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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

S1P causes longer-lasting lamellipodia protrusions.A. HUVEC expressing GFP-actin displayed frequent protrusion and withdrawal of lamellipodia, (baseline-0 min S1P) and addition of 2 μM S1P caused a coordinated increase in protrusion of lamellipodia (2 min, arrows). Within 10 min, the initial lamellipodia that had formed after S1P was added typically had withdrawn. B. S1P caused a brief, significant increase in protrusion frequency. C. Protrusion persistence also increased significantly at 10 min after S1P was added D. Withdrawal time was significantly sustained for at 10 and 20 min after S1P was added. *P<0.05 versus time 0 min (baseline). For the imaging experiments, N = 9 cells were studied.
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pone.0117970.g002: S1P causes longer-lasting lamellipodia protrusions.A. HUVEC expressing GFP-actin displayed frequent protrusion and withdrawal of lamellipodia, (baseline-0 min S1P) and addition of 2 μM S1P caused a coordinated increase in protrusion of lamellipodia (2 min, arrows). Within 10 min, the initial lamellipodia that had formed after S1P was added typically had withdrawn. B. S1P caused a brief, significant increase in protrusion frequency. C. Protrusion persistence also increased significantly at 10 min after S1P was added D. Withdrawal time was significantly sustained for at 10 and 20 min after S1P was added. *P<0.05 versus time 0 min (baseline). For the imaging experiments, N = 9 cells were studied.

Mentions: We next tested whether adding an agent that enhances endothelial barrier integrity might impact local lamellipodia in endothelial cells in a roughly opposite fashion as thrombin. We utilized S1P, a physiologically relevant bioactive lipid that reduces permeability in vivo and in vitro [41–44]. We verified that GFP-actin expression in HUVEC did not impact the ability of S1P to increase TER compared to mock-transfected cells (S3G Fig.). Addition of 2 μM S1P caused a rapid and coordinated increase in protrusion all along the edges of endothelial cells (S4 Movie), with many new local lamellipodia evident within 2 min after S1P was added (Fig. 2A). The summarized data show that protrusion significantly increased within 5 minutes of the addition of S1P, however quickly returned towards baseline after the 5-minute time point (Fig. 2B). Protrusion distance and velocity were not significantly altered by S1P treatment (S6A, B Fig.), however mean protrusion persistence, which indicates the average time for lamellipodia to continue their spreading, was significantly elevated at the 10-minute time point (Fig. 2C). Although withdrawal distance and velocity were not affected (S6C, D Fig.), S1P significantly elevated the number of lamellipodia with withdrawal times greater than 5 min, which occurs when lamellipodia withdraw more slowly or produce a net gain in surface area covered (S6E Fig.). Withdrawal time was significantly elevated compared to baseline, approximately tripled at 10 and 20 min after S1P was added (Fig. 2D). Combined, these results indicate that the rise in TER during the first five minutes after S1P addition coincides with a rise in lamellipodia protrusion frequency, and that the elevated TER that persists thereafter is associated with decreased withdrawal of local lamellipodia.


Involvement of local lamellipodia in endothelial barrier function.

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

S1P causes longer-lasting lamellipodia protrusions.A. HUVEC expressing GFP-actin displayed frequent protrusion and withdrawal of lamellipodia, (baseline-0 min S1P) and addition of 2 μM S1P caused a coordinated increase in protrusion of lamellipodia (2 min, arrows). Within 10 min, the initial lamellipodia that had formed after S1P was added typically had withdrawn. B. S1P caused a brief, significant increase in protrusion frequency. C. Protrusion persistence also increased significantly at 10 min after S1P was added D. Withdrawal time was significantly sustained for at 10 and 20 min after S1P was added. *P<0.05 versus time 0 min (baseline). For the imaging experiments, N = 9 cells were studied.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0117970.g002: S1P causes longer-lasting lamellipodia protrusions.A. HUVEC expressing GFP-actin displayed frequent protrusion and withdrawal of lamellipodia, (baseline-0 min S1P) and addition of 2 μM S1P caused a coordinated increase in protrusion of lamellipodia (2 min, arrows). Within 10 min, the initial lamellipodia that had formed after S1P was added typically had withdrawn. B. S1P caused a brief, significant increase in protrusion frequency. C. Protrusion persistence also increased significantly at 10 min after S1P was added D. Withdrawal time was significantly sustained for at 10 and 20 min after S1P was added. *P<0.05 versus time 0 min (baseline). For the imaging experiments, N = 9 cells were studied.
Mentions: We next tested whether adding an agent that enhances endothelial barrier integrity might impact local lamellipodia in endothelial cells in a roughly opposite fashion as thrombin. We utilized S1P, a physiologically relevant bioactive lipid that reduces permeability in vivo and in vitro [41–44]. We verified that GFP-actin expression in HUVEC did not impact the ability of S1P to increase TER compared to mock-transfected cells (S3G Fig.). Addition of 2 μM S1P caused a rapid and coordinated increase in protrusion all along the edges of endothelial cells (S4 Movie), with many new local lamellipodia evident within 2 min after S1P was added (Fig. 2A). The summarized data show that protrusion significantly increased within 5 minutes of the addition of S1P, however quickly returned towards baseline after the 5-minute time point (Fig. 2B). Protrusion distance and velocity were not significantly altered by S1P treatment (S6A, B Fig.), however mean protrusion persistence, which indicates the average time for lamellipodia to continue their spreading, was significantly elevated at the 10-minute time point (Fig. 2C). Although withdrawal distance and velocity were not affected (S6C, D Fig.), S1P significantly elevated the number of lamellipodia with withdrawal times greater than 5 min, which occurs when lamellipodia withdraw more slowly or produce a net gain in surface area covered (S6E Fig.). Withdrawal time was significantly elevated compared to baseline, approximately tripled at 10 and 20 min after S1P was added (Fig. 2D). Combined, these results indicate that the rise in TER during the first five minutes after S1P addition coincides with a rise in lamellipodia protrusion frequency, and that the elevated TER that persists thereafter is associated with decreased withdrawal of local lamellipodia.

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