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. 2012 Jul 10;109(28):11110-5.
doi: 10.1073/pnas.1207326109. Epub 2012 Jun 4.

Roles of Cell Confluency and Fluid Shear in 3-dimensional Intracellular Forces in Endothelial Cells

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Free PMC article

Roles of Cell Confluency and Fluid Shear in 3-dimensional Intracellular Forces in Endothelial Cells

Sung Sik Hur et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

We use a novel 3D inter-/intracellular force microscopy technique based on 3D traction force microscopy to measure the cell-cell junctional and intracellular tensions in subconfluent and confluent vascular endothelial cell (EC) monolayers under static and shear flow conditions. We found that z-direction cell-cell junctional tensions are higher in confluent EC monolayers than those in subconfluent ECs, which cannot be revealed in the previous 2D methods. Under static conditions, subconfluent cells are under spatially non-uniform tensions, whereas cells in confluent monolayers are under uniform tensions. The shear modulations of EC cytoskeletal remodeling, extracellular matrix (ECM) adhesions, and cell-cell junctions lead to significant changes in intracellular tensions. When a confluent monolayer is subjected to flow shear stresses with a high forward component comparable to that seen in the straight part of the arterial system, the intracellular and junction tensions preferentially increase along the flow direction over time, which may be related to the relocation of adherens junction proteins. The increases in intracellular tensions are shown to be a result of chemo-mechanical responses of the ECs under flow shear rather than a direct result of mechanical loading. In contrast, the intracellular tensions do not show a preferential orientation under oscillatory flow with a very low mean shear. These differences in the directionality and magnitude of intracellular tensions may modulate translation and transcription of ECs under different flow patterns, thus affecting their susceptibility for atherogenesis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Measurements of cell–cell and intracellular tensions from cell-ECM stresses. (A) A pair of ECs in contact (2 Cells). White line outlines the region on which Newton’s first principle of static equilibrium is applied. Green and orange lines indicate regions used for computation of junction tensions for cell 1 and cell 2, respectively [Eq. 1]. Yellow line indicates cell–cell junction. (B) Confluent EC monolayer (M Cells). White line indicates the region on which Newton’s first principle of static equilibrium is applied. Green and orange lines outline regions used for computation of junction for group 1 and group 2, respectively. Yellow line indicates cell–cell junction. (C) Schematic of two cells on a substrate with traction stresses (red arrows) and cell–cell tensions (red arrows). (D) Contour and vector plot of traction stresses (Top) and displacement (Bottom) of two ECs in contact. White, green, orange, and yellow lines are the same as in A. The modulus of the 3D traction stress vector underneath the cells is represented by the pseudocolor bar. Units of traction stress and displacement are Pa and μm, respectively. (E) Contour and vector plot of traction stresses (Top) and displacement (Bottom) in a confluent EC monolayer. White, green, orange, and yellow lines are the same as in B. The modulus of the 3D traction stress vector underneath the cells is represented by the pseudocolor bar. Units of traction stress and displacement are Pa and μm, respectively. (F) Plot of errors in |(JT1 - JTm)/JTm| × 100 for two ECs and EC monolayer. JT is cell–cell junctional tension. JTm = (JT1 - JT2)/2, JT1 is cell–cell force of cell 1 or group 1, and JT2 is cell–cell force of cell 2 or group 2. The numbers of data samples are 20 and 24 for 2 Cells, and M cells, respectively.
Fig. 2.
Fig. 2.
Differences between subconfluent and confluent ECs in cell-ECM stresses, cell–cell tension, and intracellular tensions. (A) Intracellular tensions of two ECs in tangential (XY) direction. J indicates cell–cell junction, I1, I2, and I3 indicate intracellular section lines with increasing distance from J. (B) Intracellular tensions of two ECs in normal (Z) direction. J indicates cell–cell junction, I1, I2, and I3 indicate intracellular section lines with increasing distance from J. (C) Intracellular tensions of EC monolayers in tangential (XY) direction. J indicates cell–cell junction, I2 indicates cell center. I1 indicates in between. (D) Intracellular tensions of ECs monolayers in normal (Z) direction. J indicates cell–cell junction, I2 indicates cell center. I1 indicates in between. (E) Ratio of normal (Z) to tangential (XY) components of traction stresses. (F) Ratio of normal (Z) to tangential (XY) components of cell–cell tensions. * P < 0.05, § p < 0.005. (Schematics) Section lines are defined as interfacial lines that divide a cell or cells into two separated groups. cell–cell tensions or intracellular tensions are calculated as average values along these section lines. The 2 Cells are pairs of cell attached to each other. M Cells are confluent cell monolayers. The numbers of data samples in AD are 12 for both two ECs and EC monolayers. The numbers of data samples in E and F are 9, 20, and 24 for single EC, two ECs, and EC monolayers, respectively.
Fig. 3.
Fig. 3.
Confluent EC monolayers respond differently to laminar vs. oscillatory flow shear. (A) EC monolayers under constant laminar flow shear of 12 dyn/cm2 at time 0, 0.5 h, and 24 h. The bottom panels show color maps of the absolute value of the 3D TS vector at each time point and arrow plots of TSxy. The white arrow indicates the flow direction. Scale bar is 10 μm. Unit of color bar is Pa. (B) EC monolayers under oscillatory laminar flow shear of 0.5 ± 4 dyn/cm2 at time 0, 0.5 h, and 24 h. The bottom panels show color maps of the absolute value of the 3D TS vector at each time point and arrow plots of TSxy. The white arrow indicates the flow direction. Scale bar is 10 μm. Unit of color bar is Pa. (C) Tangential intracellular tension after 0.5 and 24 h of laminar flow at different intracellular section angles α from 0° to 180° at 30° intervals. (D) Tangential intracellular tension after 0.5 and 24 h of oscillatory flow at different intracellular section angles α from 0° to 180° at 30° intervals. (E) Tangential intracellular tension under no flow (time point 0) at different intracellular section angles (α) from 0° to 180° at 30° intervals. (F) Tangential intracellular tension under no flow (control in Petri dish) at different intracellular section angles (α) from 0° to 180° at 30° intervals. Colors of blue, green, and red indicate 0, 0.5, and 24 h, respectively. Blue lines indicate the mean value at time 0. The inset plot is a schematic of the section line (green) where IT is measured and the angle α between this section line and the flow direction. The arrow indicates flow direction. The * P < 0.005. LS, OS, and CT denote laminar shear, oscillatory shear, and control (no flow), respectively. The numbers of data samples are 14, 36, and 57 for laminar shear, oscillatory shear, and control, respectively.

Comment in

  • Cells gain traction in 3D.
    Ruder WC, LeDuc PR. Ruder WC, et al. Proc Natl Acad Sci U S A. 2012 Jul 10;109(28):11060-1. doi: 10.1073/pnas.1208617109. Epub 2012 Jul 9. Proc Natl Acad Sci U S A. 2012. PMID: 22778421 Free PMC article. No abstract available.

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