Nitric Oxide Activates Telomerase and Delays Endothelial Cell Senescence

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).

Senescence is believed to be triggered by a critical shortening of telomere length. EC senescence correlated with a shortening of telomere length (Figure 1D). Because telomerase antagonizes telomere loss, we analyzed telomerase activity in ECs. HUVECs revealed a significant telomerase activity during early passages, which decreased with increasing passages (Figure 1A). Importantly, the reduction of telomerase activity clearly preceded the onset of EC senescence (Figure 1A).

To examine the effects of NO on the onset of EC senescence, HUVECs were incubated with the NO donor S-nitrosopenicillamine (SNAP), which was added every 48 hours. Figure 2A illustrates that the addition of SNAP significantly reduced the number of senescent β-Gal–positive cells with maximal effect after 30 population doublings (53±7% reduction, P<0.05). To exclude a potential influence of NO on cell-cycle progression, the data are expressed as a function of population doublings. For comparison, the data plotted against the passage numbers are shown in the online data supplement (Figure 2 online, available in an online-only data supplement at http://www.circresaha.org). Importantly, inhibition of EC senescence was accompanied by a significant delay in the inactivation of telomerase activity and the shortening of telomere length during continuous passages of the cells (Figures 2B and 2C; data not shown). Moreover, we analyzed the effect of short-term exposure to different NO donors for 18 hours. In both HUVECs (fourth passage) starved for 12 hours in 1% BSA and ECs at passage 11 or 13 in complete medium, incubation with the NO donors was associated with a significant increase in telomerase activity (Figure 2D; data not shown). Furthermore, inhibition of endogenous NO synthesis by N^G-mono-methyl-l-arginine induced a 2-fold increase in EC senescence and reduced telomerase activity (Figure 2B).

The results of the present study suggest a link between reduced telomerase activity and senescence of ECs in culture. A reduction in telomerase activity invariably preceded EC senescence, suggesting that downregulation of telomerase activity may be involved in the progression of senescence. These data are in line with findings showing that stable overexpression of telomerase prevents EC senescence.¹¹ The function of endogenous telomerase in somatic cells is not clear. Telomerase activity is thought to be repressed in normal somatic cells, whereas it is upregulated in embryogenesis and neoplasia.⁶ Our present data demonstrate that telomerase is active in HUVECs and in iliac and microvascular ECs (data not shown), thus complementing previous studies demonstrating low telomerase activity in several proliferating somatic cells including primary ECs.^{12 13}

Importantly, exogenous NO delays EC senescence in culture, suggesting that shortening of telomeres is not strictly a function of the number of cellular divisions but can be modulated. Indeed, NO interferes with telomerase activity thereby inhibiting telomere shortening. The mechanism by which NO stimulates telomerase activity remains to be determined. NO may react with tissue-derived oxygen radicals, thereby reducing oxidative stress, which has been shown to accelerate EC senescence.⁷ Alternatively, NO may directly upregulate telomerase activity via transcriptional and/or posttranscriptional mechanisms.

Regardless of the molecular mechanisms involved in the modulation of telomerase activity, the demonstration that NO affects telomerase activity and delays EC senescence establishes a novel endothelial protective function of NO. The integrity of EC function including the capability of ECs to proliferate and migrate is essential for angiogenesis.¹⁴ Therefore, EC senescence and the consequent reduction of their proliferative ability may contribute to compromised angiogenesis associated with advanced age.³ Additionally, senescent ECs express adhesion molecules, which may promote neutrophil adhesion and inflammation.⁷ Thus, EC senescence may also enhance the chronic inflammatory process, which importantly contributes to the progression of atherosclerotic disease.¹⁵

On the other hand, endothelial NO is essential for angiogenesis and protects against atherosclerosis.^{16 17} However, the bioavailability of endothelial-derived NO is impaired with aging.^{8 18} Therefore, it is tempting to speculate that the reduction of NO may accelerate EC senescence, thereby impairing EC function and contributing to impaired angiogenesis and atherosclerotic disease progression in advanced age.

This work was supported by the Deutsche Forschungsgemeinschaft (SFB553) and by a grant for young research scientists to K.B. by the University of Frankfurt. We would like to thank Alexandra Bittner for expert technical assistance and Prof Wolfgang Schlote (Edinger Institut, Frankfurt) and Barbara Lafferton for help with the electron microscopy.