Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells

Abstract

Replicative senescence and oxidative stress have been implicated in ageing, endothelial dysfunction and atherosclerosis. Replicative senescence is determined primarily by telomere integrity. In endothelial cells the glutathione redox-cycle plays a predominant role in the detoxification of peroxides. The aim of this study was to elucidate the role of the glutathione-dependent antioxidant system on the replicative capacity and telomere dynamics of cultured endothelial cells. Human umbilical vein endothelial cells were serially passaged while exposed to regular treatment with 0.1 μM tert-butyl hydroperoxide, a substrate of glutathione peroxidase, or 10 μM L-buthionine-[S,R]-sulphoximine, an inhibitor of glutathione synthesis. Both treatments induced intracellular oxidative stress but had no cytotoxic or cytostatic effects. Nonetheless, treated cultures entered senescence prematurely (30 versus 46 population doublings), as determined by senescence-associated β-galactosidase staining and a sharp decrease in cell density at confluence. In cultures subjected to oxidative stress terminal restriction fragment (TRF) analysis demonstrated faster telomere shortening (110 versus 55 bp/population doubling) and the appearance of distinct, long TRFs after more than 15-20 population doublings. Fluorescence in situ hybridisation analysis of metaphase spreads confirmed the presence of increased telomere length heterogeneity, and ruled out telomeric end-to-end fusions as the source of the long TRFs. The latter was also confirmed by Bal31 digestion of genomic DNA. Similarly, upregulation of telomerase could not account for the appearance of long TRFs, as oxidative stress induced a rapid and sustained decrease in this activity. These findings demonstrate a key role for glutathione-dependent redox homeostasis in the preservation of telomere function in endothelial cells and suggest that loss of telomere integrity is a major trigger for the onset of premature senescence under mild chronic oxidative stress.

Phenyl 2‐pyridyl ketoxime induces cellular senescence‐like alterations via nitric oxide production in human diploid fibroblasts

“…The free‐radical theory of aging posits that degenerative senescence is largely the result of the cumulative effect of ROS (Haendeler et al ., 2003; Kurz et al ., 2004), and the present study demonstrates that PPKO induced ROS generation in 30 min (Fig. 3) and that the antioxidants NAC and TEMPO abrogated PPKO‐induced SA‐β‐gal staining in young HDFs (Fig.…”

“…The involvement of ROS in degenerative senescence has been proposed previously (Beckman & Ames, 1998; Haendeler et al ., 2003; Kurz et al ., 2004). To investigate the molecular mechanism for PPKO‐induced cellular senescence, we examined whether PPKO induces the accumulation of ROS in young HDFs.…”

“…The molecular changes associated with senescence‐like growth arrest depend on the chemicals used. According to the free‐radical theory of aging (Beckman & Ames, 1998; Haendeler et al ., 2003; Kurz et al ., 2004), reactive oxygen species (ROS) are potential candidates for senescence induction, and ROS‐induced oxidative stress may promote cellular senescence. Because ROS are highly reactive molecules, they can damage many cellular components, and thus, they are considered a probable cause of cellular senescence and degenerative aging.…”

Rap1 induces cytokine production in pro-inflammatory macrophages through NFκB signaling and is highly expressed in human atherosclerotic lesions

“…Telomere attrition is accelerated as the cell ages or faces stress. [42][43][44] Indeed, in senescent PAECs, the amount of Rap1 in the cytosol is elevated compared with young PAECs. 37 Thus, it is tempting to speculate that as telomere length is reduced, the ratio between telomere-bound and "free" Rap1 (available for the cytoplasmic activation of the NFkB pathway) is affected.…”

Telomere-Driven Karyotypic Complexity Concurs with p16INK4a Inactivation in TP53-Competent Immortal Endothelial Cells

“…Furthermore, quantitation by real-time PCR showed that telomerase activity in BMhTERT clones was comparable with a number of tumor cell lines (additional data not shown). One likely explanation for shortening of mean telomere length in BMhTERTs is that oxidative stress overrode the lengthening capacity of telomerase (49). Mounting evidence indicates that the length of a subset of the shortest telomeres, rather than the average length of the telomeres, predicts proliferative arrest (50).…”