In Vitro Models of the Blood-Brain Barrier Prof. Pierre-Olivier Couraud

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1 Director, Institut Cochin France 1 Introduction: phenotypic characteristics of the BBB Reference in vitro models of the BBB A new model of human BBB Applications of in vitro models of the BBB 2 The Blood-Brain Barrier Intra-cardiac injection of fluorescently-labeled dextran (10 kda): No diffusion of the dye into the CNS across brain capillary walls From Furuse & Tsukita 3 1

2 The Blood-Brain Barrier (BBB) Brain endothelial cells Prolonged pedicle of astrocyte Pericyte Pericytes Basal membrane Pinocytotic v esicles Endothelial cell Mitochondria Tight j unction Astrocytes 4 The Blood-Brain Barrier (BBB) Pericytes Pericytes control CNS capillary diameter Peppiatt et al., Nature Oct The Blood-Brain Barrier (BBB): Molecular Characteristics Brain endothelial cells present a unique phenotype, characterized by a highly restrictive permeability to solutes, due to: 1. The presence of continuous intercellular tight junctions 2. The polarized expression of specific transporters 6 2

3 Introduction: phenotypic characteristics of the BBB Reference in vitro models of the BBB A new model of human BBB Applications of in vitro models of the BBB 7 In vitro Culture of Brain Microvessel Endothelial Cells Cultured brain microvascular endothelial cells 8 In vitro Culture of Brain Microvessel Endothelial Cells Phenotypic characterization PECAM-1 (CD31), vw F, VE-cadherin (Cadh-5) Lectin binding sites (BSI-4, UEA-1) Uptake of acetylated LDL No staining for GFAP, CD68, sm Actin P-glycoprotein, transferrin receptor, Tight junction proteins: occludin, claudins, JAM, ZO-1 Reduced drug permeabilit y Trans-endothelial electrical resistan ce (TEER) 9 3

4 BBB In vitro Models Cultured brain microvascul ar endotheli al cells Bov ine cells (Cecchelli et al., de Boer et al.) Porcine cells (Galla et al.) Rat cells (Deli et al., Perrière et al.) 10 Co-Culture of Brain Endothelial Cells with Glial Cells Glial cells (rat or porcine primary cultures; astrocytes, pericytes) are co-cultured with brain endothelial cells grown on semi-permeable filters (Transwell) Brain Endothelial Cells (bovine, mouse, rat) Glial Cells 11 In vitro Permeability Assay Unstirred water layer Endothelial cell Tight junction Filter Unstirred water layer Clearance(µL) = [concentration abluminal] [volume abluminal] [concentration luminal] 1 The average volume cleared is plotted versus time Permeability surface area product value for endothelial monolayer (PSe) is: PS endothelial 1 = PS time plotted 1 PS insert 1 PSe divided by the surface area is the endothelial permeability coefficient [Pe (cm/s)] 12 4

5 Trans-Endothelial Electrical Resistance (TEER) Chopstick electrodes with the Millicell ERS volt-ohmmeter: easy to use, but hardly reproducible Endohm system: large areas to cover the whole filter, more reproducible Impedance spectrosco py: resistance and capacit an ce measurement s (HJ Galla s group, Germany) cells are grown either on insert filters or directly on gold micro-electrodes Coculture with astrocytes: Enhanced TEER from to Ω.cm 2 13 Correlation Between In vitro and In vivo Drug Permeability In vitro BBB- P(cm/hr) R 2 = In vivo BBB- P [log(cbr/cbl)] Oldendorf 1974 Exp Bio Med 147: 812 Cecchelli et al., (1999) 14 Correlation Between In vitro and In vivo Drug Permeability Comparison of various in vitro models In vitro models for the blood brain barrier P. Garberg, M. Ball, N. Borg, R. Cecchelli, L. Fenart, R.D. Hurst, T. Lindmark, A. Mabondzo, J.E. Nilsson, T.J. Raub, D. Stanimirovic, T. Terasaki, J.-O. Ö Berg, T. Ö Sterberg; Toxicology in Vitro 19 (2005) Correlation between distribution to brain tissue in vivo and transport across BBEC in vitro Correlation between distribution to brain tissue in vivo and transport across Caco-2 cells in vitro log Pe in vitro (BBEC) log Papp in vitro (Caco-2) log Papp in vivo (MBUA) log Papp in vivo (MBUA) 15 5

6 Immortalization of Rat Brain Endothelial Cells RBE4 (E1A) Roux et al. (1994) GP8 (T-SV40) Greenwood et al. (1996) GPNT (T-SV40) Régina et al. (1999) Cultured brain microvascul ar endotheli al cells TR-BBBs (T-SV40) Terasaki et al. (2001) rbcec4 (Polyoma T) Blasig et al. (2001) 16 BBB In vitro Models: Primary BMECs vs. immortalized cell lines Primary BMECs Rat immortalized cell lines Bovine cells, Porcine cells Very good differentiation Limited supply Limited life-span Good differentiation Illimited supply Illimited life-span Susceptible to genetic engineerin g Towards an in vitro model of human BBB? 17 Introduction: phenotypic characteristics of the BBB Reference in vitro models of the BBB A new model of human BBB Applications of in vitro models of the BBB 18 6

7 Immortalization of Human Brain Endothelial Cells Infection of primary cultures by lentiviral vectors encoding htert and T-SV40 Selection of fully differentiated cellular clones Primary culture hcmec/d3 cell line 19 hcmec/d3: A Non-Transformed Phenotype (1) Contact-inhibited, substrate - and growth factor-dependent proliferation Normal diploid phenoty pe Absence of proliferation in soft agar 20 hcmec/d3: A Non-Transformed Phenotype (2) Formation of capillary-like structures in 3D gels 21 7

8 hcmec/d3: Expression of Junction Proteins (1) PECAM-1 β -catenin VE-cadherin γ-catenin 22 hcmec/d3: Expression of Junction Proteins (2) ZO-1 JAM-A Claudin-5 23 Correlation Between In vivo and In vitro Permeability A B diazepam R = prazosin vincristine colchicine C R = diazepam vincristine prazosin colchicine B: HCMEC/D3 vs. GPNT C: HCMEC/D3 vs. HUVEC 24 8

9 Efflux Pumps: ABC Transporters Multidrug resistance protein: MDR-1 / ABCB1 N ATP C ATP Multidrug resistance-relat ed proteins: MRP-1-MRP-7 / ABCC1-7 N C MRP-1 Breast cancer resistan ce protein: BCRP / ABCG2 C 25 hcmec/d3: Functionality of ABC Transporters Drug uptake (% control) Calcein-AM * * M K MK PSC 833 KO 143 Inhibitors Rhodamine 123 * Drug uptake experiments [ 3 H] Daunorubicin * * *: p < 0.01 * MK PSC 833 KO 4143 Inhibitors PSC 833 M K 571 KO 143 PSC 833 MK 571 Inhibitors The hcmec/d3 Cell Line Stably maintains a non-transformed endothelial phenotype, with growth factor-dependent proliferation, contact-inhibition, Expresses key tight junction-asso ciat ed proteins known to be expressed at the BBB Expresses the expected adhesion molecules (ICAM-1, VCAM-1, etc.) and chemokin e receptors involved in leukocyt e infiltration across the BBB Displays a restricted permeabilit y to small hydrophilic molecules Expresses functional ABC transporters: P-gp (MDR-1), MRPs, BCRP We believe that the hcmec/d3 cell line may constitute a unique in vitro model of human BBB Weksler et al., FASEB J

10 The hcmec/d3 Model Under Flow: Further Differentiation hcmec/d3 in a DIV-BBB device (Collaboration with Dr. D Janigro, Cleveland, USA) 28 hcmec/d3 in a DIV-BBB Device hcmec/d3 cells present high TEER, even without astrocytes 29 Hyperosmolar Opening of the BBB in DIV Models Transient TEER decrease induced by intraluminal perfusion (30 sec) with hyperosmolar mannitol (1.6M) 30 10

11 [ 14 C]-Sucrose Permeability in DIV Models hcmec/d3 cells display a v ery low permeability to sucrose: 2.11 x 10-7 ± 0.53 cm/sec i.e., 1.3 x 10-5 cm/min vs. 1.6 x 10-3 cm/min (Transwell) 31 hcmec/d3 in a DIV-BBB Device Conclusions hcmec/d3 cells hav e the intrinsic capacity to display a BBB phenotype, when grown in appropriate conditions Immortalized human brain endothelial cells and flow-based vascular modeling: a marriage of convenien ce for rational neurovascular studies Cucullo et al., J Cereb Blood Flow Metab (2007) 32 Introduction: phenotypic characteristics of the BBB Reference in vitro models of the BBB A new model of human BBB Applications of in vitro models of the BBB 33 11

12 BBB Physiological and Pathophysiological Roles What is the cellular and molecular basis of the BBB? CNS inflammation: what is the functional role of brain endothelium in leucocyte, virus or bacteria infiltration? How is brain endothelium involved in CNS tumor progression? Brain vasculature dysfunctions in stroke? How can we deliv er therapeutic drugs to the CNS through the BBB? How can we design and assay new drug candidates? 34 In vitro BBB Models and Research Axes Mechanisms of brain endothelial response to cell adhesion and transmigration: endothelial cell signaling, regulation of BBB permeability Structural and functional analysis of tight junctions, receptors, transporters: drug targeting to the CNS High throughput screenin g of drug candidates: drug discovery 35 Brain Endothelial Response to Cell Adhesion and Transmigration Leukocyte infiltration across the BBB Rolling Leucocyte Selectin ligands, α4-integrins Firm adhesion β2-integrins α4-integrins, Infiltration Tissue retention β1-integrins Endothelium E- and P-selectins, VCAM-1 ICAM-1, -2, ICAM-1, VCAM-1 PECAM-1, CD99, JAM-1, CD40, HA ECM 36 12

13 Brain Endothelial Response to Cell Adhesion and Transmigration A leucocyte migratory cup or docking structure for leukocyte firm adhesion and trans-endothelial migration ICAM-1, VCAM-1 37 Brain Endothelial Response to Cell Adhesion and Transmigration LFA-1 Mac-1 Leukocyte ICAM-1 Endothelial cell RhoGTPase Actin cytoskeleton rearrangement SHP-2 Src Cortactin phosphorylation ICAM-1 and PECAM-1 signalings control trans-endothelial migration of leukocytes P P PECAM-1 TJ AJ 38 Tight Junctions, Receptors and Transporters BBEC solo culture BBEC co-culture hcmec/d3 cond. 1 hcmec/d3 cond. 2 kda ph4.0 ph4.5ph5.0ph5.5ph6.0 ph6.5ph7.0ph Differential global proteomic analysis by 2DE - DIGE / MS-MS (Coll. R. Cecchelli & C. Flahaut, Lens, France) 39 13

14 Tight junctions, receptors and transporters Analysis of TJ Proteomes in hcmec/d3 Cells A key role for Claudin-5 at the blood brain barrier Aqueous pore Trends in Cell Biology TJ strand Claudin Impairment of the blood brain barrier in Claudin-5-deficient mice From Furuse & Tsukita, Trends Cell Biol 2006, 16: Tight junctions, receptors and transporters Proteomics: Absolute Quantification of Membrane Proteins by Multiple Reaction Monitoring (MRM) VPQVSTPTLVEVSR Select the best peptide candidate of protein of interest by in silico calculation Synthetise peptide with heavy isotopes of C or N Optimize LC-MS/MS separation protocol for best quantitation Peptide from sample Added peptide Add known quantity of synthetic peptide (internal standard) to sample Trypsin digest Reproduce LC-MS/MS separation protocol to perform absolute quantitation (integrate areas) on mass spectrum 41 Tight junctions, receptors and transporters Proteomic Analysis of BBB Transporters and Receptors by Quantitative Mass Spectrometry 100 In plasma membrane fraction of hcmec / D3 cell ( fmol / mg protein) (Coll. S. Ohtsuki & T. Terasaki, Sendai, Japan) 10 Na + / K + ATPase Glut1 Mdr1a / MDR1 1 1 Bcrp / BCRP Mrp1 / Mct1 / MCT1 MRP1 0.1 Mrp4/ MRP4 Mouse brain capillary fraction (fmol/mg protein) 42 14

15 Tight junctions, receptors and transporters Optimizing Drug Delivery to the Brain Can we modulate ABC-transporters activity? Can we take advantage of the internalization of receptors / transporters on brain endothelium? i.e., TransfR, p97, HB-EGF Can we identify new markers of brain endothelium? Can we design new vectors targeted to brain endothelium? Tight junction structural and functional organization 2. Cellular/molecular analysis of the «neurovascular unit» identification BBB of BBB-inducing In vitro Models: factors and regulators Conclusions, Perspectives 3. Understanding BBB dysfunction in CNS pathologies: stroke, multiple sclerosis, Alzheimer s disease 4. Optimizing drug delivery to the brain 44 Institut Cochin, Paris Department of Cell Biology Collaborations R. Cecchelli & C. Flahaut Univ. Artois, Lens, France J. Greenwood Inst. Ophthalmology, London D. Janigro Cleveland Clinic, USA J.M. Scherrmann Univ. Paris-Descartes, Paris T. Terasaki Sendai Univ., Japan B. W eksler Cornell University, NY, USA 45 15

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