European Journal of Nutrition

, Volume 44, Issue 2, pp 95–104

Proteome analysis for identification of target proteins of genistein in primary human endothelial cells stressed with oxidized LDL or homocysteine

  • D. Fuchs
  • S. de Pascual-Teresa
  • G. Rimbach
  • Fabio Virgili
  • Roberto Ambra
  • Rufus Turner
  • H. Daniel
  • U. Wenzel
ORIGINAL CONTRIBUTION

DOI: 10.1007/s00394-004-0499-6

Cite this article as:
Fuchs, D., de Pascual-Teresa, S., Rimbach, G. et al. Eur J Nutr (2005) 44: 95. doi:10.1007/s00394-004-0499-6

Summary

Background

Epidemiological studies suggest that soy consumption contributes to the prevention of coronary heart disease. The proposed anti–atherogenic effects of soy appear to be carried by the soy isoflavones with genistein as the most abundant compound.

Aim of the study

To identify proteins or pathways by which genistein might exert its protective activities on atherosclerosis, we analyzed the proteomic response of primary human umbilical vein endothelial cells (HUVEC) that were exposed to the pro–atherosclerotic stressors homocysteine or oxidized low–density lipoprotein (ox-LDL).

Methods

HUVEC were incubated with physiological concentrations of homocysteine or ox–LDL in the absence and presence of genistein at concentrations that can be reached in human plasma by a diet rich in soy products (2.5 µM) or by pharmacological intervention (25 µM). Proteins from HUVEC were separated by two–dimensional polyacrylamide gel electrophoresis and those that showed altered expression level upon genistein treatment were identified by peptide mass fingerprints derived from tryptic digests of the protein spots.

Results

Several proteins were found to be differentially affected by genistein. The most interesting proteins that were potently decreased by homocysteine treatment were annexin V and lamin A. Annexin V is an antithrombotic molecule and mutations in nuclear lamin A have been found to result in perturbations of plasma lipids associated with hypertension. Genistein at low and high concentrations reversed the stressor–induced decrease of these anti–atherogenic proteins. Ox–LDL treatment of HUVEC resulted in an increase in ubiquitin conjugating enzyme 12, a protein involved in foam cell formation. Treatment with genistein at both doses reversed this effect.

Conclusion

Proteome analysis allows the identification of potential interactions of dietary components in the molecular process of atherosclerosis and consequently provides a powerful tool to define biomarkers of response.

Key words

human umbilical vein endothelial cellsgenisteinox–LDLhomocysteineatherosclerosisproteomics

Abbreviations

AIP1

apoptosis–linked gene 2 interacting protein 1

CHD

Coronary heart disease

2D–PAGE

two–dimensional polyacrylamide gel–electrophoresis

FBS

Fetal bovine serum

HEPES

4–(2–hydroxyethyl)–1–piperazine ethanesulfonic acid

HUVEC

human umbilical vein endothelial cells

IEF

isoelectric focussing

IPG

immobilized pH–gradient

MALDI–TOF–MS

Matrix–assisted laser desorption ionization time–of–flight mass spectrometry

ox–LDL

oxidized low–density lipoprotein

SDS

sodium dodecylsulfate

smc1

chromosome segregation protein

Copyright information

© Steinkopff Verlag 2004

Authors and Affiliations

  • D. Fuchs
    • 1
  • S. de Pascual-Teresa
    • 2
  • G. Rimbach
    • 2
  • Fabio Virgili
    • 3
  • Roberto Ambra
    • 3
  • Rufus Turner
    • 2
  • H. Daniel
    • 1
  • U. Wenzel
    • 1
  1. 1.Dept. of Food and NutritionMolecular Nutrition Unit Technical University of MunichFreisingGermany
  2. 2.Hugh Sinclair Unit of Human NutritionSchool of Food Biosciences University of ReadingReadingUK
  3. 3.National Institute for Food and Nutrition ResearchRomeItaly