Circulating neutrophil extracellular traps in cats with hypertrophic cardiomyopathy and cardiogenic arterial thromboembolism

Abstract Background Cats with hypertrophic cardiomyopathy (HCM) are at risk of cardiogenic arterial thromboembolism (CATE). Neutrophil extracellular traps (NETs) may be a potential biomarker and therapeutic target for cardiomyopathy in cats. Hypothesis/Objectives Characterize NETs in cats with HCM or CATE. We hypothesized that circulating NETs assessed in the form of cell‐free DNA (cfDNA) and citrullinated histone H3 (citH3) are increased in cats with HCM and CATE and associated with reported predisposing factors for thrombus formation. Animals Eighty‐five cats including client‐owned cats with HCM and CATE and staff‐ and student‐owned clinically healthy cats without HCM. Methods After echocardiographic evaluations, NETs were measured as cfDNA and citH3. Results Cats with CATE had significant increases in cfDNA (11.2 ng/μL; interquartile range [IQR], 8.1 to 29.6) compared to those without HCM (8.2 ng/μL; IQR, 5.7 to 11.7 μL; P = .01) and were responsible for 75% to 83% of cases with cfDNA fragments sized 100 to 2000 base pairs. Citrullinated histone 3, detected in 52% of cats with HCM (31.1 ng/mL; IQR, 16.9 to 29.8), was significantly lower than in those with CATE (48.2 ng/mL; IQR, 34.2 to 60.2; P = .007). The citH3 concentrations correlated significantly with reported risk factors of CATE, such as left atrial auricular velocity. Conclusions and Clinical Importance Neutrophil extracellualr traps, especially citH3, are increased in cats with HCM and CATE. They may serve as a novel therapeutic target and biomarker of thrombosis in cats with HCM.


| INTRODUCTION
Hypertrophic cardiomyopathy (HCM) is the most common cardiac disease in cats affecting approximately 15% of the cat population, and characterized by highly variable disease outcomes from subclinical to severe morbidity and mortality. Cats with HCM that develop clinical signs usually succumb to congestive heart failure (CHF), fatal arrhythmias, or cardiogenic arterial thromboembolism (CATE). Recent studies identified CATE as a major contributor to morbidity and mortality in cats with HCM with an incidence of 11.3% in 1008 cats with HCM. 1 This finding suggests that the prevalence of CATE was previously underreported. 2,3 Because cats with CATE often present acutely with extreme pain and no prior warning, CATE remains a distressing emergency for both cat owners and veterinarians, with a mortality rate of up to 67%. [3][4][5][6] Despite the devastating outcome, clinicians have limited tools to recognize cats at risk of CATE. Echocardiographic assessment of risk for CATE is most commonly employed including the presence of spontaneous echo-contrast (SEC), left atrial (LA) enlargement and left atrial appendage (LAA) dysfunction. 7 Identification of these risk factors is complicated by the fact that many cats at risk of CATE appear healthy and do not have auscultatory abnormalities, and hence, may be unlikely to undergo echocardiographic screening. 5,8 Reliable and accessible techniques to recognize cats with HCM at risk of CATE have yet to be identified.
The pathophysiology of CATE is poorly understood and likely results from dysregulation of each component of Virchow's triad, which includes endothelial dysfunction, hypercoagulability and blood stasis. Formation of neutrophil extracellular traps (NETs), which are web-like fragments of cell-free DNA (cfDNA) decorated with histones and neutrophil granular proteins, is an important component of innate immunity. 9 The prothrombotic properties of NETs, which facilitate microvascular thrombosis, are an important first-line of defense because they prevent systemic dissemination of pathogens. However, excess circulating cfDNA and NETs proteins can have proinflammatory and prothrombotic consequences. In dogs, neutrophil-derived cfDNA decreases clot lysis, whereas histones on NETs accelerate clot formation. 10 Circulating cfDNA fragments also facilitate clot formation by carrying tissue factor and binding to factors XI, XII and high molecular weight kininogen, all critical for promoting thrombin generation. [11][12][13][14][15] The web-like scaffold of NETs also fortifies clots by binding to circulating erythrocytes, platelets and fibrin. 16 Increased cfDNA concentrations and fragment sizes are known to affect fibrin formation and have diagnostic and prognostic value in humans with metastatic neoplasia and ischemic stroke. [17][18][19] Recently, we identified the presence of NETs as structural components in arterial thrombi from cats with CATE. 20 Not only were NETs found in all layers of arterial thrombi, but their distribution also varied greatly in relation to their proximity to the initial site of vascular occlusion. 21 This finding suggests that NETs also may play an important role in the pathogenesis of intracardiac thrombosis and thrombus growth in cats with HCM and CATE. In humans, NETs within coronary thrombi are associated with poor outcome in myocardial infarction and contribute to resistance to thrombolytic therapy. 22 velocity as previously described. 7 Left atrial size was evaluated as a ratio to the aortic root and measured as previously described. 26 Left atrial enlargement was defined as an absolute 2-dimensional (2D) long axis value ≥1.6 cm or 2D LA:Ao in short axis ≥ 1.6. All other echocardiographic assessments were performed as previously described. 26 Diagnosis of HCM required identification of idiopathic regional or

| Blood collection
Blood was drawn from the medial saphenous vein or jugular vein using a 23G butterfly needle within 6 hours of presentation. Blood was drawn from the cephalic or jugular vein in cats with CATE. Whole blood was immediately aliquoted to tubes containing 3.2% trisodium citrate (BD Vacutainer, Franklin Lakes, New Jersey) and lithium heparin (BD Microtainer, Franklin Lakes, New Jersey), placed on ice, and processed within 1 hour of collection. After gentle inversion and inspection for blood clots, a CBC and blood smear evaluation were performed on heparinized whole blood and the remainder of the sample was used for biochemical analysis and total T4 measurement (VS2, Zoetis, Parsippany, New Jersey).
A portion of the remnant sample was used for analyzing NETs markers.
Citrated and heparinized whole blood then underwent centrifugation (2000 Â G, 10 min, 4 C). After extraction of plasma, protease inhibitor cocktail (1Â HALT, Thermo Scientific, Waltham, Massachusetts) was added to heparinized plasma to prevent histone degradation, flash-frozen in liquid nitrogen, and stored at À80 C before analysis.

| Isolation and purification of plasma cell free DNA
Citrated plasma first was thawed at room temperature before cfDNA purification using a magnetic bead-based cleanup kit (QIAamp Minelute ccfDNA mini kit, Qiagen, Germantown, Maryland) as previously described. 27 The component mixture consisting of proteinase K, magnetic bead suspension and bead binding buffer was adjusted according to the available volume of plasma from each cat. The maximum and minimum volumes of plasma processed for cfDNA purification were 1000 and 500 μL, respectively. To optimize isolated cfDNA concentration, 20 μL of 1Â tris-EDTA buffer was applied directly on the center of column, which underwent 3 additional rounds of elution by reapplying eluate to the column. Eluted cfDNA was stored at 4 C for further analysis.

| DISCUSSION
We found that circulating NETs markers were significantly increased in cats with HCM and CATE. We also found that citH3 correlated significantly with predisposing risk factors of intracardiac thrombosis and thromboembolism in cats with HCM.
NETosis, a term that describes the active cellular processes underlying the formation of NETs, is dependent on complex and coordinated signaling facilitated by extracellular stimuli such as pathogens, danger-associated molecular patterns, cytokines, reactive oxygen species and, most importantly, platelet-neutrophil interactions. [30][31][32][33] Although the underlying mechanisms of NETosis in cardiomyopathies remain poorly understood, evidence in animal models of carotid artery thrombosis suggest that NETs formation is dependent on platelet activation, histone citrullination and upregulation of the neutrophil integrin, α9β1. [34][35][36] These findings suggest that neutrophils in cats with HCM and CATE may be activated at the site of endothelial injury or via platelet-neutrophil interactions resulting in NETosis. Given that NETs previously were found to be a component of arterial thrombi in cats, the presence of circulating NET markers in plasma further supports the notion that the bidirectional feedback of inflammation and thrombosis may contribute to a prothrombotic state that further promotes thrombosis. 21 We found that circulating cfDNA was significantly higher in cats with CATE but did not correlate with selected risk factors of thrombosis on echocardiogram. There are several explanations for these findings. First, quantification of total cfDNA could not discriminate between genomic DNA contamination originating from necrotic muscle cells associated with CATE and secondary ischemic injury or active release of cfDNA from living cells. In addition, in vitro processing also could lead to cell lysis and subsequent genomic DNA contamination.
Although every effort was made to minimize genomic DNA contami- tion. This identification is complicated by the fact that many cats at risk for CATE are apparently healthy and do not have auscultatory abnormalities, hence they are unlikely to be selected for echocardiographic screening. 5,8 In human beings, a number of studies indicate that increased citH3 is associated with thrombotic risk in cardiovascular disorders such as ischemic stroke, atrial fibrillation and myocardial infarction. [46][47][48] Thus, measurement of free circulating citH3 maybe an accessible way to determine cats at risk of CATE. Unfortunately, our small sample size and an inadequate amount of residual plasma for citH3 measurements in some cats prevented us from performing subgroup analyses to further explore the associations between increased-citH3 and prothrombotic risk factors. Additonal studies are needed to assess the diagnostic utility of plasma citH3 in cats with HCM so that thromboprophylaxis can be administered sufficiently early to prevent CATE. Although histone H3 is highly conserved among species and the feline protein is found to be 100% homologous to the human amino acid sequence, we noticed that feline plasma proteins consistently interfere with the detection of human protein standards in ELISA kits. This interference may be caused by nonspecific binding to the detection or capture antibodies or formation of histone-plasma protein complexes. Given the limitations of Western blot analysis such as long processing times and its semi-quantitative nature, custom development of a feline-specific citH3 ELISA is a possible future method to accurately measure citH3 concentrations as a clinical test.
Our study had several limitations. First, although chip-based capillary electrophoresis provides a reasonable estimation of cfDNA concentration, the small amount of cfDNA might have represented false negatives and this data should be interpreted with caution. More precise cfDNA fragment concentrations will need to be obtained by digital droplet PCR in future studies. 49 Second, although correlations among NETs, especially in free citH3, were found in cats with HCM and CATE, NETs could be a marker of disease severity. The latter would require a longitudinal study in HCM cats characterizing both citH3 and cfDNA fragment size using high-sensitivity detection and represents a future direction for this work. Third, we did not assess coagulation status of our study population and hence could not confirm the associations between circulating NETs and thrombosis in cats. Finally, inadequate volume of plasma samples and the semiquantitative nature of citH3 measurements prevented advanced subgroup analyses to further assess the diagnostic and prognostic utility of circulating citH3. Hence development of a feline-specific assay to measure plasma citH3 reliably and accurately in cats is critically needed.

| CONCLUSION
We found that cats with HCM and CATE had increased concentrations of circulating NETs, in the form of cfDNA at 100 to 300 bp and citH3.
Approximately 40% and 80% of cats with HCM and CATE, respectively, had detectable plasma citH3, which correlated with thrombotic risk factors.

ACKNOWLEDGMENT
This study was supported by the Morris Animal Foundation (D20FE-805). The authors thank the collaborative efforts of the Veterinary