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. 2013 Apr 23;8(4):e62461.
doi: 10.1371/journal.pone.0062461. Print 2013.

The Focal Adhesion: A Regulated Component of Aortic Stiffness

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Free PMC article

The Focal Adhesion: A Regulated Component of Aortic Stiffness

Robert J Saphirstein et al. PLoS One. .
Free PMC article

Abstract

Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cortical stiffness measurement in VSMCs.
(A) Controlled force pulses generated by magnetic tweezers displace aortic VSMC-adherent, RGD-coated superparamagnetic beads (2.8 µm) to measure the stiffness of the bead-focal adhesion-cortical cytoskeleton linkage (see Methods). The force F exerted on a bead depends on the induced magnetic moment in the bead m and the spatial gradient of the magnetic field B, which depends critically on the sharpness of the probe’s tip, characterized by its radius of curvature R. (B) Calibration curve relating the force exerted on a bead (d = 2.8 µm) to its distance from the MT tip (R = 40 µm) and the current through the electromagnet solenoid (I = 1.5 A). The gray box denotes the operating range used for the MT experiments, i.e. the distance that is set between the MT tip and a bead before pulling commences. (C) Mean cortical stiffness increases with LPA stimulation in a PP2-attenuated manner. Right: BSA beads, which do not bind integrins but adhere nonspecifically, do not register an increase in stiffness with LPA. *p<0.05, **p<0.01, n.s. – not significant, unpaired, two-tailed Student’s t-test, assuming unequal variances.
Figure 2
Figure 2. LPA stimulation increases FA size in a PP2-sensitive manner.
Top: Deconvolution microscopy of FAs. Representative FAs are shown in an expanded view in the insets. All FAs in each cell, not just those featured in the insets, were analyzed to determine the mean FA area. Scale bars: top, 20 µm; bottom, 5 µm. Bottom: Mean FA area is significantly greater in cells stimulated with LPA (n = 82 cells). ***p<0.001, unpaired, two-tailed Student’s t-test.
Figure 3
Figure 3. Aortic tissue stiffening during contractile stimulation is decreased by inhibition of Src, FAK, or MLCK.
(A) Tissue stiffness E measured in vitro during PE-induced contraction at optimum length L O with small-amplitude (1%), high frequency (40Hz) sinusoidal stretches ΔL. Box height and width for magnified traces: 0.5 mN, 5 µm, and 0.02 s. Upper Inset: Stiffness calculation. Lower Inset: Fluorescent micrograph of aortic ring in cross-section, used to determine ring thickness for calculation of cross-sectional area A. Green: autofluorescent elastic laminae. Blue: cell nuclei. Scale bar, 100 µm. (B) PE-induced stress is significantly lower when pre-treated with MLCK inhibitor ML-9, confirming the importance of myosin activation and contraction to vascular stiffness (n = 4). PE-induced stress is also significantly reduced when pre-treated with Src inhibitor PP2 and FAK inhibitor 14. (C) PE-induced stiffening is significantly reduced when pre-treated with MLCK inhibitor ML-9, confirming the importance of myosin activation to aortic stiffness. Stiffening is also significantly lower when pre-treated with Src inhibitor PP2 and FAK inhibitor 14, indicating a role for Src, FAK, and FA proteins in aortic stiffness. n = 10 untreated, 6 ML-9, 4 PP2, 5 FI-14 rings. *p<0.05,**p<0.01, ***p<0.001, unpaired, two-tailed Student’s t-test.
Figure 4
Figure 4. PE induces FAK and Src-mediated tyrosine phosphorylation of FA proteins in dVSMCs.
(A) Typical blot, phosphotyrosine screening of mouse aorta tissue homogenates. PE increases tyrosine phosphorylation, and pre-treatment with Src inhibitor PP2 decreases tyrosine phosphorylation. (B) Mean densitometry of phosphotyrosine bands indicated. (C) Phospho-FAK Y925 increases in response to PE in a PP2-inhibitable manner, mean densitometry (n = 9 mice, 3 experiments). (D-E) Phospho-CAS Y165 and phospho-paxillin Y118 increase in response to PE in a FI-14-inhibitable manner, mean densitometry (n = 9 mice, 3 experiments). The brightness of the representative bands in Insets C-E has been uniformly altered for visual display; however, unaltered images were used for densitometry quantitation. *p<0.05, **p<0.01 vs. control, +p<0.05, ++p<0.01, +++p<0.001 vs. PP2+PE or FI-14+PE, unpaired, two-tailed Student’s t-test.
Figure 5
Figure 5. Model.
(A) Tension-induced, Src- and FAK-mediated growth and remodeling of dynamic focal adhesions in aortic VSMCs leads to cell-matrix adhesion strengthening (cortical stiffening) in response to contractile stimulus. This strengthening is required for adequate force and stiffness transmission from the VSMC to the blood vessel wall. (B) Inhibition of Src with PP2 or FAK with FI-14 inhibits FA dynamics and growth, preventing reinforcement of the cell-matrix linkage. As a result, forces and stiffness generated by the activated VSMC cannot propagate efficiently to the tissue. (C) Inhibition of MLCK with ML-9 reduces contractile force and, as a result, lessens reinforcement of the cell-matrix linkage. Consequently, force and stiffness development in the aortic wall are reduced.

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