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. Histological analyses of human vascular lesions of atherosclerosis³ and abdominal aortic aneurysm⁴ showed that neutrophil infiltration and NET formation were colocalized. In circulating blood, levels of NETs markers, such as citrullinated histones, NET-DNA, and MPO, increase with the severity of coronary artery disease⁵. In the early stages of atherosclerosis, NET formation often precedes lipid accumulation, which occurs via macrophage-driven foam cell formation^6,7. These phenomena strongly suggest that MPO and proteolytic enzymes released extracellularly during NET formation can act not only on vascular tissues but also on blood components, such as lipoproteins. Lipoproteins are fragile lipid-protein complexes that can be damaged by NET-derived proteins such as MPO⁸; hence, NET formation can cause structural and biochemical alterations in lipoproteins, thereby influencing vascular homeostasis. However, the synergistic effects of NET formation and lipoproteins on vascular cell-induced dysregulation and inflammatory responses remain unclear.

Here, this manuscript provides a method for examining the cellular responses of human vascular endothelial cells induced by treatment with NETs prepared from HL-60-derived neutrophil-like cells and low-density lipoproteins (LDL) fractionated from human plasma. Representative results demonstrate the fractionation of LDL from human plasma by ultracentrifugation and the cellular responses of human aortic endothelial cells (HAECs) stimulated with NETs after co-incubation with LDL. This method is broadly applicable to the study of vascular cells involving other lipoproteins.

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, hypertension, dyslipidemia, severe inflammatory diseases, and cancer. The reagents and the equipment used are listed in the Table of Materials.

1. Isolation of LDL from human plasma

1. Collect whole blood from a healthy human volunteer in the presence of anti-coagulant heparin (10 units/mL).
2. Centrifuge 45-50 mL of human whole blood obtained in a heparin tube (700 × g for 15 min at 4 °C). Ensure to slowly reduce the speed of the centrifuge. Collect the upper layer comprising the plasma fraction.
3. Perform centrifugation twice (700 × g, 15 min at 4 °C) at the same deceleration setting as in step 1.2 to eliminate blood cells. Add 1:1,000 volume of 250 mM EDTA (pH 7.4) to prevent divalent metal ion-mediated oxidation of lipoproteins.
4. Place 2.7 mL of plasma in a 4 mL-polycarbonate (4PC) tube, overlay 900 µL of phosphate-buffered saline (PBS) containing 250 µM EDTA (PBS/EDTA), and ultracentrifuge (600,000 × g, 7 min at 4 °C).
5. Discard 900 µL of the top layer to eliminate chylomicron. Stack 900 µL of PBS/EDTA on plasma and ultracentrifuge (600,000 × g, 2.5 h at 4 °C).
6. Discard 900 µL of the top layer to eliminate very low-density lipoprotein (VLDL). This step is important to prevent contamination of LDL with other lipoprotein fractions.
7. Add 540 µL of 0.5 g/mL KBr solution to adjust the density to d = 1.063, followed by gentle mixing with a pipette and then ultracentrifuge (600,000 x g, 2.5 h at 4 °C).
8. Collect 540 µL of the top layer containing LDL. A yellow-orange layer indicates successful LDL separation⁹. Transfer to a dialysis membrane.
9. Dialyze the LDL fraction three times against 2 L of PBS/EDTA at 4 °C in the dark to eliminate KBr.
10. Store LDL at 4 °C in the dark before use.

2. Preparation of poly-L-lysine- or gelatin-coated wells

1. Prepare a 0.01% poly-L-lysine solution¹⁰ by diluting 0.1% poly-L-lysine solution with sterile water, followed by filtration with a 0.2-µm sterile filter. Store at 4 °C before use.
2. Add 152 μL/well of 0.01% poly-L-lysine solution to each well of a 12-well plate and incubate for at least 5 min.
3. Remove the solution and then wash once with 200 μL/well of sterile water and dry for at least 2 h.
4. Store at room temperature before use in the preparation of NETs.

3. Preparation of gelatin-coated wells

1. Prepare a 0.1% gelatin solution before use by diluting 2% gelatin solution with sterile water.
2. Add 152 μL/well of the 0.1 % gelatin solution to each well of a 12-well plate and incubate for at least 5 min¹¹.
3. Remove the solution and then wash once with 200 μL/well of sterile water and dry for at least 2 h.
4. Store at room temperature before use.

4. Maintenance and preparation of human aortic endothelial cells (HAECs)

1. Culture primary HAECs in a non-coated culture flask using complete medium containing 2% fetal calf serum (FCS), 5 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor, 20 ng/mL insulin-like growth factor, 0.5 ng/mL vascular endothelial growth factor, 1 µg/mL ascorbic acid, 22.5 µg/mL heparin, and 0.2 µg/mL hydrocortisone, as indicated in the manufactures' instruction. Change the medium every alternate day until the cells are 70%-80% confluent.
2. For stimulation, seed HAECs in gelatin-coated 12-well plates. Change the medium every alternate day until confluence is reached. Perform the experiment between passages 4 and 7.

5. Preparation of HL-60-derived neutrophil-like cells followed by NETs

1. Maintain HL-60 cells with RPMI-1640 medium supplemented with 5 % fetal bovine serum and 1 % penicillin/streptomycin, using non-treated dishes.
2. For preparation of HL-60-derived neutrophil-like cells, culture 2 × 10⁶ cells per 10 mL of HL-60 cells for 4 days in RPMI-1640 medium containing 2 µM all-trans retinoic acid (AtRA).

6. Preparation of NETs with LDL

1. Collect HL-60 cells 4 days after treatment with 2 µM AtRA. After differentiation of HL-60 cells into neutrophil-like cells, the cells become smaller.
2. Centrifuge cells (220 × g, 4 min at 18-22 °C). Aspirate the supernatant and wash the cells with an equal volume of serum-free RPMI-1640.
3. Centrifuge the cells (220 × g, 4 min at 18-22 °C) and then resuspend the cells with serum-free RPMI-1640.
4. Count the cells, then resuspend the cells to 2 × 10⁶ cells/mL in serum-free RPMI-1640 and seed 0.5 mL of cell suspension in the wells of a 12-well plate pre-coated with poly-L-lysine. Culture for at least 30 min.
5. Add 100 µL of serum-free RPMI-1640 with or without 300 nM phorbol 12-myristate 13-acetate (PMA) to the final concentration of 0 or 50 nM PMA. Culture for 30 min.
6. Remove the medium and wash the cells once with serum-free RPMI-1640. Replace the medium with serum-free RPMI-1640 with or without 20 µg/mL of LDL and then culture for 2 h.
7. Collect culture medium and centrifuge (700 × g, 3 min at 18-22 °C) to remove cell debris.

7. Stimulation of HAECs with NETs and LDL

1. Replace the medium with 0.5 mL of fresh culture medium. Culture for at least 30 min before stimulation.
2. Add 167 µL of the culture medium containing NETs and LDL (step 6.7) and LDL to HAEC dishes. Check the morphological changes in HAECs induced after stimulation for 12 h or more.

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