The role of brain endothelial cells (ECs) and in particular the role of cilium, a microtubule flow sensor organelle expressed on the apical surface of ECs and facing the lumen has been poorly studied in the context of blood- brain-barrier (BBB) function. ECs and in turn cilia are the first line of contact with red blood cells (RBCs) in the blood. Studies from us and others suggest that cilia on ECs are critical for flow-mediated brain vessel stability. Thus, how flow relays signals to ECs via cilia and in turn to cells of the neurovascular (NVU) unit is not known. Without this knowledge, our ability to generate BBB models that mimic in vivo conditions will be hampered. The question of EC-cilia and its role in BBB function is clinically relevant given that patients with sickle cell disease (SCD) are predisposed to both overt and silent cerebral infarct, caused by sickle RBCs adhesion to the endothelium. The adhesion of sickle RBCs to endothelium may facilitate the physical removal of cilia (deciliation) from ECs surface, a mechanism recently identified in mammalian cilia shedding. In this multi-PI proposal, investigative team in vascular biology, sickle cell biology, human induced pluripotent stem cell (iPSC)-derived BBB and NVU models, cilia biology and rodent injury models will investigate the overarching hypothesis that disturbed cerebral blood flow triggers deciliation and release of cilia into the blood, thus resulting in aberrant signaling in the deciliated ECs and in the surrounding cells that comprise the NVU resulting in impact on NVU and BBB (Fig. 1). The objective of this proposal is to study RBCs-EC-cilia interaction and its importance to BBB model development. In the R61 phase, we will test whether RBCs from sickle cell disease (mouse models & human patients) will directly or indirectly trigger cilia shedding in brain ECs in vitro and in vivo. We will identify proteins in the cilia shed fragments, and also assess the importance of EC-cilia on BBB phenotypes in vitro and in vivo with emphasis on BBB integrity. Upon successfully establishing the milestone that RBC-EC-cilia interaction is critical for BBB function in the R61 phase, a go-decision in the R33 phase will initiate deeper probe into the underlying mechanisms and the role of the RBC-EC cilia interaction in SCD and traumatic brain injury (TBI) rodent models in vivo. The significance of this project is that EC-cilia status is an important determinant when brain ECs are included in flow-mediated BBB model development in vitro. The innovation is that until now, EC-cilia has been largely ignored in BBB protocols and accomplishing the objectives of this proposal will move the status quo in this field. This proposal will also bring us one step closer to monitoring cerebral vessel impairment using RBC-triggered EC-cilia shedding as a biomarker of vascular health, a NIH mission-related topic of research.
The proposed research is relevant to public health and RFA-HL-20-021 because it identifies blood-derived factors that influence the blood-brain-barrier (BBB) integrity. In this multi-PI application, we determine the mechanisms by which RBCs influence the removal of cilia (deciliation), a cellular organelle projecting from the endothelial cell surface into the CNS blood, which impacts BBB integrity in vitro and in vivo. The proposed research is directly relevant to NIH?s mission of employing preventive measures to block BBB leakage and integrity, and development of better fluid-based BBB models in vitro.
