A Balance of Substrate Mechanics and Matrix Chemistry Regulates Endothelial Cell Network Assembly

Article

Abstract

Driven by specific extracellular matrix cues, endothelial cells can spontaneously assemble into networks. Cell network assembly is, in part, dictated by both substrate stiffness and extracellular matrix chemistry; however, the balance between substrate mechanics and matrix chemistry in promoting cell network assembly is not well understood. Because both mechanics and chemistry can alter cell–substrate and cell–cell adhesion, we hypothesized that cell network assembly can be promoted on substrates that minimize cell–substrate adhesivity while promoting cell–cell connections. To investigate these hypotheses, bovine aortic endothelial cells (BAEC) were seeded on variably compliant polyacrylamide (PA) substrates derivatized with type I collagen and observed over time. Our results indicate that cell network assembly can be induced on substrates that are sufficiently compliant (Young's modulus, E = 200 Pa) and present significant amounts of substrate-bound ligand, and on substrates that are stiffer (E = 10,000 Pa) but which present less adhesive ligand. In both of these cases, cell–substrate adhesivity is decreased, which may enhance cell–cell adhesivity. Moreover, our data indicate that fibronectin polymerization stabilizes cell–cell contacts and is necessary for network formation to occur regardless of substrate compliance or the density of substrate-bound ligand. These data demonstrate the balance between substrate mechanics and chemistry in directing cell network assembly.

Keywords

Polyacrylamide gel Substrate compliance/stiffness Fibronectin polymerization Angiogenesis 

References

  1. 1.
    Connolly J. O., N. Simpson, L. Hewlett, A. Hall 2002 Rac regulates endothelial morphogenesis and capillary assembly. Mol. Biol. Cell. 13(7): 2474–2485CrossRefGoogle Scholar
  2. 2.
    Davis G. E., D. R. Senger 2005 Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ. Res. 97(11): 1093–1107CrossRefGoogle Scholar
  3. 3.
    Deroanne C. F., C. M. Lapiere, B. V. Nusgens 2001 In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton. Cardiovasc. Res. 49(3), 647–658CrossRefGoogle Scholar
  4. 4.
    Discher D. E., P. Janmey, Y. L. Wang 2005 Tissue cells feel and respond to the stiffness of their substrate. Science 310(5751), 1139–1143CrossRefGoogle Scholar
  5. 5.
    Engler A., L. Bacakova, C. Newman, A. Hategan, M. Griffin, D. Discher 2004 Substrate compliance versus ligand density in cell on gel responses. Biophys. J. 86(1 Pt 1), 617–628CrossRefGoogle Scholar
  6. 6.
    Fam N. P., S. Verma, M. Kutryk, D. J. Stewart 2003 Clinician guide to angiogenesis. Circulation 108(21), 2613–2618CrossRefGoogle Scholar
  7. 7.
    Feder J., J. C. Marasa, J. V. Olander 1983 The formation of capillary-like tubes by calf aortic endothelial cells grown in vitro. J. Cell. Physiol. 116(1), 1–6CrossRefGoogle Scholar
  8. 8.
    Gamble J. R., L. J. Matthias, G. Meyer, P. Kaur, G. Russ, R. Faull, M. C. Berndt, M. A. Vadas 1993 Regulation of in vitro capillary tube formation by anti-integrin antibodies. J. Cell. Biol. 121(4), 931–943CrossRefGoogle Scholar
  9. 9.
    Guo W. H., M. T. Frey, N. A. Burnham, Y. L. Wang 2006 Substrate rigidity regulates the formation and maintenance of tissues. Biophys. J. 90(6), 2213–2220CrossRefGoogle Scholar
  10. 10.
    Hocking D. C., J. Sottile, K. J. Langenbach 2000 Stimulation of integrin-mediated cell contractility by fibronectin polymerization. J. Biol. Chem. 275(14), 10673–10682CrossRefGoogle Scholar
  11. 11.
    Ingber D. E. 1990 Fibronectin controls capillary endothelial cell growth by modulating cell shape. Proc. Natl. Acad. Sci. USA 87(9), 3579–3583CrossRefGoogle Scholar
  12. 12.
    Ingber D. E., J. Folkman 1989 How does extracellular matrix control capillary morphogenesis? Cell 58(5), 803–805CrossRefGoogle Scholar
  13. 13.
    Ingber D. E., J. Folkman 1989 Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix. J. Cell. Biol. 109(1), 317–330CrossRefGoogle Scholar
  14. 14.
    Ingber D. E., D. Prusty, Z. Sun, H. Betensky, N. Wang 1995 Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis. J. Biomech. 28(12), 1471–1484CrossRefGoogle Scholar
  15. 15.
    Intengan H. D., E. L. Schiffrin 2001 Vascular remodeling in hypertension: roles of apoptosis, inflammation, and fibrosis. Hypertension 38(3 Pt 2), 581–587CrossRefGoogle Scholar
  16. 16.
    Jiang G., A. H. Huang, Y. Cai, M. Tanase, M. P. Sheetz 2006 Rigidity sensing at the leading edge through alphavbeta3 integrins and RPTPalpha. Biophys. J. 90(5), 1804–1809CrossRefGoogle Scholar
  17. 17.
    Klein E. A., Y. Yung, P. Castagnino, D. Kothapalli, R. K. Assoian 2007 Cell adhesion, cellular tension, and cell cycle control. Methods Enzymol. 426, 155–175CrossRefGoogle Scholar
  18. 18.
    Kubota Y., H. K. Kleinman, G. R. Martin, T. J. Lawley 1988 Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J. Cell. Biol. 107(4), 1589–1598CrossRefGoogle Scholar
  19. 19.
    Liu Y., D. R. Senger 2004 Matrix-specific activation of Src and Rho initiates capillary morphogenesis of endothelial cells. Faseb. J. 18(3), 457–468CrossRefGoogle Scholar
  20. 20.
    Morla A., Z. Zhang, E. Ruoslahti 1994 Superfibronectin is a functionally distinct form of fibronectin. Nature 367(6459), 193–196CrossRefGoogle Scholar
  21. 21.
    Olander J. V., M. E. Bremer, J. C. Marasa, J. Feder 1985 Fibrin-enhanced endothelial cell organization. J. Cell. Physiol. 125(1), 1–9CrossRefGoogle Scholar
  22. 22.
    Pless D. D., Y. C. Lee, S. Roseman, R. L. Schnaar 1983 Specific cell adhesion to immobilized glycoproteins demonstrated using new reagents for protein and glycoprotein immobilization. J. Biol. Chem. 258(4), 2340–2349Google Scholar
  23. 23.
    Reinhart-King, C. A., M. Dembo, and D. A. Hammer. Biophys. J. (in press)Google Scholar
  24. 24.
    Reinhart-King C. A., M. Dembo, D. A. Hammer 2005 The dynamics and mechanics of endothelial cell spreading. Biophys. J. 89(1), 676–689CrossRefGoogle Scholar
  25. 25.
    Ryan P. L., R. A. Foty, J. Kohn, M. S. Steinberg 2001 Tissue spreading on implantable substrates is a competitive outcome of cell–cell vs. cell–substratum adhesivity. Proc. Natl. Acad. Sci. USA 98(8), 4323–4327CrossRefGoogle Scholar
  26. 26.
    Schwarzbauer J. E., J. L. Sechler 1999 Fibronectin fibrillogenesis: a paradigm for extracellular matrix assembly. Curr. Opin. Cell. Biol. 11(5), 622–627CrossRefGoogle Scholar
  27. 27.
    Sieminski A. L., R. P. Hebbel, K. J. Gooch 2004 The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro. Exp. Cell. Res. 297(2), 574–584CrossRefGoogle Scholar
  28. 28.
    Sottile J., J. Chandler 2005 Fibronectin matrix turnover occurs through a caveolin-1-dependent process. Mol. Biol. Cell. 16(2), 757–768CrossRefGoogle Scholar
  29. 29.
    Sottile J., D. C. Hocking 2002 Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell–matrix adhesions. Mol. Biol. Cell. 13(10), 3546–3559CrossRefGoogle Scholar
  30. 30.
    Tomasini-Johansson B. R., N. R. Kaufman, M. G. Ensenberger, V. Ozeri, E. Hanski, D. F. Mosher 2001 A 49-residue peptide from adhesin F1 of Streptococcus pyogenes inhibits fibronectin matrix assembly. J. Biol. Chem. 276(26), 23430–23439CrossRefGoogle Scholar
  31. 31.
    Vailhe B., X. Ronot, P. Tracqui, Y. Usson, L. Tranqui 1997 In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to alpha(v)beta3 integrin localization. In vitro Cell. Dev. Biol. Anim. 33(10), 763–773CrossRefGoogle Scholar
  32. 32.
    Vernon R. B., J. C. Angello, M. L. Iruela-Arispe, T. F. Lane, E. H. Sage 1992 Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro. Lab. Invest. 66(5), 536–547Google Scholar
  33. 33.
    Vernon R. B., S. L. Lara, C. J. Drake, M. L. Iruela-Arispe, J. C. Angello, C. D. Little, T. N. Wight, E. H. Sage 1995 Organized type I collagen influences endothelial patterns during “spontaneous angiogenesis in vitro”: planar cultures as models of vascular development. In vitro Cell. Dev. Biol. Anim. 31(2), 120–131CrossRefGoogle Scholar
  34. 34.
    Wang Y. L., R. J. Pelham Jr. 1998 Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 298, 489–496CrossRefGoogle Scholar
  35. 35.
    Yeung T., P. C. Georges, L. A. Flanagan, B. Marg, M. Ortiz, M. Funaki, N. Zahir, W. Ming, V. Weaver, P. A. Janmey 2005 Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell. Motil. Cytoskeleton 60(1), 24–34CrossRefGoogle Scholar
  36. 36.
    Zhou X., R. G. Rowe, N. Hiraoka, J. P. George, D. Wirtz, D. F. Mosher, I. Virtanen, M. A. Chernousov, S. J. Weiss 2008 Fibronectin fibrillogenesis regulates three-dimensional neovessel formation. Genes Dev. 22(9), 1231–1243CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2008

Authors and Affiliations

  • Joseph P. Califano
    • 1
  • Cynthia A. Reinhart-King
    • 1
  1. 1.Department of Biomedical EngineeringCornell UniversityIthacaUSA

Personalised recommendations