Keywords
neutrophil - NETosis - atherogenic inflammation - atherothrombotic disease
Introduction
Despite current therapies, cardiovascular diseases (CVDs) have remained the leading
cause of mortality globally for many years. In addition to the major impact on personal
health, CVDs constitute a serious social and economic burden worldwide. Coronary artery
and cerebrovascular disease—causative for myocardial infarction and stroke, respectively—are
the most common and severe complications of CVDs. Atherosclerosis is recognized as
the primary pathophysiology of CVD originating from a lipid-driven chronic inflammation
of the vessel wall.[1]
[2] Hyperlipidaemia can damage endothelial cells promoting lipid deposition and plaque
formation, and represents the initial spark in atherosclerosis; however, chronic inflammation
fuels progression of the disease. Many recent studies have linked hyperlipidaemia
to atherogenic inflammation,[3]
[4]
[5]
[6] and neutrophils are likely activated during hyperlipidaemia to promote atherogenic
inflammation.[4]
[7]
[8] Indeed, hyperlipidaemia drives neutrophilia, and circulating neutrophil counts are
directly related to atherosclerosis burden.[4]
[7] Infusion of neutrophil-depleting antibody reduces atherosclerosis in animal models.[7] In addition, hypercholesterolaemia can trigger the synthesis of granulocyte colony-stimulating
factor (G-CSF), a master regulator of granulopoiesis.[4]
[9]
[10]
[11]
[12] G-CSF stimulates the proliferation of myeloid precursor cells and suppresses the
clearance of aged neutrophils.[13] Also, hypercholesterolaemia increases serum levels of CXCL1 promoting mobilization
of neutrophils.[14] Indeed, in experimental mouse models neutrophils accumulate in atherosclerotic lesions.[7]
[15]
[16]
[17]
[18]
[19] Within human atheroma, neutrophil infiltrates are detected less frequently.[20]
[21]
[22]
[23]
[24] Nevertheless, they have been detected at the sites of plaque erosion or rupture,[20]
[24]
[25]
[26] and in clinical cohort studies, neutrophil blood counts show a robust relationship
with increased risk of acute coronary events.[27]
[28]
[29]
[30] Despite the recognition that infiltration of leukocytes acts as a driving force
of atherothrombotic disease, the contribution of neutrophils to CVDs has, however,
been under-estimated.
Neutrophils are the most abundant population of leukocytes in human circulation and
form an essential part of the inflammatory response to combat invading pathogens through
their functional properties such as phagocytosis, degranulation and the generation
of reactive oxygen species (ROS).[31]
[32] Given their limited lifespan, neutrophils have in the context of chronic inflammation
long been over-shadowed by other leukocyte populations, such as monocytes and macrophages.
However, the last 10 years witnessed a revival of neutrophils as multi-functional
innate immune cells that can greatly influence the course of chronic inflammation
via their crosstalk with other immunocompetent cells.[33]
[34]
[35]
[36] Among several new neutrophil interactions discovered, the finding that neutrophils
can release threads of chromatin covered with proteins of nuclear, cytoplasmic or
granular origin—named neutrophil extracellular traps (NETs)—has placed neutrophils
back in the spotlight of cutting-edge immunological research.[37] The release of NETs by neutrophils is called NETosis, and neutrophil NETosis is
an emerging mechanism underlying atherogenic inflammation. Recent studies have highlighted
the importance of neutrophil activation and NETosis in acute coronary events,[38]
[39] while other studies have suggested a role of NETosis in atherogenesis[8]
[40]
[41]
[42] and plaque erosion.[43]
[44] Yet, few studies have examined directly the effect of NETs on the formation, development
and complication of atherosclerosis. This highlights the need to elucidate potential
inflammatory mechanisms underlying neutrophil NETosis in atherogenesis and to explore
the potential clinical and therapeutic implications of NETosis for CVDs.
Formation of Neutrophil Extracellular Traps
Formation of Neutrophil Extracellular Traps
Neutrophils contribute to an acute inflammatory cascade by several different mechanisms,
including phagocytosis, chemotaxis and degranulation.[31]
[32] In response to damage, neutrophils not only secrete inflammatory mediators, but
can also release their cytoplasmic content and extrude their deoxyribonucleic acid
(DNA) in a process named NETosis.[45] Besides other types of cell death such as necrosis and apoptosis, NETosis is an
alternative form of programmed cell death wherein neutrophils release NETs.[46] Depending on the inciting event, the host membrane receptors, signalling cascades
and effector proteins involved, NETosis unfolds in a ‘vital’ or ‘suicidal’ manner.
‘Suicidal’ NETosis is preceded by hours of oxidant generation by the multi-component
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and ends when
NETs are expelled from the neutrophil; the outcome is neutrophil cell death.[46]
[47] In contrast, without compromising membrane integrity, ‘vital’ NET formation involves
the secreted expulsion of chromatin via vesicles. The surface membrane reseals and
results in a viable anuclear neutrophil.[48]
[49]
[50]
Irrespective of the mechanism of NETosis, NETs are networks of extracellular fibrous
material composed of neutrophil DNA and granule-derived peptides and proteolytic enzymes.
Many effector mediators cover this extracellular neutrophil DNA such as histones,
multiple proteinases such as neutrophil elastase (NE), proteinase 3, cathepsin G and
gelatinase, and the pro-oxidant enzyme myeloperoxidase (MPO). Neutrophils use these
fibrous structures to trap extracellular pathogens and prevent bacterial dissemination.
NETs are released during inflammation and occur in vivo following infections.[51] Moreover, NETs can trigger coagulation, cause endothelial dysfunction and amplify
local inflammation, and NETosis not only plays a role in the elimination of pathogens,
but also contributes to sterile inflammation, cancer, autoimmunity and thrombosis.[36]
[44]
Cholesterol, oxidized low-density lipoprotein (oxLDL) and platelets as drivers of
neutrophil NET formation: The idea that NETs might mediate atherothrombotic disease by stimulating an overall
process of inflammation and thrombosis has moved studies to expand beyond the original
characterization of NETosis as a mechanism of defence against bacteria. Over the subsequent
decade, the factors controlling NET formation and the molecular underpinnings of mechanisms
linking NETosis to atherogenic inflammation have in part been revealed ([Fig. 1]). In vitro, neutrophils release NETs in response to cholesterol crystals and oxLDLs
in a manner that depends on NADPH oxidase activity[40]
[52] ([Fig. 1A]), and inhibition of mitochondrial oxidative stress reduces the formation of NETs
by cultured neutrophils when exposed to 7-ketocholesterol, an oxysterol found in human
atheroma.[53] Also, interactions with activated platelets commit neutrophils to undergo NETosis[54]
[55] ([Fig. 1A]). This can be propagated through high-mobility group box 1 (HMGB1) exposed on the
surface of activated platelets and by interactions of neutrophil P-selectin glycoprotein
ligand-1 with platelet P-selectin.[56]
[57] In addition, CCL5 and CXCL4 are chemokines stored in platelet α-granules and CCL5/CXCL4
heterodimers are potent neutrophil attractants. Upon adhesive contact with platelets,
the CCL5/CXCL4 complex also triggers neutrophils to release NETs[58] and blocking the heterophilic interaction between CCL5 and CXCL4 was recently shown
to prevent NETosis in a mouse model of myocardial infarction.[59] Vice versa, expelled NETs enhance the adhesion of additional platelets via von Willebrand
factor[60]
[61] and promote their activation, thereby coordinating a pro-thrombotic cycle of coagulation
and inflammation[56]
[60]
[62]
[63] ([Fig. 1B]). Important insights have been gained by the elucidation that neutrophil NETosis
participates in pathological thrombosis, including deep vein thrombosis[61]
[64] and atherothrombosis.[39]
[65] NETs have been suggested to participate in thrombus growth and stabilization by
providing a scaffold for fibrin formation and platelet aggregation.[60]
[66] Indeed, elevated levels of circulating DNA have been detected in patients with acute
coronary events[65] and also in situ in the thrombus mass of patients with acute myocardial infarction.[67] Moreover, mediators associated with NETs can stimulate inflammatory cells ranging
from plasmacytoid dendritic cells[68] to macrophages,[40] and recent insight suggests a role of NETs in macrophage activation, potentiating
plaque formation in murine atherosclerosis.[40] However, the importance of NETs in macrophage priming in early atherosclerosis was
not found in another study. Instead, it appeared that neutrophil-derived proteases
directly contributed to interleukin (IL)-1β maturation.[69] In sum, understanding of the role of NET formation in atherogenic inflammation remains
scant, and only recently studies emerged that established causation linking NETosis
to inflammation, plaque disruption and thrombosis with different strategies (i.e.
pharmacologically blocking NET formation, use of genetic approaches to interfere with
NETosis, etc.).
Fig. 1 Recent insights into the drivers and amplifiers of neutrophil extracellular trap
(NET) formation in atherogenic inflammation. (A) Within atheroma, neutrophil cholesterol accumulation and exposure to oxidized low-density
lipoprotein (LDL) likely trigger the formation of NETs in a manner that requires oxidant
production. Similarly, mitochondrial oxidative stress in lesional neutrophils causes
NETosis. Moreover, damage to the vessel wall leads to the activation of platelets,
with the exposure of P-selectin and high-mobility group box 1 (HMGB-1) on their surface,
and neutrophil interactions with activated platelets provoke NET release. Finally,
platelet-derived CCL5/CXCL4 heterodimers drive neutrophils to form NETs. (B) Neutrophil NETosis and coagulation go hand in hand, and multiple factors can cause
thrombin cleavage and fibrin formation on NETs. Tissue factor pathway inhibitor (TFPI),
the major extrinsic coagulation pathway inhibitor, abrogates the function of tissue
factor. However, NET-associated neutrophil serine proteases such as neutrophil elastase
(NE) locally degrade TFPI impairing the anticoagulant function of TFPI to increase
blood coagulation. Also, NETs can stimulate the coagulation cascade directly through
exposure of tissue factor or by binding and activating factor XII. Finally, the adhesion
of platelets to NETs via von Willebrand factor might lead to platelet aggregation,
an important step in the formation of a platelet-fibrin clot. (C) Within atherosclerotic vessels, the neutrophil granular peptide cathelicidin-related
antimicrobial peptide (CRAMP) acts as a chemotactic cue to propagate homing of monocytes.
Lesional extracellular deoxyribonucleic acid (DNA) accumulates in advanced plaques
promoting macrophage inflammation. Sensing of double stranded DNA (dsDNA) by macrophages
and ligation of absent in melanoma 2 (Aim2) in the cytosol nucleates an inflammasome,
with the subsequent release of interleukin (IL)-1β to further alarm the immune system.
(D) Engagement of innate immune receptors on endothelial cells (e.g. Toll-like receptor
[TLR] 2) by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular
patterns (DAMPs) can cause chronic low-grade endothelial injury, the initial spark
in atherogenic inflammation. In complicated plaques, hyaluronic acid may bind TLR2.
Neutrophils are quickly recruited to sites of damaged endothelium. Here, NETosis can
activate the complement cascade, and vice versa. For example, NETs serve as a platform
on which activation of the complement cascade occurs locally near vascular endothelial
cells, suggesting continuous aggression on the endothelium. Also, NETs promote the
formation of the anaphylatoxins C3a and C5a that, in turn, amplify inflammation by
recruiting and subsequently priming neutrophils. In addition, C5a and the membrane
attack complex (composed of C5b, C6, C7, C8 and C9) can stimulate the expression of
tissue factor on endothelial cells. Finally, activated neutrophils release proteolytic
enzymes such as matrix metalloproteinases (MMPs) that can degrade the extracellular
matrix resulting in further endothelial damage.
NETs in Atherosclerosis
Atherosclerosis is a chronic inflammatory pathology of the medium- and large-sized
arteries that underlies CVDs.[1] Activation of the vascular endothelium is the initial spark in atherosclerosis;
however, chronic inflammation fuels progression of the disease. Circulating leukocytes
anchor to and infiltrate the inflamed vessel wall. Adherent monocytes continually
migrate and accumulate inside the atherosclerotic lesion, driving progression and
enlargement of the plaque with the formation of a collagenous fibrous cap ([Fig. 2A]). Activated macrophages within the lesion damage the fibrous cap making the atherosclerotic
plaque vulnerable to rupture.[70] Plaque rupture—with subsequent exposure of tissue factor and collagen resulting
in thrombus formation ([Fig. 2B] and [C])—causes a myocardial infarction or stroke within seconds. Although plaque instability
is responsible for two-thirds of sudden coronary death,[71]
[72] plaque rupture does not always lead to major acute clinical events.[73] Indeed, large plaque burden and severe lumen narrowing are essential criteria for
the development of acute coronary events, rendering the residual lumen unable to host
the thrombus.[74]
Fig. 2 The involvement of neutrophil extracellular traps (NETs) during the different stages
of atherosclerosis and its complications. In the early stages of plaque formation
(A), neutrophils accumulate throughout the atheroma. Activated neutrophils further stimulate
the recruitment and activation of monocytes through the release of granular proteins
such as cathelicidin-related antimicrobial peptide (CRAMP). The atheroma builds up
and a core of lipids, living and dead cells and a fibrous cap with collagen expands.
Deposition of oxidized low-density lipoprotein (LDL) and cholesterol causes neutrophil
inflammasome activation, increases oxidative stress and triggers NETosis. Once the
necrotic core progresses, extracellular deoxyribonucleic acid (DNA) accumulates. Sensing
of double stranded DNA (dsDNA) by lesional macrophages through the cytosolic absent
in melanoma 2 (Aim2) inflammasome results in the release of interleukin (IL)-1β, further
amplifying local inflammation. During plaque erosion (B), activation of endothelial cells (for example, through engagement of Toll-like receptor
[TLR] 2) propagates the recruitment of neutrophils. Neutrophils localized near the
inflamed intimal surface degranulate and generate reactive oxidants, leading to endothelial
cell death and detachment. Superficial plaque erosion exposes pro-thrombotic factors,
and activated platelets trigger neutrophils to form NETs and the release of tissue
factor. Through complement, NETs induce continued endothelial erosion. Further complement
activation drives neutrophil recruitment to the site of atherothrombosis (C). Here, tissue factor-covered NETs entrap platelets and provide a nidus for platelet
aggregation and thrombus formation.
Only recently neutrophils have been forwarded as important regulators in atherosclerosis
and particularly in atherothrombosis.[34]
[75] Histological studies on murine atherosclerotic plaques in experimental models of
atherosclerosis revived the discussion on the role of neutrophils in human atherosclerotic
disease. While experimental plaques in severely hypercholesterolaemic mice contain
neutrophils,[7]
[17]
[18]
[19] investigations on human atherosclerotic plaques failed to demonstrate the presence
of large numbers of neutrophils in intact plaques.[20]
[21]
[22] Neutrophils are found only scarcely scattered as solitary cells throughout the intima,
suggesting that neutrophils do not play a prominent role during early stages of human
atherosclerosis.[20]
[23]
[24] Yet, the paucity of validated selective markers for human neutrophils has rendered
their identification in pathological studies difficult, and the extent and timing
of potential neutrophil involvement in human atherosclerosis remains unsettled.[76]
[77] In addition, neutrophils may undergo phenotypic changes, and hence, lose expression
of specific markers in response to inflammation. For example, a recent study reported
similar staining patterns for NE and MPO—generally considered as markers to identify
neutrophils in situ[17]
[19]
[20]
[67]—in complicated atherosclerotic plaques.[24] However, CD177—a neutrophil-specific antigen involved in cell migration[78]—reacted with only a sub-population of the neutrophils.[24]
In fact, the clinical relevance of neutrophil NETosis remains poorly understood and
hard to prove experimentally. The potential non-specificity of NETosis markers still
precludes understanding of the causes of NET formation in men and their contribution
to human disease. Although extracellular DNA is found in several acute and chronic,
sterile and infective disease compartments, yet it is still unclear whether free DNA
truly derives from NET formation. Also, it remains experimentally challenging to dissect
cellular origins of free extracellular DNA. Finally, even if putative NET-associated
markers, (e.g. citrullinated histones or proteases) support the presence of NETs,
their impact on disease pathologies is still hard to assess.
Neutrophils lay down the tracks in atherogenic inflammation: Several experimental mouse models of atherogenesis have demonstrated the presence
of neutrophils in arterial plaques,[18]
[79] and that neutrophil-derived factors are able to modulate murine plaque size and
composition.[80] Neutrophils may contribute to plaque formation through promoting inflammatory monocyte
recruitment, and may also participate in lesion evolution and complication. Within
atherosclerotic plaques, NET components such as cathepsin G and cathelicidins exhibit
monocyte-chemotactic activity.[81]
[82] The neutrophil granular cathelicidin-related antimicrobial peptide (CRAMP) affects
the recruitment and activation of other immune cells, including monocytes and dendritic
cells.[83] In atherosclerotic vessels, CRAMP is deposited on the inflamed endothelial surface
leading to the attachment of monocytes to the vessel wall,[84] and ApoE−/−
mice that lack CRAMP develop smaller plaques suggesting that CRAMP is involved in
plaque formation[80] ([Fig. 1C]). Moreover, neutrophil MPO triggers macrophages to release ROS and other pro-inflammatory
cytokines.[2] In turn, reactive oxidants modify LDL to generate oxLDL that drives the differentiation
of foam cells.[85]
[86]
[87]
[88]
[89]
Oxidative burden, DNA sensing and NETosis in atherogenic inflammation: Oxidative stress is present in aging and human atherosclerotic vascular diseases.[90] Recent findings underscore a relationship between mitochondrial oxidative stress
and neutrophil NETosis in animal models for atherosclerosis.[53] Irradiation of recipient animals to ablate endogenous haematopoietic tissues, followed
by reconstitution of aged atheroprone Ldlr−/−
mice with mitochondrial catalase transgenic bone marrow cells suppresses oxidative
stress and protects against atherosclerosis development. The higher oxidative burden
in these old mice correlates with enhanced NETosis. In vitro, exposure of neutrophils
to 7-ketocholesterol led to the formation of NETs ([Fig. 1A]), and suppression of mitochondrial oxidative stress reduced NETosis in response
to 7-ketocholesterol.[53] Along these lines, further studies have revealed that cholesterol crystal-triggered
NETosis in atherosclerotic lesions exerts direct pro-inflammatory effects on macrophages
leading to cytokine release such as IL-1β and IL-6, thus further amplifying local
inflammatory cascades in the artery and exacerbating and propagating arterial intimal
injury and thrombosis.[40] Finally, recent studies have shown that defective cholesterol efflux pathways lead
to neutrophil cholesterol accumulation, inflammasome activation and prominent NET
formation in atherosclerotic lesions, suggesting a novel role for cholesterol accumulation
in atherogenic inflammation.[8] Links between inflammasome activation and NETosis, however, need to be more clearly
delineated. In this regard, recent studies in Aim2−/−
mice have revealed a major role of the double stranded DNA (dsDNA), absent in melanoma
2 (Aim2) inflammasome in lesional macrophages.[91] At later stages of atherosclerosis, ApoE−/−
mice showed prominent lesional deposition of extracellular dsDNA, and this was echoed
by parallel Aim2 expression in macrophages at advanced stages of the disease ([Fig. 1C]). Aim2 deficiency on the ApoE−/−
background diminished the production of IL-1β and reduced plaque destabilization
suggesting a novel role for Aim2 in inflammation associated with atherosclerosis.
At present, the possibility that dsDNA is primarily released by accumulating dead
cells within the expanding necrotic core cannot be ruled out, and whether NETs promote
Aim2 activation in atherosclerosis remains unclear.
Peptidylarginine deiminase 4 (PAD4), a nodal intervention point to target NET formation: NETs can be detected in atherosclerosis, and given their pro-inflammatory and pro-thrombotic
properties, the presence of NETosis could potentiate atherosclerotic plaque formation
via enhanced inflammation and increased monocyte recruitment. Yet, few studies have
attempted to establish a direct link between NETosis and atherogenic inflammation.[40]
[41]
[42]
[43] The enzyme PAD4 participates in NET formation by citrullination of histones, releasing
the electrostatic bonds that constrain nuclear DNA to nucleosomes. Loss of these positive
charges due to PAD4 activity frees the chromatin to unfold and form the threads of
chromatin furnished by NETs. Indeed, NET formation in mice depends on PAD4 activity.[92] Cl−amidine, a pan-PAD inhibitor administered systemically, prevented NETosis, retarded
neutrophil and monocyte recruitment to arteries and reduced experimental atherosclerosis
and the pro-thrombotic phenotype of ApoE−/−
mice.[41] However, Cl−amidine could potentially have off-target effects,[93]
[94]
[95]
[96] and findings obtained with Cl−amidine—targeting all PAD isotypes—should be translated with caution. PAD-mediated
citrullination can drive T cell polarization and cytokine production.[96] Also, PAD4 activity can affect dendritic and smooth muscle cell activation.[93]
[94]
[95] Thus, the lack of specificity of Cl−amidine limits unambiguous probing of the role of PAD4 and NETs in atherothrombosis.
Recently, one study reported a novel selective PAD4 inhibitor to block NETosis by
human and murine neutrophils in vitro.[97] However, more work is needed to validate the enzymatic role of PAD4 in the formation
of NETs. It will therefore be critically important to test the causal contributions
of neutrophil NETosis to atherogenic inflammation at different stages of lesion development
using new tools for the detection and manipulation of NET formation in key model systems
for human atherosclerosis. Finally, given the many functions of PAD4 other than NET
formation, any phenotype in mice lacking PAD4 cannot undoubtedly be taken as an unambiguous
demonstration of the involvement of NETs.
To evaluate more rigorously the participation of PAD4 and NETosis in atherothrombotic
disease, more recent studies used mice with genetic deficiency of PAD4 in blood cells
but not in intrinsic vascular wall cells and other tissues. Backcrossing of atheroprone
ApoE−/−
mice to mice that lack PAD4 specifically in myeloid cells protects against atheromata
burden that is intimately linked to diminished NETosis and reduced atherogenic inflammation
in the artery.[42] In the same model, NETs provoked macrophages to release pro-inflammatory cytokines
such as IL-1β and CXCL1 facilitating further local inflammatory responses. These findings
are in line with previous work that suggested a link between NET formation and macrophage
inflammation in atherosclerotic plaques.[40] By contrast, other results indicate that myeloablative irradiation and reconstitution
with Pad4-deficient bone marrow cells, and hence, NETosis does not alter atherogenesis in hypercholesterolaemic
Ldlr−/−
mice, but are involved causally in endothelial erosion.[43] Previously, NETs were shown to directly induce endothelial dysfunction and to kill
endothelial cells in vitro,[98]
[99] and this was associated with endothelial damage in systemic lupus erythematosus,[100]
[101] an effect mediated in particular by matrix-degrading factors contained in NETs such
as matrix metalloproteinases.[98]
[100] Now, genetic deficiency of Pad4 in blood cells has been shown to reduce intimal damage in mice with arterial lesions,
without affecting plaque size and atherogenic inflammation.[43] On balance, while the role of NETs in early atherosclerosis and plaque erosion has
been studied intensely, their role in plaque progression is unclear and future studies
will be needed to determine the involvement of NETosis in the development of unstable
lesions.
In mice, experimental atherosclerotic plaques are usually devoid of thrombosis.[102] On the other hand, neutrophil contribution in human atherosclerotic disease appears
to be a prominent feature of complicated thrombosed plaques.[20]
[24] In contrast to intact atherosclerotic plaques, complicated plaques contain large
numbers of neutrophils, which frequently also express markers for NET formation such
as citrullinated histone H3 and PAD4.[24]
NETs in Atherothrombosis
For decades, research has focused primarily on the so-called ‘vulnerable plaque’,
a morphology associated with plaque rupture and thrombosis, which may be triggered
by neutrophil NETosis.[44]
[56] Clinical cohort studies reported that neutrophil blood counts associated with an
increased risk of acute coronary events, heart failure and death.[27]
[28]
[29]
[30] However, the involvement of neutrophils, and particularly NETosis, in plaque destabilization
and rupture remains scant, and only supported by associative data. Moreover, contradictory
findings have been observed. For example, some have reported neutrophils to accumulate
in rupture-prone human atherosclerotic lesions.[25] In contrast to those studies, others have shown neutrophils and markers for NETosis
to co-localize with apoptotic endothelial cells in lesions complicated by superficial
erosion, but not in plaques considered rupture-prone.[26] Thus, experimental studies are clearly warranted to unambiguously establish the
contribution of neutrophil NETosis to plaque destabilization, rupture and its complications.
Decades of therapies to lower exposure to traditional risk factors may have altered
human atheromata, increasing the proportion of acute coronary events by superficial
plaque erosion. Indeed, recent data indicate that up to one-third of acute coronary
events currently result from erosion rather than plaque rupture.[103] A growing body of evidence underscores that neutrophils and NETs pertain to the
propagation of thrombotic complications of atheromata prompted by superficial plaque
erosion.[24]
[26]
[43]
[104] NETs appear to be associated with eroded or erosion-prone plaques in endarterectomy
specimens of carotid arteries[26] and recently in coronary specimens from patients with acute myocardial infarction.[24] In the same study, ligation of Toll-like receptor 2 was found to activate endothelial
cells and potentiate neutrophil recruitment ([Figs. 1D] and [2B]). Participation of neutrophils led to endothelial cell death and detachment, implicating
a role for neutrophil NETosis in superficial plaque erosion. Also, cells bearing CD66b,
MPO and NE were found to localize near luminal endothelial cells within human plaques
harvested from carotid arteries supporting the presence of neutrophils at the intimal
surface of complicated plaques that required endarterectomy.[104] Finally, genetic loss of PAD4 function in haematopoietic cells and NETosis protects
against endothelial desquamation and thrombus formation in a mouse model of atherosclerosis.[43] Here, NETs trigger endothelial cell death and detachment in a manner that depends
on complement deposition ([Figs. 1D] and [2B]).
In addition, further complement activation also triggers neutrophil recruitment to
the site of atherothrombosis in acute myocardial infarction,[105] and pathological studies showed high numbers of neutrophils in coronary specimens
from patients with acute myocardial infarction[20]
[106]
[107]
[108] or with complicated thrombosed plaques.[20] Also, thrombectomy specimens retrieved from patients with acute myocardial infarction
contain neutrophils in the thrombus mass.[109] Similarly, neutrophils were found more frequently associated with the presence of
occlusive thrombi.[21] The presence of NETs has been reported in thrombectomy specimens of patients with
acute myocardial infarction[39]
[67] or with stent thrombosis.[110] Elevated levels of circulating DNA, chromatin and MPO–DNA complexes are independently
associated with severe coronary events and the pro-thrombotic state.[65] However, the possibility that DNA and nucleosomes are released as a result of other
cell death programs, for example, endothelial cell apoptosis and cardiomyocyte necrosis,
cannot be ruled out, and it remains unclear to what extent neutrophil NETosis contributes
to thrombus formation. NETs exposed to blood gather the potent pro-coagulant tissue
factor, the initiator of the extrinsic coagulation pathway ([Fig. 1B]). Local accumulation of tissue factor-covered NETs occurs at sites of coronary thrombosis,[111] and neutrophils release NETs bearing tissue factor within thrombi of infarcted regions.[39] In addition, cleavage and inactivation of the endogenous anticoagulant protein,
tissue factor pathway inhibitor by NETs-contained neutrophil proteases (such as NE
and cathepsin G) drive and amplify intravascular clot formation[54] ([Fig. 1B]). Studies with mice that lack factor XII (FXII), the starting point of the intrinsic
coagulation pathway, suggest that NETs also contribute to the propagation of intravascular
blood coagulation by promoting FXII activation.[61] Plaque rupture during acute myocardial infarction triggers platelet aggregation
and deposition of fibrin at the initial site of the vulnerable atheroma. In turn,
activated platelets present HMGB1 protein to neutrophils provoking the formation of
NETs and the release of tissue factor.[39]
[56] Together, these events may contribute to plaque rupture and subsequent thrombus
formation ([Fig. 2B] and [C]). Indeed, platelet-derived HMGB1 protein facilitates NETosis and coagulation.[62] Results from other studies suggest that the formation of NETs may promote the growth
of a thrombus mass after the onset of a rupture of the plaque[60]
[64] by providing a scaffold for erythrocytes binding and platelet aggregation. Thrombus
growth and expansion leads to the reduction of blood flow and thus the onset of ischaemic
heart failure. In line herewith, NETosis has emerged as an important contributor in
a mouse model of myocardial ischaemia-reperfusion injury.[59]
[112] Neutrophils are able to produce a large array of cytokines, chemotactic factors
and proteolytic enzymes and therefore play a role in inflammation, fibrogenesis and
angiogenesis.[75]
[79] Thus, given the array of matrix-degrading enzymes that neutrophils contain, one
can anticipate a destabilizing impact of neutrophils during thrombus evolution. Release
of these enzymes could lead to thrombus disintegration and embolization. Indeed, lytic
thrombi with features of tissue necrosis have been reported to contain highest concentrations
of NETs[67] together with matrix metalloproteinase.[113]
Clinical Perspective and Future Challenges
Clinical Perspective and Future Challenges
Despite current therapies that have successfully lowered LDL, there remains a large
burden of residual risk, and atherothrombotic disease is still the leading cause of
morbidity and mortality globally. Attempts to lower exposure to additional risk factors
such as hypertension and smoking have only met with modest success. Although diet
and lifestyle certainly contribute to atherothrombosis, these alone cannot account
for the entire burden of atherosclerosis.[114]
[115]
[116] Indeed, genetic and environmental factors (such as co-morbidity, infection and products
of the endogenous microbiome) are now emerging as risk factors to atherosclerosis.
In fact, normal aging—a process that drives a state of chronic systemic low-grade
inflammation—is receiving more attention as perhaps the greatest risk factor for a
wide variety of chronic disease, including atherothrombotic disease. Without challenging
traditional risk factors, inflammation and immunity provide pathways that connect
traditional with these emerging risk factors that give rise to the disease and its
complications.
There is a great deal of excitement about new targets linked to atherogenic inflammation
that have emerged from animal and human studies. For example, multiple studies have
shown that IL-1β plays important roles in atherosclerosis,[117]
[118] and the recent CANTOS trial strongly underscores the concept of anti-inflammatory
therapy to treat CVDs.[6] Yet, broadly immunosuppressive therapies carry the risk of excess deaths from infections
limiting the clinical impact of these strategies. This heightens the need to understand
more clearly the inflammatory mechanisms that are specific to atherogenesis. Perhaps
the most obvious candidates for future studies are inflammatory mechanisms that are
linked to hyperlipidaemia and oxidative stress. Neutrophil NETosis is an emerging
mechanism underlying atherogenic inflammation, and likely interacts with hyperlipidaemia
and oxidative stress to promote atherogenic inflammation.[8]
[53]
Neutrophils and NETosis have been implied to contribute to atherogenesis as well as
thrombotic plaque complications, and thus represent novel targets for the treatment
and/or prevention of atherothrombotic disease. Given the profound pro-inflammatory
and pro-thrombotic effects that have been identified in earlier studies, systemic
treatment with DNase I merits consideration as a therapeutic approach. Indeed, formulations
of DNase I (Pulmozyme)—approved for the treatment of cystic fibrosis[119]—exert beneficial effects in mice with experimental atherogenic inflammation and
thrombosis. Moreover, NETs appear to jeopardize normal endothelial functions. In this
regard, the complement pathway and NETosis are intimately linked. NETs can activate
the alternative complement pathway[120] and promote endothelial damage affecting glomeruli in anti-neutrophil cytoplasmic
antibody-associated vasculitis.[121] NETs could constitute a critical scaffold promoting the local activation of the
complement pathway in the vicinity of vascular endothelial cells, exacerbating endothelial
cell death, detachment and thrombosis ([Fig. 1D]). Thus, strategies that limit complement activation also merit consideration as
an adjunct to treatment of atherosclerosis and its thrombotic complications. Recent
studies have linked oxidative stress, neutrophil cholesterol accumulation and NETosis
to atherogenic inflammation. However, this area remains poorly understood, therapy
is challenging and there is a tremendous need for further research.
Pulmozyme is used in the clinic to treat patients with cystic fibrosis, where it has
a beneficial effect, suggesting that—in this setting—NETs do more harm than good.
Initially, NETosis was demonstrated to be involved in anti-bacterial responses. Yet,
the initial finding that NETs are protective for host immunity has been challenged
by recent studies, and the clinical relevance of NETosis in infective diseases, particularly
chronic infections, is hard to judge. Hence, the net clinical impact of therapeutically
preventing NET formation in NETosis-associated diseases remains the most important,
unanswered question in the field to be resolved.
Perhaps the biggest challenge facing the field is translating findings from mouse
to human into novel and effective therapies. The principles of evolution, as well
as the scientific literature, suggest that there are many similarities between both
mammal species, but also significant differences.[122] When it comes to modelling immunity and inflammation in atherothrombotic disease,
this is by no means surprising or new. Although there are surely important differences
of opinion, mouse models do provide enough similarities in their immune responses,
and clinical and histological manifestations to be of value as a relevant model organism
in understanding mechanisms of atherosclerosis. Clearly, however, the physiology and
pathophysiology of mice is also sufficiently different to mandate an awareness of
potential resulting pitfalls. Nevertheless, despite these differences between mice
and men, immunologic research in mice led—among many other relevant insights—to the
discovery of the major histocompatibility complex and, ultimately, successful organ
transplants. The challenges ahead in understanding the genetic and/or environmental
factors accounting for heterogeneity in man (e.g. age, sex and co-morbid factors)
and faithfully modelling them in pre-clinical studies to best fit the human condition
loom large. With technology and innovation, the research community will hopefully
overcome them.
Conclusion
In summary, recent work has contributed to a growing body of evidence that NETosis
participates in atherogenic inflammation and the propagation of atherothrombosis.
NETs can perpetuate activation of endothelial cells, macrophages and platelets, trigger
coagulation and complement activation and cause endothelial dysfunction. Therefore,
interfering with the formation of NETs may result in numerous beneficial clinical
effects for patients with CVD. In animal experimental models, DNase I and Cl−amidine restrict neutrophil NETosis, and thus protect against atheroma burden and
thrombotic plaque complications. By contrast, other studies indicate that neutrophils
also have beneficial effects in complications associated with atherosclerosis.[123]
[124] NETs have been found at each stage of atherothrombotic disease. Nevertheless, whether
NETosis plays different roles at different stages remains unknown. In addition, it
will be a challenge to explore whether NETs are involved in crosstalk with intrinsic
vascular wall cells or other cell types such as smooth muscle cells. The identification
of endogenous triggers of NETosis remains an interesting prospect. Thus, future studies
will be needed to establish a better understanding of the role of NETosis in atherosclerotic
plaques and will be of paramount importance for the identification of the best candidates
for therapeutic targeting.
