Synergistic induction of blood-brain barrier properties.

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell-derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/ß-catenin signaling and inhibition of the TGF-ß pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/ß-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.

Role of interleukin-6 and interleukin-10 in morphological and functional changes of the blood-brain barrier in hypertriglyceridemia.

BACKGROUND: Hypertriglyceridemia is closely linked to atherosclerosis related inflammatory processes and blood-brain barrier (BBB) dysfunction. Using apolipoprotein B-100 (APOB-100) transgenic mice, an animal model of chronic hypertriglyceridemia, we analyzed BBB function and morphology in vitro and ex vivo. Our objective was to determine which BBB characteristics are produced mainly by interleukin (IL)-6, an atherosclerosis promoting cytokine, and whether these actions can be antagonized by IL-10, an anti-inflammatory cytokine. METHODS: Brain endothelial and glial cell cultures and brain microvessels were isolated from wild type (WT) and APOB-100 transgenic mice and were treated with IL-6, IL-10 and their combination. First, IL-6 and IL-10 production was measured in WT and APOB-100 microvessels using qPCR. Then functional parameters of endothelial cell cultures were analyzed and immunocytochemistry for key BBB proteins was performed. RESULTS: IL-6 mRNA levels were higher in brain microvessels than in brain parenchyma of APOB-100 transgenic mice. Transendothelial electric resistance and P-glycoprotein activity were lower, and paracellular permeability was higher in cultured APOB-100 brain endothelial cells. These features were sensitive to both IL-6 and IL-10 treatments. A decreased P-glycoprotein immunostaining was measured in transgenic endothelial cells under control conditions and in WT cells after treating them with IL-6. This effect was antagonized by IL-10. Changes in immunostaining for tight junction proteins were observed after IL-6 exposure, which were in part antagonized by IL-10. In glial cell cultures an increase in aquaporin-4 immunolabeling in the transgenic group and an increase in microglia cell density in WT glia cultures was detected after IL-6 treatment, which was antagonized by IL-10. In isolated brain microvessels a decrease in P-glycoprotein immunolabeled area fraction was measured in APOB-100 microvessels under control conditions and in WT microvessels after every cytokine treatment. ZO-1 immunolabeling showed characteristics similar to that of P-glycoprotein. No change was seen in claudin-5 and occludin immunoreactive area fractions in microvessels. A decrease in aquaporin-4 immunoreactivity was measured in WT microvessels treated by IL-6, which was antagonized by IL-10. CONCLUSION: IL-6 produced in microvessels contributes to BBB impairment observed in the APOB-100 mice. We showed that IL-10 partly antagonizes the effects of IL-6 at the BBB.

Targeting Human Endothelial Cells with Glutathione and Alanine Increases the Crossing of a Polypeptide Nanocarrier through a Blood-Brain Barrier Model and Entry to Human Brain Organoids.

Nanoparticles (NPs) are the focus of research efforts that aim to develop successful drug delivery systems for the brain. Polypeptide nanocarriers are versatile platforms and combine high functionality with good biocompatibility and biodegradability. The key to the efficient brain delivery of NPs is the specific targeting of cerebral endothelial cells that form the blood-brain barrier (BBB). We have previously discovered that the combination of two different ligands of BBB nutrient transporters, alanine and glutathione, increases the permeability of vesicular NPs across the BBB. Our aim here was to investigate whether the combination of these molecules can also promote the efficient transfer of 3-armed poly(l-glutamic acid) NPs across a human endothelial cell and brain pericyte BBB co-culture model. Alanine and glutathione dual-targeted polypeptide NPs showed good cytocompatibility and elevated cellular uptake in a time-dependent and active manner. Targeted NPs had a higher permeability across the BBB model and could subsequently enter midbrain-like organoids derived from healthy and Parkinson's disease patient-specific stem cells. These results indicate that poly(l-glutamic acid) NPs can be used as nanocarriers for nervous system application and that the right combination of molecules that target cerebral endothelial cells, in this case alanine and glutathione, can facilitate drug delivery to the brain.

Mycobacterium bovis Bacillus Calmette-Guérin-Infected Dendritic Cells Induce TNF-α-Dependent Cell Cluster Formation That Promotes Bacterial Dissemination through an In Vitro Model of the Blood-Brain Barrier.

CNS tuberculosis (CNSTB) is the most severe manifestation of extrapulmonary tuberculosis infection, but the mechanism of how mycobacteria cross the blood-brain barrier (BBB) is not well understood. In this study, we report a novel murine in vitro BBB model combining primary brain endothelial cells, Mycobacterium bovis bacillus Calmette-Guérin-infected dendritic cells (DCs), PBMCs, and bacterial Ag-specific CD4+ T cells. We show that mycobacterial infection limits DC mobility and also induces cellular cluster formation that has a similar composition to pulmonary mycobacterial granulomas. Within the clusters, infection from DCs disseminates to the recruited monocytes, promoting bacterial expansion. Mycobacterium-induced in vitro granulomas have been described previously, but this report shows that they can form on brain endothelial cell monolayers. Cellular cluster formation leads to cluster-associated damage of the endothelial cell monolayer defined by mitochondrial stress, disorganization of the tight junction proteins ZO-1 and claudin-5, upregulation of the adhesion molecules VCAM-1 and ICAM-1, and increased transmigration of bacteria-infected cells across the BBB. TNF-α inhibition reduces cluster formation on brain endothelial cells and mitigates cluster-associated damage. These data describe a model of bacterial dissemination across the BBB shedding light on a mechanism that might contribute to CNS tuberculosis infection and facilitate treatments.

Effect of Lipopolysaccharide-Induced Inflammatory Challenge on ß-Glucuronidase Activity and the Concentration of Quercetin and Its Metabolites in the Choroid Plexus, Blood Plasma and Cerebrospinal Fluid.

Quercetin-3-glucuronide (Q3GA), the main phase II metabolite of quercetin (Q) in human plasma, is considered to be a more stable form of Q for transport with the bloodstream to tissues, where it can be potentially deconjugated by ß-glucuronidase (ß-Gluc) to Q aglycone, which easily enters the brain. This study evaluates the effect of lipopolysaccharide (LPS)-induced acute inflammation on ß-Gluc gene expression in the choroid plexus (ChP) and its activity in blood plasma, ChP and cerebrospinal fluid (CSF), and the concentration of Q and its phase II metabolites in blood plasma and CSF. Studies were performed on saline- and LPS-treated adult ewes (n = 40) receiving Q3GA intravenously (n = 16) and on primary rat ChP epithelial cells and human ChP epithelial papilloma cells. We observed that acute inflammation stimulated ß-Gluc activity in the ChP and blood plasma, but not in ChP epithelial cells and CSF, and did not affect Q and its phase II metabolite concentrations in plasma and CSF, except Q3GA, for which the plasma concentration was higher 30 min after administration (p < 0.05) in LPS- compared to saline-treated ewes. The lack of Q3GA deconjugation in the ChP observed under physiological and acute inflammatory conditions, however, does not exclude its possible role in the course of neurodegenerative diseases.

Flow induces barrier and glycocalyx-related genes and negative surface charge in a lab-on-a-chip human blood-brain barrier model.

Microfluidic lab-on-a-chip (LOC) devices allow the study of blood-brain barrier (BBB) properties in dynamic conditions. We studied a BBB model, consisting of human endothelial cells derived from hematopoietic stem cells in co-culture with brain pericytes, in an LOC device to study fluid flow in the regulation of endothelial, BBB and glycocalyx-related genes and surface charge. The highly negatively charged endothelial surface glycocalyx functions as mechano-sensor detecting shear forces generated by blood flow on the luminal side of brain endothelial cells and contributes to the physical barrier of the BBB. Despite the importance of glycocalyx in the regulation of BBB permeability in physiological conditions and in diseases, the underlying mechanisms remained unclear. The MACE-seq gene expression profiling analysis showed differentially expressed endothelial, BBB and glycocalyx core protein genes after fluid flow, as well as enriched pathways for the extracellular matrix molecules. We observed increased barrier properties, a higher intensity glycocalyx staining and a more negative surface charge of human brain-like endothelial cells (BLECs) in dynamic conditions. Our work is the first study to provide data on BBB properties and glycocalyx of BLECs in an LOC device under dynamic conditions and confirms the importance of fluid flow for BBB culture models.

Glycocalyx regulates the strength and kinetics of cancer cell adhesion revealed by biophysical models based on high resolution label-free optical data.

The glycocalyx is thought to perform a potent, but not yet defined function in cellular adhesion and signaling. Since 95% of cancer cells have altered glycocalyx structure, this role can be especially important in cancer development and metastasis. The glycocalyx layer of cancer cells directly influences cancer progression, involving the complicated kinetic process of cellular adhesion at various levels. In the present work, we investigated the effect of enzymatic digestion of specific glycocalyx components on cancer cell adhesion to RGD (arginine-glycine-aspartic acid) peptide motif displaying surfaces. High resolution kinetic data of cell adhesion was recorded by the surface sensitive label-free resonant waveguide grating (RWG) biosensor, supported by fluorescent staining of the cells and cell surface charge measurements. We found that intense removal of chondroitin sulfate (CS) and dermatan sulfate chains by chondroitinase ABC reduced the speed and decreased the strength of adhesion of HeLa cells. In contrast, mild digestion of glycocalyx resulted in faster and stronger adhesion. Control experiments on a healthy and another cancer cell line were also conducted, and the discrepancies were analysed. We developed a biophysical model which was fitted to the kinetic data of HeLa cells. Our analysis suggests that the rate of integrin receptor transport to the adhesion zone and integrin-RGD binding is strongly influenced by the presence of glycocalyx components, but the integrin-RGD dissociation is not. Moreover, based on the kinetic data we calculated the dependence of the dissociation constant of integrin-RGD binding on the enzyme concentration. We also determined the dissociation constant using a 2D receptor binding model based on saturation level static data recorded at surfaces with tuned RGD densities. We analyzed the discrepancies of the kinetic and static dissociation constants, further illuminating the role of cancer cell glycocalyx during the adhesion process. Altogether, our experimental results and modelling demonstrated that the chondroitin sulfate and dermatan sulfate chains of glycocalyx have an important regulatory function during the cellular adhesion process, mainly controlling the kinetics of integrin transport and integrin assembly into mature adhesion sites. Our results potentially open the way for novel type of cancer treatments affecting these regulatory mechanisms of cellular glycocalyx.

A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood-Brain Barrier Mouse Model.

In vitro culture models of the blood-brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to test Mycobacterium tuberculosis (Mtb) dissemination across brain endothelial cells. One-third of the global population is infected with Mtb, and in 1%-2% of cases bacteria invade the CNS through a largely unknown process. The "Trojan horse" theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB-granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of primary mouse brain astrocytes and endothelial cells Basic Protocol 2: Isolation of primary mouse bone marrow-derived dendritic cells Support Protocol 1: Validation of dendritic cell purity by flow cytometry Basic Protocol 3: Isolation of primary mouse peripheral blood mononuclear cells Support Protocol 2: Isolation of primary mouse spleen cells Support Protocol 3: Purification and validation of CD4+ T cells from PBMCs and spleen cells Basic Protocol 4: Isolation of liver granuloma supernatant and determination of organ load Support Protocol 4: In vivo and in vitro infection with mycobacteria Basic Protocol 5: Assembly of the BBB co-culture model Basic Protocol 6: Assembly of the combined in vitro granuloma and BBB model.

The effects of Vitis vinifera L. phenolic compounds on a blood-brain barrier culture model: Expression of leptin receptors and protection against cytokine-induced damage.

ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal properties of grapes (Vitis vinifera L.) are well known since ancient times. Ethnobotanical grape preparations, like the Ayurvedic Darakchasava are used as cardiotonic and for the treatment of cardiovascular diseases. Dried grape products are also applied in Iranian traditional medicine for memory problems, which are linked to the pathology of brain microvessels, a special part of the cardiovascular system. The anti-inflammatory and protective effects of these traditional preparations on the cardiovascular system are related to their bioactive phenolic compounds. AIM OF THE STUDY: The blood-brain barrier (BBB), formed by brain capillaries, is not only involved in inflammatory and other diseases of the central nervous system, but also in many systemic diseases with an inflammatory component. Dietary obesity is a systemic chronic inflammatory condition in which the peripheral and central vascular system is affected. Among the cerebrovascular changes in obesity defective leptin transport across the BBB related to central leptin resistance is observed. Our aim was to study the protective effects of grape phenolic compounds epicatechin (EC), gallic acid (GA) and resveratrol (RSV) and grape-seed proanthocyanidin-rich extract (GSPE) on a cytokine-induced vascular endothelial inflammation model. Using a culture model of the BBB we investigated cytokine-induced endothelial damage and changes in the expression of leptin receptors and leptin transfer. MATERIALS AND METHODS: For the BBB model, primary cultures of rat brain endothelial cells, glial cells and pericytes were used in co-culture. Cells were treated by tumor necrosis factor-α (TNF-α) and interleukin-1 ß (IL-1ß) (10 ng/ml each) to induce damage. Cell toxicity was evaluated by the measurement of impedance. The expression of leptin receptors was assessed by RT-qPCR and western blot. The production of reactive oxygen species (ROS) and nitric oxide (NO) were detected by fluorescent probes. RESULTS: GSPE (10 µg/ml), EC (10 µM), GA (1 µM) or RSV (10 µM) did not change the viability of brain endothelial cells. The gene expression of the short leptin receptor isoform, Ob-Ra, was up-regulated by GSPE, EC and RSV, while the mRNA levels of Lrp2 and clusterin, clu/ApoJ were not affected. The tested compounds did not change the expression of the long leptin receptor isoform, Ob-Rb. RSV protected against the cytokine-induced increase in albumin permeability of the BBB model. GSPE and EC exerted an antioxidant effect and GSPE increased NO both alone and in the presence of cytokines. The cytokine-induced nuclear translocation of transcription factor NF-κB was blocked by GSPE, GA and RSV. Cytokines increased the mRNA expression of Lrp2 which was inhibited by EC. RSV increased Ob-Ra and Clu in the presence of cytokines. Cytokines elevated leptin transfer across the BBB model, which was not modified by GSPE or RSV. CONCLUSION: Our results obtained on cell culture models confirm that natural grape compounds protect vascular endothelial cells against inflammatory damage in accordance with the ethnopharmacological use of grape preparations in cardiovascular diseases. Furthermore, grape compounds and GSPE, by exerting a beneficial effect on the BBB, may also be considered in the treatment of obesity after validation in clinical trials.

Lidocaine turns the surface charge of biological membranes more positive and changes the permeability of blood-brain barrier culture models.

The surface charge of brain endothelial cells forming the blood-brain barrier (BBB) is highly negative due to phospholipids in the plasma membrane and the glycocalyx. This negative charge is an important element of the defense systems of the BBB. Lidocaine, a cationic and lipophilic molecule which has anaesthetic and antiarrhytmic properties, exerts its actions by interacting with lipid membranes. Lidocaine when administered intravenously acts on vascular endothelial cells, but its direct effect on brain endothelial cells has not yet been studied. Our aim was to measure the effect of lidocaine on the charge of biological membranes and the barrier function of brain endothelial cells. We used the simplified membrane model, the bacteriorhodopsin (bR) containing purple membrane of Halobacterium salinarum and culture models of the BBB. We found that lidocaine turns the negative surface charge of purple membrane more positive and restores the function of the proton pump bR. Lidocaine also changed the zeta potential of brain endothelial cells in the same way. Short-term lidocaine treatment at a 10 µM therapeutically relevant concentration did not cause major BBB barrier dysfunction, substantial change in cell morphology or P-glycoprotein efflux pump inhibition. Lidocaine treatment decreased the flux of a cationic lipophilic molecule across the cell layer, but had no effect on the penetration of hydrophilic neutral or negatively charged markers. Our observations help to understand the biophysical background of the effect of lidocaine on biological membranes and draws the attention to the interaction of cationic drug molecules at the level of the BBB.

Claudin peptidomimetics modulate tissue barriers for enhanced drug delivery.

The blood-brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (≤40 kDa) and reversibly (12-48 h) increased the permeability of brain endothelial and claudin-5-transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell-cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 µmol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd-diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between ß-sheets flanked by α-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5.

The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms.

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging.

Tesmilifene modifies brain endothelial functions and opens the blood-brain/blood-glioma barrier.

Tesmilifene, a tamoxifen analog with antihistamine action, has chemopotentiating properties in experimental and clinical cancer studies. In our previous works, tesmilifene increased the permeability of the blood-brain barrier (BBB) in animal and culture models. Our aim was to investigate the effects of tesmilifene on brain microvessel permeability in the rat RG2 glioma model and to reveal its mode of action in brain endothelial cells. Tesmilifene significantly increased fluorescein extravasation in the glioma. Short-term treatment with tesmilifene reduced the resistance and increased the permeability for marker molecules in a rat triple co-culture BBB model. Tesmilifene also affected the barrier integrity in brain endothelial cells co-cultured with RG2 glioblastoma cells. Tesmilifene inhibited the activity of P-glycoprotein and multidrug resistance-associated protein-1 efflux pumps and down-regulated the mRNA expression of tight junction proteins, efflux pumps, solute carriers, and metabolic enzymes important for BBB functions. Among the possible signaling pathways that regulate BBB permeability, tesmilifene activated the early nuclear translocation of NFκB. The MAPK/ERK and PI3K/Akt kinase pathways were also involved. We demonstrate for the first time that tesmilifene increases permeability marker molecule extravasation in glioma and inhibits efflux pump activity in brain endothelial cells, which may have therapeutic relevance. Tesmilifene, a chemopotentiator in experimental and clinical cancer studies increases vascular permeability in RG2 glioma in rats and permeability for marker molecules in a culture model of the blood-brain barrier. Tesmilifene inhibits the activity of efflux pumps and down-regulates the mRNA expression of tight junction proteins, transporters, and metabolic enzymes important for the blood-brain barrier functions, which may have therapeutic relevance.

Role of the blood-brain barrier in the nutrition of the central nervous system.

The blood-brain barrier (BBB) is a dynamic and complex interface between the blood and the central nervous system regulating brain homeostasis. Major functions of the BBB include the transport of nutrients and protection of the brain from toxic compounds. This review summarizes the most important transport pathways contributing to the nutrition of the brain. Carrier-mediated transport selectively delivers small molecules like sugars, amino acids, vitamins, and trace elements. Large biomolecules, lipoproteins, peptide and protein hormones cross the BBB by receptor-mediated transport. Active efflux transporters participate in the brain efflux of endogenous metabolites as well as toxins, xenobiotics and drugs. Dysfunction in the transport of nutrients at the BBB is described in several neurological disorders and diseases. The BBB penetration of neuroprotective nutrients, especially plant polyphenols and alkaloids, their potential protective effect on brain endothelium and the interaction of nutraceuticals with active efflux transporters at the BBB are discussed. In vitro BBB models to examine nutrient transport are also presented.

Kinetic analysis of the toxicity of pharmaceutical excipients Cremophor EL and RH40 on endothelial and epithelial cells.

Cremophor EL and RH40 are widely used excipients in oral and intravenous drug formulations such as Taxol infusion to improve drug dissolution and absorption. Studies indicate that Cremophors, especially EL, have toxic side effects, but few data are available on endothelial and epithelial cells, which form biological barriers and are directly exposed to these molecules. Human hCMEC/D3 brain endothelial and Caco-2 epithelial cells were treated with Cremophor EL and RH40 in the 0.1-50 mg/mL concentration range. Cell toxicity was monitored by real-time cell microelectronic sensing and verified by lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and morphological methods. Cremophors caused dose- and time-dependent damage in both cell types. In endothelial cells, 0.1 mg/mL and higher concentrations, in epithelial cells, concentrations of 5 mg/mL and above were toxic, especially at longer incubations. Cell death was also proven by double fluorescent staining of cell nuclei. Immunostaining for tight junction proteins claudin-4 and -5 showed barrier disruption in cells treated by surfactants at 24 h. In conclusion, Cremophor EL and RH40 in concentrations corresponding to clinical doses caused endothelial and epithelial toxicity. Endothelial cells were more sensitive to surfactant treatment than epithelial cells, and Cremophor EL was more toxic than RH40 in both cell types.