Molecular mechanisms of endothelial cell sencescence

Human ageing is accompanied by the degeneration of various tissues, leading to functional impairment. Many different factors contribute to human ageing and at the molecular level there are no firmly established mechanisms known to sufficient detail. Most of our current knowledge about the ageing process has been derived from studies of simple model systems. Genetic factors that contribute to the ageing process are studied predominantly in lower organisms, such as yeast, flies, and worms, and these studies have already revealed some genetic traits involved in the ageing process. However, to study human ageing, these systems are of very limited use, as there is increasing evidence that ageing processes differ considerably between lower eukaryotes and higher organisms, e.g. mammals. Even within the mammalia, considerable differences in molecular mechanisms of ageing have been revealed, e.g. between human ageing and the ageing of rodents, such as the mouse. In the absence of any good animal models for human ageing, human cells in primary culture have been widely used as a model system to study determinants of human ageing. Cells explanted from various human tissues are grown in cell culture until their proliferation capacity is exhausted, a status referred to as replicative senescence. Many important features of human ageing have been derived from the study of senescent fibroblasts, providing compelling evidence for the usefulness of such systems. In the present project, we propose to study replicative senescence of another human cell type, namely endothelial cells from the vascular system. The aim of this project is to get insight into molecular mechanisms of vascular ageing at the cellular level and to provide the basis for corresponding in vivo studies. In particular, we will determine mechanisms of apoptotic cell death that is associated with replicative senescence of endothelial cells, and determine molecular mechanisms responsible for age-associated polyploidization of this cell type. It is also planned to determine the influence of oxidative stress on the senescence phenotype of human endothelial cells. To identify candidate genes that might be involved in age-associated apoptosis and/or polyploidization, RNA expression profiling of young vs. senescent cells was performed in a pilot experiment, addressing a total of 8300 human genes. This experiment revealed a number of genes that appear to be differentially regulated as endothelial cells enter senescence, and we propose to validate these results by independent methods. The expression of genes that are upregulated with endothelial cell senescence in vitro will also be studied in vascular biopsies from human donors of different age. Genes that are identified by this screen will be tested for their ability to induce features of the senescent phenotype (apoptosis, polyploidization, growth arrest) when ectopically expressed in young endothelial cells. A first set of experiments will be performed with the genes coding for DRAL and IGFBP-3, two known inducers of apoptosis that were found upregulated in senescent endothelial cells. The main goal of the project is to get insight into molecular mechanisms of vascular ageing in vitro and provide the basis for a systematic comparison of the in vitro findings with in vivo ageing phenotypes.