Blood-brain Barrier Model Research Articles

Abstract 1449: Targeting squalene epoxidase for breast cancer brain metastasis: The evaluation of a new therapeutic target for brain extravasation and colonization using animal models

Abstract Breast cancer brain metastasis is one of the most common forms of breast cancer metastasis and a major cause of morbidity and mortality. Patients who develop brain metastases tend to be associated with progressive neurologic deficits and short overall survival, which represents an unmet medical need. Extensive research has been conducted to elucidate the mechanism of cancer metastasis with limited information gained as to how cancer cells extravasate and colonize to the brain. Recently, we identified a novel mechanism by which squalene epoxidase (SQLE), the second rate-limiting enzyme in cholesterol biosynthesis, plays a critical role in the process of breast cancer metastasis to the brain. Thus, toward a better understanding of the underlying molecular mechanisms and applications of molecular target(s) for brain metastasis therapy, murine models were employed for investigation of brain extravasation and colonization with intracardiac or direct intracranial injections of GFP/FLuc-labeled human MDA-MB-231 metastatic breast carcinoma cells. The MDA-MB-231 cells were found to metastasize to the brain and other organs following intracardiac implantation into nude mice and were further isolated from each tissue. Interestingly, the isolated brain-metastasized tumor cells (MDA-MB-231-BrM) exhibited a significant upregulation of SQLE expression when compared with those that metastasized to other distal sites. The essential role of SQLE in the specific steps of the breast cancer-to-brain metastatic process was evaluated by ex vivo immunofluorescence analysis and immunohistochemistry staining of brain slices from the study subjects. Our data demonstrate that high SQLE expression is essential for MDA-MB-231-BrM to extravasate into the parenchyma, as well as the formation of micro- and macro-metastases in the brain. Interestingly, we found that blood vessel co-option and the surrounding neuronal cells play key roles in supporting MDA-MB-231-BrM to develop brain macro-metastases. In vitro blood-brain barrier (BBB) models further demonstrated the critical role of SQLE in promoting tumor cell invasion and penetration through the BBB. To verify SQLE as an oncogenic factor that can be selected as a potential therapeutic target in suppressing breast cancer brain metastasis, we evaluated the inhibitory effects of terbinafine (a SQLE inhibitor) in both the MDA-MB-231-BrM orthotopic and intracardiac mouse models. Pharmacologic inhibition of SQLE by terbinafine suppressed MDA-MB-231-BrM tumor growth at the mammary fat pad and distal metastases to the brain, suggesting that targeting SQLE represents a therapeutic opportunity for breast cancer brain metastasis. Citation Format: Pony Yu-Ling Lee, Kun-Yuan Lin, Chao-Di Chang, Shan-Yun Cheng, Ya-Wen Hung, Chih-Chieh Yang, Yu-Hsien Chang, Chien-Chang Shen, Jia-Yun Yeh, Yung-Lung Yu, Shu-Ping Wang. Targeting squalene epoxidase for breast cancer brain metastasis: The evaluation of a new therapeutic target for brain extravasation and colonization using animal models [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 1449.

Abstract 1550: Identification of a novel squalene epoxidase function in breast cancer brain metastasis

Abstract Brain metastases are serious complications of breast cancer, especially in triple-negative breast cancer (TNBC) patients. There is currently no effective treatment due to the unique brain microenvironment and limitation for drugs to cross the blood-brain barrier (BBB). BBB limits access of nutrients from the circulation and thereby makes the brain hypoxic and depleted of metabolites, growth factors and proteins. However, it remains poorly understood how breast cancer cells manipulate for adaption and colonization in the brain. To probe the molecular mechanism of breast cancer brain metastasis (BCBM), we compared gene signatures in primary breast tumors and BCBM tumors, and identified that SQLE (encodes squalene epoxidase, the second rate-limiting enzyme in the cholesterol biosynthesis) could be a leading-edge gene in breast cancer brain metastasis. To explore the role of SQLE in BCBM, we established the brain metastatic TNBC MDA-MB-231 (MDA231-BrM) cell line from brain metastatic subpopulation that originated from its parental MDA-MB-231 (MDA231) cell line. We found that SQLE expression was greatly upregulated in MDA231-BrM cells compared with the parental MDA231 cells. Using MDA231-BrM and MDA231 cell lines expressing SQLE shRNAs, we evaluated the effects of SQLE loss on cancer cell migration, invasion, and stemness by wound-healing, transwell invasion/migration, and tumorsphere formation assays. While loss of SQLE greatly attenuated cell invasiveness and stemness in both MDA231 and MDA231-BrM cells, loss of SQLE could only affect the cell migration activity on MDA231 cells but not MDA231-BrM cells. Our RNA-seq data further identified a subset of SQLE-affected genes that is uniquely enriched in MDA231-BrM cells and favors brain extravasation and colonization. To explore the potential function of SQLE in brain extravasation and colonization, we established in vitro BBB models and ex vivo mouse brain slice organotypic cultures. We showed that loss of SQLE inhibited the ability of MDA231-BrM cells to across the BBB-mimic astrocyte-endothelial structures as well as impaired the co-option with blood capillaries in the mouse brain slices. Although SQLE-deficient MDA231-BrM cells could still spread on the surface of the blood vessels, they seemed to undergo apoptosis. Since the ability to invade, migrate, and penetrate is critical for invasion of cancer cells, our results strongly imply the novel function of SQLE in breast cancer cell invasion, penetration, and even colonization in the brain through blood vessel co-option. In summary, our data reveal a novel role for SQLE in two critical requisites for breast-to-brain extravasation and colonization - the ability to penetrate through BBB and to co-opt brain vessels for metastatic expansion. Our findings indicate that targeting SQLE may represent a therapeutic opportunity for breast cancer brain metastases. Citation Format: Shu-Ping Wang, Jia-Yun Yeh, Yu-Ling (Pony) Lee, Kun-Yuan Lin, Chao-Di Chang, Chih-Chieh Yang, Shan-Yun Cheng, Ya-Wen Hung, Yu-Hsien Chang, Yao-Feng Li, Yung-Lung Yu. Identification of a novel squalene epoxidase function in breast cancer brain metastasis [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 1550.

Protecting the Brain: Novel Strategies for Preventing Breast Cancer Brain Metastases through Selective Estrogen Receptor β Agonists and In Vitro Blood-Brain Barrier Models.

Breast cancer brain metastasis (BCBM) is a challenging condition with limited treatment options and poor prognosis. Understanding the interactions between tumor cells and the blood-brain barrier (BBB) is critical for developing novel therapeutic strategies. One promising target is estrogen receptor β (ERβ), which promotes the expression of key tight junction proteins, sealing the BBB and reducing its permeability. In this study, we investigated the effects of 17β-estradiol (E2) and the selective ERβ agonist diarylpropionitrile (DPN) on endothelial and cancer cells. Western blot analysis revealed the expression patterns of ERs in these cell lines, and estrogen treatment upregulated claudin-5 expression in brain endothelial cells. Using in vitro models of the BBB, we found that DPN treatment significantly increased BBB tightness about suppressed BBB transmigration activity of representative Her2-positive (BT-474) and triple-negative (MDA-MB-231) breast cancer cell lines. However, the efficacy of DPN treatment decreased when cancer cells were pre-differentiated in the presence of E2. Our results support ERβ as a potential target for the prevention and treatment of BCBM and suggest that targeted vector-based approaches may be effective for future preventive and therapeutic implications.

Empirical analysis of lead neurotoxicity mode of action and its application in health risk assessment

The mode of action (MOA) framework is proposed to inform a biological link between chemical exposures and adverse health effects. Despite a significant increase in knowledge and awareness, the application of MOA in human health risk assessment (RA) remains limited. This study aims to discuss the adoption of MOA for health RA within a regulatory context, taking our previously proposed but not yet validated MOA for lead neurotoxicity as an example. We first conducted a quantitative weight of evidence (qWOE) assessment, which revealed that the MOA has a moderate confidence. Then, targeted bioassays were performed within an in vitro blood-brain barrier (BBB) model to quantitatively validate the scientific validity of key events (KEs) in terms of essentiality and concordance of empirical support (dose/temporal concordance), which increases confidence in utilizing the MOA for RA. Building upon the quantitative validation data, we further conducted benchmark dose (BMD) analysis to map dose-response relationships for the critical toxicity pathways, and the lower limit of BMD at a 5% response (BMDL5) was identified as the point of departure (POD) value for adverse health effects. Notably, perturbation of the Aryl Hydrocarbon Receptor (AHR) signaling pathway exhibited the lowest POD value, measured at 0.0062 μM. Considering bioavailability, we further calculated a provisional health-based guidance value (HBGV) for children's lead intake, determining it to be 2.56 μg/day. Finally, the health risk associated with the HBGV was assessed using the hazard quotient (HQ) approach, which indicated that the HBGV established in this study is a relative safe reference value for lead intake. In summary, our study described the procedure for utilizing MOA in health RA and set an example for MOA-based human health risk regulation.

Patient-Derived Blood-Brain Barrier Model for Screening Copper Bis(thiosemicarbazone) Complexes as Potential Therapeutics in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent cause of dementia characterized by a progressive cognitive decline. Addressing neuroinflammation represents a promising therapeutic avenue to treat AD; however, the development of effective antineuroinflammatory compounds is often hindered by their limited blood-brain barrier (BBB) permeability. Consequently, there is an urgent need for accurate, preclinical AD patient-specific BBB models to facilitate the early identification of immunomodulatory drugs capable of efficiently crossing the human AD BBB. This study presents a unique approach to BBB drug permeability screening as it utilizes the familial AD patient-derived induced brain endothelial-like cell (iBEC)-based model, which exhibits increased disease relevance and serves as an improved BBB drug permeability assessment tool when compared to traditionally employed in vitro models. To demonstrate its utility as a small molecule drug candidate screening platform, we investigated the effects of diacetylbis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(atsm)) and a library of metal bis(thiosemicarbazone) complexes─a class of compounds exhibiting antineuroinflammatory therapeutic potential in neurodegenerative disorders. By evaluating the toxicity, cellular accumulation, and permeability of those compounds in the AD patient-derived iBEC, we have identified 3,4-hexanedione bis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(dtsm)) as a candidate with good transport across the AD BBB. Furthermore, we have developed a multiplex approach where AD patient-derived iBEC were combined with immune modulators TNFα and IFNγ to establish an in vitro model representing the characteristic neuroinflammatory phenotype at the patient's BBB. Here, we observed that treatment with CuII(dtsm) not only reduced the expression of proinflammatory cytokine genes but also reversed the detrimental effects of TNFα and IFNγ on the integrity and function of the AD iBEC monolayer. This suggests a novel pathway through which copper bis(thiosemicarbazone) complexes may exert neurotherapeutic effects on AD by mitigating BBB neuroinflammation and related BBB integrity impairment. Together, the presented model provides an effective and easily scalable in vitro BBB platform for screening AD drug candidates. Its improved translational potential makes it a valuable tool for advancing the development of metal-based compounds aimed at modulating neuroinflammation in AD.

Co-Culture Models: Key Players in In Vitro Neurotoxicity, Neurodegeneration and BBB Modeling Studies.

The biological barriers existing in the human body separate the blood circulation from the interstitial fluid in tissues. The blood-brain barrier (BBB) isolates the central nervous system from the bloodstream, presenting a dual role: the protection of the human brain against potentially toxic/harmful substances coming from the blood, while providing nutrients to the brain and removing metabolites. In terms of architectural features, the presence of junctional proteins (that restrict the paracellular transport) and the existence of efflux transporters at the BBB are the two major in vivo characteristics that increase the difficulty in creating an ideal in vitro model for drug permeability studies and neurotoxicity assessments. The purpose of this work is to provide an up-to-date literature review on the current in vitro models used for BBB studies, focusing on the characteristics, advantages, and disadvantages of both primary cultures and immortalized cell lines. An accurate analysis of the more recent and emerging techniques implemented to optimize the in vitro models is also provided, based on the need of recreating as closely as possible the BBB microenvironment. In fact, the acceptance that the BBB phenotype is much more than endothelial cells in a monolayer has led to the shift from single-cell to multicellular models. Thus, in vitro co-culture models have narrowed the gap between recreating as faithfully as possible the human BBB phenotype. This is relevant for permeability and neurotoxicity assays, and for studies related to neurodegenerative diseases. Several studies with these purposes will be also presented and discussed.

A dynamic study of VEGF-A siDOX-EVs trafficking through the in-vitro insert co-culture blood-brain barrier model by digital holographic microscopy.

Modeling the blood-brain barrier has long been a challenge for pharmacological studies. Up to the present, numerous attempts have been devoted to recapitulating the endothelial barrier in vitro to assess drug delivery vehicles' efficiency for brain disorders. In the current work, we presented a new approach for analyzing the morphometric parameters of the cells of an insert co-culture blood-brain barrier model using rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes. This analytical approach could aid in getting further information on drug trafficking through the blood-brain barrier and its impact on the brain indirectly. In the current work, we cultured rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes and then used an insert well to culture the cells in contact with each other to model the blood-brain barrier. Then, the morphometric parameters of the porous membrane of the insert well, as well as each cell type were imaged by digital holographic microscopy before and after cell seeding. At last, we performed folate conjugation on the surface of the EVs we have previously tested for glioma therapy in our previous work called VEGF-A siDOX-EVs and checked how the trafficking of EVs improves after folate conjugation as a clathrin-mediated delivery setup. the trafficking and passage of EVs were assessed by flowcytometry and morphometric analysis of the digital holographic microscopy holograms. Our results indicated that EVs successfully entered through the proposed endothelial barrier assessed by flow cytometry analysis and furthermore, folate conjugation significantly improved EV passage through the blood-brain barrier. Moreover, our results indicated that the VEGF-A siDOX-EVs insert cytotoxic impact on the cells of the bottom of the culture plate. folate-conjugation on the surface of EVs improves their trafficking through the blood-brain barrier and by using digital holographic microscopy analysis, we could directly assess the morphometric changes of the blood-brain barrier cells for pharmacological purposes as an easy, label-free, and real-time analysis.

Anti-Inflammatory Action of Resveratrol in the Central Nervous System in Relation to Glucose Concentration-An In Vitro Study on a Blood-Brain Barrier Model.

Unbalanced blood glucose levels may cause inflammation within the central nervous system (CNS). This effect can be reversed by the action of a natural neuroprotective compound, resveratrol (RSV). The study aimed to investigate the anti-inflammatory effect of RSV on astrocyte cytokine profiles within an in vitro model of the blood-brain barrier (BBB) under varying glucose concentrations (2.2, 5.0, and 25.0 mmol/L), corresponding to hypo-, normo-, and hyperglycemia. The model included co-cultures of astrocytes (brain compartment, BC) and endothelial cells (microvascular compartment, MC), separated by 0.4 µm wide pores. Subsequent exposure to 0.2 μM LPS in the brain compartment (BC) and 50 μM RSV in the microvascular compartment (MC) of each well was carried out. Cytokine levels (IL-1 α, IL-1 β, IL-2, IL-4, IL-6, IL-8) in the BC were assessed using a Multi-Analyte ELISArray Kit before and after the addition of LPS and RSV. Statistical analysis was performed to determine significance levels. The results demonstrated that RSV reduced the concentration of all studied cytokines in the BC, regardless of glucose levels, with the most substantial decrease observed under normoglycemic conditions. Additionally, the concentration of RSV in the BC was highest under normoglycemic conditions compared to hypo- and hyperglycemia. These findings confirm that administration of RSV in the MC exerts anti-inflammatory effects within the BC, particularly under normoglycemia-simulating conditions. Further in vivo studies, including animal and human research, are warranted to elucidate the bioavailability of RSV within the central nervous system (CNS).

Post-translational modifications of beta-amyloid alter its transport in the blood-brain barrier in vitro model.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of beta-amyloid peptide (Aβ) leading to formation of soluble neurotoxic Aβ oligomers and insoluble amyloid plaques in various parts of the brain. Aβ undergoes post-translational modifications that alter its pathogenic properties. Aβ is produced not only in brain, but also in the peripheral tissues. Such Aβ, including its post-translationally modified forms, can enter the brain from circulation by binding to RAGE and contribute to the pathology of AD. However, the transport of modified forms of Aβ across the blood-brain barrier (BBB) has not been investigated. Here, we used a transwell BBB model as a controlled environment for permeability studies. We found that Aβ42 containing isomerized Asp7 residue (iso-Aβ42) and Aβ42 containing phosphorylated Ser8 residue (pS8-Aβ42) crossed the BBB better than unmodified Aβ42, which correlated with different contribution of endocytosis mechanisms to the transport of these isoforms. Using microscale thermophoresis, we observed that RAGE binds to iso-Aβ42 an order of magnitude weaker than to Aβ42. Thus, post-translational modifications of Aβ increase the rate of its transport across the BBB and modify the mechanisms of the transport, which may be important for AD pathology and treatment.

Modular tissue-in-a-CUBE platform to model blood-brain barrier (BBB) and brain interaction

With the advent of increasingly sophisticated organoids, there is growing demand for technology to replicate the interactions between multiple tissues or organs. This is challenging to achieve, however, due to the varying culture conditions of the different cell types that make up each tissue. Current methods often require complicated microfluidic setups, but fragile tissue samples tend not to fare well with rough handling. Furthermore, the more complicated the human system to be replicated, the more difficult the model becomes to operate. Here, we present the development of a multi-tissue chip platform that takes advantage of the modularity and convenient handling ability of a CUBE device. We first developed a blood-brain barrier-in-a-CUBE by layering astrocytes, pericytes, and brain microvascular endothelial cells in the CUBE, and confirmed the expression and function of important tight junction and transporter proteins in the blood-brain barrier model. Then, we demonstrated the application of integrating Tissue-in-a-CUBE with a chip in simulating the in vitro testing of the permeability of a drug through the blood-brain barrier to the brain and its effect on treating the glioblastoma brain cancer model. We anticipate that this platform can be adapted for use with organoids to build complex human systems in vitro by the combination of multiple simple CUBE units.

Gingipains may be one of the key virulence factors of Porphyromonas gingivalis to impair cognition and enhance blood-brain barrier permeability: An animal study.

Blood-brain barrier (BBB) disorder is one of the early findings in cognitive impairments. We have recently found that Porphyromonas gingivalis bacteraemia can cause cognitive impairment and increased BBB permeability. This study aimed to find out the possible key virulence factors of P. gingivalis contributing to the pathological process. C57/BL6 mice were infected with P. gingivalis or gingipains or P. gingivalis lipopolysaccharide (P. gingivalis LPS group) by tail vein injection for 8 weeks. The cognitive behaviour changes in mice, the histopathological changes in the hippocampus and cerebral cortex, the alternations of BBB permeability, and the changes in Mfsd2a and Cav-1 levels were measured. The mechanisms of Ddx3x-induced regulation on Mfsd2a by arginine-specific gingipain A (RgpA) in BMECs were explored. P. gingivalis and gingipains significantly promoted mice cognitive impairment, pathological changes in the hippocampus and cerebral cortex, increased BBB permeability, inhibited Mfsd2a expression and up-regulated Cav-1 expression. After RgpA stimulation, the permeability of the BBB model in vitro increased, and the Ddx3x/Mfsd2a/Cav-1 regulatory axis was activated. Gingipains may be one of the key virulence factors of P. gingivalis to impair cognition and enhance BBB permeability by the Ddx3x/Mfsd2a/Cav-1 axis.

Nanoparticles Coated with Brain Microvascular Endothelial Cell Membranes can Target and Cross the Blood-Brain Barrier to Deliver Drugs to Brain Tumors.

The blood-brain barrier (BBB) contains tightly connected brain microvascular endothelial cells (BMECs) that hinder drug delivery to the brain, which makes brain tumors difficult to treat. Previous studies have shown that nanoparticles coated with tumor cell membranes selectively target their homologous tumors. Therefore, this study investigated whether bEnd.3-line BMEC membrane-coated nanoparticles with poly(lactide-co-glycolide)-poly(ethylene glycol)-based doxorubicin-loaded cores (BM-PDs) can be used to target BMECs and cross the BBB. In vitro, the BM-PDs effectively target BMECs and cross a BBB model. The BM-PDs enter the BMECs via macropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and membrane fusion, which result in excellent cellular uptake. The BM-PDs also show excellent cellular uptake in brain tumor cells. In vivo, the BM-PDs target BMECs, cross the BBB, accumulate in brain tumors, and efficiently kill tumor cells. Therefore, the proposed strategy has great therapeutic potential owing to its ability to cross the BBB to reach brain tumors.

Inhibiting ferroptosis in brain microvascular endothelial cells: A potential strategy to mitigate polystyrene nanoplastics‒induced blood‒brain barrier dysfunction

Polystyrene nanoplastics (PS-NPs), a group of ubiquitous pollutants, may injure the central nervous system through the blood‒brain barrier (BBB). However, whether exposure to PS-NPs contributes to BBB disruption and the underlying mechanisms are still unclear. In vivo, we found that PS-NPs (25 mg/kg BW) could significantly increase BBB permeability in mice and downregulate the distribution of the tight junction-associated protein zona occludens 1 (ZO-1) in brain microvascular endothelial cells (BMECs). Using an in vitro BBB model, exposure to PS-NPs significantly reduced the transendothelial electrical resistance and altered ZO-1 expression and distribution in a dose-dependent manner. RNA-seq analysis and functional investigations were used to investigate the molecular pathways involved in the response to PS-NPs. The results revealed that the ferroptosis and glutathione metabolism signaling pathways were related to the disruption of the BBB model caused by the PS-NPs. PS-NPs treatment promoted ferroptosis in bEnd.3 cells by inducing disordered glutathione metabolism in addition to Fe2+ and lipid peroxide accumulation, while suppressing ferroptosis with ferrostatin-1 (Fer-1) suppressed ferroptosis-related changes in bEnd.3 cells subjected to PS-NPs. Importantly, Fer-1 alleviated the decrease in ZO-1 expression in bEnd.3 cells and the exacerbation of BBB damage induced by PS-NPs. Collectively, our findings suggest that inhibiting ferroptosis in BMECs may serve as a potential therapeutic target against BBB disruption induced by PS-NPs exposure.

Dahuang-Wumei decoction protects against hepatic encephalopathy in mice: Behavioural, biochemical, and molecular evidence

BackgroundDisturbance of the blood‒brain barrier (BBB) and associated inflammatory responses are observed in patients with hepatic encephalopathy (HE) and can cause long-term complications. Dahuang-Wumei decoction (DWD) is a renowned traditional Chinese herbal medicine with a long history of clinical use and has been widely employed as an effective treatment for hepatic encephalopathy (HE). Despite its established efficacy, the precise mechanisms underlying the therapeutic effects of DWD have not been fully elucidated. PurposeThe present study aimed to comprehensively explore the potential effects and underlying molecular mechanisms of DWD on HE through an integrated investigation that included both in vivo and in vitro experiments. MethodsIn the present study, carbon tetrachloride (CCl4) and thioacetamide (TAA) were used to establish an HE model in mice. The therapeutic effects of DWD on liver injury, fibrosis, brain injury, behaviour, and consciousness disorders were evaluated in vivo. C8-D1A and bEnd.3 cells were used to construct a BBB model in vitro. The effects of DWD on proinflammatory factor expression, BBB damage and the Wnt/β-catenin pathway were detected in vivo and in vitro. ResultsOur results showed that DWD can improve liver injury and fibrosis and brain damage and inhibit neurofunctional and behavioural disorders in mice with HE. Afterwards, we found that DWD decreased the levels of proinflammatory factors and suppressed BBB disruption by increasing the levels of junction proteins in vivo and vitro. Further studies verified that the Wnt/β-catenin pathway may play a pivotal role in mediating the inhibitory effect of DWD on HE. ConclusionThese results demonstrated that DWD can treat HE by preventing BBB disruption, and the underlying mechanisms involved were associated with the activation of the Wnt/β-catenin pathway and the inhibition of inflammatory responses.

Dual fluorescence images, transport pathway, and blood–brain barrier penetration of B-Met-W/O/W SE

Borneol is an aromatic traditional Chinese medicine that can improve the permeability of the blood–brain barrier (BBB), enter the brain, and promote the brain tissue distribution of many other drugs. In our previous study, borneol-metformin hydrochloride water/oil/water composite submicron emulsion (B-Met-W/O/W SE) was prepared using borneol and SE to promote BBB penetration, which significantly increased the brain distribution of Met. However, the dynamic images, transport pathway (uptake and efflux), promotion of BBB permeability, and mechanisms of B-Met-W/O/W SE before and after entering cells have not been clarified. In this study, rhodamine B and coumarin-6 were selected as water-soluble and oil-soluble fluorescent probes to prepare B-Met-W/O/W dual-fluorescent SE (B-Met-W/O/W DFSE) with concentric circle imaging. B-Met-W/O/W SE can be well taken up by brain microvascular endothelial cells (BMECs). The addition of three inhibitors (chlorpromazine hydrochloride, methyl-β-cyclodextrin, and amiloride hydrochloride) indicated that its main pathway may be clathrin-mediated and fossa protein-mediated endocytosis. Meanwhile, B-Met-W/O/W SE was obviously shown to inhibit the efflux of BMECs. Next, BMECs were cultured in the Transwell chamber to establish a BBB model, and Western blot was employed to detect the protein expressions of Occludin, Zona Occludens 1 (ZO-1), and p-glycoprotein (P-gp) after B-Met-W/O/W SE treatment. The results showed that B-Met-W/O/W SE significantly down-regulated the expression of Occludin, ZO-1, and P-gp, which increased the permeability of BBB, promoted drug entry into the brain through BBB, and inhibited BBB efflux. Furthermore, 11 differentially expressed genes (DEGs) and 7 related signaling pathways in BMECs treated with B-W/O/W SE were detected by transcriptome sequencing and verified by quantitative real-time polymerase chain reaction (qRT-PCR). These results provide a scientific experimental basis for the dynamic monitoring, transmembrane transport mode, and permeation-promoting mechanism of B-Met-W/O/W SE as a new brain-targeting drug delivery system.

Systemic delivery of mutant huntingtin lowering antisense oligonucleotides to the brain using apolipoprotein A-I nanodisks for Huntington disease

Efficient delivery of therapeutics to the central nervous system (CNS) remains a major challenge for the treatment of neurological diseases. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion mutation in the HTT gene which codes for a toxic mutant huntingtin (mHTT) protein. Pharmacological reduction of mHTT in the CNS using antisense oligonucleotides (ASO) ameliorates HD-like phenotypes in rodent models of HD, with such therapies being investigated in clinical trials for HD.In this study, we report the optimization of apolipoprotein A-I nanodisks (apoA-I NDs) as vehicles for delivery of a HTT-targeted ASO (HTT ASO) to the brain and peripheral organs for HD. We demonstrate that apoA-I wild type (WT) and the apoA-I K133C mutant incubated with a synthetic lipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, can self-assemble into monodisperse discoidal particles with diameters <20 nm that transmigrate across an in vitro blood-brain barrier model of HD. We demonstrate that apoA-I NDs are well tolerated in vivo, and that apoA-I K133C NDs show enhanced distribution to the CNS and peripheral organs compared to apoA-I WT NDs following systemic administration. ApoA-I K133C conjugated with HTT ASO forms NDs (HTT ASO NDs) that induce significant mHTT lowering in the liver, skeletal muscle and heart as well as in the brain when delivered intravenously in the BACHD mouse model of HD. Furthermore, HTT ASO NDs increase the magnitude of mHTT lowering in the striatum and cortex compared to HTT ASO alone following intracerebroventricular administration. These findings demonstrate the potential utility of apoA-I NDs as biocompatible vehicles for enhancing delivery of mutant HTT lowering ASOs to the CNS and peripheral organs for HD.

A novel study of brain microvascular endothelial cells induced by astrocyte conditioned medium for constructing blood brain barrier model in vitro: A promising tool for meningitis of teleost

The blood-brain barrier (BBB) is mainly composed of specialized endothelial cells, which can resist harmful substances, transport nutrients, and maintain the stability of the brain environment. In this study, an endothelial cell line from tilapia (Oreochromis niloticus) named TVEC-01 was successfully established. During the earlier establishment phase of the cell line, the TVEC-01 cells were persistently exposed to an astrocyte-conditioned medium (ACM). TVEC-01 cells were identified as an endothelial cell line. TVEC-01 cells retained the multiple functions of endothelial cells and were capable of performing various experiments in vitro. Furthermore, TVEC-01 cells efficiently expressed BBB-related tight junctions and key efflux transporters. From the results of the qRT-PCR, we found that the TVEC-01 cell line did not gradually lose BBB characteristics after persistent and repetitive passages, which was different from the vast majority of immortalized endothelial cells. The results showed that ACM induced up-regulation of the expression levels of multiple BBB-related genes in TVEC-01 cells. We confirmed that Streptococcus agalactiae was capable of invading the TVEC-01 cells and initiating a series of immune responses, which provided a theoretical basis for S. agalactiae to break through the BBB of teleost through the transcellular traversal pathway. In summary, we have successfully constructed an endothelial cell line of teleost, named TVEC-01, which can be used in many experiments in vitro and even for constructing BBB in vitro. Moreover, it was confirmed that S. agalactiae broke through the BBB of teleost through the transcellular traversal pathway and caused meningitis.

Enhanced macromolecular substance extravasation through the blood-brain barrier via acoustic bubble-cell interactions

The blood–brain barrier (BBB) maintains brain homeostasis, regulates influx and efflux transport, and provides protection to the brain tissue. Ultrasound (US) and microbubble (MB)-mediated blood–brain barrier opening is an effective and safe technique for drug delivery in-vitro and in-vivo. However, the exact mechanism underlying this technique is still not fully elucidated. The aim of the study is to explore the contribution of transcytosis in the BBB transient opening using an in-vitro model of BBB. Utilizing a diverse set of techniques, including Ca2+ imaging, electron microscopy, and electrophysiological recordings, our results showed that the combined use of US and MBs triggers membrane deformation within the endothelial cell membrane, a phenomenon primarily observed in the US + MBs group. This deformation facilitates the vesicles transportation of 500 kDa fluorescent Dextran via dynamin-/caveolae-/clathrin- mediated transcytosis pathway. Simultaneously, we observed increase of cytosolic Ca2+ concentration, which is related with increased permeability of the 500 kDa fluorescent Dextran in-vitro. This was found to be associated with the Ca2+-protein kinase C (PKC) signaling pathway. The insights provided by the acoustically-mediated interaction between the microbubbles and the cells delineate potential mechanisms for macromolecular substance permeability.

Borneol-Modified Schisandrin B Micelles Cross the Blood-Brain Barrier To Treat Alzheimer's Disease in Aged Mice.

Objective: Schisandrin B (Sch B) is a bioactive dibenzocyclooctadiene derizative that is prevalent in the fruit of Schisandra chinensis. Numerous studies have demonstrated that Sch B has a neuroprotective action by reducing oxidative stress and effectively preventing inflammation. It follows that Sch B is a potential treatment for Alzheimer's disease (AD). However, the drug's solubility, bioavailability, and lower permeability of the blood-brain barrier (BBB) can all reduce its efficacy during the therapy process. Therefore, this study constructed borneol-modified schisandrin B micelles (Bor-Sch B-Ms), which increase brain targeting by accurately delivering medications to the brain, effectively improving bioavailability. High therapeutic efficacy has been achieved at the pathological site. Methods: Bor-Sch B-Ms were prepared using the thin film dispersion approach in this article. On the one hand, to observe the targeting effect of borneol, we constructed a blood-brain barrier (BBB) model in vitro and studied the ability of micelles to cross the BBB. On the other hand, the distribution of micelle drugs and their related pharmacological effects on neuroinflammation, oxidative stress, and neuronal damage were studied through in vivo administration in mice. Results: In vitro studies have demonstrated that the drug uptake of bEnd.3 cells was increased by the borneol alteration on the surface of the nano micelles, implying that Bor-Sch B-Ms can promote the therapeutic effect of N2a cells. This could result in more medicines entering the BBB. In addition, in vivo studies revealed that the distribution and circulation time of medications in the brain tissue were significantly higher than those in other groups, making it more suitable for the treatment of central nervous system diseases. Conclusion: As a novel nanodrug delivery system, borneol modified schisandrin B micelles have promising research prospects in the treatment of Alzheimer's disease.

Involvement of Astrocytes in the Formation, Maintenance, and Function of the Blood-Brain Barrier.

The blood-brain barrier (BBB) is a fundamental structure that protects the composition of the brain by determining which ions, metabolites, and nutrients are allowed to enter the brain from the blood or to leave it towards the circulation. The BBB is structurally composed of a layer of brain capillary endothelial cells (BCECs) bound to each other through tight junctions (TJs). However, its development as well as maintenance and properties are controlled by the other brain cells that contact the BCECs: pericytes, glial cells, and even neurons themselves. Astrocytes seem, in particular, to have a very important role in determining and controlling most properties of the BBB. Here, we will focus on these latter cells, since the comprehension of their roles in brain physiology has been continuously expanding, even including the ability to participate in neurotransmission and in complex functions such as learning and memory. Accordingly, pathological conditions that alter astrocytic functions can alter the BBB's integrity, thus compromising many brain activities. In this review, we will also refer to different kinds of in vitro BBB models used to study the BBB's properties, evidencing its modifications under pathological conditions.