Requirement of Rac activity for maintenance of capillary endothelial barrier properties

Abstract

Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers. In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability. In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 +/- 0.6 to 100.8 +/- 18.7 x 10-7 cm x s(-1) x cm H2O(-1) after 80 min. In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and beta-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.

Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

“…However, our results indicated this was not the case (Figure 4) and therefore we did not pursue this or the interaction between Rnd3 and p190RhoGAP further. 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.…”

Ultrastructural Analysis Reveals cAMP-Dependent Enhancement of Microvascular Endothelial Barrier Functions via Rac1-Mediated Reorganization of Intercellular Junctions

“…Cells were passaged using a kit (Detach kit-30; PromoCell) and used between passages 2 and 5. Immortalized microvascular mouse myocardial endothelial cells (MyEnd) 17,18 were cultured on collagen 1-coated culture flasks and maintained in Dulbecco's modified Eagle's medium (SigmaAldrich, Munich, Germany) containing 10% fetal calf serum and 50 U/mL penicillin/50 g streptomycin. Primary porcine pulmonary artery endothelial cells (PAECs) were isolated by collagenase A treatment, as described elsewhere.…”

“…Wistar rats with a body weight ranging from 350 g to 450 g (Charles River Laboratories, Sulzfeld, Germany) were anesthetized with pentobarbital sodium (65 mg/kg body weight) and prepared as previously described. 18 Briefly, the rat was placed on a heating pad with the mesentery pulled out and spread on a pillar after a midline incision. The mesentery was constantly suffused with Ringer's solution at 37°C.…”

“…As previously described, measurements of hydraulic conductivity (L p ) were based on the modified Landis technique, which measures the volume flux per unit surface area across the wall of a perfused microvessel. 2,18,21 Measurements were performed at approximately 10-minute intervals for up to 120 minutes. All animals were used for one experiment only.…”

Allosteric Regulation of E-Cadherin Adhesion

“…The strengthening of cadherin-mediated intercellular adhesion has been attributed to several mechanisms, including GTPase activity (27)(28)(29)(30)(31), enhanced cadherin-cytoskeletal interactions (5,(32)(33)(34)(35), cadherin catch bonds (36), cadherin clustering (19,37,38), and altered cortical tension (5,6). Demonstrating that Colo 205 aggregation was caused by the allosteric regulation of E-cadherin required a demonstration that specific perturbations, which do not affect the binding site directly, caused quantitative changes in the E-cadherin affinity.…”