Skip to main content
NCBI home page
Indian Journal of Clinical Biochemistry logoLink to Indian Journal of Clinical Biochemistry
. 2022 Apr 21;38(1):67–72. doi: 10.1007/s12291-022-01034-y

Cell-free DNA Release in the Plasma of Patients with Cardiac Disease is Associated with Cell Death Processes

Junko Fujihara 1,✉, Yoshikazu Takinami 2, Kaori Kimura-Kataoka 1, Yasuyuki Kawai 3, Haruo Takeshita 1
PMCID: PMC9852365  PMID: 36684502

Abstract

Cell-free DNA (cfDNA) is released into the plasma of patients with cardiac disease. Here, the source and mechanism of plasma cfDNA release in patients with myocardial infarction (MI) and other cardiac diseases (n = 59) were investigated. Plasma levels of various markers including M30 (apoptosis), M65 (apoptosis and necrosis), cyclophilin A (CyPA) (necrosis), and myeloperoxidase (MPO) (neutrophil activation) were assayed. The plasma cfDNA concentrations in MI and other cardiac diseases were significantly higher than that in the healthy control subjects. Significant differences were not observed among the cardiac disease patients (MI and other cardiac diseases) and healthy control subjects in M30, M65, and CyPA levels. In contrast,the MPO levels were significantly elevated in cardiac disease patients when compared to control groups, and MPO levels in MI patients were significantly higher than other cardiac diseases patients. These results suggest that cfDNA is mainly released by neutrophils via NETosis in addition to apoptosis except for epithelial apoptosis in patients with cardiac disease and the degree is greater in MI patients. The results from this study provide basic information for diagnosis marker of MI.

Keywords: cfDNA, Apoptosis, M30, M65, CyPA, NETosis, MPO

Abbreviations.

AMI acute myocardial infarction.

cfDNA cell-free DNA.

CyPA cyclophilin A.

MPO myeloperoxidase.

Introduction

Cell-free DNA (cfDNA) is released into serum, plasma, urine, semen, and other body fluids [1]. Previously, we reported significantly higher plasma cfDNA concentrations in patients with myocardial infarction (MI) and cardiac angina than in the controls. Further, we found three DNA ladder fragments in the plasma cfDNA of patients with cardiac disease, with a higher 150–200 bp/500–600 bp ratio in patients with MI than in other cardiac diseases; a positive correlation was found between deoxyribonuclease I activity and cfDNA concentration [2]. Recently, we demonstrated differences between postmortem and biogenic subjects in the concentration and fragment distribution of serum cfDNA; we found that postmortem subjects had significantly higher cfDNA concentrations than biogenic subjects and that a high concentration of 150–200 bp fragments was characteristic in postmortem samples [3].

Several reports have suggested that cfDNA originates from apoptotic or necrotic processes [4–7]. Cytokeratin 18 is a cytoskeletal protein of the epithelium that is cleaved by caspases during apoptosis and non-apoptotic cell death, and caspase-cleaved cytokeratin-18 (M30) is used to detect epithelial apoptosis. Uncleaved cytokeratin-18 (M65) is also released from dying cells (both apoptotic and necrotic). Both M30 and M65 are important prognostic markers for several malignancies [8]. Cyclophilin A (CyPA) is a cytosolic peptidyl-prolyl cis-trans isomerase that is released in the early stages of necroptosis and is proposed as a biomarker for necroptosis [9]. cfDNA is also presumed to be released via NETosis, a regulated cell death pathway that releases neutrophil extracellular traps (NETs) in response to sterile inflammation, infection, or hypoxia [10, 11]. NETs are web-like networks of decondensed nuclear DNA in association with histones, granule proteins, and antimicrobial peptides. Myeloperoxidase (MPO) is a granule protein and neutrophil enzyme that is released during NETosis [7].

To our knowledge, the source and mechanism of cfDNA release in patients with cardiac disease is unclear. Therefore, in the present study, we investigated the source and mechanism of cfDNA release in patients with cardiac disease by measuring the cytokeratin 18 (M30 and M65), CyPA, and MPO levels in the plasma of patients with cardiac disease.

Materials and Methods

Study Subjects

Blood samples were collected from patients with MI and other cardiac diseases (chest pain, cardiac angina, atrial fibrillation, Takotsubo cardiomyopathy, cardiac failure, and myocardial ischemia) (n = 59) who presented at the emergency outpatient department of Shimane University Hospital (Shimane, Japan) from 2016 to 2019 (Table 1); these patients were a part of the subjects included in our previous study [2]. Patients with cancer were not enrolled in the study. At the time of emergency room admission, venous blood was drawn into tubes and plasma was separated by centrifugation at 500 × g and stored at ˗70 °C. Control blood samples were collected from 24 healthy Japanese volunteers living in Shimane Prefecture, and plasma was separated by centrifugation as described above. The study including the use of plasma and DNA derived from patients and control subjects was reviewed and approved by the Human Ethics Committee of Shimane University School of Medicine. Informed consent was obtained from all the participants and control subjects.

Table 1.

Baseline characteristics in cardiac disease patients and controls

Controls Myocardial infarction Other Cardiac diseases*
(n = 24) (n = 22) (n = 37)
Age 43.0 ± 8.95 71.2 ± 12.8 75.8 ± 11.1
Male/Female 23/1 11/11 17/20

Values are presented as mean ± S.D.

* Chest pain, cardiac angina, atrial fibrillation, Takotsubo cardiomyopathy, cardiac failure, and myocardial ischemia
Open in a new tab

Cf DNA Isolation from Plasma

Cf DNA was extracted from 1 mL of plasma using a Maxwell® RSC cfDNA plasma Kit (Promega Corp., Madison, WI, USA), as described previously [2, 3]. Following extraction, cfDNA concentrations were measured using a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Each sample (2 µL) was plated on Thermo Scientific™ µDrop™ plates, and spectrophotometric absorbance was measured at 260 nm, 280 nm, and 320 nm.

Enzyme-Linked Immunosorbent Assay (ELISA)

The M30 and M65 levels in plasma were evaluated using the M30 Apoptosense ELISA kit and M65 Epideath ELISA kit (Peviva, Bromma, Sweden) according to the manufacturer’s instructions. Plasma CyPA levels were assayed using ELISA (RayBiotech Life, Inc., GA, USA). Absorbance at 450 nm was measured using a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific Inc.).

Statistical Analysis

Differences between the groups in cfDNA concentrations, M30, M65, CyPA, MPO levels were analyzed using Scheffe’s test. Spearman’s correlation coefficient was used for the correlation analysis. These analyses were performed using Bell Curve for Excel (Social Survey Research Information Co. Ltd., Tokyo, Japan).

Results and Discussion

Plasma cfDNA Concentration

We have previously reported elevated plasma cfDNA concentrations in all cardiac diseases and significantly higher cfDNA concentrations in patients with MI and cardiac angina than that in control subjects [2]. Similar to our previous study, the plasma cfDNA concentrations in MI and other cardiac diseases were significantly higher than that in the healthy control subjects (Fig. 1). No significant differences were observed between MI and other cardiac diseases.

Fig. 1.

Fig. 1

Open in a new tab

Cell-free DNA (cfDNA) concentrations in plasma from patients with cardiac disease and healthy control subjects. The top and bottom of each box represents the 25th and 75th percentile, respectively. The line through the box is the median and the error bars indicate the 5th and 95th percentiles. Data were analyzed using Scheffe’s test. CfDNA concentrations in plasma from patients with other cardiac diseases (*p < 0.05) and myocardial infarction (**p < 0.01) are significantly higher than those of healthy control subjects

Plasma M30 and M65 Levels

The M30 and M65 levels have been reported in patients with cancer and tumors. Ueno et al. showed elevated M30 levels in patients with breast cancer [12]. Ozturk et al. demonstrated increased M30 and M65 levels in the serum of patients with head and neck tumors [13]. Oven Ustaalioglu et al. reported that serum M65 values were elevated in advanced non-small cell lung cancer compared to that in the healthy controls, and this could predict progression-free survival [8]. Further, Wei et al. indicated that M65 was helpful in diagnosing some stages of liver inflammation and fibrosis [14]. Zheng et al. reported that M30 and M65 levels were significantly increased in patients with chronic hepatitis B, and that the M30/M65 ratio in patients with chronic hepatitis B and acute-to-chronic liver failure was lower than that in the control groups [15].

As mentioned above, M30 is a selective biomarker for epithelial apoptosis [16, 17]. M65 can detect both cleaved and uncleaved CK-18, which is used as a marker of both necrosis and apoptosis [15]. Plasma M30 and M65 levels in patients with cardiac disease (MI and other cardiac diseases) and healthy control subjects are shown in Fig. 2. In both M30 and M65 levels, no significant differences were observed among the cardiac disease patients and healthy control subjects. Previously, we have reported ladder fragments of cfDNA of cardiac disease patients suggesting the occurrence of apoptosis [2]. These results suggest that cfDNA in patients with cardiac disease may be released by mechanisms other than epithelial apoptosis and necrosis.

Fig. 2.

Fig. 2

Open in a new tab

Plasma levels of (a) M30 and (b) M65 from patients with cardiac disease and healthy control subjects. The top and bottom of each box represents the 25th and 75th percentile, respectively. The line through the box is the median and the error bars indicate the 5th and 95th percentiles. Data were analyzed using Scheffe’s test. Significant differences were not observed between groups

Plasma CyPA Levels

CyPA is a cytosolic protein isomer that is released by necrotic cell death when the integrity of the plasma membrane is compromised [9]. As mentioned above, CyPA is a marker of necrosis. Recent studies have reported the relevance of CyPA in diseases. Jackson Chornenki et al. did not observe any significant difference in plasma CyPA levels between patients with sepsis and trauma [7]. Ramachandran et al. reported that patients with type 2 diabetes have higher circulating levels of CyPA compared to the normal population [18]. Satoh et al. showed that the plasma CyPA levels were significantly higher in patients with coronary artery disease than in the control subjects [19]. Similar to cytokeratin 18 (M30 and M65), no significant differences were observed in the CyPA levels among the control group and cardiac disease patients (Fig. 3). The present results thus suggest that cfDNA in patients with cardiac disease is not released from necrotic processes.

Fig. 3.

Fig. 3

Open in a new tab

Plasma levels of cyclophilin A (CyPA) from patients with cardiac disease and healthy control subjects. The top and bottom of each box represent the 25th and 75th percentile, respectively. The line through the box is the median and the error bars indicate the 5th and 95th percentiles. Data were analyzed using Scheffe’s test. Significant differences were not observed between groups

Plasma MPO Levels

MPO is a neutrophil enzyme released during NETosis and is linked with both inflammation and oxidative stress [20]. Previous studies have reported that cfDNA and MPO levels are markers of neutrophil activation and NET formation [21, 22], and that these are correlated with inflammatory diseases such as sepsis [23, 24]. Jackson Chornenki et al. reported that MPO levels were not correlated with cfDNA in trauma but were strongly correlated with cfDNA in sepsis, suggesting that cfDNA in trauma is released by necrotic cells whereas cfDNA in sepsis is likely released by activated neutrophils via NETosis [7]. Plasma MPO levels have been reported as a useful biomarker for MI [25], chest pain [26], and other cardiovascular diseases [20, 27].

In this study, plasma MPO levels were quantified in healthy control subjects and in patients with cardiac disease (Fig. 4). The MPO levels were significantly elevated in cardiac disease patients when compared to control groups (p < 0.01) (Fig. 4a), and MPO levels in MI patients were significantly higher than other cardiac diseases (p < 0.01). The MPO levels were correlated with cfDNA in all patients with cardiac disease (Fig. 4b). These results suggest that cfDNA in cardiac disease patients is released by neutrophils via NETosis and this extent is especially greater in MI than other cardiac patients.

Fig. 4.

Fig. 4

Open in a new tab

(a) Myeloperoxidase (MPO) levels in plasma from patients with cardiac disease and healthy control subjects. The top and bottom of each box represents the 25th and 75th percentile, respectively. The line through the box is the median and the error bars indicate the 5th and 95th percentiles. Data were analyzed using Scheffe’s test. MPO levels in plasma from patients with other cardiac disease and myocardial infarction are significantly higher than those of healthy control subjects (**p < 0.01) and significant difference are observed between other cardiac disease and myocardial infarction patients (**p < 0.01). (b) Relationship between MPO levels and cfDNA concentrations in the plasma of patients with cardiac disease. Spearman’s correlation coefficient was used for correlation analysis

Conclusions

In this study, the plasma levels of M30, M65, CyPA, and MPO were assayed to investigate the source and mechanism involved in the release of cfDNA to the plasma of patients with cardiac disease. The plasma cfDNA concentrations in MI and other cardiac diseases were significantly higher than that in the healthy control subjects. Significant differences were not observed among the cardiac disease (MI and other cardiac diseases) patients and healthy control subjects in M30, M65, and CyPA levels. In contrast, the MPO levels were significantly elevated in cardiac disease patients when compared to control groups, and MPO levels in MI patients were significantly higher than other cardiac diseases patients. MPO levels were correlated with cfDNA in all patients with cardiac disease. These results suggest that plasma cfDNA in cardiac disease is released by neutrophils via the NETosis process in addition to apoptosis except for epithelial apoptosis. To our knowledge, this study is the first to show the correlation of MPO levels with cfDNA concentrations in cardiac disease patients and elevated MPO levels in MI patients when compared to other cardiac disease patients. The results from this study provide basic information for diagnosis marker of MI.

Author Contributions

Conceptualization: Junko Fujihara; Methodology; Junko Fujihara; Formal analysis and investigation: Junko Fujihara; Writing - original draft preparation: Junko Fujihara; Funding acquisition: Junko Fujihara, Yasuyuki Kawai; Resources: Yoshikazu Takinami, Kaori Kimura-Kataoka, Haruo Takeshita.

Funding

This work was supported by the Shimane University Support Programs for Young Female Researchers under the MEXT Initiative for Realizing Diversity in the Research Environment (Collaboration Type) to JF and JSPS KAKENHI Grants-in-Aid for Scientific Research (B) [grant number 21H03212] to JF.

Declarations

Consent for Publication

Consent for publication was obtained in accordance with ethical guidelines.

Disclosure of Potential Conflicts of Interest

The authors declare that they have no conflicts of interest.

Research Involving Human Participants

The study including usage of plasma derived from patients and control subjects was reviewed and approved by the Human Ethics Committee of Shimane University School of Medicine (approval number: 20151214-2).

Informed Consent

Informed consent was obtained from all participants included in the present study.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Ponti G, Maccaferri M, Mandrioli M, Manfredini M, Micali S, Cotugno M, et al. Seminal cell-free DNA assessment as a novel prostate cancer biomarker. Pathol Oncol Res. 2018;24:941–945. Available from doi:10.1007/s12253-018-0416-6. [DOI] [PubMed]
  • 2.Fujihara J, Takinami Y, Ueki M, Kimura-Kataoka K, Yasuda T, Takeshita H. Circulating cell-free DNA fragment analysis by microchip electrophoresis and its relationship with DNase I in cardiac diseases. Clin Chim Acta. 2019;497:61–66. Available from doi:10.1016/j.cca.2019.07.014. [DOI] [PubMed]
  • 3.Fujihara J, Takinami Y, Kawai Y, Kimura-Kataoka K, Takeshita H. Comparison of serum cell-free DNA between postmortem and living samples. Clin Chim Acta. 2021;519:255–259. Available from doi:10.1016/j.cca.2021.05.013. [DOI] [PubMed]
  • 4.Fournié GJ, Courtin JP, Laval F, Chalé JJ, Pourrat JP, Pujazon MC, et al. Plasma DNA as a marker of cancerous cell death. Investigations in patients suffering from lung cancer and in nude mice bearing human tumours. Cancer Lett. 1995; 91(2):221–227. Available from: doi:10.1016/0304-3835(95)03742-F. [DOI] [PubMed]
  • 5.Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 2001;61(4):1659–65. [PubMed] [Google Scholar]
  • 6.Salvi S, Gurioli G, De Giorgi U, Conteduca V, Tedaldi G, Calistri D, et al. Cell-free DNA as a diagnostic marker for cancer: current insights. Onco Targets Ther. 2016;9:6549–6559. Available from doi: 10.2147/OTT.S100901. [DOI] [PMC free article] [PubMed]
  • 7.Jackson Chornenki NL, Coke R, Kwong AC, Dwivedi DJ, Xu MK, McDonald E, et al. Comparison of the source and prognostic utility of cfDNA in trauma and sepsis. Intensive Care Med Exp. 2019;7(1):29. Available from doi: 10.1186/s40635-019-0251-4. [DOI] [PMC free article] [PubMed]
  • 8.Oven Ustaalioglu B, Bilici A, Ercan S, Orcun A, Seker M, Ozkan A, et al. Serum M30 and M65 values in patients with advanced stage non-small-cell lung cancer compared with controls. Clin Transl Oncol. 2012;14:356–361. Available from doi:10.1007/s12094-012-0808-0. [DOI] [PubMed]
  • 9.Christofferson DE, Yuan J. Cyclophilin A release as a biomarker of necrotic cell death, Cell Death Differ. 2010;17:1942–1943. Available from doi:10.1038/cdd.2010.123. [DOI] [PMC free article] [PubMed]
  • 10.Gould TJ, Lysov Z, Liaw PC. Extracellular DNA and histones: double-edged swords in immunothrombosis. J Thromb Haemost. 2015;13(Suppl 1):S82-S91.Available from doi:10.1111/jth.12977. [DOI] [PubMed]
  • 11.Pokrywka A, Zembron-Lacny A, Baldy-Chudzik K, Orysiak J, Sitkowski D, Banach M. The influence of hypoxic physical activity on cfDNA as a new marker of vascular inflammation. Arch Med Sci. 2015;1(6):1156–1163. Available from doi:10.5114/aoms.2015.56341. [DOI] [PMC free article] [PubMed]
  • 12.Ueno T, Toi M, Bivén K, Bando H, Ogawa T, Linder S. Measurement of an apoptotic product in the sera of breast cancer patients. Eur J Cancer. 2003;39(6): 769–774. Available from doi:10.1016/s0959-8049(02)00865-1. [DOI] [PubMed]
  • 13.Ozturk B, Coskun U, Sancak B, Yaman E, Buyukberber S, Benekli M. Elevated serum levels of M30 and M65 in patients with locally advanced head and neck tumors. Int. Immunopharmacol. 2009;9(5):645–648. Available from doi:10.1016/j.intimp.2009.02.004. [DOI] [PubMed]
  • 14.Wei X, Wei H, Lin W, Hu Z, Zhang J. Cell death biomarker M65 is a useful indicator of liver inflammation and fibrosis in chronic hepatitis B: A cross-sectional study of diagnostic accuracy. Med. (Baltim.) 2017;96(20): e6807. Available from doi:10.1097/MD.0000000000006807. [DOI] [PMC free article] [PubMed]
  • 15.Zheng SJ, Liu S, Liu M, McCrae MA, Li JF, Han YP, et al. Prognostic value of M30/M65 for outcome of hepatitis B virus-related acute-on-chronic liver failure. World J Gastroenterol. 2014;20(9): 2403–2411. Available from doi:10.3748/wjg.v20.i9.2403. [DOI] [PMC free article] [PubMed]
  • 16.Kramer G, Erdal H, Mertens HJ, Nap M, Mauermann J, Steiner G, et al. Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res. 2004;64(4):1751–1756. Available from doi:10.1158/0008-5472.can-03-2455. [DOI] [PubMed]
  • 17.Ueno T, Toi M, Linder S. Detection of epithelial cell death in the body by cytokeratin 18 measurement, Biomed Pharmacother. 2005;59(Supplement 2) S359–S362. Available from doi:10.1016/s0753-3322(05)80078-2. [DOI] [PubMed]
  • 18.Ramachandran S, Venugopal A, Kutty VR, Chitrasree AVGD V, et al. Plasma level of cyclophilin A is increased in patients with type 2 diabetes mellitus and suggests presence of vascular disease. Cardiovasc Diabetol. 2014;13:38. Available from doi:10.1186/1475-2840-13-38. [DOI] [PMC free article] [PubMed]
  • 19.Satoh K, Fukumoto Y, Sugimura K, Miura Y, Aoki T, Nochioka K, et al. Plasma cyclophilin A is a novel biomarker for coronary artery disease, Circ J. 2016; 80(4):980–988. Available from doi:10.1253/circj.CJ-15-1212. [DOI] [PubMed]
  • 20.Schindhelm RK, van der Zwan LP, Teerlink T, Scheffer PG. Myeloperoxidase: a useful biomarker for cardiovascular disease risk stratification? Clin Chem. 2009; 55(8):1462–1470. Available from doi:10.1373/clinchem.2009.126029. [DOI] [PubMed]
  • 21.Maruchi Y, Tsuda M, Mori H, Takenaka N, Gocho T, Huq MA, et al. Plasma myeloperoxidase-conjugated DNA level predicts outcomes and organ dysfunction in patients with septic shock. Crit Care. 2018;22:176. Available from doi:10.1186/s13054-018-2109-7. [DOI] [PMC free article] [PubMed]
  • 22.Jiménez-Alcázar M, Kim N, Fuchs TA Circulating extracellular DNA: cause or consequence of thrombosis?, Semin Thromb Hemost. 2017;43(06):553–561. Available from doi:10.1055/s-0036-1597284. [DOI] [PubMed]
  • 23.Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, Kelly MM, et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med. 2007;13:463–469. Available from doi:10.1038/nm1565. [DOI] [PubMed]
  • 24.O’Brien XM, Biron BM, Reichner JS. Consequences of extracellular trap formation in sepsis. Curr Opin Hematol. 2017;24(1):66–71. Available from doi:10.1097/MOH.0000000000000303. [DOI] [PMC free article] [PubMed]
  • 25.Rudolph V, Goldmann BU, Bös C, Rudolph TK, Klinke A, Friedrichs K, et al. Diagnostic value of MPO plasma levels in patients admitted for suspected myocardial infarction, Int J Cardiol. 2011;153(3):267–271. Available from doi:10.1016/j.ijcard.2010.08.015. [DOI] [PubMed]
  • 26.Rudolph V, Keller T, Schulz A, Ojeda F, Rudolph TK, Tzikas S, et al. Diagnostic and prognostic performance of myeloperoxidase plasma levels compared with sensitive troponins in patients admitted with acute onset chest pain. Cir Cardiovasc Genet. 2012;5:561–568. Available from doi:10.1161/CIRCGENETICS.111.962290. [DOI] [PubMed]
  • 27.Loria V, Dato I, Graziani F, Biasucci LM. Myeloperoxidase: a new biomarker of inflammation in ischemic heart disease and acute coronary syndromes. Mediators Inflamm. 2008; 2008;135625. Available from doi: 10.1155/2008/135625. [DOI] [PMC free article] [PubMed]

Articles from Indian Journal of Clinical Biochemistry are provided here courtesy of Springer

RESOURCES