Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

Abstract

Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2 ~ 5 microm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of alpha(5)- and beta(3)-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800 ~ 1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to alpha(5)beta(1)- and alpha(v)beta(3)-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces.

Fig. 1.

A: mechanical circuit that simulates the passive viscoelastic property of vascular smooth muscle cells (VSMCs). The elastic components are represented by the two springs with elasticity of K₁ and K₀, and the viscous components are represented by the dashpot with viscosity of γ₀. B: schematic representation of the instant deformation of a VSMC once pulling force (F) was applied to the bead. The envelope represents a single VSMC, and the lines inside represent the cytoskeletal elements. C: typical fitting of the experimental data (○) with the viscoelastic model (solid line). The dashed line represents the initial instant displacement of the bead, which was determined by the elastic element in the model (K₁ + K₀). x is the vertical bead displacement and t is time.

Fig. 2.

A: trans-illumination image a bead-fused atomic force microscopy (AFM) probe applied to surface of a VSMC. B: force required to detach the bead from the cell surface plotted as a function of contact time. C: displacement of a fibronectin (FN)-coated bead on the VSMC surface in response to step increases of pulling force applied by AFM. The FN-coated bead fused to the AFM cantilever was brought into contact with the VSMC surface to form molecular connections. Top, step increases of pulling force (z-direction) applied onto the bead-cell connection site using AFM. Bottom, corresponding z-direction movements of the FN-coated bead. D: schematics of the VSMC response to a step increase of pulling force. E: myogenic response of an intact isolated rat cremaster skeletal muscle arteriole induced by a stepwise increase of the intraluminal pressure. Arrows depict time points of application of 15 mmHg pressure steps; vessel diameters were normalized by the vessel diameter at 70 mmHg (D/D₇₀), and are shown in a percentage scale. F: vessel responses to a step pressure change from 45 to 60 mmHg. Data represent means ± SE.

Fig. 3.

A: α₅-integrin clustering around the FN-coated bead. B: β₃-integrin clustering around the FN-coated bead. C: actin filament clustering around the FN-coated bead. FN-coated fluorescent beads were allowed to adhere to the cell surface for 2 h. Cells were then fixed and stained for integrins and actin filament, respectively, and were imaged using confocal microscopy. Scale bar = 10 μm.

Fig. 4.

A and B: effect of function-blocking antibodies for α₅β₁- and α_vβ₃ integrins (HMα5-1 and F11, respectively) on the VSMC mechanoresponse. For control, n = 7; for HMα5-1 (20 μg/ml), n = 7; for HMα5-1 (50 μg/ml), n = 6; for F11 (20 μg/ml), n = 4; and for F11 (50 μg/ml), n = 8. C and D: pulling elasticity and viscosity of control VSMCs and cells treated with HMα5-1 or F11. Arrows indicate the time points of force application. Data represent means ± SE.

Fig. 5.

Effect of cytochalasin D (CytoD; 13.3 μM) and jasplakinolide (JPK; 0.2 μM) on the force-induced VSMC mechanical response and VSMC pulling elasticity. A and C: averaged displacement of FN-coated beads by VSMCs before and after treatment with CytoD (A) or JPK (C). The bead displacement shown was recorded from 10 s before the step increase of pulling force (800 pN) and until 100∼260 s after force application. A: before CytoD, n = 5; and after CytoD, n = 6. C: before JPK, n = 9; and after JPK, n = 13. B and D: summarized effect of CytoD (B) and JPK (D) on the total displacement of FN-coated beads by VSMCs. The total bead displacement was determined as the change of bead position over 4 min after the application of pulling force. E: effect of CytoD, JPK, and DMSO on the pulling elasticity of VSMCs. Arrows indicate the time points of force application. Data represent means ± SE. *P < 0.05.

Fig. 6.

Comparison of VSMC mechanical responses, pulling elasticity, and viscosity with beads coated with collagen type I (CNI), laminin (LN), vitronectin (VN), and FN. A: displacement of CNI-, FN-, LN-, VN-, or BSA-coated beads by VSMCs. The bead displacement was recorded from 10 s before a step increase of pulling force and until 160∼260 s after force application. FN-coated bead, n = 25 and force = 800 pN; CNI-coated bead, n = 19 and force = 800 pN; VN-coated bead, n = 6 and force = 500 pN; LN-coated bead, n = 13 and force = 500∼600 pN; and BSA-coated bead, n = 2 and force = 800 pN. Arrows indicate the time points of force application. B: pulling elasticity of VSMCs with CNI-, FN-, LN-, or VN-coated beads. C: pulling viscosity of VSMCs with CNI-, FN-, LN-, or VN-coated bead. Data represent means ± SE.

Fig. 7.

Immunofluorescent microscopy of actin filament structures and α₅- and β₃-integrin clusterings formed by CNI-coated (A), VN-coated (B), and LN-coated (C) beads. Extracellular matrix protein-coated fluorescent beads were allowed to contact the cell surface for 30 min. Cells were then fixed and either stained for actin filament using Alexa 568-conjugated phalloidin or for α₅- and β₃-integrins using Cy5. Cells were visualized using confocal microscopy. Arrows depict the position of actin filament clustering and integrin clustering. Scale bars = 10 μm.

Fig. 8.

A and B: VSMC mechanoresponses to FN-coated (A) and CNI-coated (B) beads in the presence of PP2 or PP3. VSMCs were incubated with PP2 (5 μM) or PP3 (5 μM) for 30 min before the application of AFM pulling force and measurement of bead displacement. For each group, n = 5 and force = 800 pN. C: pulling elasticity of VSMCs with FN- and CNI-coated beads and with PP2 or PP3 treatment. D: pulling viscosity of VSMCs with FN- and CNI-coated beads and with PP2 or PP3 treatment. Data represent means ± SE. E and F: immunofluorescent microscopy of FAK and paxillin clusterings formed by CNI-coated (E) and FN-coated (F) beads. FN- and CNI-coated borosilicate beads were allowed to contact the cell surface for 1 h. Cells were then fixed and stained for FAK and paxillin with Alexa 488 and visualized using confocal microscopy. Scale bars = 10 μm. *P < 0.05.