Neutrophil extracellular traps (NETs) are found in patients with various diseases, including cardiovascular diseases. We previously reported that copper-oxidized low-density lipoprotein (oxLDL) promotes NET formation of neutrophils, and that the resulting NETs increase the inflammatory responses of endothelial cells. In this study, we investigated the effects of high-density lipoproteins (HDL) on NET formation. HL-60-derived neutrophils were treated with phorbol 12-myristate 13-acetate (PMA) and further incubated with oxLDL and various concentrations of HDL for 2 h. NET formation was evaluated by quantifying extracellular DNA and myeloperoxidase. We found that the addition of native HDL partially decreased NET formation of neutrophils induced by oxLDL. This effect of HDL was lost when HDL was oxidized. We showed that oxidized phosphatidylcholines and lysophosphatidylcholine, which are generated in oxLDL, promoted NET formation of PMA-primed neutrophils, and NET formation by these products was completely blocked by native HDL. Furthermore, we found that an electronegative subfraction of LDL, LDL(–), which is separated from human plasma and is thought to be an in vivo oxLDL, was capable of promoting NET formation. These results suggest that plasma lipoproteins and their oxidative modifications play multiple roles in promoting NET formation, and that HDL acts as a suppressor of this response.

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.

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.