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. 2022 Nov 3;13:6320. doi: 10.1038/s41467-022-33796-7

Fig. 3. VWF induces pro-inflammatory cytokine and chemokine expression and promotes monocyte chemotaxis.

Fig. 3

a pd-VWF (10 µg/ml) or LPS (100 ng/ml) incubation with primary human macrophages for 4 h resulted in significant increases in secretion of a TNF (control vs LPS ****P < 0.0001, control vs VWF **P = 0.0052) and b IL-6 pro-inflammatory cytokines similar to LPS (control vs LPS ****P < 0.0001, control vs VWF **P = 0.0011). c, d Although VWF binding to macrophages was associated with an increase in IL-1β mRNA and intracellular pro-IL-1β levels, a significant increase in IL-1β secretion (and concurrent decrease in intracellular pro-IL-1β) levels was only observed when VWF-treated macrophages were also subsequently exposed to ATP as a second hit (***P = 0.0003). Similarly, pd-VWF (10 μg/ml) or LPS (100 ng/ml) stimulation of primary human macrophages for 24 h resulted in a significant increase in secretion of chemokines including (e) CCL2, f CCL3 and g CCL4 with ****P < 0.0001 control vs LPS and control vs VWF for all chemokines. All experiments were performed in triplicate, and the results shown represent the mean values ± standard deviation (SD) and the significance was determined by ANOVA respect to the control. *P < 0.05, **P < 0.01, ***P < 0.001 respectively. h Primary human macrophages were incubated with pd-VWF (10 μg/ml) or LPS (100 ng/ml) for 24 h. Cell supernatants were then collected and placed in the lower chamber of a transmigration wells. Naïve human monocytes were placed in the upper chamber and allowed to migrate for 2.5 h. Migrated cell numbers were stained with calcein-AM and assessed using Image J software. Monocyte migration induced by the supernatants from macrophages incubated with pd-VWF (10 μg/ml) (*P < 0.0207), LPS (100 ng/ml) (P = 0.213) or media are represented as average fold cell-count increase over four independent experiments and presented as mean values ± SD. pd-VWF (10 μg/ml) alone had no significant effect on the migration of naïve human monocytes (ns = not significant). All scale bars are 80 μM. Source data for this figure are provided as a Source Data file.