Involvement of Local Lamellipodia in Endothelial Barrier Function

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.

Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

“…As alternatives, we investigated Rac1 and Cdc42, 2 other key Rho family small GTPases that contribute to the control of endothelial barrier function 22, 24, 77, 78, 79. Thrombin caused a rapid, significant decrease in Rac1‐GTP in agreement with several previous reports 17, 78, 80, 81. However, thrombin did not activate Cdc42 in HUVEC, in contrast to one previous report that described a delayed activation of Cdc42 in immortalized human dermal microvascular endothelial cells 71.…”

“…In addition, coordinated, cyclic activity of Rac1 and RhoA are known to control the protrusion and withdrawal of cell protrusions 83. Recently, we reported that treatment of endothelial cells with either thrombin or a selective inhibitor of Rac1 can decrease the protrusion frequency of small, local lamellipodia in conjunction with disruption of the endothelial barrier 17. These protrusions, also termed junctional‐associated intermittent lamellipodia by other investigators,84, 85 are also disrupted by histamine and appear to control rapid changes in permeability by altering the overlap of adjacent endothelial cells 18.…”

“…These were determined as previously described17 using the RhoA and Rac1G‐LISA ™ activation assay luminescence kits and Cdc42G‐LISA ™ activation assay colorimetric kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer's instructions. HUVEC were transfected with plasmid or siRNA (or appropriate controls) and cultured for 2 days on 60‐mm plates.…”

“…Experiments were performed as previously described17, 18, 39 using a Nikon Eclipse TE2000U inverted microscope system (Nikon Instruments, Melville, NY). Briefly, HUVEC expressing GFP‐actin, with or without co‐expression of mCherryRnd3, were perfused with warm (37°C) albumin–physiological salt solution (APSS; NaCl, 120 mmol/L; KCl, 4.7 mmol/L; CaCl 2 ·2H 2 O, 2 mmol/L; MgSO 4 ·7H 2 O, 1.2 mmol/L; NaH 2 PO 4 , 1.2 mmol/L; Na pyruvate, 2 mmol/L; glucose, 5 mmol/L; EDTA, 0.02 mmol/L; MOPS, 3 mmol/L; purified BSA 1 g/100 mL; pH 7.4).…”

ADAMTS13 controls vascular remodeling by modifying VWF reactivity during stroke recovery

“…Together with these findings, the BBB tight junction proteins including ZO-1, occludin, and claudin 5 in brain microvessels were reduced ( Figure 2F; supplemental Figure 2E-G), and collagen IV 1 basement membrane coverage was 54% less in Adamts13 2/2 mice ( Figure 2G). In agreement with this result, actin stress fiber formation, which is associated with irregular endothelial junctions and elevated endothelial permeability, 35 was increased by 2.2-fold in Adamts13 2/2 mice ( Figure 2H). In addition, Adamts13 …”