Skip to main content
SpringerLink
Log in

The Proteome of Endothelial Cells

  • Protocol
Cardiovascular Proteomics

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 357))

Abstract

Endothelial cells form a continuous monolayer lining the inside face of all blood vessels, and present the ability to selectively control vascular permeability. The endothelium is involved in a wide variety of normal physiological and pathological processes. The endothelial dysfunction occurs under activation conditions, with the acquisition of many new functional, inflammatory, and immune properties, and as a consequence, endothelial cells display many different transcription profiles. We describe here the isolation and culture of the most useful model of human umbilical vein endothelial cells, and undertake the proteomic analysis under both basal quiescent condition and activated by stimulation with a proinflammatory cytokine. Series of two-dimensional electrophoresis have allowed us to detect a total of close to 600 polypeptide spots using 4.0–7.0 pH range in both culture conditions. We have selected 233 proteins by cross-matching the gels, and found that 70% showed an increase and 30% a decrease of expression levels in activated cells. Subsequent identification of 35 altered peptides is made by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry, as well as a study of posttranslational modifications. These global findings may contribute to understand the effects of pathological stimuli and the mechanisms that regulate vascular diseases.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nachman, R. L. and Jaffe, E. A. (2004) Endothelial cell culture: beginnings of modern vascular biology. J. Clin. Invest. 114, 1037–1040.

    PubMed  CAS  Google Scholar 

  2. Cines, D. B., Pollak, E. S., Buck, C. A., et al. (1998) Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91, 3527–3561.

    PubMed  CAS  Google Scholar 

  3. Pasyk, K. A. and Jakobczak, B. A. (2004) Vascular endothelium: recent advances. Eur. J. Dermatol. 14, 209–213.

    PubMed  Google Scholar 

  4. Nash, G. B., Buckley, C. D., and Ed Rainger, G. (2004) The local physicochemical environment conditions the proinflammatory response of endothelial cells and thus modulates leukocyte recruitment. FEBS Lett. 569, 13–17.

    Google Scholar 

  5. Stevens, T., Garcia, J. G., Shasby, D. M., Bhattacharya, J., and Malik, A. B. (2000) Mechanisms regulating endothelial cell barrier function. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L419–L422.

    PubMed  CAS  Google Scholar 

  6. Sumpio, B. E., Riley, J. T., and Dardik, A. (2002) Cells in focus: endothelial cell. Int. J. Biochem. Cell. Biol. 34, 1508–1512.

    Article  PubMed  CAS  Google Scholar 

  7. Sato, Y. (2001) Current understanding of the biology of vascular endothelium. Cell Struct. Funct. 26, 9–10.

    Article  PubMed  CAS  Google Scholar 

  8. Bazzoni, G. and Dejana, E. (2004) Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol. Rev. 84, 869–901.

    Article  PubMed  CAS  Google Scholar 

  9. Lee, T. Y. and Gotlieb, A. I. (2003) Microfilaments and microtubules maintain endothelial integrity. Microsc. Res. Tech. 60, 115–127.

    Article  PubMed  CAS  Google Scholar 

  10. Yuan, S. Y. (2002) Protein kinase signalling in the modulation of microvascular permeability. Vasc. Pharmacol. 39, 213–223.

    Article  CAS  Google Scholar 

  11. De Caterina, R. (2000) Endothelial dysfunctions: common denominators in vascular disease. Curr. Opin. Lipidol. 11, 9–23.

    Article  PubMed  Google Scholar 

  12. Bachetti, T. and Morbidelli, L. (2000) Endothelial cells in culture: a model for studying vascular functions. Pharmacol. Res. 42, 9–19.

    Article  PubMed  CAS  Google Scholar 

  13. Blann, A. D. (2004) Assessment of endothelial dysfunction: focus on atherothrombotic disease. Pathophysiol. Haemost. Thromb. 33, 256–261.

    Article  Google Scholar 

  14. Libby, P., Ridker, P. M., and Maseri, A. (2002) Inflammation and atherosclerosis. Circulation 105, 1135–1143.

    Article  PubMed  CAS  Google Scholar 

  15. Cook-Mills, J. M. and Deem, T. L. (2005) Active participation of endothelial cells in inflammation. J. Leukoc. Biol. 77, 487–495.

    Article  PubMed  CAS  Google Scholar 

  16. Endemann, D. H. and Schiffrin, E. L. (2004) Endothelial dysfunction. J. Am. Soc. Nephrol. 15, 1983–1992.

    Article  PubMed  CAS  Google Scholar 

  17. Landmesser, U., Hornig, B., and Drexler, H. (2004) Endothelial function: a critical determinant in atherosclerosis? Circulation 109, II27–II33.

    Article  PubMed  Google Scholar 

  18. Garlanda, C. and Dejana, E. (1997) Heterogeneity of endothelial cells. Specific markers. Arterioscler. Thromb. Vasc. Biol. 17, 1193–1202.

    PubMed  CAS  Google Scholar 

  19. McIntyre, T. M., Prescott, S. M., Weyrich, A. S., and Zimmerman, G. A. (2003) Cell-cell interactions: leukocyte-endothelial interactions. Curr. Opin. Hematol. 10, 150–158.

    Article  PubMed  CAS  Google Scholar 

  20. Muller, W. A. (2003) Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends Immunol. 24, 327–334.

    PubMed  CAS  Google Scholar 

  21. Steeber, D. A., Venturi, G. M., and Tedder, T. F. (2005) A new twist to the leukocyte adhesion cascade: intimate cooperation is key. Trends Immunol. 26, 9–12.

    Article  PubMed  CAS  Google Scholar 

  22. Blankenberg, S., Barbaux, S., and Tiret, L. (2003) Adhesion molecules and atherosclerosis. Atherosclerosis 170, 191–203.

    Article  PubMed  CAS  Google Scholar 

  23. Jaffe, E. A., Nachman, R. L., Becker, C. G., and Minick, C. R. (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest. 52, 2745–2756.

    Article  PubMed  CAS  Google Scholar 

  24. Gimbrone, M. A., Jr., Cotran, R.S., and Folkman, J. (1974) Human vascular endothelial cells in culture: growth and DNA synthesis. J. Cell Biol. 60, 673–684.

    Article  PubMed  CAS  Google Scholar 

  25. Raab, M., Daxecker, H., Markovic, S., Karimi, A., Griesmacher, A., and Mueller, M. M. (2002) Variation of adhesion molecule expression on human umbilical vein endothelial cells upon multiple cytokine application. Clin. Chim. Acta 321, 11–16.

    PubMed  CAS  Google Scholar 

  26. Daxecker, H., Raab, M., Markovic, S., Karimi, A., Griesmacher, A., and Mueller, M. M. (2002) Endothelial adhesion molecule expression in an in vitro model of inflammation. Clin. Chim. Acta 325, 171–175.

    Article  PubMed  CAS  Google Scholar 

  27. Tan, P. H., Chan, C., Xue, S. A., et al. (2004) Phenotypic and functional differences between human saphenous vein (HSVEC) and umbilical vein (HUVEC) endothelial cells. Atherosclerosis 173, 171–183.

    Article  PubMed  CAS  Google Scholar 

  28. Jungblut, P. R., Zimny-Arndt, U., Zeindl-Eberhart, E., et al. (1999) Proteomics in human disease: cancer, heart and infectious diseases. Electrophoresis 20, 2100–2110.

    Article  PubMed  CAS  Google Scholar 

  29. Arrell, D. K., Neverova, I., and Van Eyk, J. E. (2001) Cardiovascular proteomics: evolution and potential. Circ. Res. 88, 763–773.

    Article  PubMed  CAS  Google Scholar 

  30. Durán, M. C., Mas, S., Martín-Ventura, J. L., et al. (2003) Proteomic analysis of human vessels: application to atherosclerotic plaques. Proteomics 3, 973–978.

    Article  PubMed  Google Scholar 

  31. Martín-Ventura, J. L., Durán, M. C., Blanco-Colio, L. M., et al. (2004) Identification by a differential proteomic approach of heat shock protein 27 as a potential marker of atherosclerosis. Circulation 110, 2216–2219.

    Article  PubMed  Google Scholar 

  32. Mayr, M., Mayr, U., Chung, Y. L., Yin, X., Griffiths, J. R., and Xu, Q. (2004) Vascular proteomics: linking proteomic and metabolomic changes. Proteomics 4, 3751–3761.

    Article  PubMed  CAS  Google Scholar 

  33. Zerkowski, H. R., Grussenmeyer, T., Matt, P., Grapow, M., Engelhardt, S., and Lefkovits, I. (2004) Proteomics strategies in cardiovascular research. J. Proteome Res. 3, 200–208.

    Article  PubMed  CAS  Google Scholar 

  34. Oh, P., Li, Y., Yu, J., et al. (2004) Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Nature 429, 629–635.

    Article  PubMed  CAS  Google Scholar 

  35. Bruneel, A., Labas, V., Mailloux, A., et al. (2003) Proteomic study of human umbilical vein endothelial cells in culture. Proteomics 3, 714–723.

    Article  PubMed  CAS  Google Scholar 

  36. Kamino, H., Hiratsuka, M., Toda, T., et al. (2003) Searching for genes involved in arteriosclerosis: proteomic analysis of cultured human umbilical vein endothelial cells undergoing replicative senescence. Cell. Struct. Funct. 28, 495–503.

    Article  PubMed  CAS  Google Scholar 

  37. Traxler, E., Bayer, E., Stockl, J., Mohr, T., Lenz, C., and Gerner, C. (2004) Towards a standardized human proteome database: quantitative proteome profiling of living cells. Proteomics 4, 1314–1323.

    Article  PubMed  CAS  Google Scholar 

  38. Sprenger, R. R., Speijer, D., Back, J. W., De Koster, C. G., Pannekoek, H., and Horrevoets, A. J. (2004) Comparative proteomics of human endothelial cell caveolae and rafts using two-dimensional gel electrophoresis and mass spectrometry. Electrophoresis 25, 156–172.

    Article  PubMed  CAS  Google Scholar 

  39. Scheurer, S. B., Rybak, J. N., Rosli, C., Neri, D., and Elia, G. (2004) Modulation of gene expression by hypoxia in human umbilical cord vein endothelial cells: a transcriptomic and proteomic study. Proteomics 4, 1737–1760.

    Article  PubMed  CAS  Google Scholar 

  40. McGregor, E., Kempster, L., Wait, R., et al. (2001) Identification and mapping of human saphenous vein medial smooth muscle proteins by two-dimensional polyacrylamide gel electrophoresis. Proteomics 1, 1405–1414.

    Article  PubMed  CAS  Google Scholar 

  41. Yan, J. X., Wait, R., Berkelman, T., et al. (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21, 3666–3672.

    Article  PubMed  CAS  Google Scholar 

  42. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858.

    Article  PubMed  CAS  Google Scholar 

  43. Westbrook, J. A., Yan, J. X., Wait, R., Welson, S. Y., and Dunn, M. J. (2001) Zooming-in on the proteome: very narrow-range immobilised pH gradients reveal more protein species and isoforms. Electrophoresis 22, 2865–2871.

    Article  PubMed  CAS  Google Scholar 

  44. Larsen, M. R., Cordwell, S. J., and Roepstorff, P. (2002) Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry. Proteomics 2, 1277–1287.

    Article  PubMed  CAS  Google Scholar 

  45. Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R., and Roepstorff, P. (1999) Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J. Mass Spectrom. 34, 105–116.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Research Unit, Renal and Vascular Pathology Laboratory, Fundación Jiménez Díaz, Madrid, Spain

    Jesús González-Cabrero, Mayte Pozo & Rosario de Nicolás

  2. Department of Immunology, Fundación Jiménez Díaz, Madrid, Spain

    Mari-Carmen Durán & Fernando Vivanco

  3. Department of Biochemistry and Molecular Biology, Protein Research Group, University of Southern Denmark, Odense, Denmark

    Mari-Carmen Durán

  4. Renal and Vascular Pathology Laboratory, Fundación Jiménez Díaz, Universidad Autónoma, Madrid, Spain

    Jesús Egido

  5. Department of Biochemistry and Molecular Biology I, Proteomics Unit, Universidad Complutense, Madrid, Spain

    Fernando Vivanco

Authors
  1. Jesús González-Cabrero
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mayte Pozo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mari-Carmen Durán
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rosario de Nicolás
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jesús Egido
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fernando Vivanco
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Immunology, Fundación Jiménez Díaz, Universidad Complutense, Madrid, Spain

    Fernando Vivanco

  2. Department of Biochemistry and Molecular Biology I, Universidad Complutense, Madrid, Spain

    Fernando Vivanco

Rights and permissions

Reprints and permissions

Copyright information

© 2007 Humana Press Inc.

About this protocol

Cite this protocol

González-Cabrero, J., Pozo, M., Durán, MC., de Nicolás, R., Egido, J., Vivanco, F. (2007). The Proteome of Endothelial Cells. In: Vivanco, F. (eds) Cardiovascular Proteomics. Methods in Molecular Biology™, vol 357. Humana Press. https://doi.org/10.1385/1-59745-214-9:181

Download citation

  • DOI: https://doi.org/10.1385/1-59745-214-9:181

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-535-4

  • Online ISBN: 978-1-59745-214-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation