Abstract P808: The Main Peptide Ssubstrates of Neurolysin Enhance Brain Microvascular Permeability in a Human in vitro Model

Abstract

Increased brain microvascular permeability and disruption of blood-brain barrier (BBB) function are among hallmarks of ischemic stroke. Recently, peptidase neurlysin (Nln) has been identified as a compensatory and cerebroprotective mechanism in the post-stroke brain that functions to process a diverse group of extracellular neuropeptides, including bradykinin (BK), neurotensin (NT) and substance P (SP). A number of studies suggest involvement of BK, NT and SP in BBB impairment and edema formation after stroke, however there is paucity of data in regards to the direct effects of these peptides on the brain microvascular endothelial cells (BMECs) and BBB. The purpose of this study was to evaluate the direct effects of BK, NT and SP on permeability of BBB in an in vitro model based on human, induced pluripotent stem cell (iPSC)-derived BMECs. Our data indicate that all three peptides increase BBB permeability in a concentration-dependent manner in an in vitro model formed from two different iPSC lines (CTR90F and CTR65M) and widely used hCMEC/D3 human BMECs. Combination of BK, NT and SP at a sub-effective concentration also resulted in increased BBB permeability in the iPSC-derived model. Furthermore, we observed abrogation of BK, NT and SP effects with pretreatment of pharmacological blockers targeting their specific receptors or in presence of recombinant neurolysin (Nln). This is the first experimental study to document increased permeability of BBB in response to direct action of NT in an in vitro model. In addition, our study confirms the expected but not well-documented, direct effect of SP on BBB permeability and adds to the well-recognized actions of BK on BBB. Lastly, we demonstrate that peptidase Nln can neutralize the effects of these peptides on BBB, suggesting potential therapeutic implications.