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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 protective effects against thrombin-induced EC barrier disruption. (A) Measurements of TER. ECs were transfected with 100 nM Asef-specific siRNA or nonspecific RNA 72 h before TER measurement. After 15-min pretreatment with vehicle or HGF, cells were stimulated with thrombin (0.5 U/ml; arrow). The TER was monitored over time. Representative data of five independent experiments. Bar graph depicts EC permeability changes at the time point corresponding to the maximal TER response. Results are represented as mean ± SD; *p < 0.01. (B) EC monolayers transfected with nonspecific RNA or Asef-specific siRNA were stimulated with thrombin (0.5 U/ml, 10 min) with or without HGF pretreatment (50 ng/ml, 15 min). Stress fiber formation and integrity of EC monolayer were monitored by immunofluorescence staining with Texas red–phalloidin. Paracellular gaps and disrupted intercellular contacts are marked by arrows. Bar, 10 μm. (C) Cells treated with nonspecific or Asef-specific siRNA were stimulated with thrombin (0.5 U/ml, 10 min) with or without HGF pretreatment (50 mg/ml, 15 min). Rho activation was evaluated by RhoGTP pull-down assay and normalized to total Rho content in cell lysates. Asef depletion was verified by Western blot. (D) Activation of Rho pathway was evaluated by Western blot analysis of phospho-MYPT and diphospho-MLC levels. Reprobing with β-actin antibody was used as the normalization control. Asef depletion was verified by Western blot. Bar graphs depict quantitative densitometry analysis of Western blot data; *p < 0.05, thrombin nonspecific RNA vs. HGF + thrombin nonspecific RNA; **p < 0.05, HGF + thrombin nonspecific RNA vs. si-Asef.
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Figure 7: Asef knockdown attenuates HGF protective effects against thrombin-induced EC barrier disruption. (A) Measurements of TER. ECs were transfected with 100 nM Asef-specific siRNA or nonspecific RNA 72 h before TER measurement. After 15-min pretreatment with vehicle or HGF, cells were stimulated with thrombin (0.5 U/ml; arrow). The TER was monitored over time. Representative data of five independent experiments. Bar graph depicts EC permeability changes at the time point corresponding to the maximal TER response. Results are represented as mean ± SD; *p < 0.01. (B) EC monolayers transfected with nonspecific RNA or Asef-specific siRNA were stimulated with thrombin (0.5 U/ml, 10 min) with or without HGF pretreatment (50 ng/ml, 15 min). Stress fiber formation and integrity of EC monolayer were monitored by immunofluorescence staining with Texas red–phalloidin. Paracellular gaps and disrupted intercellular contacts are marked by arrows. Bar, 10 μm. (C) Cells treated with nonspecific or Asef-specific siRNA were stimulated with thrombin (0.5 U/ml, 10 min) with or without HGF pretreatment (50 mg/ml, 15 min). Rho activation was evaluated by RhoGTP pull-down assay and normalized to total Rho content in cell lysates. Asef depletion was verified by Western blot. (D) Activation of Rho pathway was evaluated by Western blot analysis of phospho-MYPT and diphospho-MLC levels. Reprobing with β-actin antibody was used as the normalization control. Asef depletion was verified by Western blot. Bar graphs depict quantitative densitometry analysis of Western blot data; *p < 0.05, thrombin nonspecific RNA vs. HGF + thrombin nonspecific RNA; **p < 0.05, HGF + thrombin nonspecific RNA vs. si-Asef.

Mentions: Agonist-induced activation of Rac1 protects the vascular endothelial barrier in thrombin-stimulated ECs by inhibiting the thrombin-induced Rho pathway of barrier dysfunction (Finigan et al., 2005; Birukova et al., 2007c; Tauseef et al., 2008; Baumer et al., 2009). Involvement of Asef in HGF-induced barrier protective effects against thrombin was tested in control and Asef-depleted cells. Agonist-induced permeability responses in control cells treated with nonspecific RNA and Asef-depleted EC monolayers were monitored by TER measurements. HGF exhibited a prominent protective effect against thrombin-induced TER decline in EC monolayers treated with nonspecific RNA (Figure 7A, left). In contrast, Asef knockdown impaired HGF-induced EC barrier protection against thrombin (Figure 7A, right). Effects of Asef knockdown on agonist-induced TER changes are summarized in the bar graph in Figure 7A (bottom).


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 protective effects against thrombin-induced EC barrier disruption. (A) Measurements of TER. ECs were transfected with 100 nM Asef-specific siRNA or nonspecific RNA 72 h before TER measurement. After 15-min pretreatment with vehicle or HGF, cells were stimulated with thrombin (0.5 U/ml; arrow). The TER was monitored over time. Representative data of five independent experiments. Bar graph depicts EC permeability changes at the time point corresponding to the maximal TER response. Results are represented as mean ± SD; *p < 0.01. (B) EC monolayers transfected with nonspecific RNA or Asef-specific siRNA were stimulated with thrombin (0.5 U/ml, 10 min) with or without HGF pretreatment (50 ng/ml, 15 min). Stress fiber formation and integrity of EC monolayer were monitored by immunofluorescence staining with Texas red–phalloidin. Paracellular gaps and disrupted intercellular contacts are marked by arrows. Bar, 10 μm. (C) Cells treated with nonspecific or Asef-specific siRNA were stimulated with thrombin (0.5 U/ml, 10 min) with or without HGF pretreatment (50 mg/ml, 15 min). Rho activation was evaluated by RhoGTP pull-down assay and normalized to total Rho content in cell lysates. Asef depletion was verified by Western blot. (D) Activation of Rho pathway was evaluated by Western blot analysis of phospho-MYPT and diphospho-MLC levels. Reprobing with β-actin antibody was used as the normalization control. Asef depletion was verified by Western blot. Bar graphs depict quantitative densitometry analysis of Western blot data; *p < 0.05, thrombin nonspecific RNA vs. HGF + thrombin nonspecific RNA; **p < 0.05, HGF + thrombin nonspecific RNA vs. si-Asef.
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Figure 7: Asef knockdown attenuates HGF protective effects against thrombin-induced EC barrier disruption. (A) Measurements of TER. ECs were transfected with 100 nM Asef-specific siRNA or nonspecific RNA 72 h before TER measurement. After 15-min pretreatment with vehicle or HGF, cells were stimulated with thrombin (0.5 U/ml; arrow). The TER was monitored over time. Representative data of five independent experiments. Bar graph depicts EC permeability changes at the time point corresponding to the maximal TER response. Results are represented as mean ± SD; *p < 0.01. (B) EC monolayers transfected with nonspecific RNA or Asef-specific siRNA were stimulated with thrombin (0.5 U/ml, 10 min) with or without HGF pretreatment (50 ng/ml, 15 min). Stress fiber formation and integrity of EC monolayer were monitored by immunofluorescence staining with Texas red–phalloidin. Paracellular gaps and disrupted intercellular contacts are marked by arrows. Bar, 10 μm. (C) Cells treated with nonspecific or Asef-specific siRNA were stimulated with thrombin (0.5 U/ml, 10 min) with or without HGF pretreatment (50 mg/ml, 15 min). Rho activation was evaluated by RhoGTP pull-down assay and normalized to total Rho content in cell lysates. Asef depletion was verified by Western blot. (D) Activation of Rho pathway was evaluated by Western blot analysis of phospho-MYPT and diphospho-MLC levels. Reprobing with β-actin antibody was used as the normalization control. Asef depletion was verified by Western blot. Bar graphs depict quantitative densitometry analysis of Western blot data; *p < 0.05, thrombin nonspecific RNA vs. HGF + thrombin nonspecific RNA; **p < 0.05, HGF + thrombin nonspecific RNA vs. si-Asef.
Mentions: Agonist-induced activation of Rac1 protects the vascular endothelial barrier in thrombin-stimulated ECs by inhibiting the thrombin-induced Rho pathway of barrier dysfunction (Finigan et al., 2005; Birukova et al., 2007c; Tauseef et al., 2008; Baumer et al., 2009). Involvement of Asef in HGF-induced barrier protective effects against thrombin was tested in control and Asef-depleted cells. Agonist-induced permeability responses in control cells treated with nonspecific RNA and Asef-depleted EC monolayers were monitored by TER measurements. HGF exhibited a prominent protective effect against thrombin-induced TER decline in EC monolayers treated with nonspecific RNA (Figure 7A, left). In contrast, Asef knockdown impaired HGF-induced EC barrier protection against thrombin (Figure 7A, right). Effects of Asef knockdown on agonist-induced TER changes are summarized in the bar graph in Figure 7A (bottom).

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