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It is known that replicative senescence of endothelium in vivo contributes at least partially to age-related vascular disorders such as arteriosclerosis. However, the genes involved in this process remain to be identified. In this study, we employed a proteomics-based approach to identify candidate genes using in vitro cultured human umbilical vein endothelial cells (HUVECs) as an experimental model for replicative senescence. By comparing protein spots from young and senescent HUVECs using two-dimensional electrophoresis, we identified three up-regulated proteins and five down-regulated proteins in senescent HUVECs as compared to young HUVECs, whose alteration was not observed during replicative senescence of primary human fibroblasts. Consistent results were obtained in Western blotting analysis using specific antibodies raised against some of these proteins, whereas there were no significant changes in the mRNA levels of these genes during senescence of HUVECs. Among them, cathepsin B, a protease participating in both intracellular proteolysis and extracellular matrix remodeling was observed to be dramatically up-regulated in senescent HUVECs and whose activity is known to be up-regulated in atherosclerotic lesions with senescence-associated phenotypes in vivo. Additional proteins, including cytoskeletal proteins and proteins involved in the processes of synthesis, turnover and modification of protein, were identified, whose function in endothelium was previously unsuspected. These proteins identified by a proteomics-based approach using cultured HUVECs may be involved not only in replicative senescence but also in functional alterations in vascular endothelial cells with senescence-associated phenotypes and may serve as molecular markers for these processes.

cell structure and function

Searching for Genes Involved in Arteriosclerosis: Proteomic Analysis of Cultured Human Umbilical Vein Endothelial Cells Undergoing Replicative Senescence