The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. Endothelial nitric oxide synthase (eNOS) is the key enzyme of vascular homeostasis involved in the pathophysiology of several cardiovascular diseases. The aim of our study was to investigate whether eNOS expression and activity are regulated by the proteasome. Bovine pulmonary artery endothelial cells (CPAE cells) were treated with the proteasome inhibitor MG132. MG132 (50-250 nmol/L) dose-dependently increased mRNA and protein levels of eNOS. Comparable results were obtained with other specific proteasome inhibitors, whereas the nonproteasomal calpain and cathepsin inhibitor ALLM had no effect. Efficacy of proteasome inhibition was evidenced by accumulation of poly-ubiquitinylated proteins and by measuring proteasomal activity in cell extracts. Cycloheximide prevented up-regulation of eNOS protein, indicating that post-translational stabilization of eNOS is not involved. eNOS activity was increased up to 2.8-fold (MG132 100 nmol/L, 48 h). Incubation of rat aortic rings with MG132 significantly enhanced endothelial-dependent vasorelaxation. Single MG132 treatment (100 nmol/L) induced long-term effects in CPAE cells, with increases of eNOS protein and activity for up to 10 days. Our results indicate that low-dose proteasome inhibition enhances eNOS expression and activity, and improves endothelial function.

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The cellular adhesion molecule E-selectin is expressed on activated endothelial cells, and is involved in the process of adherence of blood cells to vessel endothelium in inflammatory events such as atherosclerosis. In a recent study we found a Ser128Arg mutation in the EGF domain as well as a Leu554Phe mutation in the membrane domain of E-selectin. We also established increased frequencies of both mutations among young patients with severe coronary atherosclerosis. In the present study we investigated the influence of these mutations on cell adhesion and on the release of soluble E-selectin. Mutants were created by site-directed mutagenesis and COS cells were transfected with E-selectin, either wild-type or mutant. Antibody-binding studies and cell-adhesion assays were performed on transfected COS cells and on interleukin-1 beta-stimulated HUVECs. Soluble E-selectin in supernatants of wild type and Leu554Phe mutant-transfected COS cells was measured by ELISA. We discovered significant differences in the strength of HL-60 cell adhesion for the Ser128Arg mutant: in comparison with the wild type, the strength of adhesion to the mutant was reduced on transfected COS cells (P < 0.01) as well as on stimulated HUVECs (P < 0.01). Significantly diminished release of soluble E-selectin was detected for the Leu554Phe membrane domain mutant, in comparison with the wild type. In summary, the mutations studied here influence the E-selectin function in vitro and may be considered as one of the risk factors involved in the complex pathogenesis of atherosclerosis.

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