Aging impairs smooth muscle-mediated regulation of aortic stiffness: a defect in shock absorption function?

Abstract

Increased aortic stiffness is an early and independent biomarker of cardiovascular disease. Here we tested the hypothesis that vascular smooth muscle cells (VSMCs) contribute significantly to aortic stiffness and investigated the mechanisms involved. The relative contributions of VSMCs, focal adhesions (FAs), and matrix to stiffness in mouse aorta preparations at optimal length and with confirmed VSMC viability were separated by the use of small-molecule inhibitors and activators. Using biomechanical methods designed for minimal perturbation of cellular function, we directly quantified changes with aging in aortic material stiffness. An alpha adrenoceptor agonist, in the presence of N(G)-nitro-l-arginine methyl ester (l-NAME) to remove interference of endothelial nitric oxide, increases stiffness by 90-200% from baseline in both young and old mice. Interestingly, increases are robustly suppressed by the Src kinase inhibitor PP2 in young but not old mice. Phosphotyrosine screening revealed, with aging, a biochemical signature of markedly impaired agonist-induced FA remodeling previously associated with Src signaling. Protein expression measurement confirmed a decrease in Src expression with aging. Thus we report here an additive model for the in vitro biomechanical components of the mouse aortic wall in which 1) VSMCs are a surprisingly large component of aortic stiffness at physiological lengths and 2) regulation of the VSMC component through FA signaling and hence plasticity is impaired with aging, diminishing the aorta's normal shock absorption function in response to stressors.

Keywords: Src; alpha agonist; focal adhesion; phosphotyrosine; smooth muscle.

Fig. 1.

In vitro steady-state stiffness methods. A: schematic of lever arm for stiffness measurements; steady-state force response measured from stretching the tissue sample (shown here with typical unloaded in vitro geometry) is that which is experienced in the circumferential direction at the center of the axial cross section (arrow). B: representative force trace with sample inset of electronic recordings for high-frequency, low-amplitude (HFLA) oscillatory stretch; sinusoidal length input (ΔL) and force output (ΔF) were used to calculate stiffness at optimal length L. Stiffness E is the ratio of change in stress (Δσ, equivalent to ΔF normalized to cross-sectional area A) to change in strain experienced by tissue (Δε).

Fig. 2.

Physiological optimal length determination. Physiological optimal length, the strain level at which maximal contractile stress (kPa) occurs within physiological ranges of aortic strain, was determined to be 80% in both young and old mice (n = 3 each).

Fig. 3.

Changes in baseline aortic mechanical parameters with aging. A: baseline steady-state force increases with aging. B: wall tension, equivalent to force normalized to axial length, increases with aging. C: stress, equivalent to tension normalized to wall thickness, does not change with aging. D: baseline aortic stiffness via HFLA measurements in the absence of agonist decreases with aging. Values are means ± SE; n = 13 young, 12 old. ***P < 0.001.

Fig. 4.

Passive aortic stiffness via mechanical stress-strain method increases with aging only beyond physiological strain range: stress (A) and stiffness (B) plotted against strain for young and old proximal thoracic aortas in calcium-free environment using ramp input from slack to failure. No significant difference is observed at low strains, including the physiological range of 50–80%. At higher strain levels, when collagen is heavily recruited because of large-amplitude stretching, old aortas are stiffer. Values are means ± SE; n = 6 young, 7 old. *P < 0.05, **P < 0.01.

Fig. 5.

Vascular smooth muscle (VSM) cells (VSMCs) contribute significantly to aortic stress and stiffness via HFLA method at optimal length. A and B: addition of phenylephrine (PE) increases aortic stress and stiffness from baseline values. Treatment with N^G-nitro-l-arginine methyl ester (l-NAME) to remove effects of endothelial nitric oxide (NO) further augments these increases. Values are means ± SE; n = 4 for both young and old. C: representative smoothed stress trace for young mouse aorta with scale for time and typical levels of stress showing individually additive components of maximal stress and stiffness. D and E: maximally activated VSM accounts for approximately half of maximal total aortic stress (D) and stiffness (E). All stiffness values were obtained via HFLA protocol. ECM, extracellular matrix. Absolute values are means ± SE; n = 3 young, 4 old. ***P < 0.001 with aging; †††P < 0.001 with addition of l-NAME.

Fig. 6.

Agonist-induced, PP2-sensitive focal adhesion signaling is impaired in old aortas. A and B: mean ± SE of densitometry for tyrosine phosphorylation (pTyr) for young and old mouse aortas, with typical blots shown. Typical gel image brightness was adjusted for visual clarity, but densitometry was based on raw data. n = 10 young unstimulated, 4 young +PE and +PE+PP2, 5 old for each treatment. *P < 0.05, **P < 0.01, ***P < 0.001. C: densitometry of c-Src immunoblots of young and old aortic homogenates (n = 3 each). Typical c-Src blot shown in inset; bands are from the same gel but have been digitally moved adjacent to each other. n = 3 young, 3 old. *P < 0.05.

Fig. 7.

Agonist-induced increases in stress and stiffness with Src kinase inhibitor pretreatment are higher with aging. Thoracic aortas pretreated with 10 μM PP2 contract and stiffen less in young but not old mice in the presence of maximal activation (PE + l-NAME). As a result, maximal active stress (A) and stiffness (B) increase >2-fold with aging under these conditions with HFLA measurements. Also shown for comparison are control maximal active data in the absence of PP2 (PP2−), reproduced from Fig. 5, A and B. Absolute values are means ± SE; n = 3 young, 4 old. ***P < 0.001 with aging; ‡P < 0.05, ‡‡‡P < 0.001 with addition of PP2.

Fig. 8.

Shock absorption via Src-mediated focal adhesion/nonmuscle cytoskeleton regulation is defective in old aortas: model of how changes in stiffness generated by cross-bridge attachment and remodeling of focal adhesions and nonmuscle cortical cytoskeleton can be regulated by a PP2-sensitive mechanism present in young but not old aortas. FA, focal adhesion; NMC, nonmuscle cytoskeleton; MLCK, myosin light chain kinase.