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Nck1, But Not Nck2, Mediates Disturbed Flow-Induced p21-Activated Kinase Activation and Endothelial Permeability.

ABSTRACT: Background Alteration in hemodynamic shear stress at atheroprone sites promotes endothelial paracellular pore formation and permeability. The molecular mechanism remains unknown. Methods and Results We show that Nck (noncatalytic region of tyrosine kinase) deletion significantly ameliorates disturbed flow-induced permeability, and selective isoform depletion suggests distinct signaling mechanisms. Only Nck1 deletion significantly reduces disturbed flow-induced paracellular pore formation and permeability, whereas Nck2 depletion has no significant effects. Additionally, Nck1 re-expression, but not Nck2, restores disturbed flow-induced permeability in Nck1/2 knockout cells, confirming the noncompensating roles. In vivo, using the partial carotid ligation model of disturbed flow, Nck1 knockout prevented the increase in vascular permeability, as assessed by Evans blue and fluorescein isothiocyanate dextran extravasations and leakage of plasma fibrinogen into the vessel wall. Domain swap experiments mixing SH2 (phosphotyrosine binding) and SH3 (proline-rich binding) domains between Nck1 and Nck2 showed a dispensable role for SH2 domains but a critical role for the Nck1 SH3 domains in rescuing disturbed flow-induced endothelial permeability. Consistent with this, both Nck1 and Nck2 bind to platelet endothelial adhesion molecule-1 (SH2 dependent) in response to shear stress, but only Nck1 ablation interferes with shear stress-induced PAK2 (p21-activated kinase) membrane translocation and activation. A single point mutation into individual Nck1 SH3 domains suggests a role for the first domain of Nck1 in PAK recruitment to platelet endothelial cell adhesion molecule-1 and activation in response to shear stress. Conclusions This work provides the first evidence that Nck1 but not the highly similar Nck2 plays a distinct role in disturbed flow-induced vascular permeability by selective p21-activated kinase activation.

SUBMITTER: Alfaidi M

PROVIDER: S-EPMC7428973 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Nck1, But Not Nck2, Mediates Disturbed Flow-Induced p21-Activated Kinase Activation and Endothelial Permeability.

Alfaidi Mabruka M Bhattarai Umesh U Orr A Wayne AW

Journal of the American Heart Association 20200529 11

Background Alteration in hemodynamic shear stress at atheroprone sites promotes endothelial paracellular pore formation and permeability. The molecular mechanism remains unknown. Methods and Results We show that Nck (noncatalytic region of tyrosine kinase) deletion significantly ameliorates disturbed flow-induced permeability, and selective isoform depletion suggests distinct signaling mechanisms. Only Nck1 deletion significantly reduces disturbed flow-induced paracellular pore formation and per ...[more]

PMID: 32468886

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Project description:The collective migration of vascular endothelial cells plays important roles in homeostasis and angiogenesis. Oxygen concentration in vivo, which is lower than in the atmosphere and changes due to diseases, is a key factor affecting the cellular dynamics of vascular endothelial cells. We previously reported that hypoxic conditions promote the internalization of vascular endothelial (VE)-cadherin, a specific cell-cell adhesion molecule, and increase the velocity of the collective migration of vascular endothelial cells. However, the mechanism through which cells regulate collective migration as affected by oxygen tension is not fully understood. Here, we investigated oxygen-dependent collective migration, focusing on intracellular protein p21-activated kinase (PAK) and hypoxia-inducing factor (HIF)-1α. A monolayer of human umbilical vein vascular endothelial cells (HUVECs) was formed in a microfluidic device with controllability of oxygen tension. The HUVECs were then exposed to various oxygen conditions in a range from 0.8% to 21% O2, with or without PAK inhibition or chemical stabilization of HIF-1α. Collective cell migration was measured by particle image velocimetry with time-lapse phase-contrast microscopic images. Localizations of VE-cadherin and HIF-1α were quantified by immunofluorescent staining. The collective migration of HUVECs varied in an oxygen-dependent fashion; the migration speed was increased by hypoxic exposure down to 1% O2, while it decreased under an extremely low oxygen tension of less than 1% O2. PAK inhibition suppressed the hypoxia-induced increase of the migration speed by preventing VE-cadherin internalization into HUVECs. A decrease in the migration speed was also obtained by chemical stabilization of HIF-1α, suggesting that excessive accumulation of HIF-1α diminishes collective cell migration. These results indicate that the oxygen-dependent variation of the migration speed of vascular endothelial cells is mediated by the regulation of VE-cadherin through the PAK pathway, as well as other mechanisms via HIF-1α, especially under extreme hypoxic conditions.

Dataset Information

Nck1, But Not Nck2, Mediates Disturbed Flow-Induced p21-Activated Kinase Activation and Endothelial Permeability.

Publications

Nck1, But Not Nck2, Mediates Disturbed Flow-Induced p21-Activated Kinase Activation and Endothelial Permeability.

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