Stimulation of Vascular Endothelial Cells Using Neutrophil Extracellular Traps in the Presence of Low-Density Lipoprotein

Neutrophil extracellular traps (NETs) have emerged as causative factors in various non-infectious diseases and have been implicated in cardiovascular disorders such as atherosclerosis and thrombosis. NET formation is observed in the vascular wall, and there is compelling evidence that plasma markers of NET formation increase with disease severity. Neutrophil-derived NET components, including myeloperoxidases and proteases, affect plasma lipoproteins and vascular homeostasis. Here, a series of methods for stimulating vascular cells with NETs formed in the presence of low-density lipoprotein (LDL) is described. LDL was fractionated from human plasma by ultracentrifugation. HL-60 cells were treated with all-trans retinoic acid to differentiate them into neutrophil-like cells and then stimulated with phorbol 12-myristate 13-acetate (PMA) to induce NET formation. Following the removal and wash-out of PMA, cells were further incubated with LDL. The collected supernatants containing NETs and LDL were immediately used to stimulate human aortic endothelial cells (HAECs). As a representative response, the morphological alteration of HAECs induced by NET treatment was enhanced by the presence of LDL, suggesting that NET formation, in combination with LDL, enhances the response of HAECs. These methods are beneficial for exploring the effects of LDL on endothelial cells under neutrophil activation and NET-associated inflammation, thus providing new insights into the mechanisms underlying cardiovascular diseases associated with increased plasma LDL.