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

Takashi Obama,Takashi Takaki,Sanju Iwamoto,Hitomi Ohinata,Hiroyuki Itabe,Naoko Sawada,Toshihiro Aiuchi,Rina Kato

doi:10.3389/fimmu.2019.01899

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.

Highlights

The biological significance of oxidatively modified low-density lipoprotein has been widely and extensively studied as a risk factor for the development and progression of cardiovascular diseases
When HL-60-derived neutrophils were stimulated with phorbol 12-myristate 13acetate (PMA), citrullinated histone H4 generated by the cells colocalized with extracellular DNA stained by SYTOX Green (Figure 2B)
Neither native low-density lipoprotein (LDL) nor oxidatively modified low-density lipoprotein (oxLDL) in the absence of PMA was sufficient for the induction of histone citrullination as well as DNA release, and this was demonstrated by the Hoechst 33342 staining of nuclei (Figure 2A)

Summary

Introduction

The biological significance of oxidatively modified low-density lipoprotein (oxLDL) has been widely and extensively studied as a risk factor for the development and progression of cardiovascular diseases. The pathological process of vascular diseases induced by oxLDL has been explored in terms of the activation of multiple cellular responses in the macrophages and vascular endothelial cells. It has been demonstrated that neutrophils (polymorphonuclear leukocytes), the most abundant form of white cells, infiltrate into the atherosclerotic and thrombotic lesions [6, 7]; the impact of neutrophils on the progression of atherosclerosis and its functional link with lipoproteins remains to be investigated. Neutrophils have diverse functions, and they are crucial as the first line of defense responses against pathogenic bacteria due to their phagocytotic activity and ability to produce bactericidal reactive oxygen species (ROS). NETs have been studied with regards to the primary immune responses elicited against pathogenic bacteria via extracellular DNA trap. A growing evidence has revealed that NETs have

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