Neutrophil extracellular traps, deoxyribonuclease and fibrosis in thoracic aortic aneurysm

Abstract Introduction Thoracic aortic aneurysm (TAA) is a rare but severe cardiovascular disease. The condition often goes undiagnosed, increasing the risk of acute aortic events. It is characterized by medial degeneration, caused by neutrophils infiltrating aortic tissue. Neutrophils can form proinflammatory and prothrombotic neutrophil extracellular traps (NETs), promoting disease progression. Purpose Recently, NETs and abdominal aortic aneurysm (AAA) were linked, while their effects on TAA development and progression remain unclear. We aimed to characterize NETs and NET-specific markers in tissue and plasma samples of TAA patients. Methods We included 171 patients with either ascending TAA (n=153) or Type A dissection (n=18). Healthy subjects were recruited as controls (n=135). Sample fiber composition was analyzed using trichrome; NET burden was quantified using immunofluorescence. Plasma NET surrogate markers including double-stranded (ds)DNA, myeloperoxidase (MPO), DNase activity and matrixmetalloproteinases (MMP) were measured using a fluorescence-based assay and ELISA, respectively. mRNA expression was assessed using qPCR. Results Median patient age was 65 years, and most were male (68.4%). Cardiovascular risk factors were common, including hypertension (77.8%), hyperlipidemia (57.9%), coronary artery disease (26.3%), and diabetes (18.7%). TAA patients were older with high BMI and impaired kidney function. Trichrome staining revealed abundant fibrotic material (median 47.99%), while collagen and elastic fiber distributions were 27.67% and 20.17%, respectively. TAA patients had increased amounts of aortic fibrotic material (47.99% vs 38.94%, p=0.001). Using immunofluorescence, we found higher NET expression in diseased tissue (0.87 [0.3, 1.85] vs 0.15 [0.03, 0.22]%, p=0.0017). This increase was primarily driven by the dissection patients (1.76 [0.89, 2.83]%, p=0.0037). Plasma MPO levels were increased in TAA compared to controls (36.93 [23.46, 57.55] vs 26.8 [19.7, 36.2]ng/mL, p<0.001), while DNase activity was decreased in patients (4.16 [2.66, 6.24] vs 6.06 [4.25, 8.11]mU/mL, p<0.001). Moreover, circulating plasma levels of MMP2 were elevated in TAA (250.36 [190.24, 351.32] vs 218.85 [171.32, 263.87]ng/mL, p=0.004). qPCR analysis of mRNA expressions in TAA cryospecimens revealed upregulations of TNFα (p=0.033) and CD45 (p=0.003), when compared to aortic disease-free controls. Conclusion Aortic fibrosis at the aneurysm site may be caused by inflammatory mechanisms involving TNFα and leucocytes (CD45+). High local NET-burden is indicative of activated neutrophils in the aortic media. TAA patients have suppressed DNase activity and higher MPO levels in venous blood, leading to dsDNA accumulation and increased neutrophil turnover. Elevated MMP2 suggests its involvement in TAA formation. Our findings could enable further research targeting NETs and inflammation in TAA, possibly finding novel therapies capable of mitigating aneurysm development.