University Hospital, Oslo, Norway
a r t i c l e
i n f o
Article history: Received 7 July 2015 Received in revised form 6 October 2015 Accepted 27 October 2015 Available online 28 October 2015 Keywords: Heart failure Nucleosomes Biomarker Circulating DNA Inflammation
Chronic heart failure (HF) is a progressive disorder characterized by a persistent, low-grade, systemic and myocardial inflammation that correlates with disease severity and prognosis. While the cause of this low grade inflammation is currently unknown, endogenous danger associated molecule-patterns (DAMPs), may be a contributing factor [1]. Among structures identified as a potential trigger of endogenous inflammation is the nucleosome, a chromatin structure consisting of at least eight histones wrapped by double stranded DNA. Several studies have shown that nucleosomes may activate the immune system, and generate an inflammatory response, but so far, there is to best of our knowledge, no data on extracellular nucleosome in HF [2–5]. Nucleosomes are normally separated from the inflammatory system in the nucleus of cells. However, in certain situations these structures may be released into the extracellular space and find their way to the circulation. Firstly, during apoptosis, DNA is cleaved into nucleosomes as a part of the apoptotic process, and if not properly digested by macrophages, these nucleosomes may be released into their surroundings [3]. Secondly, upon activation, neutrophils in the circulation may release their cellular DNA as neutrophil extracellular traps (NET)-like formations [6]. These NETs consist mainly of DNA with the histone core preserved, as well as a range of enzymes. Break down of NETs by circulating DNases may release the nucleosome component from the NETs into the extracellular space or the circulation [7]. Nucleosomes are increased in the circulation in a number of diseases, such as sepsis, ischemia–reperfusion injury and stroke [8]. In ⁎ Corresponding author. E-mail address: [email protected] (S.H. Nymo).
http://dx.doi.org/10.1016/j.ijcard.2015.10.219 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
this study, we hypothesized that nucleosomes could also be a potential DAMP in chronic HF, and that the levels of circulating nucleosomes could be a prognostic marker for these patients. To evaluate this hypothesis we measured the concentration of circulatory nucleosomes in the plasma of 179 patients with chronic HF of New York Heart Association (NYHA) functional classes II–IV, stable for at least 6 months, described elsewhere [9]. Patients were followed up for a median time of 38 months (interquartile range 26–46 months), and 30 patients died during the follow-up. Nucleosome levels were measured using a cell death detection ELISA from Roche (Indianapolis, USA). As standard curve, a stepwise dilution of a nucleosome rich solution isolated from HepG2 cells were used, and concentration was measured in arbitrary units (AU). Intra- and inter-assay variations were b10% and b15%, respectively. For all baseline variables, the relationship between nucleosomes and other variables were tested using Pearson correlation or Kruskal–Wallis test as appropriate. All survival analyses were performed using the Cox proportional hazard regression model. Nucleosome levels were divided into tertiles and included in the Cox model as a group variable. NTproBNP and CRP where included as log-transformed variables. A twosided P-value b 0.05 was considered to be significant. The median nucleosome level in HF patients was 2.56 AU (interquartile range [IQR] 1.06–6.44). Baseline nucleosome levels were higher in men (median 3.42 AU (IQR 1.17–8.26) vs 1.82 AU (0.76–2.7), p = 0.019), and in patients with higher NYHA class (median 1.17 AU (IQR
Fig. 1. Survival of patients with chronic heart failure according to levels of circulating nucleosomes. T: tertile.
Correspondence
743
Table 1 Survival according to levels of circulating nucleosomes. Variable Nucleosomes Tertile 2 Tertile 3 Age (per 10 year) Female gender NYHA-class Class III Class IV eGFR EF CRP NT-proBNP
Unadjusted
Model 1
Model 2
2.87(0.91–9.01) p = 0.071 4.16(1.38–12.54) p = 0.011
3.37 (1.06–10.69)p = 0.039 5.37 (1.73–16.70)p = 0.004 1.62(1.14–2.31)p = 0.007 2.34(1.04–5.26)p = 0.039
4.78 (0.87–26.21)p = 0.071 6.37 (1.24–32.67)p = 0.026 1.78(1.11–2.89)p = 0.019 2.77(1.04–7.37)p = 0.041 0.93(0.175–4.91)p = 0.931 2.47(0.48–12.69)p = 0.279 0.993(0.97–1.01)p = 0.441 1.03(1.01–1.07)p = 0.022 1.46(0.63–3.39)p = 0.369 2.38(0.84–6.80)p = 0.104)
All values listed as hazard ratio (95% confidence interval) p-value. NYHA-class, EF: ejection fraction, eGFR estimated glomerular filtration rate, CRP: C-reactive protein and NT-proBNP: Nterminal-pro brain natriuretic protein. All significant values are shown in bold.
0.58–7.85), 2.93 AU (1.33–5.90) and 4.45 AU (2.28–9.02) for NYHA classes II–IV, respectively, p = 0.01 for difference between NYHA II and IV). Baseline nucleosome levels were also associated with systemic inflammation (CRP; Spearman correlation r = 0.29, p b 0.001) and increased myocardial wall stress as assessed by NT-proBNP (r = 0.16, p = 0.028), but not associated with left ventricular ejection fraction (LVEF; r = −0.07, p = 0.38), or kidney function (eGFR; r = −0.07, p = 0.33). Patients with nucleosome levels in the second and third tertiles had higher risk of mortality than patients with lower nucleosome levels (HR 2.87, p = 0.07 and HR 4.16, p = 0.011 for the 2nd and 3rd tertiles, respectively, Fig. 1), also following multivariable adjustment (Table 1). Nucleosomes can be released from apoptotic and necrotic cells, as well as being an effect of increased neutrophil activity and NET production. We demonstrate that in patients with chronic HF, circulating nucleosome levels are associated with inflammation, disease severity and mortality. While circulating nucleosomes seems to be a feature of several conditions involving the inflammatory system, the extent to which nucleosomes merely circulate as markers of damaged tissue, or also represent a driving force of the inflammatory reaction that follows is not known. While nucleosomes may activate several inflammatory cells such as neutrophils and dendritic cells through interaction with membrane receptors, this is mainly studied in vitro, and may not be the case in more complex systems [3]. Our study show that circulating nucleosomes are present in chronic HF, correlate with disease severity, and could therefore be a potential player in the low grade inflammation seen in these patients. As this is a small study, with relatively few events, the data should be interpreted with caution. Our findings suggest that nucleosomes could be a potential biomarker in chronic HF, but larger studies are needed to confirm this.
Conflict of interest The authors report no relationships that could be construed as a conflict of interest.
References [1] Y. Zheng, S.E. Gardner, M.C. Clarke, Cell death, damage-associated molecular patterns, and sterile inflammation in cardiovascular disease, Arterioscler. Thromb. Vasc. Biol. 31 (2011) 2781–2786. [2] J. Xu, X. Zhang, R. Pelayo, M. Monestier, C.T. Ammollo, F. Semeraro, et al., Extracellular histones are major mediators of death in sepsis, Nat. Med. 15 (2009) 1318–1321. [3] V. Urbonaviciute, B.G. Fürnrohr, S. Meister, L. Munoz, P. Heyder, F. De Marchis, et al., Induction of inflammatory and immune responses by HMGB1–nucleosome complexes: implications for the pathogenesis of SLE, J. Exp. Med. 205 (2008) 3007–3018. [4] D.S. Pisetsky, The origin and properties of extracellular DNA: from PAMP to DAMP, Clin. Immunol. 144 (2012) 32–40. [5] H. Huang, J. Evankovich, W. Yan, G. Nace, L. Zhang, M. Ross, et al., Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice, Hepatology 54 (2011) 999–1008. [6] T.A. Fuchs, U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn, et al., Novel cell death program leads to neutrophil extracellular traps, J. Cell Biol. 176 (2007) 231–241. [7] M.J. Kaplan, M. Radic, Neutrophil extracellular traps: double-edged swords of innate immunity, J. Immunol. 189 (2012) 2689–2695. [8] S. Holdenrieder, P. Stieber, Clinical use of circulating nucleosomes, Crit. Rev. Clin. Lab. Sci. 46 (2009) 1–24. [9] T. Ueland, A. Svardal, E. Oie, E.T. Askevold, S.H. Nymoen, B. Bjorndal, et al., Disturbed carnitine regulation in chronic heart failure — increased plasma levels of palmitoylcarnitine are associated with poor prognosis, Int. J. Cardiol. 167 (2013) 1892–1899.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Correspondence
Circulating nucleosomes in chronic heart failure S.H. Nymo a,⁎, T. Ueland a, E. Askevold a, C.P. Dahl a, L. Gullestad b, P. Aukrust a, A. Yndestad a a b
Research Institute of Internal Medicine, Oslo University Hospital, Norway Department of Cardiology, Oslo University Hospital, Oslo, Norway
a r t i c l e
i n f o
Article history: Received 7 July 2015 Received in revised form 6 October 2015 Accepted 27 October 2015 Available online 28 October 2015 Keywords: Heart failure Nucleosomes Biomarker Circulating DNA Inflammation
Chronic heart failure (HF) is a progressive disorder characterized by a persistent, low-grade, systemic and myocardial inflammation that correlates with disease severity and prognosis. While the cause of this low grade inflammation is currently unknown, endogenous danger associated molecule-patterns (DAMPs), may be a contributing factor [1]. Among structures identified as a potential trigger of endogenous inflammation is the nucleosome, a chromatin structure consisting of at least eight histones wrapped by double stranded DNA. Several studies have shown that nucleosomes may activate the immune system, and generate an inflammatory response, but so far, there is to best of our knowledge, no data on extracellular nucleosome in HF [2–5]. Nucleosomes are normally separated from the inflammatory system in the nucleus of cells. However, in certain situations these structures may be released into the extracellular space and find their way to the circulation. Firstly, during apoptosis, DNA is cleaved into nucleosomes as a part of the apoptotic process, and if not properly digested by macrophages, these nucleosomes may be released into their surroundings [3]. Secondly, upon activation, neutrophils in the circulation may release their cellular DNA as neutrophil extracellular traps (NET)-like formations [6]. These NETs consist mainly of DNA with the histone core preserved, as well as a range of enzymes. Break down of NETs by circulating DNases may release the nucleosome component from the NETs into the extracellular space or the circulation [7]. Nucleosomes are increased in the circulation in a number of diseases, such as sepsis, ischemia–reperfusion injury and stroke [8]. In ⁎ Corresponding author. E-mail address: [email protected] (S.H. Nymo).
http://dx.doi.org/10.1016/j.ijcard.2015.10.219 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
this study, we hypothesized that nucleosomes could also be a potential DAMP in chronic HF, and that the levels of circulating nucleosomes could be a prognostic marker for these patients. To evaluate this hypothesis we measured the concentration of circulatory nucleosomes in the plasma of 179 patients with chronic HF of New York Heart Association (NYHA) functional classes II–IV, stable for at least 6 months, described elsewhere [9]. Patients were followed up for a median time of 38 months (interquartile range 26–46 months), and 30 patients died during the follow-up. Nucleosome levels were measured using a cell death detection ELISA from Roche (Indianapolis, USA). As standard curve, a stepwise dilution of a nucleosome rich solution isolated from HepG2 cells were used, and concentration was measured in arbitrary units (AU). Intra- and inter-assay variations were b10% and b15%, respectively. For all baseline variables, the relationship between nucleosomes and other variables were tested using Pearson correlation or Kruskal–Wallis test as appropriate. All survival analyses were performed using the Cox proportional hazard regression model. Nucleosome levels were divided into tertiles and included in the Cox model as a group variable. NTproBNP and CRP where included as log-transformed variables. A twosided P-value b 0.05 was considered to be significant. The median nucleosome level in HF patients was 2.56 AU (interquartile range [IQR] 1.06–6.44). Baseline nucleosome levels were higher in men (median 3.42 AU (IQR 1.17–8.26) vs 1.82 AU (0.76–2.7), p = 0.019), and in patients with higher NYHA class (median 1.17 AU (IQR
Fig. 1. Survival of patients with chronic heart failure according to levels of circulating nucleosomes. T: tertile.
Correspondence
743
Table 1 Survival according to levels of circulating nucleosomes. Variable Nucleosomes Tertile 2 Tertile 3 Age (per 10 year) Female gender NYHA-class Class III Class IV eGFR EF CRP NT-proBNP
Unadjusted
Model 1
Model 2
2.87(0.91–9.01) p = 0.071 4.16(1.38–12.54) p = 0.011
3.37 (1.06–10.69)p = 0.039 5.37 (1.73–16.70)p = 0.004 1.62(1.14–2.31)p = 0.007 2.34(1.04–5.26)p = 0.039
4.78 (0.87–26.21)p = 0.071 6.37 (1.24–32.67)p = 0.026 1.78(1.11–2.89)p = 0.019 2.77(1.04–7.37)p = 0.041 0.93(0.175–4.91)p = 0.931 2.47(0.48–12.69)p = 0.279 0.993(0.97–1.01)p = 0.441 1.03(1.01–1.07)p = 0.022 1.46(0.63–3.39)p = 0.369 2.38(0.84–6.80)p = 0.104)
All values listed as hazard ratio (95% confidence interval) p-value. NYHA-class, EF: ejection fraction, eGFR estimated glomerular filtration rate, CRP: C-reactive protein and NT-proBNP: Nterminal-pro brain natriuretic protein. All significant values are shown in bold.
0.58–7.85), 2.93 AU (1.33–5.90) and 4.45 AU (2.28–9.02) for NYHA classes II–IV, respectively, p = 0.01 for difference between NYHA II and IV). Baseline nucleosome levels were also associated with systemic inflammation (CRP; Spearman correlation r = 0.29, p b 0.001) and increased myocardial wall stress as assessed by NT-proBNP (r = 0.16, p = 0.028), but not associated with left ventricular ejection fraction (LVEF; r = −0.07, p = 0.38), or kidney function (eGFR; r = −0.07, p = 0.33). Patients with nucleosome levels in the second and third tertiles had higher risk of mortality than patients with lower nucleosome levels (HR 2.87, p = 0.07 and HR 4.16, p = 0.011 for the 2nd and 3rd tertiles, respectively, Fig. 1), also following multivariable adjustment (Table 1). Nucleosomes can be released from apoptotic and necrotic cells, as well as being an effect of increased neutrophil activity and NET production. We demonstrate that in patients with chronic HF, circulating nucleosome levels are associated with inflammation, disease severity and mortality. While circulating nucleosomes seems to be a feature of several conditions involving the inflammatory system, the extent to which nucleosomes merely circulate as markers of damaged tissue, or also represent a driving force of the inflammatory reaction that follows is not known. While nucleosomes may activate several inflammatory cells such as neutrophils and dendritic cells through interaction with membrane receptors, this is mainly studied in vitro, and may not be the case in more complex systems [3]. Our study show that circulating nucleosomes are present in chronic HF, correlate with disease severity, and could therefore be a potential player in the low grade inflammation seen in these patients. As this is a small study, with relatively few events, the data should be interpreted with caution. Our findings suggest that nucleosomes could be a potential biomarker in chronic HF, but larger studies are needed to confirm this.
Conflict of interest The authors report no relationships that could be construed as a conflict of interest.
References [1] Y. Zheng, S.E. Gardner, M.C. Clarke, Cell death, damage-associated molecular patterns, and sterile inflammation in cardiovascular disease, Arterioscler. Thromb. Vasc. Biol. 31 (2011) 2781–2786. [2] J. Xu, X. Zhang, R. Pelayo, M. Monestier, C.T. Ammollo, F. Semeraro, et al., Extracellular histones are major mediators of death in sepsis, Nat. Med. 15 (2009) 1318–1321. [3] V. Urbonaviciute, B.G. Fürnrohr, S. Meister, L. Munoz, P. Heyder, F. De Marchis, et al., Induction of inflammatory and immune responses by HMGB1–nucleosome complexes: implications for the pathogenesis of SLE, J. Exp. Med. 205 (2008) 3007–3018. [4] D.S. Pisetsky, The origin and properties of extracellular DNA: from PAMP to DAMP, Clin. Immunol. 144 (2012) 32–40. [5] H. Huang, J. Evankovich, W. Yan, G. Nace, L. Zhang, M. Ross, et al., Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice, Hepatology 54 (2011) 999–1008. [6] T.A. Fuchs, U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn, et al., Novel cell death program leads to neutrophil extracellular traps, J. Cell Biol. 176 (2007) 231–241. [7] M.J. Kaplan, M. Radic, Neutrophil extracellular traps: double-edged swords of innate immunity, J. Immunol. 189 (2012) 2689–2695. [8] S. Holdenrieder, P. Stieber, Clinical use of circulating nucleosomes, Crit. Rev. Clin. Lab. Sci. 46 (2009) 1–24. [9] T. Ueland, A. Svardal, E. Oie, E.T. Askevold, S.H. Nymoen, B. Bjorndal, et al., Disturbed carnitine regulation in chronic heart failure — increased plasma levels of palmitoylcarnitine are associated with poor prognosis, Int. J. Cardiol. 167 (2013) 1892–1899.