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

Summary

A series of methods is described, including the isolation of low-density lipoprotein (LDL) from human plasma, differentiation of HL-60 cells into neutrophil-like cells, preparation of neutrophil extracellular traps (NETs) in the presence of LDL, and stimulation of human aortic endothelial cells with a mixture of NETs and LDL.

Abstract

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.

Introduction

Neutrophil extracellular traps (NETs), produced by activated neutrophils upon bacterial infection, as well as under non-infectious physiologically plausible conditions, release decondensed DNA and proteins, such as histones, myeloperoxidase (MPO), and neutrophil elastase¹. Because these components can dismantle marginal cells and tissues, causing persistent inflammation, NET formation has emerged as a causative factor in the initiation and progression of various acute and chronic diseases, including rheumatoid arthritis, psoriasis, diabetes, Alzheimer's disease, cancer metastasis, and cardiovascular diseases². Histologic....

Protocol

The protocol for LDL preparation was approved by the ethics committee of Showa Medical University School of Pharmacy (No. 231). Written informed consent was obtained in accordance with the Declaration of Helsinki, and all participants voluntarily provided their signatures for participation in this study. The cells were aseptically manipulated using a laminar flow hood. The inclusion criterion was an age range of 22-65 years. Exclusion criteria included smoking habits, pre-existing cardiovascular diseases (such as coronary heart diseases, stroke, and peripheral artery diseases), severe liver dysfunction, and uncontrolled chronic conditions, including diabetes, hyperten....

Results

After removal of the chylomicrons, followed by VLDL fractions from human plasma through sequential ultracentrifugation, human plasma was mixed with a KBr solution to adjust the density to d = 1.063. In this step, the remaining precipitant was not completely dissolved in the solution, and severe mixing, which created gas bubbles, was avoided to keep the LDL intact. After ultracentrifugation, LDL was visually assessed on the top layer by its yellow-orange color⁹ (Figure.......

Discussion

This protocol describes the preparation of NETs from dHL-60 cells induced by the coexistence of LDL, which are subsequently used to analyze HAEC responses. The significance of this protocol is the use of fresh medium containing NETs and LDL for the stimulation of the cells, even though many other studies utilize a stock solution of NETs frozen at -20 °C. As lipoproteins are lipid-protein complexes, their intact structures and characteristics are lost during freeze-thawing, causing denaturation and aggregation of LDL.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1% Poly-L-Lysine	SigmaAldrich	P8920-100ML
10 cm dish	Corning	430591
12-well Tissue Culture Plates	Falcon	353043
2% Gelatin	SigmaAldrich	G1393-20ML
4PC tube	Himac	S404332A
all-trans retinoic acid	Wako	188-01113	Can be substituted with any vendor
Cellurose dialysis tubing	MISUMI Group Inc.	0327-23-37-07 (UC18-32-100)	Can be substituted with any vendor
EDTA	Dojindo	345-01865	Can be substituted with any vendor
Endothelial Cell Growth Medium 2 Kit	PromoCell	C-22111
Fetal Bovine Serum	Gibco	10270-106	Can be substituted with any vendor
Heparin Sodium (5,000 units/5 mL)	Mochida Pharmaceutical Co., Ltd.		Can be substituted with any vendor
High Speed Refrigerated Centrifuge	TOMY	SRX-201	Acceleration: 9, Deceleration: 7
human aortic endothelial cells	Lonza	cc-2535
KBr	Wako	168-03475	Can be substituted with any vendor
KEYENCE Microscopy software	KEYENCE	BZ-II Analyzer
Micro Ultracentrifuge	Himac	CS150GX
Microscopy	OLYMPUS	CK40
Microscopy	KEYENCE	BZ-9000	For time-lapse imaging (option)
Microscopy camera	Nikon	DS-Fi3
Microscopy camera Software	Nikon	DS-L4
Penicillin/Streptomycin	Gibco	15140-122 100ML	Can be substituted with any vendor
phosphate buffer saline (×10)			Dissolve 2.6 g of NaH2PO4·2H2O, 29 g of Na2HPO4·12H2O, 80 g of NaCl and 2.0 g of KCl in distilled water and adjust to the volume of 1 L.  (Can be substituted with any vendor)
PMA(Phorbol 12-Myristate 13-Acetate)	Wako	162-23591	Can be substituted with any vendor
ReagentPack	Lonza	CC-5034	(CC-5022: HEPES Buffered Saline Solution, CC-5012: Trypsin/EDTA, CC-5002: Trypsin Neutralizing Solution)
Rotor	Himac	S100AT6-0242
RPMI-1640 (with Phenol red)	Wako	189-02025	Can be substituted with any vendor
RPMI-1640 (without Phenol red)	Wako	186-02155	Can be substituted with any vendor
T-25 flask	Corning	3275