Abstract 24: NETosis is Associated with Abdominal Aortic Aneurysm Rupture

Abstract

Objectives: Abdominal aortic aneurysm (AAA) rupture (RAAA) is associated with high mortality. Intraluminal thrombus and associated inflammation have been implicated in RAAA. We sought to characterize neutrophil extracellular trap (NET) formation, or NETosis, associated with a rat RAAA model that we previously developed. Methods: To stimulate AAA formation, infrarenal abdominal aortas of male SD rats were infused with porcine pancreatic elastase (PPE): active (AAA, n=21) and heat-inactivated (control, n=14). Aortas were harvested 3, 6, and 14d post PPE exposure. To stimulate RAAA, a separate group of animals was administered β-aminopropionitrile (BAPN, n=12) daily. Those animals that did not rupture (NRAAA-14d, n=6) were harvested 14d post PPE exposure. Paraffin embedded sections were analyzed using immunofluorescence. To evaluate evidence of primarily neutrophil activity and NET generation, myeloperoxidase (MPO) and peptidylarginine deiminase 4 (PAD4) were quantified utilizing Cy3 and Alexa-488 secondary antibodies, respectively. To determine the presence of NETs, citrullinated histone H3 (H3Cit, Cy3) was visualized. DAPI was utilized to detect nuclei. Aortic sections were imaged with fluorescent microscopy, and quantification of percent area stained (PAS) was performed by threshold analysis. Results: Greater MPO PAS was observed for AAA-6d compared with control-6d (p=0.03). Greater PAD4 PAS was observed for AAA-3d (p=0.02) and -6d (p=0.05) compared with control-3d and -6d, respectively. Greater H3Cit PAS was observed for AAA-6d (p<0.0001) and -14d (p=0.001) compared with control-6d and -14d, respectively. In the setting of rupture stimulation, greater H3Cit PAS was observed for RAAA compared with AAA-14d (p=0.001) and NRAAA-14d (p=0.0002). Conclusions: Increased PAD4 expression by AAAs compared with controls at 3 and 6d post PPE exposure, results in increased H3Cit expression at 6 and 14d post PPE exposure. Comparable H3Cit expression by AAA-6d and RAAA demonstrates the acute nature of this rupture model; however, greater H3Cit expression by RAAAs suggests a role for NETosis in RAAA. Further evaluation of NETosis as it pertains to human RAAA is necessary. Therapeutic inhibition of NETosis may slow AAA progression and prevent RAAA.