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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 peripheral actin remodeling and EC barrier enhancement. (A, B) Control and Asef-depleted EC were stimulated with HGF (50 ng/ml, 10 min). Immunofluorescence visualization of actin cytoskeleton was performed in subconfluent (A; bar; 5 μm) or dense (B; bar, 10 μm) EC culture using TR-phalloidin staining. HGF-induced peripheral accumulation of polymerized actin was abolished by Asef knockdown. Magnified images (insets) show details of actin structure. (C) Permeability measurements. Control and Asef-depleted ECs were stimulated with HGF (50 ng/ml), and TER was monitored over time. Cumulative data of five independent experiments. Results are represented as mean ± SD. (D) HPAECs grown on glass coverslips with immobilized biotinylated gelatin (0.25 mg/ml) and transfected with Asef-specific siRNA or nonspecific RNA and then stimulated with vehicle or HGF (50 ng/ml, 10 min), followed by addition of FITC-avidin (25 μg/ml, 3 min). Unbound FITC-avidin was removed, and FITC fluorescence signal was visualized by fluorescence microscopy. Bar, 10 μm.
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Figure 4: Asef knockdown attenuates HGF-induced peripheral actin remodeling and EC barrier enhancement. (A, B) Control and Asef-depleted EC were stimulated with HGF (50 ng/ml, 10 min). Immunofluorescence visualization of actin cytoskeleton was performed in subconfluent (A; bar; 5 μm) or dense (B; bar, 10 μm) EC culture using TR-phalloidin staining. HGF-induced peripheral accumulation of polymerized actin was abolished by Asef knockdown. Magnified images (insets) show details of actin structure. (C) Permeability measurements. Control and Asef-depleted ECs were stimulated with HGF (50 ng/ml), and TER was monitored over time. Cumulative data of five independent experiments. Results are represented as mean ± SD. (D) HPAECs grown on glass coverslips with immobilized biotinylated gelatin (0.25 mg/ml) and transfected with Asef-specific siRNA or nonspecific RNA and then stimulated with vehicle or HGF (50 ng/ml, 10 min), followed by addition of FITC-avidin (25 μg/ml, 3 min). Unbound FITC-avidin was removed, and FITC fluorescence signal was visualized by fluorescence microscopy. Bar, 10 μm.

Mentions: Potential effect of EC confluence on Asef-dependent peripheral cortactin activation and actin cytoskeletal remodeling was further tested. Activation of cortactin in response to HGF was reflected by cortactin phosphorylation and accumulation in cell-peripheral lamellipodia-like structures and was observed in both sparse and dense EC monolayers (Figure 3, D and E). These effects were markedly attenuated by Asef knockdown in both sparse and dense EC cultures. Cortactin translocation was also accompanied by increased F-actin accumulation at cell peripheral compartments (Figure 4A). Inspection of subcortical F-actin accumulation at different cell regions revealed that HGF-induced cortical F-actin accumulation in lamellipodia-like structures was observed in regions with intercellular gaps (inset 1 in Figure 4A) but also in the regions of established cell–cell contacts (inset 2 in Figure 4, A and B). HGF-induced cortical actin dynamics was inhibited by Asef knockdown (Figure 4, A and B). These data show that HGF-induced Asef activity stimulates cortical cytoskeletal dynamics in a cell contact–independent manner.


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 peripheral actin remodeling and EC barrier enhancement. (A, B) Control and Asef-depleted EC were stimulated with HGF (50 ng/ml, 10 min). Immunofluorescence visualization of actin cytoskeleton was performed in subconfluent (A; bar; 5 μm) or dense (B; bar, 10 μm) EC culture using TR-phalloidin staining. HGF-induced peripheral accumulation of polymerized actin was abolished by Asef knockdown. Magnified images (insets) show details of actin structure. (C) Permeability measurements. Control and Asef-depleted ECs were stimulated with HGF (50 ng/ml), and TER was monitored over time. Cumulative data of five independent experiments. Results are represented as mean ± SD. (D) HPAECs grown on glass coverslips with immobilized biotinylated gelatin (0.25 mg/ml) and transfected with Asef-specific siRNA or nonspecific RNA and then stimulated with vehicle or HGF (50 ng/ml, 10 min), followed by addition of FITC-avidin (25 μg/ml, 3 min). Unbound FITC-avidin was removed, and FITC fluorescence signal was visualized by fluorescence microscopy. Bar, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Asef knockdown attenuates HGF-induced peripheral actin remodeling and EC barrier enhancement. (A, B) Control and Asef-depleted EC were stimulated with HGF (50 ng/ml, 10 min). Immunofluorescence visualization of actin cytoskeleton was performed in subconfluent (A; bar; 5 μm) or dense (B; bar, 10 μm) EC culture using TR-phalloidin staining. HGF-induced peripheral accumulation of polymerized actin was abolished by Asef knockdown. Magnified images (insets) show details of actin structure. (C) Permeability measurements. Control and Asef-depleted ECs were stimulated with HGF (50 ng/ml), and TER was monitored over time. Cumulative data of five independent experiments. Results are represented as mean ± SD. (D) HPAECs grown on glass coverslips with immobilized biotinylated gelatin (0.25 mg/ml) and transfected with Asef-specific siRNA or nonspecific RNA and then stimulated with vehicle or HGF (50 ng/ml, 10 min), followed by addition of FITC-avidin (25 μg/ml, 3 min). Unbound FITC-avidin was removed, and FITC fluorescence signal was visualized by fluorescence microscopy. Bar, 10 μm.
Mentions: Potential effect of EC confluence on Asef-dependent peripheral cortactin activation and actin cytoskeletal remodeling was further tested. Activation of cortactin in response to HGF was reflected by cortactin phosphorylation and accumulation in cell-peripheral lamellipodia-like structures and was observed in both sparse and dense EC monolayers (Figure 3, D and E). These effects were markedly attenuated by Asef knockdown in both sparse and dense EC cultures. Cortactin translocation was also accompanied by increased F-actin accumulation at cell peripheral compartments (Figure 4A). Inspection of subcortical F-actin accumulation at different cell regions revealed that HGF-induced cortical F-actin accumulation in lamellipodia-like structures was observed in regions with intercellular gaps (inset 1 in Figure 4A) but also in the regions of established cell–cell contacts (inset 2 in Figure 4, A and B). HGF-induced cortical actin dynamics was inhibited by Asef knockdown (Figure 4, A and B). These data show that HGF-induced Asef activity stimulates cortical cytoskeletal dynamics in a cell contact–independent manner.

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