Coordination of fibronectin adhesion with contraction and relaxation in microvascular smooth muscle

Abstract

Aims: The regulation of vascular diameter by vasoconstrictors and vasodilators requires that vascular smooth muscle cells (VSMCs) be physically coupled to extracellular matrix (ECM) and neighbouring cells in order for a vessel to mechanically function and transfer force. The hypothesis was tested that integrin-mediated adhesion to the ECM is dynamically up-regulated in VSMCs during contractile activation in response to a vasoconstrictor and likewise down-regulated during relaxation in response to a vasodilator.

Methods and results: VSMCs were isolated from the Sprague-Dawley rat cremaster muscles. Atomic force microscopy (AFM) with fibronectin (FN)-functionalized probes was employed to investigate the biomechanical responses and adhesion of VSMCs. Responses to angiotensin II (Ang II; 10(-6) M) and adenosine (Ado; 10(-4) M) were recorded by measurements of cell cortical elasticity and cell adhesion. The results showed that Ang II caused an immediate increase in adhesion (+27%) between the probe and cell. Cell stiffness increased (+70%) in parallel with the adhesion change. Ado decreased adhesion (-15%) to FN and reduced (-30%) stiffness.

Conclusion: Changes in the receptor-mediated activation of the contractile apparatus cause parallel alterations in cell adhesion and cell cortical elasticity. These studies support the hypothesis that the regulation of cell adhesion is coordinated with contraction and demonstrate the dynamic nature of cell adhesion to the ECM. It is proposed that coordination of adhesion and VSMC contraction is an important mechanism that allows for an efficient transfer of force between the contractile apparatus of the cell and the extracellular environment.

Figure 1

Continuous real-time VSMC stiffness recordings following Ang II or Ado. (A) Representative single cell record of VSMC elastic modulus before and after addition of Ang II (10⁻⁶ M). (B) Group average of VSMC elastic modulus alteration before and after addition of Ang II (n = 10). (C) Average elastic modulus summed across all time points for the group of VSMCs before and after the addition of Ang II (n = 10, *P <0.05). (D) Representative single-cell measurement of elastic modulus before and after the addition of cell buffer only. (E) Group average of VSMC elastic modulus alteration after treatment with buffer vehicle only (n = 10). (F) Average elastic modulus summed for all time points of VSMC before and after treatment with buffer (n = 10). (G) Representative alteration in VSMC elastic modulus before and after the addition of Ado (10⁻⁴ M). (H) Group average of VSMC elastic modulus alteration before and after the addition of Ado (n = 8). (I) Average elastic modulus summed for all time points of VSMC before and after addition of Ado (n = 8, *P <0.05). Data were acquired at 0.1 Hz of indentation frequency and are presented as mean ± SEM.

Figure 2

Continuous real-time recordings of adhesion events between VSMCs and FN-coated AFM probe following Ang II or Ado. (A) Representative alteration in the number of adhesion events per AFM retraction curve before and after stimulation with Ang II. (B) The alteration of group average number of adhesion event per retraction curve before and after the addition of Ang II (n = 10). (C) Average number of adhesion events per retraction curve before and after addition of Ang II summed over 1800 s (n = 10, *P < 0.05). (D) Representative change in the number of adhesion event per AFM retraction curve before and after the administration of buffer in cell bath. (E) The alteration of group average number of adhesion event per retraction curve before and after the addition of buffer (n = 10). (F) The average number of adhesion event per retraction curve before and after the addition of buffer summed over 1800 s (n = 10, *P < 0.05). (G) Representative alteration in the number of adhesion event per AFM retraction curve before and after stimulation with Ado. (H) The alteration of group average number of adhesion event per retraction curve before and after the addition of Ado (n = 8). (I) The average number of adhesion event per retraction curve before and after the addition of Ado summed over 1800s (n = 8, *P < 0.05). Data were acquired at 0.1 Hz of indentation frequency and are presented as mean ± SEM.

Figure 3

Adhesion rupture forces between VSMCs and FN following Ang II or Ado. (A) Representative real-time recording of the force required to rupture an adhesion between FN and VSMCs on a single VSMCs before and after Ang II treatment. (B) Average adhesion force between FN and VSMCs before and after Ang II stimulation summed over 1800s (n = 10, *P < 0.05). (C) Representative real-time record of the adhesion between FN and VSMCs on single VSMCs before and after the administration of buffer in cell bath. (D) Average adhesion force between FN and VSMCs before and after the addition of buffer in cell bath summed over 1800 s (n = 10). (E) Representative real-time record of the adhesion force between FN and VSMCs on single VSMCs before and after Ado treatment. (F) Average adhesion force between FN and VSMCs before and after Ado stimulation summed over 1800 s (n = 8, *P < 0.05). Data were acquired at 0.1 Hz of indentation frequency. Data for bar graphs are presented as mean ± SEM.

Figure 4

Analyses of relationship between retraction distances and occurrence adhesion rupture. (A) Distribution of retraction distances at the time of adhesion rupture. (group/n = 10, only the last 600 s post-Ang II treatment data were included for comparison with the 600 s control recording to normalize for the recording time period). (B) The adhesion rupture force vs. retraction distance at the time of rupture (group/n = 10, only the last 600 s post-Ang II treatment data were included to normalize for the recording time period). (C) Illustration of possible adhesion models. (1) During short retraction from the cell the recorded rupture force is higher because of stronger attachment within the FN–integrin–cytoskeletal complex; (2) When the pulling force exerted on the cell exceeds the adhesion rupture force necessary to break connections within the FN–integrin–cytoskeletal complex the cell membrane is believed to deform into a lipid nanotube resulting in a second population of lower rupture force adhesion that break at and longer distances. The deflection signal (nm) of force curve was transformed to force signal (pN) by multiplying with the spring constant of the AFM cantilever. (D) Average adhesion rupture force between FN and VSMCs at long- (<400 nm) and short- (>400 nm) pulling distance before and after Ang II stimulation (n = 10, *P < 0.05). (E) Average adhesion rupture force between FN and VSMCs at long- and short-pulling distance before and after the addition of buffer in cell bath (n = 10). (F) Average adhesion rupture force between FN and VSMCs at long- and short-pulling distance before and after Ado stimulation (n = 8, *P <0.05). Data were acquired at 0.1 Hz of indentation frequency. Data for bar graphs are presented as mean ± SEM.