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In vitro Assays for Endothelial Cell Functions Related to Angiogenesis: Proliferation, Motility, Tubular Differentiation and Proteolysis

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Angiogenesis Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 467))

Abstract

This chapter covers the breakdown of the process of angiogenesis into simple assays to measure discrete endothelial cell functions. The techniques described are suitable for studying stimulators or inhibitors of angiogenesis and determining which aspect of the process is modulated. The procedures outlined are robust and straightforward but cannot cover the complexity of the angiogenic process as a whole, incorporating as it does myriad positive and negative signals, three-dimensional interactions with host tissues and many accessory cells, including fibroblasts, macrophages, pericytes, and platelets. The extent to which in vitro assays predict responses in vivo (e.g., wound healing, tumour angiogenesis, or surrogate techniques such as Matrigel plugs, sponge implants, corneal assays, etc.) remains to be determined.

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References

  1. Folkman, J. (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285, 1182–1186.

    Article  PubMed  CAS  Google Scholar 

  2. Alitalo, K., Tammela, T., Petrova, T. V. (2005) Lymphangiogenesis in development and human disease. Nature 438, 946–953.

    Article  PubMed  CAS  Google Scholar 

  3. Benelli, R., Lorusso, G., Albini, A., Noonan, D. M. (2006) Cytokines and chemokines as regulators of angiogenesis in health and disease. Curr Pharm Des 12, 3101–3115.

    Article  PubMed  CAS  Google Scholar 

  4. Carmeliet, P. (2005) Angiogenesis in life, disease and medicine. Nature 438, 932–936.

    Article  PubMed  CAS  Google Scholar 

  5. Folkman, J. (2007). Is angiogenesis an organizing principle in biology and medicine? J Pediatr Surg 42, 1–11.

    Article  PubMed  Google Scholar 

  6. Luttun, A. and Carmeliet, P. (2004).Angiogenesis and lymphangiogenesis: highlights of the past year. Curr Opin Hematol 11, 262–271.

    Article  PubMed  Google Scholar 

  7. Shaked, Y., Bertolini, F., Man, S., (2005) Genetic heterogeneity of the vasculogenic phenotype parallels angiogenesis; implications for cellular surrogate marker analysis of antiangiogenesis. Cancer Cell 7, 101–111.

    PubMed  CAS  Google Scholar 

  8. Tammela, T., Enholm, B., Alitalo, K., Paavonen, K. (2005) The biology of vascular endothelial growth factors. Cardiovasc Res, 65, 550–563.

    Article  PubMed  CAS  Google Scholar 

  9. Tammela, T., Petrova, T. V., Alitalo, K. (2005) Molecular lymphangiogenesis: new players. Trends Cell Biol 15, 434–441.

    Article  PubMed  CAS  Google Scholar 

  10. Wissmann, C., Detmar, M. (2006) Pathways targeting tumor lymphangiogenesis. Clin Cancer Res 12, 6865–6868.

    Article  PubMed  CAS  Google Scholar 

  11. Bisacchi, D., Benelli, R., Vanzetto, C., Ferrari, N., Tosetti, F., Albini, A. (2003) Anti-angiogenesis and angioprevention: mechanisms, problems and perspectives. Cancer Detect Prev 27, 229–238.

    Article  PubMed  CAS  Google Scholar 

  12. Carmeliet, P. (2005) VEGF as a key mediator of angiogenesis in cancer. Oncology 69 Suppl 3, 4–10.

    Article  PubMed  CAS  Google Scholar 

  13. Ferrara, N., Kerbel, R. S. (2005) Angiogenesis as a therapeutic target. Nature 438, 967–974.

    Article  PubMed  CAS  Google Scholar 

  14. Ferretti, C., Bruni, L., Dangles-Marie, V., Pecking, A. P., Bellet, D. (2007) Molecular circuits shared by placental and cancer cells, and their implications in the proliferative, invasive and migratory capacities of trophoblasts. Hum Reprod Update 13, 121–141.

    Article  PubMed  CAS  Google Scholar 

  15. Folkman, J. (2003) Angiogenesis inhibitors: a new class of drugs. Cancer Biol Ther 2, S127–S133.

    PubMed  CAS  Google Scholar 

  16. He, Y., Karpanen, T., Alitalo, K. (2004) Role of lymphangiogenic factors in tumor metastasis. Biochim Biophys Acta 1654, 3–12.

    PubMed  CAS  Google Scholar 

  17. Hirakawa, S., Brown, L. F., Kodama, S., Paavonen, K., Alitalo, K., Detmar, M. (2007) VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites. Blood 109, 1010–1017.

    Article  PubMed  CAS  Google Scholar 

  18. Kerbel, R. S. (2004) Antiangiogenic drugs and current strategies for the treatment of lung cancer. Semin Oncol 31, 54–60.

    Article  PubMed  CAS  Google Scholar 

  19. Munoz, R., Shaked, Y., Bertolini, F., Emmenegger, U., Man, S., Kerbel, R. S. (2005) Anti-angiogenic treatment of breast cancer using metronomic low-dose chemotherapy. Breast 14, 466–479.

    Article  PubMed  Google Scholar 

  20. Naumov, G. N., Akslen, L. A., Folkman, J. (2006) Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch. Cell Cycle 5, 1779–1787.

    Article  PubMed  CAS  Google Scholar 

  21. Saharinen, P., Tammela, T., Karkkainen, M. J., Alitalo, K. (2004) Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation. Trends Immunol, 25, 387–395.

    Article  PubMed  CAS  Google Scholar 

  22. Sanz, L., Alvarez-Vallina, L. (2005) Antibody-based antiangiogenic cancer therapy. Expert Opin Ther Targets 9, 1235–1245.

    Article  PubMed  CAS  Google Scholar 

  23. Shields, J. D., Emmett, M. S., Dunn, D. B., (2007) Chemokine-mediated migration of melanoma cells towards lymphatics—a mechanism contributing to metastasis. Oncogene 26, 2997–3005.

    Article  PubMed  CAS  Google Scholar 

  24. Sridhar, S. S., Shepherd, F. A. (2003) Targeting angiogenesis: a review of angiogenesis inhibitors in the treatment of lung cancer. Lung Cancer 42 Suppl 1, S81–S91.

    Article  PubMed  Google Scholar 

  25. Verhoef, C., de Wilt, J. H., Verheul, H. M. (2006) Angiogenesis inhibitors: perspectives for medical, surgical and radiation oncology. Curr Pharm Des 12, 2623–2630.

    Article  PubMed  CAS  Google Scholar 

  26. Kerbel, R. S. (2006) Antiangiogenic therapy: a universal chemosensitization strategy for cancer? Science, 312, 1171–1175.

    Article  PubMed  CAS  Google Scholar 

  27. Kerbel, R. S., Kamen, B. A. (2004) The anti-angiogenic basis of metronomic chemotherapy. Nat Rev Cancer 4, 423–436.

    Article  PubMed  CAS  Google Scholar 

  28. Lavie, G., Mandel, M., Hazan, S., et al. (2005) Anti-angiogenic activities of hypericin in vivo: potential for ophthalmologic applications. Angiogenesis, 8, 35–42.

    Article  PubMed  CAS  Google Scholar 

  29. Griggs, J., Skepper, J. N., Smith, G. A., Brindle, K. M., Metcalfe, J. C., Hesketh, R. (2002) Inhibition of proliferative retinopathy by the anti-vascular agent combretastatin-A4. Am J Pathol 160, 1097–1103.

    Article  PubMed  CAS  Google Scholar 

  30. Stellmach, V., Crawford, S. E., Zhou, W., Bouck, N. (2001) Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. Proc Natl Acad Sci U S A 98, 2593–2597.

    Article  PubMed  CAS  Google Scholar 

  31. Tong, Y., Zhang, X., Zhao, W., (2004) Anti-angiogenic effects of Shiraiachrome A, a compound isolated from a Chinese folk medicine used to treat rheumatoid arthritis. Eur J Pharmacol 494, 101–109.

    Article  PubMed  CAS  Google Scholar 

  32. Dupont, E., Savard, P. E., Jourdain, C., (1998) Antiangiogenic properties of a novel shark cartilage extract: potential role in the treatment of psoriasis. J Cutan Med Surg 2, 146–152.

    PubMed  CAS  Google Scholar 

  33. Sauder, D. N., Dekoven, J., Champagne, P., Croteau, D., Dupont, E. (2002) Neovastat (AE-941), an inhibitor of angiogenesis: randomized phase I/II clinical trial results in patients with plaque psoriasis. J Am Acad Dermatol 47, 535–541.

    Article  PubMed  Google Scholar 

  34. Griffin, R. J., Molema, G., Dings, R. P. (2006) Angiogenesis treatment, new concepts on the horizon. Angiogenesis 9, 67–72.

    Article  PubMed  Google Scholar 

  35. Saaristo, A., Tammela, T., Farkkila, A., (2006) Vascular endothelial growth factor-C accelerates diabetic wound healing. Am J Pathol 169, 1080–1087.

    Article  PubMed  CAS  Google Scholar 

  36. Detillieux, K. A., Cattini, P. A., Kardami, E. (2004) Beyond angiogenesis: the cardioprotective potential of fibroblast growth factor-2. Can J Physiol Pharmacol 82, 1044–1052.

    Article  PubMed  CAS  Google Scholar 

  37. Levy, A. P., Levy, N. S., Loscalzo, J., et al. (1995). Regulation of vascular endothelial growth factor in cardiac myocytes. Circ Res 76, 758–766.

    PubMed  CAS  Google Scholar 

  38. Eccles, S. A. (2004) Parallels in invasion and angiogenesis provide pivotal points for therapeutic intervention. Int J Dev Biol 48, 583–598.

    Article  PubMed  CAS  Google Scholar 

  39. Albini, A., Benelli, R., Noonan, D. M., Brigati, C. (2004) The “chemoinvasion assay”: a tool to study tumor and endothelial cell invasion of basement membranes. Int J Dev Biol 48, 563–571.

    Article  PubMed  CAS  Google Scholar 

  40. Benelli, R., Albini, A. (1999) In vitro models of angiogenesis: the use of Matrigel. Int J Biol Markers, 14, 243–246.

    PubMed  CAS  Google Scholar 

  41. Mastyugin, V., McWhinnie, E., Labow, M., Buxton, F. (2004) A quantitative high-throughput endothelial cell migration assay. J Biomol Screen 9, 712–718.

    Article  PubMed  Google Scholar 

  42. Montanez, E., Casaroli-Marano, R. P., Vilaro, S., Pagan, R. (2002) Comparative study of tube assembly in three-dimensional collagen matrix and on Matrigel coats. Angiogenesis 5, 167–172.

    Article  PubMed  CAS  Google Scholar 

  43. Oberringer, M., Meins, C., Bubel, M., Pohlemann, T. (2007) A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts. Biol Cell 99, 197–207.

    Article  PubMed  CAS  Google Scholar 

  44. Schneider, M., Tjwa, M., Carmeliet, P. (2005) A surrogate marker to monitor angiogenesis at last. Cancer Cell 7, 3–4.

    Article  PubMed  CAS  Google Scholar 

  45. Brkovic, A., Pelletier, M., Girard, D., Sirois, M. G. (2007) Angiopoietin chemotactic activities on neutrophils are regulated by PI-3K activation. J Leukoc Biol 81, 1093–1101.

    Article  PubMed  CAS  Google Scholar 

  46. Chang, L. K., Garcia-Cardena, G., Farnebo, F., (2004) Dose-dependent response of FGF-2 for lymphangiogenesis. Proc Natl Acad Sci U S A 101, 11658–11663.

    Article  PubMed  CAS  Google Scholar 

  47. Gualandris, A., Lopez Conejo, T., Giunciuglio, D., (1997) Urokinase-type plasminogen activator overexpression enhances the invasive capacity of endothelial cells. Microvasc Res 53, 254–260.

    Article  PubMed  CAS  Google Scholar 

  48. Rak, J., Kerbel, R. S. (1998) Basic fibroblast growth factor and the complexity of tumour angiogenesis. Expert Opin Investig Drugs 7, 797–801.

    Article  PubMed  CAS  Google Scholar 

  49. Rosenkilde, M. M., Schwartz, T. W. (2004) The chemokine system—a major regulator of angiogenesis in health and disease. APMIS 112, 481–495.

    Article  PubMed  CAS  Google Scholar 

  50. Strieter, R. M., Burdick, M. D., Mestas, J., Gomperts, B., Keane, M. P., Belperio, J. A. (2006) Cancer CXC chemokine networks and tumour angiogenesis. Eur J Cancer 42, 768–778.

    Article  PubMed  CAS  Google Scholar 

  51. Alghisi, G. C., Ruegg, C. (2006) Vascular integrins in tumor angiogenesis: mediators and therapeutic targets. Endothelium 13, 113–135.

    Article  PubMed  CAS  Google Scholar 

  52. Cai, W., Chen, X. (2006) Anti-angiogenic cancer therapy based on integrin alphavbeta3 antagonism. Anticancer Agents Med Chem 6, 407–428.

    Article  PubMed  CAS  Google Scholar 

  53. Mettouchi, A., Meneguzzi, G. (2006) Distinct roles of beta1 integrins during angiogenesis. Eur J Cell Biol 85, 243–247.

    Article  PubMed  CAS  Google Scholar 

  54. Serini, G., Valdembri, D., Bussolino, F. (2006) Integrins and angiogenesis: a sticky business. Exp Cell Res 312, 651–658.

    Article  PubMed  CAS  Google Scholar 

  55. Lakka, S. S., Gondi, C. S., Rao, J. S. (2005) Proteases and glioma angiogenesis. Brain Pathol 15, 327–341.

    Article  PubMed  CAS  Google Scholar 

  56. Rundhaug, J. E. (2005) Matrix metalloproteinases and angiogenesis. J Cell Mol Med 9, 267–285.

    Article  PubMed  CAS  Google Scholar 

  57. van Hinsbergh, V. W., Engelse, M. A., Quax, P. H. (2006) Pericellular proteases in angiogenesis and vasculogenesis. Arterioscler Thromb Vasc Biol 26, 716–728.

    Article  PubMed  CAS  Google Scholar 

  58. Rabbani, S. A., Mazar, A. P. (2001) The role of the plasminogen activation system in angiogenesis and metastasis. Surg Oncol Clin N Am 10, 393–415, x.

    PubMed  CAS  Google Scholar 

  59. Stefansson, S., McMahon, G. A., Petitclerc, E., Lawrence, D. A. (2003) Plasminogen activator inhibitor-1 in tumor growth, angiogenesis and vascular remodeling. Curr Pharm Des 9, 1545–1564.

    Article  PubMed  CAS  Google Scholar 

  60. Chantrain, C. F., Henriet, P., Jodele, S., (2006) Mechanisms of pericyte recruitment in tumour angiogenesis: a new role for metalloproteinases. Eur J Cancer 42, 310–318.

    Article  PubMed  CAS  Google Scholar 

  61. Kiselyov, A., Balakin, K. V., Tkachenko, S. E. (2007) VEGF/VEGFR signalling as a target for inhibiting angiogenesis. Expert Opin Investig Drugs 16, 83–107.

    Article  PubMed  CAS  Google Scholar 

  62. Liu, C. C., Shen, Z., Kung, H. F., Lin, M. C. (2006) Cancer gene therapy targeting angiogenesis: an updated review. World J Gastroenterol 12, 6941–6948.

    PubMed  CAS  Google Scholar 

  63. O’Dwyer, P. J. (2006) The present and future of angiogenesis-directed treatments of colorectal cancer. Oncologist 11, 992–998.

    Article  PubMed  Google Scholar 

  64. Telliez, A., Furman, C., Pommery, N., Henichart, J. P. (2006) Mechanisms leading to COX-2 expression and COX-2 induced tumorigenesis: topical therapeutic strategies targeting COX-2 expression and activity. Anticancer Agents Med Chem 6, 187–208.

    Article  PubMed  CAS  Google Scholar 

  65. Trachsel, E., Neri, D. (2006) Antibodies for angiogenesis inhibition, vascular targeting and endothelial cell transcytosis. Adv Drug Deliv Rev 58, 735–754.

    Article  PubMed  CAS  Google Scholar 

  66. Wanebo, H. J., Argiris, A., Bergsland, E., Agarwala, S., Rugo, H. (2006) Targeting growth factors and angiogenesis; using small molecules in malignancy. Cancer Metastasis Rev, 25, 279–292.

    Article  PubMed  Google Scholar 

  67. Eccles, S. A., Box, C. (2005) Court W Cell migration assays and their application in cancer drug discovery. Biotech Annu Rev 11, 391–421

    Article  CAS  Google Scholar 

  68. Sanderson, S., Valenti, M., Gowan, S., (2006) Benzoquinone ansamycin heat shock protein 90 inhibitors modulate multiple functions required for tumor angiogenesis. Mol Cancer Ther 5, 522–532.

    Article  PubMed  CAS  Google Scholar 

  69. Connolly, D. T., Knight, M. B., Harakas, N. K., Wittwer, A. J., Feder, J. (1986) Determination of the number of endothelial cells in culture using an acid phosphatase assay. Anal Biochem 152, 136–140.

    Article  PubMed  CAS  Google Scholar 

  70. Jackson, S. J., Venema, R. C. (2006) Quercetin inhibits eNOS, microtubule polymerization, and mitotic progression in bovine aortic endothelial cells. J Nutr 136, 1178–1184.

    PubMed  CAS  Google Scholar 

  71. Michaelis, U. R., Fisslthaler, B., Barbosa-Sicard, E., Falck, J. R., Fleming, I., Busse, R. (2005) Cytochrome P450 epoxygenases 2C8 and 2C9 are implicated in hypoxia-induced endothelial cell migration and angiogenesis. J Cell Sci 118, 5489–5498.

    Article  PubMed  CAS  Google Scholar 

  72. Alessandri, G., Chirivi, R. G., Castellani, P., Nicolo, G., Giavazzi, R., Zardi, L. (1998) Isolation and characterization of human tumor-derived capillary endothelial cells: role of oncofetal fibronectin. Lab Invest 78, 127–128.

    PubMed  CAS  Google Scholar 

  73. Garrafa, E., Alessandri, G., Benetti, A., (2006) Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils. J Cell Physiol 207, 107–113.

    Article  PubMed  CAS  Google Scholar 

  74. Invernici, G., Ponti, D., Corsini, E., (2005) Human microvascular endothelial cells from different fetal organs demonstrate organ-specific CAM expression. Exp Cell Res 308, 273–282.

    Article  PubMed  CAS  Google Scholar 

  75. Park, H. J., Zhang, Y., Georgescu, S. P., Johnson, K. L., Kong, D., Galper, J. B. (2006) Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis. Stem Cell Rev 2, 93–102.

    Article  PubMed  CAS  Google Scholar 

  76. Gu, W., Pellegrino, T., Parak, W. J., (2007) Measuring cell motility using quantum dot probes. Methods Mol Biol 374, 125–132.

    PubMed  Google Scholar 

  77. Gildea, J. J., Harding, M. A., Gulding, K. M., Theodorescu, D. (2000) Transmembrane motility assay of transiently transfected cells by fluorescent cell counting and luciferase measurement. Biotechniques 29, 81–86.

    PubMed  CAS  Google Scholar 

  78. Spessotto, P., Giacomello, E., Perri, R. (2002) Improving fluorescence-based assays for the in vitro analysis of cell adhesion and migration. Mol Biotechnol 20, 285–304.

    Article  PubMed  CAS  Google Scholar 

  79. Spessotto, P., Giacomello, E., Perris, R. (2000) Fluorescence assays to study cell adhesion and migration in vitro. Methods Mol Biol 139, 321–343.

    PubMed  CAS  Google Scholar 

  80. Albini, A. (1998) Tumor and endothelial cell invasion of basement membranes. The Matrigel chemoinvasion assay as a tool for dissecting molecular mechanisms. Pathol Oncol Res 4, 230–241.

    Article  PubMed  CAS  Google Scholar 

  81. Ranta, V., Mikkola, T., Ylikorkala, O., Viinikka, L., Orpana, A. (1998) Reduced viability of human vascular endothelial cells cultured on Matrigel. J Cell Physiol, 176, 92–98.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Cancer Research UK Centre for Cancer Therapeutics, McElwain Laboratories, Institute of Cancer Research, Surrey, UK

    Suzanne A Eccles PHD, William Court MSC & Lisa Patterson BSC

  2. Cancer Research UK Molecular Angiogenesis Laboratory, Weatherhall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

    Sharon Sanderson PHD

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  1. Suzanne A Eccles PHD
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  2. William Court MSC
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  3. Lisa Patterson BSC
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  4. Sharon Sanderson PHD
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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Eccles, S.A., Court, W., Patterson, L., Sanderson, S. (2009). In vitro Assays for Endothelial Cell Functions Related to Angiogenesis: Proliferation, Motility, Tubular Differentiation and Proteolysis. In: Murray, C., Martin, S. (eds) Angiogenesis Protocols. Methods in Molecular Biology, vol 467. Humana Press. https://doi.org/10.1007/978-1-59745-241-0_9

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    • DOI: https://doi.org/10.1007/978-1-59745-241-0_9

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