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

Christopher Mehlin¹

¹University 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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Abstract

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

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Materials

Basic Protocol: Measurement of VLA-4/CS-1 Adhesion Inhibition

Materials

Biotinylated CS-1 peptide: biotin-(CH₂)₃CO-GPEILDVPST-NH₃
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, 1997)

Alternate Protocol: Measurement of VLA-4/VCAM Adhesion Inhibition

Additional Materials (also see Basic Protocol)

HUVEC cells (e.g., Clonetics)
HUVEC medium (e.g., EGM from Clonetics)
TNF- (e.g., Calbiochem)
96-well tissue culture plates

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Figures

Figure 12.7.1

Standard RAMOS binding assay to CS-1 peptide and inhibition by cyclic peptide. The histogram on the left illustrates the binding of RAMOS cells to the Neutravidin-CS-1 complex in the absence of an inhibitor or cyclic peptide (hatched bar) or to Neutravidin alone (solid bar). All values are five replicates ±SEM.

View Image
Figure 12.7.2

Standard expected results using RAMOS cells binding to TNF--stimulated HUVEC cells and inhibition by cyclic peptide. The histogram on the left illustrates the binding of RAMOS cells to TNF-stimulated HUVEC cells (solid bar) and to HUVEC cells which were not stimulated with TNF (hatched bar). All values are five replicates ±SEM.

View Image

Literature Cited

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 4 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 4 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.