Abstract
Introduction: Sickle cell disease (SCD) is a genetic hemoglobinopathy associated with increased red blood cell (RBC) rigidity and susceptibility to hemolysis. Hemolysis-derived products including heme and RBC-derived extracellular vesicles (REVs) drive endothelial activation, triggering aberrant blood cell adhesion. REVs are known to carry heme and expose surface phosphatidylserine (PS), with their endothelial-activating potential primarily attributed to their heme content. Independently, REVs have also been shown to mediate complement system (CS) activation, a process linked to both their heme cargo and PS exposure. During intravascular hemolysis, free heme likely reaches the endothelium more rapidly due to its small molecular size and unrestricted diffusion, whereas REVs exhibit slower transport kinetics. Here, we aim to establish an in vitro model to investigate the ‘second-hit’ effect of REVs on heme-primed endothelium in the context of CS activation.
Methods: We employed our SCD-EV-BioChip microfluidic in vitro assay to test this second-hit effect by evaluating REV-mediated activation of heme-primed HUVECs, using aberrant monocyte (THP-1) adhesion as a functional biomarker. To ensure consistency across experiments, we first quantified the heme content of REVs using a commercial heme assay kit and diluted them in basal medium to match a final heme concentration of 5 µM, which was used in all subsequent experiments. HUVECs were primed with 5 µM heme in 25% normal human serum (NHS) diluted in basal medium for 30 minutes, followed by a second 30mins incubation with REVs in the same medium. To evaluate the second-hit impact, control experiments included sequential exposure to 0.5 µM heme followed by 0.5 µM heme, REVs followed by REVs, and a no-treatment control. To assess the specific contribution of complement activation, these experimental groups were also tested in basal medium lacking NHS. Following the 1-hour total incubation, monocyte adhesion was assessed under a shear stress of 1 dyne/cm², followed by rinsing and quantification of adherent THP-1 cells. Complement activation was evaluated by measuring C5a levels using a commercial ELISA kit. Statistical comparisons were performed using two-tailed t-tests, with significance defined as p < 0.05.
Results and Discussion: ELISA results confirmed that both SCD REVs and heme mediated enhanced CS activation. The SCD-EV-BioChip assay demonstrated that the highest level of monocyte adhesion occurred in endothelial cells primed with 5 μM heme followed by REVs in NHS-containing medium (mean [SD] = 9813 [763]). This was significantly higher than heme followed by heme (1600 [231]) or the no-treatment control (213 [37]). Notably, heme priming followed by REV exposure also resulted in significantly greater adhesion than REVs followed by REVs (3285 [1052]). In contrast, adhesion values were significantly reduced in basal medium without NHS: REVs followed by REVs (1472 [111]), heme followed by REVs (1643 [296]), and heme followed by heme (448 [111]). The untreated control yielded 341 [37], showing no significant difference from the control group with NHS.
Together, these findings provide preliminary evidence REVs cause second hit to heme-primed endothelial cells in CS activation relevant manner. The enhanced monocyte adhesion observed in endothelial cells sequentially treated with heme followed by REVs, compared to sequential heme treatments alone, indicates that REVs carry additional pro-inflammatory factors beyond heme. However, the precise mechanisms remain unclear, necessitating further studies to identify and characterize these REV-associated components。
Conclusions: We used our SCD-EV-BioChip and developed a REV-mediated second hit model. Our results suggest that initial priming by heme sensitizes endothelial cells to subsequent REV-mediated CS activation. Furthermore, our findings suggest that REVs may contain additional pro-inflammatory components beyond heme that contribute to endothelial activation through CS activation. However, the exact mechanisms underlying this increased endothelial susceptibility remain unclear and warrant further investigation.
