The effect of cyclic stretch on aortic viscoelasticity and the putative role of smooth muscle focal adhesion

FIGURE 1. Analyzing viscoelastic properties of aortic segments using pressure-diameter tracings, generated from the ROTSAC setup. (A) A raw “pressure-diameter” tracing of a single distension cycle of an aortic segment between 80 and 120 mmHg. The tracing depicts a viscous component (i.e., hysteresis) as well as an inertial component. (B) Removal of the inertial component of the raw tracing by manual iteration of the inertial modulus (EM). This results in a pressure-diameter curve with clear hysteresis. (C) Removal of the viscous component (/hysteresis) by manual iteration of the viscous modulus (Eη) after having removed the inertial component. When the minimum area of hysteresis is reached, a pure elastic pressure-diameter is obtained. The slope at 100 mmHg of the resulting curve is considered as the elastic modulus (EE). The values of EM and Eη at the minimum area of hysteresis are measures of the total amount of inertia and wall viscosity, respectively.

FIGURE 2. The viscoelastic properties of central aortic tissue. The viscoelastic properties of central aortic tissue were determined via an adapted Kelvin-Voigt model using pressure-diameter tracings as described in the method section. There were no significant differences in (A) overall stiffness (quantified using the Peterson’s elastic modulus) and (B, C) viscoelastic properties between the thoracic ascending and thoracic descending aorta. However, the abdominal infrarenal aorta had a significantly higher Ep as compared to both thoracic tissues and had also a significantly higher viscous and elastic modulus. Statistical analysis was performed by using a Repeated Measures One-way ANOVA with a Tukey post hoc test for multiple comparisons (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ep = Peterson’s Modulus of Elasticity; TAA = Thoracic Ascending Aorta; TDA = Thoracic Descending Aorta; AIA = Abdominal Infrarenal Aorta.

FIGURE 3. Increased cyclic stretch, either through pulse pressure or pulse frequency, modulates aortic viscoelasticity. The changes in viscoelastic properties due to altered cyclic stretch were measured in aortic segments from the thoracic ascending aorta (TAA), thoracic descending aorta (TDA) and the abdominal infrarenal aorta (AIA). Increasing the pulse frequency from 1 Hz to 25 Hz (100 mmHg mean pressure, 40 mmHg pulse pressure) decreased (A) the viscous modulus, increased (B) the elastic modulus and decreased (C) the Ep in all types of aortic segments. Increasing the pulse pressure from 10 mmHg to 100 mmHg (100 mmHg mean pressure, 10 Hz) decreased (D) the viscous modulus. (E) A biphasic response of the elastic modulus was observed when increasing pulse pressure from 10 mmHg to 100 mmHg. (F) Ep decreased with increasing pulse pressure. Statistical analysis was performed using a Two-Way ANOVA with a Tukey post hoc test for multiple comparisons. n = 4 per group. **p < 0.01, ***p < 0.001, ****p < 0.0001. Ep = Peterson’s Modulus of Elasticity; Eη = Viscous Modulus; EE = Elastic Modulus; TAA = Thoracic Ascending Aorta; TDA = Thoracic Descending Aorta; AIA = Abdominal Infrarenal Aorta.

FIGURE 4. Strain rate is inversely correlated with aortic viscosity in the thoracic ascending aorta. The parameters “pulse frequency” and “pulse pressure” were each transformed to another parameter: strain rate. Strain rate was calculated as the change in diameter from diastolic pressure to systolic pressure, divided by Dt. There was an inverse relationship between strain rate and both (A) Ep and (B) the viscous modulus. There was no correlation between strain rate and (C) the elastic modulus. Statistical analysis: Pearson correlation. Ep = Peterson’s Modulus of Elasticity; Eη = Viscous Modulus; EE = Elastic Modulus.

FIGURE 5. Vascular smooth muscle cell contraction induces elastin fibre tortuosity and increases aortic viscoelasticity. (A, B) Indeed, VSMC contraction increases the tortuosity of elastin fibers in the medial layer of the aorta. (C) Additionally, the circularity of the nuclei is increased after VSMC contraction. Finally, acute contraction with phenylephrine increases both (D) the viscous and the (E) elastic properties of the aortic segment. Statistical analyses: (B–C) Unpaired T-test, (D–E) paired T-test. (B–E) n = 6. *p < 0.05, **p < 0.01, ***p < 0.001. PE = phenylephrine.

FIGURE 6. Acute high pulsatile bout decreases viscoelastic properties and decreases elastin tortuosity in pre-contracted aortic segments. Two out of four aortic segments (originating from the same mouse) were pre-contracted with 2 µM phenylephrine while subjected to high frequency cyclic stretch (80–120 mmHg). After 30 min, one contracted and one uncontracted segment were subjected to a high pulsatile bout for 4 minutes (increase in systolic pressure to 170 mmHg). VSMC contraction-increased viscous and elastic moduli were attenuated after an acute high pulsatile bout (A, B). Furthermore, VSMC-contraction induced elastin fiber tortuosity was also decreased after an acute high pulsatile bout (C, D). Statistical analyses; (A–B) One-way ANOVA with a Tukey post hoc test for multiple comparisons, n = 4; (C) Unpaired T-test, n = 3. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 7. Composition of focal adhesion proteins along the thoracic and abdominal aorta and the differential cyclic stretch- and contraction-mediated viscoelastic responses. The thoracic descending and the abdominal infrarenal aorta were excised from mice and cleaned from adipose tissue. Afterwards, a series of immunoblots were performed for different focal adhesion proteins (A–F). Furthermore, a proximity ligation assay was performed to analyze the amount of medial talin-vinculin bonds (G, H). The white lines denote the medial layer of the aortic sections. Scale bar = 20 µm. Lastly, viscoelastic responses to strain rate (I) and smooth muscle cell contraction (J) were compared between the TDA and the AIA. Statistical analyses: (A–H, J) paired Student’s t-test, n = 5-6 per group; (I) Least squares regression plus extra sum-of-squares F test to compare best fit values (i.e., the slope). *p < 0.05, **p < 0.01, ***p < 0.001. TDA = Thoracic Descending Aorta; AIA = Abdominal Infrarenal Aorta; Eη = Viscous Modulus.

FIGURE 8. Disruption of either Focal adhesion activity or the F-actin cytoskeleton integrity attenuates the frequency-mediated changes in viscous properties. The role of focal adhesion and F-actin was further investigated by treating thoracic ascending aorta (TAA) (A) and abdominal intrarenal aorta (B) segments with 10 µM PP2 or 10 µM Cytochalasin D. Afterwards, pulse frequency was increased from 1 Hz to 25 Hz at 100 mmHg mean pressure (80/120 mmHg, diastolic and systolic pressure). The difference in viscous modulus between 25 Hz and 1 Hz (denoted as “ηf”) was smaller after either treatment, indicating that focal adhesion and F-actin are mechanosensors which are sensitive to pulse frequency. The relative effect of PP2 and Cytochalasin D on ηf are shown in (C). Statistical analysis: (A, B) repeated measures One-Way ANOVA with a Holm-Sidak post hoc test for multiple comparisons; (C) Two-way ANOVA with a Sidak post hoc test for multiple comparisons. n = 4. *p < 0.05. η f = Frequency-mediated reduction in the viscous modulus (=Eη 25 Hz–Eη 1Hz); Eη = Viscous Modulus; TAA = Thoracic ascending aorta; AIA = Abdominal infrarenal aorta.