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(Circulation Research. 2000;87:540.)
© 2000 American Heart Association, Inc.

Reports

Nitric Oxide Activates Telomerase and Delays Endothelial Cell Senescence

Mariuca Vasa, Kristin Breitschopf, Andreas M. Zeiher, Stefanie Dimmeler

From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Germany.

Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. E-mail Dimmeler{at}em.uni-frankfurt.de

Key Words: nitric oxide • aging • endothelial cells • telomerase

	Introduction

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Endothelial cells (ECs) undergo a limited number of cell divisions, ultimately stop dividing, and enter a state that is designated replicative senescence. Shortening of telomeres is believed to be a molecular clock that triggers senescence. Telomerase, a RNA-directed DNA polymerase, extends telomeres of eukaryotic chromosomes and delays the development of senescence. In this study, we examined telomere length and the activity of telomerase during aging of human ECs in culture and elucidated the effect of nitric oxide (NO). A significant increase in senescent cells as detected by acidic ß-galactosidase expression and a reduction of telomere length were found after 11 passages. Telomerase activity was reduced after the seventh passage, thereby preceding the development of EC senescence. The repeated addition of the NO donor S-nitroso-penicillamine significantly reduced EC senescence and delayed age-dependent inhibition of telomerase activity, whereas inhibition of endogenous NO synthesis had an adverse effect. Taken together, our results demonstrate that telomerase inactivation precedes EC aging. NO prevents age-related downregulation of telomerase activity and delays EC senescence.

The incidence of atherosclerosis increases with age. Aging is associated not only with endothelial dysfunction, a key pathogenic factor in atherosclerotic disease progression,^{1 2} but also impairs angiogenesis,³ suggesting that the process of aging itself affects endothelial cell (EC) function.

On a cellular level, aging leads to an irreversible state of cell cycle arrest known as replicative senescence.⁴ It is generally believed that a relevant factor in regulating cellular life span is the telomere length.⁵ Telomerase, a ribonucleoprotein with reverse transcriptase activity, synthesizes the telomeric repeats at the linear ends of eukaryotic chromosomes.⁵ Telomerase is active in germline cells and cancer cells, whereas it is repressed in most somatic cells, resulting in the progressive telomere shortening with each cell division.⁶

A recent study demonstrated that homocysteine, a proatherosclerotic factor, accelerates the rate of EC senescence in vitro, suggesting an association between EC senescence and atherosclerosis.⁷ Importantly, EC nitric oxide (NO) bioavailability, a pivotal atheroprotective mechanism, decreases as a function of age.⁸ Therefore, we investigated the role of telomerase activity for EC senescence and the potential effects of NO on the age-related changes in telomerase activity.

	Materials and Methods

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Cell Culture
Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Systems/Clonetics and cultivated as previously described.⁹

Acidic ß-Galactosidase (ß-Gal) Staining
Cells were fixed and incubated for 12 hours at 37°C with ß-Gal staining solution.¹⁰

Telomerase Assay
HUVECs were washed in PBS and lysed, and telomerase activity was measured by the Telo TAGGG Telomerase PCR ELISA^PLUS kit (Roche Molecular Biochemicals). Alternatively, the telomerase products were separated on an acrylamide gel (12%) and detected with the Biotin Luminescent Detection kit (Roche Molecular Biochemicals).

Telomere Length Assay
Genomic DNA was isolated, and 4 µg was digested overnight at 37°C with HinfI/RsaI. The resulting DNA fragments were separated onto a 0.7% agarose gel, blotted, and hybridized with radioactively labeled oligonucleotides (5'-CCCTAA-3')₄ at 50°C for 8 hours. The products were detected by phosphoimaging.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Prolonged passage of ECs leads to the induction of acidic ß-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).

View larger version (37K): [in this window] [in a new window]   Figure 1. Senescence, telomere length, and telomerase activity in aging HUVECs. A and B, HUVECs were continuously passaged, and acidic ß-Gal staining and telomerase activity were determined, n=4. B, Representative figure. C, Detection of von Willebrand factor with FITC-linked antibodies. Cells were counterstained with DAPI. Representative result is shown. D, Reduction of telomere length in HUVECs during aging by Southern blot analysis. Representative result is shown (n=3).

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 ß-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).

View larger version (35K): [in this window] [in a new window]   Figure 2. Effect of NO on senescence and telomerase activity. A, SNAP (20 µmol/L) was added every 48 hours starting at the eighth passage, and HUVEC senescence was detected by acidic ß-Gal staining (A). NG-mono-methyl-L-arginine (LNMA, 1 mmol/L) was added every 12 hours starting at the sixth passage. Telomerase activity was detected by ELISA (percentage compared with samples at population doubling 14; *P<0.05 vs control) (B) or by separation of telomerase-synthesized DNA ladders (C). Representative figures are shown (n=4 to 6). D, Short-term effect of SNAP (50 µmol/L), sodium nitroprusside (SNP, 50 µmol/L), or S-nitrosoglutathione (GSNO, 20 µmol/L) on telomerase activity. HUVECs (fourth passage) were starved for 12 hours in medium containing 1% BSA before incubation with the NO donors for 18 hours. Telomerase activity was detected by ELISA (n=4 to 6).

	Discussion

Top Introduction Materials and Methods Results Discussion References

 
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.

	Acknowledgments

 
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.

Received July 20, 2000; revision received August 15, 2000; accepted August 23, 2000.

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