Cell contractility and focal adhesion kinase control circumferential arterial stiffness

Figures

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  Figure 1. Reduced smooth muscle contraction with blebbistatin selectively affects circumferential arterial mechanics in WT carotid arteries. Right carotid arteries isolated from male WT (C57BL/6J) mice (n = 6) were analyzed by pressure myography and then immediately reanalyzed after treatment with blebbistatin (see Methods). (A) In vitro stretch as determined from axial force-length tests. (B) Axial stress–stretch curves obtained at 100 mmHg. (C) Changes in outer diameter with increasing pressure. (D and E) Changes in vessel inner radius and wall thickness, respectively, with pressure. (F) Circumferential stress–stretch curves with data points showing results at 0–140 mmHg in increments of 10 mm. Statistical significance was determined by two-tailed t-test (panel A) or two-way ANOVA (panels B-F). In F, the indicated statistical significance was the same for both stretch and stress. The axial stress–stretch curves shown in panel B and in Supplementary Fig. 1A, B and C were taken from the same mice and biomechanical testing but at different pressures.

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  Figure 2. Effect of EGTA on passive arterial mechanics in WT carotid arteries. Right carotid arteries isolated from male WT (C57BL/6J) mice (n = 5) were analyzed by pressure myography and then immediately reanalyzed after treatment with 2 mM EGTA. (A) In vitro stretch as determined from axial force-length tests. (B) Axial stress–stretch curve obtained at 100 mmHg. (C, D and E) Changes in vessel outer diameter, inner radius, and wall thickness, respectively, with pressure. (F) Circumferential stress–stretch curves with data points showing results at 0–140 mmHg in increments of 10 mm. Statistical significance was determined by two-tailed t-test (panel A) or two-way ANOVA (panels B-F). In F, the indicated statistical significance was the same for both stretch and stress. The axial stress–stretch curves shown in panel B and in Supplementary Fig. 1D, E and F were taken from the same mice and biomechanical testing but at different pressures.

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  Figure 3. Deletion of SMC FAK. (A) Images of carotid artery cross-sections from FAKf/f;iCre mice that had been treated with vehicle (ethanol) or tamoxifen and stained with H&E and an antibody to FAK. Scale bars = 100 μm. Dashed lines in panel A approximate the positions of the external and internal elastic laminae, which separate the medial (M) and adventitial (Adv) layers. (B) The immunostaining results in A were quantified and graphed as box plots with Tukey whiskers; n = 6–7. Statistical significance was determined by two-tailed Mann–Whitney tests comparing ethanol to tamoxifen within the media and adventitia.

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  Figure 4. Deletion of SMC FAK attenuates circumferential arterial stiffness. Freshly isolated right carotid arteries from FAKff-iCre mice were treated with ethanol (EtOH) or tamoxifen (Tamox) and analyzed by pressure myography (n = 5 per condition). (A) In vitro stretch as determined from axial force-length tests. (B) Axial stress–stretch curves obtained at 100 mmHg. (C) Changes in outer diameter with increasing pressure. (D and E) Changes in vessel inner radius and wall thickness, respectively, with pressure. (F) Circumferential stress–stretch curves with data points showing results at 0–140 mmHg in increments of 10 mm. Statistical significance was determined by two-tailed t-test (panel A) or two-way ANOVA (panels B-F). In F, the indicated statistical significance was the same for both stretch and stress. The axial stress–stretch curves shown in panel B and in Supplementary Fig. 1G, H and I were taken from the same mice and biomechanical testing but at different pressures.

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  Figure 5. Arterial fibrillar collagens abundance is not affected by deletion of SMC FAK. (A) Images of carotid artery cross-sections from FAKf/f;iCre mice that had been treated with vehicle (ethanol) or tamoxifen and stained with antibodies to collagen-I, collagen-III, or collagen-V. Scale bars = 100 µm. Dashed lines approximate the positions of the external and internal elastic laminae, which separate the medial (M) and adventitial (Adv) layers. (B) The immunostaining results in A were quantified and graphed as box plots with Tukey whiskers; n = 5–7. Statistically significant differences were not detected when comparing (by two-tailed Mann–Whitney tests) ethanol vs tamoxifen treatment within each tissue layer.

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  Figure 6. Similar expression of SMC differentiation markers upon deletion of SMC FAK. (A) Images of carotid artery cross-sections from FAKf/f;iCre mice that had been treated with vehicle (ethanol) or tamoxifen and immunostained with antibodies to SM-MHC or SMA show medial (M) localization of the two contractile markers. Scale bars = 100 μm. (B) Immunostaining results in A were quantified and graphed as box plots with Tukey whiskers; n = 7. Statistically significant differences were not detected when comparing (by two-tailed Mann–Whitney tests) ethanol vs tamoxifen treatment within the media and adventitia (Adv).

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  Figure 7. Deletion of SMC FAK reduces carotid arterial contraction. FAKff-iCre mice were treated with ethanol or tamoxifen. Freshly isolated left carotid arteries were mounted on the pressure myograph and submerged in HBSS. Mounted vessels were preconditioned by stretching them axially to 1.15 times their UVL at 40 mmHg for 15 min and then 1.3 times their UVL at 60 mmHg for 15 min. The preconditioned vessels were brought to their IVS and pressurized to 100 mmHg. Increasing concentrations of KCl (10−4 M to 10−1 M in HBSS; 5 mL) were added to the vessel chamber sequentially at 10-min intervals or once the constriction plateau had been reached. Each new KCl solution was added after removing the prior KCl solution, and the extent of contraction to each KCl solution (defined as the change in outer diameter relative to baseline) was measured when the vessel’s outer diameter had reached its plateau (~5 min after each treatment). The baseline (dashed line) was defined as the contraction seen with 10−4 M KCl (this concentration approximated the KCl concentration in HBSS) and set to zero. Data were passed through a Grubb's test and then analyzed for statistical significance by one-way ANOVA; n = 6…

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  Figure 8. Blebbistatin eliminates the effect of SMC FAK deletion on arterial wall thickness and circumferential stiffness. Each carotid artery tested in Fig. 4 was immediately treated with blebbistatin and reanalyzed. (A) In vitro stretch as determined from axial force-length tests. (B) Axial stress–stretch curves obtained at 100 mmHg. (C) Changes in outer diameter with increasing pressure. (D-E) Changes in vessel inner radius and wall thickness, respectively, with pressure. (F) Circumferential stress–stretch curves with data points showing results at 0–140 mmHg in increments of 10 mm. Statistical significance was determined as in Fig. 4. In F, statistical significance is shown for stretch; differences in stress were not significant. The axial stress–stretch curves shown in panel B and in Supplementary Fig. 1J, K and L were taken from the same mice and biomechanical testing but at different pressures.