Nucleotide excision DNA repair is associated with age-related vascular dysfunction

Abstract

Background: Vascular dysfunction in atherosclerosis and diabetes mellitus, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction.

Methods and results: In mice with genomic instability resulting from the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1(d/-) and Xpd(TTD) mice), we explored age-dependent vascular function compared with that in wild-type mice. Ercc1(d/-) mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness, and elevated blood pressure at a very young age. The vasodilator dysfunction was due to decreased endothelial nitric oxide synthase levels and impaired smooth muscle cell function, which involved phosphodiesterase activity. Similar to Ercc1(d/-) mice, age-related endothelium-dependent vasodilator dysfunction in Xpd(TTD) animals was increased. To investigate the implications for human vascular disease, we explored associations between single-nucleotide polymorphisms of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium and found a significant association of a single-nucleotide polymorphism (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity.

Conclusions: Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans but with an accelerated progression compared with wild-type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness, which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease.

Figure 1

Senescence in Ercc1^d/− mice vascular tissue. The thoracic aorta wall of Ercc1^d/− and WT mice stained with SA-β-gal staining (A). Quantification of SA-β-gal positive cells in the lamina media of thoracic aorta (B). Aortic RNA levels of senescence markers p21 (C) and p53 (D). Percentage of SA-β-gal positive cells after prolonged culture of isolated lung endothelial cells in Ercc1^d/− compared to WT mice (E, F). †= p<0.05 (Mann-Whitney U test).

Figure 2

Measures of central and peripheral hemodynamics in 8 week old Ercc1^d/− and WT animals. Functional Differences between skin reperfusion after 2 minutes of occlusion between WT (A) and Ercc1^d/− (B), average maximum response (C), as well as area under the curve (D). Differences in systolic, mean, diastolic blood pressure (E, F, G) and pulse pressure (H) between 8 week old Ercc1^d/− and their WT. *= p<0.05 (t-test).

Figure 3

Ex-vivo vascular function in progeria models and aging models. Vasodilatations to (A) acetylcholine and (B) sodium nitroprusside in U46619-precontracted isolated aortic rings of 8 and 16 week old Ercc1^d/− vs. WT, measured in organ bath setups. To express the contribution of the endothelium, acetylcholine responses were expressed as % of the response to sodium nitroprusside (C). Dilator responses to acetylcholine (D) and sodium nitroprusside (E) of WT mouse isolated aortic rings precontracted with 3×10⁻⁸ mol/L U46619, measured at 16, 26 and 52 weeks of age. Vasodilatations to (F) acetylcholine and (G) sodium nitroprusside in U46619-precontracted isolated aortic rings of 26 and 52 week old Xpd^TTD vs. WT. #,*= p<0.05 (t-test on log transformed values, general linear model repeated – measures).

Figure 4

eNOS in aorta and myocardium of Ercc1^d/− mice. Protein levels of eNOS in 16 week old Ercc1^d/− animals and their WT littermates, in (A) aorta, and (B, C) cardiac ventricles. Phosphorylation of Ser1177-eNOS in lungs of 16 week old Ercc1^d/− animals, and their WT littermates on baseline (D), and upon stimulation with 10⁻⁵ mol/L Ach (E). The difference in phosphorylation before and after stimulation (F). # = p<0.05 (t-test on log transformed values). Lanes were run on the same gel but are noncontiguous.

Figure 5

Effects of ROS scavenging and prevention of eNOS uncoupling on ex-vivo vascular function. Effects of acute BH4 supplementation on responses to acetylcholine (A) and sodium nitroprusside (B) in aortic tissue of 16 week old Ercc1^d/− and WT mice. The acetylcholine responses after correction for individual responses to sodium nitroprusside (E). The effect of acute NAC supplementation on responses of Ercc1^d/− to acetylcholine (C) and sodium nitroprusside (D). Acetylcholine responses expressed as % of the sodium nitroprusside response (F). †= p<0.05 (Mann-Whitney U test).

Figure 6

Effect of PDE inhibition vinpocetine on endothelium-independent vasodilatation. Ercc1^d/− mice show reduced vasodilatory response to sodium nitroprusside 10⁻⁴ mol/L, when compared with WT littermates. PDE blockade abolishes this difference. * = p<0.05 (t-test).

Figure 7

Mechanical properties of conductive vessels in Ercc1^d/−. Relationship of the diameter of carotid artery to the internal pressure (A). Reduced strain of the tissue of Ercc1^d/−mice (B) is accompanied by no difference in stress on the wall (C). * = p<0.05 (general linear model repeated – measures)