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      The role of shear stress in Blood-Brain Barrier endothelial physiology

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          One of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells (ECs) into a blood-brain barrier (BBB) phenotype is shear stress (SS). With the use of a well established humanized dynamic in vitro BBB model and cDNA microarrays, we have profiled the effect of SS in the induction/suppression of ECs genes and related functions.


          Specifically, we found a significant upregulation of tight and adherens junctions proteins and genes. Trans-endothelial electrical resistance (TEER) and permeability measurements to know substances have shown that SS promoted the formation of a tight and highly selective BBB. SS also increased the RNA level of multidrug resistance transporters, ion channels, and several p450 enzymes. The RNA level of a number of specialized carrier-mediated transport systems (e.g., glucose, monocarboxylic acid, etc.) was also upregulated.

          RNA levels of modulatory enzymes of the glycolytic pathway (e.g., lactate dehydrogenase) were downregulated by SS while those involved in the Krebs cycle (e.g., lactate and other dehydrogenases) were upregulated. Measurements of glucose consumption versus lactate production showed that SS negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration. BBB ECs are responsive to inflammatory stimuli. Our data showed that SS increased the RNA levels of integrins and vascular adhesion molecules. SS also inhibited endothelial cell cycle via regulation of BTG family proteins encoding genes. This was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. Furthermore, analysis of 2D gel electrophoresis (which allows identifying a large number of proteins per sample) of EC proteins extracted from membrane sub-cellular endothelial fractions showed that SS increased the expression levels of tight junction proteins. In addition, regulatory enzymes of the Krebb's cycle (aerobic glucose metabolism) were also upregulated. Furthermore, the expression pattern of key protein regulators of the cell cycle and parallel gene array data supported a cell proliferation inhibitory role for SS.


          Genomic and proteomic analyses are currently used to examine BBB function in healthy and diseased brain and characterize this dynamic interface. In this study we showed that SS plays a key role in promoting the differentiation of vascular endothelial cells into a truly BBB phenotype. SS affected multiple aspect of the endothelial physiology spanning from tight junctions formation to cell division as well as the expression of multidrug resistance transporters. BBB dysfunction has been observed in many neurological diseases, but the causes are generally unknown. Our study provides essential insights to understand the role played by SS in the BBB formation and maintenance.

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          Most cited references 46

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          The dynamic response of vascular endothelial cells to fluid shear stress.

          We have developed an in-vitro system for studying the dynamic response of vascular endothelial cells to controlled levels of fluid shear stress. Cultured monolayers of bovine aortic endothelial cells are placed in a cone-plate apparatus that produces a uniform fluid shear stress on replicate samples. Subconfluent endothelial cultures continuously exposed to 1-5 dynes/cm2 shear proliferate at a rate comparable to that of static cultures and reach the same saturation density (congruent to 1.0-1.5 X 10(5) cells/cm2). When exposed to a laminar shear stress of 5-10 dynes/cm2, confluent monolayers undergo a time-dependent change in cell shape from polygonal to ellipsoidal and become uniformly oriented with flow. Regeneration of linear "wounds" in confluent monolayer appears to be influenced by the direction of the applied force. Preliminary studies indicate that certain endothelial cell functions, including fluid endocytosis, cytoskeletal assembly and nonthrombogenic surface properties, also are sensitive to shear stress. These observations suggest that fluid mechanical forces can directly influence endothelial cell structure and function. Modulation of endothelial behavior by fluid shear stresses may be relevant to normal vessel wall physiology, as well as the pathogenesis of vascular diseases, such as atherosclerosis.
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            Tight junctions of the blood-brain barrier: development, composition and regulation.

            1. The blood-brain barrier is essential for the maintenance and regulation of the neural microenvironment. The main characteristic features of blood-brain barrier endothelial cells are an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. 2. Endothelial blood-brain barrier tight junctions differ from epithelial tight junctions, not only by distinct morphological and molecular properties, but also by the fact that endothelial tight junctions are more sensitive to microenvironmental than epithelial factors. 3. Many ubiquitous molecular tight junction components have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin and 7H6. Signaling pathways involved in tight junction regulation include G-proteins, serine-, threonine- and tyrosine-kinases, extra and intracellular calcium levels, cAMP levels, proteases and cytokines. Common to most of these pathways is the modulation of cytoskeletal elements and the connection of tight junction transmembrane molecules to the cytoskeleton. Additionally, crosstalk between components of the tight junction- and the cadherin-catenin system of the adherens junction suggests a close functional interdependence of the two cell-cell contact systems. 4. Important new molecular aspects of tight junction regulation were recently elucidated. This review provides an integration of these new results.
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              The mammalian anti-proliferative BTG/Tob protein family.

              The mammalian BTG/Tob family comprises six proteins (BTG1, BTG2/PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob and Tob2), which regulate cell cycle progression in a variety of cell types. They are characterised by the conserved N-terminal domain spanning 104-106 amino acids. Recent biochemical and structural data indicate that the conserved BTG domain is a protein-protein interaction module, which is capable of binding to DNA-binding transcription factors as well as the paralogues CNOT7 (human Caf1/Caf1a) and CNOT8 (human Pop2/Calif/Caf1b), two deadenylase subunits of the Ccr4-Not complex. Consistent with this finding, several members of the BTG/Tob family are shown to be implicated in transcription in the nucleus and cytoplasmic mRNA deadenylation and turnover. The C-terminal regions are less conserved and appear to mediate protein-protein interactions that are unique to each family member. The human and mouse BTG/Tob proteins will be the focus of this review and structural aspects of BTG/Tob interactions with components of the Ccr4-Not complex, and the role of the BTG/Tob proteins in the regulation of gene expression, tumourigenesis and cancer will be discussed.

                Author and article information

                BMC Neurosci
                BMC Neuroscience
                BioMed Central
                11 May 2011
                : 12
                : 40
                [1 ]Cerebrovascular Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195 USA
                [2 ]Dept. of Cell Biology, Cleveland Clinic, Cleveland, OH 44195 USA
                [3 ]Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Cleveland, OH 44195 USA
                Copyright ©2011 Cucullo et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Research Article


                cell cycle, alternative, shear stress, cerebral blood flow, inflammation, in vitro


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