Involvement of Local Lamellipodia in Endothelial Barrier Function

<div><p>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.</p></div

Impact of the myosin II inhibitor blebbistatin on endothelial lamellipodia and barrier function.

<p><i>A</i>. Local lamellipodia protrusion frequency in HUVEC expressing GFP-actin. At time = 0 min, cells were treated with 100 μM of (-)blebbistatin, or the inactive control, (+)blebbistatin. *P<0.05 between groups, same time point. N = 9 cells per group. <i>B</i>. Changes in TER in response to 100 μM (-) or (+)blebbistatin, added at time = 0 min (arrow). N = 4 each group. <i>C</i>. Time course of changes in <i>P</i>_s^albumin in response to 100 μM (-) or (+)blebbistatin. *P<0.05 between groups, same time point.</p

S1P causes longer-lasting lamellipodia protrusions.

<p><i>A</i>. 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. <i>B</i>. S1P caused a brief, significant increase in protrusion frequency. <i>C</i>. Protrusion persistence also increased significantly at 10 min after S1P was added <i>D</i>. 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.</p

Inhibition of Rac1 decreases lamellipodia formation and increases endothelial permeability.

<p><i>A</i>. Rac1-GTP levels in control HUVEC and cells treated with 50 μM NSC23766 for 30 min. <i>B</i>. Time course of mean changes in TER caused by 50 μM NSC23766 (N = 8), compared to control (N = 8). <i>C</i>. Time course of changes in lamellipodia protrusion frequency after the addition of 50 μM NSC23766 (N = 9 cells studied. *P<0.05 versus baseline (BL). <i>D</i>. Time course of changes in the permeability of isolated rat mesenteric venules to albumin in response to 50 μM NSC23766 (N = 4) compared to control (N = 4). *P<0.05 versus control, same time point.</p

Impact of overexpression of wild-type (WT) or dominant-negative (DN) Rac1 on endothelial barrier function and local lamellipodia dynamics.

<p><i>A</i>. <i>P</i>_s^albumin of HUVEC monolayers expressing GFP, GFP-Rac1-WT, or GFP-Rac1-DN (N = 4 for each group) ~16 h after transfection. Panels <i>B</i>, <i>C</i>, and <i>D</i> show expression of each construct, also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117970#pone.0117970.s022" target="_blank">S11 Movie</a>. These images were obtained ~16 h after transfection. The small arrows indicate lamellipodia, while the arrows with wider arrowheads show filopodia that were prevalent in cells expressing GFP-Rac1-DN. Lamellipodia parameters were also evaluated over a 10-min period: <i>E</i>. Protrusion frequency, <i>F</i>. Protrusion distance, <i>G</i>. Withdrawal Time, <i>H</i>. %Protrusions with a withdrawal time > 5 min. *P<0.05 between the indicated groups. N = 9 cells studied in each group.</p

Impact of S1P during thrombin-induced endothelial barrier dysfunction.

<p><i>A</i>. Time course of changes in TER of HUVEC monolayers treated with 1 U/ml thrombin or vehicle at the indicated time point, followed by addition of 2 μM S1P or vehicle 20 min. later. The TER tracings are an average for N = 8 electrode wells in each group. <i>B</i>. Protrusion frequency of HUVEC expressing GFP-actin treated with 1 U/ml thrombin, followed by 2 μM S1P 20 min later. *P<0.05 versus the 0 min time point when thrombin was added. †P<0.05 versus the 20 min time point when S1P was added. N = 9 cells studied.</p

Thrombin-induced decreased lamellipodia protrusion frequency and actin stress fiber formation.

<p><i>A</i>. HUVEC expressing GFP-actin displayed frequent formation and withdrawal of local lamellipodia (small arrows) and actin “clouds” (large arrowhead) during baseline recording. Shortly after addition of thrombin (1 U/ml; 3 min image) there was a coordinated increase in actin at the periphery of cells (thin arrowheads) and around vesicles (very small arrows) followed by a sharp decline in protrusive activity. Later (27.5 min image), ventral stress fibers (SF) formed de novo in the cells, and cortical actin migrated to become transverse arc (TA) stress fibers. Also, in this image a transient opening at a tricellular junction (*) is apparent. <i>B</i>. Thrombin initially decreased the mean protrusion frequency of local lamellipodia. <i>C</i>. The number of actin fibers significantly increased at 30 min after the addition of 1 U/ml thrombin. *P<0.05 versus the zero-minute time point. N = 9 cells studied in the imaging experiments.</p

GTP-bound RhoA, Rac1, and Cdc42 levels in response to thrombin and S1P.

<p><i>A</i>. Impact of thrombin on GTP-bound RhoA, Rac1, and Cdc42 in cultured HUVEC. Untreated cells served as control. <i>B</i>. Impact of S1P on GTP-bound RhoA, Rac1, and Cdc42 in cultured HUVEC. Untreated cells served as control, and vehicle controls were also tested at the 0.5-min and 10-min time points. The numbers in parentheses indicate the number of replicates for each group. *P<0.05 compared to control (no treatment).</p

Thrombin and S1P increase phosphorylation of MLC on Thr-18/Ser-19.

<p>Confocal images of immunofluorescence labeling of dually phosphorylated myosin within HUVEC monolayers are shown. The cells were either untreated controls (<i>A</i>) or treated with 1 U/ml thrombin (<i>B</i>, <i>C</i>) or 2 μM S1P (<i>D</i>, <i>E</i>, <i>F</i>) for the durations indicated in each panel. Scale bar = 50 μm. Representative of three separate experiments.</p

Discovery of a Series of Thiazole Derivatives as Novel Inhibitors of Metastatic Cancer Cell Migration and Invasion

Effective inhibitors of cancer cell migration and invasion can potentially lead to clinical applications as a therapy to block tumor metastasis, the primary cause of death in cancer patients. To this end, we have designed and synthesized a series of thiazole derivatives that showed potent efficacy against cell migration and invasion in metastatic cancer cells. The most effective compound, <b>5k</b>, was found to have an IC₅₀ value of 176 nM in the dose-dependent transwell migration assays in MDA-MB-231cells. At a dose of 10 μM, <b>5k</b> also blocked about 80% of migration in HeLa and A549 cells and 60% of invasion of MDA-MB-231 cells. Importantly, the majority of the derivatives exhibited no apparent cytotoxicity in the clonogenic assays. The low to negligible inhibition of cell proliferation is a desirable property of these antimigration derivatives because they hold promise of low toxicity to healthy cells as potential therapeutic agents. Mechanistic studies analyzing the actin cytoskeleton by microscopy demonstrate that compound <b>5k</b> substantially reduced cellular f-actin and prevented localization of fascin to actin-rich membrane protrusions. These results suggest that the antimigration activity may result from impaired actin structures in protrusions that are necessary to drive migration