Abstract TP478: Mechanisms of Brain Endothelial Erythrophagocytosis: Insights Into the Pathogenesis of Cerebral Microbleeds

Abstract

Background: Cerebral microbleeds, MRI signatures of cerebral microhemorrhage, are thought to arise from brain microvessel disruption. Our prior work suggests an alternate origin for some microbleeds, emphasizing brain endothelial erythrophagocytosis (BEE) producing microhemorrhage-like lesions (“pseudo-microbleeds”) in absence of vascular disruption (Frontiers Cell Neurosci 9-18; PMID: 30237761). We demonstrated an erythrophagocytic phenotype of brain microvascular endothelium for oxidatively-stressed red blood cells (RBC), and migration of iron-rich hemoglobin across the brain endothelium. However, cellular mechanisms underlying BEE and hemoglobin passage across brain endothelium are not defined. Herein we investigated a) role of oxidative stress and RBC phosphatidylserine (PS) exposure in BEE and b) intracellular endosomal trafficking of RBC within brain endothelial cells. Methods: Murine brain endothelial cells (bEnd.3 cells) were incubated with 2x10 6 mouse RBC treated with 3mM tert-butylhydroperoxide (t-BHP, an oxidative stressor) or sterile PBS (control) for 48h at 37°C, in the presence of 7.5 μg annexin V to cloak PS, or vitamin C (15μM-1500μM) to reduce reactive oxygen species (ROS). BEE was evaluated by hematoxylin & eosin stain and diaminofluorene assay for hemoglobin. RBC ROS levels, PS exposure, and cell viability were measured. bEnd.3 cells were immunostained to visualize RBC in early and late endosomes, the latter being the presumed site of RBC degradation post-erythrophagocytosis. Results: tBHP induced both ROS production and PS exposure in RBC. There was a 9-fold increase (p<0.001) in BEE of t-BHP-RBC compared with control. Vitamin C reduced RBC ROS levels (70%, p<0.001) and PS exposure (27%, p<0.001), while annexin V blocked RBC PS exposure (65%, p<0.001). BEE was significantly attenuated by annexin V (63%, p<0.001) and vitamin C (39%, p<0.001). No change in bEnd.3 viability was observed and t-BHP-RBC localized to both early and late endosomes. Conclusions: These data demonstrate the importance of RBC PS exposure in BEE, and describe the intracellular trafficking of RBC in brain endothelial cells. These findings provide insights into the development of microhemorrhage-like lesions and cerebral microbleeds.