PB0830 - Mechanisms of 4-Hydroxy-2-nonenal (HNE)-induced thromboinflammation

Background: HNE is the most stable and abundant α,β-unsaturated aldehyde generated during lipid peroxidation. Elevated HNE is associated with cardiovascular disease. We recently showed that administration of HNE to mice increases perivascular cells-derived tissue factor (TF)-positive extracellular vesicles (EVs), elaboration of the proinflammatory cytokine IL-8, and disruption of the endothelial barrier.

Aims: To delineate the mechanisms underlying HNE-induced thromboinflammation.

Methods: Primary cells, cell lines, and CRISPR knockout cells were treated with HNE, and the generation of TF+ EVs and expression of IL-8 were determined. The underlying signaling pathways responsible for HNE-mediated effects were investigated.

Results: (1) HNE treatment increased cell surface TF activity without increasing antigenic TF expression in all cells tested (smooth muscle, fibroblast, and monocytic THP-1). By contrast, HNE only stimulated release of TF+ EVs from perivascular but not monocytic cells. A 5-8-fold increase in EVs FXa generation and a 2-3-fold increase in prothrombinase activity was observed. Nanoparticle tracking analysis revealed that the HNE-induced increase in EVs procoagulant properties stemmed from increases in both EVs count and TF-specific activity. Pretreatment of cells with the antioxidant N-acetylcysteine or the mitochondrial ROS scavenger Mito-Tempo significantly attenuated HNE-induced EVs procoagulant activity. Pharmacological inhibition of the Rho-ROCK pathway markedly reduced TF+ EV generation. (2) HNE treatment caused endothelial barrier disruption, a process mediated by the p38 MAPK-dependent pathway. (3) HNE induced a 10-20-fold increase in IL-8 expression by monocytic THP-1 cells and human monocyte-derived macrophages. Pharmacologic inhibition of PKC-delta or a dominant-negative PKC-delta mutant attenuated HNE-induced IL-8 expression independent of NFkB. PKC-delta activates ERK1/2, which in turn facilitates nuclear translocation of the transcription factor cJUN to regulate IL-8 expression. Inhibition of ERK1/2 or CRISPR knockout of cJUN significantly reduced IL-8 gene expression.

Conclusion(s): HNE induces thromboinflammatory cell signaling. Inhibition of HNE-mediated signaling may be a therapeutic opportunity to alleviate cardiovascular complications of thromboinflammation.