Cell-based Blood-brain Barrier Model Research Articles

Identification of a novel squalene epoxidase function in breast cancer brain metastasis.

e13101 Background: Brain metastases are serious complications of breast cancer and there is no effective treatment. The major therapeutic issues can be attributed to the unique brain microenvironment as well as the difficulty of drug delivery to the brain due to the blood-brain barrier (BBB). BBB limits access of nutrients from the circulation and thereby makes the brain in a special condition that is hypoxic and composed of depleted metabolites, growth factors, and proteins. Nevertheless, tumor cells that are able to metastasize to the brain can necessitate metabolic adaptations for their nutrient availability and thereby succeed to colonize in the brain. However, it is still poorly understood how breast cancer cells alter their metabolic systems to manipulate brain metastasis by overcoming nutritional limitations. Methods: To probe the molecular mechanism of breast cancer to brain metastasis, we compared gene signatures in primary breast tumors and breast-to-brain metastatic 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-to-brain metastasis. Using the brain metastatic MDA-MB-231-BR (231-BR) and its parental MDA-MB-231 (231) cell line 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. The in vitro cell-based BBB model and in vivo mouse models that develop 231-BR brain metastases were established to verify our hypothesis. Results: While loss of SQLE greatly attenuated cell invasiveness and stemness in both 231 and 231-BR cells, loss of SQLE could only affect the cell migration activity on 231 cells but not 231-BR cells. 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 colonization in the brain. Our RNA-seq data further identified a subset of SQLE-affected genes that is uniquely enriched in 231-BR cells and favors brain extravasation and colonization. The in vitro cell-based BBB model and in vivo mouse models that develop 231-BR brain extravasation and colonization finally verified our hypothesis that SQLE may play a unique function to promote breast cancer metastasis into the brain. Conclusions: Our findings provide new insights into contributions by SQLE in breast-to-brain extravasation and colonization and indicate that targeting SQLE may represent a therapeutic opportunity for breast cancer brain metastases.

Blood-Brain Barrier Disruption Increases Amyloid-Related Pathology in TgSwDI Mice.

In Alzheimer’s disease (AD), several studies have reported blood-brain barrier (BBB) breakdown with compromised function. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are transport proteins localized at the BBB luminal membrane and play an important role in the clearance of amyloid-β (Aβ). The purpose of this study was to investigate the effect of pharmacological inhibition of Aβ efflux transporters on BBB function and Aβ accumulation and related pathology. Recently, we have developed an in vitro high-throughput screening assay to screen for compounds that modulate the integrity of a cell-based BBB model, which identified elacridar as a disruptor of the monolayer integrity. Elacridar, an investigational compound known for its P-gp and BCRP inhibitory effect and widely used in cancer research. Therefore, it was used as a model compound for further evaluation in a mouse model of AD, namely TgSwDI. TgSwDI mouse is also used as a model for cerebral amyloid angiopathy (CAA). Results showed that P-gp and BCRP inhibition by elacridar disrupted the BBB integrity as measured by increased IgG extravasation and reduced expression of tight junction proteins, increased amyloid deposition due to P-gp, and BCRP downregulation and receptor for advanced glycation end products (RAGE) upregulation, increased CAA and astrogliosis. Further studies revealed the effect was mediated by activation of NF-κB pathway. In conclusion, results suggest that BBB disruption by inhibiting P-gp and BCRP exacerbates AD pathology in a mouse model of AD, and indicate that therapeutic drugs that inhibit P-gp and BCRP could increase the risk for AD.

Comparison of the rate of dedifferentiation with increasing passages among cell sources for an in vitro model of the blood-brain barrier.

Cell culture-based blood-brain barrier (BBB) models are useful experimental tools for developing central nervous system drugs. Several endothelial cell sources exist for BBB models, including primary cultured brain endothelial cells and immortalized cell lines. Among them, primary cell-based models are considered suitable for the functional analysis of the BBB; however, little is known about the utility of low-passage brain endothelial cells for this purpose. In this study, we investigated the effect of passage on brain endothelial cells from human, mouse and rat brain tissue as BBB models. We established in vitro BBB models using primary brain endothelial cells (Passage 1-Passage 4) from humans, mice, and rats. To analyze the effect of cell type on BBB function, we evaluated transendothelial electrical resistance (TEER) and performed immunofluorescence staining of tight junction proteins. Among the brain endothelial cell models, TEER was highest in the Passage 1 (P1) cell-based BBB model. There was no adequate increase in TEER in other low-passage cultures (P2-P4). A confluent, non-overlapping, uniform monolayer of cells in all P1 cell-based models was visible on immunostaining of tight junction proteins, whereas it was weak or undetectable in more passaged cultures. Increasing passages cultured of brain endothelial cells did not exhibit restrictive BBB function regardless of the cell source and despite culturing with pericytes and astrocytes. Among the tested culture models, only the lowest cultured cell-based models are suitable for functional analysis of the BBB.

Granisetron Alleviates Alzheimer's Disease Pathology in TgSwDI Mice Through Calmodulin-Dependent Protein Kinase II/cAMP-Response Element Binding Protein Pathway.

Alzheimer's disease (AD) is characterized by a compromised blood-brain barrier (BBB) and disrupted intracellular calcium homeostasis in the brain. Therefore, rectifying the BBB integrity and restoring calcium homeostasis could provide an effective strategy to treat AD. Recently, we developed a high throughput-screening assay to screen for compounds that enhance a cell-based BBB model integrity, which identified multiple hits among which is granisetron, a Food and Drug Administration approved drug. Here, we evaluated the therapeutic potential of granisetron against AD. Granisetron was tested in C57Bl/6J young and aged wild-type mice, and in a transgenic mouse model of AD namely TgSwDI for its effect on BBB intactness and amyloid-β (Aβ)-related pathology. Our study findings showed that granisetron enhanced BBB integrity in both aged and TgSwDI mice. This effect was associated with an overall reduction in Aβ load and neuroinflammation in TgSwDI mice brains. In addition, and supported by proteomics analysis, granisetron significantly reduced Aβ induced calcium influx in vitro, and rectified calcium dyshomeostasis in TgSwDI mice brains by restoring calmodulin-dependent protein kinase II/cAMP-response element binding protein pathway, which was associated with cognitive improvement. These results support granisetron repurposing as a potential drug to hold, slow, and/or treat AD.

Plasma Rich in Growth Factors (PRGF) Disrupt the Blood-Brain Barrier Integrity and Elevate Amyloid Pathology in the Brains of 5XFAD Mice.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder affecting 5.4 million people in the United States. Currently approved pharmacologic interventions for AD are limited to symptomatic improvement, not affecting the underlying pathology. Therefore, the search for novel therapeutic strategies is ongoing. A hallmark of AD is the compromised blood-brain barrier (BBB); thus, developing drugs that target the BBB to enhance its integrity and function could be a novel approach to prevent and/or treat AD. Previous evidence has shown the beneficial effects of growth factors in the treatment of AD pathology. Based on reported positive results obtained with the product Endoret®, the objective of this study was to investigate the effect of plasma rich in growth factors (PRGF) on the BBB integrity and function, initially in a cell-based BBB model and in 5x Familial Alzheimer’s Disease (5xFAD) mice. Our results showed that while PRGF demonstrated a positive effect in the cell-based BBB model with the enhanced integrity and function of the model, the in-vivo findings showed that PRGF exacerbated amyloid pathology in 5xFAD brains. At 10 and 100% doses, PRGF increased amyloid deposition associated with increased apoptosis and neuroinflammation. In conclusion, our results suggest PRGF may not provide beneficial effects against AD and the consideration to utilize growth factors should further be investigated.

Multi-faceted therapeutic strategy for treatment of Alzheimer's disease by concurrent administration of etodolac and α-tocopherol

Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple dysfunctional pathways. Therefore, a sophisticated treatment strategy that simultaneously targets multiple brain cell types and disease pathways could be advantageous for effective intervention. To elucidate an effective treatment, we developed an in vitro high-throughput screening (HTS) assay to evaluate candidate drugs for their ability to enhance the integrity of the blood-brain barrier (BBB) and improve clearance of amyloid-β (Aβ) using a cell-based BBB model. Results from HTS identified etodolac and α-tocopherol as promising drugs for further investigation. Both drugs were tested separately and in combination for the purpose of targeting multiple pathways including neuroinflammation and oxidative stress. In vitro studies assessed the effects of etodolac and α-tocopherol individually and collectively for BBB integrity and Aβ transport, synaptic markers and Aβ production in APP-transfected neuronal cells, as well as effects on inflammation and oxidative stress in astrocytes. Transgenic 5XFAD mice were used to translate in vitro results of etodolac and α-tocopherol independently and with concurrent administration. Compared to either drug alone, the combination significantly enhanced the BBB function, decreased total Aβ load correlated with increased expression of major transport proteins, promoted APP processing towards the neuroprotective and non-amyloidogenic pathway, induced synaptic markers expression, and significantly reduced neuroinflammation and oxidative stress both in vitro and in vivo. Collective findings demonstrated the combination produced mixed interaction showing additive, less than additive or synergistic effects on the evaluated markers. In conclusion, this study highlights the significance of combination therapy to simultaneously target multiple disease pathways, and suggest the repurposing and combination of etodolac and α-tocopherol as a novel therapeutic strategy against AD.

Characterization of Hit Compounds Identified from High-throughput Screening for their Effect on Blood–brain Barrier Integrity and Amyloid-β Clearance: In Vitro and In Vivo Studies

In Alzheimer’s disease (AD) the blood–brain barrier (BBB) is compromised, thus therapeutic targeting of the BBB to enhance its integrity and function could be a unique approach to treat, slow or hold the progression of AD. Recently, we have developed an in vitro high-throughput screening assay to screen for compounds that increase the integrity of a cell-based BBB model. Results from primary screen identified multiple hit compounds that enhanced the monolayer integrity. Herein, further characterization of selected hit compounds, namely 8-bromoguanosine cyclic monophosphate, JW74, 1,10-phenanthroline monohydrate, SB216763 and α-tocopherol was performed. Compounds were subjected to concentration-dependent studies to determine their EC50 and potency to enhance the cell-based model integrity by the Lucifer Yellow permeability and amyloid-beta (Aβ) transport across the monolayer. The compounds demonstrated different EC50s to enhance the monolayer integrity ranging from 0.4 to 12.8 µM, and different effect on enhancing Aβ transport with highest transport observed for α-tocopherol (2.2-fold increase). Such effects were associated with increased levels of tight junction proteins such as claudin-5 and/or ZO-1, and Aβ major transport proteins LRP1 and P-glycoprotein. In vivo studies for α-tocopherol were performed in AD mouse model; consistent with the in vitro results α-tocopherol significantly increased BBB integrity measured by IgG extravasation, and reduced brain Aβ levels. In conclusion, findings support our developed cell-based BBB model as a functional predictive in vivo tool to select hit compounds, and suggest that enhancing BBB tightness and function has the potential to reduce Aβ pathology associated with AD.

Mixed oligomers and monomeric amyloid-β disrupts endothelial cells integrity and reduces monomeric amyloid-β transport across hCMEC/D3 cell line as an in vitro blood–brain barrier model

Senile amyloid plaques are one of the diagnostic hallmarks of Alzheimer's disease (AD). However, the severity of clinical symptoms of AD is weakly correlated with the plaque load. AD symptoms severity is reported to be more strongly correlated with the level of soluble amyloid-β (Aβ) assemblies. Formation of soluble Aβ assemblies is stimulated by monomeric Aβ accumulation in the brain, which has been related to its faulty cerebral clearance. Studies tend to focus on the neurotoxicity of specific Aβ species. There are relatively few studies investigating toxic effects of Aβ on the endothelial cells of the blood–brain barrier (BBB). We hypothesized that a soluble Aβ pool more closely resembling the in vivo situation composed of a mixture of Aβ40 monomer and Aβ42 oligomer would exert higher toxicity against hCMEC/D3 cells as an in vitro BBB model than either component alone. We observed that, in addition to a disruptive effect on the endothelial cells integrity due to enhancement of the paracellular permeability of the hCMEC/D3 monolayer, the Aβ mixture significantly decreased monomeric Aβ transport across the cell culture model. Consistent with its effect on Aβ transport, Aβ mixture treatment for 24h resulted in LRP1 down-regulation and RAGE up-regulation in hCMEC/D3 cells. The individual Aβ species separately failed to alter Aβ clearance or the cell-based BBB model integrity. Our study offers, for the first time, evidence that a mixture of soluble Aβ species, at nanomolar concentrations, disrupts endothelial cells integrity and its own transport across an in vitro model of the BBB.

In vitro evidence of dysregulation of blood–brain barrier function after acute and repeated/long-term exposure to TiO 2 nanoparticles

The effects of titanium dioxide nanoparticles (TiO 2 NPs) on blood–brain barrier (BBB) function are unknown. Here, we report such evidence of adverse effects after in vitro exposure of a rat primary cell-based BBB model to NPs. BBB integrity was studied by measuring the flux of sucrose through the monolayer. P-glycoprotein (P-gp) activity was assessed by measuring the passage of vinblastine. Transcription profiles of P-gp and other ABC transporters as well as of cytokines were investigated by real-time PCR. Electron microscopy and particle-induced X-ray emission measurements were performed. We compared several exposure modalities, from early to chronic, mimicking a brain-to-blood transport or a systemic contamination. In the first case, BBB integrity was preserved, but P-gp activity of endothelial cells (BECs) was reduced. In the second case, BBB integrity and P-gp function were impaired from 5 μg/mL for 24 h and expression of tight junction proteins and efflux transporters was modulated. An inflammatory response had repercussions on ABC transporter expression of glial cells. We demonstrate that NPs accumulated in BECs and crossed the cell monolayer. These findings suggest that there is an immunoregulatory loop between inflammatory components, BECs and glial cells in the dysfunction of the BBB during exposure to TiO 2 NPs.