Short regulatory DNA sequences to target brain endothelial cells for gene therapy

2021 ◽  
pp. 0271678X2110396 ◽  
Author(s):  
Hanna Graßhoff ◽  
Helge Müller-Fielitz ◽  
Godwin K Dogbevia ◽  
Jakob Körbelin ◽  
Jacqueline Bannach ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Regulatory Elements ◽  
Brain Barrier ◽  
Regulatory Sequences ◽  
Brain Endothelial Cells ◽  
Enhancer Element ◽  
Blood Brain

Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters ( Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C- Ocln-W and C- Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

Neural precursor cell influences on blood–brain barrier characteristics in rat brain endothelial cells

2007 ◽  
Vol 1159 ◽  
pp. 67-76 ◽  
Author(s):  
Joseph C. Lim ◽  
Adam J. Wolpaw ◽  
Maeve A. Caldwell ◽  
Stephen B. Hladky ◽  
Margery A. Barrand
Keyword(s):  
Endothelial Cells ◽  
Rat Brain ◽  
Blood Brain Barrier ◽  
Precursor Cell ◽  
Brain Barrier ◽  
Neural Precursor ◽  
Brain Endothelial Cells ◽  
Neural Precursor Cell ◽  
Blood Brain ◽  
Rat Brain Endothelial Cells

scholarly journals Characterization of a Primate Blood-Brain Barrier Co-Culture Model Prepared from Primary Brain Endothelial Cells, Pericytes and Astrocytes

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1484
Author(s):  
Daisuke Watanabe ◽  
Shinsuke Nakagawa ◽  
Yoichi Morofuji ◽  
Andrea E. Tóth ◽  
Monika Vastag ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Transport Systems ◽  
Brain Endothelial Cells ◽  
Monkey Brain ◽  
Blood Brain ◽  
Culture Model ◽  
Culture Models ◽  
Human Primate

Culture models of the blood-brain barrier (BBB) are important research tools. Their role in the preclinical phase of drug development to estimate the permeability for potential neuropharmaceuticals is especially relevant. Since species differences in BBB transport systems exist, primate models are considered as predictive for drug transport to brain in humans. Based on our previous expertise we have developed and characterized a non-human primate co-culture BBB model using primary cultures of monkey brain endothelial cells, rat brain pericytes, and rat astrocytes. Monkey brain endothelial cells in the presence of both pericytes and astrocytes (EPA model) expressed enhanced barrier properties and increased levels of tight junction proteins occludin, claudin-5, and ZO-1. Co-culture conditions also elevated the expression of key BBB influx and efflux transporters, including glucose transporter-1, MFSD2A, ABCB1, and ABCG2. The correlation between the endothelial permeability coefficients of 10 well known drugs was higher (R2 = 0.8788) when the monkey and rat BBB culture models were compared than when the monkey culture model was compared to mouse in vivo data (R2 = 0.6619), hinting at transporter differences. The applicability of the new non-human primate model in drug discovery has been proven in several studies.

Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes

1994 ◽  
Vol 107 (5) ◽  
pp. 1347-1357 ◽  
Author(s):  
H. Wolburg ◽  
J. Neuhaus ◽  
U. Kniesel ◽  
B. Krauss ◽  
E.M. Schmid ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Blood Brain Barrier ◽  
Barrier Function ◽  
Primary Cultures ◽  
Brain Barrier ◽  
Brain Endothelial Cells ◽  
Blood Brain ◽  
Camp Levels

Tight junctions between endothelial cells of brain capillaries are the most important structural elements of the blood-brain barrier. Cultured brain endothelial cells are known to loose tight junction-dependent blood-brain barrier characteristics such as macromolecular impermeability and high electrical resistance. We have directly analyzed the structure and function of tight junctions in primary cultures of bovine brain endothelial cells using quantitative freeze-fracture electron microscopy, and ion and inulin permeability. The complexity of tight junctions, defined as the number of branch points per unit length of tight junctional strands, decreased 5 hours after culture but thereafter remained almost constant. In contrast, the association of tight junction particles with the cytoplasmic leaflet of the endothelial membrane bilayer (P-face) decreased continuously with a major drop between 16 hours and 24 hours. The complexity of tight junctions could be increased by elevation of intracellular cAMP levels while phorbol esters had the opposite effect. On the other hand, the P-face association of tight junction particles was enhanced by elevation of cAMP levels and by coculture of endothelial cells with astrocytes or exposure to astrocyte-conditioned medium. The latter effect on P-face association was induced by astrocytes but not fibroblasts. Elevation of cAMP levels together with astrocyte-conditioned medium synergistically increased transendothelial electrical resistance and decreased inulin permeability of primary cultures, thus confirming the effects on tight junction structure and barrier function. P-face association of tight junction particles in brain endothelial cells may therefore be a critical feature of blood-brain barrier function that can be specifically modulated by astrocytes and cAMP levels. Our results suggest an important functional role for the cytoplasmic anchorage of tight junction particles for brain endothelial barrier function in particular and probably paracellular permeability in general.

scholarly journals Encephalitic Alphaviruses Exploit Caveola-Mediated Transcytosis at the Blood-Brain Barrier for Central Nervous System Entry

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hamid Salimi ◽  
Matthew D. Cain ◽  
Xiaoping Jiang ◽  
Robyn A. Roth ◽  
Wandy L. Beatty ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Endothelial Cells ◽  
Nervous System ◽  
Viral Replication ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Type I ◽  
Brain Endothelial Cells ◽  
Blood Brain ◽  
Deficient Mice

ABSTRACT Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB. IMPORTANCE VEEV, WEEV, and eastern equine encephalitis virus (EEEV) are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the bloodstream; however, the underlying molecular mechanisms have remained largely unknown. Here, we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPases within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens in the brain than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.

scholarly journals Modulation of Wnt/β-catenin signaling promotes blood-brain barrier phenotype in cultured brain endothelial cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marlyn D. Laksitorini ◽  
Vinith Yathindranath ◽  
Wei Xiong ◽  
Sabine Hombach-Klonisch ◽  
Donald W. Miller
Keyword(s):  
Endothelial Cells ◽  
Wnt Signaling ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Brain Endothelial Cells ◽  
Cancer Resistance ◽  
Blood Brain ◽  
Culture Model ◽  
External Activation

AbstractWnt/β-catenin signaling is important for blood-brain barrier (BBB) development and is implicated in BBB breakdown under various pathophysiological conditions. In the present study, a comprehensive characterization of the relevant genes, transport and permeability processes influenced by both the autocrine and external activation of Wnt signaling in human brain endothelial cells was examined using hCMEC/D3 culture model. The hCMEC/D3 expressed a full complement of Wnt ligands and receptors. Preventing Wnt ligand release from hCMEC/D3 produced minimal changes in brain endothelial function, while inhibition of intrinsic/autocrine Wnt/β-catenin activity through blocking β-catenin binding to Wnt transcription factor caused more modest changes. In contrast, activation of Wnt signaling using exogenous Wnt ligand (Wnt3a) or LiCl (GSK3 inhibitor) improved the BBB phenotypes of the hCMEC/D3 culture model, resulting in reduced paracellular permeability, and increased P-glycoprotein (P-gp) and breast cancer resistance associated protein (BCRP) efflux transporter activity. Further, Wnt3a reduced plasmalemma vesicle associated protein (PLVAP) and vesicular transport activity in hCMEC/D3. Our data suggest that this in vitro model of the BBB has a more robust response to exogenous activation of Wnt/β-catenin signaling compared to autocrine activation, suggesting that BBB regulation may be more dependent on external activation of Wnt signaling within the brain microvasculature.

scholarly journals Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier

2017 ◽  
Vol 38 (4) ◽  
pp. 727-740 ◽  
Author(s):  
Arsalan S Haqqani ◽  
Christie E Delaney ◽  
Eric Brunette ◽  
Ewa Baumann ◽  
Graham K Farrington ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Early Endosome ◽  
Brain Barrier ◽  
Endosomal Trafficking ◽  
Brain Endothelial Cells ◽  
Density Fractions ◽  
Blood Brain ◽  
Late Endosomes ◽  
Early Endosomes

Current methods for examining antibody trafficking are either non-quantitative such as immunocytochemistry or require antibody labeling with tracers. We have developed a multiplexed quantitative method for antibody ‘tracking’ in endosomal compartments of brain endothelial cells. Rat brain endothelial cells were co-incubated with blood-brain barrier (BBB)-crossing FC5, monovalent FC5Fc or bivalent FC5Fc fusion antibodies and control antibodies. Endosomes were separated using sucrose-density gradient ultracentrifugation and analyzed using multiplexed mass spectrometry to simultaneously quantify endosomal markers, receptor-mediated transcytosis (RMT) receptors and the co-incubated antibodies in each fraction. The quantitation showed that markers of early endosomes were enriched in high-density fractions (HDF), whereas markers of late endosomes and lysosomes were enriched in low-density fractions (LDF). RMT receptors, including transferrin receptor, showed a profile similar to that of early endosome markers. The in vitro BBB transcytosis rates of antibodies were directly proportional to their partition into early endosome fractions of brain endothelial cells. Addition of the Fc domain resulted in facilitated antibody ‘redistribution’ from LDF into HDF and additionally into multivesicular bodies (MVB). Sorting of various FC5 antibody formats away from late endosomes and lysosomes and into early endosomes and a subset of MVB results in increased antibody transcytosis at the abluminal side of the BBB.

scholarly journals Mouse Adenovirus Type 1-Induced Breakdown of the Blood-Brain Barrier

2009 ◽  
Vol 83 (18) ◽  
pp. 9398-9410 ◽  
Author(s):  
Lisa E. Gralinski ◽  
Shanna L. Ashley ◽  
Shandee D. Dixon ◽  
Katherine R. Spindler
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Mouse Brain ◽  
Adenovirus Type ◽  
Brain Barrier ◽  
Brain Endothelial Cells ◽  
Blood Brain ◽  
Mouse Brain Endothelial Cells ◽  
Primary Mouse ◽  
Infected Cells

ABSTRACT Infection with mouse adenovirus type 1 (MAV-1) results in fatal acute encephalomyelitis in susceptible mouse strains via infection of brain endothelial cells. Wild-type (wt) MAV-1 causes less brain inflammation than an early region 3 (E3) null virus in C57BL/6 mice. A mouse brain microvascular endothelial cell line infected with wt MAV-1 had higher expression of mRNAs for the proinflammatory chemokines CCL2 and CCL5 than mock- and E3 null virus-infected cells. Primary mouse brain endothelial cells infected with wt virus had elevated levels of CCL2 compared to mock- or E3 null virus-infected cells. Infection of C57BL/6 mice with wt MAV-1 or the E3 null virus caused a dose-dependent breakdown of the blood-brain barrier, primarily due to direct effects of virus infection rather than inflammation. The tight junction proteins claudin-5 and occludin showed reduced surface expression on primary mouse brain endothelial cells following infection with either wt MAV-1 or the E3 null virus. mRNAs and protein for claudin-5, occludin, and zona occludens 2 were also reduced in infected cells. MAV-1 infection caused a loss of transendothelial electrical resistance in primary mouse brain endothelial cells that was not dependent on E3 or on MAV-1-induced CCL2 expression. Taken together, these results demonstrate that MAV-1 infection caused breakdown of the blood-brain barrier accompanied by decreased surface expression of tight junction proteins. Furthermore, while the MAV-1-induced pathogenesis and inflammation were dependent on E3, MAV-1-induced breakdown of the blood-brain barrier and alteration of endothelial cell function were not dependent on E3 or CCL2.

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