S266: different molecular vaso-occlusive triggers stimulate distinct vaso-occlusive profiles: in vivo results from mice with sickle cell anemia

Background: Microvascular vaso-occlusion (VO) characterizes sickle cell anemia (SCA) pathophysiology and is the result of inflammatory mechanisms that promote interactions of leukocytes and erythrocytes with the endothelium, with consequent obstruction of blood flow. However, a number of events can induce VO and it is unclear whether these triggers drive different pathways of VO. Aims: This study aims to determine whether different vaso-occlusive stimuli provoke similar or distinct mechanisms of cellular and inflammatory interactions in the vasculature of a mouse model of SCA. Methods: Male Townes mice, aged 20 weeks, were used to determine whether different stimuli trigger distinct vaso-occlusive mechanisms. Mice were randomly distributed into 5 groups, according to stimuli: Vehicle (equivalent times for each stimulus, total n = 20); tumor necrosis factor-α (TNF; proinflammatory cytokine) (0.5 µg, i.p., 3h, n = 5); lipopolysaccharide (LPS; bacterial endotoxin) (2 mg/kg, i.p., 2h, n = 5); hemin (DAMP released during hemolysis) (32 µmol/kg, i.v., 1h, n = 4) and PB1-F2 (Influenza A virus protein) (1 nmol, i.n., 3h, n = 6). Optimal dosages and administration times were previously determined in C57BL/6 mice (data not shown). After molecular trigger administration, the animals were anesthetized and submitted to laser Doppler perfusion monitoring (LDPM) of the of pelvis skin, to determine microvascular perfusion and blood flow. Cremaster muscle exteriorization was also performed for analysis of the microvasculature by intravital microscopy (8 microvessels per mouse were filmed during 1 min each and analyzed for leukocyte rolling, adhesion and extravasation). Blood samples from each animal were also collected to monitor the formation of neutrophil-platelet aggregates (NPA; CD45+/Ly6G+/CD41+) by flow cytometry. All animal procedures were carried out with approval of the local Commission for Ethics in Animal Experimentation (University of Campinas; protocols 5790-1/6078-1). Results: LDPM showed that all four stimuli triggered VO in the microcirculation of the pelvic region, significantly decreasing skin blood perfusion and flow, compared to the Vehicle groups (Fig.1A, 1B). Intravital microscopy demonstrated that TNF, Hemin and PB1-F2 all accelerate the rolling of leukocytes along cremaster venule walls compared to Vehicle, while LPS does not (Fig.1C). In contrast, while the TNF and hemin both induced increased venular leukocyte adhesion, the adhesion incurred by LPS was more evident (Fig.1D). Increased leukocyte extravasation to local tissue was observed only for the TNF and LPS stimuli, compared to Vehicle (Fig.1E). Similarly, NPA in peripheral blood were elevated only by TNF and LPS, compared to Vehicle, and not by hemin nor PB1-F2 (Fig.1F). Summary/Conclusion: These data suggest that the four molecular stimuli all induced the VO process in SCD mice, decreasing blood perfusion and flow velocity. However, different cellular interactions triggered this VO, with the participation of increased leukocyte adhesion to venule walls and of NPA formation in TNF and LPS-triggered VO, in contrast to a more prominent role for leukocyte rolling in Hemin and PB1-F2-induced VO. Preliminary experiments using flow adhesion with blood from SCA patients confirm that different cellular and molecular mechanisms are involved in the vaso-occlusive process, depending on the trigger. These results suggest that different therapeutic approaches for reversing VO could be warranted in SCD, depending on the triggering event. Grants #2021/11851-8, #2019/18886-1, FAPESP.Keywords: Sickle cell adhesion, Sickle cell disease