Extracellular DNA as potential contributors to pathological calcification

Some biomolecules involved in pathological calcification have been identified. However, the exact mechanism in which this intricate process occurs remains unknown. Extracellular DNA (exDNA) has recently been recognized as a partaker in this ectopic phenomenon. Extracellular DNA acts as an intercellular messenger that transmits information and orchestrates complex inflammatory responses. Changes in the morphology or function of exDNA may trigger calcification under pathological conditions. In the present review, recent advances on how DNA is released into the extracellular milieu to become exDNA will be highlighted in conjunction with how exDNA directly and indirectly contributes to the progression of pathological calcification. Emphasis is placed on the “gluing” effect of neutrophil extracellular traps that act as a bridge between inflammation and pathological calcification. Manipulation of exDNA may open new vistas for the development of enterprising strategies that prevent or treat pathological calcification.

behind pathological calcification. 2 Despite more than 2 decades of intensive research, the mechanism of pathological calcification remains obscure.
Pathological calcification shares common features with physiological calcification. 3,4The most common composition of physiological and pathological calcification is calcium phosphate.However, some pathological calcifications contain calcium oxalate or cholesterol. 5It is known that biomacromolecules such as protein, 6 polysaccharide 7 and lipids 8 regulate the process of physiological calcification.Collagen, which serves as the protein skeleton and the template for guiding mineralization, 9 occupies 22% of the weight of bone. 10 Non-collagenous proteins (NCPs), which occupy a small percentage of the bone weight, control the process of mineralization. 11hospholipids have been shown to affect the growth of hydroxyapatite in vitro. 12With intensive research, the role of deoxyribonucleic acids (DNA) in pathological calcification is gradually unveiled.
As an indispensable intracellular component of eukaryotic cells, DNA serves as a carrier of genetic instruction within cells.When released into the the extracellular milieu, DNA acts as an intercellular messenger.There are also DNA remnants from dead cells or other sources.The DNA-containing materials that are present in the extracellular environment are collectively called extracellular DNA (exDNA).Usually, these DNA messengers are received by cells in a timely manner and the residual exDNA are eliminated by macrophages and nucleases present in the body fluids. 13However, under pathological conditions, DNA is abnormally released into the extracellular milieu.The functions of macrophages and nucleases are blocked and the exDNA is not cleared appropriately. 14Therefore, exDNA will accumulate in the extracellular milieu and cause diseases such as autoimmune diseases, 15 tumors 16 and inflammation. 17The authors recently identified that exDNA function as stabilizers of mineralization precursors during the intrafibrillar mineralization of collagen fibrils. 18Because different forms of DNA have been identified in the calcified nodules of gallstone, 19 salivary calculi 20 and atherosclerotic plaques, 21 the authors hypothesize that exDNA has a strong correlation with pathological calcification.
The present review commences with a concise review of the previously proposed mechanisms of pathological calcification.This is followed by providing evidence to support the hypothesis that exDNA is involved in pathological calcification.These pieces of evidence include the routes taken by exDNA from their origin to the ultimate sites of pathological calcification, as well as the underlying mechanisms.Strategies aiming at clearing exDNA to arrest the progression of pathological calcification are also covered.By summarizing current knowledge on these issues, the present review aims to provide a new perspective on the regulatory effects of exDNA on pathological calcification, with the goal of inspiring more intensive research on targeted therapy against the different forms of ectopic calcification.

MECHANISMS OF PATHOLOGICAL CALCIFICATION
Prior efforts on unveiling the mechanism of pathological calcification have been nothing short of phenomenal.These studies mostly focused on deranged ionic concentrations, 22 abnormal osteoblastic activities, as well as the various components that regulate the nucleation, growth and aggregation of pathological calcification (Figure 1). 26imilar to these widely accepted theories, it was found that exDNA is also capable of inducing pathological calcification by providing nucleation sites and promoting local supersaturation of calcium and phosphorus. 27Apart from their high molecular weight, DNA molecules also possess significantly different structures compared with other amino acid oligomers and macro-molecules.The main structural unit of DNA is deoxyribonucleic acid, while other macro-molecules in living organisms are mostly monosaccharides or amino acids.In addition, the double helix structure of DNA is also different from other macro-molecules. 28To date, the role of exDNA in biomineralization remains largely unexplored.

| FORMS AND ORIGIN OF exDNA
Elucidation of the forms and origin of exDNA helps to enhance the understanding of the underlying mechanisms of DNA-induced calcification.DNA exists in the extracellular matrix and plasma in two forms: free DNA and extracellular DNA traps (ETs) (Figure 2A).Free DNA is derived from multiple sources.Endogenous sources of free DNA include active secretion, erythrocyte nucleation, necrosis and degradation of ETs.Living cells secret free DNA through an amphisome-dependent mechanism, which usually occurs during autophagy. 29Under normal condition, enucleation of red blood cells does not result in the release of DNA to the extracellular milieu.This is because macrophages will engulf the nuclei in a timely manner.However, if there are too many erythrocytes or when the function of macrophage is impaired, DNA fragments will be released into the extracellular matrix and plasma. 30In necrosis, the length of DNA varies due to random breaks.The maximum length of DNA may be more than 10000 base pairs. 31Exogenous free DNA is mainly derived from microbes and food substrates.The source of ETs is relatively limited.Although ETs are found in many types of cells, they are mostly derived from neutrophils.The ETs maintain their maximum length until they are degraded by deoxyribonucleases. 32,33The proteins on ETs function as regulators of DNA degradation F I G U R E 1 Mechanisms of pathological calcification.(A) Osteogenic differentiation acts as the trigger in some pathological calcifications.During vascular calcification, many cells differentiate into osteoblast-like cells or chondrocyte-like cells.The sources of altered cells include vascular 0smooth muscle cells, pericytes, mesenchymal stem cells, endothelial cells and fibroblasts. 23Osteoblast-like cells may also be derived from epithelial cells in cancer or bone metastasis. 24(B) Imbalance of calcium inhibitors, supersaturation of ions in matrix vesicles or mitochondria, and the supersaturation of systematic or local ions cause calcium and phosphorous enrichment and calcification. 5Some environmental factors potentially contribute to these changes, including improper diet, high fluoride levels in water, and unhealthy lifestyle habits. 5(C) The biomolecules sequester calcium ions to produce prenucleation clusters.These stable clusters aggregate and result in nucleation of crystalline phases. 25The figures were partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

F I G U R E 2
The origin of exDNA.(A) Free DNA is derived from multiple sources.Although ETs are found in many types of cells, they are mostly derived from neutrophils.(B) DNA in extracellular vesicles may be classified into three types according to the sizes of vesicle and their origin: exosomes, microvesicles and ABs.ABs, apoptotic bodies; DNA, deoxyribonucleic acid; ETs, extracellular traps.and DNA-mediated calcification.DNA in extracellular vesicles may be classified into three types according to the sizes of vesicle and their origin (Figure 2B): exosomes (30-150 nm in diameter), microvesicles (100-1000 nm in diameter) and apoptotic bodies (ABs) (1000-5000 nm in diameter). 34Exosomes are derived from endosomes, which are formed from invaginations of the plasma membrane.As the early endosomes develop into late endosomes, exosomes are formed as intraluminal vesicles inside multivesicular bodies.Exosomes are released after the multivesicular bodies fuse with the plasma membrane. 35Histones are absent in exosomes, 29 suggesting that exsosomal DNA are not derived from genes.Microvesicles arise from the bubbling of plasma membrane.Compared to exosomes, microvesicles carry more DNA. 36poptotic bodies are derived from the blebbing of apoptotic cells.Being protected by histones, the nuclear DNA in ABs are cleaved into nucleosome units (167 base pairs). 37

| Extracellular matrix and plasma
Extracellular DNA within the extracellular matrix and plasma exists as free DNA or exDNA traps (Extracellular Traps (ETs)).Free DNA has diverse origins.They may be derived from endogenous or exogenous sources.Endogenous free DNA are products of living cells via active secretion or enucleation of red blood cells. 29,30,38They may also be produced as by-products of cell death processes such as necrosis or ETosis. 39The latter is a unique form of cell death that is characterized by the release of decondensed chromatin and granular contents into the extracellular space.Extracellular DNA traps released by ETosis will be severed by nucleases to form free DNA.Exogenous free DNA may be derived from microbes or food products. 40xtracellular DNA traps represent reticular DNA backbones that are derived from nuclear chromatin with decoration of various proteins. 41,42The ETs released from neutrophils are coined neutrophil extracellular traps (NETs).These NETs are the most common form of exDNA. 43Under physiological conditions, the ETs are subsequently cleaved into DNA fragments by deoxyribonucleases (DNases). 35

| Extracellular vesicles
The current mainstream classification categorizes EVs into exosomes, microvesicles and apoptotic bodies (ABs) according to their origin and sizes. 44The membranes of exosomes are derived from endosomes.Exosomes are intraluminal vesicles consisting of intracellular multivesicular bodies that are secreted from the cells via exocytosis. 45Both microvesicles and ABs are derived from plasma membranes. 46,47The difference between these two entities is that microvesicles originate from living cells, whereas apoptotic cells originate from apoptotic cells.However, this classification is imperfect because under pathological conditions, other vesicles may be derived from autophagosomes via blockage of the autophagy flux. 48Furthermore, DNA have different binding patterns in EVs.Some DNA molecules are bound to the outer surface of EVs while most of the DNA are located within the lumina of the EVs. 49,50

| EXTRACELLUALR DNA-INDUCED PATHOLOGICAL CALCIFICATION AND THE UNDERLYING MECHANISM
Table 1 summarizes the different forms of pathological calcification associated with exDNA.A wide range of pathological calcification is covered to illustrate the connection between these conditions.Different sources of exDNA and the composition of the calcification nodules are also included.These issues help readers recognize why exDNA accumulates within regions with pathological calcification.The co-occurrence of exDNA and different ions also contributes to the understanding of interaction between these entities.Because of their anionic nature, exDNA may be used as a negative "charge pool" to attract and stabilize calcium ions.This property had been successfully employed in the authors' previous study to prepare DNA-stabilized amorphous calcium phosphate (ACP), 18 an important mineralization precursor.In solution, DNA-ACP exists as electron-dense granules.When examined by cryogenic electron microscopy, the DNA-ACP consists of nano-sized prenucleation clusters (Figure 3A).Under physiological conditions, these mineralization precursors alone promote bone formation. 57As pathological conditions are much more complex, the nucleation sites for DNA-ACP may involve entangling of proteins with DNA as well as the interaction of DNA with other contents within the extracellular matrix. 5The composition of ETs includes DNA and protein binding, while apoptosis also generates exDNA that interacts with the extracellular matrix to form nucleation sites. 58These factors may be the driving forces that cause DNAstabilized calcium phosphate to be nucleated as disordered calcifications.Similar to NCP-stabilized calcium phosphate precursors, the DNA-ACP precursors also form prenucleation clusters that promote intrafibrillar mineralization.Thus, DNA mimics the functions of NCPs that are involved in physiological calcification (Figure 3B). 27,59According to Columbic attraction theory, 60 NCPs rely on their acidic nature to stabilize Ca 2þ and bind electrostatically to the positively-charged areas of a collagen fibril.This enables amorphous mineralization precursors to infiltrate into the internal milieu of the fibril for intrafibrillar calcification.Such a phenomenon has also been identified for DNA and accounts for the high efficacy of DNA-induced intrafibrillar collagen mineralization.The exDNA and collagen fibrils were found to be co-localized along the same sites in various pathological calcifications in vivo (Table 1).These observations suggest that exDNA may be the inducer of intrafibrillar collagen calcification in diseases involving pathological calcification.
Besides stabilizing calcium ions and promoting intrafibrillar mineralization, DNA may also guide the formation of mineralization in pathological calcification.A unique property of DNA as a template in biomineralization has been leveraged in the field of nanomaterials. 61n the context of pathological calcification, the structure of DNA is altered by the presence of organic material.Consequently, this alteration influences the structure of (ii) DNA-ACP at different growth stages.Reproduced with permission. 18Copyright 2021, Wiley-VCH GmbH.(B) Reconstituted collagen fibrils immersed in DNA-ACP solution for 3, 5 and 24 h (scale bar = 200 nm).Minerals began to form at 3 h.Complete intrafibrillar and extrafibrillar mineralization was achieved at 24 h.Reproduced with permission. 18Copyright 2021, Wiley-VCH GmbH.pathological calcification.For instance, in the extracellular polymeric substance of bacterial biofilm, the DNA-BII protein family has a strong affinity for DNA, 62 and they act as linchpin proteins, stabilizing the crossedstrand structure of exDNA.These cross-linked exDNA functionally resemble Holliday junctions and enhance the stability of the biofilm. 63This phenomenon may explain why bacteria-driven calculus tends to be larger in size compared with other forms of pathological calcification.Furthermore, exDNA in the form of ETs is also reinforced by linked proteins.The significance of ETs in pathological calcification will be discussed in a later section. 4.1.2| "Gluing" effect of extracellular DNA traps Neutrophil ETs are the most common form of ETs.Being the sentinel cells in inflammation, neutrophils extrude their chromatin called NETs via a suicidal pathway to limit and combat pathogens. 64NETs possess reticular structures and are mainly composed of DNA and proteins.The average area of the pore on the reticular structure is 0.03 � 0.04 μm 2 .The small dimensions of these pores enable the NETs to entrap microbes and limit the spread of infection (Figure 4A).Recently, NETs have been identified in pathological calcifications (Figure 4C).The co-existence of 14 of NETs and calcification suggests a potential link between NETs and pathological calcification.
When exDNA is presented in the form of ETs, they function as "glue" to agglomerate nanoscopical calciumcontaining crystallites into larger pathological calcification entities.This phenomenon is supported by three cues, as depicted in Figure 4Dii.Firstly, NET-DNA serves as a pool of negative electric charges that enable calcium ions to aggregate, nucleate and form microcrystals.The pores on NETs confine these microcrystals within the ETs in a manner that is analogous to the limitation of infection.Secondly, different cellderived ETs entangle with each other mutually.This further augments the reticular structure of DNA to provide the morphological foundation for calcification. 20hirdly, the nano-crystallites trigger further ETosis, 66 which causes the exponential growth of ETs.The soformed web-like structure enables the ETs to adhere easily to the calculi, precipitating more calcium ions on the outer layer of these calcified masses.This causes the original microcalcification to gradually enlarge into larger lithiasis.This phenomenon may explain the onion-like structure of sialolithiasis.Therefore, NETs play an important role in the initiation and enlargement of pathological calcification.
Apart from their "gluing" effects, the DNA-binding proteins within ETs also potentially participate in mineralization.These DNA-binding proteins play an important role in maintaining the reticular structure of NETs.Once these proteins are removed, the network structure on NETs disappears (Figure 4B).What remains are several DNA branches which are incapable of effectively entrapping microcrystals..1.3| Indirect effect of exDNA Apart from direct induction of mineralization, exDNA also induces oxidative stress. 17These detrimental effects play a significant role in some forms of pathological calcification, such as cartilage calcification and vascular calcification. 70,71Oxidative stress induces endoplasmic reticulum stress-related apoptosis of endplate chondrocytes.The released ABs regulate the metabolism of pyrophosphates, which results in the calcification of endplate chondrocytes. 72Oxidative stress also induces vascular endothelial damage, which causes lipid infiltration of the heart valves. 73The latter is the culprit of atherosclerosis.
Another aspect is the immunomodulatory properties of exDNA.exDNA can be recognized by receptors on the surface of or within immune cells.Upon recognition, the DNAs ultimately trigger the recruitment of immune cells that elicit immune responses. 74A recent study suggests that exDNA influences metabolic reprogramming and the differentiation of T regulatory cells, which in turn promotes osteogenic differentiation and calcification. 75nflammation-induced recruitment of inflammatory cells and the correlated process of osteogenic differentiation contribute to calcification at sites of inflammation. 76nother way that exDNA contributes to pathological calcification is by regulating tumor behavior.This regulation leads to calcification through transfection of adjacent or distant cells.Fragmented DNA is capable of entering normal cells and transforming them into neoplastic cells. 77In calcification-related cancer types such as papillary carcinoma of the thyroid and breast, the DNA fragments secreted by neoplastic cells may transfect adjacent cells or remote cells via the circulation. 78ransfection mediated by exDNA induces calcification within the sites of cancer metastasis.
While recent studies have provided some clues for the indirect effect of exDNA in pathological calcification, there are still several gaps in our knowledge that require further exploration.One area of interest is the specific mechanisms by which exDNA induces oxidative stress and triggers immune responses in the context of calcification..1.4| Disruption of exDNA scavenging Disruption of DNA clearance in pathological sites may trigger the initiation of pathological calcification (Figure 5).Under physiological conditions, long exDNA fragments are cleaved by DNase to form DNA with appropriate lengths 14 that are easy for macrophages to ingest and break down. 33This process is likely to be blocked in pathological calcification.
In bacterial biofilms such as dental plaque, bacteria secret DNABII proteins, one of the DNA-binding protein families, to connect exDNA fragments as the structure components of a biofilm. 63A recent study reported that these proteins are also capable of converting right-handed B-DNA into left-handed Z-DNA.Since the Z-DNA possesses nuclease resistance, 79 the exDNA will linger longer to precipitate calcium phosphate from saliva and gingival crevicular fluid.This may play a role in the formation of dental calculus.To date, there is no evidence that residue DNA in the dental plaque induces dental calculus formation.If indeed the residue DNA in dental plaque is capable of inducing biomineralization, small molecule drugs which revert DNA configuration or degrade DNA fragments may be designed to intervene in the formation of dental calculus.This targeting drug may help resolve the problem of high recurrence of dental calculi.
The phagocytosis of macrophages is impaired in pathological calcification such as atherosclerotic plaques.The induced functional defect in the macrophages causes incomplete clearance of apoptotic cells and consequently secondary necrosis. 80The accumulation of large clusters of necrotic cells will generate an overflow of fragmented nuclear DNA.These DNA fragments cannot be effectively removed by macrophages and may precipitate atherosclerosis.In addition, macrophages exhibit different degrees of clearance when different forms of cell death are encountered.Compared with apoptotic cells, the debris of necrotic cells or cells undergoing other nonclassical programmed cell death is more difficult to be cleared by endocytosis. 81,82This accounts for the accumulation of exDNA under vicious stimulation.

| DNA in extracellular vesicles
In pathological calcification, EV DNA (evDNA) provides suitable sites for the formation of ACP within the EVs.
The EVs have long been recognized as the carriers of mineral precursors in biomineralization. 83Calcium and phosphate ions are abundant in the microenvironments of certain EVs, such as ABs and the calcified matrix vesicles secreted by osteoblasts. 84Because these EVs are rich in DNA, they are capable of stabilizing calcium and phosphate ions as ACP. 34orizontal genetic transfection mediated by evDNA may indirectly induce pathological calcification.Compared to free DNA, evDNA is easier to transfect because the membrane of EVs contains proteins that are readily recognized by receptors on the target cells. 85Besides, EVs contain DNA that has the capacity to penetrate the intact brain-blood barrier to expand its territorial invasiveness. 86By incorporating secreted evDNA into the genome of normal cells, breast cancer is capable of evading hormone therapy to expand its territory and preserve its microcalcifications. 87

| APPLICATIONS AND OUTLOOK
Extracellular DNA-induced pathological calcification increases the risk of cardiovascular disease and the mortality in diabetes mellitus and end-stage renal disease. 88Hence, exDNA clearance may be an effective strategy to arrest or slow the progress of pathological calcification.

| Mechanisms for exDNA clearance
Methods for exDNA elimination include enzymemediated degradation, cationic binding of exDNA, complement inhibition, or promotion of macrophage phagocytosis of deceased cells. 42exDNA traps play a dual role in the human body.On the one hand, ETs play a bacteriostatic and bactericidal role in infection.On the other hand, ETs play an important role in the induction of atherosclerosis, tophi, gallstones and sialoliths. 19,20,65Nase I is capable of efficiently degrading doublestranded DNA, thereby facilitating the degradation of NETs.The low serum stability of DNase I makes it unsuitable for systemic administration.Therefore, local application of DNase I may inhibit the formation of pathological calcification.The utilization of positively charged particles to bind with exDNA is an efficient strategy for the removal of exDNA. 89In addition, complements also play a regulatory role in NETs recruitment of neutrophils and the formation of NETs can be enhanced by activating the complement system.The presence of anti-complement components can also attenuate neutrophil recruitment, thereby inhibiting the formation of NETs. 90

| Applications
Recent studies reported the development of experimental DNase I-based microgel.This microgel effectively eliminated ETs in the serum even after exposure to ETreleasing neutrophils.Since the microgel improves the serum stability of DNase, it may be introduced into the blood circulatory system (Figure 6A). 89In addition, chloroquine converts the DNase-resistant Z-DNA to DNase-sensitive B-DNA.Hence, chloroquine facilitates the clearance of exDNA and dampens the initiation of pathologic calcification (Figure 6B). 79n addition to direct degradation, extensive research has been conducted on the utilization of nanomaterials to competitively eliminate exDNA.The reduction of exDNA and the treatment of diseases such as inflammatory bone loss in periodontitis, acute kidney injury, rheumatoid arthritis can be achieved through this approach. 89,91owever, there is a scarcity of studies on the treatment of pathological calcification using cationic binding exDNA.This suggests that the field possesses substantial research prospects in the forthcoming future.
The homogeneous polysaccharide HD-PS-3 possesses anti-complement activity and is isolated from Oldenlandia herbacea, an herb of Chinese medicine.HD-PS-3 inhibits ET formation by inhibiting complement formation. 92However, it should be noted that some clinical studies on herbal medicines are incomplete or lacking.In Reproduced with permission. 89Copyright 2022, Royal Society of Chemistry.(B) In mature biofilms, B-DNA is converted to Z-DNA.DNase can break down B-DNA but not Z-DNA.CeCl 3 promotes this kind of conversion, while chloroquine and α-DNABII inhibit the conversion of B-DNA to Z-DNA.Reproduced with permission. 79Copyright 2021, Elsevier.ETs, extracellular Traps.addition, ETs have important anti-infection effects in vivo.The timing and extent of ET elimination need to be further investigated in preclinical and clinical studies.

| CONCLUSION
Pathological calcification is a common complication in many diseases and has a long duration and high incidence.The mechanisms of pathological calcification are complex.Among the various molecules that participate in biomineralization, exDNA provides a previouslyneglected cue in the formation of mineralization in many diseases that involve pathological calcification.In the present review, we analyzed previously-established opinions on pathological calcification and put forward some deficiencies.We then cited the literature that supports the notion that exDNA is involved in inducing pathological calcification.Failure in clearing exDNA from those pathological sites accounts for the aggregation of exDNA.In addition, evDNA may be an important entity for stabilizing ACP or calcium oxalate in EVs.This enables the EVs to induce pathological calcification.However, the hypothesis that exDNA induces pathological calcification still lacks direct proof.There are some pathological calcifications in which exDNA has not been found.This may be due to the lack of extensive research or this mechanism only plays a role in some pathological calcifications.Therefore, more research should be conducted on the related mechanisms.
Treatments that interfere with pathological calcification by manipulating exDNA are still in the infancy stage of development.By elucidating the underlying mechanisms of pathological calcification, more interventions may be developed to treat this category of diseases.A study of the relationship between exDNA and calcification deposition will help improvise effective treatment modalities for controlling pathological calcification.

4. 1 |
Functions of exDNA within the extracellular matrix and plasma 4.1.1| Direct induction of mineralization

F I G U R E 3
The mechanism of DNA-mediated calcification.(A) Features of DNA-ACP.(i) Schematic of the preparation of DNA-ACP.

F I G U R E 4
Overview of ET-mediated calcification.(A) The network structure of NETs when examined with atomic force microscopy (scale bar = 2 μm).Reproduced with permission.41Copyright 2016, Royal Society of Chemistry.(B) NETs after proteolytic digestion, as examined with atomic force microscopy (scale bar = 2 μm).Reproduced with permission.41Copyright 2016, Royal Society of Chemistry.(C) NETs are detected by immunofluorescence from different pathological calcifications.Neutrophil elastase (NE) is used as a marker of NETs.exDNA (red) and NE (green) co-localize (Yellow) in (a) tophi (scale bar = 100 μm), (b) gallstones (scale bar = 4 mm) and (c) sialoliths (scale bar = 400 μm).The white arrows indicate areas where extranuclear DNA and NE co-localize in a tophus.This indicates the relevance of NETs in pathological calcifications.Reproduced with permissions. 19,20,65Copyright 2019, Elsevier and Copyright 2020, Multidisciplinary Digital Publishing Institute; Copyright 2014, Springer Nature.(D) Growth of calculus mediated by ETs.(i) The interaction between the DNA skeleton and decorated proteins.The polyanionic nature of DNA attracts calcium ions, while the polycationic proteins decorated on the surface of DNA attract anions such as phosphate.This property of ETs initiates the formation of microcrystals.(ii) The "gluing" effects of ETs in the formation of large calculus.NETs, neutrophil extracellular traps; exDNA, extracellular DNA.NIU ET AL.

F I G U R E 5
The degradation of DNA.(A) The process of DNA degradation in physiological condition.(B) Potential inhibitors that block the process of DNA degradation and cause DNA accumulation.DNA, deoxyribonucleic acid.The figures were partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

F I G U R E 6
The impact of DNase I on ETs.(A) DNase I can decompose ETs in the internal environment for a long time.
Summary of the pathological calcification associated with extracellular DNA.
T A B L E 1