Neurolysin and its extracellular substrates: A novel mechanism to modulate blood-brain barrier (BBB) permeability and consequences of ischemic stroke

Abstract ID 98637 Poster Board 329 Stroke is currently one of the leading cause of death and long-term disability with an unmet need for new treatments. Despite advances that have been made in research of understanding the mechanisms and pathophysiology of stroke and coming up with new treatment interventions in preclinical stage, there is a gap in successful implementation of those findings in clinical setting. Therefore, the perspective of research in this field is being shifted to study and understand more about the cerebroprotective mechanisms of the brain itself in case of an ischemic event. In this respect, the functions of the neuropeptides are of particular interest as they have been found to play diverse roles in maintaining the homeostasis of the central nervous system. Limited research showed that ischemic attack leads to upregulation of neurolysin, a brain endopeptidase, and enables it to act in a manner of providing neuroprotection by hydrolyzing the neuropeptides that are cerebrotoxic in nature. Amongst many substrates of neurolysin, the major substrates that are of interest in light of ischemic stroke are vasoactive peptides bradykinin, neurotensin and substance P that are shown to modulate blood-brain barrier (BBB) permeability. The objective of this study is to observe the effects of the neurolysin substrates bradykinin, neurotensin and substance P in an induced pluripotent stem cell (iPSC) derived in-vitro model of BBB exposed to ischemic stress via oxygen glucose deprivation (OGD). The study is designed by differentiating iPSCs to obtain human brain microvascular endothelial cells (BMECs), the primary constituent of BBB and then exposing the BMECs to OGD (1% O₂, 5% CO₂, 37°C, 6 hours in glucose free DMEM) to mimic acute cerebral ischemic stress followed by treatment with 1μM of the three neuropeptides for 1 hour. After treatments, the barrier function (TEER) and paracellular permeability (fluorescein diffusion) of the BMECs monolayer were assessed along with mechanistic study of the tight junction proteins (claudin-5, occludin, ZO-1) and actin cytoskeleton distribution. The OGD stress damaged the BMECs monolayer shown by decreased cell viability, decreased TEER, and increased fluorescein permeability which is further worsened by the neuropeptide treatments (Substance P treatment resulting in highest decrease of 93% in TEER and bradykinin treatment leading to highest increase of 75% in fluorescein clearance per minute when compared to control OGD BMECs with no neuropeptide treatments). While tight junction proteins showed minimal morphological changes, the neuropeptide treatments led to formation of actin stress fibers. The primary findings of the study suggest that bradykinin, neurotensin and substance P have detrimental effects on the bio-properties of the BMECs constituting the BBB in an ischemic setting. The study is in process of further investigation into how neurolysin will come in action and play a neuroprotective role following acute and chronic ischemic stress in BMECs along with other major brain cells like astrocytes and neurons.