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

Neutrophil extracellular traps are involved in a variety of diseases, including cardiovascular diseases. However, the synergistic effects of NET formation and lipoproteins on vascular cells remain unclear. Utilizing this protocol, mechanistic analysis are ongoing to this date how inflammatory responses are induced in human aortic endothelial cells treated with neutrophil extracellular traps and low-density lipoprotein.

We have demonstrated that high-density lipoprotein acts as a suppressor of NET formation. That is promoted by oxidized low-density lipoprotein, oxidized phospholipids, and lysophospholipids. To begin, centrifuge 45 to 50 milliliters of human whole blood obtained in a heparin tube at 700 G for 15 minutes at four degrees Celsius.

Slowly reduce the centrifuge speed after the spin, then collect the upper layer containing the plasma fraction. Centrifuge the plasma fraction two times at 700 G for 15 minutes at four degrees Celsius to remove blood cells completely, maintaining the same deceleration setting. Then add one microliter of 250 millimolar EDTA solution per one milliliter of plasma to prevent divalent metal ion-mediated oxidation of lipoproteins.

Place 2.7 milliliters of the plasma into the ultracentrifuge tube. Overlay with 900 microliters of PBS containing 250 micromolar EDTA. Then ultracentrifuge the tube at 600, 000 G for seven minutes at four degrees Celsius.

Next, discard 900 microliters from the top layer to eliminate the chylomicron fraction. Add 900 microliters of PBS containing EDTA on top of the remaining plasma and ultracentrifuge at 600, 000 G for 2.5 hours at four degrees Celsius. Discard 900 microliters of the top layer to eliminate the very low-density lipoprotein fraction.

Then add 540 microliters of 0.5 grams per milliliter potassium bromide solution to the plasma to adjust the density to 1.063. Mix the contents gently using a pipette and ultracentrifuge at 600, 000 G for 2.5 hours at four degrees Celsius. Collect 540 microliters of the top layer containing low-density lipoprotein.

Transfer the collected fraction into a dialysis membrane and dialyze against two liters of PBS containing EDTA at four degrees Celsius in the dark to eliminate potassium bromide. Add 152 microliters of 0.01%polylysine, or 0.1%gelatin solution to each well of a 12-well plate and incubate at room temperature for at least five minutes. Then remove the solution from the wells, wash once with 200 microliters of sterile water, and allow the wells to dry completely at room temperature.

Store the prepared plate at room temperature until further use. Prepare the gelatin-coated plates following the same procedure as demonstrated for polylysine. Culture two times ten to the power of six HL60 cells per 10 milliliters in RPMI-1640 medium, supplemented with two micromolar all-trans retinoic acid.

After four days of incubation with all-trans retinoic acid, collect the HL60 cells. Centrifuge the collected cells at 220 G for four minutes at 18 to 22 degrees Celsius. Aspirate the supernatant, then wash the cells with an equal volume of serum-free RPMI-1640 medium.

Centrifuge the washed cells again at 220 G for four minutes at 18 to 22 degrees Celsius and resuspend the cells in serum-free RPMI-1640 medium. After adjusting the cell concentration to two times ten to the power of six cells per milliliter, seed 0.5 milliliter of the suspension into each well of a 12-well plate pre-coated with polylysine. Add 100 microliters of serum-free RPMI-1640 medium with or without 300 nanomolar phorbol 12-myristate 13-acetate or PMA.

Culture the cells for 30 minutes. Next, remove the medium from the wells. Wash the cells once with serum-free RPMI-1640 medium and replace it with serum-free RPMI-1640 containing either zero or 20 micrograms per milliliter of low-density lipoprotein.

Continue culturing the cells for two hours. Collect the culture medium from each well into a tube and centrifuge at 700 G for three minutes at 18 to 22 degrees Celsius to remove cellular debris. For stimulation, obtain the human aortic endothelial cell culture and replace the spent medium with 0.5 milliliter of fresh culture medium, and incubate the cells for 30 minutes.

Finally, add 167 microliters of the culture medium collected from neutrophil-like cells containing neutrophil extracellular traps and LDL to human aortic endothelial cell dishes. Stimulation of human aortic endothelial cells with neutrophil extracellular traps led to dose-dependent morphological changes from a cobblestone-like to an elongated shape. Morphological changes in human aortic endothelial cells were enhanced when stimulated with LDL-induced neutrophil extracellular traps, with visible elongation apparent as early as six hours.