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.
Glucose is an important source of energy for the central nervous system. Its uptake at the blood-brain barrier (BBB) is mostly mediated via glucose transporter 1 (GLUT1), a facilitated transporter encoded by the SLC2A1 gene. GLUT1 Deficiency Syndrome (GLUT1DS) is a haploinsufficiency characterized by mutations in the SLC2A1 gene, resulting in impaired glucose uptake at the BBB and clinically characterized by epileptic seizures and movement disorder. A major limitation is an absence of in vitro models of the BBB reproducing the disease. This study aimed to characterize an in vitro model of GLUT1DS using human pluripotent stem cells (iPSCs). Two GLUT1DS clones were generated (GLUT1-iPSC) from their original parental clone iPS(IMR90)-c4 by CRISPR/ Cas9 and differentiated into brain microvascular endothelial cells (iBMECs). Cells were characterized in terms of SLC2A1 expression, changes in the barrier function, glucose uptake and metabolism, and angiogenesis. GLUT1DS iPSCs and iBMECs showed comparable phenotype to their parental control, with exception of reduced GLUT1 expression at the protein level. Although no major disruption in the barrier function was reported in the two clones, a significant reduction in glucose uptake accompanied by an increase in glycolysis and mitochondrial respiration was reported in both GLUT1DS-iBMECs. Finally, impaired angiogenic features were reported in such clones compared to the parental clone. Our study provides the first documented characterization of GLUT1DS-iBMECs generated by CRISPR-Cas9, suggesting that GLUT1 truncation appears detrimental to brain angiogenesis and brain endothelial bioenergetics, but maybe not be detrimental to iBMECs differentiation and barriergenesis. Our future direction is to further characterize the functional outcome of such truncated product, as well as its impact on other cells of the neurovascular unit.
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