Neurolysin substrates bradykinin, neurotensin and substance P 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 several acute neurodegenerative disorders, including stroke. Numerous studies suggest the involvement of bradykinin (BK), neurotensin (NT) and substance P (SP) in BBB impairment and oedema formation after stroke; however, there is paucity of data in regard to the direct effects of these peptides on the brain microvascular endothelial cells (BMECs) and BBB. The present study aimed to evaluate the direct effects of BK, NT and SP on the 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. The combination of BK, NT and SP at a sub-effective concentration also resulted in increased BBB permeability in the iPSC-derived model indicating potentiation of their action. Furthermore, we observed abrogation of BK, NT and SP effects with pretreatment of pharmacological blockers targeting their specific receptors. Additional mechanistic studies indicate that the short-term effects of these peptides are not mediated through alteration of tight-junction proteins claudin-5 and occludin, but likely involve redistribution of F-actin and secretion of vascular endothelial growth factor. This is the first experimental study to document the increased permeability of the 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-recognised actions of BK on BBB. Lastly, we demonstrate that peptidase neurolysin can neutralise the effects of these peptides on BBB, suggesting potential therapeutic implications.

Discovery of First-in-Class Peptidomimetic Neurolysin Activators Possessing Enhanced Brain Penetration and Stability

“…In a reverse experiment, which utilized viral vector-driven overexpression of Nln in the brain, a substantial attenuation of stroke outcomes, including brain infarction, neuroinflammation, vascular permeability, brain edema, and neurological impairment, was documented in animals . Notably, the observed cerebroprotective role of Nln has been linked to its fundamental function as a peptidase, suggesting that in the poststroke brain, Nln inactivates several cerebrotoxic neuropeptides [bradykinin (BK), neurotensin (NT), substance P, and angiotensin II] and generates at least three [angiotensin-(1–7), Met-, and Leu-enkephalins] cerebroprotective neuropeptides. , …”

Potentials of Neuropeptides as Therapeutic Agents for Neurological Diseases

“…As previously mentioned, the regulation of acid-sensing ion channels (ASICs) is altered during ischaemic stroke, contributing to neuronal death, and has been shown to depend on neuropeptide function (nocistatin, big dynorphin, and RFamide). Additionally, the upregulation of substance P, bradykinin, and neurotensin have all recently been reported to worsen stroke pathology by increasing BBB permeability [ 217 ], although their control and neutralization via actions of neurolysin appears to reduce resultant oedema [ 218 ]. Finally, studies have reported that polymorphisms in the NPY gene promoter may increase risk of ischaemic stroke [ 219 , 220 , 221 ], and upregulation of the neuropeptide has been suggested to contribute towards mechanisms underlying haemorrhagic stroke onset [ 222 ], whilst lower levels have been linked with post-ischaemic stroke epilepsy [ 223 ].…”

Modeling the blood-brain barrier for treatment of central nervous system (CNS) diseases

“…It has been previously proven that there are three neuropeptides, bradykinin (BK), neurotensin (NT), and substrate P (SP), that cause barrier weakening in ischemic stroke patients and there is now evidence that all three peptides have an impact on the permeability of the BBB. 125 In this study, two separate monolayers were explored, the first being the hCMEC/D3 cell line and then human induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells, which were differentiated using conditioned media. Once properly cultured, both cell types were seeded on a TCPS and Transwell inserts which were coated with collagen and fibronectin to promote cell adhesion.…”