Effects of oxidative stress and Ca2+ agonists on molecular chaperones in human umbilical vein endothelial cells

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Abstract
Endothelial cell dysfunction is a key factor in oxidative stress-related pathology. Disruption of Ca2+ homeostasis is thought to be responsible for much of the endothelial cell dysfunction in oxidative stress. The expression of molecular chaperones (MC), which stabilize protein structures in normal and in stress conditions, reflects the Ca2+ -dependent and -independent stress effects in the different cell compartments. By two-dimensional (2-D) gel electrophoresis, combined with immunoblotting or microsequencing, we have identified 12 major MC in human umbilical vein endothelial cells (HUVEC): (i) the endoplasmic reticulum-located MC GRP78, GRP94, protein disulfide isomerase, and calreticulin; (ii) the mitochondrial MC HSP65 and GRP75; and (iii) the cytosolic/nuclear MC HSP27, HSC70, HSP70, HSP90, cyclophilin, and ubiquitin. To differentiate oxidative stress- and Ca2+ -mediated effects, HUVEC were exposed to 1) xanthine oxidase plus hypoxanthine to generate oxidative stress, 2) ionomycin plus ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) to deplete intracellular Ca2+ stores, or 3) thrombin to increase cytosolic Ca2+. De novo protein synthesis after exposure was quantified by the incorporation of [35S]methionine. Image processing with the MELANIE system was used to create and compare the 2-D maps of [35S]methionine-labeled proteins under conditions 1)–3) with those of the controls. In a total of 24 2-D gels, 9 different MC were detected in at least 5 out 6 experimental replicates and were subjected to numeric analysis. The statistics showed a > 10% increase in GRP78 (p < 0.05), HSP27, cyclophilin, and ubiquitin after oxidative stress. Ionomycin plus EGTA influenced the expression of GRP78 (p < 0.01), cyclophilin, and ubiquitin in the same direction, but in addition increased GRP94 (p < 0.01) and calreticulin. Thrombin upregulated calreticulin (p < 0.05), HSC70, and cyclophilin. In conclusion, the computer analysis of MC expression on 2-D maps under different stress conditions reveals a differential correlation between the oxidative stress and the depletion of Ca2+ stores in HUVEC.