Reducing Abdominal Aortic Aneurysm Progression by Blocking Neutrophil Extracellular Traps Depends on Thrombus Formation

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SUMMARY
Neutrophil extracellular traps (NETs) are implicated in the pathogenesis of abdominal aortic aneurysm (AAA), located in adventitia and intraluminal thrombus.We compared the therapeutic potential of targeting upstream or downstream effector molecules of NET formation in 2 murine AAA models based on angiotensin II or peri-adventitial elastase application.In both models, NETs were detected in formed aneurysms at treatment start.Although NET inhibitors failed in the elastase model, they prevented progression of angiotensin IIinduced aneurysms with thrombus, which resembles established human disease (including thrombus development).Blockade of upstream NET mediators was more effective than interference with downstream NET molecules.(J Am Coll Cardiol Basic Trans Science 2024;9:342-360) © 2024 The Authors.Published by Elsevier on behalf of the American College of Cardiology Foundation.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A bdominal aortic aneurysm (AAA) is a progres- sive dilatation of the aorta due to weakening of the wall, which is often accompanied by an intraluminal thrombus (ILT). 1 Although mostly asymptomatic, AAAs tend to slowly grow with an eventual risk of rupture, which confers high mortality.Surgical repair remains the only treatment to date, with a lack of pharmaceutical drug options for the management of aneurysm progression or lowering of rupture risk. 2 AAA pathology involves an interplay of endothelial cells, smooth muscle cells (SMCs), and extensive immune cell infiltration that drives vessel wall degeneration through inflammation. 3Neutrophils in particular are found in abundance in the adventitia and ILT. 4 In an experimental animal model, depletion of neutrophils inhibited AAA formation. 5Neutrophil mediators such as myeloperoxidase (MPO), matrix metalloproteinases (MMPs), and neutrophil elastase were found to contribute to AAA development by matrix destruction. 6,7 part of the immune defense against pathogens, neutrophils may release their content (decondensed DNA strands, decorated with nuclear, cytosolic, and granular components such as histones, elastase, MPO, and cathepsin G) into the extracellular space as neutrophil extracellular traps (NETs). 8This process can be initiated via multiple pathways.In the NADPH oxidase 2 (Nox2)dependent pathway, the production of reactive oxygen species and the nuclear translocation of MPO as well as elastase trigger chromatin decondensation. 9Conversely, in a Nox2-independent pathway, the activation of protein arginine deiminase 4 (PADI4) leads to histone citrullination and NET release. 10The pathways can be triggered separately in vitro; however, in vivo, they likely occur concomitantly. 11 note, citrullinated histones (CitH3/CitH4) are rarely observed in other cellular processes and are therefore used as a characteristic marker of NET formation. 12e exposure of neutrophil DNA and intracellular proteins may have detrimental consequences, and studies have reported the presence of NETs in various diseases such as rheumatoid arthritis and cancer, where they contribute to pro-inflammatory processes. 11Of note, NETs have also been implicated in the pathogenesis of cardiovascular diseases and are known to promote atherosclerosis and thrombosis. 13xtracellular histone H4 released from NETs reportedly triggers a form of lytic cell death, whereby histone H4 mediates SMC membrane lysis. 14Preventing this interaction between histones and membranes by a novel histone inhibitory peptide (HIPe) averted SMC death and stabilized atherosclerotic lesions in mice. 14NETs also contribute to venous and arterial thrombus formation 15,16 via the cell-free DNA acting as a scaffold for platelet and red blood cell aggregation and via histones increasing thrombin generation by activating platelets. 16though most AAAs display severe atherosclerosis, several studies have investigated the specific role of NETs in AAA disease in which they were mainly observed in the adventitia and the luminal side of the ILT. 17 Comparably, treatment with chloramidine, an irreversible pan-PAD inhibitor, significantly mitigated AAA formation in this mouse model. 19cause patients with AAA present with established disease, we have previously addressed the role of NETs in aneurysm progression as opposed to formation, with a particular emphasis on histone citrullination.In line with earlier reports, we found a marked deposition of CitH3 in the ILT and the aortic wall tissue of patients with AAA compared with healthy control subjects as well as in their plasma samples. 20Our results suggested that circulating CitH3 holds prognostic information to predict rapid aneurysm growth in patients with AAA.Moreover, we found that histone citrullination was a promising therapeutic target to block disease progression, as the specific PADI4 inhibitor GSK484 10 prevented further AAA growth when applied to established aneurysmatic lesions. 20These findings were recently confirmed. 21 therefore hypothesized that the efficacy of   whereas thrombus formation strongly depends on the location; that is, hematomas are mostly observed when the ostium of the left suprarenal artery is affected. 28trasound measurements (Figure 1A) revealed that on day 8, the abdominal aortic volume had doubled from BL (100%) to an average of 213% AE 26.3%,

A B B R E V I A T I O N S
For mice with intramural thrombus formation, at the peak of NET prevalence (day 8), CitH4-positive staining was primarily located in the aortic wall at the interface between the thrombus and thickened remodeling adventitia, where the medial wall disruption had occurred.In contrast, at day 28, the remaining CitH4 signal was primarily located within the thrombus in all mice.Here, a significant trend was detected regarding the primary location of NETs in the vessel wall at day 8 as opposed to the thrombus at day 28 (Fisher exact test, P ¼ 0.048) (Supplemental Figure 1).In the 2 mice without thrombus formation, little neutrophil or CitH4 signal was remaining at day 28.
To target the NADPH oxidase activation pathway of NET formation, Nox2ds-tat peptide was used. 29eatment significantly attenuated aneurysm progression compared with the control (mean at day 27: 190% AE 10% vs PBS 288% AE 47%; Wilcoxon signed rank test, P ¼ 0.008) (Figure 3B).When evaluating maximum aortic diameter, the results were similar (Wilcoxon signed rank analysis, P ¼ 0.023) (Supplemental Figure 4B).Downstream of the NET formation pathway, NET products were targeted to investigate their contribution to aneurysm growth.HIPe was applied to neutralize the toxic activity of cell-free histone H4, 14 3D).In line with aortic volume growth, these 2 treatment groups did not differ significantly from PBS control regarding maximum aortic diameter increase (Supplemental Figures 4C and 4D).
To assess the impact of the inhibitors on the frequency of neutrophils, NETs, SMCs, and macrophages in treated aneurysms, aortic tissue was stained for Ly6G and CitH4, SMA, and CD68, and it was scored (0-3) for semi-quantitative analysis (Figure 4, Supplemental Figures 5 and 6).In the AngII model, the infiltration of neutrophils and the deposition of     rank sum test, treatment effect at day 27 was significant for experimental animals with thrombus (P ¼ 0.035) but not for mice without AAA-associated thrombus (P ¼ 0.56).Of note, data are also given independently for the GSK484 and Nox2ds-tat cohorts in Supplemental Figure 7, and they confirmed the same tendency in mice with thrombus formation for either treatment.
The downstream NET inhibitors (Figure 5B The presence of an intramural thrombus seemed to determine the efficacy of anti-NET therapy in the AngII model more markedly than the choice of upstream or downstream inhibitor.We therefore further pooled all anti-NET approaches and obtained a high significance level (P ¼ 0.017) for the difference between treatment and control within the thrombus subgroup (data not shown).Again, the no-thrombus subgroup had no substantial therapeutic impact at day 27 (Wilcoxon rank sum test, P ¼ 0.32).
To assess the impact of the inhibitors in relation to thrombus presence on the frequency of neutrophils and NETs, SMCs, and macrophages in treated aneurysms, a similar subanalysis was also conducted by using semi-quantitative tissue scoring (Figures 5C to 5F, Supplemental Figures 5 and 6).When focusing on mice with a thrombus, the deposition of CitH4 was lowest in the aortic tissue of animals that were given the effective upstream inhibitors compared with the PBS cohort (71% of aortas without detectable CitH4 signal vs 14% in the PBS cohort) or the downstream inhibitors, which were also effective in the thrombus cohort (44% of aortas with no detectable NETs).In terms of SMC colonization in the thrombus group, the anti-NET inhibitors showed a high SMA-positive aortic coverage (tissue scoring ¼ 3) compared with the PBS cohort (57% in the upstream inhibitor group and 56% in the downstream inhibitor group vs 14% in the PBS group).Similarly, the frequency of vimentin (Supplemental Figures 5 and 6), an intermediate filament that is up-regulated in SMC transition from the contractile to the synthetic phenotype, 30   thione peroxidase 4 (GPX4). 32Conversely, upstream NET inhibitors resulted in significantly higher transcript levels of GPX4 in aortic tissue than observed for downstream NET blockade (Figure 6A).
Remarkably, parameters of inflammation such as monocyte-chemotactic protein-1, CD68, interleukin-6, or interferon gamma did not differ significantly between treatment groups (Figure 6D).When evaluated separately for mice with or without thrombus development, the described regulatory effects on gene expression in AAA tissue were predominantly observed for the thrombus group (Supplemental Figure 8).Furthermore, comparison between the 4 distinct compounds revealed a more pronounced preservation of contractile SMC genes by Nox2ds-tat than GSK484 treatment and a predominant GSK484 impact on GPX4 and interferon gamma gene expression (Supplemental Figure 9).Of note, there is generally no aortic rupture and no thrombus occurrence in the elastase-induced aneurysms.
None of the treatments had a significant effect in terms of attenuating aneurysm progression in the EPPE mouse model.As we showed before in a smaller cohort, 20 GSK484 treatment was not effective in reducing aneurysm growth in this model (mean aortic volume increase at day 13: 367% AE 31% vs PBS 352% AE 15%; Wilcoxon signed rank analysis, P ¼ 0.58) (Figure 7A).For the Nox2ds-tat cohort, the mean volume at day 13 was 314% AE 29% and did not significantly differ from the PBS cohort (309% AE 17%; P > 0.99) (Figure 7B).
The downstream inhibitors showed a slight but nonsignificant trend for reduction.The mean volume growth at day 13 for the HIPe cohort was 298% AE 13% compared with the matched PBS group (325% AE 21%; Wilcoxon signed rank test, P ¼ 0.43) (Figure 7C).This trend was also reflected in the maximum diameter of the HIPe cohort with a mean of 1.18 AE 0.059 mm vs the PBS cohort (1.33 AE 0.067 mm; Wilcoxon signed rank analysis, P ¼ 0.16) (Supplemental Figure 10).DNase I treatment showed a similar trend with mean aortic volume growth of 261% AE 24% vs 316% AE 28% in the PBS group (Wilcoxon signed rank analysis, P ¼ 0.22) (Figure 7D).The absolute diameter, however, showed no difference between DNase and PBS treatment.
Semi-quantitative tissue scoring (Figure 8, Supplemental Figure 11) revealed that GSK484, Nox2ds-tat, and HIPe treatment resulted in a similar   In human AAA disease, the ILT plays a role in mechanically stabilizing the aneurysm by decreasing peak wall stress. 34It was also found to be a predictor of AAA growth 35 and to increase the risk for rupture through dedifferentiation and apoptosis of SMCs by promoting degradation of the extracellular matrix and by increasing the number of infiltrating inflammatory cells. 36This observation is in agreement with our published findings in human AAA aortic tissue: we found high levels of neutrophil and NET deposition in both the adventitia and the ILT of human aneurysms.When CitH3 was measured in conditioned medium of fresh-frozen tissue (n ¼ 27 per group), the levels ranged at a median of 50 ng/mg in the AAA wall (vs 1.5 ng/mg in healthy aortic tissue, P < 0.001) and at 629 ng/mg in the ILT (P ¼ 0.002 vs the AAA wall). 20Ts have been previously implicated in thrombosis, 16,37 and the accumulation of NETs in the thrombus in our human and murine aneurysmatic aorta samples supports a potential involvement in pathogenesis.Importantly, we could confirm this functional role; that is, these data showed that NET inhibition was effective in regulating AngII-induced aneurysm growth, in particular in mice that devel- Regarding clinical translation, it should be noted that the 2 upstream inhibitors performed comparably in blocking AAA progression in the AngII model.
However, the targeted approach of PADI4 inhibition might be preferable in clinical application, thereby avoiding potential unwanted side effects that are associated with Nox2 inactivation. 39In contrast, human genomic studies have indicated that loss of PADI4 does not lead to substantial impairment (ie, arguing for a safe application of PADI4 inhibitors). 40 note, the first established PADI inhibitors (eg, Fand Cl-amidine) bound irreversibly and with similar potency to several PADI family members, including PADI4, and recognized the calcium-activated enzyme form. 41Among the second-generation compounds, GSK484 shows high selectivity for PADI4 and binds to the calcium-deficient protein in a reversible manner. 10Therapeutic interference with several family members may be preferred in conditions such as cancer or rheumatoid arthritis in which several PAD enzymes are involved. 41 The present analysis focused on neutrophils.However, other immune cells certainly play an essential role in AAA pathogenesis 3 ; in particular eosinophils, basophils/mast cells, and monocytes/macrophages are also capable of extracellular trap formation. 42though 10% to 30% of CitH4-positive Ly6Gnegative cells were detected in aneurysms at treatment start, the contribution of these non-neutrophil sources of extracellular traps has not been selectively addressed in this study but would be represented in the deposited CitH4 levels per square millimeter of investigated AAA area.[45][46][47] Of note, a possible cross-talk between PADI4 and Nox2 and hence mutual interference by GSK484 and Nox2ds-tat cannot be excluded and has been addressed by several in vitro studies, with highly controversial results.Although GSK484 does not seem to block the Nox2-dependent pathway of NET release, 48 PADI4 inhibition by GSK484 was proposed to enhance reactive oxygen species release by Nox2. 49 contrast, others reported that PADI4 is physically associated with Nox2 subunits and GSK484 disrupts the complex, thereby reducing Nox2 activity.

2 PADI4 = protein arginine deiminase 4 PBS
= phosphate-buffered saline SMA = smooth muscle alpha actin SMC = smooth muscle cell From the a Division of Vascular Surgery, Department of General Surgery, Medical University of Vienna and University Hospital Vienna, Vienna, Austria; b Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna and University Hospital Vienna, Vienna, Austria; c Division of Cardiovascular and Interventional Radiology, Division of Molecular and Gender Imaging, Department of Biomedical Imaging and Image Guided Therapy, Medical University of Vienna and University Hospital Vienna, Vienna, Austria; d Division of Visceral Surgery, Department of General Surgery, Medical University of Vienna and University Hospital Vienna, Vienna, Austria; e Skin and Endothelium Research Division, Department of Dermatology, Medical University of Vienna and University Hospital Vienna, Vienna, Austria; f Department for Visceral, Thoracic and Vascular Surgery, Technical University of Dresden and University Hospital Carl-Gustav Carus, Dresden, Germany; g Leeds Institute for Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Leeds, United Kingdom; and the h Leeds Vascular Institute, Leeds General Infirmary, Leeds, United Kingdom.The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and Food and Drug Administration guidelines, including patient consent where appropriate.For more information, visit the Author Center.Manuscript received May 3, 2023; revised manuscript received October 2, 2023, accepted November 1, 2023.
Yan et al 18 reported that NETs promote AAA development through the recruitment of dendritic cells and their type I interferon production.Administration of DNase I to dismantle NETs was effective in preventing aneurysm formation when given during the early induction phase of an AAA mouse model based on aorta elastase perfusion.
blocking AAA progression might vary with the targeted pathway and molecule involved in NET formation.The aim of the present study was to compare the therapeutic impact of distinct inhibitors on established AAA disease by either blocking upstream signaling events to prevent further NET induction (via Nox2ds-tat for the Nox2-dependent pathway or GSK484 for the PADI4-dependent pathway) or by destroying and inactivating toxic components of already formed NETs (via DNase I for cell-free DNA or HIPe for extracellular histones).Two mouse models were used: aneurysms induced by angiotensin II (AngII) frequently involve an intramural thrombus and enter a progression stage resembling human established disease, whereas acute aorta injury by peri-adventitial elastase is considered a model more closely reflecting disease formation and initial expansion.22 25th and 75th percentiles.For AAA growth, data are expressed in percentage of aneurysm volume from BL (100%) or in maximum aortic diameter (millimeters).The Wilcoxon signed rank test was applied for comparisons within each group vs baseline, whereas the Wilcoxon rank sum test was used to compare unmatched mouse groups at the individual time points of the time course.Treatment mice were matched 1:1 for percent aortic volume growth at day 8 (AngII model) or day 4 (EPPE model) with the closest sized control mouse in the respective PBS cohort; differences between groups were evaluated with the Wilcoxon signed rank test at the experimental endpoint.For analysis of thrombus impact, treatment effect was determined for the thrombus or no thrombus groups at the experimental endpoint by using the Wilcoxon rank sum test.The Fisher exact test was used for categorical data; that is, to examine the association between NET location (thrombus or wall) with experimental time point (day 8 and day 28).Statistical analyses were conducted with GraphPad Prism version 9.0.0 for Windows (GraphPad Software), SPSS 27.0 software (IBM SPSS Statistics, IBM Corporation), or Python version 3.11 (Python Software Foundation), and a significance level of P < 0.05 was applied.Because the analysis was exploratory in nature, no adjustments for multiple testing were made or considered in sample size calculation.RESULTS 2 MOUSE MODELS EXHIBIT NET FORMATION IN THE TIME COURSE OF AAA DEVELOPMENT.Two established mouse models of AAA induction (the AngII model and the EPPE model) were investigated for NET deposition within the AAA.As previously described, 23 35% of AngII-infused mice experienced early aortic rupture (<10 days).In the remaining animals, aneurysms formed suprarenally over 4 weeks, as well as in the thoracic aorta, frequently accompanied by aortic dissection and intramural thrombus formation.It has previously been documented for the AngII model that medial tears occur in all mice,

FIGURE 1 AAA
FIGURE 1 AAA Progression and NET Presence in the AngII Model

FIGURE 2 AAA
FIGURE 2 AAA Progression and NET Presence in the EPPE Model CitH4 in the aortic tissue of mice administered upstream inhibitors was lower than in the PBS cohort (56% with detectable signal of Ly6G or CitH4 in the PBS group vs 14% of aortas in the GSK484 group and 25% of aortas in the Nox2ds-tat cohort).In contrast, neutrophil and NET accumulation in aneurysms of HIPe-treated mice resembled the frequency of control mice and reached a level of 71%.Of note, although no therapeutic impact of DNase I application on AAA progression was observed, it showed an effect on the NET content of treated aneurysms, which ranged at 29% of aortas with detectable Ly6G and 43% of aortas with CitH4 signal.All treatment groups showed high scores (2-3) for SMA expression, whereas only the PBS cohort had 18% of aortas with low SMC coverage.Conversely, the highest scores (3) for macrophage infiltration were recorded for the PBS and HIPe groups.NET BLOCKADE IS EFFECTIVE IN MICE THAT DEVELOP AN INTRAMURAL THROMBUS IN THE AngIIMODEL.Because analysis of NET distribution at day 28 had revealed that NETs were primarily detected in the intramural thrombus, we investigated whether the presence of thrombus influenced the outcome in the treatment groups.In the full PBS cohort (n ¼ 30), 11 mice did not have a thrombus, whereas 19 mice presented with a thrombus.To increase the

FIGURE 3
FIGURE 3 Upstream vs Downstream NET Inhibition in the AngII Model

FIGURE 4 NET
FIGURE 4 NET Accumulation at d28 in the AngII Model

FIGURE 5 NET
FIGURE 5 NET Inhibition in AngII Mice Without or With AAA-Associated Thrombus Although neutrophils are known to contribute to AAA formation by the production of reactive oxygen species, the secretion of inflammatory mediators, and matrix-degrading proteinases, NETs have been proposed to primarily act on SMCs via inducing transdifferentiation or ferroptosis.31,32Because immunostaining analysis was semi-quantitative, we further aimed to elucidate the mechanism of effective anti-NET blockade on AAA progression by comparing beneficial upstream vs ineffective downstream NET inhibitors in tissue gene expression analysis.Aortas retrieved on experimental day 28 were subjected to RNA isolation, complementary DNA generation, and real-time polymerase chain reaction of a selected gene panel (Figure6, Supplemental Methods).Aneurysms that had been treated with upstream NET inhibitors showed significantly lower levels of MPO, indicating reduced neutrophil accumulation and activation (Figure6A).Furthermore, expression of MMP-9 but not MMP-2 was substantially reduced, and elastin transcript levels were significantly higher, in AAA tissue of mice responsive to upstream vs downstream NET blockade (Figure6C).In line with the proposed NET effect on SMC plasticity,31 markers of the contractile SMC phenotype such as SMA, myosin 11, transgelin, and calponin 1 were more preserved in the upstream inhibitor group (Figure 6B).A recent report further suggested that NETs promote AAA formation by inducing ferroptosis in smooth muscle cells, as evidenced by the decreased expression of cystine/glutamate antiporter solute carrier family 7 member 11 and gluta- NET BLOCKADE DOES NOT REDUCE DISEASE PROGRESSION IN THE ELASTASE-TRIGGERED AAA MODEL.Similar to the AngII model, anti-NET treatment in the EPPE model was administered after establishment of disease (day 5) to test the drug effects on AAA progression until the endpoint at day 14.

FIGURE 6 2 :
FIGURE 6 Impact of Upstream vs Downstream NET Inhibitors on Mediators of Inflammation, Matrix Remodeling, and SMC Transdifferentiation in the AngII Model

FIGURE 7
FIGURE 7 Upstream vs Downstream NET Inhibition in the EPPE Model

FIGURE 8 NET
FIGURE 8 NET Accumulation at d14 in the EPPE Model oped an intramural thrombus.The comparison of upstream and downstream NET inhibitors in the AngII model further revealed that inhibiting the NET induction process was more effective than neutralizing or dismantling already formed NET products.Although the downstream NET inhibitors failed to achieve a significant reduction of aneurysm size in statistical analysis based on the combination of AngIItreated mice with and without thrombus, a subgroup analysis revealed a moderate therapeutic effect in mice with thrombus compared with the PBS control.However, the protective effect of targeting already formed NETs was lower than for NET prevention.Whether NETs promote the thrombus formation and thereby enhance the local accumulation of tissuedestructive components, or whether the formation of a thrombus allows for prolonged NET deposition and NET-mediated vascular toxicity to drive AAA progression, remains to be determined.Of interest, a direct effect of NETs on SMC plasticity has recently been proposed.31Clonal expansion of SMCs and phenotypic switching from a contractile to a synthetic and more phagocyte-like phenotype has previously been observed during the development of AAAs (and the AngII mouse model).Medial SMCs were reported to clonally expand into the adventitia and hemorrhagic areas and undergo down-regulation of differentiation markers such as SMA while up-regulating markers such as vimentin and CD68.38 NETs in AAA have been suggested to promote SMC switching from a contractile to a synthetic phenotype via the Hippo-YAP pathway.31Our immunofluorescence results of mice with a thrombus are in agreement with this notion (ie, a lower number of SMA-positive cells were observed in the PBS vs treatment groups).However, the highest frequency of vimentin-positive cells as well as high levels of CD68-positive cells were detected in the absence of anti-NET treatment in vicinity to or within the remodeling thrombus itself.Taken together, these results suggest an effect of NETs on SMC (de-) differentiation in association with the hemorrhagic thrombus area.When further comparing gene in aneurysms with successful upstream vs ineffective downstream NET blockade in AngII-treated mice, it became more evident that SMC plasticity is indeed a prime target of NET regulation in AAA.Transcript levels of the contractile SMC phenotype were significantly preserved by the upstream inhibitors GSK484 and Nox2ds-tat compared with the downstream effector molecules HIPe and DNase I. Of note, the recently proposed ferroptosis induction in SMCs by NETs, as evidenced by decreased GPX4 messenger RNA levels, 32 was more prominent in downstream than upstream anti-NET approaches.Little difference between the treatment groups was observed for transcript levels of monocyte/macrophage markers, whereas genes expressed by neutrophils (eg, MPO, MMP-9) were more potently reduced in upstream NET blockade.Of note, the described gene regulation pattern was mainly recorded for mice with thrombus formation.

J
A C C : B A S I C T O T R A N S L A T I O N A L SCIENCE VOL. 9, NO. 3, 2024In addition, it should be noted that GSK484 and HIPe can be considered specific anti-NET treatment approaches as they are unlikely to substantially affect other physiological or pathologic processes.In contrast, DNase I and Nox2ds-tat will not only interfere with NETs but exhibit a broader impact on DNA released by other mechanisms or on additional cell types and functions regulated by Nox2, respectively.However, the observation that both upstream inhibitors GSK484 and Nox2ds-tat outperformed the downstream-acting factors (HIPe and DNase I) in AAA growth inhibition argues for a predominance of drug action via NET regulation.CONCLUSIONSThe findings of this study indicate that in AAA, there is a total "NET impact" on disease progression that is composed of both newly forming NETs and the deposited toxic components of NETs.As shown by the distinct outcomes of the different treatments in each of the AAA models, the balance between the contributions of either arm may depend on the pathologic trigger and the chronic setting in advanced disease.Although a combination of both an upstream and downstream inhibitor might be even more effective, a wider spectrum of side effects would also be expected, thus arguing for a targeted approach.Importantly, our analysis reveals that the thrombus in particular, which plays a crucial role in AAA progression and rupture, presents as the prime site of action for NET inhibitors in the pharmaceutical management of AAA.ACKNOWLEDGMENTS The authors thank Prof Bruno Podesser's team (Department of Biomedical Research) and the Core Facility for Laboratory Animal Breeding, Medical University of Vienna, for support in mouse experiments.The authors further acknowledge the Core Facility Genomics, a member of Vienna Life-Science Instruments, for the analysis of RNA quality.Monika Weiss (Department of Dermatology, Medical University of Vienna) kindly assisted with trichrome staining.They also thank Prof Oliver Söhnlein (University of Münster) for advice in HIPe application.PERSPECTIVES COMPETENCY IN MEDICAL KNOWLEDGE: Although NETs have previously been shown to contribute to the development of AAAs, our study has specifically addressed the role of NETs in disease progression, which resembles established human disease and thus highlights the potential for clinical translation.The results from our preclinical study indicate that blocking NET formation holds substantial promise for controlling aneurysm progression, which would meet a long-standing need for a pharmaceutical drug option for patients with AAA.TRANSLATIONAL OUTLOOK: NETs have been identified as a potential therapeutic target in various acute and chronic diseases.To our knowledge, this study is the first to compare the efficacy of anti-NET approaches targeting upstream vs downstream effector molecules.Both the dependence of therapeutic impact on the formation of a local thrombus as well as the superior efficacy of upstream NET inhibitors may thus be of interest and relevance to other NET-driven conditions and the current efforts to develop NET-targeting drugs for clinical application.
the EPPE model, percent NET area (0.26% AE 0.12%), neutrophils (1,488 AE 715 N/mm 2 ), and ingly, CitH4 was detectable in areas in which elastin fibers were completely absent or mostly broken; areas with intact elastin fibers did not show any CitH4.Of note, in both mouse models, CitH4-positive cells negative for Ly6G were detected, suggesting that cell types other than neutrophils might contribute to extracellular trap formation.As shown in Supplemental Figure3, Ly6G-positive cells constituted the majority of CitH4-positive cells, thus arguing for neutrophils as the predominant source of extracellular traps in the AAA models.UPSTREAM NET INHIBITORS ARE MORE EFFECTIVE THAN DOWNSTREAM NET BLOCKADE IN PREVENTING DISEASE PROGRESSION IN THE AngII AAA MODEL.To test the effect of NET inhibitors on AAA progression, treatment was administered after establishment of disease.Based on an initial ultrasound assessment of 4 time points per model (data not shown), the treatment was started after day 8 for the AngII model and day 4 for the EPPE model; this is when the aortic volume growth had reached an average value of approximately 200% and when NETs were found to be present in the tissue (Figures1 and 2).First, GSK484 was administered to target histone citrullination.At stratification (day 8), both groups showed a comparable degree of established disease; that is, AAA growth was to 205% AE 30% (GSK484) and 199% AE 28% (PBS).The AAAs in mice that were subsequently given GSK484 treatment did not progress compared with the PBS group (mean at day 27: 180% AE 16% vs PBS 277% AE 53%; Wilcoxon signed rank test,