Remanent cell traction force in renal vascular smooth muscle cells induced by integrin-mediated mechanotransduction

Abstract

It was previously demonstrated in isolated renal vascular smooth muscle cells (VSMCs) that integrin-mediated mechanotransduction triggers intracellular Ca(2+) mobilization, which is the hallmark of myogenic response in VSMCs. To test directly whether integrin-mediated mechanotransduction results in the myogenic response-like behavior in renal VSMCs, cell traction force microscopy was used to monitor cell traction force when the cells were pulled with fibronectin-coated or low density lipoprotein (LDL)-coated paramagnetic beads. LDL-coated beads were used as a control for nonintegrin-mediated mechanotransduction. Pulling with LDL-coated beads increased the cell traction force by 61 ± 12% (9 cells), which returned to the prepull level after the pulling process was terminated. Pulling with noncoated beads had a minimal increase in the cell traction force (12 ± 9%, 8 cells). Pulling with fibronectin-coated beads increased the cell traction force by 56 ± 20% (7 cells). However, the cell traction force was still elevated by 23 ± 14% after the pulling process was terminated. This behavior is analogous to the changes of vascular resistance in pressure-induced myogenic response, in which vascular resistance remains elevated after myogenic constriction. Fibronectin is a native ligand for α(5)β(1)-integrins in VSMCs. Similar remanent cell traction force was found when cells were pulled with beads coated with β(1)-integrin antibody (Ha2/5). Activation of β(1)-integrin with soluble antibody also triggered variations of cell traction force and Ca(2+) mobilization, which were abolished by the Src inhibitor. In conclusion, mechanical force transduced by α(5)β(1)-integrins triggered a myogenic response-like behavior in isolated renal VSMCs.

Fig. 1.

Changes in mean normalized total cell traction force before and after attachment of paramagnetic beads to renal vascular smooth muscle cells (VSMCs), and when renal VSMCs were pulled with fibronectin-coated (FN-coated), LDL-coated, and noncoated paramagnetic beads. Cell traction force was normalized with the traction force measured after paramagnetic beads attached (prepull). The mean total cell traction force is 0.28 ± 0.09 dyn/cell at prepull (24 cells). Cell traction force was restored to prepull level after the pulling process was terminated except in cells pulled by fibronectin-coated paramagnetic beads. Error bars are SE. *Significant difference compared with the prepull control (P < 0.05).

Fig. 2.

Force maps of a renal VSMC pulled by uncoated paramagnetic beads (A, B, and C), LDL-coated paramagnetic beads (D, E, and F), and fibronectin-coated paramagnetic beads (G, H, I). Traction forces were measured before pulling (A, D, and G), 30 s after initiation of pulling (B, E, and H), and 30 s after termination of pulling (C, F, and I). Scale bars = 90 μm. Arrows indicate the direction of pulling force and the position of the electromagnet. Colored scales of cell traction force are in Pascal.

Fig. 3.

Mean time courses of changes in cell traction force induced by pulling paramagnetic beads coated with β₁-integrin antibody (A), paramagnetic beads coated with control isotype antibody (B), and soluble β₁-integrin antibody alone without paramagnetic beads (D). Time courses from individual cells are shown in insets. Horizontal lines indicate duration of pulling. Dotted lines are ± SE. Cell traction force was normalized with the traction force measured after paramagnetic beads attached (prepull). Mean normalized data are shown in histograms (C). Solid bars and opened bars are β₁-integrin antibody and control isotype antibody, respectively. Error bars are SE. *Significant difference compared with the prepull control (P < 0.05, n = 6 cells). The β₁-integrin antibody was added to the bath at t = 0 at (D). Preincubation of Src inhibitor PP2 (5 μM) abolished the increase of cell traction force induced by soluble β₁-integrin antibody ( n = 6 cells).

Fig. 4.

Rhythmic variations of remanent cell traction force. A sequence of consecutive force maps (1–14) sampled at 0.2-Hz display rhythmic variations in cell traction force induced by pulling with β₁-integrin antibody-coated beads. Images shown were collected after the pulling process was terminated. Cell traction force in the colored scale is 1.35E + 04 Pascal. Scale bar = 50 μm.

Fig. 5.

Ca²⁺ mobilization in renal VSMCs induced by soluble β₁-integrin antibody. Normalized time courses of fluo-4 emission in a single cultured renal VSMC (A) and the mean normalized power spectra of oscillations in fluo-4 emission (B). The β₁-integrin antibody was added to the bath at t = 0. Solid lines are mean normalized power spectra (45 cells/3 experiments). Dotted lines are ± SE. Preincubation of Src inhibitor (5 μM PP2) attenuates β₁-integrin antibody induced Ca²⁺ oscillations.

Fig. 6.

Occurrence and characterization of Ca²⁺ sparks induced by soluble integrin antibody. XT line scan image of a single VSMC exhibiting Ca²⁺ sparks (white arrowheads) in the presence of β₁-integrin antibody (A). The whole image is composed of 512 scan lines collected at 500 Hz. Distribution of full width at half the maximum (FWHM, amplitude) and full duration at half maximum (FDHM, duration) of Ca²⁺ sparks in the presence of α₅-integrin antibody (B and C, 83 Ca²⁺ sparks), β₁-integrin antibody (D and E, 71 Ca²⁺ sparks), and β₂-integrin antibody (F and G, 33 Ca²⁺ sparks) were similar. Medians of FWHM are 0.9, 1.2, and 0.8 μm, respectively. Corresponding medians FDHM are 20, 22, and 18 μs.

Fig. 7.

Immunofluorescence of activated β₁-integrin detected with antibody HUTS-4 in freshly isolated renal VSMCs after activation by antibody Ha2/5 (A) and control isotype antibody (B). Corresponding transmitted light images are shown in C and D, respectively. Strong immunofluorescence of activated β₁-integrin was localized on the plasma membrane after activation. Scale bars in C and D = 10 μm. Mean immunofluorescence intensity was increased by 110 ± 8% (3 experiments, 42 cells) after activation compared with the control.