Human Blood-brain Barrier Model Research Articles

Enterovirus A71 crosses a human blood-brain barrier model through infected immune cells.

Enterovirus A71 (EV-A71) is associated with neurological conditions such as acute meningitis and encephalitis. The virus is detected in the bloodstream, and high blood viral loads are associated with central nervous system (CNS) manifestations. We used an in vitro blood-brain barrier (BBB) model made up of human brain-like endothelial cells (hBLECs) and brain pericytes grown in transwell systems to investigate whether three genetically distinct EV-A71 strains (subgenogroups C1, C1-like, and C4) can cross the human BBB. EV-A71 poorly replicated in hBLECs, which released moderate amounts of infectious viruses from their luminal side and trace amounts of infectious viruses from their basolateral side. The barrier properties of hBLECs were not impaired by EV-A71 infection. We investigated the passage through hBLECs of EV-A71-infected white blood cells. EV-A71 strains efficiently replicated in immune cells, including monocytes, neutrophils, and NK/T cells. Attachment to hBLECs of immune cells infected with the C1-like virus was higher than attachment of cells infected with C1-06. EV-A71 infection did not impair the transmigration of immune cells through hBLECs. Overall, EV-A71 targets different white blood cell populations that have the potential to be used as a Trojan horse to cross hBLECs more efficiently than cell-free EV-A71 particles.IMPORTANCEEnterovirus A71 (EV-A71) was first reported in the USA, and numerous outbreaks have since occurred in Asia and Europe. EV-A71 re-emerged as a new multirecombinant strain in 2015 in Europe and is now widespread. The virus causes hand-foot-and-mouth disease in young children and is involved in nervous system infections. How the virus spreads to the nervous system is unclear. We investigated whether white blood cells could be infected by EV-A71 and transmit it across human endothelial cells mimicking the blood-brain barrier protecting the brain from adverse effects. We found that endothelial cells provide a strong roadblock to prevent the passage of free virus particles but allow the migration of infected immune cells, including monocytes, neutrophils, and NK/T cells. Our data are consistent with the potential role of immune cells in the pathogenesis of EV-A71 infections by spreading the virus in the blood and across the human blood-brain barrier.

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/β-catenin signaling and inhibition of the TGF-β 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/β-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.

Novel simulation of the blood–brain barrier using the polyacrylonitrile electrospun nanofibrous membrane on 3D‐printed Transwell

AbstractThe blood–brain barrier (BBB) possesses an intricate structure and set of characteristics that effectively restricts the transportation of substances to the central nervous system. Consequently, the development of pharmaceuticals for brain disorders poses significant hurdles. Numerous attempts have been made to produce innovative simulates of BBB that exhibit a striking resemblance to BBB and are suitable for efficient drug screening, as well as being user‐friendly and easily reproducible. One of the models available is the commercial Transwell® system. In this system, chosen endothelial cells form a single layer on the rigid membrane of the upper chamber. In the present study, the two‐dimensional structure of this membrane was exchanged with a three‐dimensional membrane using the 3D printing method and developed a novel approach to fabricate the scaffold in one step on the fabricated Transwell. This novel method of horizontal electrospinning has reduced the mechanical damage associated with conventional vertical electrospinning, resulting in suitable mechanical properties for cell culture. The lower diameter of the nanofibers, the thickness of the mesh, bulk Young's modulus and hydrophobicity compared to conventional mesh‐based counterparts are characteristics which make it more similar to the natural basement membrane and improve cell culture necessities on the scaffold. The designed BBB model was characterized via cell morphology, trans‐endothelial electrical resistance, and permeability value. This human BBB model formed robust cell integrity and achieved the acceptable range of TEER and low permeability that confirm the polyacrylonitrile (PAN) scaffold as an in vitro cell culture platform with promising cell culture results of the brain microvascular endothelial cell line hCMEC/D3.

Abstract 2823: Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological malignancy, is characterized by abnormal thymocyte proliferation, frequently with mutations of the Notch-1 gene and has an overall 10-year survival rate of ~27%. A marker of poor prognosis is the presence of T-ALL within the central nervous system (CNS), which is treated with intrathecal chemotherapy and cranial irradiation; two toxic treatments with high morbidities. Gene-expression profiling of Notch-1-induced adhesion regulators showed that C-C chemokine receptor 7 (CCR7) was highly expressed and was associated with leukemic T-cell invasion of the CNS. Normal functions of CCR7 include lymph node homing and immune cell trafficking in response to CCR7 ligands, CC-chemokine ligand 19 (CCL19) or CCL21. In several studies CCL19 has been linked to T-cell invasion of the CNS across the blood-brain barrier (BBB). We hypothesize that antagonizing CCR7 binding to CCL19 will block migration of T-ALL into the CNS and thereby alleviate the need for toxic therapies. To test our hypothesis, we are using receptor internalization assays, calcium mobilization assays, transwell migration assays and an in vitro model of an intact human BBB. We have used flow cytometry-based CCR7 internalization assays ± CCR7 antagonist and found that we can completely block CCL19-induced receptor internalization in human CCR7-expressing CCRF-CEM, and HuT-78 T-ALL cells, and chemotaxis via β1 integrins across a fibronectin-coated transwell membrane. Our BBB employs HBEC-5i, human cerebral microvascular endothelial cells, in transwell chambers coated with gelatin and fibronectin for the adhesion of endothelial cells. HBEC-5i cells are a suitable cell line to make a 3D model of the BBB, as they form an endothelial monolayer with tight junctions (TJ), which is crucial to maintaining the integrity and inhibiting permeability of the BBB. We are establishing a transwell migration assay to assess the migration of CCRF-CEM T-ALL cells across the HBEC-5i cell monolayer. The integrity of the HBEC-5i cell monolayer is assessed by trans-epithelial electrical resistance (TEER), which measures the electrical resistance across the HBEC-5i monolayer, a measure of TJ integrity. Using TEER measurements, we observe that HBEC-5i form a monolayer that reaches an intact monolayer functioning with TJs over 72-96 hours. We are using this model system to investigate the migration of CCR7-expressing CCRF-CEM cells through the BBB ± CCL19. Subsequently, we will add the CCL19 antagonist (CCL197-77) to determine if inhibiting CCR7 is sufficient to eliminate migration of the CCRF-CEM cells through the BBB. If successful, this model will allow us to test the contribution of CCR7 signaling to integrins, the contribution of other immune cell types and the overall molecular mechanisms that mediate invasion of the CNS in a 3-D model of the BBB. Citation Format: Alondra Rodriguez, Cesar I. Cardona, Colin A. Bill, Charlotte M. Vines. Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2823.

Rebuilding insight into the pathophysiology of Alzheimer's disease through new blood-brain barrier models.

The blood-brain barrier is a unique function of the microvasculature in the brain parenchyma that maintains homeostasis in the central nervous system. Blood-brain barrier breakdown is a common pathology in various neurological diseases, such as Alzheimer's disease, stroke, multiple sclerosis, and Parkinson's disease. Traditionally, it has been considered a consequence of neuroinflammation or neurodegeneration, but recent advanced imaging techniques and detailed studies in animal models show that blood-brain barrier breakdown occurs early in the disease process and may precede neuronal loss. Thus, the blood-brain barrier is attractive as a potential therapeutic target for neurological diseases that lack effective therapeutics. To elucidate the molecular mechanism underlying blood-brain barrier breakdown and translate them into therapeutic strategies for neurological diseases, there is a growing demand for experimental models of human origin that allow for functional assessments. Recently, several human induced pluripotent stem cell-derived blood-brain barrier models have been established and various in vitro blood-brain barrier models using microdevices have been proposed. Especially in the Alzheimer's disease field, the human evidence for blood-brain barrier dysfunction has been demonstrated and human induced pluripotent stem cell-derived blood-brain barrier models have suggested the putative molecular mechanisms of pathological blood-brain barrier. In this review, we summarize recent evidence of blood-brain barrier dysfunction in Alzheimer's disease from pathological analyses, imaging studies, animal models, and stem cell sources. Additionally, we discuss the potential future directions for blood-brain barrier research.

An Induced Pluripotent Stem Cell-Derived Human Blood-Brain Barrier (BBB) Model to Test the Crossing by Adeno-Associated Virus (AAV) Vectors and Antisense Oligonucleotides.

The blood-brain barrier (BBB) is the specialised microvasculature system that shields the central nervous system (CNS) from potentially toxic agents. Attempts to develop therapeutic agents targeting the CNS have been hindered by the lack of predictive models of BBB crossing. In vitro models mimicking the human BBB are of great interest, and advances in induced pluripotent stem cell (iPSC) technologies and the availability of reproducible differentiation protocols have facilitated progress. In this study, we present the efficient differentiation of three different wild-type iPSC lines into brain microvascular endothelial cells (BMECs). Once differentiated, cells displayed several features of BMECs and exhibited significant barrier tightness as measured by trans-endothelial electrical resistance (TEER), ranging from 1500 to >6000 Ωcm2. To assess the functionality of our BBB models, we analysed the crossing efficiency of adeno-associated virus (AAV) vectors and peptide-conjugated antisense oligonucleotides, both currently used in genetic approaches for the treatment of rare diseases. We demonstrated superior barrier crossing by AAV serotype 9 compared to serotype 8, and no crossing by a cell-penetrating peptide-conjugated antisense oligonucleotide. In conclusion, our study shows that iPSC-based models of the human BBB display robust phenotypes and could be used to screen drugs for CNS penetration in culture.

TGF-β and SHH Regulate Pluripotent Stem Cell Differentiation into Brain Microvascular Endothelial Cells in Generating an In Vitro Blood-Brain Barrier Model.

Blood-brain barrier (BBB) models are important tools for studying CNS drug delivery, brain development, and brain disease. In vitro BBB models have been obtained from animals and immortalized cell lines; however, brain microvascular endothelial cells (BMECs) derived from them have several limitations. Furthermore, obtaining mature brain microvascular endothelial-like cells (BME-like cells) from human pluripotent stem cells (hPSCs) with desirable properties for establishing BBB models has been challenging. Here, we developed an efficient method for differentiating hPSCs into BMECs that are amenable to the development and application of human BBB models. The established conditions provided an environment similar to that occurring during BBB differentiation in the presence of the co-differentiating neural cell population by the modulation of TGF-β and SHH signaling. The developed BME-like cells showed well-organized tight junctions, appropriate expression of nutrient transporters, and polarized efflux transporter activity. In addition, BME-like cells responded to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance. Moreover, the BME-like cells exhibited an immune quiescent property of BBB endothelial cells by decreasing the expression of adhesion molecules. Therefore, our novel cellular platform could be useful for drug screening and the development of brain-permeable pharmaceuticals.

Pathogenic bacteria exploit transferrin receptor transcytosis to penetrate the blood–brain barrier

The human blood-brain barrier (BBB) comprises a single layer of brain microvascular endothelial cells (HBMECs) protecting the brain from bloodborne pathogens. Meningitis is among the most serious diseases, but the mechanisms by which major meningitis-causing bacterial pathogens cross the BBB to reach the brain remain poorly understood. We found that Streptococcus pneumoniae, group B Streptococcus, and neonatal meningitis Escherichia coli commonly exploit a unique vesicle fusion mechanism to hitchhike on transferrin receptor (TfR) transcytosis to cross the BBB and illustrated the details of this process in human BBB model in vitro and mouse model. Toll-like receptor signals emanating from bacteria-containing vesicles (BCVs) trigger K33-linked polyubiquitination at Lys168 and Lys181 of the innate immune regulator TRAF3 and then activate the formation of a protein complex containing the guanine nucleotide exchange factor RCC2, the small GTPase RalA and exocyst subcomplex I (SC I) on BCVs. The distinct function of SEC6 in SC I, interacting directly with RalA on BCVs and the SNARE protein SNAP23 on TfR vesicles, tethers these two vesicles and initiates the fusion. Our results reveal that innate immunity triggers a unique modification of TRAF3 and the formation of the HBMEC-specific protein complex on BCVs to authenticate the precise recognition and selection of TfR vesicles to fuse with and facilitate bacterial penetration of the BBB.

P16-21: Applications of an isogenic human iPSC-derived blood-brain barrier (BBB) model

P16-21: Applications of an isogenic human iPSC-derived blood-brain barrier (BBB) model

Overcoming the blood-brain barrier? - prediction of blood-brain permeability of hydrophobically modified polyethylenimine polyplexes for siRNA delivery into the brain with in vitro and in vivo models

The blood-brain barrier (BBB) is a highly selective biological barrier that represents a major bottleneck in the treatment of all types of central nervous system (CNS) disorders. Small interfering RNA (siRNA) offers in principle a promising therapeutic approach, e.g., for brain tumors, by downregulating brain tumor-related genes and inhibiting tumor growth via RNA interference. In an effort to develop efficient siRNA nanocarriers for crossing the BBB, we utilized polyethyleneimine (PEI) polymers hydrophobically modified with either stearic-acid (SA) or dodecylacrylamide (DAA) subunits and evaluated their suitability for delivering siRNA across the BBB in in vitro and in vivo BBB models depending on their structure. Physicochemical characteristics of siRNA-polymer complexes (polyplexes (PXs)), e.g., particle size and surface charge, were measured by dynamic light scattering and laser Doppler anemometry, whereas siRNA condensation ability of polymers and polyplex stability was evaluated by spectrophotometric methods. The composition of the biomolecule corona that absorbs on polyplexes upon encountering physiological fluids was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. Cellular internalization abilities of PXs into brain endothelial cells (hCMEC/D3) was confirmed, and a BBB permeation assay using a human induced pluripotent stem cell (hiPSC)–derived BBB model revealed similar abilities to cross the BBB for all formulations under physiological conditions. However, biodistribution studies of radiolabeled PXs in mice were inconsistent with in vitro results as the detected amount of radiolabeled siRNA in the brain delivered with PEI PXs was higher compared to PEI-SA PXs. Taken together, PEI PXs were shown to be a suitable nanocarrier to deliver small amounts of siRNA across the BBB into the brain but more sophisticated human BBB models that better represent physiological conditions and biodistribution are required to provide highly predictive in vitro data for human CNS drug development in the future.

TNF-α evokes blood-brain barrier dysfunction through activation of Rho-kinase and neurokinin 1 receptor

Ischaemic stroke, accompanied by neuroinflammation, impairs blood–brain barrier (BBB) integrity through a complex mechanism involving activation of both RhoA/Rho kinase/myosin light chain-2 and neurokinin 1 receptor (NK1R). Using an in vitro model of human BBB composed of brain microvascular endothelial cells (BMEC), astrocytes and pericytes, this study examined the potential contributions of these elements to BBB damage induced by elevated availability of pro-inflammatory cytokine, TNF-α. Treatment of human BMECs with TNF-α significantly enhanced RhoA activity and the protein expressions of Rho kinase and phosphorylated Ser19MLC-2 while decreasing that of NK1R. Pharmacological inhibition of Rho kinase by Y-27632 and NK1R by CP96345 neutralised the disruptive effects of TNF-α on BBB integrity and function as ascertained by reversal of decreases in transendothelial electrical resistance and increases in paracellular flux of low molecular weight permeability marker, sodium fluorescein, respectively. Suppression of RhoA activation, mitigation of actin stress fibre formation and restoration of plasma membrane localisation of tight junction protein zonula occludens-1 appeared to contribute to the barrier-protective effects of both Y-27632 and CP96345. Attenuation of TNF-α-mediated increases in NK1R protein expression in BMEC by Y-27632 suggests that RhoA/Rho kinase pathway acts upstream to NK1R. In conclusion, specific inhibition of Rho kinase in cerebrovascular conditions, accompanied by excessive release of pro-inflammatory cytokine TNF-α, helps preserve endothelial cell morphology and inter-endothelial cell barrier formation and may serve as an important therapeutic target.

Substance P reversibly compromises the integrity and function of blood-brain barrier

BackgroundSubstance P (SP) plays a role in vasodilatation and tissue integrity through its receptor, neurokinin 1 (NK1R). However, its specific effect on blood-brain barrier (BBB) remains unknown. MethodsThe impact of SP on the integrity/function of human BBB model in vitro, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, was assessed by measurements of transendothelial electrical resistance and paracellular flux of sodium fluorescein (NaF), respectively in the absence/presence of specific inhibitors targeting NK1R (CP96345), Rho-associated protein kinase (ROCK; Y27632) and nitric oxide synthase (NOS; N(G)-nitro-L-arginine methyl ester). Sodium nitroprusside (SNP), a NO donor, was employed as a positive control. The levels of tight junction proteins, zonula occludens-1, occludin and claudin-5 alongside RhoA/ROCK/myosin regulatory light chain-2 (MLC2) and extracellular signal‑regulated protein kinase (Erk1/2) proteins were detected by western analyses. Subcellular localisations of F-actin and tight junction proteins were visualized by immunocytochemistry. Flow cytometry was used to detect transient calcium release. ResultsExposure to SP increased RhoA, ROCK2 and phosphorylated serine-19 MLC2 protein levels and Erk1/2 phosphorylation in BMECs which were abolished by CP96345. These increases were independent of the changes in intracellular calcium availability. SP perturbed BBB in a time-dependent fashion through induction of stress fibres. Changes in tight junction protein dissolution or relocalisation were not involved in SP-mediated BBB breakdown. Inhibition of NOS, ROCK and NK1R mitigated the effect of SP on BBB characteristics and stress fibre formation. ConclusionSP promoted a reversible decline in BBB integrity independent of tight junction proteins expression or localisation.

Non-Invasive, Targeted Nanoparticle-Mediated Drug Delivery across a Novel Human BBB Model.

The blood-brain barrier (BBB) is a highly sophisticated system with the ability to regulate compounds transporting through the barrier and reaching the central nervous system (CNS). The BBB protects the CNS from toxins and pathogens but can cause major issues when developing novel therapeutics to treat neurological disorders. PLGA nanoparticles have been developed to successfully encapsulate large hydrophilic compounds for drug delivery. Within this paper, we discuss the encapsulation of a model compound Fitc-dextran, a large molecular weight (70 kDa), hydrophilic compound, with over 60% encapsulation efficiency (EE) within a PLGA nanoparticle (NP). The NP surface was chemically modified with DAS peptide, a ligand that we designed which has an affinity for nicotinic receptors, specifically alpha 7 nicotinic receptors, found on the surface of brain endothelial cells. The attachment of DAS transports the NP across the BBB by receptor-mediated transcytosis (RMT). Assessment of the delivery efficacy of the DAS-conjugated Fitc-dextran-loaded PLGA NP was studied in vitro using our optimal triculture in vitro BBB model, which successfully replicates the in vivo BBB environment, producing high TEER (≥230 ) and high expression of ZO1 protein. Utilising our optimal BBB model, we successfully transported fourteen times the concentration of DAS-Fitc-dextran-PLGA NP compared to non-conjugated Fitc-dextran-PLGA NP. Our novel in vitro model is a viable method of high-throughput screening of potential therapeutic delivery systems to the CNS, such as our receptor-targeted DAS ligand-conjugated NP, whereby only lead therapeutic compounds will progress to in vivo studies.

Antiretroviral drugs efavirenz, dolutegravir and bictegravir dysregulate blood-brain barrier integrity and function.

The implementation of combined antiretroviral therapy (cART) significantly reduces the mortality associated with human immunodeficiency virus (HIV) infection. However, complications such as HIV-associated neurocognitive disorders (HAND) remain a major health concern. We hypothesized that the toxicity of antiretroviral drugs (ARVs) may contribute to the pathogenesis of HAND in addition to cerebral viral infection. To address this question, we evaluated the impact of HIV integrase strand transfer inhibitors (dolutegravir and bictegravir), and a non-nucleoside reverse transcriptase inhibitor (efavirenz) on the integrity and permeability of various human and mouse blood-brain barrier (BBB) models, in vitro, ex vivo and in vivo. We observed a significant downregulation of tight junction proteins (TJP1/Tjp1, OCLN/Ocln and CLDN5/Cldn5), upregulation of proinflammatory cytokines (IL6/Il6, IL8/Il8, IL1β/Il1β) and NOS2/Nos2, and alteration of membrane-associated transporters (ABCB1/Abcb1a, ABCG2/Abcg2 and SLC2A1/Slc2a1) mRNA expression, in vitro, in human (hCMEC/D3) and primary cultures of mouse microvascular endothelial cells, and ex vivo in isolated mouse brain capillaries treated with efavirenz, dolutegravir, and/or bictegravir. We also observed a significant increase in BBB permeability in vivo following treatment with the selected ARVs in mice applying NaF permeability assay. Taken together, these results suggest that clinically recommended integrase strand transfer inhibitors such as dolutegravir may exacerbate HIV-associated cerebrovascular pathology, which may contribute to the associated short-term neuropsychiatric side effects and the high incidence of mild forms of HAND reported in the clinical setting.

High and low permeability of human pluripotent stem cell-derived blood-brain barrier models depend on epithelial or endothelial features.

The search for reliable human blood-brain barrier (BBB) models represents a challenge for the development/testing of strategies aiming to enhance brain delivery of drugs. Human-induced pluripotent stem cells (hiPSCs) have raised hopes in the development of predictive BBB models. Differentiating strategies are thus required to generate endothelial cells (ECs), a major component of the BBB. Several hiPSC-based protocols have reported the generation of in vitro models with significant differences in barrier properties. We studied in depth the properties of iPSCs byproducts from two protocols that have been established to yield these in vitro barrier models. Our analysis/study reveals that iPSCs derivatives endowed with EC features yield high permeability models while the cells that exhibit outstanding barrier properties show principally epithelial cell-like (EpC) features. We found that models containing EpC-like cells express tight junction proteins, transporters/efflux pumps and display a high functional tightness with very low permeability, which are features commonly shared between BBB and epithelial barriers. Our study demonstrates that hiPSC-based BBB models need extensive characterization beforehand and that a reliable human BBB model containing EC-like cells and displaying low permeability is still needed.

Manufactured tissue-to-tissue barrier chip for modeling the human blood-brain barrier and regulation of cellular trafficking.

Microphysiological system or organ-on-a-chip technologies can replicate the key structure and function of 3D human tissues with higher reproducibility than less controllable 3D cell aggregate models, providing great potential to become advanced drug toxicity and efficacy test platforms alternative to animal models. However, these organ chip models remain to be manufactured and standardized in a highly reproducible manner for reliable drug screening and mechanism of action research. Herein, we present a manufactured form of 'micro-engineered physiological system-tissue barrier chip' called MEPS-TBC for the highly replicable modeling of the human blood-brain barrier (BBB) with a 3D perivascular space. The perivascular region was controlled by tunable aspiration, where human astrocytes reside in 3D, create a network, and communicate with human pericytes facing human vascular endothelial cells, thereby replicating the 3D BBB. The lower channel structure of MEPS-TBC was designed and optimized using a computational simulation to facilitate aspiration while maintaining multicellular construction. Our human BBB model of the 3D perivascular unit and the endothelium perfused by physiological shear stress secured significantly enhanced barrier function exhibiting greater TEER and lower permeability, compared to the only endothelial model, indicating that the cellular interactions between BBB cells significantly contribute to the BBB formation. Importantly, our BBB model showed the cellular barrier function for homeostatic trafficking regulation against inflammatory peripheral immune cells, as well as for molecular transport control across the BBB. We believe our manufactured chip technology will construct reliable and standardized organ-chip models for disease mechanism research and predictive drug screening.

Human iPS-derived blood-brain barrier model exhibiting enhanced barrier properties empowered by engineered basement membrane

The basement membrane (BM) of the blood-brain barrier (BBB), a thin extracellular matrix (ECM) sheet underneath the brain microvascular endothelial cells (BMECs), plays crucial roles in regulating the unique physiological barrier function of the BBB, which represents a major obstacle for brain drug delivery. Owing to the difficulty in mimicking the unique biophysical and chemical features of BM in in vitro systems, current in vitro BBB models have suffered from poor physiological relevance. Here, we describe a highly ameliorated human BBB model accomplished by an ultra-thin ECM hydrogel-based engineered basement membrane (nEBM), which is supported by a sparse electrospun nanofiber scaffold that offers in vivo BM-like microenvironment to BMECs. BBB model reconstituted on a nEBM recapitulates the physical barrier function of the in vivo human BBB through ECM mechano-response to physiological relevant stiffness (∼500 kPa) and exhibits high efflux pump activity. These features of the proposed BBB model enable modelling of ischemic stroke, reproducing the dynamic changes of BBB, immune cell infiltration, and drug response. Therefore, the proposed BBB model represents a powerful tool for predicting the BBB permeation of drugs and developing therapeutic strategies for brain diseases.

Quantitative Targeted Absolute Proteomics for Better Characterization of an In Vitro Human Blood-Brain Barrier Model Derived from Hematopoietic Stem Cells.

We previously developed an in vitro model of the human blood-brain barrier (BBB) based on the use of endothelial cells derived from CD34+-hematopoietic stem cells and cultured with brain pericytes. The purpose of the present study was to provide information on the protein expression levels of the transporters, receptors, tight junction/adherence junction molecules, and transporter-associated molecules of human brain-like endothelial cells (hBLECs). The absolute protein expression levels were determined by liquid chromatography-mass spectrometry-based quantitative targeted absolute proteomics and compared with those from human brain microvessels (hBMVs). The protein levels of CD144, CD147, MRP4, Annexin A6 and caveolin-1 showed more than 3-fold abundance in hBLECs, those of MCT1, Connexin 43, TfR1, and claudin-5 showed less than 3-fold differences, and the protein levels of other drug efflux transporters and nutrient transporters were less represented in hBLECs than in hBMVs. It is noteworthy that BCRP was more expressed than MDR1 in hBLECs, as this was the case for hBMVs. These results suggest that transports mediated by MCT1, TfR1, and claudin-5-related tight junction function reflect the in vivo BBB situation. The present study provided a better characterization of hBLECs and clarified the equivalence of the transport characteristics between in vitro BBB models and in vivo BBB models using LC-MS/MS-based protein quantification.

Pioglitazone Attenuates the Effects of Peripheral Inflammation in a Human In Vitro Blood–Brain Barrier Model

Biological mediators secreted during peripheral chronic inflammation reach the bloodstream and may damage the blood-brain barrier (BBB), triggering central nervous system (CNS) disorders. Full-fledged human BBB models are efficient tools to investigate pharmacological pathways and mechanisms of injury at the BBB. We here employed a human in vitro BBB model to investigate the effects of either plasma from inflammatory bowel disease (IBD) patients or tumor necrosis factor α (TNFα), a cytokine commonly released in periphery during IBD, and the anti-inflammatory role of pioglitazone, a peroxisome proliferator-activated receptor γ agonist (PPARγ). The BBB model was treated with either 10% plasma from healthy and IBD donors or 5 ng/mL TNFα, following treatment with 10 µM pioglitazone. Patient plasma did not alter BBB parameters, but TNFα levels in plasma from all donors were associated with varying expression of claudin-5, claudin-3 and ICAM-1. TNFα treatment increased BBB permeability, claudin-5 disarrangement, VCAM-1 and ICAM-1 expression, MCP1 secretion and monocyte transmigration. These effects were attenuated by pioglitazone. Plasma from IBD patients, which evoked higher BBB permeability, also increased ICAM-1 expression, this effect being reversed by pioglitazone. Our findings evidence how pioglitazone controls periphery-elicited BBB inflammation and supports its repurposing for prevention/treating of such inflammatory conditions.

The SARS-CoV-2 envelope protein disrupts barrier function in an in vitro human blood-brain barrier model.

Patients with coronavirus disease 2019 (COVID-19) have been frequently reported to exhibit neurological manifestations and disruption of the blood-brain barrier (BBB). Among the risk factors for BBB breakdown, the loss of endothelial cells and pericytes has caused widespread concern. Recent studies have revealed that severe acute respiratory syndrome coronavirus 2 envelope (S2E) protein caused cell death. We tested the hypothesis that the S2E protein alone could induce BBB dysfunction. The S2E protein bound to human BBB-related cells and inhibited cell viability in a dose- and time-dependent manner. Importantly, the S2E protein disrupted barrier function in an in vitro BBB model composed of HCMEC/D3 (brain endothelial cell line), HBVP (brain vascular pericyte), and U87MG (astrocyte cell line) cells and suppressed the expression of major genes involved in maintaining endothelial permeability and function. In addition, the S2E protein crossed the HCMEC/D3 monolayer. The S2E protein triggered inflammatory responses in HCMEC/D3 and U87MG cells. Taken together, these results show for the first time that the S2E protein has a negative impact on the BBB. Therapies targeting the S2E protein could protect against and treat central nervous system manifestations in COVID-19 patients.