Vascular Ageing Features Caused by Selective DNA Damage in Smooth Muscle Cell

Abstract

Persistently unrepaired DNA damage has been identified as a causative factor for vascular ageing. We have previously shown that a defect in the function or expression of the DNA repair endonuclease ERCC1 (excision repair cross complement 1) in mice leads to accelerated, nonatherosclerotic ageing of the vascular system from as early as 8 weeks after birth. Removal of ERCC1 from endothelial alone partly explains this ageing, as shown in endothelial-specific Ercc1 knockout mice. In this study, we determined vascular ageing due to DNA damage in vascular smooth muscle cells, as achieved by smooth muscle-selective genetic removal of ERCC1 DNA repair in mice (SMC-KO: SM22αCre+ Ercc1fl/-). Vascular ageing features in SMC-KO and their wild-type littermates (WT: SM22αCre+ Ercc1fl/+) were examined at the age of 14 weeks and 25 weeks. Both SMC-KO and WT mice were normotensive. Compared to WT, SMC-KO showed a reduced heart rate, fractional shortening, and cardiac output. SMC-KO showed progressive features of nonatherosclerotic vascular ageing as they aged from 14 to 25 weeks. Decreased subcutaneous microvascular dilatation and increased carotid artery stiffness were observed. Vasodilator responses measured in aortic rings in organ baths showed decreased endothelium-dependent and endothelium-independent responses, mostly due to decreased NO-cGMP signaling. NADPH oxidase 2 and phosphodiesterase 1 inhibition improved dilations. SMC-KO mice showed elevated levels of various cytokines that indicate a balance shift in pro- and anti-inflammatory pathways. In conclusion, SMC-KO mice showed a progressive vascular ageing phenotype in resistant and conduit arteries that is associated with cardiac remodeling and contractile dysfunction. The changes induced by DNA damage might be limited to VSMC but eventually affect EC-mediated responses. The fact that NADPH oxidase 2 as wells as phosphodiesterase 1 inhibition restores vasodilation suggests that both decreased NO bioavailability and cGMP degradation play a role in local vascular smooth muscle cell ageing induced by DNA damage.

Figure 1

Systolic BP (a) and diastolic BP (b) and functional differences between skin reperfusion after 2 minutes of occlusion in the calculated area under the curve (c) and average maximum response (d) between WT and SMC-KO at ET and LT. The number in each column represents the number of animals in the corresponding group. Statistical differences were analyzed two-way ANOVA followed by Bonferroni's post hoc test (^∗p < 0.05).

Figure 2

Strain difference at ET (a) and LT (b) and stress differences at ET (c) and LT (d) of the carotid arteries of SMC-KO vs. WT. Statistical difference was analyzed by general linear model repeated measures (^∗p < 0.05).

Figure 3

Cardiac function comparison between WT and SMC-KO at ET and LT for heart rate (a), fractional shortening (b), cardiac output (c), LV wall thickness (d), LV mass (e), relative LV weight to body weight at LT (f), LV free wall collagen content (g), and LT free wall cardiomyocyte size (h). The number in each column represents the number of animals in the corresponding group. Statistical differences were analyzed by two-way ANOVA followed by Bonferroni's post hoc test for (a–e) and two-tailed t-test for (f–h) (^∗p < 0.05).

Figure 4

Vasorelaxation in aortic rings of SMC-KO and WT mice for both time-points in response to ACh (10⁻⁹ to 10⁻⁵ mol/L) (a). The contribution of NO-cGMP and EDH pathway in WT ET (b), WT LT (c), SMC-KO ET (d), and SMC-KO LT (e). Vasorelaxation in aortic rings of SMC-KO and WT mice for both time-points in response to SNP (10⁻¹¹ to 10⁻⁴ mol/L) (f), SNP (0.1 mmol/L) after ACh CRC (g), and ACh (10⁻⁹ to 10⁻⁵ mol/L) corrected to SNP (0.1 mmol/L) (h). The number in each column represents the number of animals in the corresponding group. Statistical differences were analyzed by general linear model repeated measures for (a–f) and (h) and two-way ANOVA followed by Bonferroni's post hoc test for (g) (^∗p < 0.05).

Figure 5

Vasorelaxation response to ACh (10⁻⁹ to 10⁻⁵ mol/L) in aortic rings either without inhibitor or with apocynin or GSK279503 preincubation in WT LT (a), SMC-KO LT (b), ACh (10 μmol/L) Emax (c), and SNP (0.1 mmol/L) Emax (d) at LT. Vasorelaxation response to SNP (10⁻¹¹ to 10⁻⁴ mol/L) in aortic rings either without inhibitor or with sildenafil or lenrispodun preincubation at ET (e) and LT (f). The number in each column represents the number of animals in the corresponding group. Statistical differences were analyzed by general linear model repeated measures for (a), (b), (e), and (f) and one-way ANOVA followed by Bonferroni's post hoc test for (c) and (d) (^∗p < 0.05).

Figure 6

MSD analysis of plasma samples in LT WT and SMC-KO for IL-6 (a), IFN-γ (b), and IL-10 (c). qPCR analysis in WT and SMC-KO for Il-6 in LV (d), Il-6 in kidney (e), and Pde1a in the abdominal aorta (f). The number in each column represents the number of animals in the corresponding group. Statistical differences were analyzed by two-tailed t-test (^∗p < 0.05).