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Asef controls vascular endothelial permeability and barrier recovery in the lung.

Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA - Mol. Biol. Cell (2014)

Bottom Line: Molecular inhibition of Asef attenuated HGF-induced peripheral accumulation of cortactin, formation of lamellipodia-like structures, and enhancement of VE-cadherin adherens junctions and compromised HGF-protective effect against thrombin-induced RhoA GTPase activation, Rho-dependent cytoskeleton remodeling, and EC permeability.This effect was lost in Asef(-/-) mice.This study shows for the first time the role of Asef in HGF-mediated protection against endothelial hyperpermeability and lung injury.

View Article: PubMed Central - PubMed

Affiliation: Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637.

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Asef knockdown attenuates HGF-induced Rac1 activation. (A) Endothelial cells were transfected with control or 100 nM Asef-specific siRNA, and after 24 h, changes in TER were monitored over time. (B) FRET analysis of HGF-induced Rac1 activation. HPAECs were transfected with nonspecific or Asef-specific siRNA duplexes for 24 h, followed by transfection with CFP/YPet-Rac1 biosensor for an additional 24 h. FRET analysis of time-dependent Rac activation was performed in HGF-stimulated control and Asef-knockdown cells. Images represent a ratio of activated Rac1 to the total Rac1 content. Areas of Rac1 activation appear in red. Bar, 5 μm. (C) Quantitative analysis of HGF-induced Rac1 activation at the cell periphery. Bar graphs represent normalized CFP/YPet emission ratio. Rac1 activation in cells before HGF stimulation was compared with Rac1 activation after 6 min of HGF addition. Data are expressed as mean ± SD of four independent experiments, five to seven cells for each experiment; *p < 0.05. (D–F) Subconfluent (D), sparse (E), or dense (F) HPAECs were transfected with Asef-specific siRNA or nonspecific RNA and stimulated with HGF (50 ng/ml). Left, Rac1 activation determined by Rac-GTP pull-down assay. The content of activated Rac1 was normalized to the total Rac1 content in EC lysates. *p <0.01 vs. nonstimulated cells treated with nonspecific (ns) RNA; **p <0.01 vs. nsRNA. Right, representative phase-contrast images of HPAECs used for experiments.
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Figure 2: Asef knockdown attenuates HGF-induced Rac1 activation. (A) Endothelial cells were transfected with control or 100 nM Asef-specific siRNA, and after 24 h, changes in TER were monitored over time. (B) FRET analysis of HGF-induced Rac1 activation. HPAECs were transfected with nonspecific or Asef-specific siRNA duplexes for 24 h, followed by transfection with CFP/YPet-Rac1 biosensor for an additional 24 h. FRET analysis of time-dependent Rac activation was performed in HGF-stimulated control and Asef-knockdown cells. Images represent a ratio of activated Rac1 to the total Rac1 content. Areas of Rac1 activation appear in red. Bar, 5 μm. (C) Quantitative analysis of HGF-induced Rac1 activation at the cell periphery. Bar graphs represent normalized CFP/YPet emission ratio. Rac1 activation in cells before HGF stimulation was compared with Rac1 activation after 6 min of HGF addition. Data are expressed as mean ± SD of four independent experiments, five to seven cells for each experiment; *p < 0.05. (D–F) Subconfluent (D), sparse (E), or dense (F) HPAECs were transfected with Asef-specific siRNA or nonspecific RNA and stimulated with HGF (50 ng/ml). Left, Rac1 activation determined by Rac-GTP pull-down assay. The content of activated Rac1 was normalized to the total Rac1 content in EC lysates. *p <0.01 vs. nonstimulated cells treated with nonspecific (ns) RNA; **p <0.01 vs. nsRNA. Right, representative phase-contrast images of HPAECs used for experiments.

Mentions: Molecular inhibition of Asef in pulmonary ECs using small interfering RNA (siRNA)–mediated knockdown delayed establishment of endothelial monolayers as monitored by measurements of transendothelial electrical resistance (TER; Figure 2A). Using the Rac1 fluorescence resonance energy transfer (FRET) biosensor (Birukova et al., 2012), we evaluated the time course of regional Rac1 activation in subconfluent pulmonary EC cultures. HGF induced rapid activation of Rac1 (Figure 2B), whereas Asef knockdown significantly suppressed HGF-induced Rac1 activation. The quantitative analysis of FRET data is summarized in Figure 2C. Because this study investigated the ability of HGF to restore endothelial monolayer integrity, the subconfluent EC cultures were used in the following experiments. Asef knockdown abolished HGF-induced Rac1 activation, monitored by Rac-GTP pull-down assay (Figure 2D). To test whether Asef-mediated Rac1 activation is additionally regulated by cell–cell contacts, we also evaluated HGF-induced Rac1 activity in sparse (Figure 2E) and dense (confluent; Figure 2F) EC cultures. Representative phase-contrast microscopy images in Figures 2, D–F, depict cell density of human pulmonary artery endothelial cell (HPAEC) cultures used in these experiments, although actual densities could slightly vary between experiments. HGF activated Rac1 GTPase at all three cell density conditions, and the effect was abolished by Asef knockdown.


Asef controls vascular endothelial permeability and barrier recovery in the lung.

Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA - Mol. Biol. Cell (2014)

Asef knockdown attenuates HGF-induced Rac1 activation. (A) Endothelial cells were transfected with control or 100 nM Asef-specific siRNA, and after 24 h, changes in TER were monitored over time. (B) FRET analysis of HGF-induced Rac1 activation. HPAECs were transfected with nonspecific or Asef-specific siRNA duplexes for 24 h, followed by transfection with CFP/YPet-Rac1 biosensor for an additional 24 h. FRET analysis of time-dependent Rac activation was performed in HGF-stimulated control and Asef-knockdown cells. Images represent a ratio of activated Rac1 to the total Rac1 content. Areas of Rac1 activation appear in red. Bar, 5 μm. (C) Quantitative analysis of HGF-induced Rac1 activation at the cell periphery. Bar graphs represent normalized CFP/YPet emission ratio. Rac1 activation in cells before HGF stimulation was compared with Rac1 activation after 6 min of HGF addition. Data are expressed as mean ± SD of four independent experiments, five to seven cells for each experiment; *p < 0.05. (D–F) Subconfluent (D), sparse (E), or dense (F) HPAECs were transfected with Asef-specific siRNA or nonspecific RNA and stimulated with HGF (50 ng/ml). Left, Rac1 activation determined by Rac-GTP pull-down assay. The content of activated Rac1 was normalized to the total Rac1 content in EC lysates. *p <0.01 vs. nonstimulated cells treated with nonspecific (ns) RNA; **p <0.01 vs. nsRNA. Right, representative phase-contrast images of HPAECs used for experiments.
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Figure 2: Asef knockdown attenuates HGF-induced Rac1 activation. (A) Endothelial cells were transfected with control or 100 nM Asef-specific siRNA, and after 24 h, changes in TER were monitored over time. (B) FRET analysis of HGF-induced Rac1 activation. HPAECs were transfected with nonspecific or Asef-specific siRNA duplexes for 24 h, followed by transfection with CFP/YPet-Rac1 biosensor for an additional 24 h. FRET analysis of time-dependent Rac activation was performed in HGF-stimulated control and Asef-knockdown cells. Images represent a ratio of activated Rac1 to the total Rac1 content. Areas of Rac1 activation appear in red. Bar, 5 μm. (C) Quantitative analysis of HGF-induced Rac1 activation at the cell periphery. Bar graphs represent normalized CFP/YPet emission ratio. Rac1 activation in cells before HGF stimulation was compared with Rac1 activation after 6 min of HGF addition. Data are expressed as mean ± SD of four independent experiments, five to seven cells for each experiment; *p < 0.05. (D–F) Subconfluent (D), sparse (E), or dense (F) HPAECs were transfected with Asef-specific siRNA or nonspecific RNA and stimulated with HGF (50 ng/ml). Left, Rac1 activation determined by Rac-GTP pull-down assay. The content of activated Rac1 was normalized to the total Rac1 content in EC lysates. *p <0.01 vs. nonstimulated cells treated with nonspecific (ns) RNA; **p <0.01 vs. nsRNA. Right, representative phase-contrast images of HPAECs used for experiments.
Mentions: Molecular inhibition of Asef in pulmonary ECs using small interfering RNA (siRNA)–mediated knockdown delayed establishment of endothelial monolayers as monitored by measurements of transendothelial electrical resistance (TER; Figure 2A). Using the Rac1 fluorescence resonance energy transfer (FRET) biosensor (Birukova et al., 2012), we evaluated the time course of regional Rac1 activation in subconfluent pulmonary EC cultures. HGF induced rapid activation of Rac1 (Figure 2B), whereas Asef knockdown significantly suppressed HGF-induced Rac1 activation. The quantitative analysis of FRET data is summarized in Figure 2C. Because this study investigated the ability of HGF to restore endothelial monolayer integrity, the subconfluent EC cultures were used in the following experiments. Asef knockdown abolished HGF-induced Rac1 activation, monitored by Rac-GTP pull-down assay (Figure 2D). To test whether Asef-mediated Rac1 activation is additionally regulated by cell–cell contacts, we also evaluated HGF-induced Rac1 activity in sparse (Figure 2E) and dense (confluent; Figure 2F) EC cultures. Representative phase-contrast microscopy images in Figures 2, D–F, depict cell density of human pulmonary artery endothelial cell (HPAEC) cultures used in these experiments, although actual densities could slightly vary between experiments. HGF activated Rac1 GTPase at all three cell density conditions, and the effect was abolished by Asef knockdown.

Bottom Line: Molecular inhibition of Asef attenuated HGF-induced peripheral accumulation of cortactin, formation of lamellipodia-like structures, and enhancement of VE-cadherin adherens junctions and compromised HGF-protective effect against thrombin-induced RhoA GTPase activation, Rho-dependent cytoskeleton remodeling, and EC permeability.This effect was lost in Asef(-/-) mice.This study shows for the first time the role of Asef in HGF-mediated protection against endothelial hyperpermeability and lung injury.

View Article: PubMed Central - PubMed

Affiliation: Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637.

Show MeSH
Related in: MedlinePlus