Results

Prolonged passage of ECs leads to the induction of acidic β-Gal as a marker protein for senescence with maximal effects observed at passage 14 (Figures 1A and 1B). Light microscopy confirmed the typical features of replicative senescence including an increased cell size and cytoplasmic granularity (Figure 1B). Moreover, ultrastructural analysis revealed convoluted nuclei and vacuolization of cytoplasm (Figure 1 online, available in an online-only data supplement at http://www. circresaha. org). At passage 14, senescent ECs still express the EC marker protein von Willebrand factor (Figure 1C), whereas increasing passages lead to a loss of von Willebrand factor expression indicating a dedifferentiation of ECs (data not shown). Control experiments confirmed that no increase in apoptosis occurred during the observation period (data not shown).