The Role of BAR Proteins and the Glycocalyx in Brain Endothelium Transcytosis

Within the brain, endothelial cells lining the blood vessels meticulously coordinate the transport of nutrients, energy metabolites and other macromolecules essential in maintaining an appropriate activity of the brain. While small molecules are pumped across specialised molecular transporters, large macromolecular cargos are shuttled from one side to the other through membrane-bound carriers formed by endocytosis on one side, trafficked to the other side and released by exocytosis. Such a process is collectively known as transcytosis. The brain endothelium is recognised to possess an intricate vesicular endosomal network that mediates the transcellular transport of cargos from blood-to-brain and brain-to-blood. However, mounting evidence suggests that brain endothelial cells (BECs) employ a more direct route via tubular carriers for a fast and efficient transport from the blood to the brain. Here, we compile the mechanism of transcytosis in BECs, in which we highlight intracellular trafficking mediated by tubulation, and emphasise the possible role in transcytosis of the Bin/Amphiphysin/Rvs (BAR) proteins and glycocalyx (GC)—a layer of sugars covering BECs, in transcytosis. Both BAR proteins and the GC are intrinsically associated with cell membranes and involved in the modulation and shaping of these membranes. Hence, we aim to summarise the machinery involved in transcytosis in BECs and highlight an uncovered role of BAR proteins and the GC at the brain endothelium.

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Abstract W P238: Selective Deletion of Pi3-kinase Gamma in the Brain Inhibits Tissue Plasminogen Activator-induced Brain Hemorrhage

Stroke ◽

10.1161/str.46.suppl_1.wp238 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

Rong Jin ◽

Xiaolei Zhu ◽

Cuiping Wang ◽

Anil Nanda ◽

D Neil Granger ◽

...

Keyword(s):

Endothelial Cells ◽

Ischemic Stroke ◽

Plasminogen Activator ◽

Brain Hemorrhage ◽

Brain Endothelial Cells ◽

Brain Endothelium ◽

Chimeric Mice ◽

Ischemic Brain ◽

Tissue Plasminogen ◽

The Brain

Background: PI3-kinase gamma (PI3Kγ), a key modulator in inflammation, is involved in many inflammatory diseases. We recently reported that PI3Kγ deficiency protects the brain from ischemic injury in PI3Kγ-/- mice by reducing blood-brain barrier damage and tissue infarction through inhibition of NF-kB-dependent inflammation, mainly involved in reduced MMP-9 activity in ischemic brain endothelium, microglia activation, and neutrophil infiltration into the ischemic brain. Meanwhile, we reported that ischemic brain endothelium, activated microglia, and infiltrating neutrophils represent the major cellular sources of PI3Kγ expressed in the brain within 24 h after cerebral ischemia. Brain hemorrhage is a serious complication of recombinant tissue plasminogen activator (tPA) therapy for ischemic stroke. In this study, we further investigated potential involvement of PI3Kγ, in particular the expression of PI3Kγ in the brain, in tPA-induced brain hemorrhage after ischemic stroke. Methods and Results: Focal ischemic stroke was induced by transient middle cerebral artery occlusion and reperfusion. PI3Kγ deficiency in PI3Kγ-/- mice not only reduced tissue infarction but also almost completely blocked brain hemorrhage induced by delayed tPA treatment at 6 hours after stroke. Bone marrow chimeras reveal that chimeric mice lacking PI3Kγ in the brain (WT BM →KO) blocked the delayed tPA-induced brain hemorrhage effectively as found in PI3Kγ-/- mice while chimeric mice lacking PI3Kγ expressed in leukocytes (KO BM →WT) had minimal effect on the tPA-induced brain hemorrhage. These effects could be attributed to a profound inhibition of MMP-9 activity in ischemic brain endothelium found in both PI3Kγ-/- mice and (WT BM →KO) chimeric mice but not in (KO BM →WT) chimeric mice, as determined by double IHC staining of MMP-9 in brain endothelial cells and by zymographic analysis of MMP-9 activity using microvessels isolated from the brain. Conclusion: Our findings suggest that PI3Kγ in the brain may play a predominant role in delayed tPA-induced brain hemorrhage after ischemic stroke. Our future study will define the role of PI3Kγ derived from brain endothelial cells versus microglia by generation of tissue/cell-specific knockout mice using the Cre-loxP system.

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TAK1 in brain endothelial cells mediates fever and lethargy

Journal of Experimental Medicine ◽

10.1084/jem.20110398 ◽

2011 ◽

Vol 208 (13) ◽

pp. 2615-2623 ◽

Cited By ~ 57

Author(s):

Dirk A. Ridder ◽

Ming-Fei Lang ◽

Sergei Salinin ◽

Jan-Peter Röderer ◽

Marcel Struss ◽

...

Keyword(s):

Endothelial Cells ◽

Sickness Behavior ◽

Brain Endothelial Cells ◽

Brain Endothelium ◽

Endogenous Pyrogen ◽

Inflammatory Stimuli ◽

Cox 2 ◽

Fever Response ◽

The Brain ◽

Pituitary Adrenal Axis

Systemic inflammation affects the brain, resulting in fever, anorexia, lethargy, and activation of the hypothalamus–pituitary–adrenal axis. How peripheral inflammatory signals reach the brain is still a matter of debate. One possibility is that, in response to inflammatory stimuli, brain endothelial cells in proximity to the thermoregulatory centers produce cyclooxygenase 2 (COX-2) and release prostaglandin E2, causing fever and sickness behavior. We show that expression of the MAP kinase kinase kinase TAK1 in brain endothelial cells is needed for interleukin 1β (IL-1β)–induced COX-2 production. Exploiting the selective expression of the thyroxine transporter Slco1c1 in brain endothelial cells, we generated a mouse line allowing inducible deletion of Tak1 specifically in brain endothelium. Mice lacking the Tak1 gene in brain endothelial cells showed a blunted fever response and reduced lethargy upon intravenous injection of the endogenous pyrogen IL-1β. In conclusion, we demonstrate that TAK1 in brain endothelial cells induces COX-2, most likely by activating p38 MAPK and c-Jun, and is necessary for fever and sickness behavior.

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Targeting nanoparticles to the brain by exploiting the blood–brain barrier impermeability to selectively label the brain endothelium

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2002016117 ◽

2020 ◽

Vol 117 (32) ◽

pp. 19141-19150 ◽

Cited By ~ 5

Author(s):

Daniel Gonzalez-Carter ◽

Xueying Liu ◽

Theofilus A. Tockary ◽

Anjaneyulu Dirisala ◽

Kazuko Toh ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Brain Targeting ◽

Brain Endothelial Cells ◽

Brain Endothelium ◽

Target Proteins ◽

Blood Brain ◽

Brain Microvasculature ◽

The Brain

Current strategies to direct therapy-loaded nanoparticles to the brain rely on functionalizing nanoparticles with ligands which bind target proteins associated with the blood–brain barrier (BBB). However, such strategies have significant brain-specificity limitations, as target proteins are not exclusively expressed at the brain microvasculature. Therefore, novel strategies which exploit alternative characteristics of the BBB are required to overcome nonspecific nanoparticle targeting to the periphery, thereby increasing drug efficacy and reducing detrimental peripheral side effects. Here, we present a simple, yet counterintuitive, brain-targeting strategy which exploits the higher impermeability of the BBB to selectively label the brain endothelium. This is achieved by harnessing the lower endocytic rate of brain endothelial cells (a key feature of the high BBB impermeability) to promote selective retention of free, unconjugated protein-binding ligands on the surface of brain endothelial cells compared to peripheral endothelial cells. Nanoparticles capable of efficiently binding to the displayed ligands (i.e., labeled endothelium) are consequently targeted specifically to the brain microvasculature with minimal “off-target” accumulation in peripheral organs. This approach therefore revolutionizes brain-targeting strategies by implementing a two-step targeting method which exploits the physiology of the BBB to generate the required brain specificity for nanoparticle delivery, paving the way to overcome targeting limitations and achieve clinical translation of neurological therapies. In addition, this work demonstrates that protein targets for brain delivery may be identified based not on differential tissue expression, but on differential endocytic rates between the brain and periphery.

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Cryptococcus neoformans Promotes Its Transmigration into the Central Nervous System by Inducing Molecular and Cellular Changes in Brain Endothelial Cells

Infection and Immunity ◽

10.1128/iai.00554-13 ◽

2013 ◽

Vol 81 (9) ◽

pp. 3139-3147 ◽

Cited By ~ 36

Author(s):

Kiem Vu ◽

Richard A. Eigenheer ◽

Brett S. Phinney ◽

Angie Gelli

Keyword(s):

Central Nervous System ◽

Endothelial Cells ◽

Nervous System ◽

Human Brain ◽

Cryptococcus Neoformans ◽

Brain Endothelial Cells ◽

Brain Endothelium ◽

Content Type ◽

The Central Nervous System ◽

The Brain

ABSTRACTCryptococcusspp. cause fungal meningitis, a life-threatening infection that occurs predominately in immunocompromised individuals. In order forCryptococcus neoformansto invade the central nervous system (CNS), it must first penetrate the brain endothelium, also known as the blood-brain barrier (BBB). Despite the importance of the interrelation betweenC. neoformansand the brain endothelium in establishing CNS infection, very little is known about this microenvironment. Here we sought to resolve the cellular and molecular basis that defines the fungal-BBB interface during cryptococcal attachment to, and internalization by, the human brain endothelium. In order to accomplish this by a systems-wide approach, the proteomic profile of human brain endothelial cells challenged withC. neoformanswas resolved using a label-free differential quantitative mass spectrometry method known as spectral counting (SC). Here, we demonstrate that as brain endothelial cells associate with, and internalize, cryptococci, they upregulate the expression of several proteins involved with cytoskeleton, metabolism, signaling, and inflammation, suggesting that they are actively signaling and undergoing cytoskeleton remodeling via annexin A2, S100A10, transgelin, and myosin. Transmission electronic microscopy (TEM) analysis demonstrates dramatic structural changes in nuclei, mitochondria, the endoplasmic reticulum (ER), and the plasma membrane that are indicative of cell stress and cell damage. The translocation of HMGB1, a marker of cell injury, the downregulation of proteins that function in transcription, energy production, protein processing, and the upregulation of cyclophilin A further support the notion thatC. neoformanselicits changes in brain endothelial cells that facilitate the migration of cryptococci across the BBB and ultimately induce endothelial cell necrosis.

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The Possible Role of Microbial Proteases in Facilitating SARS-CoV-2 Brain Invasion

Biology ◽

10.3390/biology10100966 ◽

2021 ◽

Vol 10 (10) ◽

pp. 966

Author(s):

Nozethu Mjokane ◽

Olufemi S. Folorunso ◽

Adepemi O. Ogundeji ◽

Olihile M. Sebolai

Keyword(s):

Endothelial Cells ◽

Angiotensin Converting Enzyme ◽

Converting Enzyme ◽

Brain Endothelial Cells ◽

Limited Information ◽

Brain Invasion ◽

Invasion Mechanism ◽

Intervention Measures ◽

The Brain

SARS-CoV-2 has been shown to display proclivity towards organs bearing angiotensin-converting enzyme (ACE2) expression cells. Of interest herein is the ability of the virus to exhibit neurotropism. However, there is limited information on how this virus invades the brain. With this contribution, we explore how, in the context of a microbial co-infection using a cryptococcal co-infection as a model, SARS-CoV-2 could reach the brain. We theorise that the secretion of proteases by disseminated fungal cells might also activate the S2 domain of the viral spike glycoprotein for membrane fusion with brain endothelial cells leading to endocytosis. Understanding this potential invasion mechanism could lead to better SARS-CoV-2 intervention measures, which may also be applicable in instances of co-infection, especially with protease-secreting pathogens.

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Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

10.1101/2021.02.18.431829 ◽

2021 ◽

Author(s):

Robert W. Robey ◽

Andrea N. Robinson ◽

Fatima Ali-Rahmani ◽

Lyn M. Huff ◽

Sabrina Lusvarghi ◽

...

Keyword(s):

Endothelial Cells ◽

Multidrug Transporter ◽

Glycoprotein P ◽

Tissue Localization ◽

Brain Vasculature ◽

P Glycoprotein ◽

Capillary Endothelial Cells ◽

The Brain ◽

Viable Model

ABSTRACTGiven its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.

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Two peptides targeting endothelial receptors are internalized into murine brain endothelial cells

PLoS ONE ◽

10.1371/journal.pone.0249686 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0249686

Author(s):

Diána Hudecz ◽

Sara Björk Sigurdardóttir ◽

Sarah Christine Christensen ◽

Casper Hempel ◽

Andrew J. Urquhart ◽

...

Keyword(s):

Endothelial Cells ◽

Transferrin Receptor ◽

Vesicular Transport ◽

Brain Diseases ◽

Brain Endothelial Cells ◽

Binding Studies ◽

Wide Range ◽

Murine Brain ◽

The Brain

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

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The brain development of infants with intrauterine growth restriction: role of glucocorticoids

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2019-0016 ◽

2019 ◽

Vol 39 (1) ◽

Cited By ~ 2

Author(s):

Ying-xue Ding ◽

Hong Cui

Keyword(s):

Endothelial Cells ◽

Brain Injury ◽

Intrauterine Growth Restriction ◽

Growth And Development ◽

Growth Restriction ◽

Microvascular Endothelial Cells ◽

Intrauterine Growth ◽

Growth Restrictions ◽

The Brain

Abstract Brain injury is a serious complication of intrauterine growth restriction (IUGR), but the exact mechanism remains unclear. While glucocorticoids (GCs) play an important role in intrauterine growth and development, GCs also have a damaging effect on microvascular endothelial cells. Moreover, intrauterine adverse environments lead to fetal growth restriction and the hypothalamus-pituitary-adrenal (HPA) axis resetting. In addition, chronic stress can cause a decrease in the number and volume of astrocytes in the hippocampus and glial cells play an important role in neuronal differentiation. Therefore, it is speculated that the effect of GCs on cerebral neurovascular units under chronic intrauterine stimulation is an important mechanism leading to brain injury in infants with growth restrictions.

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Endogenous THBD (Thrombomodulin) Mediates Angiogenesis in the Ischemic Brain—Brief Report

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.315061 ◽

2020 ◽

Vol 40 (12) ◽

pp. 2837-2844 ◽

Cited By ~ 1

Author(s):

Jan Wenzel ◽

Dimitrios Spyropoulos ◽

Julian Christopher Assmann ◽

Mahtab Ahmad Khan ◽

Ines Stölting ◽

...

Keyword(s):

Endothelial Cells ◽

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Cerebral Artery ◽

Soluble Form ◽

Stroke Outcome ◽

Brain Endothelial Cells ◽

Ischemic Brain ◽

Infarct Area ◽

The Brain

Objective: THBD (thrombomodulin) is part of the anticoagulant protein C-system that acts at the endothelium and is involved in anti-inflammatory and barrier-stabilizing processes. A recombinant soluble form of THBD was shown to have protective effects in different organs, but how the endogenous THBD is regulated during ischemia, particularly in the brain is not known to date. The aim of this study was to investigate the role of THBD, especially in brain endothelial cells, during ischemic stroke. Approach and Results: To induce ischemic brain damage, we occluded the middle cerebral artery of mice. We found an increased endothelial expression of Thbd in the peri-infarct area, whereas in the core of the ischemic tissue Thbd expression was decreased compared with the contralateral side. We generated a novel Cre/loxP-based mouse line that allows for the inducible deletion of Thbd specifically in brain endothelial cells, which worsened stroke outcome 48 hours after middle cerebral artery occlusion. Unexpectedly, we found no signs of increased coagulation, thrombosis, or inflammation in the brain but decreased vessel diameters and impaired angiogenesis in the peri-infarct area that led to a reduced overall vessel length 1 week after stroke induction. Conclusions: Endogenous THBD acts as a protective factor in the brain during ischemic stroke and enhances vessel diameter and proliferation. These previously unknown properties of THBD could offer new opportunities to affect vessel function after ischemia and thereby improve stroke outcome.

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Perception is far from perfection: The role of the brain and mind in constructing realities

Behavioral and Brain Sciences ◽

10.1017/s0140525x05270139 ◽

2005 ◽

Vol 28 (6) ◽

pp. 763-763 ◽

Cited By ~ 18

Author(s):

Itiel E. Dror

Keyword(s):

The Other ◽

Objective Reality ◽

The Brain

Dichotomizing perceptions, by those that have an objective reality and those that do not, is rejected. Perceptions are suggested to fall along a multidimensional continuum in which neither end is totally “pure.” At the extreme ends, perceptions neither have an objective reality without some subjectivity, nor, at the other end, even as hallucinations, are they totally dissociated from reality.

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