Proteomics and aging : studying the influence of aging on endothelial cells and human plasma

Abstract

In general, human aging is considered one of the most complex and less-well understood process in biology. In this thesis the possibilities of proteomics technology in the field of aging were explored. The complexity of the aging process was supposed to accompanied by relatively subtle proteome variations, which made the application of the proteomics technology a real challenge. The use of two-dimensional In Gel Electrophoresis (2D-DIGE) technology enabled a reduction of the analytical variation and allowed the use of biological replicates, which increased the significance of annotated biological processes inducing the observed proteome differences. A part of the work described in this thesis was to analyse the influence of cellular aging (i.e replicative senescence) on protein expression in an endothelial cell model system, called HUVECS. Cellular aging of endothelial cells has been detected in vivo in atherosclerotic lesions and is therefore considered to be important in the development of the age-related disease atherosclerosis. This study showed that endothelial replicative senescence might be accompanied with increased cellular stress, protein biosynthesis and reduction in DNA repair and maintenance. Also remarkable differences were observed concerning the cellular architecture, which could be confirmed with electron microscopy. Next to cellular aging we were also interested in age-related changes on the organismal level and for that purpose pooled human plasma samples from three different healthy age groups were subjected also to 2D-DIGE. Functional analysis revealed that during healthy aging, a pro-inflammatory as well as an increased pro-coagulant state becomes evident. The most remarkable observation was the high upregulation of complement C3 splicing products, which suggests that the immune response becomes enhanced in the healthy elderly group. Apparently, a pro-inflammatory as well as pro-coagulant state might be necessary for healthy aging. In a second explorative 2D-DIGE study, in which we have compared plasma samples from healthy and unhealthy individuals at the age of >85, these states appeared to be reduced in the unhealthy population. In conclusion, the application of 2D DIGE resulted in the discovery of changes in the expression of several proteins during cellular and human aging.