Blood-brain Barrier Model Research Articles

Engineering extracellular vesicles to transiently permeabilize the blood–brain barrier

BackgroundDrug delivery to the brain is challenging due to the restrict permeability of the blood brain barrier (BBB). Recent studies indicate that BBB permeability increases over time during physiological aging likely due to factors (including extracellular vesicles (EVs)) that exist in the bloodstream. Therefore, inspiration can be taken from aging to develop new strategies for the transient opening of the BBB for drug delivery to the brain.ResultsHere, we evaluated the impact of small EVs (sEVs) enriched with microRNAs (miRNAs) overexpressed during aging, with the capacity to interfere transiently with the BBB. Initially, we investigated whether the miRNAs were overexpressed in sEVs collected from plasma of aged individuals. Next, we evaluated the opening properties of the miRNA-enriched sEVs in a static or dynamic (under flow) human in vitro BBB model. Our results showed that miR-383-3p-enriched sEVs significantly increased BBB permeability in a reversible manner by decreasing the expression of claudin 5, an important tight junction protein of brain endothelial cells (BECs) of the BBB, mediated in part by the knockdown of activating transcription factor 4 (ATF4).ConclusionsOur findings suggest that engineered sEVs have potential as a strategy for the temporary BBB opening, making it easier for drugs to reach the brain when injected into the bloodstream.Graphical abstract

VCPP2319 interacts with metastatic breast cancer extracellular vesicles (EVs) and transposes a human blood-brain barrier model

Brain metastases (BM) are frequently found in cancer patients and, though their precise incidence is difficult to estimate, there is evidence for a correlation between BM and specific primary cancers, such as lung, breast, and skin (melanoma). Among all these, breast cancer is the most frequently diagnosed among women and, in this case, BM cause a critical reduction of the overall survival (OS), especially in triple negative breast cancer (TNBC) patients. The main challenge of BM treatment is the impermeable nature of the blood-brain barrier (BBB), which shields the central nervous systems (CNS) from chemotherapeutic drugs. Extracellular vesicles (EVs) have been proposed as ideal natural drug carriers and these may exhibit some advantages over synthetic nanoparticles (NPs). In this work, we isolate breast cancer-derived EVs and study their ability to carry vCPP2319, a peptide with dual cell-penetration and anticancer activities. The selective cytotoxicity of anticancer peptide-loaded EVs towards breast cancer cells and their ability to translocate an in vitro BBB model are also addressed. Overall, it was possible to conclude that vCPP2319 naturally interacts with breast cancer-derived EVs, being retained at the surface of these vesicles. Moreover, the results revealed a cytotoxic activity for peptide-loaded EVs similar to that obtained with the peptide alone and the ability of peptide-loaded EVs to translocate an in vitro BBB model, which contrasts with the results obtained with the peptide alone. In conclusion, this work supports the use of EVs in the development of biological drug-delivery systems (DDS) capable of translocating the BBB.

Snake venom weakens neurovascular integrity and promotes vulnerability to neuroinflammation

Snake envenomation poses significant medical challenges, particularly in subtropical and tropical regions, with long-term impacts on neurovascular integrity and neuroinflammation remaining underexplored. This study investigates the effects of venom from four species of venomous snakes in southern China—Zhoushan Cobra (Naja atra, NA), Many-banded Krait (Bungarus multicinctus, BM), Five-paced Pit Viper (Deinagkistrodon acutus, DA), and Chinese Moccasin (Protobothrops mucrosquamatus, PM) — on the blood–brain barrier (BBB) and chronic neuroinflammation. Using mass spectrometry, we analyzed venom protein compositions, while cytotoxic effects on mouse brain endothelial cells (bEND.3) were evaluated to determine IC50 values. In vitro BBB models and in vivo experiments in C57BL/6J mice revealed that NA venom, in particular, significantly compromised BBB integrity without inducing large-scale apoptosis, leading to persistent BBB disruption characterized by increased permeability and selective degradation of extracellular matrix and tight junction proteins. Moreover, to simulate secondary infections that often occur following snakebites, we combined venom exposure with lipopolysaccharide (LPS) treatment, which exacerbated neuroinflammatory responses by intensifying microglial activation and promoting a pro-inflammatory phenotype. These findings highlight the role of snake venom in compromising neurovascular integrity and promoting vulnerability to chronic neuroinflammation, emphasizing the need for further research into venom-induced neuroinflammatory pathways and their potential as therapeutic targets.

Selection of LRP1 ligand phage-displayed single domain antibody that transmigrates BBB

Effective drug delivery to the central nervous system (CNS) remains a challenge due to the blood–brain barrier (BBB). Macromolecules such as proteins and peptides are unable to cross BBB and have poor therapeutic efficacy due to little or no drug distribution. A promising alternative is the conjugation of a drug to a shuttle molecule that can reach the CNS via receptor-mediated transcytosis (RMT). Several receptors have been described for RMT, such as low-density lipoprotein receptor-related protein 1 (LRP1). We used phage display technology combined with an in vitro BBB model to identify LRP1 ligands. A single domain antibody (dAb) library was used to enrich for species that selectively bind to immobilised LRP1 ligand. We obtained a novel nanobody, dAb D11, that selectively binds to LRP1 receptor and mediates in vitro internalisation of phage particles in brain endothelial cells, with a dissociation constant Kd of 183.1 ± 85.8 nM. The high permeability of D11 was demonstrated by an in vivo biodistribution assay in mice. We discovered D11, the first LRP1 binding dAb with BBB permeability. Our findings will contribute to the development of RMT-based drugs for the treatment of CNS diseases.

Penetratin and Mannose-Functionalized Cannabidiol Lipid Nanoparticles Encapsulating the BDNF Gene Reduce Amyloid-Induced Inflammation.

Inflammation is emerging as a critical player in the disease progression of Alzheimer's disease (AD) by its interaction with amyloid beta plaques in a feed-forward loop. There is also a decline in the nourishment and enriching neurotrophic factor, brain-derived neurotrophic factor (BDNF), in the brain. Therefore, supplementing the brain with BDNF by gene delivery and delivering the anti-inflammatory agent, cannabidiol (CBD) in this case, to mitigate inflammation-induced disease cascade offers an attractive treatment strategy. To achieve the brain localization of CBD and pBDNF, lipid nanoparticles (LNPs) functionalized with mannose and penetratin were utilized. CBD and pBDNF were successfully encapsulated in the LNPs (more than 80%) with a size less than 180 nm, polydispersity index less than 0.25, and zeta potential of 23 mV. CBD was released from the formulation over a period of a week. The dual-functionalized LNPs demonstrated higher cellular uptake of CBD and expressed a significantly higher amount of BDNF (p-value <0.05) after transfection than their nonmodified counterparts in four brain cell lines, i.e., brain endothelial cells (b.END3), immortalized microglia cells (IMGs), primary astrocytes, and primary neurons. Similarly, the permeation of CBD through the dual-modified LNPs across the in vitro coculture blood-brain barrier model was significantly higher (p-value <0.05) compared to free CBD or nonfunctionalized nanoparticles. The LNPs demonstrated anti-inflammatory activity against lipopolysaccharides and human amyloid beta1-42 oligomer induction as they reduced the protein and mRNA expression of pro-inflammatory cytokines TNF-α (p < 0.05) and IL-1β (p < 0.05) in IMG cells. In summary, the penetratin and mannose-functionalized LNPs encapsulating CBD and pBDNF could serve as a promising therapy in AD, requiring further validation in animal models.

Electroacupuncture Serum Protects Blood-brain Barrier Damage after Ischemic Stroke BY Regulating the Pericytes in vitro.

Electroacupuncture (EA) exerts a protective role in Blood-Brain Barrier (BBB) damage after ischemic stroke, but whether this effect involves the regulation of the pericytes in vitro is unclear. The in vitro BBB models were established with brain microvascular endothelial cells (BMECs) and pericytes, and the co-cultured cells were randomly divided into three groups: the control group, oxygen-glucose deprivation/reoxygenation (OGD/R) group and EA group. OGD/R was performed to simulate cerebral ischemia-reperfusion in vitro. EA serum was prepared by EA treatment at the "Renzhong" (GV26) and "Baihui" (GV20) acupoints in middle cerebral artery occlusion/ reperfusion rats. Furthermore, the characteristics of BMECs and pericytes were identified with immunohistochemistry staining. The cell morphology of the BBB model was observed using an inverted microscope. The function of BBB was measured with transendothelial electrical resistance (TEER) and sodium fluorescein, and the viability, apoptosis, and migration of pericytes were detected by cell counting kit-8, flow cytometry, and Transwell migration assay. BMECs were positive staining for Factor-VIII, and pericytes were positive staining for the α-SMA and NG2. EA serum improved cell morphology of the BBB model increased TEER, and decreased sodium fluorescein in OGD/R condition. Besides, EA serum alleviated pericytes" apoptosis rate and migration number, and enhanced pericytes' viability rate in OGD/R condition. EA serum protects against BBB damage induced by OGD/R in vitro, and this protection might be achieved by attenuating pericytes apoptosis and migration, as well as enhancing pericytes viability. The findings provided new evidence for EA as a medical therapy for ischemic stroke.

Hybrid-integrated devices for mimicking malignant brain tumors ("tumor-on-a-chip") for in vitro development of targeted drug delivery and personalized therapy approaches.

Acute and requiring attention problem of oncotheranostics is a necessity for the urgent development of operative and precise diagnostics methods, followed by efficient therapy, to significantly reduce disability and mortality of citizens. A perspective way to achieve efficient personalized treatment is to use methods for operative evaluation of the individual drug load, properties of specific tumors and the effectiveness of selected therapy, and other actual features of pathology. Among the vast diversity of tumor types-brain tumors are the most invasive and malignant in humans with poor survival after diagnosis. Among brain tumors glioblastoma shows exceptionally high mortality. More studies are urgently needed to understand the risk factors and improve therapy approaches. One of the actively developing approaches is the tumor-on-a-chip (ToC) concept. This review examines the achievements of recent years in the field of ToC system developments. The basics of microfluidic chips technologies are considered in the context of their applications in solving oncological problems. Then the basic principles of tumors cultivation are considered to evaluate the main challengers in implementation of microfluidic devices, for growing cell cultures and possibilities of their treatment and observation. The main achievements in the culture types diversity approaches and their advantages are being analyzed. The modeling of angiogenesis and blood-brain barrier (BBB) on a chip, being a principally important elements of the life system, were considered in detail. The most interesting examples and achievements in the field of tumor-on-a-chip developments have been presented.

The regulatory effects of mitragynine on P-glycoprotein transporter.

Kratom preparation containing Mitragyna speciosa Korth plant is frequently used as a recreational drug. Mitragynine, a major alkaloid isolated from M. speciosa, is often detected concurrently with other drugs during forensic analysis, indicating a safety concern. P-glycoprotein (P-gp) is a multidrug transporter. Modulation of P-gp transport activity by drugs or herbal compounds in the brain may lead to drug-herb interactions, resulting in neurotoxicity. We aim to determine the effects of mitragynine on the P-gp regulation and possible neurotoxicity. The effects of mitragynine on the P-gp regulation were investigated in human brain capillary endothelial cells (hCMEC/D3) using molecular docking and dynamic simulation and an optimized bidirectional transport assay, respectively. Repeated-dose treatment and neurotoxicity assessment were carried out using a blood-brain barrier model and polimerase chain reaction (PCR) array. Mitragynine inhibits the P-gp transport activity via binding onto the nucleotide-binding domain site and forms a stable interaction with the P-gp protein complex. Nontoxic concentrations of mitragynine (<4 μM) and substrate drugs (0.001 μM) in the cells significantly enhanced endothelial cell permeability and elicited signs of neurotoxicity in PC-12 cells. Mitragynine is likely a P-gp inhibitor, hence concurrent administration of kratom products with P-gp substrates may lead to clinically significant interactions and neurotoxicity.

Atomically Precise Fluorescent Gold Nanocluster as a Barrier-Permeable and Brain-Specific Imaging Probe.

Photonic nanomaterials play a crucial role in facilitating the necessary signal for optical brain imaging, presenting a promising avenue for early diagnosis of brain-related disorders. However, the blood-brain barrier (BBB) presents a significant challenge, blocking the entry of most molecules or materials from the bloodstream into the brain. To overcome this, photonic nanocrystals in the form of gold clusters (LAuC) with size less than 3 nm, have been developed, with Levodopa conjugated to LAuC (Dop@LAuC) for targeted brain imaging. Dop@LAuC crosses the BBB and emits in the near-infrared (NIR) wavelength, enabling real-time optical brain imaging. An in vitro BBB model using brain endothelial cells showed that 50 % of Dop@LAuC crossed the barrier within 3 hours, compared to only 10 % of LAuC, highlighting the enhanced ability of L-dopa-conjugated gold clusters to penetrate the BBB. In vivo optical imaging in healthy mice further confirmed the material's efficacy to cross BBB without compromising the barrier integrity.

A guide for blood-brain barrier models.

Understanding the intricate mechanisms underlying brain-related diseases hinges on unraveling the pivotal role of the blood-brain barrier (BBB), an essential dynamic interface crucial for maintaining brain equilibrium. This review offers a comprehensive analysis of BBB physiology, delving into its cellular and molecular components while exploring a wide range of in vivo and in vitro BBB models. Notably, recent advancements in 3D cell culture techniques are explicitly discussed, as they have significantly improved the fidelity of BBB modeling by enabling the replication of physiologically relevant environments under flow conditions. Special attention is given to the cellular aspects of in vitro BBB models, alongside discussions on advances in stem cell technologies, providing valuable insights into generating robust cellular systems for BBB modeling. The diverse array of cell types used in BBB modeling, depending on their sources, is meticulously examined in this comprehensive review, scrutinizing their respective derivation protocols and implications. By synthesizing diverse approaches, this review sheds light on the improvements of BBB models to capture physiological conditions, aiding in understanding BBB interactions in health and disease conditions to foster clinical developments.

EXTH-56. INVESTIGATING THE EFFECTS OF INDIRUBINS IN PEDIATRIC DIFFUSE HIGH GRADE GLIOMA MODELS

Abstract Diffuse midline glioma (DMG) is a highly invasive and fatal pediatric brain tumor that arises from glial cells within the pons of the brainstem. Despite focal radiation treatment, DMG continues to have a poor prognosis, with a median survival of less than 10 months. A significant challenge in treating DMG is the blood-brain barrier (BBB), which tightly restricts access to therapeutic agents, preventing drugs from reaching the brain at effective concentrations. Previous studies from the Lawler lab have shown that the indirubin derivative 6-bromoindirubin-3′-acetoxime (BIO-acetoxime/BIA) has anti-invasive properties and can enhance survival in glioblastoma xenograft models. We investigated the effects of BIA on pediatric glioma models using various assays. In an in vitro scratch migration assay, BIA significantly slowed cell migration at a concentration of 1µM in pediatric glioma cells over 72 hours. Additionally, BIA was shown to modulate tumor vasculature and improve drug delivery to tumors by targeting tight junctions in tumor-associated endothelium. BIA treatment increased dextran uptake into 3D BBB models and lowered endothelial cell permeability in vitro by reducing the expression of tight junction proteins, as shown by staining and a decrease in TEER values. Furthermore, BIA’s anti-angiogenic effects were demonstrated through staining, showing significant alterations in angiogenesis-related protein expression. To assess potential synergistic interactions, a drug screen was performed on a panel of pediatric glioma cell lines identifying several FDA-approved drugs that combine well with BIA. Drugs identified in the screen are currently under investigation. Our hypothesis is that BIA could be an effective therapeutic agent for DMG by targeting tumor invasion, angiogenesis, and improving drug potency and delivery through modulation of endothelial cell tight junctions. Future studies will focus on elucidating the underlying mechanisms and developing drug formulations for in vivo studies to assess the translational potential of this approach.

Activation of glial cells induces proinflammatory properties in brain capillary endothelial cells in vitro

Neurodegenerative diseases are often accompanied by neuroinflammation and impairment of the blood-brain barrier (BBB) mediated by activated glial cells through their release of proinflammatory molecules. To study the effects of glial cells on mouse brain endothelial cells (mBECs), we developed an in vitro BBB model with inflammation by preactivating mixed glial cells (MGCs) with lipopolysaccharide (LPS) before co-culturing with mBECs to study the influence of molecules released by activated MGCs. The response of the mBECs to activated MGCs was compared to direct stimulation with LPS. The cytokine profile of activated MGCs was analyzed together with their effects on the mBEC’s integrity, expression of tight junction proteins, adhesion molecules, and BBB-specific transport proteins. Stimulation of MGCs significantly upregulated mRNA expression and secretion of several pro-inflammatory cytokines. Co-culturing mBECs with pre-stimulated MGCs significantly affected the barrier integrity of mBECs similar to direct stimulation with LPS. The gene expression levels of tight junction proteins were unaltered, but tight junction proteins revealed rearrangements with respect to subcellular distribution. Compared to direct stimulation with LPS, the expression of cell-adhesion molecules was significantly increased when mBECs were co-cultured with prestimulated MGCs and thus pre-activating MGCs transforms mBECs into a proinflammatory phenotype.

WITHDRAWN: Tumor cell extracellular vesicles derived long non-coding RNA X-inactive specific transcript regulates the blood–brain barrier and non-small cell lung cancer brain metastasis via miR-19b-3p/EPN2

ObjectiveTo investigate the function of tumor cell extracellular vesicles (EVs) derived long non-coding RNA X-inactive specific transcript (XIST) in non-small cell lung cancer (NSCLC) brain metastasis and to clarify its specific mechanism. MethodsEVs were isolated from A549 cells, and transmission electron microscopy, nanoparticle tracking, and western blotting were used to detect morphology and particle size of EVs. Human brain microvascular endothelial cells (HBMEC) and astrocytes were used to construct an in vitro blood–brain barrier (BBB) model. The BBB model was treated with EVs for 24 h. PKH67 staining was performed to examine EV engulfment by HBMEC and astrocytes. RT-qPCR was used to examine XIST, miR-19b-3p, and epsin 2 (EPN2) mRNA expression. Expression of EPN2 and tight junction (TJ) proteins such as claudin-5, occludin, and ZO-1 were measured using western blotting. Transepithelial electrical resistance (TEER), rhodamine-dextran, and immunofluorescence staining were performed to explore the effects of EVs and XIST on the BBB. Transwell assay was used to evaluate the invasive ability of A549 and H1299 cells. Dual-luciferase and immunoprecipitation assays were used to verify binding between miR-19b-3p, XIST, and EPN2. The Interactive Video Information System was used to observe brain metastasis of xenografts in BALB/c nude mice. ResultsA549 cell-derived EVs exhibited typical characteristics of EVs. HBMEC ingested EVs in the BBB model. EVs treatment decreased TEER and TJ protein level and increased the permeability to rhodamine-dextran. XIST was overexpressed in A549 and H1299 cells. Knockdown of XIST in A549 cell-derived EVs alleviated BBB damage and reduced NSCLC cell invasion. In vivo, XIST inhibition repressed NSCLC brain metastasis by ameliorating the BBB. Mechanistically, XIST sponged miR-19b-3p and positively regulated EPN2 expression. miR-19b-3p inhibition weakened the protective effect of XIST inhibition on the BBB. ConclusionA549 cells EVs-derived XIST inhibition ameliorates BBB injury and inhibits NSCLC brain metastasis by regulating the miR-19b-3p/EPN2 axis.

Blood-brain barrier permeability increases with the differentiation of glioblastoma cells in vitro

BackgroundGlioblastoma multiforme (GBM) is an aggressive tumor, difficult to treat pharmacologically because of the blood-brain barrier (BBB), which is rich in ATP-binding cassette (ABC) transporters and tight junction (TJ) proteins. The BBB is disrupted within GBM bulk, but it is competent in brain-adjacent-to-tumor areas, where eventual GBM foci can trigger tumor relapse. How GBM cells influence the permeability of BBB is poorly investigated.MethodsTo clarify this point, we co-cultured human BBB models with 3 patient-derived GBM cells, after separating from each tumor the stem cell/neurosphere (SC/NS) and the differentiated/adherent cell (AC) components. Also, we set up cultures of BBB cells with the conditioned medium of NS or AC, enriched or depleted of IL-6. Extracellular cytokines were measured by protein arrays and ELISA. The intracellular signaling in BBB cells was measured by immunoblotting, in the presence of STAT3 pharmacological inhibitor or specific PROTAC. The competence of BBB was evaluated by permeability assays and TEER measurement.ResultsThe presence of GBM cells or their conditioned medium increased the permeability to doxorubicin, mitoxantrone and dextran-70, decreased TEER, down-regulated ABC transporters and TJ proteins at the transcriptional level. These effects were higher with AC or their medium than with NS. The secretome analysis identified IL-6 as significantly more produced by AC than by NS. Notably, AC-conditioned medium treated with an IL-6 neutralizing antibody reduced the BBB permeability to NS levels, while NS-conditioned medium enriched with IL-6 increased BBB permeability to AC levels. Mechanistically, IL-6 released by AC GBM cells activated STAT3 in BBB cells. In turn, STAT3 down-regulated ABC transporter and TJ expression, increased permeability and decreased TEER. The same effects were obtained in BBB cells treated with STA-21, a pharmacological inhibitor of STAT3, or with a PROTAC targeting STAT3.ConclusionsOur work demonstrates for the first time that the degree of GBM differentiation influences BBB permeability. The crosstalk between GBM cells that release IL-6 and BBB cells that respond by activating STAT3, controls the expression of ABC transporters and TJ proteins on BBB. These results may pave the way for novel therapeutic tools to tune BBB permeability and improve drug delivery to GBM.

In vitro characterization of taurine transport using the human brain microvascular endothelial cell line as a human blood-brain barrier model

Taurine, a sulfur-containing β-amino acid, has various roles in the brain including cellular osmoregulation and neuroprotection. For adequate supply to the brain, taurine has to pass through the blood-brain barrier (BBB); however, the associated mechanism behind crossing the human BBB is not fully understood. Therefore, we characterized taurine transport in vitro using the human brain microvascular endothelial (hCMEC/D3) cell line, a model of human BBB function. [3H]Taurine uptake by hCMEC/D3 cells exhibited time-, as well as extracellular Na+- and Cl−-dependence. The uptake was saturable with a Km of 19 μM and was inhibited by GABA at an IC50 of 328 μM, which were similar to Km values of taurine transporter (TauT)-mediated transport of taurine and GABA, respectively, suggesting that TauT is a major contributor to taurine uptake. For distribution to the brain, taurine must undergo cellular efflux after uptake. Taurine efflux from hCMEC/D3 cells increased for at least 60 min, and monocarboxylate transporter 7 (MCT7)-targeted siRNA significantly reduced MCT7 mRNA levels and [3H]taurine efflux by 93% and 12%, respectively, suggesting that MCT7 partly contributes to taurine efflux from hCMEC/D3 cells. Taken together, these results suggest that TauT and MCT7 function cooperatively in the human BBB.

Collagen I Microfiber Promotes Brain Capillary Network Formation in Three-Dimensional Blood-Brain Barrier Microphysiological Systems.

The blood-brain barrier (BBB) strictly regulates the penetration of substances into the brain, which, although important for maintaining brain homeostasis, may delay drug development because of the difficulties in predicting pharmacokinetics/pharmacodynamics (PKPD), toxicokinetics/toxicodynamics (TKTD), toxicity, safety, and efficacy in the central nervous system (CNS). Moreover, BBB functional proteins show species differences; therefore, humanized in vitro BBB models are urgently needed to improve the predictability of preclinical studies. Recently, international trends in the 3Rs in animal experiments and the approval of the FDA Modernization Act 2.0 have accelerated the application of microphysiological systems (MPSs) in preclinical studies, and in vitro BBB models have become synonymous with BBB-MPSs. Recently, we developed an industrialized humanized BBB-MPS, BBB-NET. In our previous report, we reproduced transferrin receptor (TfR)-mediated transcytosis with high efficiency and robustness, using hydrogels including fibrin and collagen I microfibers (CMFs). We investigated how adding CMFs to the fibrin gel benefits BBB-NETs. We showed that CMFs accelerate capillary network formation and maturation by promoting astrocyte (AC) survival, and clarified that integrin β1 is involved in the mechanism of CMFs. Our data suggest that the quality control (QC) of CMFs is important for ensuring the stable production of BBB-NETs.

Neurons enhance blood–brain barrier function via upregulating claudin-5 and VE-cadherin expression due to glial cell line-derived neurotrophic factor secretion

Blood–brain barrier (BBB) prevents neurotoxins from entering central nervous system. We aimed to establish and characterize an in vitro triple co-culture BBB model consisting of brain endothelial cells hCMEC/D3, astrocytoma U251 cells, and neuroblastoma SH-SY5Y cells. Co-culture of SH-SY5Y and U251 cells markedly enhanced claudin-5 and VE-cadherin expression in hCMEC/D3 cells, accompanied by increased transendothelial electrical resistance and decreased permeability. Conditioned medium (CM) from SH-SY5Y cells (S-CM), U251 cells (U-CM), and co-culture of SH-SY5Y and U251 cells (US-CM) also promoted claudin-5 and VE-cadherin expression. Glial cell line-derived neurotrophic factor (GDNF) levels in S-CM and US-CM were significantly higher than CMs from hCMEC/D3 and U-CM. Both GDNF and US-CM upregulated claudin-5 and VE-cadherin expression, which were attenuated by anti-GDNF antibody and GDNF signaling inhibitors. GDNF increased claudin-5 expression via the PI3K/AKT/FOXO1 and MAPK/ERK pathways. Meanwhile, GDNF promoted VE-cadherin expression by activating PI3K/AKT/ETS1 and MAPK/ERK/ETS1 signaling. The roles of GDNF in BBB integrity were validated using brain-specific Gdnf silencing mice. The developed triple co-culture BBB model was successfully applied to predict BBB permeability. In conclusion, neurons enhance BBB integrity by upregulating claudin-5 and VE-cadherin expression through GDNF secretion and established triple co-culture BBB model may be used to predict drugs’ BBB permeability.

Neurons enhance blood-brain barrier function via upregulating claudin-5 and VE-cadherin expression due to glial cell line-derived neurotrophic factor secretion.

Blood-brain barrier (BBB) prevents neurotoxins from entering central nervous system. We aimed to establish and characterize an in vitro triple co-culture BBB model consisting of brain endothelial cells hCMEC/D3, astrocytoma U251 cells, and neuroblastoma SH-SY5Y cells. Co-culture of SH-SY5Y and U251 cells markedly enhanced claudin-5 and VE-cadherin expression in hCMEC/D3 cells, accompanied by increased transendothelial electrical resistance and decreased permeability. Conditioned medium (CM) from SH-SY5Y cells (S-CM), U251 cells (U-CM), and co-culture of SH-SY5Y and U251 cells (US-CM) also promoted claudin-5 and VE-cadherin expression. Glial cell line-derived neurotrophic factor (GDNF) levels in S-CM and US-CM were significantly higher than CMs from hCMEC/D3 and U-CM. Both GDNF and US-CM upregulated claudin-5 and VE-cadherin expression, which were attenuated by anti-GDNF antibody and GDNF signaling inhibitors. GDNF increased claudin-5 expression via the PI3K/AKT/FOXO1 and MAPK/ERK pathways. Meanwhile, GDNF promoted VE-cadherin expression by activating PI3K/AKT/ETS1 and MAPK/ERK/ETS1 signaling. The roles of GDNF in BBB integrity were validated using brain-specific Gdnf silencing mice. The developed triple co-culture BBB model was successfully applied to predict BBB permeability. In conclusion, neurons enhance BBB integrity by upregulating claudin-5 and VE-cadherin expression through GDNF secretion and established triple co-culture BBB model may be used to predict drugs' BBB permeability.

Oxime Linked Doxorubicin Glycoconjugates Improve the Specific Targeting of Glioblastoma in High-Grade Glioma Therapy.

The treatment of glioblastoma (GBM) represents an urgent challenge for public health due to the inability to effectively deliver anticancer agents, such as doxorubicin (DOX), through the blood-brain barrier (BBB). Herein we report the synthesis of two novel DOX glycoconjugates using an oxime linkage that maintained the intercalation capability of the planar anthracycline ring of DOX, as demonstrated by UV-vis and fluorescence experiments in the presence of DNA. The biological effect of DOX glycoconjugates was evaluated in GBM cell lines, showing an enhanced cytotoxic and pro-apoptotic effect of 7 as compared to 4 and to conventional DOX. These data were confirmed in an in vitro coculture BBB model in which DOX glycoconjugate 7 showed high capability to cross a cellular monolayer and exert its cytotoxic effect on GBM cells. The results show that conjugation with glucose may represent a helpful tool to increase chemotherapy effectiveness in poor-responding GBM patients.

Involvement of Proton-Coupled Organic Cation Antiporter in Human Blood-Brain Barrier Transport of Mesoridazine and Metoclopramide.

Mesoridazine and metoclopramide are cationic drugs that are distributed in the human brain despite being substrates of multidrug resistance protein 1 (MDR1), an efflux transporter expressed at the blood-brain barrier (BBB). We investigated their transport mechanisms at the BBB using hCMEC/D3, a human cerebral microvascular endothelial cell line often used as an in vitro BBB model. The cells exhibited time- and concentration-dependent uptake of mesoridazine and metoclopramide, with Km values of 34 and 277 µM, respectively. The uptake of both drugs significantly decreased in the presence of typical inhibitors and/or substrates of the H+-coupled organic cation (H+/OC) antiporter but not in the presence of inhibitors or substrates of organic cation transporters (OCTs), OCTN2, OATPs, SLC35F2, or the plasma membrane monoamine transporter (PMAT). Furthermore, metoclopramide uptake by hCMEC/D3 cells was pH- and energy-dependent, whereas mesoridazine uptake was unaffected by intracellular acidification and treatment with metabolic inhibitors. These results suggest that the H+/OC antiporter is involved in the influx of mesoridazine and metoclopramide into the brain across the BBB.