Impact of hemolysate on HUVECs in a fluidic model mimicking intracellular hemolysis. (A) Three-step fluidic model reproducing intracellular hemolysis. HUVECs cultured in a flow system (step 1, flow culture of HUVECs) were exposed to either AA hemolysate, TNFα, or serum for 4 hours (step 2, HUVEC preconditioning). Step 3, functional assays were performed by infusing heparinized WB from either AA donors or patients with SCD on hemolysate-preconditioned HUVECs from step 2 for 10 minutes. The infusion flow rate in the fluidic model was adjusted to reach a shear stress 1 dyne·cm−2. (B) Hemolysate induced activation and damage of HUVECs after preconditioning: HUVECs cultured in flow system were exposed to either AA hemolysate, TNFα, or serum for 4 hours, then stained for activation and apoptosis markers by PECAM-1 Alexa Fluor 647 (red), ICAM-1 PE (orange), VCAM-1 Alexa Fluor 488 (green), Hoechst 34442 (blue), and annexin-V Alexa Fluor 594 (purple) staining. Images representative of 5 different experiments. Mean fluorescence intensity (MFI) of PECAM-1, ICAM-1, VCAM-1, and annexin-V markers were quantified by ImageJ. Data represent mean ± SEM (n = 5), the Kruskal-Wallis test with ∗P < .05 and ∗∗∗P < .001. (C) Hemolysate upregulates inflammation and acute response genes on HUVECs. HUVECs cultured in a flow system were exposed to either serum or hemolysate for 4 hours. Cells were then recuperated to analyze gene expression profiles by messenger RNA sequencing as described in supplemental Data. Example of genes with a global fold change of >1.5 and a false discovery rate (FDR) P value < .05 induced by hemolysate vs serum from 7 AA donors.
