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      Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs)

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          Abstract

          Background

          The endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood–brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells.

          Methods

          To assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm −2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood–brain barrier were performed.

          Results

          Human brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood–brain barrier markers.

          Conclusions

          The characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood–brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation of tight junctions decreases cell motility and prevents any morphological transitions, (3) flow serves to increase the contact area between cells, resulting in very low cell displacement in the monolayer, (4) since tight junctions are already formed under static conditions, increasing the contact area between cells does not cause upregulation in protein and gene expression of BBB markers, and (5) the increase in contact area induced by flow makes barrier function more robust.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12987-017-0068-z) contains supplementary material, which is available to authorized users.

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          Most cited references63

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          In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform

          In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment.
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            FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images.

            Cell biology heavily relies on the behavior of fibrillar structures, such as the cytoskeleton, yet the analysis of their behavior in tissues often remains qualitative. Image analysis tools have been developed to quantify this behavior, but they often involve an image pre-processing stage that may bias the output and/or they require specific software. Here we describe FibrilTool, an ImageJ plug-in based on the concept of nematic tensor, which can provide a quantitative description of the anisotropy of fiber arrays and their average orientation in cells, directly from raw images obtained by any form of microscopy. FibrilTool has been validated on microtubules, actin and cellulose microfibrils, but it may also help analyze other fibrillar structures, such as collagen, or the texture of various materials. The tool is ImageJ-based, and it is therefore freely accessible to the scientific community and does not require specific computational setup. The tool provides the average orientation and anisotropy of fiber arrays in a given region of interest (ROI) in a few seconds.
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              A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes.

              Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400Omegacm(2) on average, while the endothelial permeability coefficients (P(e)) for fluorescein was in the range of 3x10(-6)cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R(2)=0.89) was found between in vitroP(e) values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs.
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                Author and article information

                Contributors
                jdestef@jhu.edu
                zxu20@jhmi.edu
                awill35@illinois.edu
                nahomyimam@yahoo.com
                (410) 516-8774 , searson@jhu.edu
                Journal
                Fluids Barriers CNS
                Fluids Barriers CNS
                Fluids and Barriers of the CNS
                BioMed Central (London )
                2045-8118
                4 August 2017
                4 August 2017
                2017
                : 14
                : 20
                Affiliations
                [1 ]ISNI 0000 0001 2171 9311, GRID grid.21107.35, Institute for Nanobiotechnology, , Johns Hopkins University, ; 100 Croft Hall, 3400 North Charles Street, Baltimore, MD 21218 USA
                [2 ]ISNI 0000 0001 2171 9311, GRID grid.21107.35, Department of Materials Science and Engineering, , Johns Hopkins University, ; Baltimore, MD 21218 USA
                [3 ]ISNI 0000 0001 2171 9311, GRID grid.21107.35, Department of Biomedical Engineering, , Johns Hopkins University, ; 720 Rutland Avenue, Baltimore, MD 21205 USA
                Author information
                http://orcid.org/0000-0002-5417-0828
                Article
                68
                10.1186/s12987-017-0068-z
                5543552
                28774343
                fa4b7f29-72c3-4614-a69a-4ab2953ce8de
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 30 December 2016
                : 20 July 2017
                Funding
                Funded by: Defense Threat Reduction Agency (US)
                Award ID: HDTRA1-15-1-0046
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000968, American Heart Association;
                Award ID: 15GRNT25090122
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Neurology
                shear stress,brain microvascular endothelial cells (bmecs),human endothelial cell line,blood–brain barrier,endothelial turnover,cell morphology,cell motility,stem cells

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