Insights Into the Mechanisms of Brain Endothelial Erythrophagocytosis

Abstract

The endothelial cells which form the inner cellular lining of the vasculature can act as non-professional phagocytes to ingest and remove emboli and aged/injured red blood cells (RBCs) from circulation. We previously demonstrated an erythrophagocytic phenotype of the brain endothelium for oxidatively stressed RBCs with subsequent migration of iron-rich RBCs and RBC degradation products across the brain endothelium in vivo and in vitro, in the absence of brain endothelium disruption. However, the mechanisms contributing to brain endothelial erythrophagocytosis are not well defined, and herein we elucidate the cellular mechanisms underlying brain endothelial erythrophagocytosis. Murine brain microvascular endothelial cells (bEnd.3 cells) were incubated with tert-butyl hydroperoxide (tBHP, oxidative stressor to induce RBC aging in vitro)- or PBS (control)-treated mouse RBCs. tBHP increased the reactive oxygen species (ROS) formation and phosphatidylserine exposure in RBCs, which were associated with robust brain endothelial erythrophagocytosis. TNFα treatment potentiated the brain endothelial erythrophagocytosis of tBHP-RBCs in vitro. Brain endothelial erythrophagocytosis was significantly reduced by RBC phosphatidylserine cloaking with annexin-V and with RBC-ROS and phosphatidylserine reduction with vitamin C. Brain endothelial erythrophagocytosis did not alter the bEnd.3 viability, and tBHP-RBCs were localized with early and late endosomes. Brain endothelial erythrophagocytosis increased the bEnd.3 total iron pool, abluminal iron levels without causing brain endothelial monolayer disruption, and ferroportin levels. In vivo, intravenous tBHP-RBC injection in aged (17–18 months old) male C57BL/6 mice significantly increased the Prussian blue-positive iron-rich lesion load compared with PBS-RBC-injected mice. In conclusion, RBC phosphatidylserine exposure and ROS are key mediators of brain endothelial erythrophagocytosis, a process which is associated with increased abluminal iron in vitro. tBHP-RBCs result in Prussian blue-positive iron-rich lesions in vivo. Brain endothelial erythrophagocytosis may provide a new route for RBC/RBC degradation product entry into the brain to produce iron-rich cerebral microhemorrhage-like lesions.

Comparative studies between the murine immortalized brain endothelial cell line (bEnd.3) and induced pluripotent stem cell-derived human brain endothelial cells for paracellular transport

“…All the studies demonstrating the phagocytic phenotype of the vascular endothelium were done using immortalized cells lines or in vivo rodent models. Our previous work, along with other studies, shows that the bEnd.3 cells [ 27 , 32 ] and HCMEC/D3 human brain microvascular endothelial cells [ 28 ] exhibit an erythrophagocytic phenotype for aged/damaged red blood cells. Since the red blood cell adhesion and phagocytic repertoire of the ihBMECs had not been reported, we determined the erythrophagocytic phenotype of the ihBMECs.…”

“…In accordance with the reported phagocytic phenotype of vascular endothelial cells, including the brain microvascular endothelial cells, our results show that ihBMECs also demonstrate cell adhesion and phagocytic characteristics ( Fig 4 ). This is the first study to show that the ihBMECs recapitulate the erythrophagocytic phenotype shown by the bEnd.3 cells [ 27 , 32 ] and human brain microvascular cells [ 28 ], making them a suitable in vitro model to study brain microvascular endothelial cell-red blood cell interactions.…”

“…However, despite the many-fold higher TEER measurements across the ihBMECs, the bEnd.3 monolayer restricted > 80% passage of free FITC-dextran. Our results, therefore, suggest that the bEnd.3 cells may be a suitable model for transport studies of large molecules and for cell interaction and migration studies despite their low TEER [ 27 ]. Modification of the culture conditions, e.g., serum-free conditions and co-cultures, can be adapted to further enhance bEnd.3 barrier properties [ 42 ].…”

“…At 24 h after the re-plating, ihBMECs were cultured in hESFM with 1X B27 for culture and replaced with fresh medium daily. For initial experiments, monolayers growing on Transwell inserts were stained with hematoxylin and eosin (H&E), as described previously [ 27 , 32 ], to confirm monolayer formation on the Transwell inserts.…”

“…Red blood cells were treated either with sterile phosphate-buffered saline (PBS, Invitrogen, Waltham, MA, United States) as control or 3 mM tert-butylhydroperoxide (tBHP, Sigma-Aldrich, St. Louis, MO, United States) at 37°C for 30 min. tBHP is an oxidative stressor used to expose phosphatidylserine on the outer leaflet of the red blood cell membrane to mimic aged red blood cells in vitro [ 27 ]. PBS- and tBHP-treated red blood cells (5 x 10 6 ) were incubated with ihBMECs seeded onto collagen and fibronectin-coated coverslips in a 24-well plate for 48 h. Cells were stained with H&E to detect attachment and engulfment of red blood cells by ihBMECs as described previously [ 27 ], and H&E-stained slides were imaged using a confocal microscope to confirm red blood cell internalization.…”