Abstract Topic: 26. Sickle cell disease
Background: Acute vaso-occlusive crises (VOCs) are the leading cause of hospitalization for patients with sickle cell disease (SCD). VOCs are characterized by microvascular occlusion that is initiated and progressed by adhesion of sickle red blood cells (RBCs), neutrophils and platelets to the vascular endothelium. Epeleuton is 15- hydroxy eicosapentaenoic acid (15(S)-HEPE) ethyl ester, a novel synthetic second generation ω-3 fatty acid which targets key drivers of multicellular adhesion. In Townes humanized SCD mice, epeleuton acts as a disease-modifying agent, decreasing (a) systemic and local inflammatory activation, (b) vascular dysfunction including adhesion molecule expression, (c) organ damage and (d) intravascular hemolysis and sickling.
Aims: To evaluate the effect of 15(S)-HEPE, the active moiety of epeleuton, on SCD patient RBCs’ adhesion to heme-activated endothelium using a novel endothelialized microfluidic system.
Methods: Whole blood samples were collected from 7 SCD subjects with HbSS genotype at University Hospitals Cleveland Medical Center, Cleveland, OH, USA. RBCs were isolated after centrifugation of whole blood collected in EDTA and were resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) with 10 mM of HEPES to reach 20% hematocrit. Human umbilical vein endothelial cells (HUVECs; Lonza, Morristown, NJ, USA) were seeded within the microfluidic channels and cultured for at least 72 hours at 15 dyne/cm2 prior to experiments. Tested concentrations of 15(S)-HEPE were selected based on clinical pharmacokinetics for 15(S)-HEPE at 2g/day and 4g/day epeleuton doses. Incubation of RBCs or HUVECs with 15(S)-HEPE was conducted at 37°C for 6 hours. In one experiment 15(S)-HEPE-treated tor vehicle-treated RBCs flowed on untreated HUVECs and in the second experiment untreated RBCs flowed on 15(S)-HEPE-treated or vehicle-treated RBCs. In both cases a short-term acute activation was conducted where RBCs were supplemented with 40 µM heme and injected through the microfluidic channels for 15 minutes. Non-adherent RBCs were washed with basal cell culture medium. Phase-contrast images of the remaining RBCs were recorded using inverted microscope (DMi8 Leica Microsystems Inc. Deerfield, IL, USA) and the images were analyzed based on previous published methods. Statistical significance was assessed using paired t-test.
Results: In a standardized endothelium-on-a-chip microfluidic platform treatment of both RBCs or endothelium with 15(S)-HEPE decreased RBC adhesion to heme-activated endothelium under physiological flow conditions. Treatment of heme-activated HUVECs with 15(S)-HEPE significantly decreased adhesion of SCD patient RBCs to endothelium at both 50µM (p=0.03) and 100 µM concentrations (p=0.01). Similarly, treatment of RBCs with 15(S)-HEPE significantly decreased their adhesion to heme-activated endothelium at both 50µM (p=0.01) and 100 µM concentrations (p=0.001).
Summary/Conclusion: Under physiological flow conditions, 15(S)-HEPE decreased adhesion of SCD patient RBCs at clinically relevant concentrations by affecting both the endothelium and RBCs independently. Assessment of 15(S)-HEPE’s effects on whole blood adhesion and pre-treatment of both blood and endothelium in the same experiment may allow a more complete assessment of 15(S)-HEPE’s effects on multicellular adhesion.
These findings support the clinical development of epeleuton as a potential novel therapeutic option for patients with SCD.
Keywords: Sickle cell disease, Vasoocclusive crisis, Inflammation, Sickle cell adhesion