Correlation of NETs to Atherosclerosis and Implication: Comparison
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Neutrophil extracellular traps (NETs) have attracted much attention recently, beyond elemental host immunity, due to their fundamental implication in a variety of pathologic conditions and widespread impactful diseases. Atherosclerotic cardiovascular disease (ASCVD) is one of them, and a major cause of mortality and disability worldwide. Consequently, years of basic and clinical research were dedicated to shedding light on every possible pathophysiologic mechanism that could be used as an effective prevention and treatment tool to ameliorate its burden. This led to the development of complex and prevention protocols and regimens that are now widely used, with lipid-lowering treatment being the current cornerstone; however, this is not adequate to alleviate the residual cardiovascular risk, which remains prominent. 

  • atherosclerosis
  • dyslipidemia
  • lipid-lowering therapies
  • neutrophils

1. Atherosclerosis, Immunity and Inflammation

The recent advancements in understanding molecular and cellular mechanisms in atherosclerosis, as well as future perspectives, have been described [2][1]. Accumulating evidence suggests that inflammation is the key component linking risk factors with atherosclerosis [18][2]. While oxidized LDL particles are well-studied drivers of atherogenesis [18[2][3],48], TGRLs are correlated to inflammatory status more effectively than LDL particles [49[4][5],50], as reflected by levels of high-sensitivity C-reactive protein (hsCRP) [51][6]. Consistent links are also documented with hypertension [52][7], obesity [53][8], and diabetes [54][9]. Importantly, many studies document the participation of innate and adaptive immunity to atherosclerosis pathophysiologically [55][10] and as a promising therapeutic target [18][2].
Under physiological conditions, macrophages reside in the vascular cell wall, specifically in the adventitia or under the endothelium, where they contribute to the maintenance of vascular homeostasis by interacting with SMCs and endothelium [26,56,57][11][12][13]. Endothelial cells damaged by well-studied stimuli like hypercholesterolemia, hypertension, diabetes, and oxidative stress attract monocytes with the contribution of activated platelets, by expressing leukocyte adhesion molecules like VCAM-1 [18,27][2][14] and excreting chemokines and guide them in the vascular wall intima via expression of leukocyte adhesion molecules, mostly integrins, on their surface. This marks the beginning of atheroma formation. Monocyte/macrophage recruitment and local proliferation make them the cornerstone of the atherosclerotic process [26][11]. Neutrophils and activated SMCs aid the monocyte infiltration by excreting chemokines like cathelicidin, cathepsin G, CCL2, and CCL5 [26][11]. SMCs also migrate to the developing fibrous cap and undergo apoptosis there after their metaplasia to SMC foam cells induced by lipid uptake. Macrophages also uptake lipids and transform into foam cells that comprise the lipid core of atherosclerotic plaque [18,58][2][15]. This uptake, particularly of oxidized LDL [59][16], along with reduced cholesterol efflux [60][17], triggers the activation of inflammasome NLRP3, which, in turn, leads to maturation of IL-1β and IL-18 [26,59][11][16]. Neutrophils also excrete their neutrophil extracellular traps NETs(NETs), which enhance inflammasome priming and exert cytotoxic effects on SMCs via histone H4 [26][11]. Conversely, inflammasome activation also causes the production of NETs via IL-18, as documented here [61][18]. The fundamental role of inflammasome in the atherosclerotic process is underlined by a study that documented improvement in plaque stability after either genetic or pharmacologic inhibition of absent in melanoma 2 (AIM2), a DNA-sensing cytosolic part of the inflammasome [62][19]. Activated T-helper1 lymphocytes also co-orchestrate and propagate the fluctuating imbalance of proinflammatory and anti-inflammatory molecules that eventually, through years of process, lead to atherosclerosis [18][2]. Ultimately, atherosclerosis appears to be the result of failure to counteract the aforementioned inflammatory mediators by their counterpart anti-inflammatory molecules excreted by B1, T-helper 2, and regulatory T lymphocytes like IL-10 and TGFβ [18][2]. Notably, inadequate clearance of cellular debris and dying cells by mononuclear phagocytes, a process called efferocytosis, leads to their accumulation and the formation of the lipid core of the atherosclerotic plaque [18][2].

2. Complications Leading to AStherosclerotic CVDardiovascular Disease Events

Rupture, superficial erosion, and increase in size of the atherosclerotic plaque become clinically apparent as cardiovascular disease (CVD) events [18][2]. In the past, the major mechanism for plaque disruption was thought to be the thinning and rupturing of the fibrous cap of the plaque due to collagen degradation by ongoing inflammation [63][20]. This results in exposing thrombogenic components to the circulation, priming the coagulation cascade, and leading to concurrent thrombotic events [64][21]. However, imaging studies have demonstrated that the most vulnerable, thin-capped plaques are the least clinically overt [18,65,66,67][2][22][23][24]. The most likely mechanism causing plaque vulnerability and concurrent CVD events appears to be the superficial erosion of the plaque, with neutrophils and their extracellular traps being the major components initiating and propagating this process [68,69,70][25][26][27].

3. The Role of Neutrophil ETxtracellular Traps in Atherosclerosis

As previously indicated, the atherosclerotic process is grounded on an intricate interplay between vascular homeostasis and the immune system. Research underscores the pivotal role of neutrophils and NETs throughout every phase of the atherosclerosis timeline, from initiation to the clinically evident thrombotic complications [10][28]. Initial indications of NETs’ involvement in atherosclerosis surfaced through experimental investigations on plaques derived from mice and humans [71][29]. Apolipoprotein E deficient mice were subjected to either a high-fat or a high-fiber diet for four weeks and atherosclerotic plaques derived from both groups were analyzed afterwards. The presence of luminally adhered neutrophils excreting NETs was determined in 57% of the first group’s atherosclerotic lesions while no neutrophils were observed in the second group specimens [71][29]. Similar findings were replicated in human atherosclerotic plaques post-carotid endarterectomy within the same study [71][29]. Subsequent research documented neutrophil and NETs abundance mostly in complicated plaques either with thrombosis or rupture [72][30]. Among 64 autopsy-derived specimens from post-myocardial infarction patients, 44 contained complicated and 20 intact atherosclerotic plaques. Neutrophils and NETs were predominantly observed in complicated plaques with ruptures, erosions, and intraplaque hemorrhage (p < 0.05) in similar amounts at each complication type and mostly in early stages of thrombus formation and plaque hemorrhage [72][30]. This observation was corroborated by the finding that NETs’ histone H4 favors inflammatory process and plaque destabilization by affecting smooth muscle cells. In this study, both atherosclerotic mouse models, as well as human endarterectomy samples, were employed to investigate the connection and participation of neutrophils and NETs in this context. Overall plaque vulnerability was significant in neutrophilic but not neutropenic mice [73][31]. Moreover, observations of the exact location where neutrophils are concentrated and of their interactions with smooth-muscle cells were documented. In summary, the well-designed experiments demonstrated that activated smooth-muscle cells attracted neutrophils on-site through excretion of chemokines, particularly CCL7, as well as ROS production by SMCs, triggering significant NETs release. CCL7 blockade resulted in reduced NETosis [73][31]. On the other hand, NETs exhibited strong cytotoxic effect on SMCs, mostly via H4 histone, resulting in reduced amounts of the latter in atherosclerotic lesions and increased plaque vulnerability due to thinning of plaque’s fibrous cap [26,73][11][31]. Notably, extranuclear histone H4 showed a significant positive correlation with intimal neutrophil counts. When the researchers neutralized H4 histone using specific antibodies, SMCs numbers and plaque stability remained intact [73][31]. Similarly, when they blocked NETosis using knockout mice for the PAD4 enzyme or through pharmacologic inhibition using chloramidine, plaque stability was preserved [73][31]. In another elegantly designed series of experiments, the induction of ROS-dependent NETosis by cholesterol crystals was demonstrated [74][32]. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and NE, fundamental in ROS-dependent NETosis, inhibition efficiently blocked cholesterol-induced NETosis, specifically while cloramidine (a PAD4 inhibitor) failed, obviously due to different NETosis molecular pathways [74][32]. Consequently, researchers employed mouse models of atherosclerosis to elaborate on the roles of neutrophils and NETs in the process. They noted that ApoE-deficient mice after DNase injection and ApoE/PR3/NE-deficient mice, namely incapable of producing or using NETs, experienced a significant reduction in atherosclerotic lesion size after eight weeks of a high-fat diet, compared to control ApoE-deficient counterparts without any treatment [74][32]. Moreover, NETosis-inactivated models demonstrated lower levels of circulating cytokines in the same time period. Notably, DNase administration caused a reduction in plasma cytokines in ApoE-deficient but not in ApoE/PR3/NE-deficient mice where interleukines (IL) IL-1α,-1β,-6 were already absent [74][32]. In particular IL-1β, a fundamental cytokine triggered by activated macrophages to recruit neutrophils, was significantly reduced to being absent in the latter model lesions. Naturally, cytokine regulation by NETs affects all immune cell communication, and that is also documented hitherto [74][32]. Monocytes exposed to supernatants containing NETs were more sensitive to cholesterol stimulation and produced larger amounts of cytokines [74][32]. Finally, IL-1β-regulated T-cells, which also promote neutrophil recruitment, and total immune cell counts in atherosclerotic lesions were also significantly less in ApoE/PR3/NE-deficient mice than ApoE-deficient controls. Thus, the aforementioned study [74][32] proved the NETs priming and amplifying effect in the complex cellular interplay between macrophages, neutrophils, and T-cells in the setting of atherosclerosis. PAD4, a fundamental enzyme for histone citrullination and chromatin decondensation [75][33], has successfully been targeted by chloramidine, thus inhibiting NETs release in atherosclerotic murine models and alleviating atherosclerosis by decreasing lesion size [26,76][11][34]. However, as clearly stated here [26][11], chloramidine’s incapability of targeted PAD4 isoform inhibition renders it unsuitable for clinical use. In a model of PAD4 and ApoE-deficient mice atherosclerosis burden was diminished in accordance with reduced inflammatory status and NETs formation [77][35]. Interestingly, PAD4 deletion in a murine model of LDLR-deficient animals failed to improve plaque size or composition after ten weeks of a high-fat diet, despite documented limited NETosis on-site. However, it benefited plaque stability by reducing intimal injury and thrombus formation [75][33]. Furthermore, in the same study, NET components like NE and citH4 were localized vastly in superficial erosion plaques compared to rupture-prone ones in human samples derived from endarterectomy procedures, implying NETs involvement in the specific type of plaque complication [75][33]. Importantly, NETosis has been triggered also by stimuli and pathways that do not implicate PAD4 in the process [78][36], meaning that PAD4 inhibition alone might not cause sufficient NETosis suppression in clinical practice.
There is also a considerable amount of clinical evidence on NETosis engagement in atherosclerosis, which has been concisely summarized recently by Doring et al. [10][28]. For instance, in a prospective, observational, cross-sectional cohort study of 282 patients with possible coronary artery disease (CAD), increased NETosis biomarkers-dsDNA, nucleosomes, and MPO–DNA complexes were identified in the plasma of individuals with severe coronary atherosclerosis compared to those without significant coronary disease [43][37]. Nucleosomes emerged as an independent marker of severe coronary stenosis (OR = 2.14, 95% CI 1.26–3.63; p = 0.005), while MPO–DNA complexes predicted major CVD events during the study [43][37]. Moreover, dsDNA levels were higher in those with severe (CAD) (p = 0.003) or increased coronary artery calcification (p < 0.001) compared with patients without CAD [43][37]. Luminal stenosis was also positively associated with circulating dsDNA (Spearman’s ρ = 0.271; p < 0.001) and the number of pathological coronary artery segments with plasma dsDNA (Spearman’s ρ = 0.242; p < 0.001), nucleosomes (Spearman’s ρ = 0.219; p = 0.001), and MPO–DNA complexes (Spearman’s ρ = 0.337; p < 0.001) [43][37]. Importantly, baseline levels of the aforementioned circulating NETosis biomarkers emerged as sufficient predictive tools for the occurrence of major adverse cardiovascular events (MACE) during a median follow-up period of 545 days [43][37]. Finally, NETs are believed to be a new source of TF in atherothrombosis [79][38]. A plethora of evidence implicating NETs in thrombotic complications following atherosclerosis has been analyzed here [30][39].

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