Blood-brain Barrier Endothelial Cells Research Articles

Pericyte Control of Gene Expression in the Blood-Brain Barrier Endothelium: Implications for Alzheimer's Disease.

A strong body of evidence suggests that cerebrovascular pathologies augment the onset and progression of Alzheimer's disease (AD). One distinctive aspect of this cerebrovascular dysfunction is the degeneration of brain pericytes-often overlooked supporting cells of blood-brain barrier endothelium. The current study investigates the influence of pericytes on gene and protein expressions in the blood-brain barrier endothelium, which is expected to facilitate the identification of pathophysiological pathways that are triggered by pericyte loss and lead to blood-brain barrier dysfunction in AD. Bioinformatics analysis was conducted on the RNA-Seq expression counts matrix (GSE144474), which compared solo-cultured human blood-brain barrier endothelial cells against endothelial cells co-cultured with human brain pericytes in a non-contact model. We constructed a similar cell culture model to verify protein expression using western blots. The insulin resistance and ferroptosis pathways were found to be enriched. Western blots of the insulin receptor and heme oxygenase expressions were consistent with those observed in RNA-Seq data. Additionally, we observed more than 5-fold upregulation of several genes associated with neuroprotection, including insulin-like growth factor 2 and brain-derived neurotrophic factor. Results suggest that pericyte influence on blood-brain barrier endothelial gene expression confers protection from insulin resistance, iron accumulation, oxidative stress, and amyloid deposition. Since these are conditions associated with AD pathophysiology, they imply mechanisms by which pericyte degeneration could contribute to disease progression.

Acupuncture Extended the Thrombolysis Window by Suppressing Blood-Brain Barrier Disruption and Regulating Autophagy-Apoptosis Balance after Ischemic Stroke.

Ischemic stroke (IS) is one of the leading causes of death and disability worldwide. The narrow therapeutic window (within 4.5 h) and severe hemorrhagic potential limits therapeutic efficacy of recombinant tissue type plasminogen activator (rt-PA) intravenous thrombolysis for patients. Xingnao Kaiqiao (XNKQ) acupuncture is an integral part of traditional Chinese medicine, specifically designed to address acute ischemic stroke by targeting key acupoints such as Shuigou (GV26) and Neiguan (PC6). In this study, we explored the therapeutic potential of XNKQ acupuncture in extending the time window for thrombolysis and interrogated the molecular mechanisms responsible for this effect. The effect of extending the thrombolysis window by acupuncture was evaluated via TTC staining, neuronal score evaluation, hemorrhagic transformation assay, and H&E staining. RNA sequencing (RNA-seq) technology was performed to identify the therapeutic targets and intervention mechanisms of acupuncture. Evans blue staining and transmission electron microscopy were used to assess blood-brain barrier (BBB) integrity. Immunofluorescence staining and co-immunoprecipitation were performed to evaluate the level of autophagy and apoptosis and validate their interactions with BBB endothelial cells. Acupuncture alleviated infarction and neurological deficits and extended the thrombolysis window to 6 h. The RNA-seq revealed 16 potential therapeutic predictors for acupuncture intervention, which related to suppressing inflammation and restoring the function of BBB and blood vessels. Furthermore, acupuncture suppressed BBB leakage and preserved tight junction protein expression. The protective effect was associated with regulation of the autophagy-apoptosis balance in BBB endothelial cells. Acupuncture intervention dissociated the Beclin1/Bcl-2 complex, thereby promoting autophagy and reducing apoptosis. XNKQ acupuncture could serve as an adjunctive therapy for rt-PA thrombolysis, aiming to extend the therapeutic time window and mitigate ischemia-reperfusion injury. Acupuncture suppressed BBB disruption by regulating the autophagy-apoptosis balance, which in turn extended the therapeutic window of rt-PA in IS. These findings provide a rationale for further exploration of acupuncture as a complementary candidate co-administered with rt-PA.

Rapamycin rescues loss of function in blood-brain barrier-interacting Tregs.

In autoimmunity, FOXP3+ Tregs skew toward a proinflammatory, nonsuppressive phenotype and are, therefore, unable to control the exaggerated autoimmune response. This largely affects the success of autologous Treg therapy, which is currently under investigation for autoimmune diseases, including multiple sclerosis (MS). There is a need to ensure in vivo Treg stability before successful application of Treg therapy. Using genetic fate-mapping mice, we demonstrate that inflammatory, cytokine-expressing exFOXP3 T cells accumulate in the CNS during experimental autoimmune encephalomyelitis. In a human in vitro model, we discovered that interaction with inflamed blood-brain barrier endothelial cells (BBB-ECs) induces loss of function by Tregs. Transcriptome and cytokine analysis revealed that in vitro migrated Tregs have disrupted regenerative potential and a proinflammatory Th1/17 signature, and they upregulate the mTORC1 signaling pathway. In vitro treatment of migrated human Tregs with the clinically approved mTORC1 inhibitor rapamycin restored suppression. Finally, flow cytometric analysis indicated an enrichment of inflammatory, less-suppressive CD49d+ Tregs in the cerebrospinal fluid of people with MS. In summary, interaction with BBB-ECs is sufficient to affect Treg function, and transmigration triggers an additive proinflammatory phenotype switch. These insights help improve the efficacy of autologous Treg therapy of MS.

FGF17 protects cerebral ischemia reperfusion-induced blood-brain barrier disruption via FGF receptor 3-mediated PI3K/AKT signaling pathway

Maintaining blood-brain barrier (BBB) integrity is critical components of therapeutic approach for ischemic stroke. Fibroblast growth factor 17 (FGF17), a member of FGF8 superfamily, exhibits the strongest expression throughout the wall of all major arteries during development. However, its molecular action and potential protective role on brain endothelial cells after stroke remains unclear. Here, we observed reduced levels of FGF17 in the serum of patients with ischemic stroke, as well as in the brains of mice subjected to middle cerebral artery occlusion (MCAO) injury and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain microvascular endothelial cells (bEnd.3) cells. Moreover, treatment with exogenous recombinant human FGF17 (rhFGF17) decreased infarct volume, improved neurological deficits, reduced Evans Blue leakage and upregulated the expression of tight junctions in MCAO-injured mice. Meanwhile, rhFGF17 increased cell viability, enhanced trans-endothelial electrical resistance, reduced sodium fluorescein leakage, and alleviated reactive oxygen species (ROS) generation in OGD/R-induced bEnd.3 cells. Mechanistically, the treatment with rhFGF17 resulted in nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear accumulation and upregulation of heme oxygenase-1 (HO-1) expression. Additionally, based on in-vivo and in-vitro research, rhFGF17 exerted protective effects against ischemia/reperfusion (I/R) -induced BBB disruption and endothelial cell apoptosis through the activation of the FGF receptor 3/PI3K/AKT signaling pathway. Overall, our findings indicated that FGF17 may hold promise as a novel therapeutic strategy for ischemic stroke patients.

Abstract 1500: Breast cancer-derived extracellular vesicles modulate the endocytic metabolism and morphodynamics of the endothelial cells of the blood-brain barrier

Abstract Breast cancer, particularly the triple-negative subtype, is the primary cause of brain metastasis in women, affecting 10-20% of breast cancer patients. Breast cancer patients who develop brain metastasis have few treatment options and a dismal prognosis with a median survival time of 6-16 months. We demonstrated, for the first time, that EVs derived from brain-seeking breast cancer cells (BrEVs) breach the intact blood-brain barrier (BBB) via transcytosis and cause a significantly increased incidence of breast-to-brain metastasis. Within this context, we are now studying the interactions between BrEVs and the endothelial cells (ECs) of the BBB. BrEVs were isolated by ultracentrifugation, characterized following the latest guidelines published by the International Society of Extracellular Vesicles (MISEV 2018) and tested in vitro using primary human brain ECs and in vivo using our mouse model of breast-to-brain metastasis. ECs were treated with BrEVs and analyzed using antibody-based assays, impedance measurements, genetic engineering and fixed and live cell imaging. The imaging results were analyzed using novel machine learning approaches to detect and describe, for the first time, EV-mediated morphodynamic changes at the brain EC barrier level. In vivo, we administered BrEVs intravenously to our models of breast-to-brain metastasis, followed by the analysis of mouse brains and their cerebral microvessels to examine microscopic structure and protein expression. Our studies show that (1) BrEVs target BBB ECs both in vitro and in vivo, causing significant variations of multiple proteins involved in the intracytoplasmic vesicles' long-recycling loop, (2) BrEVs are responsible for specific EC barrier function and morphodynamic changes and (3) BrEVs mediate, at least in part, their effect(s) through the delivery of specific micro RNAs. Taken together, our studies demonstrate that BrEVs have a significant impact on brain EC endocytic metabolism and morphodynamics, promoting transcytosis and sustaining breast-to-brain metastasis. Our findings increase our understanding of the early events that facilitate brain metastasis and have the potential to identify key regulators of brain metastasis formation. These results also inform and contribute to the development of EV-based therapeutic and diagnostic strategies for brain metastasis. (This work was supported by NIH R21 CA253051-01, the Breast Cancer Research Foundation, NIH T32 5T32HL007917-22 and the Nile Albright Research Foundation.) Citation Format: Sara Busatto, Golnaz Morad, Marsha A Moses. Breast cancer-derived extracellular vesicles modulate the endocytic metabolism and morphodynamics of the endothelial cells of the blood-brain barrier [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 1500.

Viral Infections, Are They a Trigger and Risk Factor of Alzheimer's Disease?

Alzheimer's Disease (AD), a progressive and debilitating condition, is reported to be the most common type of dementia, with at least 55 million people believed to be currently affected. Many causation hypotheses of AD exist, yet the intriguing link between viral infection and its possible contribution to the known etiology of AD has become an attractive focal point of research for the field and a challenging study task. In this review, we will explore the historical perspective and milestones that led the field to investigate the viral connection to AD. Specifically, several viruses such as Herpes Simplex Virus 1 (HSV-1), Zika virus (ZIKV), and severe cute respiratory syndrome coronavirus 2 (SARS-CoV-2), along with several others mentioned, include the various viruses presently considered within the field. We delve into the strong evidence implicating these viruses in the development of AD such as the lytic replication and axonal transport of HSV-1, the various mechanisms of ZIKV neurotropism through the human protein Musashi-1 (MSI1), and the spread of SARS-CoV-2 through the transfer of the virus through the BBB endothelial cells to glial cells and then to neurons via transsynaptic transfer. We will also explore beyond these mere associations by carefully analyzing the potential mechanisms by which these viruses may contribute to AD pathology. This includes but is not limited to direct neuronal infections, the dysregulation of immune responses, and the impact on protein processing (Aβ42 and hyperphosphorylated tau). Controversies and challenges of the virus-AD relationship emerge as we tease out these potential mechanisms. Looking forward, we emphasize future directions, such as distinct questions and proposed experimentations to explore, that the field should take to tackle the remaining unanswered questions and the glaring research gaps that persist. Overall, this review aims to provide a comprehensive survey of the past, present, and future of the potential link between viral infections and their association with AD development while encouraging further discussion.

Selective Uptake of Macromolecules to the Brain in Microfluidics and Animal Models Using the HAVN1 Peptide as a Blood-Brain Barrier Modulator.

Monoclonal antibodies (mAbs) possess favorable pharmacokinetic properties, high binding specificity and affinity, and minimal off-target effects, making them promising therapeutic agents for central nervous system (CNS) disorders. However, their development as effective therapeutic and diagnostic agents for brain disorders is hindered by their limited ability to efficiently penetrate the blood-brain barrier (BBB). Therefore, it is crucial to develop efficient delivery methods that enhance the penetration of antibodies into the brain. Previous studies have demonstrated the potential of cadherin-derived peptides (i.e., ADTC5, HAVN1 peptides) as BBB modulators (BBBMs) to increase paracellular porosities for penetration of molecules across the BBB. Here, we test the effectiveness of the leading BBBM peptide, HAVN1 (Cyclo(1,6)SHAVSS), in enhancing the permeation of various monoclonal antibodies through the BBB using both in vitro and in vivo systems. In vitro, HAVN1 has been shown to increase the permeability of fluorescently labeled macromolecules, such as a 70 kDa dextran, 50 kDa Fab1, and 150 kDa mAb1, by 4- to 9-fold in a three-dimensional blood-brain barrier (3D-BBB) microfluidics model using a human BBB endothelial cell line (i.e., hCMEC/D3). HAVN1 was selective in modulating the BBB endothelial cell, compared to the pulmonary vascular endothelial (PVE) cell barrier. Co-administration of HAVN1 significantly improved brain depositions of mAb1, mAb2, and Fab1 in C57BL/6 mice after 15 min in the systemic circulation. Furthermore, HAVN1 still significantly enhanced brain deposition of mAb2 when it was administered 24 h after the administration of the mAb. Lastly, we observed that multiple doses of HAVN1 may have a cumulative effect on the brain deposition of mAb2 within a 24-h period. These findings offer promising insights into optimizing HAVN1 and mAb dosing regimens to control or modulate mAb brain deposition for achieving desired mAb dose in the brain to provide its therapeutic effects.

Cerebral microvascular endothelial cell-derived extracellular vesicles regulate blood − brain barrier function

Autoreactive T lymphocytes crossing the blood-brain barrier (BBB) into the central nervous system (CNS) play a crucial role in the initiation of demyelination and neurodegeneration in multiple sclerosis (MS). Recently, extracellular vesicles (EV) secreted by BBB endothelial cells (BBB-EC) have emerged as a unique form of cell-to-cell communication that contributes to cerebrovascular dysfunction. However, the precise impact of different size-based subpopulations of BBB-EC-derived EV (BBB-EV) on the early stages of MS remains unclear. Therefore, our objective was to investigate the content and function of distinct BBB-EV subpopulations in regulating BBB integrity and their role in T cell transendothelial migration, both in vitro and in vivo. Our study reveals that BBB-ECs release two distinct size based EV populations, namely small EV (sEV; 30-150 nm) and large EV (lEV; 150-300 nm), with a significantly higher secretion of sEV during inflammation. Notably, the expression patterns of cytokines and adhesion markers differ significantly between these BBB-EV subsets, indicating specific functional differences in the regulation of T cell migration. Through in vitro experiments, we demonstrate that lEV, which predominantly reflect their cellular source, play a major role in BBB integrity loss and the enhanced migration of pro-inflammatory Th1 and Th17.1 cells. Conversely, sEV appear to protect BBB function by inducing an anti-inflammatory phenotype in BBB-EC. These findings align with our in vivo data, where the administration of sEV to mice with experimental autoimmune encephalomyelitis (EAE) results in lower disease severity compared to the administration of lEV, which exacerbates disease symptoms. In conclusion, our study highlights the distinct and opposing effects of BBB-EV subpopulations on the BBB, both in vitro and in vivo. These findings underscore the need for further investigation into the diagnostic and therapeutic potential of BBB-EV in the context of MS.

Escin avoids hemorrhagic transformation in ischemic stroke by protecting BBB through the AMPK/Cav-1/MMP-9 pathway.

Hemorrhagic transformation (HT) seriously affects the clinical application of recombinant tissue plasminogen activator (rt-PA). The main strategy for combating HT is to keep the blood-brain barrier (BBB) stable. Escin is the active ingredient of Aesculus hippocastanum and a natural mixture of triterpene saponins, and may play a part in mitigation of HT. This study sought to investigate the effect of Escin in improving rt-PA-induced HT, explore possible mechanisms, and provide new ideas for the treatment of clinical HT. In in vivo experiments, transient middle cerebral artery occlusion (tMCAO) was undertaken in 6-week-old and 12-month-old mice, and rt-PA was administered to induce HT injury. The inhibitory effect of Escin on HT and its protective effect on neurobehavior, the BBB, and cerebrovascular endothelial cells was determined. In in vitro experiments, bEnd.3 cells were injured by oxygen-glucose deprivation/reperfusion (OGD/R) and rt-PA. The protective effect of Escin was measured by the CCK8 assay, release of lactate dehydrogenase (LDH), and expression of tight junction (TJ) proteins. In mechanistic studies, the effect of Escin on the adenosine monophosphate-activated kinase / caveolin-1 / matrix metalloprotease-9 (AMPK/Cav-1/MMP-9) pathway was investigated by employing AMPK inhibitor and Cav-1 siRNA. In mice suffering from ischemia, rt-PA caused HT as well as damage to the BBB and cerebrovascular endothelial cells. Escin reduced the infarct volume, cerebral hemorrhage, improved neurobehavioral deficits, and maintained BBB integrity in rt-PA-treated tMCAO mice while attenuating bEnd.3 cells damage caused by rt-PA and OGD/R injury. Under physiological and pathological conditions, Escin increased the expression of p-AMPK and Cav-1, leading to decreased expression of MMP-9, which further attenuated damage to cerebrovascular endothelial cells, and these effects were verified with AMPK inhibitor and Cav-1 siRNA. We revealed important details of how Escin protects cerebrovascular endothelial cells from HT, these effects were associated with the AMPK/Cav-1/MMP-9 pathway. This study provides experimental foundation for the development of new drugs to mitigate rt-PA-induced HT and the discovery of new clinical application for Escin.

Measurement of Transendothelial Electrical Resistance in Blood-Brain Barrier Endothelial Cells.

The integrity of the blood-brain barrier (BBB), the protective barrier of the brain, is key to maintaining normal microvascular permeability and brain homeostasis. Brain microvascular endothelial cells are primary components of the blood-brain barrier. Transendothelial electrical resistance (TEER) is the electrical resistance across a cellular monolayer such as the brain microvascular endothelial cell monolayers. Measurement of TEER is considered a sensitive, reliable, and noninvasive method for evaluating barrier integrity and permeability of an endothelial cell monolayer under in vitro conditions. Measurement of TEER is also helpful for studying various cellular and molecular changes and signaling events that regulate barrier functions in endothelial monolayers. One of the in vitro endothelial cell barrier models that have been commonly used for measuring TEER is the BBB model using human or rodent brain microvascular endothelial cells grown as a monolayer. In this protocol, we describe how TEER is measured in brain microvascular endothelial cell monolayers, to determine blood-brain barrier integrity under in vitro conditions.

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.

Regulation of the ATP-binding cassette transporters ABCB1, ABCG2 and ABCC5 by nuclear receptors in porcine blood-brain barrier endothelial cells.

Blood-brain barrier (BBB) ABCB1, ABCG2 and ABCC5 transporters influence central therapeutic drug distribution. Transporter expression is regulated by the NR3C1, NR1I3 and NR1I2 nuclear receptors, but their precise roles in brain are poorly understood. We investigated the effects of selective ligand-based activation of NR3C1, NR1I3, NR1I2 and NR2B1 in porcine brain endothelial cells (PBECs). Primary cultures of PBECs were exposed to NR3C1, NR1I3 and NR1I2 ligands and ABCB1, ABCG2 and ABCC5 transporter activities determined by measuring intracellular accumulation of fluorescent probes. Western blotting was used to determine the effects of receptor ligands on expression of ABCB1, ABCG2, ABCC5, NR1I2, NR1I3, NR3C1 and NR2B1. Fluorescent immunocytochemistry was employed to assess the effects of receptor ligands on the cellular localisation of NR1I2 and NR1I3. The NR1I2 agonist rifampicin significantly up-regulated ABCG2 activity, which is counteracted by co-treatment with NR1I2 antagonist l-sulforaphane. The NR1I3 agonist 6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde and inverse agonist meclizine significantly down-regulated ABCB1, ABCG2 and ABCC5 activity. NR3C1 agonist dexamethasone significantly increased ABCB1, ABCG2 and ABCC5 activity and ABCG2 and ABCC5 protein expression, which was counteracted by co-treatment with the NR3C1 antagonist mifepristone. This first study demonstrates that NR1I3 and NR3C1 regulate ABCC5 activity and protein expression in BBB endothelial cells. In PBECs, expression of key ATP-binding cassette (ABC) transporters and nuclear receptors is differentially regulated by NR1I3, NR1I2, NR3C1 and NR2B1. This will help to better understand the response of the BBB to physiological and pharmacological activation of nuclear receptors.

Amyloid-beta peptides 40 and 42 empl oy distinct molecular pathways for cell entry and intracellular transit at the blood-brain barrier (BBB) endothelium.

The blood-brain barrier (BBB) plays a critical role in maintaining the equilibrium between amyloid beta (Aβ) levels in blood and the brain by regulating Aβ transport. Our previous publications demonstrated that BBB trafficking of Aβ42 and Aβ40 is distinct and is disrupted under various pathophysiological conditions. However, the intracellular mechanisms that allow BBB endothelium to differentially handle Aβ40 and Aβ42 have not been clearly elucidated. In this study, we identified mechanisms of Aβ endocytosis in polarized human cerebral microvascular endothelial (hCMEC/D3) cell monolayers. Our studies demonstrated that Aβ peptides with fluorescent label (F-Aβ) were internalized by BBB endothelial cells via energy, dynamin and actin dependent endocytosis. Interestingly, endocytosis of F-Aβ40 but not F-Aβ42 was substantially reduced by clathrin inhibition, whereas F-Aβ42 but not F-Aβ40 endocytosis was reduced by half after inhibiting the caveolae-mediated pathway. Following endocytosis, both isoforms were sorted by the endo-lysosomal system. While Aβ42 was shown to accumulate more in the lysosomes, which could lead to its higher degradation and/or aggregation at lower lysosomal pH, Aβ40 demonstrated robust accumulation in recycling endosomes, which may facilitate its exocytosis by the endothelial cells. These results provide a mechanistic insight into the selective ability of BBB endothelium to transport Aβ40 versus Aβ42. This knowledge contributes to the understanding of molecular pathways underlying Aβ accumulation in the BBB endothelium and associated BBB dysfunction. Moreover, it allows us to establish mechanistic rationale for altered Aβ40:Aβ42 ratios and anomalous amyloid deposition in the cerebral vasculature as well as brain parenchyma during AD progression. Significance Statement Differential interaction of Aβ40 and Aβ42 isoforms with the blood-brain barrier (BBB) endothelium may contribute to perturbation in Aβ42:Aβ40 ratio, which is associated with Alzheimer's disease (AD) progression and severity. The current study identified distinct molecular pathways by which Aβ40 and Aβ42 are trafficked at the BBB, which regulates equilibrium between blood and brain Aβ levels. These findings provide molecular insights into mechanisms that engender BBB dysfunction and promote Aβ accumulation in AD brain.

SONIC HEDGEHOG (SHH) PROMOTES BLOOD-BRAIN BARRIER (BBB) INTEGRITY IN DIFFUSE MIDLINE GLIOMA OF THE PONS

Abstract Diffuse Midline Glioma of the Pons (DMG-P) is the most common brainstem tumor in children and is universally lethal in this age group. The intact Blood-Brain Barrier (BBB) in these tumors are believed to play a major role in preventing systemically administered therapeutic agents from reaching therapeutic concentrations in the tumor. The factors that contribute to the integrity of BBB in DMG-P is not well understood. Sonic Hedgehog (SHH) signaling promotes BBB integrity in the developing and adult brain and plays an important role in the formation and maintenance of DMG-P tumors. In the present study, we show that DMG-P tumor cells secrete SHH ligand. Secreted SHH present in the tumor conditioned media (TCM) tightens the BBB by increasing electrical impedance and decreased paracellular and transcellular permeability in BBB Endothelial cells (ECs). Mechanistically, SHH in TCM causes tightening of the BBB by increasing the expression of junctional proteins in BBB-ECs, likely by signaling through non- canonical pathways. SHH in the TCM also increased the expression of ATP-binding cassette (ABC) transporter proteins ABCB1(P- glycoprotein) and ABCG2 (BCRP- Breast cancer resistance protein) in the Endothelial cells. These pro-barrier functions of SHH in TCM can be reversed by pharmacological inhibition of the S-acylation of the SHH ligand using Hedgehog acyltransferase (HHAT) inhibitor RUSKI-201. Our study demonstrates a potential therapeutic strategy of altering the BBB permeability to increase the efficacy of systemic therapies in DMG-P tumors. Ongoing studies will validate these findings in orthotopic murine models of DMG-P.

The use of a multicellular in vitro model to investigate uptake and migration of bacterial extracellular vesicles derived from the human gut commensal Bacteroides thetaiotaomicron

AbstractBacterial extracellular vesicles (BEVs) are increasingly seen as key signalling mediators between the gut microbiota and the host. Recent studies have provided evidence of BEVs ability to transmigrate across cellular barriers to elicit responses in other tissues, such as the central nervous system (CNS). Here we use a combination of single‐, two‐ and three‐cell culture systems to demonstrate the transmigration of Bacteroides thetaiotaomicron derived BEVs (Bt‐BEVs) across gut epithelium and blood brain barrier (BBB) endothelium, and their subsequent acquisition and downstream effects in neuronal cells. Bt‐BEVs were shown to traffic to the CNS in vivo after intravenous administration to mice, and in multi‐cell in vitro culture systems to transmigrate across gut epithelial and BBB endothelial cell barriers, where they were acquired by both microglia and immature neuronal cells. No significant activation/inflammatory effects were induced in non‐differentiated neurons, in contrast to that observed in microglia cells, although this was notably less than that induced by lipopolysaccharide (LPS). Overall, our findings provide evidence for transmigration of Bt‐BEVs across gut‐epithelial and BBB endothelial cell barriers in vivo and in vitro, and their downstream responses in neural cells. This study sheds light onto how commensal bacteria‐derived BEV transport across the gut‐brain axis and can be exploited for the development of targeted drug delivery.

Microglia and the Blood-Brain Barrier: An External Player in Acute and Chronic Neuroinflammatory Conditions.

Microglia are the resident immune cells of the central nervous system that guarantee immune surveillance and exert also a modulating role on neuronal synaptic development and function. Upon injury, microglia get activated and modify their morphology acquiring an ameboid phenotype and pro- or anti-inflammatory features. The active role of microglia in blood-brain barrier (BBB) function and their interaction with different cellular components of the BBB-endothelial cells, astrocytes and pericytes-are described. Here, we report the specific crosstalk of microglia with all the BBB cell types focusing in particular on the involvement of microglia in the modulation of BBB function in neuroinflammatory conditions that occur in conjunction with an acute event, such as a stroke, or in a slow neurodegenerative disease, such as Alzheimer's disease. The potential of microglia to exert a dual role, either protective or detrimental, depending on disease stages and environmental conditioning factors is also discussed.

Regulation of Blood-Brain Barrier Endothelial Cell Hyperpermeability by NLRP3 Inflammasome Inhibition

The blood-brain barrier (BBB) is a semi-permeable and protective barrier of the brain, primarily composed of endothelial cells interconnected by tight junction proteins, that regulates the movement of ions and molecules between the blood and the neural matter. In pathological conditions such as traumatic brain injury (TBI), disruption of the BBB contributes to the leakage of solutes and fluids into the brain parenchyma resulting in the onset of cerebral edema and elevation of intracranial pressure. Recent studies from our lab have demonstrated that the release of the proinflammatory cytokine interleukin 1-β (IL-1β) followed by the activation of matrixmetalloproteinase-9 (MMP-9) is critical to BBB dysfunction following TBI. NLRP3 inflammasome is a multiprotein complex that is critical to the formation of IL-1β. The role of NLRP3 inflammasome in modulating BBB integrity has been recently investigated in our lab. Our results suggest that NLRP3 inflammasome activation is critical to BBB dysfunction and hyperpermeability following TBI. Once activated, the NLRP3 inflammasome is a critical component in the regulation of BBB hyperpermeability, recruiting enzymes such as caspase-1 and subsequent activation of MMP-9, which disrupt tight junction proteins necessary for BBB integrity. Thus, the NLRP3 inflammasome pathway remains a potential target for TBI therapeutics. The objective of this study was to determine the upstream regulators of NLRP3 signaling and BBB hyperpermeability, particularly to determine if extracellular ATP via P2X7R, a purinergic receptor promotes NLRP3 inflammasome activation. Extracellular ATP is a major contributor of secondary injuries following TBI. NLRP3 inflammasome pathway and its impact on BBB endothelial monolayer permeability, tight junction protein expression, and caspase-1 and MMP-9 activation following extracellular ATP-mediated barrier dysfunction was investigated in in human brain microvascular endothelial cells. The effect of benzyl ATP (BzATP)-treatment on tight junction integrity was investigated via zonula occludens-1 (ZO-1) immunofluorescence localization, ZO-1 gene expression by RT-PCR, and caspase-1 and MMP-9 activities by fluorometric assays. Pharmacological inhibition of NLRP3 (using MCC950) resulted in a decrease in BzATP-induced monolayer hyperpermeability, caspase-1 and MMP-9 activities. These effects were independent of changes in cell viability. Our results suggest that extracellular ATP promotes NLRP3 inflammasome activation and subsequent caspase-1 and MMP-9-mediated tight junction disorganization are major pathways that lead to BBB dysfunction and hyperpermeability following conditions such as traumatic brain injury. This project would not have been possible without funding from the National Institute of Health and guidance from Dr. Travis Hein and other collaborators at Texas A&M. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

DH5α Outer Membrane-Coated Biomimetic Nanocapsules Deliver Drugs to Brain Metastases but not Normal Brain Cells via Targeting GRP94.

Receptor-mediated vesicular transport has been extensively developed to penetrate the blood-brain barrier (BBB) and has emerged as a class of powerful brain-targeting delivery technologies. However, commonly used BBB receptors such as transferrin receptor and low-density lipoprotein receptor-related protein 1, are also expressed in normal brain parenchymal cells and can cause drug distribution in normal brain tissues and subsequent neuroinflammation and cognitive impairment. Here, the endoplasmic reticulum residing protein GRP94 is found upregulated and relocated to the cell membrane of both BBB endothelial cells and brain metastatic breast cancer cells (BMBCCs) by preclinical and clinical investigations. Inspired by that Escherichia coli penetrates the BBB via the binding of its outer membrane proteins with GRP94, avirulent DH5α outer membrane protein-coated nanocapsules (Omp@NCs) are developed to cross the BBB, avert normal brain cells, and target BMBCCs via recognizing GRP94. Embelin (EMB)-loaded Omp@EMB specifically reduce neuroserpin in BMBCCs, which inhibits vascular cooption growth and induces apoptosis of BMBCCs by restoring plasmin. Omp@EMB plus anti-angiogenic therapy prolongs the survival of mice with brain metastases. This platform holds the translational potential to maximize therapeutic effects on GRP94-positive brain diseases.

Reduction in cardiolipin reduces expression of creatine transporter-1 and creatine transport in growing hCMEC/D3 human brain microvessel endothelial cells

The phospholipid cardiolipin (CL) regulates mitochondrial energy production. Endothelial cells of the blood-brain barrier (BBB) play a vital role in uptake of metabolites into the brain and are enriched in mitochondria. We examined how deficiency in BBB endothelial cell CL regulates the expression of selected drug and metabolite transporters and their function. Cardiolipin synthase-1 (hCLS1) was knocked down in a human brain microvessel endothelial cell line, hCMEC/D3, and CL levels and the mRNA expression of selected BBB drug and metabolite transporters examined. Mock transfected hCMEC/D3 cells served as controls. Incorporation of (14C)creatine and (14C)oleate into hCMEC/D3 cells was determined as a measure of solute metabolite transport. In addition, protein expression of the creatine transporter was determined. Knockdown of hCLS1 in hCMEC/D3 reduced CL and the mRNA expression of creatine transporter-1, p-glycoprotein and breast cancer resistance protein compared to controls. In contrast, mRNA expression of ATP binding cassette subfamily C members-1, -3, multidrug resistance-associated protein-4 variants 1, -2, and fatty acid transport protein-1 were unaltered. Although ATP production was unaltered by hCLS1 knockdown, incorporation of (14C)creatine into hCMEC/D3 cells was reduced compared to controls. The reduction in (14C)creatine incorporation was associated with a reduction in creatine transporter-1 protein expression. In contrast, incorporation of (14C)oleic acid into hCMEC/D3 cells and the mRNA expression of fatty acid transport protein-1 was unaltered by knockdown of hCLS1 compared to controls. Thus, knockdown of hCLS1 in hCMEC/D3, with a corresponding reduction in CL, results in alteration in expression of specific solute membrane transporters.

Protective Effect of Ergothioneine against 7-Ketocholesterol-Induced Mitochondrial Damage in hCMEC/D3 Human Brain Endothelial Cells.

Recent findings have suggested that the natural compound ergothioneine (ET), which is synthesised by certain fungi and bacteria, has considerable cytoprotective potential. We previously demonstrated the anti-inflammatory effects of ET on 7-ketocholesterol (7KC)-induced endothelial injury in human blood-brain barrier endothelial cells (hCMEC/D3). 7KC is an oxidised form of cholesterol present in atheromatous plaques and the sera of patients with hypercholesterolaemia and diabetes mellitus. The aim of this study was to elucidate the protective effect of ET on 7KC-induced mitochondrial damage. Exposure of human brain endothelial cells to 7KC led to a loss of cell viability, together with an increase in intracellular free calcium levels, increased cellular and mitochondrial reactive oxygen species, a decrease in mitochondrial membrane potential, reductions in ATP levels, and increases in mRNA expression of TFAM, Nrf2, IL-1β, IL-6 and IL-8. These effects were significantly decreased by ET. Protective effects of ET were diminished when endothelial cells were coincubated with verapamil hydrochloride (VHCL), a nonspecific inhibitor of the ET transporter OCTN1 (SLC22A4). This outcome demonstrates that ET-mediated protection against 7KC-induced mitochondrial damage occurred intracellularly and not through direct interaction with 7KC. OCTN1 mRNA expression itself was significantly increased in endothelial cells after 7KC treatment, consistent with the notion that stress and injury may increase ET uptake. Our results indicate that ET can protect against 7KC-induced mitochondrial injury in brain endothelial cells.