Measurement of VLA‐4/CS‐1 and VLA‐4/VCAM Adhesion Inhibition

Christopher Mehlin1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.7
DOI:  10.1002/0471141755.ph1207s24
Online Posting Date:  May, 2004
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Cell adhesion, a critical early step in the inflammatory process, has increasingly become the target of drug discovery efforts. Described in this unit are techniques for measuring inhibitors of VLA‐4‐mediated adhesion to either VCAM or the connecting segment (CS‐1) of fibronectin.

Keywords: VLA‐4; VCAM; CS‐1; cell adhesion; inflammation

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Table of Contents

  • Basic Protocol 1: Measurement of VLA‐4/CS‐1 Adhesion Inhibition
  • Alternate Protocol 1: Measurement of VLA‐4/VCAM Adhesion Inhibition
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Measurement of VLA‐4/CS‐1 Adhesion Inhibition

  • Biotinylated CS‐1 peptide: biotin‐(CH 2) 3CO‐GPEILDVPST‐NH 3
  • DPBS with calcium and magnesium (Life Technologies)
  • BSA (e.g., Sigma)
  • Binding buffer (see recipe)
  • Test compounds
  • Cyclic control peptide (C‐ss‐CWLDV)
  • RAMOS cells (ATCC #CRL‐1596), grown in RPMI with 10% FBS and 10 mM HEPES
  • 1 µg/µl calcein‐AM (Molecular Probes) in DMSO
  • Lysis buffer (see recipe)
  • 96‐well Neutravidin‐coated plates (Pierce)
  • Gentle ELISA plate–washing apparatus (e.g., Molecular Devices' Skanwasher 400; for automatic washing) or 12‐channel pipettor and tips (for manual washing)
  • 96‐well polypropylene plates
  • Fluorescence plate reader (e.g., BioTek Synergy HT)
  • Nonlinear regression software (e.g., Prism, GraphPad)
  • Additional reagents and equipment for counting cells and measuring viability (Phelan, )

Alternate Protocol 1: Measurement of VLA‐4/VCAM Adhesion Inhibition

  • HUVEC cells (e.g., Clonetics)
  • HUVEC medium (e.g., EGM from Clonetics)
  • TNF‐α (e.g., Calbiochem)
  • 96‐well tissue culture plates
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Literature Cited

   Hocking, D.C. 2002. Fibronectin matrix deposition and cell contractility. Chest 122:275S‐278S.
   Lin, K., Ateeq, H.S., Hsiung, S.H., Chong, L.T., Zimmerman, C.N., Castro, A., Lee, W.C., Hammond, C.E., Kalkunte, S., Chen, L.L., Pepinsky, R.B., Leone, D.R., Sprague, A.G., Abraham, W.M., Gill, A., Lobb, R.R., Adams, S.P. 1999. Selective, tight‐binding inhibitors of integrin 41 that inhibit allergic airway responses. J. Med. Chem. 42(5):920‐34.
   Lobb, R.R. and Adams, S.P. 1999. Small molecule antagonists of α4 integrins: Novel drugs for asthma. Exp. Opin. Invest. Drugs. 8(7):935‐945.
   Lobb, R.R., Abraham, W.M., Burkly, L.C., Gill, A., Ma, W., Knight, J.A., Leone, D.R., Antognetti, G., and Pepinsky, R.B. 1996. Pathophysiologic role of alpha4 integrins in the lung. Ann. N.Y. Acad. Sci. 796:113‐123.
   Masumoto, A. and Hemler, M.E. 1993. Multiple activation states of VLA‐4. J. Biol. Chem. 268:228‐234.
   Phelan, M.C. 1997. Techniques for mammalian cell tissue culture. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, P.T. Wingfield, eds.) pp. A.3C.1‐A.3C.14. John Wiley & Sons, Hoboken, N.J.
   Swerlick, R.A., Lee, K.H., Li, L., Sepp, N.T., Caughman, S.W. and Lawley, T.J. 1992. Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells. J. Immunol. 149:698‐705.
   van der Flier, A. and Sonnenberg, A. 2001. Function and interactions of integrins. Cell Tissue Res. 305:285‐298.
   Vanderslice, P. Ren, K., Revelle, J.K., Kim, D.C., Scott, D., Bjercke, R.J., Yeh, E.T.H., Beck, P.J., and Kogan, T.P. 1997. A cyclic hexapeptide is a potent antagonist of α4 integrins. J. Immunol. 158:1710‐1718.
   Yang, G.X. and Hagman, W.K. 2003. VLA‐4 antagonists: Potent inhibitors of lymphocyte migration. Med. Res. Rev. 23:369‐392.
   Yusuf‐Makagiansar, H., Anderson, M.E., Yakovleva, T.V., Murray, J.S., and Siahaan, T.J. 2002. Inhibition of LFA‐1/ICAM‐1 and VLA‐4/VCAM‐1 as a therapeutic approach to inflammation and autoimmune disease. Med. Res. Rev. 22(2):146‐67.
Key Reference
   Yusuf‐Makagiansar et al., 2002. See above.
  This is a broad review covering cell adhesion as a therapeutic target with specific reference to VLA‐4.
   Yang et al., 2003. See above.
  This review focuses on compounds developed as VLA‐4 inhibitors.
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