Identification of cultivation-independent markers of human endothelial cell senescence in vitro

Human aging processes are regulated by many divergent pathways and on many levels. Thus, to understand such a complex system and define conserved mechanisms of aging, the use of cell culture-based models is a widespread practice. An often stated advantage of in vitro aging of primary cells is the high reproducibility compared to the much more intricate aging of organisms. However, the aging process of cultured cells is, like aging of organisms, not only defined by genetic but also by environmental factors, making it difficult to distinguish between cell culture condition-induced artefacts and true aspects of aging. Therefore we investigated aging of HUVEC (human umbilical vascular endothelial cells), a well-known and widely used model system for in vitro aging, with different, already well-established cell culture protocols. Culturing conditions had indeed a strong impact on cell proliferation, the replicative lifespan and apoptosis rates. However, despite these significant differences, we found also various robust markers that define senescent HUVEC: morphological changes, increased senescence-associated beta-galactosidase staining, cell cycle arrest in the G1 phase, lowered mitochondrial membrane potential and increased oxidatively modified proteins were displayed independent of cell culture protocols and could therefore be considered also as markers for in vivo aging.