Mitochondrial oxidative stress in aortic stiffening with age: the role of smooth muscle cell function

Abstract

Objective: Age-related aortic stiffness is an independent risk factor for cardiovascular diseases. Although oxidative stress is implicated in aortic stiffness, the underlying molecular mechanisms remain unelucidated. Here, we examined the source of oxidative stress in aging and its effect on smooth muscle cell (SMC) function and aortic compliance using mutant mouse models.

Methods and results: Pulse wave velocity, determined using Doppler, increased with age in superoxide dismutase 2 (SOD2)+/- but not in wild-type, p47phox-/- and SOD1+/- mice. Echocardiography showed impaired cardiac function in these mice. Increased collagen I expression, impaired elastic lamellae integrity, and increased medial SMC apoptosis were observed in the aortic wall of aged SOD2+/- versus wild-type (16-month-old) mice. Aortic SMCs from aged SOD2+/- mice showed increased collagen I and decreased elastin expression, increased matrix metalloproteinase-2 expression and activity, and increased sensitivity to staurosporine-induced apoptosis versus aged wild-type and young (4-month-old) SOD2+/- mice. Smooth muscle α-actin levels were increased with age in SOD2+/- versus wild-type SMCs. Aged SOD2+/- SMCs had attenuated insulin-like growth factor-1-induced Akt and Forkhead box O3a phosphorylation and prolonged tumor necrosis factor-α-induced Jun N-terminal kinase 1 activation. Aged SOD2+/- SMCs had increased mitochondrial superoxide but decreased hydrogen peroxide levels. Finally, dominant-negative Forkhead box O3a overexpression attenuated staurosporine-induced apoptosis in aged SOD2+/- SMCs.

Conclusion: Mitochondrial oxidative stress over a lifetime causes aortic stiffening, in part by inducing vascular wall remodeling, intrinsic changes in SMC stiffness, and aortic SMC apoptosis.

Figure 1

Age-dependent changes in arterial compliance and cardiac function in wild-type and SOD2^+/− mice fed normal chow (ND) or Western diet (WD). Aortic pulse wave velocity (PWV, A), ejection fraction (EF, B), left ventricle end diastolic volume (LVEDV, C), left ventricle posterior wall thickness (LVPW, D) and calculated left ventricle mass (LV mass, E) are presented as mean±SE (n=10).

Figure 2

SOD2 deficiency increases collagen I synthesis and disrupts elastic laminae in aortas of aged mice, increases collagen and decreases elastin levels in aged aortic SMC and enhances MMP-2 activity in SMC of young and aged mice. A, Representative sections from fresh frozen aortas were stained for collagen I and elastin. B, Aortic SMC lysates were analyzed by Western blotting with anti-collagen I, anti-elastin and anti-GAPDH antibodies. Densitometric analysis of collagen I and elastin levels was shown in the lower panel (mean±SE, n=3). C, A representative gelatin zymogram showing MMP-2 activity in aortic SMC lysates. Densitometric analysis of MMP-2 activity was shown in the lower panel (mean±SE, n=3).

Figure 3

SOD2 deficiency enhances medial SMC apoptosis in the aorta of aged mice and sensitizes aortic SMC from aged mice to staurosporine-induced apoptosis. A, Dual immunofluorescent staining of cleaved caspase-3 (red) and α-smooth muscle actin (green) demonstrated colocalization (yellow) in SMC. Nuclei were counterstained with DAPI (blue). B, Lysates from aortic SMC treated or without 1.0 μmol/L staurosporine for 6 h were analyzed by Western blotting with anti-caspase-3, anti-PARP and anti-β-actin antibodies. Data shown represent an experiment that was repeated at least twice with similar results. C, Aged SMC treated with staurosporine (0.1 μmol/L) for 12 h were analyzed by fluorescent (green) TUNEL staining. Nuclei were stained with DAPI (blue). Quantitation of apoptotic cells presented as % TUNEL-positive cells in each field of view (mean ± SE, n=3).

Figure 4

SOD2 deficiency decreases IGF-1-induced Akt and FoxO3a phosphorylation and increases TNF-α-induced JNK1 phosphorylation in aortic SMC from aged mice and α-actin levels in SMC from young and aged mice. Lysates from growth-arrested and IGF-1 (100 ng/ml) treated aged SMC were analyzed by Western blotting with anti-phosphospecific Akt or Akt (A) or anti-phosphospecific FoxO3a or FoxO3a antibodies (B). Lysates from growth-arrested and TNF-α (100 ng/ml) treated SMC were analyzed by Western blotting with anti-phosphospecific JNK1 or JNK1 antibodies (C). Densitometric analysis of phosphorylated proteins was shown in the lower panel of each figure (mean±SE, n=3). A representative Western blot of SMC lysates probed with anti-α-actin or GAPDH antibodies (D). Densitometric analysis of α-actin levels in the lower panel (mean±SE, n=3).

Figure 5

Mitochondrial superoxide generation is increased whereas extracellular H₂O₂ levels are decreased with age in aortic SMC from SOD2^+/− mice. A, Confocal laser-scanning microscopy showing colocalization of mitochondria-targeting fluorescent probe MitoSOX Red with the mitochondria-selective dye, MitoTracker Green. Yellow fluorescence indicates localization of superoxide in mitochondria. B, H₂O₂ production was measured using Amplex Red fluorescence assay (mean±SE, n=9).

Figure 6

DN-FoxO3a overexpression attenuates increase in cleaved PARP levels induced by staurosporine in aortic SMC from aged SOD2^+/− mice. A, Aged aortic SMC infected with AdGFP or AdDN-FoxO3a were either untreated or treated with 1.0 μmol/L staurosporine for 6 h and cell lysates were analyzed by Western blotting with anti-PARP, anti-HA or anti-β-actin antibodies. Densitometric analysis of β-actin levels in the lower panel (mean±SE, n=3).