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Phenotyping replicative senescence of human endothelial cells cultured from atherosclerotic patients
Cellular senescence is characterised by the limited ability of primary cultured cells to divide. It is associated with specific changes in cell morphology and gene expression and lead to reduced endothelial cell (EC) function (Wagner et al., 2001). It normally occurs in vivo during the ageing process and cardiovascular risks factors could lead to premature senescence (Minamino et al., 2002). To further characterise this phenotype, we induced replicative senescence in cultured internal mammary artery (hIMA) EC isolated from atherosclerotic patients. ECs were isolated from explants of segments of hIMA (n=50, 35 males and 15 females, from 40 to 80 y.o) from patients undergoing coronary artery bypass. Cells were serially passaged and maintained in culture until replicative senescence. Cytochemical detection of a senescence-associated β-Galactosidase (X-Gal) at pH 6 was used as a marker of senescence (Dimri et al., 1995). X-Gal positive cells (%) were counted at each passage. Telomere shortening, a consequence of cell division, was quantified by Southern blot at the initial passage (Zhang et al., 2000). Cell proliferation was estimated by cumulative PDL through the culture and by 3H-thymidine incorporation. Cell viability was assessed by trypan blue exclusion. Nuclear expression of p53 and the level of lipid peroxydation (HNE) were observed by immunofluorescence by confocal imaging. The time course of senescence was exponential (P<0.05) and specific to each patient tested. From 55 ± 4 to 126 ± 7 days of culture were needed to reach 50% of X-Gal positive cells, suggesting strong heterogeneity in the development of senescence in ECs. Initial telomere length ranged from 6.6 to 11.6 kbp. Despite individual heterogeneity, the shorter the initial telomere length, the faster senescence appeared subsequently (P=0.03). A loss of replicative potential (∑PDL) was correlated with senescence (P=0.01). Similarly, in senescent EC, DNA synthesis was abolished (174 ± 72 dpm versus 18590±849 dpm, senescent versus non senescent ECs, P<0.01). Senescence was associated with a significant decrease in cell viability (P<0.0001). Nuclear expression of p53 and expression of HNE were positively correlated with the level of X-Gal (P<0.05). Neither senescence, nor cell growth or telomere length were dependent on the age of the donor (P>0.05). In contrast, an increased duration of exposure to risk factors for cardiovascular disease (years) was associated with an accelerated appearance of senescence (P=0.015). In conclusion, long exposure to cardiovascular risks factors led to premature replicative senescence of EC, characterised by a loss in replicative potential, short telomere and senescence markers such as nuclear expression of p53 and high levels of HNE, a consequence of oxidative stress-induced damage.
Dimri GP, et al. (1995). Proc. Natl. Acad. Sci.USA. 92, 9363-9367. |
