In 2004, Brinkmann et al (11) first demonstrated the existence of NETs and showed that they contain neutrophil elastase (NE), cathepsin G, myeloperoxidase (MPO), lactoferrin, gelatinase, histones H1, H2A, H2B, H3 and H4 and H2A-H2B-DNA complexes. Subsequent research has generated extensive proteomic data on NETs. Dwyer et al (12) found that NETs induced by both nonmucoid and mucoid Pseudomonas aeruginosa strains contain a conserved group of proteins, irrespective of the inducing factor, which they termed the 'core NET signature'. This conserved protein set includes nucleosome-associated proteins (such as histones), structural proteins (such as coronin-1, β-actin and α-actinin), enzymes (such as NE, transaldolase, thymidine kinase, glyceraldehyde-3-phosphate dehydrogenase and α-enolase) and antimicrobial proteins (such as MPO, lysozyme C, lactoferrin and azurocidin).

However, NETs are not structurally uniform and their protein composition varies according to the inducing stimulus and tissue microenvironment. Proteomic analyses have identified stimulus-specific NET signatures. For example, phorbol 12-myristate 13-acetate (PMA)-induced NETs are enriched in heat shock proteins, whereas lipopolysaccharide (LPS)-induced NETs contain higher levels of lysosomal proteins, such as LAMP2 (13).

NETs are released by activated neutrophils through a process termed NETosis. The currently recognized pathways of NET formation are broadly classified as NADPH oxidase (NOX)-dependent NETosis, also known as suicidal NETosis and NOX-independent NETosis, also known as vital NETosis. Vital NETosis can be further divided into nuclear DNA-derived and mitochondrial DNA-derived NET (mtNET) subtypes. The specific mechanisms are shown in Fig. 1.

i) NOX-dependent NETosis (classical pathway). Also known as suicidal NETosis, this process typically occurs within 2-4 h and culminates in the complete rupture of the neutrophil nuclear envelope and plasma membrane, accompanied by cell death. It can be initiated by high concentrations of LPS, pathogens such as Staphylococcus aureus, PMA, high concentrations of complement component C5a, immune complexes and inflammatory mediators such as tumor necrosis factor (TNF)-α and interleukin (IL)-8. These stimuli activate neutrophil Toll-like receptors (TLRs), Fcγ receptors and complement receptors (14). Following receptor activation, calcium is initially released from endoplasmic reticulum stores, followed by substantial extracellular Ca²⁺ influx through store-operated calcium channels. The resulting increase in cytosolic calcium activates protein kinase C (PKC), which subsequently initiates the Raf-MEK-ERK signaling pathway (15). This pathway phosphorylates and activates the NOX complex, resulting in the sustained generation of large amounts of reactive oxygen species (ROS), which further increase intracellular calcium levels (16). Calcium-dependent peptidylarginine deiminase 4 (PAD4) is subsequently activated and catalyzes the citrullination of arginine residues in the tails of histones H1, H2A, H3 and H4. Citrullination neutralizes the positive charges of histones, weakens their electrostatic interactions with DNA and initiates chromatin decondensation (17). Concurrently, NOX-derived ROS promote the release of MPO and NE from azurophilic granules into the cytoplasm, after which these enzymes translocate to the nucleus. NE cleaves histones through its proteolytic activity, whereas MPO enhances oxidative signaling and facilitates chromatin relaxation; together, these processes promote complete chromatin decondensation. Downstream ROS and calcium signaling activate gasdermin D (GSDMD), which oligomerizes and forms membrane pores. The nuclear envelope subsequently disintegrates, allowing decondensed chromatin to mix with granule proteins. Finally, the plasma membrane ruptures, releasing large quantities of NETs into the extracellular space (18).

ii) NOX-independent NETosis. Also referred to as vital NETosis, this pathway occurs rapidly, typically within 15-60 min. The plasma membrane remains intact throughout the process, allowing neutrophils to survive and retain their phagocytic and chemotactic functions. This pathway does not depend on plasma membrane-associated NOX-derived ROS, although certain subtypes require mitochondrial ROS (mtROS). It can be divided into two principal categories: i) Nuclear DNA-derived vital NETs: Classical inducers include the calcium ionophore A23187, low concentrations of C5a, S. aureus and low-dose LPS presented by activated platelets. Following stimulation, rapid and substantial extracellular Ca²⁺ influx directly activates PAD4, which catalyzes histone citrullination and promotes nuclear chromatin decondensation (14). Decondensed chromatin and granule proteins are packaged into vesicles that bud from the nuclear envelope and are released into the extracellular space through exocytosis. Once outside the cell, granule proteins bind to DNA to form web-like NETs. The resulting enucleated neutrophils retain phagocytic vacuoles and remain capable of phagocytosis (19). Notably, stimulation with Candida albicans can induce PAD4-independent vital NETosis, in which chromatin decondensation is mediated solely by NE-dependent histone cleavage without requiring histone citrullination (20). ii) Mitochondrial DNA (mtDNA)-derived vital NETs: When neutrophils are primed with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequently stimulated with low-dose LPS or C5a, increased calcium signaling induces opening of the mitochondrial permeability transition pore, resulting in mitochondrial damage, mtROS production and the release of mtDNA (14). After oxidative modification, mtDNA associates with granule proteins, including MPO and NE, to form complexes that are released through mitochondrial vesicle secretion or local fusion of mitochondria with the plasma membrane (21). This process generates mtNETs with mtDNA as their structural backbone, while the neutrophil remains intact and does not undergo lysis.

Although NETs were initially identified in the context of microbial infection, numerous stimuli relevant to cardiovascular pathology can also induce NETosis. Within the cardiovascular system, NET-inducing stimuli predominantly include lipid metabolites, damage-associated molecular patterns (DAMPs) and intercellular signaling, rather than direct stimulation by exogenous pathogens. First, among lipid-related triggers, oxidized low-density lipoprotein (oxLDL) and endogenous cholesterol crystals can engage TLR4 and the receptor for advanced glycation end products on the neutrophil surface, thereby promoting sustained neutrophil activation and acting as major chemical initiators of NETosis in lipid-associated CVDs (22,23). Metabolic abnormalities, including hyperglycemia and elevated levels of free fatty acids, can also function as persistent stimuli and induce sustained NET release from perivascular neutrophils (14,24). Second, DAMPs released following tissue injury contribute substantially to NET formation. After myocardial infarction, ischemic stroke, or organ ischemia-reperfusion injury, necrotic endothelial and parenchymal cells release high-mobility group box 1 (HMGB1), cell-free mtDNA and extracellular histones (25). These molecules can establish a positive-feedback loop that continuously stimulates circulating neutrophils to generate additional NETs. Third, intercellular signaling associated with vascular injury can induce NETosis. P-selectin expressed on activated platelets binds to P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils, thereby promoting NET formation (26). The complement activation product C5a regulates NETosis in a concentration-dependent manner: Low concentrations of C5a primarily induce calcium influx and mtNET formation, whereas high concentrations activate the NOX complex and promote suicidal NETosis (14). Furthermore, cardiovascular risk factors, including monosodium urate crystals, tobacco smoke and fine particulate matter with an aerodynamic diameter of ≤2.5 µm (PM2.5), can chronically stimulate neutrophils within the vascular wall, thereby sustaining persistent, low-grade NET release (14).

NETs induced in the cardiovascular milieu are enriched with a variety of disease-specific functional molecules that form the molecular basis of their prothrombotic and vascular-damaging effects. First, NETs contain procoagulant proteins: NET fibers display abundant tissue factor (TF) and von Willebrand factor (vWF) on their surfaces. TF directly activates the extrinsic coagulation pathway, whereas vWF captures circulating platelets; moreover, NET-bound NE can degrade tissue factor pathway inhibitor, thereby disrupting the anticoagulant balance and promoting immunothrombosis (27,28). Second, NETs contain matrix-degrading proteases: matrix metalloproteinase (MMP)-9 is abundantly associated with plaque-derived NETs and degrades collagen within the arterial fibrous cap, resulting in cap thinning and increased plaque vulnerability to rupture, thereby precipitating acute cardiovascular and cerebrovascular events (29). Third, NETs contain inflammatory chemotactic mediators: NET-associated chemokines such as IL-8 continuously recruit peripheral neutrophils to vascular lesions, thereby perpetuating chronic inflammation (30). At the clinical level, plasma MPO-DNA complexes, circulating citrullinated histone H3 (CitH3) and cell-free mtDNA are specific biomarkers of NET formation in cardiovascular disease; their levels are markedly elevated in patients with coronary artery disease, ischemic stroke and deep vein thrombosis and can be used to assess disease severity (31). However, direct comparative proteomic analyses of NETs across different CVDs remain scarce. Defining disease-specific NET signatures will not only deepen mechanistic understanding but also facilitate the development of targeted diagnostic biomarkers and selective therapeutic strategies.

At sites of cardiovascular injury, NETs shape and amplify the local inflammatory microenvironment by acting on macrophages, monocytes and endothelial cells. These effects occur at three interconnected levels: Induction of pro-inflammatory macrophage polarization, chemotactic recruitment of monocytes and maintenance of their pro-inflammatory phenotype and pro-inflammatory activation of endothelial cells (Fig. 2).

NETs drive immunothrombosis by inducing procoagulant activity in endothelial cells and activating platelets, thereby forming a key interface at which inflammation and thrombosis reinforce each other in cardiovascular disease. In this pathological sequence, NETs first shift endothelial cells from an anticoagulant to a procoagulant phenotype, providing the initiating signal for subsequent platelet adhesion and activation. Activated platelets and NETs then establish a bidirectional amplification loop that markedly augments the initial thrombotic signal and ultimately promotes the formation of stable immunothrombi.

NETs promote pathological fibrosis in cardiovascular tissues through three principal mechanisms: Direct activation of fibroblasts, induction of monocyte-to-fibroblast-like transdifferentiation and promotion of endothelial-to-mesenchymal transition (EndoMT). The pro-fibrotic matrix deposition and tissue-invasive capacity conferred on monocytes by NETs provide an important link between inflammation and fibrosis.

Growing evidence indicates that inflammation, thrombosis and fibrosis are not isolated pathological events in cardiovascular disease but instead form an interconnected network of mutually reinforcing and sequential processes. NETs serve as a central integrative node within this triad.

NETs establish a bidirectional amplification loop between inflammation and thrombosis. NET-associated IL-1α and cathepsin G, together with the activation of endothelial TLR2 and downstream cGAS-STING signaling, synergistically upregulate TF and adhesion molecules, shifting the endothelium toward a pro-inflammatory and procoagulant phenotype (46,60). Activated platelets, in turn, potently induce NETosis through multiple pathways, including P-selectin/PSGL-1, TLR4 and αIIbβ3-SLC44A2. Conversely, NET components such as S100A8/A9 and dsDNA activate platelets; S100A8/A9 induces platelet pyroptosis, whereas dsDNA elicits cGAS-STING-dependent platelet activation. Together, these reciprocal interactions form a bidirectional platelet-NET amplification loop that continuously reinforces the local immunothrombotic microenvironment (48-51). During acute events such as myocardial infarction and plaque erosion, this inflammation-thrombosis axis is particularly prominent. NET-rich thrombi not only exacerbate ischemic tissue injury but also promote further recruitment of inflammatory cells.

NETs also link early inflammation to subsequent fibrosis. NET-recruited monocytes can transdifferentiate into fibroblast-like cells and directly contribute to ECM production (40). At the endothelial level, NET-associated elastase cleaves VE-cadherin and activates β-catenin signaling, inducing EndoMT and the acquisition of a myofibroblast-like phenotype (68). Furthermore, NETs directly activate cardiac fibroblasts through the TGF-β/SMAD, MAPK and RhoA signaling axes, promoting collagen deposition and myofibroblast differentiation (58,59).

A bidirectional relationship also exists between thrombosis and fibrosis. The thrombotic network formed by NETs and fibrin provides a structural scaffold for fibroblast migration and matrix deposition (69,70). Meanwhile, thrombin and other coagulation proteases generated through the NET-driven coagulation cascade can activate protease-activated receptors on the fibroblast surface, further reinforcing the fibrotic response and promoting the gradual progression of thrombotic lesions toward fibrosis (71).

Collectively, these findings support a unified model in which NETs act as a central hub that integrates and propagates the inflammation-thrombosis-fibrosis cascade in cardiovascular pathology. This framework has potential therapeutic implications: targeting NETs may attenuate all three arms of the cascade and therefore offer broader clinical benefits than inhibiting a single pathological component.

In AMI, NETs are not merely markers of sterile inflammation but also constitute an important mechanistic link between the initial inflammatory burst and subsequent reparative fibrosis. Clinical studies have consistently demonstrated that coronary thrombi and infarcted myocardial tissue from patients with ST-segment elevation myocardial infarction (STEMI) are enriched in NETs and that a greater NET burden is closely associated with microvascular obstruction, adverse left ventricular remodeling and worse clinical outcomes (72-75).

The intense sterile inflammatory response triggered by myocardial infarction makes NET-monocyte interactions a major determinant of repair quality. NETs stimulate vascular endothelial cells to secrete large amounts of MCP-1. High local concentrations of MCP-1, in turn, prime neutrophils for NETosis, establishing a positive-feedback loop between NETs and MCP-1 in the infarct zone that directs monocyte infiltration (40). Following NET stimulation, infiltrating monocytes transdifferentiate into fibroblast-like cells through TLR4 signaling, directly participate in collagen deposition and become an important cellular source of post-infarction fibrosis (75). These findings suggest that NETs function as a molecular switch in AMI, redirecting the acute defensive response toward pro-fibrotic remodeling.

Studies have further clarified the upstream triggers and downstream amplification mechanisms of NETosis. Zhang et al (76) found that post-infarction hypoxia induces high S100A12 expression in neutrophils through HIF1α. S100A12 then binds to ANXA5, triggering calcium influx and activating the NOX4-ROS-ERK pathway to drive 'suicidal' NETosis, thereby aggravating cardiac dysfunction. In parallel, dsDNA released from NETs activates the cGAS-STING-NLRP3 pathway in platelets, enhancing platelet activation and thrombus formation and further worsening myocardial ischemia (52). Together, these studies delineate a pathogenic sequence in which the S100A12-ANXA5-Ca²⁺ axis initiates NETosis, whereas the dsDNA-cGAS-platelet pathway amplifies thrombotic injury following NET release. These mechanisms operate sequentially as upstream and downstream components of the same pathogenic cascade. Notably, during the later stages of infarct healing, moderate NET levels may facilitate the clearance of necrotic tissue by promoting Mertk+ reparative macrophage polarization (77), suggesting that NETs exert stage-dependent effects in AMI. Therefore, transient suppression of excessive NETosis during the early phase, while preserving the physiological functions of NETs during tissue repair, may represent a more rational therapeutic strategy. However, the longitudinal dynamics of NET activity in humans following myocardial infarction remain poorly characterized. Identifying the signals that terminate NETosis and define the repair window is therefore an important priority for translational research.

AF is the most common sustained tachyarrhythmia and atrial remodeling is central to its initiation and maintenance. Early histological studies showed that approximately two-thirds of patients with 'lone AF' exhibited localized atrial inflammation that met the criteria for myocarditis (78). NETosis has been shown to participate in the inflammatory response associated with AF (79), making NETs an emerging focus of AF pathophysiology research.

Emerging evidence indicates that NETs are not merely an inflammatory epiphenomenon in AF but instead function as a central hub driving atrial remodeling. He et al (80) found that elevated angiotensin II (Ang II) levels in AF increase AKT phosphorylation in neutrophils through the AT1 receptor. However, because the mTOR pathway is simultaneously inhibited, Ang II does not induce autophagy and is insufficient to initiate NETosis alone; instead, it synergizes with inflammatory stimuli to enhance NET release. Meanwhile, mtDNA and HMGB1 released following rapid pacing-induced mitochondrial damage in cardiomyocytes can act directly as DAMPs and trigger NETosis. Meng et al (81) further showed that chronic stress induces high S100A8/A9 expression in atrial tissue. S100A8/A9 recruits neutrophils and activates the TLR4-NLRP3 inflammatory pathway, providing another upstream trigger of NETosis. Downstream, NETs exert distinct effects on cardiomyocytes and fibroblasts. In cardiomyocytes, NETs induce autophagic apoptosis by upregulating p62/Beclin-1/LC3B signaling and cause irreversible mitochondrial damage through mitochondrial membrane depolarization and a burst of mtROS. In fibroblasts, NETs activate Smad/p-p38 MAPK signaling, promote fibroblast-to-myofibroblast transition and exacerbate atrial fibrosis and electrical remodeling (80). Collectively, these findings delineate a self-sustaining pathogenic circuit in AF: Ang II-mediated synergistic signaling and the S100A8/A9-TLR4 axis initiate NETosis; DAMPs such as mtDNA and HMGB1 sustain NET formation; and NET-induced mitochondrial damage promotes the release of additional mtDNA. These three components establish a positive-feedback loop that can persist independently of the initial stressor, thereby driving the perpetuation and recurrence of AF.

However, some gaps remain in AF research. Although NETs induce mitochondrial damage and mtDNA release, direct experimental evidence that the classical mtDNA-cGAS-STING cytosolic DNA-sensing pathway is activated in AF and amplifies the inflammatory cycle is currently lacking.

VMC is myocardial inflammation caused by viral infection and may progress to dilated cardiomyopathy or HF. In a mouse model of coxsackievirus B3 (CVB3)-induced VMC, NETs were detected in both serum and myocardial tissue and NET degradation markedly attenuated the severity of inflammation (82). The attenuation of inflammation following NET degradation provides causal evidence that NETs participate in the inflammatory pathology of VMC.

Recent research using the CVB3 myocarditis model has identified a previously unrecognized mechanism through which CVB3 infection regulates NETosis. CVB3 infection markedly upregulates calpain small subunit 1 (CAPNS1) expression in neutrophils. CAPNS1 binds to the catalytic subunit calpain-1, which then directly cleaves the nuclear envelope protein nesprin-1. This disrupts nuclear envelope integrity, permits chromatin release and thereby drives NETosis (83). However, the specific mechanisms through which NETs contribute to VMC-associated inflammation remain unexplored. Subsequent studies showed that CVB3 infection induces the mitochondrial accumulation of calpain-1, leading sequentially to ATP5A1 cleavage, excessive mitochondrial ROS production, mitochondrial dysfunction, NLRP3 inflammasome activation and cardiomyocyte pyroptosis (84). These findings raise the possibility that NETs promote VMC-associated inflammation by disrupting mitochondrial function and activating NLRP3, a hypothesis that warrants further investigation.

Importantly, NETs may exert context-dependent protective and pathological effects in VMC. NETs can restrict the intercellular spread of viruses; therefore, indiscriminate NET depletion may increase the viral load and promote tissue dissemination. This trade-off represents a broader challenge for NET-targeted anti-inflammatory strategies in infectious diseases. Future interventions should selectively suppress the pathological effects of NETs without compromising host defense.

Endocarditis is inflammation of the inner lining of the heart and its valves and may present acutely or chronically. It is broadly categorized as infective endocarditis (IE) or non-IE. IE is a severe, potentially fatal cardiac disease characterized by direct pathogen invasion of the heart valves, resulting in valvular damage, vegetation formation and inflammation. Li et al (85) detected NETs in the peripheral blood of patients with IE and found that elevated NET levels were markedly associated with poor prognosis. During IE pathogenesis, NETs do not function as host bactericidal effectors; instead, pathogens 'hijack' them to create a central platform for vegetation formation and immune evasion. This process may occur through both classical and nonclassical pathways. In the classical pathway, Streptococcus mutans bridges to platelet Fcγ receptors via specific IgG, thereby upregulating platelet P-selectin expression through the Src family kinase/Syk/PI3K/p38 MAPK signaling pathway (86). Activated platelets subsequently bind to PSGL-1 on the neutrophil surface via P-selectin, providing one of the key signals that trigger NETosis. Simultaneously, bacteria stimulate neutrophil ROS production and induce histone H3 citrullination via TLR2/FcγR signaling. Both signals are indispensable and act together to drive NET release (86). The nonclassical pathway is mediated directly by EsxA, an effector protein secreted through the type VII secretion system of Streptococcus gordonii. EsxA can induce NETosis at a distance and in a dose-dependent manner, independently of platelets, thereby enabling bacteria to evade phagocytosis during the early stages of bloodstream infection (87). Through their DNA scaffolds and histone components, the released NETs physically entrap and immobilize bacteria-platelet aggregates, promoting the formation of multilayered, biofilm-like vegetations. They also activate the coagulation cascade by promoting thrombin generation and enhance platelet aggregation, thereby establishing a self-amplifying bacteria-platelet-NET feedback loop that drives continued vegetation growth (88). Notably, in the aforementioned studies, NETs exhibited no direct bactericidal activity against the pathogens tested. Instead, they encased the bacteria and consolidated free-floating organisms into biofilm-like vegetations that were difficult to eradicate.

Regarding non-IE, a case report involving patients with systemic lupus erythematosus suggested that Libman-Sacks endocarditis may be associated with NETs (89). However, mechanistic evidence supporting this association remains extremely limited and currently consists only of isolated case observations. Systematic studies are therefore needed to determine whether NETs have a causal role in non-IE.

HF is a clinical syndrome resulting from structural and functional abnormalities of the heart and is among the leading causes of death worldwide (90). Persistent low-grade inflammation is recognized as a central mechanism underlying the pathogenesis and progression of HF (91) and NETs are emerging as important mediators linking immune-mediated inflammation to myocardial energy failure. Clinical studies have shown that serum NET levels are markedly elevated in patients with HF and are associated with poor prognosis (92,93).

Studies have identified NETs as active drivers of HF progression rather than merely inflammatory biomarkers. Tang et al (94) found that elevated Ang II levels in HF downregulate the transcription factor KLF2 in neutrophils and relieve suppression of the HIF1α pathway, thereby promoting neutrophil infiltration into the heart and NET release. Zhang et al (95) further showed that HMGB1 released following pressure overload-induced myocardial injury acts as a DAMP, directly recruiting neutrophils and inducing NETosis. Furthermore, Mang et al (96) demonstrated that vWF/ADAMTS13 imbalance in HF results in excessive vWF accumulation, which activates NET formation through SLC44A2 on the neutrophil surface. Zhao et al (97) found that downregulation of the endogenous anti-inflammatory protein DEL-1 relieves inhibition of p38 signaling in neutrophils, providing another upstream mechanism for initiating NETosis. Downstream, NET-associated NE activates TLR4-p38 signaling in cardiomyocytes, suppresses peroxisome pro liferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-mediated mitochondrial biogenesis and causes mitochondrial respiratory dysfunction and cardiomyocyte apoptosis (96). NETs also serve as procoagulant scaffolds that induce immunothrombosis in the myocardial microvasculature, resulting in episodes of ischemia, hypoxia and microcirculatory dysfunction (94). In addition, NETs promote macrophage infiltration and exacerbate cardiac inflammation (95,97). Together, these pathways define the mechanistic framework through which NETs contribute to HF. Neurohormonal activation mediated by Ang II, the release of damage-associated signals such as HMGB1 and coagulation-factor imbalance involving VWF initiate NETosis. NET-induced mitochondrial injury and microthrombosis then promote further DAMP release and aggravate ischemia. These mechanisms establish a self-sustaining positive-feedback loop that persists independently of the initial stressor and drives the progression and deterioration of HF. Notably, empagliflozin can disrupt the HMGB1-NET axis, suggesting that the clinical benefits of SGLT2 inhibitors may be mediated partly through NET-related pathways. This possibility warrants further validation (Fig. 3).

AS is the most common underlying pathology leading to myocardial infarction, stroke and peripheral vascular disease and is fundamentally a lipid-driven chronic inflammatory disease affecting the intima of large- and medium-sized arteries (98). In 2012, Megens et al (99) first directly visualized NET deposition in human and murine AS lesions. Subsequent studies have shown that serum NET levels in patients with coronary artery disease are positively correlated with the severity of coronary stenosis (100). Evidence accumulated in recent years indicates that the mechanisms through which NETs contribute to AS can be organized into three stages: NETosis induction, impaired NET clearance and downstream effector activity (Fig. 4).

The initiating signals for NETosis are highly dependent on the pathological context. At the hemodynamic level, low shear stress at sites such as vascular bifurcations downregulates the mechanosensitive ion channel Piezo1, thereby reducing Ca²⁺ influx and upregulating histone deacetylase 2. These changes promote NOX-mediated ROS generation and directly induce NETosis (101), helping to explain the predilection of AS for vascular bifurcations. At the metabolic level, oxLDL can induce NETosis through several pathways. First, oxLDL-stimulated macrophages release exosomes enriched in miR-146a that induce NETosis at a distance by targeting and suppressing neutrophil SOD2, resulting in a burst of mitochondrial ROS (102). Second, oxLDL stimulates vascular endothelial cells to secrete exosomes enriched in miR-505. Following uptake by neutrophils, these exosomes target and suppress the mitochondrial deacetylase SIRT3, causing a burst of mitochondrial ROS and NET release (103). Finally, oxLDL downregulates the cystic fibrosis transmembrane conductance regulator in neutrophils, increasing intracellular Cl⁻ concentrations and activating the serum and glucocorticoid inducible protein kinase 1 (SGK1)-ROS pathway to promote NETosis (104). Aging represents another important contributing factor. Myeloid cells from aged mice exhibit markedly greater mitochondrial oxidative stress (mitoOS) and 7-ketocholesterol induces NET release more potently in aged neutrophils, whereas mitoOS scavenging effectively inhibits this process (105). Thus, NETosis in AS arises from the convergence of mechanical, metabolic and age-related stimuli.

The pathological effects of NETs depend not only on their rate of formation but, more importantly, on the efficiency of their clearance. Under homeostatic conditions, deoxyribonuclease (DNase) I and DNase1L3 rapidly degrade NETs and restore homeostasis. However, AS-associated metabolic abnormalities can severely impair this clearance system. Hypercholesterolemia systemically suppresses the NET-induced DNase feedback response. In ApoE⁻/⁻ mice, severe hypercholesterolemia impairs the secretory responses of hepatic DNase I and intestinal DNase1L3, delaying NET clearance and preventing the resolution of inflammation (106). Within plaques, macrophages are the principal source of DNase. However, lipid loading-induced endoplasmic reticulum stress activates the PERK-ATF4 signaling axis, which markedly inhibits macrophage DNase secretion and promotes persistent NET accumulation (107). Diabetes further aggravates this clearance defect. In STZ-induced diabetic Ldlr⁻/⁻ mice, NETs persist within plaques and fail to regress spontaneously following lipid lowering, unlike those in nondiabetic mice, thereby sustaining the inflammatory microenvironment (108). Importantly, these clearance defects do not occur in isolation. Hypercholesterolemia frequently coexists with diabetes and advanced age and their synergistic effects can lead to widespread disruption of the NET clearance system.

Once NETs persist within plaques, they disrupt the functions of multiple cell types and establish a self-reinforcing pathological cycle. Their effects on macrophages are multifaceted. NETs directly increase macrophage uptake of oxLDL and promote MMP-9 expression and secretion, thereby exacerbating plaque instability (39). A recent study has further shown that NET-associated HMGB1 activates ADAM17 through TLR4. ADAM17 then cleaves the efferocytosis receptor MerTK on the macrophage surface, impairing apoptotic-cell clearance and consequently promoting expansion of the necrotic core (107). In vascular smooth muscle cells (VSMCs), NE cleaves TLR4 and activates the downstream MyD88-IRAK1-TRAF6-NF-κB pathway, promoting VSMC proliferation, migration and inflammatory cytokine release. These effects drive neointimal hyperplasia and contribute to plaque inflammation, growth and instability (109). In infection-associated AS, cationic histone H2A on the NET surface captures negatively charged monocytes through receptor-independent electrostatic attraction, facilitating their infiltration into plaques and markedly accelerating plaque progression (110). In foam cells, NET-associated CitH3 directly binds to and activates the NLRP3 inflammasome, promoting IL-1β and IL-18 release (111). Collectively, these findings demonstrate that NETs affect virtually all major cell types within plaques through diverse molecular mechanisms.

Overall, NET-driven AS can be conceptualized as a systemic pathological process involving multisource induction, multistep failure of clearance and multicellular effector activity. However, most existing studies rely on endpoint analyses and the specific contributions of NETs to plaque initiation, progression, regression and rupture have not been longitudinally characterized. Furthermore, the functional differences among NETs induced by distinct stimuli have not been systematically compared. LSS-induced NETosis may primarily determine lesion localization, whereas metabolic and aging-related factors may be more important in sustaining plaque progression. Defining these spatiotemporal regulatory differences will be essential for developing stage-specific NET-targeted interventions.

Hypertension is one of the most common chronic diseases and is characterized by persistently elevated arterial blood pressure. Its pathogenesis involves inflammation-driven oxidative stress, endothelial dysfunction, VSMC phenotypic switching and ECM remodeling (6). Serum levels of the NET markers cfDNA and MPO are markedly elevated in patients with hypertension (112), whereas the Ang II-induced increase in blood pressure is markedly attenuated in NET-deficient mice (113), providing functional evidence for the involvement of NETs in hypertension.

Studies have delineated a pathogenic cascade extending from upstream stimuli and NET release to downstream effects and target-organ damage. Under hypertensive conditions, vascular mechanical stretch and neurohumoral activation constitute the two principal triggers of NETosis. Krishnan et al (113) demonstrated that cyclic mechanical stretch of the vessel wall activates the mechanosensitive calcium channel TRPV4 on the neutrophil membrane, triggering calcium influx and robust NET release. A stretch magnitude of 10%, simulating hypertension, induced markedly greater NETosis than 5% stretch, which simulated normotension. Concurrently, Chrysanthopoulou et al (114) showed that Ang II directly induces NET formation through the AT1 receptor in a process requiring NOX-derived ROS production, autophagy activation and PAD4-mediated histone citrullination. These pathways do not operate independently but converge on ROS as a common mediator. In addition to causing direct cellular damage, ROS generate isolevuglandins (isoLGs). IsoLGs covalently modify lysine residues in histones and disrupt higher-order nucleosome structures, thereby promoting chromatin decondensation and providing the structural basis for NET release (115).

Following NET release, NET-associated histones, particularly CitH3 and TF increase blood pressure through two distinct mechanisms: Vascular functional dysregulation and structural remodeling. At the functional level, CitH3 directly activates endothelial cells, upregulates adhesion molecules such as ICAM-1, VCAM-1 and E-selectin and impairs acetylcholine-induced endothelium-dependent vasodilation (113). At the vascular structural level, Fang et al (116) showed that NETs activate PI3K-Akt signaling in VSMCs, leading to downregulation of the cyclin-dependent kinase inhibitor CDKN1b. This relieves G₁/S checkpoint arrest and promotes cell-cycle progression. The response is accompanied by marked upregulation of thymidine kinase 1 (TK1), a rate-limiting enzyme in DNA synthesis that directly promotes VSMC proliferation. Moreover, NET-stimulated VSMCs can transmit proliferative signals horizontally to neighboring VSMCs not directly exposed to NETs by releasing TK1-containing exosomes, thereby extending the spatial range of vascular remodeling (116).

The pathological effects of NETs ultimately converge on fibrosis and functional decompensation in hypertensive target organs. At the cardiac level, Tu et al (117) first linked NETs to ferroptosis, demonstrating that NETs activate NF-κB signaling, upregulate the pro-ferroptotic protein ACSL4 and simultaneously downregulate ferritin heavy chain FTH1 and the antioxidant enzyme GPX4. These changes induce iron-dependent lipid peroxidation and ferroptotic cardiomyocyte death. This process not only directly promotes cardiomyocyte loss but also upregulates pro-fibrotic markers, including α-smooth muscle actin, fibronectin and collagen II, through pro-inflammatory and pro-fibrotic signaling, thereby driving myocardial interstitial fibrosis and cardiac dysfunction.

AAA is a degenerative vascular disease characterized by chronic vessel-wall inflammation, ECM degradation and disrupted vascular smooth muscle cell homeostasis (118). Multiple clinical studies have shown that circulating and aortic tissue levels of NET-specific markers, including CitH3, cell-free DNA, nucleosomes, MPO and NE, are markedly higher in patients with AAA than in healthy controls. The concentrations of these markers are positively correlated with aneurysm diameter, expansion rate and rupture risk, suggesting that NET burden may reflect AAA severity and adverse prognosis (119,120).

NET formation is tightly regulated by upstream signaling mechanisms. Phosphoinositide 3-kinase γ promotes noncanonical pyroptosis, specifically activation of the caspase-11/GSDMD pathway, through cAMP/PKA signaling, thereby enhancing NET release and aggravating AAA (121). Studies have identified another important upstream mechanism involving the gut microbiota. Patients with AAA exhibit marked gut dysbiosis characterized by a reduced abundance of the butyrate-producing bacterium Roseburia intestinalis and a concomitant decrease in butyrate levels (122). Reduced butyrate availability diminishes its inhibitory effect on neutrophil NOX2 expression, resulting in excessive NET release. Supplementation with R. intestinalis or butyrate markedly reduces neutrophil infiltration and NET deposition in the aortic wall and attenuates AAA progression (122). These findings link the gut microbiota-metabolite axis to NET formation and provide a rationale for microbiota-targeted interventions in AAA.

NETs promote AAA formation and progression through multiple convergent mechanisms. First, NETs directly induce ferroptosis, a form of programmed cell death driven by iron-dependent lipid peroxidation, in VSMCs. NETs inhibit PI3K/AKT signaling in VSMCs, downregulating SLC7A11 and GPX4 while upregulating ACSL4 and TFR1, thereby triggering lipid peroxidation and ferroptosis (123). Additionally, NETs also destabilize the dimeric form of the mitochondrial glutathione transporter SLC25A11, leading to mitochondrial glutathione (mitoGSH) depletion and further inducing ferroptosis in SMCs (124). Second, NETs suppress Hippo signaling through TLR9, thereby increasing YAP nuclear translocation and driving VSMC phenotypic switching from a contractile state to a synthetic and pro-inflammatory phenotype. This transition is characterized by downregulation of contractile marker genes, including ACTA2, MYH11 and CNN1 and upregulation of pro-inflammatory and matrix metalloproteinase genes, including MMP2, MMP9, IL-1β and TNF-α, thereby exacerbating arterial-wall inflammation and matrix degradation (125). Third, NETs activate p38/JNK signaling and induce VSMC apoptosis, aggravating medial VSMC depletion and elastic fiber fragmentation and promoting AAA rupture (126). Furthermore, the DNA component of NETs forms complexes with cathelicidin-related antimicrobial peptide (CRAMP/LL-37), which recruit and activate plasmacytoid dendritic cells (pDCs), induce robust type I interferon production and subsequently activate T cells to amplify the inflammatory cascade within the aortic wall (127).

In summary, NETs act through multiple mechanisms to drive AAA formation and progression, including the induction of SMC ferroptosis, phenotypic switching and apoptosis and the activation of pDC-mediated immune amplification. Potential therapeutic approaches therefore include inhibiting upstream NET formation, modulating gut microbial metabolites to reduce NET generation and targeting downstream effector mechanisms, such as ferroptosis, YAP signaling and pDC activation. These strategies may have translational potential for the treatment of AAA.

Aortic valve stenosis is the most common valvular heart disease, with a prevalence exceeding 2% among individuals older than 60 years and a two-year mortality rate of ~50% among those with severe stenosis (128). The disease is primarily driven by progressive inflammatory and fibrocalcific processes involving interactions among circulating inflammatory mediators, valvular endothelial cells and valvular interstitial cells (129,130).

At the tissue level, abundant NET-specific markers, including CitH3 and cfDNA, have been detected in human stenotic aortic valve tissue (131,132), confirming the presence of NETs in valvular pathology. oxLDL is considered an important mediator of inflammation in aortic valve stenosis (133) and Awasthi et al (134) further showed that oxLDL induces NET formation through the TLR-PKC-IRAK-MAPK pathway and NADPH oxidase activation, establishing a molecular basis for the functional relationship between oxLDL and NETs. Building on these observations, evidence increasingly implicates NF-κB as a key downstream effector of NETs. NF-κB is a well-established pro-inflammatory signaling hub and contributes to the calcification associated with aortic valve stenosis (135). As NETs can activate NF-κB through multiple pathways, the present study proposed that NETs within stenotic aortic valves may convert the initial inflammatory signal induced by oxLDL into an osteogenic differentiation program in valvular interstitial cells through NF-κB signaling. NETs may simultaneously promote ECM remodeling through NET-associated elastase and cathepsins, thereby providing a collagen scaffold for calcific nodule formation. However, this hypothesis currently lacks direct functional validation. No studies have yet used NET inhibitors or PAD4-deficient animals in aortic valve stenosis models to evaluate changes in valvular NF-κB activity, calcification markers and valve function.

DNA forms the structural backbone of NETs, making enzymatic degradation the most direct approach for dismantling preformed NET structures.

DNase I is the most extensively investigated enzyme for NET degradation. Across multiple cardiovascular disease models, DNase I attenuates NET-mediated pathological changes. For example, in myocardial infarction models, DNase I dissolves NET-rich microthrombi, thereby improving microvascular perfusion and reducing infarct size (73). Recombinant human DNase I is clinically approved for cystic fibrosis, in which it reduces sputum viscosity when administered by aerosol inhalation, bronchoscopic instillation, or other routes (136,137). However, no relevant clinical studies have evaluated DNase I in cardiovascular disease and its translation to cardiovascular indications faces several challenges. First, NETs consist of a DNA backbone decorated with histones and granular proteins. DNase I cleaves DNA but does not neutralize histones. DNA degradation may therefore release substantial quantities of positively charged histones into the circulation. Extracellular histones are highly cytotoxic and can directly damage endothelial cells, activate platelets, promote microthrombus formation and trigger further sterile inflammation (138). Second, blood contains G-actin, a high-affinity inhibitor of DNase I. Plasma G-actin concentrations are sufficient to eliminate most of the activity of administered DNase I within minutes. Although modified DNase I variants, including actin-resistant mutants, are under investigation, none have entered cardiovascular clinical trials (139). Third, NETs in cardiovascular disease are predominantly localized within vascular-wall plaques, thrombi and the microcirculation. Neither intravenous nor intra-arterial administration achieves efficient lesion-specific accumulation of DNase I. Instead, DNase I nonspecifically degrades circulating NET structures, potentially disrupting the physiological roles of NETs in host defense and vascular homeostasis. Consequently, the translational potential of DNase I monotherapy in cardiovascular disease remains limited. Potential strategies for overcoming these limitations include combining DNase I with histone-neutralizing agents, developing actin-resistant DNase I variants and using nanocarriers for lesion-targeted delivery.

Other nucleases, including three-prime repair exonuclease 1 (TREX1), contribute to the physiological clearance of NETs by macrophages (140). TREX1-deficient mice develop inflammatory myocarditis that gradually progresses to dilated cardiomyopathy and circulatory failure, possibly because of impaired NET degradation (141). However, the therapeutic potential of pharmacologically enhancing TREX1 activity remains unexplored and is currently theoretical.