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


OBJECTIVE
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 microM) or by pharmacological intervention (25 microM). 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.


CONCLUSIONS
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.
Dept. of Food and Nutrition, Molecular Nutrition Unit Technical University of Munich, Hochfeldweg 2, 85350, Freising, Germany.
Dagmar
D
Fuchs
Sonia
S
de Pascual-Teresa
Gerald
G
Rimbach
Fabio
F
Virgili
Roberto
R
Ambra
Rufus
R
Turner
Hannelore
H
Daniel
Uwe
U
Wenzel
D017304:Annexin A5 / Q000378:metabolism*; D001161:Arteriosclerosis / Q000517:prevention & control; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D042783:Endothelial Cells; D004730:Endothelium, Vascular / Q000378:metabolism*; D004791:Enzyme Inhibitors; D019833:Genistein / Q000494:pharmacology*; D006710:Homocysteine / Q000494:pharmacology; D006801:Humans; D034904:Lamin Type A / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000494:pharmacology; D010084:Oxidation-Reduction; D020543:Proteome / Q000032:analysis*; D030262:Soybean Proteins / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014470:Umbilical Cord / Q000166:cytology
D017304:Annexin A5; D004791:Enzyme Inhibitors; D034904:Lamin Type A; D008077:Lipoproteins, LDL; D020543:Proteome; D030262:Soybean Proteins; C099252:oxidized low density lipoprotein; D006710:Homocysteine; D019833:Genistein