Aging results in endothelial cell telomere uncapping that induces senescence, arterial stiffening, and reduced nitric oxide bioavailability

Abstract

Advancing age results in arterial dysfunction that contributes to the development of cardiovascular disease. One plausible cause of arterial dysfunction in advanced age is elevated inflammation and oxidative stress derived from senescent cells, as senescent cells accumulate with age and display a pro-inflammatory, pro-oxidative phenotype. Telomeres are repeat DNA sequences that cap chromosome ends, shorten with each cell division, and are highly susceptible to oxidative damage. Importantly, when critically short or damaged, telomeres become uncapped, leading to cellular senescence. Here, we test the hypothesis that endothelial cell (EC) telomere uncapping occurs with advancing age, resulting in senescence-induced arterial dysfunction. To evaluate telomere uncapping, we examined colocalization of a DNA damage marker, 53BP1, and telomeres, using immunofluorescence-in situ hybridization in human lung ECs (HLECs). Indeed, HLECs from older donors (67±1 yr, N=2, 78 cells analyzed) had ~4.5 fold more cells containing ≥1 uncapped compared to HLECs from young sex matched donors (29±1 yr, N=2, 64 cells analyzed, p<0.01, Fig. 1). To investigate if EC telomere uncapping drives senescence and arterial dysfunction, we generated an EC-specific knockout mouse model of TRF2 (EC TRF2 KO), a telomeric binding protein that mediates telomere capping. We compared young (3.3 ± 0.2 mo) EC TRF2 KO mice to wild type, littermate controls (WT). Mouse lung ECs (MLECs) isolated from EC TRF2 KO mice had ~3 fold less TRF2 mRNA expression (N=6-7, p<0.001) compared to WT controls, resulting in increased telomere uncapping (N=5, p<0.001) and senescence, as demonstrated by a ~4.3 fold increase in the percentage of senescence associated β-galactosidase positive cells (N=3, p<0.01) and ~3 fold higher mRNA expression of the senescence marker, p21, in carotid artery ECs (N=3-8, p<0.001). Next, we assessed pulse wave velocity (PWV), a measure of large artery stiffness. PWV was increased by ~27% in EC TRF2 KO mice vs. WT controls (N=6-13, p<0.001, Fig. 2). Finally, we assessed endothelium-dependent dilation (EDD) to insulin in mesenteric arterioles and found that EC TRF2 KO mice displayed a ~62% reduction in EDD to insulin compared to WT controls (N=12-14, p<0.001, Fig. 3). This was nitric oxide (NO)-dependent, as there were no differences in insulin EDD when eNOS was inhibited with LNAME (N=12, p>0.05, Fig. 3). Endothelium-independent dilation to sodium nitroprusside was not different between EC TRF2 KO and WT mice (N=12-16, p>0.05). In summary, advancing age results in EC telomere uncapping in humans. In mice, genetically induced telomere uncapping in ECs results in senescence, increases arterial stiffness and attenuates endothelial-mediated vasodilation via reduced NO bioavailability.