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Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases.

Wojciak-Stothard B, Ridley AJ - J. Cell Biol. (2003)

Bottom Line: Instead, Rho and Rac1 regulated directionality of cell movement.Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement.Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House St., London W1W 7BS, UK. beata@ludwig.ucl.ac.uk

ABSTRACT
Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

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Cell shape remodelling induced by 3 dyn/cm2 shear stress. A–F show F-actin staining in growing endothelial cells stimulated with shear stress. The direction of flow is indicated by an arrow (E). A shows cells in control (static) conditions, whereas B–F show cells stimulated with shear stress shear for 5 min, 15 min, 1 h, 2 h, and 4 h, respectively. Bar, 20 μm. G shows changes in the cell spread area; H shows changes in cell elongation; and I shows changes in cell alignment (orientation) over 24 h of stimulation with shear stress. Cell alignment (I) is shown as a percentage of cells that aligned within 10° of the shear direction. *, P ≤ 0.05; **, P ≤ 0.01, comparisons with static control, t test.
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fig1: Cell shape remodelling induced by 3 dyn/cm2 shear stress. A–F show F-actin staining in growing endothelial cells stimulated with shear stress. The direction of flow is indicated by an arrow (E). A shows cells in control (static) conditions, whereas B–F show cells stimulated with shear stress shear for 5 min, 15 min, 1 h, 2 h, and 4 h, respectively. Bar, 20 μm. G shows changes in the cell spread area; H shows changes in cell elongation; and I shows changes in cell alignment (orientation) over 24 h of stimulation with shear stress. Cell alignment (I) is shown as a percentage of cells that aligned within 10° of the shear direction. *, P ≤ 0.05; **, P ≤ 0.01, comparisons with static control, t test.

Mentions: Subconfluent human umbilical vein endothelial cells (HUVECs) showed a two-phase response to shear stress. In the first phase, within 5 min of stimulation, there was a rapid increase in the number of actin stress fibers (Fig. 1, A and B) . During the next 15–30 min, the number of stress fibers decreased, the cells retracted (Fig. 1 C), and started to polarize within the direction of shear stress (Fig. 1 I). The degree of cell retraction correlated with the level of shear stress (unpublished data). In the second phase, between 30 min and 24 h, the cells respread and elongated within the direction of the flow (Fig. 1 D). The most dynamic changes in cell alignment were noticed between 30 min and 4 h of stimulation, when ∼40% of cells became aligned in the direction of shear stress (Fig. 1, D–F and I).


Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases.

Wojciak-Stothard B, Ridley AJ - J. Cell Biol. (2003)

Cell shape remodelling induced by 3 dyn/cm2 shear stress. A–F show F-actin staining in growing endothelial cells stimulated with shear stress. The direction of flow is indicated by an arrow (E). A shows cells in control (static) conditions, whereas B–F show cells stimulated with shear stress shear for 5 min, 15 min, 1 h, 2 h, and 4 h, respectively. Bar, 20 μm. G shows changes in the cell spread area; H shows changes in cell elongation; and I shows changes in cell alignment (orientation) over 24 h of stimulation with shear stress. Cell alignment (I) is shown as a percentage of cells that aligned within 10° of the shear direction. *, P ≤ 0.05; **, P ≤ 0.01, comparisons with static control, t test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172912&req=5

fig1: Cell shape remodelling induced by 3 dyn/cm2 shear stress. A–F show F-actin staining in growing endothelial cells stimulated with shear stress. The direction of flow is indicated by an arrow (E). A shows cells in control (static) conditions, whereas B–F show cells stimulated with shear stress shear for 5 min, 15 min, 1 h, 2 h, and 4 h, respectively. Bar, 20 μm. G shows changes in the cell spread area; H shows changes in cell elongation; and I shows changes in cell alignment (orientation) over 24 h of stimulation with shear stress. Cell alignment (I) is shown as a percentage of cells that aligned within 10° of the shear direction. *, P ≤ 0.05; **, P ≤ 0.01, comparisons with static control, t test.
Mentions: Subconfluent human umbilical vein endothelial cells (HUVECs) showed a two-phase response to shear stress. In the first phase, within 5 min of stimulation, there was a rapid increase in the number of actin stress fibers (Fig. 1, A and B) . During the next 15–30 min, the number of stress fibers decreased, the cells retracted (Fig. 1 C), and started to polarize within the direction of shear stress (Fig. 1 I). The degree of cell retraction correlated with the level of shear stress (unpublished data). In the second phase, between 30 min and 24 h, the cells respread and elongated within the direction of the flow (Fig. 1 D). The most dynamic changes in cell alignment were noticed between 30 min and 4 h of stimulation, when ∼40% of cells became aligned in the direction of shear stress (Fig. 1, D–F and I).

Bottom Line: Instead, Rho and Rac1 regulated directionality of cell movement.Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement.Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House St., London W1W 7BS, UK. beata@ludwig.ucl.ac.uk

ABSTRACT
Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

Show MeSH
Related in: MedlinePlus