Target Discovery and Validation Reviews and Protocols

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12 The HUVEC/Matrigel Assay An In Vivo Assay of Human Angiogenesis Suitable for Drug Validation Dag K. Skovseth, Axel M. Küchler, and Guttorm Haraldsen Summary The future ability to manipulate the growth of new blood vessels (angiogenesis) holds great promise for treating ischemic disease and cancer. Several models of human in vivo angiogenesis have been described, but they seem to depend on transgenic support and have not been validated in a therapeutic context. Here, we describe an in vivo assay that mimics human angiogenesis in which native human umbilical vein-derived endothelial cells are suspended in a liquid laminin/collagen gel (Matrigel), injected into immunodeficient mice, and develop into mature, functional vessels that vascularize the Matrigel plug in the course of 30 d. Moreover, we demonstrate how to target this process therapeutically by sustained delivery of the angiogenesis inhibitor endostatin from subcutaneously implanted microosmotic pumps. Key Words: Adoptive transfer; angiogenesis; endostatin; human umbilical vein-derived endothelial cells; HUVECs; Matrigel; RAG2 mice. 1. Introduction Angiogenesis is the growth of new capillaries from preexisting blood vessels (1,2). This multistep process begins with the activation of quiescent endothelial cells (ECs) leading to vessel dilation and permeabilization accompanied by degradation of the basement membrane. ECs then migrate toward the angiogenic stimulus and proliferate to form new patent tubules surrounded by a new basement membrane. The ECs of the sprouting, new vessels adapt to the local demand of the tissue by differentiation into capillaries, veins, or arteries, and they are subsequently stabilized by recruitment of perivascular cells (reviewed in ref. 3). From: Methods in Molecular Biology, vol. 360, Target Discovery and Validation Reviews and Protocols Volume I, Emerging Strategies for Targets and Biomarker Discovery Edited by: M. Sioud © Humana Press Inc., Totowa, NJ 253
254 Skovseth, Küchler, and Haraldsen In the healthy adult, the vasculature remains quiescent, except in response to exercise-induced muscle hypertrophy and during the transient neovascularization of the uterus. However, angiogenesis is a prominent feature of wound repair and has the potential to become an important therapeutic modality in ischemic disease (4). Angiogenesis is also an attractive target of neoplastic tumor growth, in particular because ECs are genetically stable and therefore less likely to accumulate mutations that allow them to develop drug resistance (5). Indeed, the antiangiogenic approach to treat cancer has made substantial progress reflected in the recent approval of Avastin (a humanized antibody to vascular endothelial growth factor-A) for support therapy of colorectal cancer (6). However, it should be noted that Avastin’s effect is restricted to prolonging progression-free survival times with an average of few months (7). Thus, although this achievement is a milestone toward antiangiogenic treatment of human cancer, it is still far from copying the full potential that Avastin ® displayed in animal experiments. In our view, this discrepancy reflects the lack of both standardized tools and models that reliably mirror the complexity of tumor angiogenesis mechanisms in humans. Characterization of candidates for treatment of angiogenesis-dependent diseases has traditionally been performed using a variety of in vitro and in vivo models (reviewed in ref. 8). Here, we briefly describe the most commonly used in vivo models based on host vessel growth. One classical in vivo approach has been the chick chorioallantoic membrane (CAM) assay (9) where angiogenesis can be monitored in response to implanted slow-release polymer pellets or organ grafts onto the developing CAM. Another assay exploits the avascular cornea in which slow release pellets induce vascularization from the limbus and offer another elegant way to study and manipulate angiogenesis in vivo (10). A third, frequently used in vivo assay is based on the injection of Matrigel into the abdominal wall of mice where it polymerizes at body temperature. Matrigel is a commercially available, solubilized basement membrane preparation extracted from the Engelbreth–Holm–Swarm mouse sarcoma-derived cell line. It contains laminin, collagen IV, heparan sulfate proteoglycans, entactin, and nidogen, and it gels rapidly at 22–35°C, forming a solid mass upon subcutaneous injection Fig. 1. (Opposite page) Development of functional human vessels in Matrigel and inhibition of angiogenesis after endostatin treatment. (A) H&E staining of Matrigel plug (MP) positioned between the skin (S) and abdominal muscle layer (M). (B–F, H, and I) Immunofluorescence staining for α-smooth muscle actin (red) and Ulex lectin staining (green) of Matrigel sections from control and endostatin-treated animals on days as indicated. (G) Immunofluorescence staining for hCD31 with luconyl blue inside the vessels indicating functional vessels (phase contrast and immunofluorescent image of identical fields). Bars = 500 µm (A), 50 µm (B–I). Images (A, E, G, and G).
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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66 Beaty et al. 2.3.3. Protein Stai
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68 Beaty et al. 3.1.3. Labeling of
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70 Beaty et al. 1 µL of the anneal
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72 Beaty et al. 4. Phenol:cholorfor
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74 Beaty et al. 19. Wash twice with
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76 Beaty et al. The Following Volum
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78 Beaty et al. using a protein ass
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80 Beaty et al. 6. Shake the gel fo
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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146 Sano and Taira 5. 10X L (low-sa
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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Transfected Cell Arrays 159 2. The
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Transfected Cell Arrays 161 5. Simp
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164 Houdebine Fig. 1. Different met
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166 Houdebine concentrations become
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168 Houdebine Fig. 2. Major methods
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170 Houdebine integrate into the me
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172 Houdebine 2.2.3. Knock-In Into
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174 Houdebine as insulators. The co
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176 Houdebine Fig. 5. Different met
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178 Houdebine systems. Moreover, st
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180 Houdebine contribute to generat
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182 Houdebine Fig.7. Example of a v
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184 Houdebine more extensively. Tra
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186 Houdebine and stroma. Several o
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188 Houdebine explained at the mole
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190 Houdebine of the intestinal epi
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192 Houdebine interference of the g
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194 Houdebine 17. Whitelaw, C. B. A
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196 Houdebine 51. Bell, A. C., West
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198 Houdebine 86. Carmichael, G. G.
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200 Houdebine 121. Pailhoux, E., Vi
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Overview of Immune System 303 Fig.
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Overview of Immune System 307 some
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Overview of Immune System 309 sera
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Overview of Immune System 313 measu
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Overview of Immune System 315 42. H
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Overview of Immune System 317 74. D
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15 Potential Target Antigens for Im
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Tumor Antigen Discovery 321 develop
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Tumor Antigen Discovery 323 panel b
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Tumor Antigen Discovery 325 16. Joc
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16 Identification of Tumor Antigens
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Immunoproteomics 329 by “shot gun
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Immunoproteomics 331 isoelectric fo
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Immunoproteomics 333 3. 2D-PAGE is
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17 Protein Arrays A Versatile Toolb
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Protein Arrays 337 Table 1 Classifi
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Protein Arrays 339 fractions from c
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Protein Arrays 341 combinatorial sc
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Protein Arrays 343 The ideal proteo
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Protein Arrays 345 18. Cekaite, H.
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Protein Arrays 347 49. Yuko Kawahas
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Index 349 Index A Acetyl-CoA carbox
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Index 351 conditional by using FRT
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Index 353 Recombinant tumor antigen
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