Preface
Contents
Contributors
Chapter 1: Opposing Roles of Vascular Endothelial Growth Factor C in Metastatic Dissemination and Resistance to Radio/Chemothe...
1 Introduction
1.1 Increased Aggressiveness of Tumors Depending on Their Metastatic Stage
1.2 A Focus on Lymphatic Vessels: Key Players in Metastatic Spread?
2 The Antagonistic Roles of VEGFC
3 Lymphatic Vessels and VEGFC-Dependent Aggressiveness of BC, RCC, HNSCC, and MDB
3.1 Lymphatic Vessels, VEGFC, and Prognosis in BC
3.2 Lymphatic Vessels, VEGFC, and Prognosis in HNSCC
3.3 Lymphatic Vessels, VEGFC, and Prognosis in MDB
3.4 Lymphatic Vessels, VEGFC, and Prognosis in RCC
4 Conclusions and TreatmentsΒ΄ Options
References
Chapter 2: Identification of VEGF Isoforms in Mouse, Rat, and Zebrafish Using RT-qPCR
1 Introduction
2 Materials
2.1 Nuclease-Free Water
2.2 Tissue Homogenization, Cell Lysis, and RNA Extraction
2.3 RNA Quantification and Purity
2.4 RNA Integrity
2.5 Reverse Transcription
2.6 Validation of qPCR Assays
2.7 qPCR
3 Methods
3.1 Primer Design
3.2 Tissue Homogenization, Cell Lysis, and RNA Extraction
3.3 Quantification of RNA by Fluorimetry
3.4 RNA Spectrophotometry
3.5 Determination of RNA Integrity Using an Agilent Bioanalyzer
3.6 Reverse Transcription
3.7 Validation of qPCR Primer Specificity and Production of Known Copy Number Standards
3.8 qPCR of Experimental Samples
3.9 Data Normalization
4 Notes
References
Chapter 3: Multiparameter Fluorescence-Activated Cell Sorting of Human Lymphatic Endothelial Cells
1 Introduction
2 Materials
2.1 Culture Medium and Solutions for Maintaining Foreskin and Lymphatic Malformation LEC Cultures
2.2 Enzymatic Digestion
2.3 Antibodies and Buffers
2.4 Equipment
3 Methods
3.1 Sample Collection
3.2 Sample Processing and Enzymatic Digestion
3.3 Sample Staining
3.4 Instrument Set Up and Cell Sorting
3.5 Cell Culture Post FACS Sorting
3.6 Subculturing LECs
3.7 Cryopreservation
4 Notes
References
Chapter 4: Absolute Quantification of Plasma Membrane Receptors Via Quantitative Flow Cytometry
1 Introduction
2 Materials
2.1 Stain Buffer Preparation
2.2 Antibodies and Reagents
3 Methods
3.1 Single-Cell Suspension Preparation
3.2 Fluorophore Panel Design and Controls
3.3 Immunostaining
3.4 Flow Cytometer Setup
3.5 Flow Cytometry Data Acquisition
4 Notes
References
Chapter 5: Measurement of VEGF Content in Exosomes and Subsequent Tumor Tubulogenesis and In Vivo Angiogenesis Functional Assa...
1 Introduction
2 Materials
2.1 Differential Centrifugation
2.2 Nano-flow Cytometry Analysis
2.3 Enzyme-Linked Immunosorbent Assay (ELISA) Detection of VEGF
2.4 Western Blotting Detection of VEGF
2.5 Effect of VEGF-Exosomes on Colony Formation of Tumor Cells
2.6 Coculture (HUVECs/HCC) Assay
2.7 Tubulogenesis Assay of VEGF-Exosome Treated HCC Cells
2.8 Matrigel Plug Assay for Evaluating the Role of VEGF-Exosomes In Vivo Angiogenesis
3 Methods
3.1 Exosomes Isolation by Differential Centrifugation
3.2 Nano-flow Cytometry Analysis of VEGF on Exosomes
3.3 Measurement of VEGF Content in Exosomes by ELISA
3.4 Measurement of VEGF Content in Exosomes by Western Blotting
3.5 Detection of the Effect of VEGF-Exosomes on Colony Formation of Tumor Cells
3.6 Role of HUVECs-Derived Exosomes on Tumor Cells Tubulogenesis By Coculture Assay
3.7 Role of VEGF-Exosomes Derived from Tumor ECs on Tumor Cell Tubulogenesis In Vitro
3.8 Role of Exosomal VEGF in Angiogenesis In Vivo
4 Notes
References
Chapter 6: SH2-Domain Protein Isolation Using Synthetic Phosphorylated Peptides to Study VEGFR2 Signaling
1 Introduction
2 Materials
2.1 Synthetic Peptides
2.2 Peptide Dissolving Reagents
2.3 Magnetic Beads and Reagents
2.4 Cell Culture
2.5 Hypotonic Lysis Buffer and Protein Lysate (See Note 1)
2.6 Determination of Protein Concentration
2.7 Western Blot Reagents
3 Methods
3.1 Peptide Design and Hydration
3.2 Cell Culture
3.3 Optimization of the Cell Lysate Protocol and Hypotonic Lysis Buffer Solution
3.4 Optimization of the Protein Precipitation Protocol
3.4.1 Preparation of Sera-Mag Speed Streptavidin-Coated Beads
3.4.2 Immobilization of Peptides to the Magnetic Beads
3.4.3 Precipitation of Peptide-Binding Proteins
3.4.4 Release of Immobilized Biotinylated Molecules
3.5 Mass Spectrometry
3.6 Determination of Protein Concentration
3.7 Western Blot
4 Notes
References
Chapter 7: Monitoring VEGF-Stimulated Calcium Ion Flux in Endothelial Cells
1 Introduction
2 Materials
2.1 Endothelial Cell Culture
2.2 Calcium Flux Assay
2.3 NFATc2 Localization
2.4 Equipment and Software
3 Methods
3.1 Primary Endothelial Cell Culture
3.2 Preparation of HUVECs and Loading Cells with Fluorescent Dye
3.3 Cytosolic Calcium Flux Assay
3.4 Immunofluorescence Analysis of Endothelial NFATc2 Localization
4 Notes
References
Chapter 8: Co-immunoprecipitation Assays
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Preparation of Cell Extracts
2.3 Pre-clearing the Lysates
2.4 Purification and Isolation of Protein Complexes
2.5 Western Blot for Protein Detection of Co-IP Complexes
3 Methods
3.1 Cell Culture
3.2 Cell Lysis
3.3 Pre-clearing the Lysates (See Note 11)
3.4 Purification and Isolation of Protein Complexes
3.5 SDS-PAGE and Western Blot for Protein Detection of Co-IP Complexes
4 Notes
References
Chapter 9: Using Immortalized Endothelial Cells to Study the Roles of Adhesion Molecules in VEGF-Induced Signaling
1 Introduction
2 Materials
2.1 Nucleofection of siRNA or TAT.Cre
2.2 VEGF-Induced Signaling Assay
3 Methods
3.1 siRNA Knockdown or TAT.Cre-Induced Deletion of Target Genes by Nucleofection
3.1.1 siRNA-Mediated Knockdown of Target Genes in PyMT Immortalized Microvascular ECs
3.1.2 TAT.Cre-Mediated Deletion of Target Genes in PyMT immortalized Microvascular ECs
3.2 Assessing the Effects of Target Genes Knockdown/Deletion on VEGF-Induced Signaling by Western Blot
4 Notes
References
Chapter 10: RNAscope for VEGF-A Detection in Human Tumor Bioptic Specimens
1 Introduction
2 Materials
2.1 Basic Laboratory Supplies
2.2 Histology Laboratory Supplies
2.3 RNAscope Equipment Supplied by Advanced Cell Diagnostic (ACD)
2.4 RNAscope Solutions
2.5 Image Acquisition and Analysis Systems
3 Methods
3.1 Sample Preparation
3.2 RNAscope Protocol
3.3 Image Acquisition
3.4 Image Analysis
4 Notes
References
Chapter 11: Global MicroRNA Profiling of Vascular Endothelial Cells
1 Introduction
2 Materials
2.1 Endothelial Cell Isolation/Culturing
2.2 miRNA Library Preparation
2.3 Gel Size Selection (Only if Library QC Fails, See Subheading 3.8)
2.4 For Troubleshooting with qPCR (Only if Library QC Fails, See Subheading 3.8)
3 Methods
3.1 Endothelial Cell Isolation (See Note 2)
3.2 RNA Extraction (See Note 7)
3.3 Library Preparation: 3β² Ligation
3.4 5β² Ligation
3.5 Reverse Transcription
3.6 Preparation of Beads
3.7 cDNA Cleanup
3.8 Library Amplification
3.9 miRNA Library Pre-sequencing QC
3.10 Real-Time PCR Troubleshooting
3.11 Size Selection of Library
3.12 Library Concentration and Read Allocation
3.13 Sequencing
3.14 Data Analysis
3.14.1 Preprocessing
3.14.2 Obtaining the Annotations for Hairpins and Mature miRNA Sequences
3.14.3 Method 1: Identification of Known Canonical miRNA Species
3.14.4 Method 1: Identification of Potential Novel miRNAs
3.14.5 Method 2, Approach 1: Identification of isomiRs and Preprocessing (miRge2.0, See Note 27)
3.14.6 Method 2, Approach 2: Identification of isomiRs and Preprocessing (Prost!, See Note 30)
3.14.7 Differential Expression Analysis (all Code Is in R)
4 Notes
References
Chapter 12: Endothelial Cell Tube Formation Assay: An In Vitro Model for Angiogenesis
1 Introduction
2 Materials
2.1 Cell Preparation
2.2 Tube Formation Assay
2.3 Visualization and Quantification of Tube Network
3 Methods
3.1 Preparation of Endothelial Cells
3.2 Tube Formation Assay
3.3 Quantification of Network
4 Notes
References
Chapter 13: Use of a Thin Layer Assay for Assessing the Angiogenic Potential of Endothelial Cells In Vitro
1 Introduction
2 Materials
3 Methods
3.1 Thin Layer Tube Formation Assay
3.2 PFA Fixation and Fluorescent Staining
3.3 Image Analysis and Quantification
3.4 RNA Extraction for qPCR
4 Notes
References
Chapter 14: VEGF-A165 -Induced Endothelial Cells Chemotactic Migration and Invasion Assays
1 Introduction
2 Materials
3 Methods
3.1 Chemotaxis Assay
3.2 Invasion Assay
3.3 Analysis
4 Notes
References
Chapter 15: Measuring Mitochondrial Calcium Fluxes in Cardiomyocytes upon Mechanical Stretch-Induced Hypertrophy
1 Introduction
2 Materials
2.1 Cells
2.2 Cyclic Mechanical Stretch
2.3 Mitochondrial Calcium Measurements
3 Methods
3.1 Cyclic Stretch
3.2 Probe Infection to Measure Ca2+ Flux
4 Notes
References
Chapter 16: Simultaneous Measurement of Endothelial Cell Proliferation and Cell Cycle Stage Using Flow Cytometry
1 Introduction
2 Materials
3 Methods
3.1 Carboxyfluorescein Succinimidyl Ester (CFSE) Staining
3.2 Propidium Iodide Staining and Flow Cytometric Analysis
4 Notes
References
Chapter 17: Ex Vivo Mouse Aortic Ring Angiogenesis Assay
1 Introduction
2 Materials
3 Methods
3.1 Harvesting the Aorta and Preparing the Rings (Day 1)
3.2 Embedding the Rings and Treatment with Agents of Interest (Days 2 to 10/12 When the Rings Are Fixed for Staining)
3.3 Immunofluorescent Staining
4 Notes
References
Chapter 18: Retinal Microvasculature-on-a-Chip for Modeling VEGF-Induced Permeability
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Collagen Matrix Preparation and Formation of the Retinal Microvasculature-on-a-Chip
2.3 Permeability Assay
2.4 Immunostaining
2.5 Particular Equipment
3 Methods
3.1 Formation of the Retinal Microvasculature-on-a-Chip
3.2 VEGF-Mediated Induction of Vascular Leakage and Quantitative Assessment of Vascular Permeability
3.2.1 VEGF-Mediated Induction of Vascular Permeability
3.2.2 Leakage Assay
3.2.3 Quantitative Assessment of Permeability
3.3 Immunostaining and Confocal Imaging of the Microvasculature-on-a-Chip
3.3.1 Immunostaining
3.3.2 Automated Confocal Imaging of the Microvasculature-on-a-Chip
4 Notes
References
Chapter 19: Preventing VEGF-Mediated Vascular Permeability by Experimentally Potentiating BBB Characteristics in Endothelial C...
1 Introduction
2 Materials
2.1 Stock Solution Preparation
2.2 Endothelial Cell Culture
2.3 Transendothelial Electrical Resistance (TEER) Assay
2.4 Fluorescein Isothiocyanate (FITC)-Dextran Permeability Assays
2.5 Immunocytochemistry
2.6 Working Stock Preparation for Promoting Barrier Resistance
3 Methods
3.1 Endothelial Cell Maintenance (see Note 13)
3.2 TEER Assay
3.3 FITC-Dextran Permeability Assays
3.4 Immunocytochemistry
4 Notes
References
Chapter 20: The Embryonic Mouse Hindbrain and Postnatal Retina as In Vivo Models to Study Angiogenesis
1 Introduction
1.1 The Mouse Embryo Hindbrain
1.2 The Postnatal Mouse Retina
2 Materials
2.1 Equipment
2.2 Reagents
3 Methods
3.1 Hindbrain Dissection and Fixation
3.2 Retina Dissection and Fixation
3.3 Whole-Mount Staining
3.4 Imaging
3.5 Quantitation of Angiogenesis
4 Notes
References
Chapter 21: Evaluating VEGF-Induced Vascular Leakage Using the Miles Assay
1 Introduction
2 Materials
2.1 Mouse Preparation
2.2 Injections
2.3 Dye Extraction/Quantification
3 Methods
3.1 Mouse Preparation
3.2 Injection of Evans Blue Dye
3.3 Induction of Vascular Leakage
3.4 Quantification of Vascular Leakage
4 Notes
References
Chapter 22: Modulation of VEGFA Signaling During Heart Regeneration in Zebrafish
1 Introduction
2 Cardiac Regeneration in Zebrafish
3 Mechanisms of Revascularization in Cardiac Repair and Regeneration
4 VEGFA and Heart Regeneration
5 Genetic Tools for Modulating VEGFA Signaling During Zebrafish Heart Regeneration
5.1 vegfaa-/-
5.2 Tg(hsp70l:vegfaa121-F17A)
5.3 Tg(hsp70l:loxp-Stop-loxp-mTom-codOptP2A-T46Avegfaa)
5.4 flt1-/-
5.5 nrp1a-/-
5.6 Tg(Ξ²actin2:loxP-mTagBFP-STOP-loxP-vegfaa)
6 Concluding Remarks
7 Transgenic Lines Acronyms and Abbreviations
References
Chapter 23: Three-Dimensional Visualization of Blood and Lymphatic Vessels in the Adult Zebrafish Heart by Chemical Clearing
1 Introduction
2 Materials
2.1 Chemical Clearing Reagents
2.2 Solutions
2.3 Antibodies
2.4 Consumables
2.5 Equipment
3 Methods
3.1 Tissue Collection and Preparation
3.2 Tissue Clearing: Fluorescent Reporter Lines
3.3 Tissue Clearing: Immunofluorescent Antibody Staining
3.4 Imaging
4 Notes
References
Chapter 24: Fluorescence-Activated Cell Sorting and Quantitative Real-Time PCR to Reveal VEGF-Expressing Macrophage Population...
1 Introduction
2 Materials
2.1 Needle Stab Injury
2.2 Cell Dissociation and FACS
2.3 RNA/cDNA Prep and qPCR
3 Methods
3.1 Needle Stab Injury
3.2 Cell Dissociation
3.3 FACS Sorting
3.4 RNA and cDNA Preparation
3.5 qPCR
4 Notes
References
Chapter 25: Assessing Molecular Regulation of Vascular Permeability Using a VEGF-Inducible Zebrafish Model
1 Introduction
2 Materials
2.1 Reagents for Generation of Cre recombinase mRNA
2.2 Tricaine (MS-222) Solution
2.3 2.5% Methylcellulose Solution
2.4 Equipment
3 Methods
3.1 Generation of Zebrafish Embryos
3.2 Linearization of pT3TS-Cre Plasmid DNA and Generation of Cre recombinase mRNA
3.3 Cre recombinase mRNA Injections
3.4 Morpholino Injections (Optional)
3.5 1-Phenyl-2-Thiourea (PTU) Treatment to Inhibition of Pigment Formation (Optional)
3.6 Selection of VEGF Transgenic Zebrafish
3.7 Dextran Injections
3.8 Heat Shock Induction of the VEGF Transgene
3.9 Imaging and Quantification of VEGF-Induced Vascular Permeability in Zebrafish
4 Notes
References
Index