Angiogenesis is the growth of new blood vessels and is an important natural process in the body. A healthy body maintains a perfect balance of angiogenesis modulators. In many serious disease states, however, the body loses control over antiogenesis. Diseases that are angiogensis-dependent result when blood vessels either grow excessively or insufficiently. * Tried-and-tested techniques written by researchers that developed them, used them, and brought them to fruition * Provides the "builder's manual" for essential techniques--a one-stop shop that eliminates needless searching among untested techniques * Includes step-by-step methods for understanding the cell and molecular basis of wound healing, vascular integrin signaling, mechanical signaling in blood vessels, and vascular proteomics
Author(s): David A. Cheresh
Series: Methods in Enzymology 443
Edition: 1
Publisher: Academic Press
Year: 2008
Language: English
Pages: 382
Contributors to Volume 443......Page 1
Preface - A Tribute to Dr. Judah Folkman......Page 5
Volume in Series......Page 7
Approaches for Studying Angiogenesis-Related Signal Transduction......Page 33
Angiogenesis......Page 34
VEGF signaling......Page 35
Stimulation of Akt phosphorylation in cell lysates......Page 36
Detection of Akt phosphorylation......Page 37
Use of dominant negative mutants to dissect signaling pathways......Page 38
Use of RNA interference to interrogate signaling pathways......Page 39
Phosphorylation of eNOS on Ser1179......Page 40
Detection of eNOS phosphorylation by use of phospho-specific antibodies......Page 41
Sample preparation from BAEC in culture......Page 42
Citrulline assay for eNOS activity......Page 43
Preparation of BAEC for detection of NO......Page 44
NO (nitrite) accumulation before and after agonist stimulation......Page 45
VEGF-induced stress fiber formation......Page 46
VEGF-induced Rac activation......Page 47
Stress fiber formation assay......Page 48
Approaches to dissect Rac1 function in cells......Page 49
Concluding Remarks......Page 50
References......Page 51
Physiologic Stress-Mediated Signaling in the Endothelium......Page 56
Introduction......Page 57
Protocol for Isolating Pericytes......Page 60
Design of a parallel plate flow chamber......Page 61
Calculation of the imposed fluid shear stress in a parallel plate flow chamber......Page 62
Materials......Page 63
Culture of aortic rings in 96-well plates and thick gels......Page 158
Disturbed flow chamber......Page 64
Materials......Page 65
Cone and Plate Flow System......Page 66
Endothelial wound healing assay......Page 67
Immunodetection of blood vessels......Page 68
Cyclic Stretching of the Endothelium......Page 69
Methods......Page 71
Western blotting......Page 72
References......Page 73
Endothelial Cell Adhesion and Migration......Page 76
Introduction......Page 77
Cell Preparation......Page 79
Preparation of Well-Defined Surfaces......Page 80
Preparation of Polyacrylamide Substrates for Cell Adhesion Studies......Page 81
Observation of endothelial cell spreading dynamics......Page 85
Centrifugation assay......Page 86
Quantifying Endothelial Cell Migration......Page 87
Collective cell migration: The wound-healing assay......Page 88
Individual cell motions: Calculation of cell speed......Page 90
References......Page 92
An Optimized Three-Dimensional In Vitro Model for the Analysis of Angiogenesis......Page 96
Angiogenesis......Page 97
In vitro angiogenesis models......Page 98
Introduction......Page 100
Cytodex 3 bead preparation......Page 102
Embedding coated beads in fibrin gel (Day 0)......Page 103
Immunocytochemistry of Angiogenic Sprouts In Vitro......Page 104
Retroviral Transduction of EC......Page 106
Protocol for 3-dimensional Pericyte-EC Coculture......Page 353
Discussion......Page 109
References......Page 252
In Vitro Three Dimensional Collagen Matrix Models of Endothelial Lumen Formation During Vasculogenesis and Angiogenesis......Page 114
Introduction......Page 115
In Vitro Models of Vasculogenesis and Angiogenesis in 3D Collagen Matrices......Page 117
Study of EC lumen and tube morphogenesis with an in vitro EC vasculogenesis model......Page 118
Critical points for success......Page 208
Study of EC tube regression with an in vitro EC or EC-pericyte vasculogenesis model......Page 121
Experimental considerations......Page 122
Molecular/Genetic Manipulation of ECs......Page 124
Microcontact printing of microarrays of focal adhesions sized ECM islands......Page 125
Rho GTPase Activation Assay During EC Tube Morphogenesis in 3D Collagen Matrices......Page 126
Biochemical Analysis of Interactions Between Cdc42 and its Downstream Effectors During EC Lumen and Tube Formation in 3D Colla......Page 127
The S-tag pull-down assay......Page 128
Conclusions......Page 129
References......Page 130
In Vitro Differentiation of Mouse Embryonic Stem Cells Into Primitive Blood Vessels......Page 133
Introduction......Page 134
Maintenance of ES Cells......Page 137
Procedure......Page 231
Materials......Page 138
Protocol #2: Generation of EBs for in vitro differentiation with dissociation by dispase......Page 139
Quantification......Page 140
Generation of Transgenic ES Cell Lines......Page 141
3D Coculture in a collagen sandwich......Page 143
Protocol #5: Visualization of ES cell-derived blood vessels with antibodies against PECAM or ICAM-2......Page 144
Acknowledgments......Page 145
The Aortic Ring Model of Angiogenesis......Page 148
Introduction......Page 312
Cell culture......Page 151
Preparing aortic rings......Page 152
Embedding aortic rings in collagen gels......Page 153
Quantification of angiogenesis......Page 154
Whole mount preparations......Page 156
Horseradish peroxidase immunohistochemistry......Page 157
Cultures of quiescent aortic rings......Page 160
Troubleshooting......Page 161
Concluding Remarks......Page 162
References......Page 356
An Assay System for In Vitro Detection of Permeability in Human "Endothelium"......Page 166
Introduction......Page 167
Procedure......Page 169
Procedure......Page 170
Flow chamber studies......Page 198
Procedure for PECAM immunolabeling......Page 173
Establishment of permanent SEC lines......Page 194
Use of this in vitro permeability assay to answer biologic questions......Page 174
Procedure for in vitro assay of permeability......Page 175
Procedure for in vivo assay......Page 176
Conclusion......Page 178
References......Page 180
Assays of Transendothelial Migration In Vitro......Page 183
Methods for Investigating Leukocyte Transendothelial Migration Under Static or Flow Conditions In Vitro......Page 184
Reagents......Page 188
Procedure......Page 189
Procedure......Page 191
Preparation of the column......Page 330
Reagents......Page 195
Procedure......Page 196
Preparation for the flow chamber assay......Page 200
Analyses of leukocyte-endothelial adhesion events......Page 201
References......Page 203
Cell Mechanics at Multiple Scales......Page 205
Introduction......Page 206
Large-Scale Cell Stretching......Page 207
Magnetic Micromanipulation......Page 209
Instrument design......Page 210
Calibration......Page 211
Cell-based experiments......Page 212
Fluorescence Laser Tracking Microrheology......Page 214
Instrument design......Page 215
Theoretical principles of FLTM......Page 216
Cell sample preparation......Page 218
Calibration......Page 219
Illustrative results......Page 220
General Summary and Conclusion......Page 224
References......Page 225
Introduction......Page 227
Integrin-Mediated Cell Adhesion......Page 230
Analysis of Integrin-Dependent Cell Migration......Page 232
Recombinant SEMA3F protein purification......Page 331
Procedure......Page 233
Quantification......Page 234
Procedure......Page 235
Materials......Page 236
Procedure......Page 237
Roles of Integrins in Endothelial Angiogenic Programming......Page 238
Procedure......Page 240
Procedure......Page 241
Equipment......Page 243
Procedure......Page 244
Integrin Affinity Modulation and Its Analysis......Page 245
Procedure......Page 247
Integrin and ECM Roles in Endothelial Cell Proliferation and Apoptosis......Page 248
Procedure......Page 249
Equipment......Page 250
Procedure......Page 251
Methods for Studying Mechanical Control of Angiogenesis by the Cytoskeleton and Extracellular Matrix......Page 255
Introduction......Page 256
Control of CE Cell Behavior with Different ECM Coating Densities......Page 257
Analytical methods......Page 259
SEMA3F-induced Endothelial Cell Collapse......Page 260
Microcontact printing......Page 263
Analysis of cell cycle progression......Page 265
Analysis of directional lamellipodia extension......Page 266
Control of Cell Fate Switching by Modulating the Cytoskeleton......Page 268
Control of cell shape and function with adenoviral CaD......Page 269
Analysis of the Mechanism of Cell Shape-Dependent Growth Control......Page 271
Cell-cycle analysis......Page 272
Determination of cell cycle position......Page 273
Methods for Analyzing Rho-Dependent Control of Cell Growth and Movement......Page 274
Preparation and delivery of recombinant Rho proteins......Page 275
Rho-Dependent Control of Angiogenesis in Whole Organ Culture......Page 276
Whole embryonic lung rudiment culture......Page 278
Rho-Dependent Control of Vascular Permeability......Page 279
In vitro transendothelial permeability assays......Page 280
Conclusion......Page 281
References......Page 282
VEGF Receptor Signal Transduction......Page 288
Introduction......Page 289
Preparation of the preperfusion solution (Sodium phosphate buffer with EDTA) and collagenase solution (Sodium phosphate buffer......Page 291
Collagenase perfusion......Page 292
Effects of VEGF on the proliferation of SECs......Page 293
VEGF-induced VEGFR2 signaling in rat SECs......Page 294
Transfection of DNA into NIH3T3 Cells and selection of geneticin G418-resistant clones......Page 295
ES cell lines......Page 296
Routine culture of ES cells......Page 297
Embryoid body 3D sprouting assay in collagen I Gel......Page 298
First Day......Page 299
The secondary day......Page 300
Prepare S1P methanol solution (maximal storage time, 1 year)......Page 302
Procedure......Page 303
Collecting matrigel plugs......Page 304
Fluorescent staining of matrigel plug sections......Page 306
Preparation of VEGFR1 (flt-1) genomic DNA from the mouse 129Sv-strain genomic DNA library (Stratagene)......Page 307
Isolation of VEGFR2 (flk-1) genomic DNA from a mouse genomic DNA library and preparation of the target vector DNA......Page 308
Conclusions......Page 309
References......Page 310
Minimize handling time......Page 314
Bead preparation protocol......Page 315
Pull-down protocol (Rho)......Page 316
Bead preparation protocol......Page 318
Rap1 activity assay lysis and wash buffer......Page 319
Rap1 activity assay protocol......Page 320
Cell density......Page 321
Serum starvation conditions......Page 322
References......Page 324
Semaphorin-Induced Cytoskeletal Collapse and Repulsion of Endothelial Cells......Page 326
Introduction......Page 327
Purification of Human Recombinant SEMA3F......Page 328
Pericyte isolation and culture......Page 329
Cell culture of HUVEC and HMVEC......Page 332
Pretreatment of cover glasses (for confocal imaging of SEMA3F-induced collapse)......Page 333
Endothelial Cell Repulsion Assay......Page 335
Preparation of the repulsion assay......Page 336
Immunocytochemistry (ICC)......Page 338
References......Page 340
Pericyte Isolation and Use in Endothelial/Pericyte Coculture Models......Page 342
Introduction......Page 343
Protocol for Multipotent Mesenchymal Cells as Pericyte Precursors......Page 348
Protocol for Two-dimensional Pericyte-EC Coculture......Page 349
Coculture with direct contact......Page 350
Coculture without direct cell contact by use of the under-agarose assay......Page 351
PKH67/PKH26 fluorescent labeling of endothelial cells and pericytes......Page 355
Author Index......Page 359
Subject Index......Page 377
