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Effects of Morphology vs. Cell–Cell Interactions on Endothelial Cell Stiffness

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Abstract

Biological processes such as atherogenesis, wound healing, cancer cell metastasis, and immune cell transmigration rely on a delicate balance between cell–cell and cell–substrate adhesion. Cell mechanics have been shown to depend on substrate factors such as stiffness and ligand presentation, while the effects of cell–cell interactions on the mechanical properties of cells has received little attention. Here, we use atomic force microscopy to measure the Young’s modulus of live human umbilical vein endothelial cells (HUVECs). In varying the degree of cell–cell contact in HUVECs (single cells, groups, and monolayers), we observe that increased cell stiffness correlates with an increase in cell area. Further, we observe that HUVECs stiffen as they spread onto a glass substrate. When we weaken cell–cell junctions (i.e., through a low dose of cytochalasin B or treatment with a VE-cadherin antibody), we observe that cell–substrate adhesion increases, as measured by focal adhesion size and density, and the stiffness of cells within the monolayer approaches that of single cells. Our results suggest that while morphology can roughly be used to predict cell stiffness, cell–cell interactions may play a significant role in determining the mechanical properties of individual cells in tissues by careful maintenance of cell tension homeostasis.

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References

  1. Adams, C. L., and W. J. Nelson. Cytomechanics of cadherin-mediated cell–cell adhesion. Curr. Opin. Cell Biol. 10:572–577, 1998.

    Article  Google Scholar 

  2. Angst, B. D., C. Marcozzi, and A. I. Magee. The cadherin superfamily: diversity in form and function. J. Cell Sci. 114:629–641, 2001.

    Google Scholar 

  3. Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Traffic 2:764–774, 2001.

    Article  Google Scholar 

  4. Balaban, N. Q., U. S. Schwarz, D. Riveline, P. Goichberg, G. Tzur, et al. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat. Cell Biol. 3:466–472, 2001.

    Article  Google Scholar 

  5. Bellin, R. M., J. D. Kubicek, M. J. Frigault, A. J. Kamien, R. L. Steward, et al. Defining the role of syndecan-4 in mechanotransduction using surface-modification approaches. Proc. Natl Acad. Sci. USA 106:22102–22107, 2009.

    Article  Google Scholar 

  6. Bereiterhahn, J., M. Luck, T. Miebach, H. K. Stelzer, and M. Voth. Spreading of trypsinized cells—cytoskeletal dynamics and energy-requirements. J. Cell Sci. 96:171–188, 1990.

    Google Scholar 

  7. Bhadriraju, K., and L. K. Hansen. Extracellular matrix- and cytoskeleton-dependent changes in cell shape and stiffness. Exp. Cell Res. 278:92–100, 2002.

    Article  Google Scholar 

  8. Blacher, J., R. Asmar, S. Djane, G. M. London, and M. E. Safar. Aortic pulse wave velocity as a marker of cardiovascular risk in hypertensive patients. Hypertension 33:1111–1117, 1999.

    Google Scholar 

  9. Blaschuk, O. W., and E. Devemy. Cadherins as novel targets for anti-cancer therapy. Eur. J. Pharmacol. 625:195–198, 2009.

    Article  Google Scholar 

  10. Boutouyrie, P., A. I. Tropeano, R. Asmar, I. Gautier, A. Benetos, et al. Aortic stiffness is an independent predictor of primary coronary events in hypertensive patients—a longitudinal study. Hypertension 39:10–15, 2002.

    Article  Google Scholar 

  11. Butt, H. J., and M. Jaschke. Calculation of thermal noise in atomic-force microscopy. Nanotechnology 6:1–7, 1995.

    Article  Google Scholar 

  12. Byfield, F. J., R. K. Reen, T. P. Shentu, I. Levitan, and K. J. Gooch. Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D. J. Biomech. 42:1114–1119, 2009.

    Article  Google Scholar 

  13. Cai, X. F., X. B. Xing, J. Y. Cai, Q. Chen, S. X. Wu, and F. C. Huang. Connection between biomechanics and cytoskeleton structure of lymphocyte and Jurkat cells: an AFM study. Micron 41:257–262, 2010.

    Article  Google Scholar 

  14. Califano, J. P., and C. A. Reinhart-King. A balance of substrate mechanics and matrix chemistry regulates endothelial cell network assembly. Cel. Mol. Bioeng. 1:122–132, 2008.

    Article  Google Scholar 

  15. Califano, J. P., and C. A. Reinhart-King. Substrate stiffness and cell area predict cellular traction stresses in single cells and cells in contact. Cel. Mol. Bioeng. 3:68–75, 2010.

    Article  Google Scholar 

  16. Chen, C. S., M. Mrksich, S. Huang, G. M. Whitesides, and D. E. Ingber. Geometric control of cell life and death. Science 276:1425–1428, 1997.

    Article  Google Scholar 

  17. Chouinard, J. A., G. Grenier, A. Khalil, and P. Vermette. Oxidized-LDL induce morphological changes and increase stiffness of endothelial cells. Exp. Cell Res. 314:3007–3016, 2008.

    Article  Google Scholar 

  18. Corada, M., M. Mariotti, G. Thurston, K. Smith, R. Kunkel, et al. Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo. Proc. Natl Acad. Sci. USA 96:9815–9820, 1999.

    Article  Google Scholar 

  19. Dahl, K. N., A. J. S. Ribeiro, and J. Lammerding. Nuclear shape, mechanics, and mechanotransduction. Circ. Res. 102:1307–1318, 2008.

    Article  Google Scholar 

  20. Davies, P. F., K. A. Barbee, M. V. Volin, A. Robotewskyj, J. Chen, et al. Spatial relationships in early signaling events of flow-mediated endothelial mechanotransduction. Annu. Rev. Physiol. 59:527–549, 1997.

    Article  Google Scholar 

  21. de Rooij, J., A. Kerstens, G. Danuser, M. A. Schwartz, and C. M. Waterman-Storer. Integrin-dependent actomyosin contraction regulates epithelial cell scattering. J. Cell Biol. 171:153–164, 2005.

    Article  Google Scholar 

  22. Dejana, E. Endothelial cell–cell junctions: happy together. Nat. Rev. Mol. Cell Biol. 5:261–270, 2004.

    Article  Google Scholar 

  23. Engler, A., L. Bacakova, C. Newman, A. Hategan, M. Griffin, and D. Discher. Substrate compliance versus ligand density in cell on gel responses. Biophys. J. 86:617–628, 2004.

    Article  Google Scholar 

  24. Etienne-Manneville, S., and A. Hall. Rho GTPases in cell biology. Nature 420:629–635, 2002.

    Article  Google Scholar 

  25. Fagotto, F., and B. M. Gumbiner. Cell contact-dependent signaling. Dev. Biol. 180:445–454, 1996.

    Article  Google Scholar 

  26. Farhadifar, R., J. C. Roper, B. Algouy, S. Eaton, and F. Julicher. The influence of cell mechanics, cell–cell interactions, and proliferation on epithelial packing. Curr. Biol. 17:2095–2104, 2007.

    Article  Google Scholar 

  27. Foty, R. A., C. M. Pfleger, G. Forgacs, and M. S. Steinberg. Surface tensions of embryonic tissues predict their mutual envelopment behavior. Development 122:1611–1620, 1996.

    Google Scholar 

  28. Gardel, M. L., F. Nakamura, J. H. Hartwig, J. C. Crocker, T. P. Stossel, and D. A. Weitz. Prestressed F-actin networks cross-linked by hinged filamins replicate mechanical properties of cells. Proc. Natl Acad. Sci. USA 103:1762–1767, 2006.

    Article  Google Scholar 

  29. Gauthier, N. C., O. M. Rossier, A. Mathur, J. C. Hone, and M. P. Sheetz. Plasma membrane area increases with spread area by exocytosis of a GPI-anchored protein compartment. Mol. Biol. Cell 20:3261–3272, 2009.

    Article  Google Scholar 

  30. Geiger, B., and A. Bershadsky. Assembly and mechanosensory function of focal contacts. Curr. Opin. Cell Biol. 13:584–592, 2001.

    Article  Google Scholar 

  31. Ghosh, K., Z. Pan, E. Guan, S. Ge, Y. Liu, et al. Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties. Biomaterials 28:671–679, 2007.

    Article  Google Scholar 

  32. Gotsch, U., E. Borges, R. Bosse, E. Boggemeyer, M. Simon, et al. VE-cadherin antibody accelerates neutrophil recruitment in vivo. J. Cell Sci. 110:583–588, 1997.

    Google Scholar 

  33. Helmke, B. P., R. D. Goldman, and P. F. Davies. Rapid displacement of vimentin intermediate filaments in living endothelial cells exposed to flow. Circ. Res. 86:745–752, 2000.

    Google Scholar 

  34. Hoffman, B. D., and J. C. Crocker. Cell mechanics: dissecting the physical responses of cells to force. Annu. Rev. Biomed. Eng. 11:259–288, 2009.

    Article  Google Scholar 

  35. Hordijk, P. L., E. Anthony, F. P. J. Mul, R. Rientsma, L. C. J. M. Oomen, and D. Roos. Vascular-endothelial-cadherin modulates endothelial monolayer permeability. J. Cell Sci. 112:1915–1923, 1999.

    Google Scholar 

  36. Hutson, M. S., Y. Tokutake, M. S. Chang, J. W. Bloor, S. Venakides, et al. Forces for morphogenesis investigated with laser microsurgery and quantitative modeling. Science 300:145–149, 2003.

    Article  Google Scholar 

  37. Hutter, J. L., and J. Bechhoefer. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64:1868–1873, 1993.

    Article  Google Scholar 

  38. Isenberg, B. C., P. A. DiMilla, M. Walker, S. Kim, and J. Y. Wong. Vascular smooth muscle cell durotaxis depends on substrate stiffness gradient strength. Biophys. J. 97:1313–1322, 2009.

    Article  Google Scholar 

  39. Juliano, R. L. Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annu. Rev. Pharmacol. Toxicol. 42:283–323, 2002.

    Article  Google Scholar 

  40. Kasas, S., X. Wang, H. Hirling, R. Marsault, B. Huni, et al. Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly. Cell Motil. Cytoskeleton 62:124–132, 2005.

    Article  Google Scholar 

  41. Ko, K. S., P. D. Arora, and C. A. McCulloch. Cadherins mediate intercellular mechanical signaling in fibroblasts by activation of stretch-sensitive calcium-permeable channels. J. Biol. Chem. 276:35967–35977, 2001.

    Article  Google Scholar 

  42. Ladoux, B., E. Anon, M. Lambert, A. Rabodzey, P. Hersen, et al. Strength dependence of cadherin-mediated adhesions. Biophys. J. 98:534–542, 2010.

    Article  Google Scholar 

  43. Lampugnani, M. G., A. Zanetti, F. Breviario, G. Balconi, F. Orsenigo, et al. VE-cadherin regulates endothelial actin activating Rac and increasing membrane association of Tiam. Mol. Biol. Cell 13:1175–1189, 2002.

    Article  Google Scholar 

  44. Lauffenburger, D. A., and L. G. Griffith. Who’s got pull around here? Cell organization in development and tissue engineering. Proc. Natl Acad. Sci. USA 98:4282–4284, 2001.

    Article  Google Scholar 

  45. Levenberg, S., B. Z. Katz, K. M. Yamada, and B. Geiger. Long-range and selective autoregulation of cell–cell or cell–matrix adhesions by cadherin or integrin ligands. J. Cell Sci. 111:347–357, 1998.

    Google Scholar 

  46. Lin, L. A. G., A. Q. Liu, Y. F. Yu, C. Zhang, C. S. Lim, et al. Cell compressibility studies utilizing noncontact hydrostatic pressure measurements on single living cells in a microchamber. Appl. Phys. Lett. 92:233901–233903, 2008.

    Article  Google Scholar 

  47. Majno, G., and I. Joris. Cells, Tissues, and Disease: Principles of General Pathology. Worcester, MA: Blackwell Science, 974, pp. 1996.

  48. Martens, J. C., and M. Radmacher. Softening of the actin cytoskeleton by inhibition of myosin II. Pflugers Arch. 456:95–100, 2008.

    Article  Google Scholar 

  49. Nelson, C. M., and C. S. Chen. Cell–cell signaling by direct contact increases cell proliferation via a PI3K-dependent signal. FEBS Lett. 514:238–242, 2002.

    Article  Google Scholar 

  50. Nelson, C. M., and C. S. Chen. VE-cadherin simultaneously stimulates and inhibits cell proliferation by altering cytoskeletal structure and tension. J. Cell Sci. 116:3571–3581, 2003.

    Article  Google Scholar 

  51. Nelson, C. M., D. M. Pirone, J. L. Tan, and C. S. Chen. Vascular endothelial-cadherin regulates cytoskeletal tension, cell spreading, and focal adhesions by stimulating RhoA. Mol. Biol. Cell 15:2943–2953, 2004.

    Article  Google Scholar 

  52. Norman, L. L., R. J. Oetama, M. Dembo, F. Byfield, D. A. Hammer, et al. Modification of cellular cholesterol content affects traction force, adhesion and cell spreading. Cel. Mol. Bioeng. 3:151–162, 2010.

    Article  Google Scholar 

  53. Oberleithner, H., C. Callies, K. Kusche-Vihrog, H. Schillers, V. Shahin, et al. Potassium softens vascular endothelium and increases nitric oxide release. Proc. Natl Acad. Sci. USA 106:2829–2834, 2009.

    Article  Google Scholar 

  54. Oberleithner, H., C. Riethmuller, H. Schillers, G. A. MacGregor, H. E. de Wardener, and M. Hausberg. Plasma sodium stiffens vascular endothelium and reduces nitric oxide release. Proc. Natl Acad. Sci. USA 104:16281–16286, 2007.

    Article  Google Scholar 

  55. Paszek, M. J., N. Zahir, K. R. Johnson, J. N. Lakins, G. I. Rozenberg, et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8:241–254, 2005.

    Article  Google Scholar 

  56. Rawicz, W., K. C. Olbrich, T. McIntosh, D. Needham, and E. Evans. Effect of chain length and unsaturation on elasticity of lipid bilayers. Biophys. J. 79:328–339, 2000.

    Article  Google Scholar 

  57. Reinhart-King, C. A. Endothelial cell adhesion and migration. Methods Enzymol. 443:45–64, 2008.

    Article  Google Scholar 

  58. Reinhart-King, C. A., M. Dembo, and D. A. Hammer. The dynamics and mechanics of endothelial cell spreading. Biophys. J. 89:676–689, 2005.

    Article  Google Scholar 

  59. Reinhart-King, C. A., M. Dembo, and D. A. Hammer. Cell–cell mechanical communication through compliant substrates. Biophys. J. 95:6044–6051, 2008.

    Article  Google Scholar 

  60. Rico, F., P. Roca-Cusachs, N. Gavara, R. Farre, M. Rotger, and D. Navajas. Probing mechanical properties of living cells by atomic force microscopy with blunted pyramidal cantilever tips. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 72:021914, 2005.

    Article  Google Scholar 

  61. Roca-Cusachs, P., J. Alcaraz, R. Sunyer, J. Samitier, R. Farre, and D. Navajas. Micropatterning of single endothelial cell shape reveals a tight coupling between nuclear volume in G1 and proliferation. Biophys. J. 94:4984–4995, 2008.

    Article  Google Scholar 

  62. Rotsch, C., and M. Radmacher. Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study. Biophys. J. 78:520–535, 2000.

    Article  Google Scholar 

  63. Ryan, P. L., R. A. Foty, J. Kohn, and M. S. Steinberg. Tissue spreading on implantable substrates is a competitive outcome of cell–cell vs. cell–substratum adhesivity. Proc. Natl Acad. Sci. USA 98:4323–4327, 2001.

    Article  Google Scholar 

  64. Sato, M., K. Nagayama, N. Kataoka, M. Sasaki, and K. Hane. Local mechanical properties measured by atomic force microscopy for cultured bovine endothelial cells exposed to shear stress. J. Biomech. 33:127–135, 2000.

    Article  Google Scholar 

  65. Shay-Salit, A., M. Shushy, E. Wolfovitz, H. Yahav, F. Breviario, et al. VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial cells. Proc. Natl Acad. Sci. USA 99:9462–9467, 2002.

    Article  Google Scholar 

  66. Sneddon, I. N. The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile. Int. J. Eng. Sci. 3:47–57, 1965.

    Article  MATH  MathSciNet  Google Scholar 

  67. Solon, J., I. Levental, K. Sengupta, P. C. Georges, and P. A. Janmey. Fibroblast adaptation and stiffness matching to soft elastic substrates. Biophys. J. 93:4453–4461, 2007.

    Article  Google Scholar 

  68. Stroka, K. M., and H. Aranda-Espinoza. Neutrophils display biphasic relationship between migration and substrate stiffness. Cell Motil. Cytoskeleton 66:328–341, 2009.

    Article  Google Scholar 

  69. Stroka, K. M., and H. Aranda-Espinoza. A biophysical view of the interplay between mechanical forces and signaling pathways during transendothelial cell migration. FEBS J. 277:1145–1158, 2010.

    Article  Google Scholar 

  70. Svaldo Lanero, T., O. Cavalleri, S. Krol, R. Rolandi, and A. Gliozzi. Mechanical properties of single living cells encapsulated in polyelectrolyte matrixes. J. Biotechnol. 124:723–731, 2006.

    Article  Google Scholar 

  71. Trepat, X., M. R. Wasserman, T. E. Angelini, E. Millet, D. A. Weitz, et al. Physical forces during collective cell migration. Nat. Phys. 5:426–430, 2009.

    Article  Google Scholar 

  72. Ueki, Y., N. Sakamoto, T. Ohashi, and M. Sato. Morphological responses of vascular endothelial cells induced by local stretch transmitted through intercellular junctions. Exp. Mech. 49:125–134, 2009.

    Article  Google Scholar 

  73. Vinckier, A., and G. Semenza. Measuring elasticity of biological materials by atomic force microscopy. FEBS Lett. 430:12–16, 1998.

    Article  Google Scholar 

  74. Vitorino, P., and T. Meyer. Modular control of endothelial sheet migration. Genes Dev. 22:3268–3281, 2008.

    Article  Google Scholar 

  75. Wakatsuki, T., B. Schwab, N. C. Thompson, and E. L. Elson. Effects of cytochalasin D and latrunculin B on mechanical properties of cells. J. Cell Sci. 114:1025–1036, 2001.

    Google Scholar 

  76. Wang, N., and D. Stamenovic. Mechanics of vimentin intermediate filaments. J. Muscle Res. Cell Motil. 23:535–540, 2002.

    Article  Google Scholar 

  77. Wang, N., I. M. Tolic-Norrelykke, J. Chen, S. M. Mijailovich, J. P. Butler, et al. Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells. Am. J. Physiol. Cell Physiol. 282:C606–C616, 2002.

    Google Scholar 

  78. Waterman-Storer, C. M., W. C. Salmon, and E. D. Salmon. Feedback interactions between cell–cell adherens junctions and cytoskeletal dynamics in Newt lung epithelial cells. Mol. Biol. Cell 11:2471–2483, 2000.

    Google Scholar 

  79. Weisenhorn, A. L., M. Khorsandit, S. Kasast, V. Gotzost, and H.-J. Butt. Deformation and height anomaly of soft surfaces studied with an AFM. Nanotechnology 4:106–113, 1993.

    Article  Google Scholar 

  80. Yeung, T., P. C. Georges, L. A. Flanagan, B. Marg, M. Ortiz, et al. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil. Cytoskeleton 60:24–34, 2005.

    Article  Google Scholar 

  81. Zhu, A. J., and F. M. Watt. Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes. J. Cell Sci. 109:3013–3023, 1996.

    Google Scholar 

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Acknowledgments

This work was completed under an NSF Graduate Research Fellowship to KMS, NIH NRSA fellowship to KMS (NINDS Award Number F31NS068028) and NSF Award CMMI-0643783 to HAE. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health.

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Stroka, K.M., Aranda-Espinoza, H. Effects of Morphology vs. Cell–Cell Interactions on Endothelial Cell Stiffness. Cel. Mol. Bioeng. 4, 9–27 (2011). https://doi.org/10.1007/s12195-010-0142-y

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