Evidence against the involvement of nitric oxide in the modulation of telomerase activity or replicative capacity of human endothelial cells.

Telomerase, a reverse transcriptase involved in the maintenance of telomere function and cellular replicative capacity, is thought to be regulated by nitric oxide (NO). Here, we have used pharmacological tools and RNA interference to re-assess the role of NO in the regulation of telomerase and senescence of human umbilical vein endothelial cells. Acute or chronic treatment of these cells with the NO donors diethylenetriamine/NO (DETA-NO) or S-nitroso-N-acetylpenicillamine (SNAP) at concentrations which generated NO in the 1-300 nM range did not modulate telomerase activity. Similarly these agents did not affect cellular replicative capacity during long-term sub-cultivation. The NO synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (1 mM) reduced basal levels of c-GMP by 50% but had no effect on telomerase activity or replicative capacity. Withdrawal of ascorbic acid increased the intracellular pro-oxidant capacity, reduced telomerase activity and increased the accumulation of senescent cells upon serial passage in culture. However, this shift to a more oxidative redox state did not unmask the putative capacity of NO to modulate telomerase or senescence. Infection of cells with a lentiviral vector expressing a small hairpin RNA targeted against endothelial NOS inhibited endogenous NO production completely but failed to affect the decrease of telomerase activity or the accumulation of senescent cells observed with passage in culture. Our findings suggest that physiological concentrations of NO do not modulate telomerase levels or replicative capacity of endothelial cells, regardless of their cellular oxidative status.

Fibroblast growth factor-2, but not vascular endothelial growth factor, upregulates telomerase activity in human endothelial cells.

OBJECTIVE: Telomerase plays a major role in the control of replicative capacity, a critical property for successful angiogenesis and maintenance of endothelial integrity. In this study, we examined the relationship between telomerase activity and endothelial cell proliferation as well as the regulation of this enzyme by fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF). METHODS AND RESULTS: Telomerase was repressed in endothelial cells freshly derived from intact endothelium, whereas activity was present during logarithmic growth in culture. In cultured human umbilical vein endothelial cells (HUVECs), mRNA levels of hTERT-the catalytic subunit of telomerase-and enzyme activity decreased reversibly on induction of quiescence. Treatment of quiescent HUVECs with FGF-2 restored telomerase activity in a time- and dose-dependent manner, whereas VEGF had no such effect, although both factors induced comparable mitogenic responses. FGF-2, but not VEGF, upregulated the mRNA levels for hTERT and for the hTERT gene transactivation factor Sp1. Serial passage in the presence of individual growth factors accelerated the accumulation of senescent cells in VEGF-treated cultures compared with cultures treated with FGF-2. CONCLUSIONS: FGF-2, but not VEGF, restores telomerase activity and maintains the replicative capacity of endothelial cells.