2:02 PM Abstract No. 96 - Neutrophil extracellular traps in ruptured carotid artery plaques

Abstract

Purpose Death of macrophage-foam cells and the accumulation of lipoproteins and lipids is a pivotal step in the process of atherogenesis. It is unknown whether these macrophages undergo a process of NETosis where the genomic DNA is released into the extracellular matrix (ECM). NETs can stimulate the immune system by activating the Toll-like receptor (TLR) family of proteins and promote cellular infiltration of inflammatory cells such as neutrophils. More importantly, the ionic DNA backbone of NETs may act as a nidus to bind lipoproteins and promote lipid core development. Our aim, therefore, was to investigate the presence of NETs in atherosclerotic plaques and whether they are spatially associated with plaque rupture. Materials and Methods Following IRB approval, 7 human carotid arteries containing segments of ruptured plaque were collected during surgical endarterectomy. Sequential 5-micron tissue sections were immunostained using monoclonal anti-H2A/H2B/DNA complex antibody to detect NETs and polyclonal anti-myeloperoxidase (MPO) antibody to detect neutrophil and monocytes. To demonstrate the specificity of the antibody to detect NETs, the adjacent tissue section was pretreated with 10 units of DNase prior to NETs immunostaining. Samples were also evaluated by hematoxylin and eosin (H&E) and Masson’s trichrome staining. Results All seven carotid arteries demonstrated advanced atherosclerotic lesions and evidence of plaque rupture. Extensive NETs immunostaining was detected predominantly within the acellular lipid core. Along the edges of the lipid core, confocal microscopy demonstrated active release of NETs from MPO positive cells. Pretreatment of tissue sections with 10 units of DNAse-1 led to undetectable NETs signal in the extracellular matrix but persistent signal within the cells along the margins of the core as the permeabilization step in the protocol was omitted. Conclusion NETosis may represent a key step in the development of the lipid core. Furthermore, it is possible that NETs may activate the TLR proteins in MPO positive cells along the margin of the lipid core and lead to progressive weakening of the plaque and increase susceptibility to rupture.