In Vitro Models for Studying Angiogenesis - …

1 In VitroModels for Studying AngiogenesisSuparna SanyalMay 12, 2010 Overview Angiogenesis Important considerations for Studying Angiogenesis in Vitro BD in Vitro Angiogenesis Models EC tube formation EC migration EC invasionAngiogenesis Angiogenesis formation of new blood vessels (sprouting, branching etc.) from existing vessels ( wound healing, fertility etc.) Vasculogenesis formation of new blood vessels from angioblasts or progenitor stem cells ( de novovessel synthesis)ExcessiveInsufficientANGIOGENE SISC ancerRheumatoid ArthritisBlindness (Diabetic Retinopathy)PsoriasisAids ComplicationsStrokeHeart DiseaseUlcersSclerodermaInfertilityDisea ses Associated With AngiogenesisTumor AngiogenesisLocalized solid tumor Necessary for tumor growth and metastasis Inhibition of Angiogenesis represents a critical point of intervention for cancer treatment Circumvents problems of cellular toxicity and tolerance development seen with chemotherapyTumor AngiogenesisAngiogenesis Research Basic research to elucidate molecular mechanisms of Angiogenesis .

2 -Identify and characterize regulatory pathways that mediate various steps of Angiogenesis such as endothelial cell migration, invasion, and tubulogenesis To develop treatments for cancer and other diseases associated with Angiogenesis - Identification of compounds that inhibit or stimulate key steps in the Angiogenesis processPro-angiogenic factors released from hypoxic cellsVEGFbFGF1 Factors Bind to EC receptorsEndothelial Cell2 Tube formationHighly permeable1094567 ProliferationInvasionAdhesionMigrationMM ps activated and releasedBasement membranedegradation3 Pericyte recruitment Vessel stabilization11 Progression of AngiogenesisAnti-angiogenic Drugs Approved for Clinical Use and Phase of Clinical Trials for Other IndicationsFolkman, J. Nature Reviews Drug Discovery6:273-286 (2007).Clinical Relevance of Endothelial Cell Based in vitroAngiogenesis AssaysImportant Considerations for Developing Angiogenesis Studies Incorporate appropriate extracellular matrix (ECM) protein(s) to facilitate cell functionality and assay outcome Choose appropriate endothelial cell source Choice of Angiogenesis assay Establish acceptable dynamic range to measure stimulation and/or inhibition of Angiogenesis Extracellular MatrixECM provides a physiological substrate that supports key cellular functions Structural organization of cells and tissue Cell attachment, survival, and proliferation Induction and maintenance of cell differentiation Can influence signal transduction and regulation of gene expressionExamples.

3 Gelatin, fibronectin, vitronectin, laminin, collagen, BD Matrigel matrix HUVEC most commonly studied human EC type in Angiogenesis Source, isolation procedure, and initial culturing conditions can influence response to pro-angiogenic factors ( VEGF, bFGF)Endothelial CellsSources of Endothelial Cells Large vessel aortic ( , HAEC) umbilical vein ( , HUVEC) pulmonary artery Microvascular ( , HMVEC) brain lung dermis ( , HDMEC) myocardiumHuman Umbilical Vein Endothelial Cells Most commonly used human EC type for studies of Angiogenesis Source, isolation procedure, and initial culturing conditions can influence response to pro-angiogenic factors ( VEGF, bFGF) BD Human Umbilical Vein Endothelial Cells (HUVEC-2)(cat. no. 354151) Pre-qualified for responsiveness to VEGF in endothelial cell migration assay Tested for presence of von Willebrand factor (vWf), CD31, uptake of Dil-Ac-LDL, and absence of alpha actin Shipped on dry ice, store at -20 CDo not store in frost free or -70 C freezerBD BioCoat Angiogenesis SystemsBD HUVEC-2 cells Pre-qualified for VEGF responsiveness and for use with endothelial cell migration assayEndothelial Cell Tube Formation Composed of a 96-well black/clear plate coated with BD Matrigel matrix (non-insert system)Endothelial Cell Migration 24- or 96-Multiwell BD FluoroBlok insert (3 m pore size) Coated with human fibronectinEndothelial Cell Invasion 24-Multiwell BD FluoroBlok insert (3 m pore size) Coated with BD Matrigel matrixBD Fluorescent DyesAngiogenesis Tube Formation Assay1.

4 EC lysis of basement membrane and extracellular matrix2. EC invasion/migration3. EC proliferation4. Capillary tube formationand differentiationAngiogenic StimulatorsVEGF, FGFA ngiogenesis Tube Formation AssaySimplified Steps in Angiogenesis ProcessBD BioCoat Angiogenesis System: Endothelial Cell Tube FormationBD Falcon 96-well black/clear microplateBD Matrigel matrix coating Optimized manufacturing process generates flat surface; elimination of meniscus Screened for ability to promote tube formationCompatible with automated image acquisition and data processingReproducible Consistent endothelial cell tube formation: well-to-well, lot-to-lot, assay-to-assay Z' value = , suitable for compound screeningMat Cover and Lid Ensures product stabilityShipped on dry ice, store at -20 CDo not store in frost free or -70 C freezerEndothelial cellsAutomated Image Acquisition& Data ProcessingDay 1 Day 237 C/5% CO2incubationFluorescent dye labelingBD Matrigel MatrixFrozen 96-well tube formation microplatePro-angiogenic factorsTrypsinize and resuspend cells in assay mediumThaw, 6-24 hoursGel, 30 Pathway 855 BioimagerAssay Flow ChartBD Matrigel MatrixBright FieldBD Matrigel MatrixFluorescenceCollagen I2 DHuman Microvascular Endothelial CellsEndothelial Cell Tube FormationBD Tube Formation ModuleBD Image Data Explorer (Software)Image Analysis on BD Pathway BioimagerNon-confocal single plane Confocal single plane Confocal collapsed stack BD HUVEC-2 cells, stained with calcein AM, 4 Xconfocal images on BD Pathway BioimagerEndothelial Cell Tubulogenesis.

5 High Resolution Detection Using Confocal Collapsed Stack Imaging Angiogenesis Segmentation Confocal improves images resulting in better tubule detection Fewer broken tubules More accurate count of tube length and number (arrow)Non-confocalConfocal, collapsed stackCalcein staining, 4X confocal0 160 MAngiogenesis Dose Response to Suramin IC50 = 10-3010002000300040005000600070008000 Log [M] SuraminTotal Tube Length(pixels) 10-3050100150200250300350 Log [M] SuraminTube Complexity(total # of segments) 10-3010000200003000040000500006000070000 Log [M] SuraminTotal Tube Area(pixels)Different Analysis ParametersSimilar ResultsHUVEC Cell Number Titration101420242840 Number of cells per well (x 1000)HMEC-1 Cell Number Titration0200040006000800010000120001400 01600018000 Total tubule length (pixels)101420242840 Number of cells per well (x 1000)Effect of Cell Number Endothelial Cell Tube tube length (pixel)Tube Formation UsingVarious Human Endothelial CellsHMVEC are seeded onto BD Matrigel matrix-coated 96-well plates on three different days and incubated at 37 C for 24 hours.

6 Images are obtained using a 4X objective. Tube area is calculated for each plate (n=90).HMVEC Tubule Formation0500100015002000250030003500400 04500 Day 1 Day 2 Day 3 Tube Area (Fluorescent Units)ReproducibilityFigure 5 Resistin promotes endothelial cell differentiation into tube-like structures. Representative experiment (200X) showing capillary-like structures formed at 22h of culture. Suramin is the negative Simone, et al., Journal of Endocrinology189:691 699 (2006).Stimulation and Inhibition of Tube FormationEndothelial Cell Tube Formation Assay setupto data within 24 hours Validated Protocols High-throughput: 96-well format Automated image acquisition and data processing Labeling with fluorescent dye Morphometric analysis Can use fluorescent microscope Reproducible Consistent endothelial cell tube formation: Well-to-well, lot-to-lot, assay-to-assay Z' value = , good for screeningBD BioCoat Angiogenesis SystemsShipped on dry ice, store at -20 CDo not store in frost free or -70 C freezerBD HUVEC-2 cells Pre-qualified for VEGF responsiveness and for use with endothelial cell migration assayEndothelial Cell Tube Formation Composed of a 96-well black/clear plate coated with BD Matrigel matrix (non-insert system)Endothelial Cell Migration 24- or 96-Multiwell BD FluoroBlok insert (3 m pore size) Coated with human fibronectinEndothelial Cell Invasion 24-Multiwell BD FluoroBlok insert (3 m pore size)

7 Coated with BD Matrigel matrixBD Falcon FluoroBlok 24- and 96-Multiwell Insert Systems Unique fluorescence-blocking PET membrane Available in , , and m pore sizes Ease-of-use Real-time kinetic analyses Automation compatibleThe dyed membrane physically and visually separates cells and/ormolecules on top of membrane from those below the is BD FluoroBlok?Cross section of an insert system not to scaleBase plateBD FluoroBlok membraneInsert (individual or multiwell)Apical chamberAnalysis of Cell Migration and Invasion Using Fluorescence Blocking PET Membrane Cell Culture InsertsDetection Instrument A fluorescent plate reader with bottom-reading capability, and an inverted fluorescent microscope for confirmation and troubleshooting A fluorescence imager Set Up Guidelines and Dimensional Templates for Fluorescence Plate Readers Used With BD Falcon HTS FluoroBlok Insert Systems and BD BioCoat Multiwell Insert Cell-Based Assays, Technical Bulletin # 436 Any fluorescent dye derived from the fluorescein, rhodamine, and cyanine families can be used with this system Ultraviolet-inducible dyes tend to be incompatible with the BD FluoroBlok Insert since they tend to emit light in the blue range For more information on spectra and alternative fluorophore choices, consult the BD FluoroBlok Insert Cross Reference Chart.

8 Technical Bulletin #451 Cell Labeling Dyes emission wavelength must be between 490-700 nmSpectrum image from under GNU free documentation or Real-Time Kinetic AssayPOST-LABELINGPRE-LABELING Calcein AMCell migrationFor this, cell may bepre-labeled or intrinsically express a fluorescent proteinBD HUVEC-2 Cells Exhibit Concentration-Dependent Migration Towards VEGFVEGF-induced cell migration assessed using the BD BioCoat Angiogenesis system: endothelial cell migration (fibronectin-coated BD FluoroBlok membrane, 96-Multiwell format). Human fibronectin-coated BD FluoroBlok 24- and 96-Multiwell 3 m pore insert systems; non-occluded pores Optimum endothelial cell migrationBD BioCoat 96-well Endothelial CellMigration SystemMigration Activity Using Different EC Types This assay is well suited for medium throughput drug screening log [Inhibitor] (M)-13-12-11-10-9-8-7-6-5024681012 HMEC-1EC50= nMlog CHEMOATTRACTANT (M)Fold increase in migrationover control (mean+SE)-13-12-11-10-9-8-7-6-5024681012 HMEC-1EC50= nMlog CHEMOATTRACTANT (M)Fold increase in migrationover control (mean+SE)-9-8-7-6-5020406080100IC50= 44 nMCell migration(% inhibition)

9 Chemotaxis Stimulation and Inhibition in HMEC-1 CellsPore occludingBD Matrigel matrix layerBD FluoroBlok membrane, 3 m pores, 24-Multiwell insert plates Cross section of one of 24 inserts of a Multiwell Insert plateBD BioCoat Endothelial Cell Invasion SystemControlChemoattractantHuman Primary Vascular Endothelial Cell Invasion through BD Matrigel MatrixEffe ct of MMP inhibitor 1'10' Phe na nthroline on HMVEC Invasion02004006008001000120014001600180 0 ControlVEGF(4ng/ml) EGF(4ng/ml)+ 1'10' PhenathrolineFluorescent Units BD Matrigel matrix-coated BD FluoroBlok 24-Multiwell 3 m pore size insert system optimized for endothelial cell invasionBD BioCoat Endothelial Cell Invasion SystemABQuantitative analysis of HUVEC (A) migration or (B) invasion toward hMSC-conditioned media. Data presented are the mean of four inserts SD., p .05 compared with hMSCs; #, p .05 compared with hMSC SPH1. Abbreviations: DMEM, Dulbecco. Potpova, , et al., Stem Cells25:1761-1768 (2007).

10 Mesenchymal Stem Cells Support Migration and Extracellular Matrix Invasion of Endothelial Cells In VitroTime Course of MCP-1 Induced Chemotaxisin THP-1 and Primary 101520253035456090 Time (mins)Fold increase over 101520253045 Time of incubation (mins)Fold increase over control Pre-labeled cells in the inserts were incubated with 25 nMMCP-1 in the bottom chamber Bottom fluorescence was measured at varying time points Data on the graph are means from a typical experiment(n=4 wells) BD Biosciences Technical Bulletin #457 Criteria for a Robust Migration/Invasion Assay on the BD FluoroBlok Insert System Definition: Positive control: Cells with chemoattractant Negative control: Cells without chemoattractant Background fluorescence: Pre-labeled cells: zero time point Post-labeled cells: Unlabeled cell Assay criteria: % CV Dynamic range (signal-to-noise ratio) Z = 1-(3SD of POS + 3SD of NEG/(Mean of POS-Mean of NEG)* > = is preferred *Zhang, etc.)