Stress-induced senescence exaggerates postinjury neointimal formation in the old vasculature.

This study aims to demonstrate the role of stress-induced senescence in aged-related neointimal formation. We demonstrated that aging increases senescence-associated beta-galactosidase activity (SA-beta-Gal) after vascular injury and the subsequent neointimal formation (neointima-to-media ratio: 0.8 +/- 0.2 vs. 0.54 +/- 0.15) in rats. We found that senescent cells (SA-beta-Gal(+) p21(+)) were scattered throughout the media and adventitia of the vascular wall at day 7 after injury and reached their maximum number at day 14. However, senescent cells only persisted in the injured arteries of aged animals until day 30. No senescent cells were observed in the noninjured, contralateral artery. Interestingly, vascular senescent cells accumulated genomic 8-oxo-7,8-dihydrodeoxyguanine, indicating that these cells were under intense oxidative stress. To demonstrate whether senescence worsens intimal hyperplasia after injury, we seeded matrigel-embedded senescent and nonsenescent vascular smooth muscle cells around injured vessels. The neointima was thicker in arteries treated with senescent cells with respect to those that received normal cells (neointima-to-media ratio: 0.41 +/- 0.105 vs. 0.26 +/- 0.04). In conclusion, these results demonstrate that vascular senescence is not only a consequence of postinjury oxidative stress but is also a worsening factor for neointimal development in the aging vasculature.

Interactions of TRF2 with model telomeric ends.

Telomeres are DNA-protein complexes at the ends of eukaryotic chromosomes, the integrity of which is essential for chromosome stability. An important telomere binding protein, TTAGGG repeat factor 2 (TRF2), is thought to protect telomere ends by remodeling them into T-loops. We show that TRF2 specifically interacts with telomeric ss/ds DNA junctions and binding is sensitive to the sequence of the 3', guanine-strand (G-strand) overhang and double-stranded DNA sequence at the junction. Association of TRF2 with DNA junctions hinders cleavage by exonuclease T. TRF2 interactions with the G-strand overhang do not involve the TRF2 DNA binding domain or the linker region. However, mobility shifts and atomic force microscopy show that the previously uncharacterized linker region is involved in DNA-specific, TRF2 oligomerization. We suggest that T-loop formation at telomere ends involves TRF2 binding to the G-strand overhang and oligomerization through both the known TRFH domain and the linker region.

DNA structure-dependent recruitment of telomeric proteins to single-stranded/double-stranded DNA junctions.

Telomeres protect chromosome ends by assembling unique protein-DNA complexes. TRF2 is a telomere binding protein that is involved in protecting the G-strand overhang, a 3', guanine-rich, overhang at the telomere terminus. TRF2 may protect the G-strand overhang by recognizing some organizational aspect of the telomeric single-stranded/double-stranded (ss/ds) DNA junction. This work demonstrates that TRF2, purified or in crude extracts, recognizes telomeric ss/ds DNA junctions containing wild type telomeric sequence in the ds region and a G-strand overhang with at least one telomeric repeat. Telomeric complexes containing TRF2 and pot1 assemble less efficiently when the G-strand overhang is in the form of an intramolecular G-quadruplex. However, recruitment of the DNA repair proteins, WRN, Mre11, and Ku86, is not inhibited by a G-quadruplex. This suggests that an intramolecular G-quadruplex has the potential to disrupt certain telomeric assemblies, but efficient recruitment of appropriate DNA repair proteins provides the means to overcome this obstacle.