Cooperative Action of Oxidized Low-Density Lipoproteins and Neutrophils on Endothelial Inflammatory Responses Through Neutrophil Extracellular Trap Formation

Abstract

The function of oxidatively modified low-density lipoprotein (oxLDL) in the progression of cardiovascular diseases has been extensively investigated and well-characterized with regards to the activation of multiple cellular responses in macrophages and endothelial cells. Although accumulated evidence has revealed the presence of neutrophils in vascular lesions, the effect of oxLDL on neutrophil function has not been properly investigated. In the present decade, neutrophil extracellular traps (NETs) gained immense attention not only as a primary response against pathogenic bacteria but also due to their pathological roles in tissue damage in various diseases, such as atherosclerosis and thrombosis. In this study, we investigated if oxLDL affects NET formation and if it is a risk factor for inflammatory reactions in endothelial cells. HL-60-derived neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA) for 30 min to induce NET formation, followed by incubation with 20 μg/mL native or oxidized LDL for additional 2 h. Culture media of the stimulated cells containing released NETs components were collected to evaluate NET formation by fluorometric quantitation of released DNA and detection of myeloperoxidase (MPO) by western blot analysis. NET formation of HL-60-derived neutrophils induced by PMA was significantly enhanced by additional incubation with oxLDL but not with native LDL. Treatment of HL-60-derived neutrophils with oxLDL alone in the absence of PMA did not induce NET formation. Furthermore, the culture media of HL-60-derived neutrophils after NET formation were then transferred to human aortic endothelial cell (HAECs) culture. Treatment of HAECs with the culture media containing NETs formed by HL-60-derived neutrophils increased the expression of metalloproteinase-1 protein in HAECs when HL-60-derived neutrophils were incubated with native LDL, and the expression was accelerated in the case of oxLDL. In addition, the culture media from NETs formed by HL-60-derived neutrophils caused the elongation of HAECs, which was immensely enhanced by coincubation with native LDL or oxLDL. These data suggest that oxLDL may act synergistically with neutrophils to form NETs and promote vascular endothelial inflammation.

Neutrophils as a Novel Target of Modified Low-Density Lipoproteins and an Accelerator of Cardiovascular Diseases

“…These findings prompted us to examine whether oxLDL influences NET formation and subsequent endothelial inflammatory responses. PMA-induced NET formation in both HL-60-derived neutrophils and human PMNs is remarkably accelerated upon coincubation with oxLDL, but not with native LDL [119]. Moreover, extracellular components, after induction of NET formation by PMA with oxLDL, are capable of inducing morphological changes and MMP-1 expression in human aortic ECs (Figure 3).…”

“…MPO and proteases released from neutrophils by NET formation could act on native low-density lipoprotein (LDL) to facilitate modification of LDL. Coexistence of NETs and oxLDL or native LDL promotes endothelial inflammation to cause cell elongation and enhanced MMP-1 production in endothelial cells[119]. MPO: myeloperoxidase, MMP-1: matrix metalloproteinase-1.…”

Differentiation of HL‑60 cells in serum‑free hematopoietic cell media enhances the production of neutrophil extracellular traps

“…HL-60 cells may be differentiated into granulocyte-like cells using all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). A model based on a stable cell line has been used to reduce dissonant results due to different experimental settings or conditions (13)(14)(15) and differentiated HL-60 (dHL-60) cells have been used to study neutrophil functions, including NETosis (16)(17)(18). However, the inefficiency of dHL-60 cells in generating NETs makes it challenging to completely replace neutrophils in the analysis of NET formation.…”

Cross-Talk between Lipoproteins and Inflammation: The Role of Microvesicles

“…Activated endothelial cells then release cytokines, chemokines, and adhesion molecules attracting circulating leukocytes, and more specifically monocytes, into the atherosclerotic lesion, inducing the differentiation of monocytes into proinflammatory macrophages and finally foam cells [18]. Although the primary origin of foam cells in atherosclerotic lesions are leukocytes [19], smooth muscle cells have been shown to differentiate to foam cells [20]. The rupture of the atherosclerotic plaque is the most common trigger of thrombosis, leading to an infarction.…”