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. 2021 Feb;33(2):e12931.
doi: 10.1111/jne.12931. Epub 2021 Jan 28.

Neurolysin substrates bradykinin, neurotensin and substance P enhance brain microvascular permeability in a human in vitro model

Abraham J Al-Ahmad  1 , Iqra Pervaiz  1 , Vardan T Karamyan  1
Affiliations

Neurolysin substrates bradykinin, neurotensin and substance P enhance brain microvascular permeability in a human in vitro model

Abraham J Al-Ahmad et al. J Neuroendocrinol. 2021 Feb.

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.

Keywords: blood-brain barrier; microvascular permeability; neuropeptide; oedema formation; peptidase neurolysin.

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Figures

FIGURE 1
FIGURE 1
Bradykinin (BK), neurotensin (NT) and substance P (SP) increase the barrier permeability in hCMEC/D3 and induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs). Changes in the barrier function were assessed in real-time in hCMEC/D3 and two iPSC-derived BMECs (CTR90F and CTR65M). Sampling for transendothelial electrical resistance (TEER) measurements (A) and paracellular permeability of fluorescein (B) were carried out every 15 minutes for 60 minutes. Baseline TEER was measured immediately before the start of treatments to normalise the experimental data. The apical compartment conditioned medium was replaced with fresh EC−/− containing nothing additional (control), 1 µmol L−1 BK (blue), 1 µmol L−1 NT (green) or 1 µmol L−1 SP (red) (n = 3 or 4 per group; *P < 0.05, **P < 0.01 compared to control group)
FIGURE 2
FIGURE 2
Induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) show a concentration-dependent response to bradykinin (BK), neurotensin (NT) and substance P (SP). Concentration-dependent (0.1, 0.5 and 1 µmol L−1) effect of BK (blue), NT (green) and SP (red) on transendothelial electrical resistance (TEER) values in CTR90F (A) and CTR65M (B) iPSC-BMECs, with effects being observed as early as 15 minutes after treatment. Difference in TEER values (ΔTEER) measured in CTR90F and CTR65M BMECs in response to different concentrations of the peptides (C). Fluorescein permeability measured in the iPSC-derived BMECs monolayers (D) (n = 9 for no peptide, ie ‘0’ group and 3–4 for all other groups; *P < 0.05, **P < 0.01 compared to no peptide group)
FIGURE 3
FIGURE 3
The effect of bradykinin (BK), neurotensin (NT) and substance P (SP) on tight junction complexes and actin cytoskeleton. Representative micrograph images of claudin-5 (A), occludin (B) and F-actin (C) localization in induced pluripotent stem cell-derived brain microvascular endothelial cells exposed to 1 µmol L−1 BK, NT or SP for 60 min. Note the subtle change in F-actin immunopositive staining and delocalisation from the cell border into the cytosol (asterisks). Scale bar = 20 µm
FIGURE 4
FIGURE 4
Bradykinin (BK), neurotensin (NT) and substance P (SP) induce secretion of vascular endothelial growth factor (VEGF) in brain microvascular endothelial cells (BMECs). The cell culture medium of induced pluripotent stem cell-derived BMECs exposed to 1 µmol L−1 BK, NT or SP for 60 min was used to measure the level of VEGF using an enzyme-linked immunosorbent assay (n = 6 per group; *P < 0.05, **P < 0.01 compared to control [ie, no peptide treatment] group)
FIGURE 5
FIGURE 5
The effects of bradykinin (BK), neurotensin (NT) and substance P (SP) on barrier function are mediated through their respective receptors. CTR90F (A) and CTR65M (B) brain microvascular endothelial cells were pre-treated with pharmacological antagonists of BK B1 (Des), B2 (Ica), NT NT1 (SR48692) or SP NK1 (Aprep or L735) receptor antagonists followed by evaluation of the effects of BK, NT and SP on fluorescein permeability (n = 3–4 per group; *P < 0.05, **P < 0.01; compared to the respective peptide treatment alone; Des, des-Arg9-[Leu8]-BK; Ica, icatibant acetate; Aprep, Aprepitant; L735, L-760735)
FIGURE 6
FIGURE 6
A combination of bradykinin (BK), neurotensin (NT) and substance P (SP) results in a potentiated effect. CTR90F (A) and CTR65M (B) brain microvascular endothelial cells (BMECs) were treated with a cocktail of BK, NT and SP at sub-effective concentrations (0.1 and 0.5 µmol L−1) followed by transendothelial electrical resistance (TEER) and fluorescein permeability measurements. To dissect the contribution of each peptide CTR90F (C) and CTR65M (D) BMECs were treated with a cocktail of BK + NT, NT + SP or BK + SP (each peptide at a concentration of 0.5 µmol L−1) followed by TEER and fluorescein permeability measurements (n = 9–13 for no peptide, ie ‘0’ group and 3–5 for all other groups; *P < 0.05, **P < 0.01 compared to no peptide treatment)
FIGURE 7
FIGURE 7
Human recombinant neurolysin has a negligible effect on barrier function. CTR90F and CTR65M brain microvascular endothelial cells were treated with different concentrations of recombinant neurolysin (rNln) (0.1–1 µg mL−1) followed by transendothelial electrical resistance (TEER) (A) and fluorescein permeability (B) measurements (n = 6–13 for no rNln, ie, ‘0’ group and 3–4 for all other groups; **P < 0.01 compared to no rNln group)
FIGURE 8
FIGURE 8
Human recombinant neurolysin neutralises the effects of bradykinin (BK), neurotensin (NT) and substance P (SP) on barrier function. The peptides were pre-treated with rNln (hashed bars) followed by evaluation of their effects on transendothelial electrical resistance (TEER) (A) and fluorescein permeability (B) in CTR90F and CTR65M brain microvascular endothelial cells (n = 14–17 for no peptide, ie, ‘0’ group and 3–6 for all other groups; *P < 0.05, **P < 0.01 compared to the respective peptide alone; ie, plain bar, group)
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References

    1. Benjamin EJ, Virani SS, Callaway CW, et al. Heart Disease and Stroke Statistics-2018 Update: A Report From the American Heart Association. Circulation. 2018;137:e67–e492. - PubMed
    1. Yang Y, Estrada EY, Thompson JF, Liu W, Rosenberg GA. Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat. J Cereb Blood Flow Metab. 2007;27:697–709. - PubMed
    1. van Bruggen N, Thibodeaux H, Palmer JT, et al. VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain. J Clin Invest. 1999;104:1613–1620. - PMC - PubMed
    1. Zhang ZG, Zhang L, Jiang Q, et al. VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest. 2000;106:829–838. - PMC - PubMed
    1. Roberts J, de Hoog L, Bix GJ. Mice deficient in endothelial alpha5 integrin are profoundly resistant to experimental ischemic stroke. J Cereb Blood Flow Metab. 2017;37:85–96. - PMC - PubMed

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