Here we report on the development of a breakthrough microfluidic human in vitro cerebrovascular barrier (CVB) model featuring stem cell-derived brain-like endothelial cells (BLECs) and nanoporous ...silicon nitride (NPN) membranes (µSiM-CVB). The nanoscale thinness of NPN membranes combined with their high permeability and optical transparency makes them an ideal scaffold for the assembly of an in vitro microfluidic model of the blood–brain barrier (BBB) featuring cellular elements of the neurovascular unit (NVU). Dual-chamber devices divided by NPN membranes yield tight barrier properties in BLECs and allow an abluminal pericyte-co-culture to be replaced with pericyte-conditioned media. With the benefit of physiological flow and superior imaging quality, the µSiM-CVB platform captures each phase of the multi-step T-cell migration across the BBB in live cell imaging. The small volume of <100 µL of the µSiM-CVB will enable in vitro investigations of rare patient-derived immune cells with the human BBB. The µSiM-CVB is a breakthrough in vitro human BBB model to enable live and high-quality imaging of human immune cell interactions with the BBB under physiological flow. We expect it to become a valuable new tool for the study of cerebrovascular pathologies ranging from neuroinflammation to metastatic cancer.
Increase in blood-brain barrier (BBB) permeability is a crucial step in neuroinflammatory processes. We previously showed that N Methyl D Aspartate Receptor (NMDARs), expressed on cerebral ...endothelial cells forming the BBB, regulate immune cell infiltration across this barrier in the mouse. Here, we describe the mechanism responsible for the action of NMDARs on BBB permeabilization. We report that mouse CNS endothelial NMDARs display the regulatory GluN3A subunit. This composition confers to NMDARs' unconventional properties: these receptors do not induce Ca
influx but rather show nonionotropic properties. In inflammatory conditions, costimulation of human brain endothelial cells by NMDA agonists (NMDA or glycine) and the serine protease tissue plasminogen activator, previously shown to potentiate NMDAR activity, induces metabotropic signaling via the Rho/ROCK pathway. This pathway leads to an increase in permeability via phosphorylation of myosin light chain and subsequent shrinkage of human brain endothelial cells. Together, these data draw a link between NMDARs and the cytoskeleton in brain endothelial cells that regulates BBB permeability in inflammatory conditions.
The authors describe how NMDARs expressed on endothelial cells regulate blood-brain barrier function via myosin light chain phosphorylation and increase in permeability. They report that these non-neuronal NMDARs display distinct structural, functional, and pharmacological features than their neuronal counterparts.
Alzheimer’s disease (AD) is characterized by the abnormal accumulation of amyloid-β (Aβ) peptides in the brain. The pathological process has not yet been clarified, although dysfunctional transport ...of Aβ across the blood–brain barrier (BBB) appears to be integral to disease development. At present, no effective therapeutic treatment against AD exists, and the adoption of a ketogenic diet (KD) or ketone body (KB) supplements have been investigated as potential new therapeutic approaches. Despite experimental evidence supporting the hypothesis that KBs reduce the Aβ load in the AD brain, little information is available about the effect of KBs on BBB and their effect on Aβ transport. Therefore, we used a human in vitro BBB model, brain-like endothelial cells (BLECs), to investigate the effect of KBs on the BBB and on Aβ transport. Our results show that KBs do not modify BBB integrity and do not cause toxicity to BLECs. Furthermore, the presence of KBs in the culture media was combined with higher MCT1 and GLUT1 protein levels in BLECs. In addition, KBs significantly enhanced the protein levels of LRP1, P-gp, and PICALM, described to be involved in Aβ clearance. Finally, the combined use of KBs promotes Aβ efflux across the BBB. Inhibition experiments demonstrated the involvement of LRP1 and P-gp in the efflux. This work provides evidence that KBs promote Aβ clearance from the brain to blood in addition to exciting perspectives for studying the use of KBs in therapeutic approaches.
Accumulating evidence indicates that chronic exposure to a low level of pesticides found in diet affects the human gut-microbiota-blood-brain barrier (BBB) axis. This axis describes the physiological ...and bidirectional connection between the microbiota, the intestinal barrier (IB), and the BBB. Preclinical observations reported a gut microbial alteration induced by pesticides, also known as dysbiosis, a condition associated not only with gastrointestinal disorders but also with diseases affecting other distal organs, such as the BBB. However, the interplay between pesticides, microbiota, the IB, and the BBB is still not fully explored. In this review, we first consider the similarities/differences between these two physiological barriers and the different pathways that link the gut microbiota and the BBB to better understand the dialogue between bacteria and the brain. We then discuss the effects of chronic oral pesticide exposure on the gut-microbiota-BBB axis and raise awareness of the danger of chronic exposure, especially during the perinatal period (pregnant women and offspring).
Characterizing interaction of newly synthetized molecules with efflux pumps remains essential to improve their efficacy and safety. Caco-2 cell line cultivated on inserts is widely used for measuring ...apparent permeability of drugs across biological barriers, and for estimating their interaction with efflux pumps such as P-gp, BCRP and MRPs. However, this method remains time consuming and expensive. In addition, detection method is required for measuring molecule passage across cell monolayer and false results can be generated if drugs concentrations used are too high as demonstrated with quinidine. For this reason, we developed a new protocol based on the use of Caco-2 cell directly seeded on 96- or 384-well plates and the use of fluorescent substrates for efflux pumps. We clearly observed that the new method reduces costs for molecule screening and leads to higher throughput compared to traditional use of Caco-2 cell model. This accelerated model could provide quick feedback regarding the molecule design during the early stage of drug discovery and therefore reduce the number of compounds to be further evaluated using the traditional Caco-2 insert method.
Excessive inflammation might activate and injure the blood-brain barrier (BBB), a common feature of many central nervous system (CNS) disorders. We previously developed an in vitro BBB injury model ...in which the organophosphate paraoxon (PX) affects the BBB endothelium by attenuating junctional protein expression leading to weakened barrier integrity. The objective of this study was to investigate the inflammatory cellular response at the BBB to elucidate critical pathways that might lead to effective treatment in CNS pathologies in which the BBB is compromised. We hypothesized that caspase-1, a core component of the inflammasome complex, might have important role in BBB function since accumulating evidence indicates its involvement in brain inflammation and pathophysiology.
An in vitro human BBB model was employed to investigate BBB functions related to inflammation, primarily adhesion and transmigration of peripheral blood mononuclear cells (PBMCs). Caspase-1 pathway was studied by measurements of its activation state and its role in PBMCs adhesion, transmigration, and BBB permeability were investigated using the specific caspase-1 inhibitor, VX-765. Expression level of adhesion and junctional molecules and the secretion of pro-inflammatory cytokines were measured in vitro and in vivo at the BBB endothelium after exposure to PX. The potential repair effect of blocking caspase-1 and downstream molecules was evaluated by immunocytochemistry, ELISA, and Nanostring technology.
PX affected the BBB in vitro by elevating the expression of the adhesion molecules E-selectin and ICAM-1 leading to increased adhesion of PBMCs to endothelial monolayer, followed by elevated transendothelial-migration which was ICAM-1 and LFA-1 dependent. Blocking caspase-8 and 9 rescued the viability of the endothelial cells but not the elevated transmigration of PBMCs. Inhibition of caspase-1, on the other hand, robustly restored all of barrier insults tested including PBMCs adhesion and transmigration, permeability, and VE-cadherin protein levels. The in vitro inflammatory response induced by PX and the role of caspase-1 in BBB injury were corroborated in vivo in isolated blood vessels from hippocampi of mice exposed to PX and treated with VX-765.
These results shed light on the important role of caspase-1 in BBB insult in general and specifically in the inflamed endothelium, and suggest therapeutic potential for various CNS disorders, by targeting caspase-1 in the injured BBB.
The ABCB1 transporter also known as P-glycoprotein (P-gp) is a transmembrane protein belonging to the ATP binding cassette super-family of transporters; it is a xenobiotic efflux pump that limits ...intracellular drug accumulation by pumping the compounds out of cells. P-gp contributes to a decrease of toxicity and possesses broad substrate specificity. It is involved in the failure of numerous anticancer and antiviral chemotherapies due to the multidrug resistance (MDR) phenomenon, where it removes the chemotherapeutics out of the targeted cells. Understanding the details of the ligand-P-gp interaction is therefore crucial for the development of drugs that might overcome the MRD phenomenon and for obtaining a more effective prediction of the toxicity of certain compounds. In this work, an in silico modeling was performed using homology modeling and molecular docking methods with the aim of better understanding the ligand-P-gp interactions. Based on different mouse P-gp structural templates from the PDB repository, a 3D model of the human P-gp (
P-gp) was constructed by means of protein homology modeling. The homology model was then used to perform molecular docking calculations on a set of thirteen compounds, including some well-known compounds that interact with P-gp as substrates, inhibitors, or both. The sum of ranking differences (SRD) was employed for the comparison of the different scoring functions used in the docking calculations. A consensus-ranking scheme was employed for the selection of the top-ranked pose for each docked ligand. The docking results showed that a high number of π interactions, mainly π-sigma, π-alkyl, and π-π type of interactions, together with the simultaneous presence of hydrogen bond interactions contribute to the stability of the ligand-protein complex in the binding site. It was also observed that some interacting residues in
P-gp are the same when compared to those observed in a co-crystallized ligand (PBDE-100) with mouse P-gp (PDB ID: 4XWK). Our in silico approach is consistent with available experimental results regarding P-gp efflux transport assay; therefore it could be useful in the prediction of the role of new compounds in systemic toxicity.
The construction of the blood-brain barrier (BBB), which is a natural barrier for maintaining brain homeostasis, is the result of a meticulous organisation in space and time of cell-cell ...communication processes between the endothelial cells that carry the BBB phenotype, the brain pericytes, the glial cells (mainly the astrocytes), and the neurons. The importance of these communications for the establishment, maturation and maintenance of this unique phenotype had already been suggested in the pioneering work to identify and demonstrate the BBB. As for the history of the BBB, the evolution of analytical techniques has allowed knowledge to evolve on the cell-cell communication pathways involved, as well as on the role played by the cells constituting the neurovascular unit in the maintenance of the BBB phenotype, and more particularly the brain pericytes. This review summarises the key points of the history of the BBB, from its origin to the current knowledge of its physiology, as well as the cell-cell communication pathways identified so far during its development, maintenance, and pathophysiological alteration.
Neuroinflammation and brain lipid imbalances are observed in Alzheimer's disease (AD). Tumor necrosis factor-α (TNFα) and the liver X receptor (
) signaling pathways are involved in both processes. ...However, limited information is currently available regarding their relationships in human brain pericytes (HBP) of the neurovascular unit. In cultivated HBP, TNFα activates the
pathway and increases the expression of one of its target genes, the transporter ATP-binding cassette family A member 1 (
), while
is not expressed. Apolipoprotein E (
) synthesis and release are diminished. The cholesterol efflux is promoted, but is not inhibited, when
or
are blocked. Moreover, as for TNFα, direct
activation by the agonist (T0901317) increases
expression and the associated cholesterol efflux. However, this process is abolished when
/
are both inhibited. Neither the other ABC transporters nor the SR-BI are involved in this TNFα-mediated lipid efflux regulation. We also report that inflammation increases
expression and function. In conclusion, our data suggest that inflammation increases HBP protection against xenobiotics and triggers an
/
independent cholesterol release. Understanding the molecular mechanisms regulating this efflux at the level of the neurovascular unit remains fundamental to the characterization of links between neuroinflammation, cholesterol and HBP function in neurodegenerative disorders.
Synovitis is characterized by a distinctmetabolic profile featuring the accumulation of lactate, a byproduct of cellular metabolism within inflamed joints. This study reveals that the activation of ...the CD31 signal by lactate instigates a metabolic shift, specifically initiating endothelial cell autophagy. This adaptive process plays a pivotal role in fulfilling the augmented energy and biomolecule demands associated with the formation of new blood vessels in the synovium of Rheumatoid Arthritis (RA). Additionally, the amino acid substitutions in the CD31 cytoplasmic tail at the Y663F and Y686F sites of the immunoreceptor tyrosine-based inhibitory motifs (ITIM) alleviate RA. Mechanistically, this results in the downregulation of glycolysis and autophagy pathways. These findings significantly advance our understanding of potential therapeutic strategies for modulating these processes in synovitis and, potentially, other autoimmune diseases.
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