Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro

  • Derrick S. Grant
  • Peter I. Lelkes
  • Katsunori Fukuda
  • Hynda K. Kleinman
Regular Papers


The extracellular matrix, particularly basement membranes, plays an important role in angiogenesis (blood vessel formation). Previous work has demonstrated that a basement membranelike substrate (Matrigel) induces human umbilical vein endothelial cells to rapidly form vessel-like tubes (Kubota, et al., 1988; Grant et al., 1989b); however, the precise mechanism of tube formation is unclear. Using this in vitro model, we have investigated morphologic changes occurring during tube formation and the cytoskeletal and protein synthesis requirements of this process. Electron microscopy showed that endothelial cells attach to the Matrigel surface, align, and form cylindrical structures that contain a lumen and polarized cytoplasmic organelles. The cytoskeleton is reorganized into bundles of actin filaments oriented along the axis of the tubes and is located at the periphery of the cells. The addition of colchicine or cytochalasin D blocked tube formation, indicating that both microfilaments and microtubules are involved in this process. Cycloheximide blocked tube formation by 100%, indicating that the process also required protein synthesis. In particular, collagen synthesis seems to be required for tube formation because cis-hydroxyproline inhibited tube formation, whereas either the presence of ascorbic acid or the addition of exogenous collagen IV to the Matrigel increased tube formation. Our results indicate that endothelial cell attachment to Matrigel induces the reorganization of the cytoskeleton and elicits the synthesis of specific proteins required for the differentiated phenotype of the cells.

Key words

endothelium basement membrane laminin differentiation blood vessel mechanisms 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bussolino, F.; Camussi, G.; Baglioni, C. Synthesis and release of platelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin la. J. Biol. Chem. 263:11856–11861; 1988.PubMedGoogle Scholar
  2. Clement, B.; Segui-Real, B.; Hassell, J. R., et al. Identification of a cell surface-binding protein for the core protein of basement membrane proteoglycan. J. Biol. Chem. 264(21):12467–12471; 1989.PubMedGoogle Scholar
  3. David, S.; LaCorbiere, M. The specificity of extracellular glycoprotein complexes in mediating cellular adhesion. J. Neurosci. 2(1):82–89; 1982.Google Scholar
  4. deGroot, P. G.; Reinders, J. H.; Sixma, J. J. Perturbation of human endothelial cells by thrombin or PMA changes the reactivity of their extracellular matrix. J. Cell Biol. 104:697–704; 1987.CrossRefGoogle Scholar
  5. Folkman, J. Toward an understanding of angiogenesis: search and discovery. Perspect. Biol. Med. 29:10–36; 1985.PubMedGoogle Scholar
  6. Folkman, J.; Haudenschild, C. Angiogenesis in vitro. Nature 288:551–556; 1980.PubMedCrossRefGoogle Scholar
  7. Folkman, J.; Klagsbrun, M. Angiogenic factors. Science 235:442–447; 1987.PubMedCrossRefGoogle Scholar
  8. Fukuda, K.; Imamura, Y.; Koshihara, Y., et al. Establishment of human mucosal microvascular endothelial cells from inferior turbinate in culture. Am. J. Otolaryngol. 10(2):85–91; 1989.PubMedCrossRefGoogle Scholar
  9. Fukuda, K.; Koshihara, Y.; Oda, H., et al. Type V collagen selectively inhibits human endothelial cell proliferation. Biochem. Biophys. Res. Commun. 151:1060–1068; 1988.PubMedCrossRefGoogle Scholar
  10. Furcht, L. T. Critical factors controlling angiogenesis: cell products, cell matrix, and growth factors. Lab. Invest. 55:505–509; 1986.PubMedGoogle Scholar
  11. Giltay, J. C.; Mourik, J. A. Structure and function of endothelial cell integrins. Haemostasis 18:376–389; 1988.PubMedGoogle Scholar
  12. Gosposdarowicz, D.; Greenburg, G.; Birdwell, C. R. Determination of cellular shape by the extracellular matrix and its correlation with the control of cellular growth. Cancer Res. 38:4155–4171; 1978.Google Scholar
  13. Grabel, L. B.; Watts, T. D. The role of extracellular matrix in the migration and differentiation of parietal endoderm from teratocarcinoma embryoid bodies. J. Cell Biol. 105:441–448; 1987.PubMedCrossRefGoogle Scholar
  14. Grant, D. S.; Kleinman, H. K.; Martin, G. R. The role of basement membranes in vascular development. NY Acad. Sci. Vol. 588:61–72; 1989a.CrossRefGoogle Scholar
  15. Grant, D. S.; Tashiro, K.-I.; Segui-Real, B., et al. Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 58:933–943; 1989b.PubMedCrossRefGoogle Scholar
  16. Howard, B. V.; Macarak, E. J.; Gunson, D., et al. Characterization of the collagen synthesized by endothelial cells in culture. Proc. Natl. Acad. Sci. USA 73:2361–2364; 1976.PubMedCrossRefGoogle Scholar
  17. Ingber, D. E.; Folkman, J. How does extracellular matrix control capillary morphogenesis? Cell 58(Sept. 8):803–805; 1989a.PubMedCrossRefGoogle Scholar
  18. Ingber, D. E.; Folkman, J. Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix. J. Cell Biol. 109:317–330; 1989b.PubMedCrossRefGoogle Scholar
  19. Ingber, D. E.; Madri, J. A.; Folkman, J. Endothelial growth factors and extracellular matrix regulate DNA synthesis through modulation of cell and nuclear expansion. In Vitro Cell. Dev. Biol. 23:387–394; 1987.PubMedCrossRefGoogle Scholar
  20. Jaffe, E. A.; Nachman, R. L.; Becker, C. G., et al. Culture of human endothelial cells derived from umbilical veins-identification by morphological and immunological criteria. J. Clin. Invest. 52:2745–2756; 1973.PubMedCrossRefGoogle Scholar
  21. Kleinman, H. K.; Klebe, R. J.; Martin, G. R. Role of collagenous matrices in the adhesion and growth of the cells. J. Cell Biol. 88:473–485; 1981.PubMedCrossRefGoogle Scholar
  22. Kleinman, H. K.; McGarvey, M. L.; Liotta, L. A., et al. Isolation and characterization of type IV procollagen, laminin and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 24:6188; 1982.CrossRefGoogle Scholar
  23. Kleinman, H. K.; Cannon, F. B.; Laurie, G. W., et al. Biological activities of laminin. J. Cell Biol. 27:317–325; 1985.Google Scholar
  24. Kleinman, H. K.; McGarvey, M. L.; Hassell, J. R., et al. Basement membrane complexes with biological activity. Biochemistry 25:312–318; 1986.PubMedCrossRefGoogle Scholar
  25. Knedler, A.; Ham, R. G. Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum. In Vitro Cell. Dev. Biol. 23:481–491; 1987.PubMedCrossRefGoogle Scholar
  26. Kramer, R. H. Extracellular matrix interactions with the apical surface of vascular endothelial cells. J. Cell. Sci. 76:1–16; 1985.PubMedGoogle Scholar
  27. Kramer, R. H.; Fuh, G. M. Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition in the subendothelial basement membrane. Biochemistry 24:7423–7430; 1985.PubMedCrossRefGoogle Scholar
  28. Kubota, Y.; Kleinman, H. K.; Martin, G. R., et al. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J. Cell Biol. 107:1589–1598; 1988.PubMedCrossRefGoogle Scholar
  29. Lawley, T. J.; Kubota, Y. Induction of morphologic differentiation of endothelial cells in culture. J. Invest. Dermatol. 93(2 suppl):59S-61S; 1989.PubMedCrossRefGoogle Scholar
  30. Li, M. J.; Aggeler, J.; Farson, D. A., et al. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc. Natl. Acad. Sci. USA 84:136–140; 1987.PubMedCrossRefGoogle Scholar
  31. Lioté, F.; Setiadi, H.; Wautier, J. L. Facteurs plasmatiques et cellulaires réulant la prolifération des cellules endothéliales. Arch. Mal. Coeur. 80:23–29; 1987.PubMedGoogle Scholar
  32. Maciag, T. Molecular and cellular mechanisms of angiogenesis. In: DeVita, V. T.; Hellman, S.; Rosenberg, S. A., eds. Advances in oncology; cancer: principles and practice of oncology, 3rd ed. Philadelphia, PA: Lippincott. In press; 1989.Google Scholar
  33. Maciag, T.; Kadish, J.; Wilkins, L., et al. Organization behavior of human umbilical vein endothelial cells. J. Cell Biol. 94:511–520; 1982.PubMedCrossRefGoogle Scholar
  34. Madri, J. A.; Dryer, B.; Pitlick, F., et al. The collagenous components of the subendothelium: correlation of structure and function. Lab. Invest. 43:303–315; 1980.PubMedGoogle Scholar
  35. Madri, J. A.; Pratt, B. M. Endothelial cell-matrix interactions: in vitro models of angiogensis. J. Histochem. Cytochem. 34:85–91; 1986.PubMedGoogle Scholar
  36. Madri, J. A.; Pratt, B. M. Angiogenesis. In: Clark, R. F.; Henson, P., eds. Molecular and Cellular Biology of Wound Healing. New York: Plenum Press; 1987:337–358.Google Scholar
  37. Madri, J. A.; Pratt, B. M.; Tucker, A. M. Phenotypic modulation of endothelial cells by transforming growth factor-b depends upon the composition and organization of the extracellular matrix. J. Cell Biol. 106:1375–1384; 1988.PubMedCrossRefGoogle Scholar
  38. Madri, J. A.; Williams, S. K.; Wyatt, T., et al. Capillary endothelial cell cultures: phenotypic modulation by matrix components. J. Cell Biol. 97:1648–1652; 1983.CrossRefGoogle Scholar
  39. Mann, K.; Deutzmann, R.; Aumailley, M., et al. Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells. EMBO J. 8:65–75; 1989.PubMedGoogle Scholar
  40. Maragoudakis, M. E. Sarmonika, M.; Panuotsacoupoulou, M. Inhibition of basement membrane biosynthesis prevents angiogenesis. J. Pharmacol. Exp. Ther. 244(2):729–733; 1988.PubMedGoogle Scholar
  41. McAuslan, B. R.; Reilly, W.; Hannan, G. N., et al. Induction of endothelial cell migration by proline analogs and its relevance to angiogenesis. Exp. Cell Res. 176:248–257; 1988.PubMedCrossRefGoogle Scholar
  42. Montesano, R. Cell-extracellular matrix interactions in morphogenesis: an in vitro approach. Experientia 42(9):977–985; 1986.PubMedCrossRefGoogle Scholar
  43. Montesano, R.; Orci, L.; Vassali, P. In vitro rapid organization of endothelial cells in to capillary-like networks is promoted by collagen matrices. J. Cell Biol. 97:1648–1652; 1983.PubMedCrossRefGoogle Scholar
  44. Montesano, E.; Pepper, M. S.; Vassalli, J.-D., et al. Phorbol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors. J. Cell. Phys. 132:509–516; 1987.CrossRefGoogle Scholar
  45. Mori, M.; Sadahira, Y.; Kawasaki, S., et al. Capillary growth from reversed rat aortic segments cultured in collagen gel. Acta Pathol. Jpn. 38(12):1503–1512; 1988.PubMedGoogle Scholar
  46. Muller, W. A.; Gimbrone, M. A. Plasmalemma proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination. J. Cell Biol. 103(6):2389–2402; 1986.PubMedCrossRefGoogle Scholar
  47. Nicosia, R. F.; McCormick, J. F.; Bielunas, J. The formation of endothelial webs and channels in plasma clot culture. Scand. Elect. Microsc. 2:793–799; 1984.Google Scholar
  48. Panayotou, G.; End, P.; Aumailley, M., et al. Domains of laminin with growth-factor activity. Cell 93–101; 1989.Google Scholar
  49. Risau, W.; Lemmon, V. Changes in the vascular extracellular matrix during embryonic vasculogenesis and angiogenesis. Dev. Biol. 125:441–450; 1988.PubMedCrossRefGoogle Scholar
  50. Ruoslahti, E.; Pierschbacher, M. D. New perspectives in cell adhesion: RGD and integrins. Science 238:491–497; 1987.PubMedCrossRefGoogle Scholar
  51. Sato, Y.; Rifkin, D. B. Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, DNA synthesis. J. Cell Biol. 107:1199–1205; 1988.PubMedCrossRefGoogle Scholar
  52. Taub, M.; Wang, Y.; Szcesney, T. M., et al. Transforming growth factor alpha is required for kidney tubulogenesis in Matrigel cultures in serum-free medium. Proc. Natl. Acad. Sci. USA 87:4002–4006; 1990.PubMedCrossRefGoogle Scholar
  53. Unemori, E. N.; Bouhana, K. S.; Werb, Z. Vectorial secretion of extracellular matrix proteins, matrix degrading proteinases, and tissue inhibitor of metallo-proteinases by endothelial cells. J. Biol. Chem. 256:445–451; 1990.Google Scholar
  54. Williams, S. K. Isolation and culture of microvessel and large vessel endothelial cells: their use in transport in clincial studies. In: McDonagh, P., ed. Microvascular perfusion and transport in health and disease. Basel: S. Karger; 1987:204–245.Google Scholar

Copyright information

© Tissue Culture Association 1991

Authors and Affiliations

  • Derrick S. Grant
    • 1
  • Peter I. Lelkes
    • 2
  • Katsunori Fukuda
    • 1
  • Hynda K. Kleinman
    • 1
  1. 1.Laboratory of Developmental Biology and AnomaliesNational Institute of Dental Research, NIHBethesda
  2. 2.Laboratory of Cell Biology, Department of MedicineUniversity of Wisconsin Medical SchoolMilwaukee

Personalised recommendations