Introduction: Endothelial dysfunction and microvascular injury are major drivers of organ failure in critical illness. Hemolytic conditions—seen in sepsis, trauma, sickle cell disease, and transfusion of stored red blood cells—release red cell–derived damage-associated molecular patterns (DAMPs), including cell-free hemoglobin (CFH) and free heme. These DAMPs promote oxidative injury, inflammation, and endothelial activation. While their effects on leukocyte and platelet adhesion are well described, their impact on red blood cell (RBC) adhesion to the endothelium is less understood. Enhanced RBC-endothelial interactions may contribute to microvascular occlusion, tissue ischemia, and organ dysfunction. We hypothesized that CFH and free heme increase RBC adhesion to human endothelial cells in a dose-dependent manner. Methods: Human aortic endothelial cells (HAECs) were exposed for 6 hours to CFH (10 or 20 µM), free heme (1–80 µM), lipopolysaccharide (LPS; 100 ng/mL, positive control), or vehicle (PBS). After washing, HAECs were incubated with isolated RBCs or fresh whole blood for 5 minutes. Adherent RBCs were imaged using EVOS M500 microscopy and quantified as RBCs per endothelial nucleus via MATLAB. Experiments were performed in triplicate; data were analyzed by ANOVA with Dunnett’s post-hoc test. Results: CFH and free heme significantly increased RBC adhesion compared to vehicle in a dose- dependent fashion. In the whole blood assay, vehicle-treated cells showed 1.1 ± 0.2 RBCs/nucleus. CFH at 10 µM and 20 µM increased adhesion to 4.5 ± 0.4 and 5.7 ± 0.3, respectively (p 10-fold increase. LPS increased adhesion (3.8 ± 0.3) but was less potent than CFH or high-dose heme. Similar trends were seen with isolated RBCs, suggesting direct RBC-endothelial interaction. Conclusions: Red cell–derived DAMPs, particularly CFH and free heme, markedly enhance RBC adhesion to human endothelial cells. These findings suggest a mechanistic link between hemolysis and microvascular dysfunction in critical illness. Therapeutic targeting of DAMP-induced endothelial activation may mitigate tissue hypoxia and organ injury in hemolytic disease states.
Lathker et al. (Sun,) studied this question.