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Involvement of local lamellipodia in endothelial barrier function.

Breslin JW, Zhang XE, Worthylake RA, Souza-Smith FM - PLoS ONE (2015)

Bottom Line: Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules.Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity.Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

No MeSH data available.


Related in: MedlinePlus

Thrombin and S1P increase phosphorylation of MLC on Thr-18/Ser-19.Confocal images of immunofluorescence labeling of dually phosphorylated myosin within HUVEC monolayers are shown. The cells were either untreated controls (A) or treated with 1 U/ml thrombin (B, C) or 2 μM S1P (D, E, F) for the durations indicated in each panel. Scale bar = 50 μm. Representative of three separate experiments.
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pone.0117970.g006: Thrombin and S1P increase phosphorylation of MLC on Thr-18/Ser-19.Confocal images of immunofluorescence labeling of dually phosphorylated myosin within HUVEC monolayers are shown. The cells were either untreated controls (A) or treated with 1 U/ml thrombin (B, C) or 2 μM S1P (D, E, F) for the durations indicated in each panel. Scale bar = 50 μm. Representative of three separate experiments.

Mentions: Increased phosphorylation of MLC has previously been reported to contribute to microvascular hyperpermeability in response to certain inflammatory stimuli [11,48], but also may participate in barrier enhancement [49]. Thus, we evaluated whether the differential impacts of thrombin and S1P on endothelial barrier integrity may be due to their impacts on MLC phosphorylation, particularly localization. We found, however, that both thrombin and S1P increased phosphorylation of MLC on T18/S19 (Fig. 6). For both thrombin and S1P, most of the phosphorylated MLC localized on actin fibers. These were mostly the cortical actin fibers parallel to edges of cells, although thrombin also caused some appearance of stress fibers as well (Fig. 6). This was confirmed with co-labeling using Texas-Red phalloidin (data not shown). From these data there does not appear to be a correlation between MLC phosphorylation and barrier function.


Involvement of local lamellipodia in endothelial barrier function.

Breslin JW, Zhang XE, Worthylake RA, Souza-Smith FM - PLoS ONE (2015)

Thrombin and S1P increase phosphorylation of MLC on Thr-18/Ser-19.Confocal images of immunofluorescence labeling of dually phosphorylated myosin within HUVEC monolayers are shown. The cells were either untreated controls (A) or treated with 1 U/ml thrombin (B, C) or 2 μM S1P (D, E, F) for the durations indicated in each panel. Scale bar = 50 μm. Representative of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320108&req=5

pone.0117970.g006: Thrombin and S1P increase phosphorylation of MLC on Thr-18/Ser-19.Confocal images of immunofluorescence labeling of dually phosphorylated myosin within HUVEC monolayers are shown. The cells were either untreated controls (A) or treated with 1 U/ml thrombin (B, C) or 2 μM S1P (D, E, F) for the durations indicated in each panel. Scale bar = 50 μm. Representative of three separate experiments.
Mentions: Increased phosphorylation of MLC has previously been reported to contribute to microvascular hyperpermeability in response to certain inflammatory stimuli [11,48], but also may participate in barrier enhancement [49]. Thus, we evaluated whether the differential impacts of thrombin and S1P on endothelial barrier integrity may be due to their impacts on MLC phosphorylation, particularly localization. We found, however, that both thrombin and S1P increased phosphorylation of MLC on T18/S19 (Fig. 6). For both thrombin and S1P, most of the phosphorylated MLC localized on actin fibers. These were mostly the cortical actin fibers parallel to edges of cells, although thrombin also caused some appearance of stress fibers as well (Fig. 6). This was confirmed with co-labeling using Texas-Red phalloidin (data not shown). From these data there does not appear to be a correlation between MLC phosphorylation and barrier function.

Bottom Line: Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules.Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity.Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

No MeSH data available.


Related in: MedlinePlus