- Open Access
Neutrophil extracellular traps in acute coronary syndrome
Journal of Inflammation volume 20, Article number: 17 (2023)
Acute coronary syndrome (ACS) is a group of clinical syndromes caused by acute myocardial ischemia, which can cause heart failure, arrhythmia and even sudden death. It is the major cause of disability and death worldwide. Neutrophil extracellular traps (NETs) are reticular structures released by neutrophils activation and have various biological functions. NETs are closely related to the occurrence and development of ACS and also the subsequent damage after myocardial infarction. The mechanisms are complex and interdependent on various pathways, which require further exploration. This article reviewed the role and mechanism of NETs in ACS, thereby providing a valuable reference for the diagnosis and clinical treatment of ACS.
Acute coronary syndrome (ACS) is an internationally recognized acute cardiovascular disease characterized by rapid onset and progression with a high mortality rate . The pathogenesis of ACS is divided into three stages. In the early stage, the disease is mainly characterized by the rupture or invasion of coronary atherosclerotic plaque, followed by rational thrombosis. During the middle and late stages, ischemic myocardium necrosis in the diseased coronary arteries leads to myocardial infarction (MI) and, eventually, heart failure or even death. Recent studies have confirmed that ACS is the leading mortality-causing clinical outcome in patients with cardiac diseases . Therefore, it is vital to explore practical strategies for the management of ACS and elucidate the inducers and mechanisms of ACS, thereby improving the prognosis of patients.
Neutrophils, immune cells in the innate immune system, are the body’s first line of defence against pathogen invasion, inducing various pathological processes such as the induction of a series of responses to inflammatory stimuli. Recently, neutrophils have been reported to function through the NETosis defence mechanism, which releases a reticulum called neutrophil extracellular traps (NETs) . The NETs comprise DNA and histones released by activated neutrophils, which play a role in the neutrophil-mediated intrinsic immune . The NETs were initially considered as effector proteins that protect the human body against pathogens, immobilizing pathogens and exposing them to high local concentrations of a bactericidal environment . This review systematically analyzed and summarized the mechanism of NETs in ACS. First, we introduce the structure and function of NETs. Second, we describe the impact of NETs on ACS from the point of view of atherosclerosis, MI and heart failure. Finally, we discuss the role of NETs as a disease prediction marker of ACS, which could aid in its clinical intervention and treatment.
Introduction to NETs
Structure and function of NETs
In 2004, Brinkmann et al.  first observed the structure of NETs on stimulating neutrophils with phenyl propyl acetate myristate (PMA), lipopolysaccharide (LPS) and interleukin 8 (IL-8). The core components of NETs are depolymerized DNA, histones, particle components and other related proteins that are assembled on a chromatin scaffold , eventually forming a lethal environment that prevents the participation of neutrophils in the body’s immune defence system, which consequently prevents the invasion of pathogens and destruction of the invaded pathogens. Studies [8, 9] report that DNase can cleave NETs, degrade their essential skeletal DNA and inhibit the extracellular bactericidal function of neutrophils. Therefore, the DNA backbone and histones are considered essential and indispensable components of NETs. Furthermore, factors that induce NETs are extracellular pathogens, viruses , fungi , cholesterol and urate crystals, lipids, activated platelets  and complements such as C5a (Fig. 1).
Formation of NETs
The production of NETs and NETosis, which have potential roles in various diseases, is considered an effective weapon in sterile inflammation . NETs have long been considered a critical pathogen barrier that induces microbial death by binding to microorganisms, inhibiting their dissemination and maintaining a locally high-concentration antimicrobial environment . Moreover, NETs are also crucial in non-pathogenic infections and have been associated with several cardiovascular diseases and corresponding risk factors . The release of NETs is premised on the rupture of the plasma membrane, wherein the activation of neutrophils leads to changes in their morphological structure, resulting in chromatin fragmentation, nuclear rupture and ultimately the release of NETs . The process of neutrophils secreting NETs is known as NETosis, which is also known as the inflammatory death mode of neutrophils. NETosis involves multiple signal pathways and molecular mechanisms; however, studies on its formation mechanism at home and abroad are scarce.
Based on current studies, NETs are secreted via three pathways. (1) Suicidal and lytic NETosis [5, 17]: neutrophils upon stimulation by PMA or IL-8, activate protein kinase C and RAF-mitogen-activated protein kinase-extracellular signal-regulated kinase pathways in the cell. This process lasts for a long time, usually, 2 to 4 h. PMA, LPS and various bacterial-related activation stimulate the RAF-MEK-ERK pathway through the NADPH oxidase 2 (NOX2) complex, producing reactive oxygen species (ROS) that act as secondary messengers to promote the detachment of the nuclear envelope and finally leading to the rupture of the plasma membrane and release of decondensed chromatin. (2) Non-lytic NETosis : this form occurs for a shorter period, generally within 30–60 min and is induced by the activation of neutrophils mediated by toll-like receptors. Notably, it is independent of NADPH oxidase activation. Moreover, cell function can still be preserved as it does not disrupt the nuclear envelope and cell membrane. Herein, the release of nuclear material is mainly achieved by vesicular export. (3) Mitochondrial DNA releases NETs : this method was recently discovered by Yousefi et al. This process is also dependent on the ROS pathway; however, the released DNA originates from the mitochondria rather than the nucleus. Notably, this process can last for 15 min (Fig. 2).
However, controversies and inconsistencies over describing the formation mechanism of NETs with respect to NETosis remain as NETosis initially only represents the release of soluble NETs and can be distinguished from cell death and lysis with respect to this aspect . However, numerous studies have shown that not all NETs formation pathways lead to cell lysis and death. The NETs formation is a complex process regulated by multiple pathways, with differences in the formation mechanism of NETs under varying stimulatory substances. However, the exact mechanism of NETs formation remains unclear and requires further exploration.
Biomarkers of NETs and detection methods
Assessing the ability of neutrophils to form NETs in peripheral blood would be a promising method for predicting ACS. Several possible methods for the detection of NETs will be discussed below.
In situ or in vivo detection methods
To evaluate the formation ability of NETs, it is necessary to evaluate peripheral blood neutrophils, which can be non-invasive detected through characteristic in situ or in vivo detection methods: (1) In vivo imaging techniques: In vivo imaging refers to the application of imaging methods at the cellular and molecular levels in the living state for the qualitative and quantitative analysis of biological processes and temporal. In 2006, Buchanan et al. used DNA embedding dyes to demonstrate a significant correlation between DNA enzymes, NETs degradation, and pathogenicity . Fuchs et al. used live cell imaging combined with phase contrast, survival markers, death markers, and histone staining to find that PMA activates neutrophils and releases NETs by neutrophils are accompanied by their death [22, 23]. Imaging techniques of different living cells have been widely used to detect a variety of diseases; (2) In vivo microscopy techniques: In vivo, microscopy can visualize protein activity, gene expression, cellular transport, cell-cell/cell-microenvironment interactions, and a range of physiological responses to stimuli in vivo, facilitating dynamic 3D in vivo cellular-level imaging of a range of biological processes in living animals. Clark et al. observed NETs produced by activated platelets in the liver sinusoids by in vivo microscopy. As living organisms continue to deepen, various types of microscopes are used to detect the formation of NETs in different tissues and organs .
In vitro assays
There is no reliable and standardized method to measure the formation of NETs in vivo, and an in vitro neutrophil function test may be a potential option. As used in COVID-19, neutrophils can be isolated from peripheral blood and then stimulated with inflammatory triggers used to induce the formation of NETs, and the resulting NETs can then be quantified. Some in vitro assays for NETs are presented next .
Co-localization of neutrophil derived proteins and extracellular DNA
Co-localization of neutrophil-derived proteins and extracellular DNA is a standard method for in vitro detection of NETs, where neutrophils are inoculated on slides, incubated for several hours with or without stimulation, fixed, and then immunostained for neutrophil-derived proteins and DNA, and co-localization of proteins and DNA is indicative of the presence of NETs [26, 27]. This method is easy to perform but requires certain precautions to permeabilize the plasma membrane while fixing the cells. Usually, these reagents induce the formation of artificial NETs, which may impact the results. In addition, the method lacks objectivity, and the investigator needs to autonomously assess whether the derived proteins and DNA are distributed inside or outside the neutrophils, with a particular subjectivity [28, 29].
Flow cytometry detection
The following flow cytometry assays are commonly used − (1) Indirect immunofluorescence assay: cfDNA is the essential backbone of NETs, and DNA staining can be used to visualize NETs. Antibodies to two significant components of NETs, citrullinated histone (CitH3) and myeloperoxidase (MPO), are combined with DNA dyes and detected by an indirect immunofluorescence method . Since this method is based on CitH3 antibody staining, it cannot be used in CitH3-deficient mice even though their NETs formation capacity is still present ; (2) Image-based flow cytometry detection: in a characteristic swelling assay on neutrophil nuclei, Zhao et al. used a Ficoll density gradient to isolate neutrophils from whole blood samples granulocytes, which were then separated from the erythrocyte layer by dextran sedimentation . After effective stimulation, neutrophils were fixed in a 2% PFA solution containing 1:1000 dilution of Hoechst, nuclear labeling was performed, and MPO staining was performed. The results showed an approximately 3-fold increase in the mean nuclear area of neutrophils compared to unstimulated cells, confirming that NETs can be detected using multispectral imaging flow cytometry; (3) High-speed multispectral imaging flow cytometry : effective cellular figuration, i.e., volume, morphology, and associated sub localization of cellular structures. In this approach, it is possible to distinguish between lysogenic and non-lysogenic NETs, and this approach also has the drawback that the imaging system only shows NETs formed in the immediate state and cannot detect NETs subsequently released from cells that have started lysis. Therefore, several flow cytometric techniques allow for specific, quantitative, and rapid detection of neutrophil morphology, providing feasibility for further studies of NETs as bioindicators in clinical medicine [30, 32].
Immunostaining analysis of guanine histones
The presence of guanosine histones specifically indicates that PAD4 mediates the induction of NETs. PAD4 is expressed mainly in hematopoietic cells and induces guanosylation of arginine, which de-agglutinates chromatin from activated neutrophils and forms NETs. The presence of guanosine histones, as determined by immunostaining, is evidence of NET formation in vitro and in vivo . However, it has been shown that the involvement of PAD4 in NET formation depends on the nature of the stimulus; therefore, PAD4 is controversial in the NET formation process. Moreover, the method is only applicable to PAD4-dependent NETosis [33, 34].
Enzyme-linked immunosorbent assay
The basic principle of enzyme-linked immunosorbent assay (ELISA) is that an enzyme couple with an antibody or antigen to form a complex, and when the antigen-antibody binds explicitly, the complex can catalyze the conversion of a colorless substrate molecule into an easily detectable substance, and the change in the substrate signal can determine whether the immune reaction has occurred and thus analyze the concentration of the target substance . This method detects CitH3, MPO, and DNA, the main components of NETs, to indirectly prove the presence of NETs and to quantify them [35, 36].
In summary, NETs formation is a specific manifestation of neutrophils after responding to infection and inflammatory reactions. Various NETs detection techniques have been implemented in recent years to visualize NETs. However, the current technical means of visualization still have different degrees of defects. In addition, for in vitro NETs testing, although promising results have been shown, challenges remain and optimization of experimental protocols and measurement techniques may be required. Thus, even though neutrophil function assays, especially in vitro assays, have great potential in predicting ACS episodes, more studies are needed to determine their validity and utility in the clinical setting.
NETs in ACS
NETs and atherosclerosis and thrombosis
Atherosclerotic lesions start from the intima, accompanied by arterial medial degeneration and calcification and ultimately lead to the thickening and hardening of the arterial wall and narrowing of the vessel lumen, which is the pre-lesion stage of ACS. Megen et al. first reported  that neutrophils and NETs are present in atherosclerotic lesions in mice and humans. Additionally, Kartika et al.  reported that ACS could be caused by the rupture of the fibrous cap of atherosclerotic plaques, plaque erosion, or intraplaque hemorrhage in patients. Moreover, at the thrombus, hemorrhage and thrombus-plaque interface, neutrophils and NETs were abundantly present in all types of complex plaques, indicating the potential role of NETs in the formation of atherosclerosis. Furthermore, NETs can damage the endothelium through the combined action of IL-1 and cathepsin G, which promotes the endothelial expression of ICAM-1, VCAM-1 and tissue factors and is associated with thrombus formation owing to the erosion of the atherosclerotic plaque surface . Silvestre et al.  observed that the externalized histone H4 on NETs could lead to the lysis of smooth muscle cells, wherein the plaques become fragile and can be easily sloughed off. Many studies have revealed the mechanism by which NETs accelerate thrombosis. Döring Y et al.  reported that interacting neutrophils and platelets at the site of plaque rupture can promote NETs formation and stimulate active tissue factors to accelerate thrombus formation. Nicoletta et al. [42, 43] reported that, in APOE-deficient mice. NETs formed in the early stage of atherosclerotic lesions. Meanwhile, relevant experimental data also suggest that NETs can promote erosive effects on the plaque surface, thereby exposing thrombotic material in the plaque to locally high concentrations of NETs and accelerating thrombus formation . Additionally, cholesterol crystals formed during atherosclerotic lesions could also stimulate the release of NETs, which can induce macrophages to secrete cellular inflammatory factors and thereby promote the migration of neutrophils to inflammatory sites . Thus, the above findings indicate that NETs play an essential role in the inflammatory response.
NETs are significant contributors to the formation of pathological thrombi. In 2010, Fuchs et al.  demonstrated that NETs provided a physical scaffold for thrombus growth by binding to platelets and erythrocytes. The primary mechanism is speculated to be that NETs can promote the adhesion and aggregation of platelets and the binding of red blood cells, consequently promoting the formation of blood clots. Conversely, NETs can promote thrombin generation by activating platelets and coagulation factors XI and XII , which activate intrinsic and extrinsic coagulation pathways and accelerate thrombus formation. However, the procoagulant effect of the NETs complex does not compare to that of DNA or histone components alone . The process by which histones and DNA are tightly bound to nucleosomes could reduce their ability to interact with the coagulation system, thus making them more potent procoagulants. However, studies report that histones cannot directly drive the coagulation cascade but can induce thrombin generation by activating platelets [48, 49].
Additionally, NETs present associated prothrombotic molecules that can directly bind to fibrinogen and accelerate fibrin deposition, which forms a fibrin network that acts as a scaffold for capturing platelets and red blood cells . Additionally, neutrophil elastase can bind to NETs, aiding thrombin in promoting fibrin formation in the presence of platelets and leading to thrombus formation. Furthermore, the degradation of histones with heparin could lead to the increased instability of NETs . Therefore, it can be concluded that NETs maintain thrombus stability (Fig. 3).
NETs and MI
MI refers to the ischemic necrosis of the myocardium, wherein the blood flow of the coronary artery is sharply reduced or interrupted at the time of the lesion. This leads to the corresponding supplied myocardium being severely and persistently acutely ischemic and eventually to myocardial ischemic necrosis. Numerous studies have demonstrated that MI is a complex2 continuous process, which comprises inflammatory responses, cardiomyocyte necrosis and scar formation. The interaction of the aforementioned factors determines the final disease condition and prognosis of patients with MI. Studies have shown that NETs play a deleterious role in cardiovascular disease . NETs formation not only provides a scaffold for thrombus formation but also aggravates endothelial cell damage, which could be a significant contributor to the development of MI. Furthermore, histidine decarboxylase (HDC), protein arginine methyltransferase 1 (PRMT), peptidyl arginine deaminase 4 (PAD4) and apolipoprotein E (APOE) act as upstream regulatory signals of NETs and exert damaging effects in cardiomyocytes (Fig. 4).
HDC exacerbates MI via NETs
HDC is a type of amino acid decarboxylase that mainly catalyzes the decarboxylation of histidine to produce histamine. Regarding MI, studies have found that HDC is associated with ROS and myeloid cell differentiation. Zhang et al. investigated the effect of HDC on ROS production in neutrophils with PMA treatment and observed that the level of ROS produced by neutrophils in the absence of HDC was significantly higher than that in the HDC group, irrespective of PMA treatment  Moreover, previous studies have demonstrated that excess ROS promotes NET production, thus highlighting that HDC plays a crucial role in regulating neutrophils function, especially cellular activity. Additionally, NETs isolated from HDC bone marrow were co-cultured with cardiomyocytes under hypoxic conditions for 24 h after PMA activation and revealed that HDC-deficient neutrophils caused more cardiomyocyte-related death than controls. Thus a positive correlation between NETs and cardiomyocyte death was observed. It is also speculated that HDC deficiency in neutrophils would increase cardiomyocyte death through NETs with cardiac fibroblast proliferation and migration, thereby aggravating MI.
PRMT1 exacerbates MI via NETs
PRMT is a group of enzymes that catalyze the methylation modification of arginine in proteins, mainly on histone H4, which is involved in gene transcription regulation, protein stability regulation, DNA damage signal and other responses. The inhibition of PRMT1 transcription by histamine significantly reduced myocardial fibrosis in HDC-deficient mice and rescued myocardial injury due to MI. Thus, PRMT1 mediates ROS enhancement and NETs generation due to HDC deficiency in neutrophils. In conclusion, neutrophils are recruited to the ischemic injury site of the myocardium when MI occurs, wherein point HDC deficiency leads to attenuated neutrophil adhesion but enhanced migration. Furthermore, PRMT1 increases the production of ROS and NETs through the H1R-SWI/SNF-PRMT1-ROS pathway , which in turn promotes cardiomyocyte death and cardiac fibroblast proliferation, ultimately leading to the aggravation of MI.
PAD4 exacerbates MI via NETs
During MI, the innate immune response and rapid recruitment of albumin play a critical role in regulating inflammation and subsequent healing. PAD4 mainly relies on calcium ions to catalyze the conversion of arginine residues to citrulline residues in target proteins, also known as citrullination. Studies have shown that PAD4 is mainly involved in chromosome decondensation during the release of NETs from activated neutrophils . At sites of inflammatory injury, in addition to recruiting neutrophils to release NETs, the ischemic site rapidly recruits monocyte macrophages. In the early stage of inflammation, monocytes are polarized into M1-type cells and gradually replaced by M2-type cells, which promote the resolution of inflammation and tissue remodelling and thereby induce beneficial healing effects on the injured myocardium [52, 53]. Additionally, by stimulating bone marrow-derived macrophages in wild-type (WT) mice with NETs, Kaveh et al. showed that NETs in the presence of INF-δ and LPS and under hypoxic conditions significantly inhibit IL-6 and TNF-α expression but can upregulate IL-10 levels. Therefore, NETs are speculated to drive macrophage polarization toward an anti-inflammatory phenotype. In addition, NETs can also form autoantigens that induce certain diseases, for example, in systemic lupus erythematosus (SLE), NETs can activate plasmacytoid dendritic cells (pDCs) and trigger type I interferon (IFN) production and drive autoimmune pathology [54, 55]. Parackova et al.  determined that NETs can be used in the development of diabetes mellitus by stimulating with PMA NETs from type 1 diabetes mellitus (T1D) patients and NETs fragments isolated from peripheral blood of healthy control donors, cleavage of NETs with a restriction endonuclease mixture, and co-culture and further compositional analysis using fractions consisting of large DNA fragments and NETs-associated proteins, showed that the presence of NETs induces IFN-γ-producing T cells in vitro and activates T cells toward IFN-γ-producing CD4 and CD8 polarization. Therefore, we speculate that NETs may exacerbate inflammation by inducing Th1 cell polarization, triggering T cell-mediated immune responses, and directly or indirectly regulating inflammatory cytokines. However, PAD4-deficient mice were unable to release NETs but produce higher levels of ROS and display higher levels of cfDNA, cTNT and pro-inflammatory mediators in the post-MI period, wherein cfDNA was widely considered to be a consequence of MI-associated cell death and neutrophil activation . Furthermore, Zhou et al.  reported that NETs could activate macrophages to constantly secrete inflammatory cytokines, thereby promoting the inflammatory response to a certain extent and further aggravating the occurrence and development of diseases. Additionally, cfDNA was observed to increase the expression of IL-10 in cells, thus highlighting the role of NETs in further acting on the myocardial ischemic site and aggravating MI.
APOE exacerbates MI via NETs
APOE is a polymorphic protein involved in the metabolism of lipoproteins. The APOE genes can regulate many biological functions and participate in various responses. Moreover, the APOE genotype-phenotype correlates with the occurrence of MI . Zhou et al.  established an MI model by performing permanent coronary artery ligation surgery on APOE−/− and WT mice. The results of TTC staining showed that APOE−/− mice had larger infarcts than WT mice, and serum assays revealed that APOE−/− mice had significantly higher serum cTnI and CK-MB levels. Thus, these findings indicate that APOE deficiency can aggravate ischemic injury after MI. Additionally, APOE deficiency could exacerbate the activation of neutrophils after MI through an NADPH oxidase-ROS-dependent pathway, promoting the formation of NETs, acting on the site of myocardial ischemic injury, promoting the proliferation of fibroblasts and leading to the exacerbation of myocardial injury in MI. Furthermore, PAD4 could provide a novel therapeutic strategy for protecting the myocardium by aiding in the inhibition of NETs formation via NADPH oxidase inhibition. Therefore, there exists a clear correlation between NETs and poor prognosis of MI. Refining the role and mechanism of NETs in MI and further validating the feasibility of NETs in the treatment of MI has promising diagnostic and therapeutic potential.
NETs and heart failure
Heart failure refers to a clinical syndrome wherein the sizeable venous return volume cannot be fully discharged out of the heart due to cardiac systolic and/or diastolic dysfunction, resulting in blood stasis in the venous system and insufficient blood perfusion in the arterial system. This syndrome is considered the terminal stage in the development of all cardiovascular diseases. Midkine (MK), which mainly mediates the formation of NETs in vitro, can attenuate the formation of NETs and neutrophil infiltration in vivo. Using experimental autoimmune myocarditis (EAM) mouse model to induce the generation of NETs, Ludwig T et al.  observed that the treatment of mice with DNase or protein arginine deamination inhibitor induced leukocyte infiltration in EAM. Furthermore, compared with the untreated model, NETs promoted myocardial inflammation during EAM. This illustrated that targeting MK could reduce neutrophil infiltration and NETs formation in the myocardium. Additionally, the inhibition of MK has also been reported to reduce the infiltration of neutrophils in cardiac tissue and release NETs, thereby reducing myocardial fibrosis . However, the exact mechanisms by which MK promotes the recruitment of NETs and the induction of heart failure by NETs remains unclear. Therefore, serum MK level may have the potential as a diagnostic indicator in patients with heart failure, reflecting the severity of heart failure and the degree of impairment of cardiac function. Thus, targeting MK could reduce neutrophil infiltration and NETs formation in the inflamed myocardium. Furthermore, the inhibition of MK or NETs is a potential therapeutic target for patients with heart failure (Fig. 5).
NETs as therapeutic targets for ACS
Recent studies have revealed that NETs play an essential role in cardiovascular diseases. NETs accelerate the development of ACS through several pathways, with the individual components acting as autoantigens, interacting with multiple cell types, activating inflammasomes and accelerating the atherosclerotic process . Previous studies also report that the level of NETs was negatively correlated with ST-segment regression and positively correlated with infarct size in patients with ST-segment elevation myocardial infarction [61, 62]. Zhou et al.  reported that the inhibition of NETs production attenuated the extent of myocardial injury in APOE−/− mice with reduced infarct size and neutrophil infiltration. Therefore, NETs burden could be considered a predictor of infarct size in ST-segment elevation coronary syndrome. In patients with heart failure, the MK level increases with the heart failure index, thus the serum MK level reflects the severity of heart failure and the course of the disease in patients [63, 64]. Numerous data also suggest that the number of neutrophils and the formation of NETs regulate the early progression of cardiovascular disease. Thus, NETs could provide a potential action target for the clinical treatment of ACS. However, correlation does not necessarily imply causation, and since inflammatory responses precede the formation of NETs, inhibition of NETs may not serve as prophylactic treatment for ACS episodes. In addition, long-term inhibition of inflammation may have other complications, such as possible inhibition of M2 macrophage induction, which may affect tissue repair and regeneration after inflammatory injury. However, in some cases, inhibition of NETs may be beneficial in reducing the inflammatory response and preventing further injury, so the effectiveness of this approach may depend on the stage of the disease and the severity of the inflammatory process, and the optimal use of NETs inhibition as a treatment for ACS may need to focus on the patient’s disease stage, comorbidities, and other factors. Liu et al. [65,66,67] demonstrated that neutrophil granulocytes generate NETs during the acute inflammatory response phase of the disease, and high NET levels polarize macrophages to the M1 phenotype; therefore, it is speculated that NETs may play an important role in the interaction between neutrophils and macrophages during the acute phase, and that NETs inhibition during the acute inflammatory response phase could be used to treat ACS.
Potential therapeutics targeting NETs
An increasing number of studies have found that the formation of neutrophil activation-associated NETs is closely related to the pathogenesis of ACS. The study of NETs and their relevance to ACS may be a therapeutic target for preventing and treating ACS . Several practical therapeutic approaches will be discussed below (Table 1).
PAD4 is a protease that catalyzes the guanylation of arginine residues and is involved in NETosis. CL-amidine, a PAD4 mimetic peptide, competitively binds to PAD4 and inhibits its action . Therefore, inhibition of PAD4 by drugs such as chloro-amidine is a possible target for treating NET-related diseases. CL-amidine exerts its inhibitory effect mainly by covalently modifying conserved cysteine residues in the active site of PAD645 and is an irreversible PAD4 inhibitor that is highly selective for PAD4 . Kinght et al.  found that the PAD4 inhibitor chloro-amidine blocked the formation of NETs in APOE-/- mice fed a high-fat diet, reduced atherosclerosis lesion size, and delayed post-injury thrombosis, leading to the treatment of ACS. In acute carotid injury experiments, Franck et al.  found that PAD4 deficiency protected mice from plaque erosion. Furthermore, Du et al.  found that myocardial ischemic injury significantly increased PAD4 expression activity and that direct inhibition of PAD4 protected the myocardium from inflammation. PAD4 inhibitors prevented the formation of NETs in plaques, reduced the number of endothelial macrophages, decreased neutrophil recruitment to the vessel wall, and reduced levels of inflammatory mediators, ultimately significantly reducing atherosclerotic plaque formation and thrombosis and decreasing the risk of myocardial infarction .
DNase I is a deoxyribonuclease that lyses the DNA component of NETs and has been used as a pharmacological intervention to treat various diseases. Since DNA is a significant component of NETs, inhibition of NETs formation with DNase I is highly likely for the treatment of ACS. DNase I treatment is an effective therapeutic tool for neutrophil-derived diseases caused by NETs. Warnatsch et al.  demonstrated that systemic injection of DNase I reduced the size of atherosclerotic plaques in mice and decreased the number of NETs and the number of inflammatory cells. In addition, Frank et al.  administered DNase I in a PAD4-/- mouse model and found reduced surface erosion of endothelial cells and an increase in surviving endothelial cells while limiting the recruitment of neutrophils. It has been shown that DNase I can partially lyse NETs. However, whether the degradation of NETs by DNase I leads to the breakdown of histones with coagulant activity, which in turn increases the risk of thrombosis, needs further investigation .
The NADPH oxidase-derived active enzyme ROS is essential for the formation of NETs. ROS has been shown to induce different levels of DNA damage and activate neutrophil elastase, which contributes to chromatin cleavage and NETs formation . Through a PAM-induced ROS model, Zeng et al.  found that antioxidants inhibit ROS in activated neutrophils production and dsDNA release and that the inhibition of NETs by antioxidants was achieved by inhibiting ROS if the cells were pretreated with NADPH inhibitors, which did not result in dsDNA release. It was shown that antioxidant substances that significantly inhibit ROS release from neutrophils likewise inhibit the formation of ROS-dependent NETs, suggesting a clear correlation between the two .
Heparin is a multifunctional glycosaminoglycan with anticoagulant activity and antithrombotic effects that inhibits the activation of neutrophils to produce NETs while promoting the breakdown of NETs, releasing histones from the chromatin backbone and destabilizing DNA, thereby disrupting the coagulation effect of NETs and preventing thrombosis [79,80,81]. Angelo et al.  showed that treating NET-related diseases with heparin depends in part on the induction of a “refractory” state, leading to a decrease in the stimulation of the inflammatory response and a decrease in the release of NETs. In addition, NETs can induce endothelial cell damage. The degree of damage is positively correlated with the concentration of NETs. Heparin can reduce NET-induced endothelial damage, which may be a potential mechanism for the protective effect of heparin on ACS .
Inflammatory mediator receptor blockers
Colchicine, a novel therapeutic agent, has demonstrated its safety and efficacy by inhibiting the inflammatory cascade response in patients with extracranial or intracranial atherosclerosis or slight arteriosclerosis, thereby reducing vascular events [84, 85]. The CALCOT trial [86,87,88] showed that compared with a placebo, low doses of colchicine significantly reduced the risk of recurrence in patients with ischemic cardiovascular disease risk. The EQUALTRCT trial [89,90,91] demonstrated that colchicine inhibits the activity of inflammatory vesicles, which can reduce the load of NETs in cardiovascular disease and effectively prevent the development of ACS.
Conclusions and future directions
Recently, the incidence of cardiovascular disease has been increasing worldwide, with ACS posing a significant threat to human health. As an important immune cell, neutrophils are vital to the study of cardiovascular diseases. Notably, NETs have become a popular research direction in the search for therapeutic targets in cardiovascular diseases. Since their discovery in 2004, various experimental and clinical literature has confirmed the role of NETs in various cardiovascular diseases and their correlation with the poor prognosis of diseases. However, the role and mechanism of NETs in various diseases remain to be fully elucidated. Additionally, controversies about the translation of NETs in clinical settings remain. Nonetheless, new insights from the study of NETs in cardiovascular diseases are expected to open new avenues for diagnosing and treating of ACS. This review explored the impact of NETs on ACS, identifying that NETs play a novel and essential role in ACS and highlighting its potential as a novel target for the prevention and treatment of ACS.
Xinchao Zhang, Xuezhong Yu, Fengying Chen, et al. Guidelines for rapid emergency diagnosis and treatment of acute coronary syndrome (2021). J Clin Emerg. 2019;20(4):253–62.
Juan Zhao, Renjie Chai, Shibo Song, et al. Clinical study of spot tracking echocardiography combined with plasma miR-30a to evaluate the short-term prognosis of patients with acute coronary syndrome. China New Clinic Med. 2022;15(1):45–50.
Fusheng Cai, Yun Hu. Research progress of neutrophil extracellular trap net in acute myocardial infarction. China Med Innov. 2021;18(9):1674–4985.
Linxin Wang, Yuan Chen, Lingze Zeng, et al. Research progress on neutrophil extracellular trap net and cardiovascular disease thrombosis. Chin J Evid Based Cardiovasc Med. 2019;9(11):1674–4055.
Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol. 2018;18:134–47.
Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–5.
Döring Y, Libby P, Soehnlein O. Neutrophil Extracellular Traps Participate in Cardiovascular Diseases: recent experimental and clinical insights. Circ Res. 2020;126(9):1228–41.
Zhang J, Dai Y, Wei C, et al. DNase I improves corneal epithelial and nerve regeneration in diabetic mice. J Cell Mol Med. 2020;24(8):4547–56.
Xia Y, He J, Zhang H, et al. AAV-mediated gene transfer of DNase I in the liver of mice with colorectal cancer reduces liver metastasis and restores local innate and adaptive immune response. Mol Oncol. 2020;14(11):2920–35.
Sung PS, Hsieh SL. C-type lectins and extracellular vesicles in virus-induced NETosis. J Biomed Sci. 2021;28(1):46.
Silva JC, Thompson-Souza GA, Barroso MV, et al. Neutrophil and eosinophil DNA extracellular trap formation: lessons from pathogenic fungi. Front Microbiol. 2021;12:634043.
Jiao Y, Li W, Wang W, et al. Platelet-derived exosomes promote neutrophil extracellular trap formation during septic shock. Crit Care. 2020;24(1):380.
Aldo Bonaventura F, Montecucco F, Dallegri, et al. Novel findings in neutrophil biology and their impact on cardiovascular disease. Cardiovascular Res. 2019;15(8):1266–85.
Milena Michalska, Tadeusz Grochowiecki, Tomasz Jakimowicz, et al. A review of the impact of neutrophils and Neutrophil Extracellular Traps (NETs) on the development of aortic aneurysms in animal and human studies. Med Sci Monitor. 2021;27:e935134.
Mozzini C, Pagani M. Cardiovascular diseases: consider netosis. Curr Probl Cardiol. 2022;47(10):100929.
Sollberger G, Tilley DO, Zychlinsky A. Neutrophil Extracellular Traps: the Biology of chromatin externalization. Dev Cell. 2018;44:542–53.
Ming W, Yanting S, Baoqi Y, et al. The role of neutrophil extracellular trap in cardiovascular disease. Prog Physiol. 2020;51(5):1994–2022.
Yipp BG, Kubes P. NETosis: how vital is it? Blood. 2013;12216:2784–94.
Reithofer M, Karacs J, Strobl J, et al. Alum triggers infiltration of human neutrophils ex vivo and causes lysosomal destabilization and mitochondrial membrane potential-dependent NET-formation. FASEB J. 2020;34(10):14024–41.
Steinberg BE, Grinstein S. Unconventional roles of the NADPH oxidase: signaling, ion homeostasis, and cell death. Sci STKE. 2007;2007:pe11.
Buchanan JT, Simpson AJ, Aziz RK, et al. DNase expression allows the pathogen group a Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol. 2006;16(4):396–400.
FuchsTA AbedU. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol. 2007;176(2):231–41.
YousefiS MihalacheC. Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps. Cell Death Differ. 2009;16(11):1438–44.
Clark SR, Ma AC, Tavener SA, et al. Platelet TLR4activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med. 2007;13(4):463–9.
Masso-Silva JA, Moshensky A, Lam MTY, et al. Increased peripheral blood neutrophil activation phenotypes and Neutrophil Extracellular trap formation in critically ill coronavirus Disease 2019 (COVID-19) patients: a Case Series and Review of the literature. Clin Infect Dis. 2022;74(3):479–89.
Nakazawa D, Tomaru U, Suzuki A, et al. Abnormal conformation and impaired degradation of propylthiouracil-induced neutrophil extracellular traps: implications of disordered neutrophil extracellular traps in a rat model of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis. Arthritis Rheum. 2012;64:3779–87.
Kessenbrock K, Krumbholz M, Schonermarck U, et al. Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med. 2009;15:623–5.
Nakazawa D, Shida H, Tomaru U, et al. Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCA-associated microscopic polyangiitis. J Am Soc Nephrol. 2014;25:990–7.
Nakazawa D, Shida H, Kusunoki Y, et al. The responses of macrophages in interaction with neutrophils that undergo NETosis. J Autoimmun. 2015;67:19–28.
GavilletM MartinodK. Flow cytometric assay for direct quantification of neutrophil extracellular traps in blood samples. Am J Hematol. 2015;90(12):1155–8.
Haozhe Q, Shuofei Y, Kejia K, et al. Research progress in neutrophil extracellular trap detection technology. Chin Med J. 2017;97(28):2234–6.
Zhao W, Fogg DK, Kaplan MJ. A novel image-based quantitative method for the characterization of NETosis. J Immunol Methods. 2015;423:104–10.
Li P, Li M, Lindberg MR, et al. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med. 2010;207:1853–62.
Hemmers S, Teijaro JR, Arandjelovic S, et al. PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection. PLoS ONE. 2011;6:e22043.
Perdomo J, Leunghh L, Ahmadi Z, et al. Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia. Nat Commun. 2019;10(1):1322.
Saha P, Yeoh BS, Xiao X, et al. PAD4-dependent NETs generation are indispensable for intestinal clearance of citrobacter rodentium. Mucosal Immunol. 2019;12(3):761–71.
Megens RT, Vijayan S, Lievens D, et al. Presence of luminal neutrophil extracellular traps in atherosclerosis. Thromb Haemost. 2012;107(3):597–8.
Pertiwi KR, Ac VDW, Pabittei DR, et al. Neutrophil Extracellular Traps participate in all different types of thrombotic and haemorrhagic complications of coronary atherosclerosis. Thromb Haemostasis. 2018;118(6):1078–87.
Folco EJ, Mawson TL, Vromman A, et al. Neutrophil Extracellular Traps induce endothelial cell activation and tissue factor production through Interleukin-1α and cathepsin G. Arterioscler Thromb Vasc Biol. 2018;38(8):1901–12.
Silvestre-Roig C, Braster Q, Wichapong K, et al. Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death. Nature. 2019;569:236–40.
Eliason JL, Hannawa KK, Ailawadi G, et al. Neutrophil depletion inhibits experimental abdominal aortic aneurysm formation. Circulation. 2005;112:232–40.
Sorvillo N, Cherpokova D, Martinod K, et al. Extracellular DNA NET-Works with dire consequences for Health. Circ Res. 2019;125(4):470–88.
Liu Y, Carmona-Rivera C, Moore E, et al. Myeloid-specific deletion of Peptidylarginine Deiminase 4 mitigates atherosclerosis. Front Immunol. 2018;9:1680.
Franck G, Mawson TL, Folco EJ, et al. Roles of PAD4 and NETosis in experimental atherosclerosis and arterial Injury: implications for superficial Erosion. Circ Res. 2018;123:33–42.
Tall AR, Westerterp M. Inflammasomes, neutrophil extracellular traps, and cholesterol. J Lipid Res. 2019;60:721–7.
Fuchs TA, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci USA. 2010;107:15880–5.
Thalin C, Hisada Y, Lundstrom S, et al. Neutrophil Extracellular Traps: villains and targets in arterial, venous, and Cancer-Associated hrombosis. Arterioscler Thromb Vasc Biol. 2019;39:1724–38.
Zucoloto AZ, Jenne CN. Platelet-neutrophil interplay: insights into neutrophil extracellular trap (NET)-driven coagulation in infection. Front Cardiovasc Med. 2019;6:85.
Mutua V, Getshwinl J. A review of neutrophil extracellular traps(NETs) in disease: potential anti-NETs therapeutics. Clin Rev Allergy Immunol. 2021;61(2):194–211.
Huaqing W, Hao Z, Donghai , et al. Research progress on the relationship between neutrophil extracellular trap net and ischemia-reperfusion injury. Chin J Cardiol. 2020;25(6):591–4.
ZhangZ DingS. Prmt1 upregulated by Hdc deficiency aggravates acute myocardial infarction via NETosis. Acta Pharm Sin B. 2022;12(4):1840–55.
Kaveh Eghbalzadeh L, Georgi T, Louis, et al. Compromised anti-inflammatory action of Neutrophil Extracellular Traps in PAD4-Deficient mice contributes to aggravated acute inflammation after myocardial infarction. Front Immunol. 2019;10:2313.
Mouton AJ, DeLeon-Pennell KY, Rivera Gonzalez OJ, et al. Mapping macrophage polarization over the myocardial infarction time continuum. Basic Res Cardiol. 2018;113:26.
Garcia-Romo GS, Caielli S, Vega B, et al. Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med. 2011;3(73):73ra20.
Lande R, Gregorio J, Facchinetti V, et al. Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature. 2007;449(7162):564–9.
Parackova Z, Zentsova I, Vrabcova P, et al. Neutrophil Extracellular Trap Induced dendritic cell activation leads to Th1 polarization in type 1 diabetes. Front Immunol. 2020;11:661.
Huaihai Z, Zhiqiang Q, Shaojia Q, et al. The relationship between inflammatory indicators and CT pulmonary artery occlusion index in patients with pulmonary embolism. Int J Respirat Sci. 2020;40(1):19–24.
Huimin Z, Qing G, Xue H, et al. ApoE gene phenotype and risk assessment of cardiovascular disease in patients with diabetes. J Pharm Forum. 2022;43(10):26–9.
Zhou Z, Zhang S, Ding S et al. Excessive Neutrophil Extracellular Trap Formation Aggravates Acute Myocardial Infarction Injury in Apolipoprotein E Deficiency Mice via the ROS-Dependent Pathway. Oxid Med Cell Longev. 2019; 2019: 1209307.
Weckbach LT, Grabmaier U, Uhl A, et al. Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis. J Exp Med. 2019;216(2):350–68.
Liu J, Yang D, Wang X, et al. Neutrophil extracellular traps and dsDNA predict outcomes among patients with ST-elevation myocardial infarction. Sci Rep. 2019;9:11599.
Pertiwi KR, van der Wal AC, Pabittei DR, et al. Neutrophil Extracellular Traps participate in all different types of thrombotic and haemorrhagic complications of coronary atherosclerosis. Thromb Haemost. 2018;118:1078–87.
Yang LY, Luo Q, Lu L, et al. Increased neutrophil extracellular traps promote metastasis potential of hepatocellular carcinoma via provoking tumorous inflammatory response. J Hematol Oncol. 2020;13(1):3.
Rivera-Franco MM, Leon-Rodriguez E, Torres-Ruiz JJ, et al. Neutrophil extracellular traps associate with clinical stages in breast cancer. Pathol Oncol Res. 2020;26(3):1781–5.
Liu S, Su X, Pan P, et al. Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury. Sci Rep. 2016;6:37252.
Li H, Zhou X, Tan H, et al. Neutrophil extracellular traps contribute to the pathogenesis of acid-aspiration-induced ALI/ARDS. Oncotarget. 2018;9(2):1772–84.
Song C, Li H, Li Y, et al. NETs promote ALI/ARDS inflammation by regulating alveolar macrophage polarization. Exp Cell Res. 2019;382(2):0014–4827.
Dong Y, Zhang Y, Yang X, et al. Recent insights into Neutrophil Extracellular Traps in Cardiovascular Diseases. J Clin Med. 2022;11(22):6662.
Luo Y, Arita K, Bhatia M, et al. Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization. Biochemistry. 2006;45:11727–36.
Knight JS, Luo W, O’Dell AA, et al. Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis. Circ Res. 2014;114:947–56.
Franck G, Mawson TL, Folco EJ, et al. Roles of PAD4 and NETosis in experimental atherosclerosis and arterial Injury: implications for superficial Erosion. Circ Res. 2018;123:33–42.
Mingjun D, Wenang Y, Schmull S, et al. Inhibition of peptidyl arginine deiminase-4 protects against myocardial infarction induced cardiac dysfunction. Int Immunopharmacol. 2020;78:1567–5769.
Warnatsch A, Ioannou M, Wang Q, et al. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis. Science. 2015;349:316–20.
Franck G, Mawson TL, Folco EJ, et al. Roles of PAD4 and netosis in experimental atherosclerosis and arterial injury implications for superfcial erosion. Circ Res. 2018;123:33–42.
Zhou Y, Xu Z, Liu Z. Impact of Neutrophil Extracellular Traps on thrombosis formation: New Findings and Future Perspective. Front Cell Infect Microbiol. 2022;12:910908.
Papayannopoulos V, Metzler KD, Hakkim A, et al. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol. 2010;191:677–91.
Zeng J, Xu H, Fan PZ, et al. Kaempferol blocks neutrophil extracellular traps formation and reduces tumour metastasis by inhibiting ROS-PAD4 pathway. J Cell Mol Med. 2020;24(13):7590–9.
Kirchner T et al. “Flavonoids and 5-aminosalicylic acid inhibit the formation of neutrophil extracellular traps.” Mediators of inflammation vol. 2013; (2013): 710239.
Fuchs TA, Brill A, Duerschmied D et al. Extracellular DNA traps promote thrombosis. Proc. Natl. Acad. Sci. U. S. A. 2010; 107 : 15880–15885.
Apostolidou E, Skendros P, Kambas K, et al. Neutrophil extracellular traps regulate IL-1beta-mediated inflammation in familial Mediterranean fever. Ann Rheumat Dis. 2016;75:269–77.
Rovere-Querini APatrizia. Low molecular weight heparins prevent the induction of autophagy of activated neutrophils and the formation of neutrophil extracellular traps. Pharmacol Res vol. 2017;123:146–56.
Sanchez J. Low Molecular Weight Heparins—A new tool to disetangle from the NETs. Pharm Res. 2017;123:157.
Tsivgoulis G, Katsanos AH, Giannopoulos G, et al. The role of colchicine in the prevention of cerebrovascular ischemia. Curr Pharm Des. 2018;24:668–74.
Kelly PJ, Murphy S, Coveney S, et al. Anti-inflammatory approaches to ischaemic stroke prevention. J Neurol Neurosurg Psychiatry. 2018;89:211–8.
Jorch SK, Kubes P. An emerging role for neutrophil extracellular traps in noninfectious disease. Nat Med. 2017;23:279–87.
Van Avondt K, Maegdefessel L, Soehnlein O. Therapeutic targeting of Neutrophil Extracellular Traps in atherogenic inflammation. Thromb Haemost. 2019;119(4):542–52.
Moschonas IC, Tselepis AD. The pathway of neutrophil extracellular traps towards atherosclerosis and thrombosis. Atherosclerosis. 2019;288:9–16.
Tsivgoulis G, Katsanos AH, Giannopoulos G, et al. The role of Colchicine in the Prevention of Cerebrovascular Ischemia. Curr Pharm Design. 2018;24:668–74.
Kelly PJ, Murphy S, Coveney S, et al. Anti-inflammatory approaches to ischaemic stroke prevention. J Neurol Neurosurg Psychiatry. 2018;89:211–8.
Hoss F, Latz E. Inhibitory effects of colchicine on inflammasomes. Atherosclerosis. 2018;273:153–4.
Chirivi RGS, van Rosmalen JWG, van der Linden M, et al. Therapeutic ACPA inhibits NET formation: a potential therapy for neutrophil-mediated inflammatory diseases. Cell Mol Immunol. 2021;18(6):1528–44.
Sollberger G, Choidas A, Burn GL, et al. Gasdermin D plays a vital role in the generation of neutrophil extracellular traps. Sci Immunol. 2018;3(26):eaar6689.
The study was funded by the Natural Sciences Foundation of Fujian (No. 2022J05105); the Cuiying Scientific and Technological Innovation Program of Lanzhou University Second Hospital (No. CY2019-HL06, CY2019-QN01); the Talent Introduction Plan of the Lanzhou University Second Hospital (No. YJRCKYQDJ-2021-02); the Science and Technology Planning Project of Chengguan District (2022RCCX0023).
Institutional Review Board Statement
Informed consent Statement
Conflict of interest
The authors declare no conflict of interest.
Consent for publication
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Wu, Y., Wei, S., Wu, X. et al. Neutrophil extracellular traps in acute coronary syndrome. J Inflamm 20, 17 (2023). https://doi.org/10.1186/s12950-023-00344-z
- Neutrophil extracellular traps
- Acute coronary syndrome
- Myocardial infarction
- Heart failure