Effects of Cytotoxic Necrotizing Factor 1 and Lethal Toxin on Actin Cytoskeleton and VE-Cadherin Localization in Human Endothelial Cell Monolayers

Abstract

Integrity of the vascular endothelium is largely dependent on endothelial cell shape and establishment of intercellular junctions. Certain pathogenic bacterial toxins alter the cytoskeletal architecture of intoxicated cells by modulating the GTPase activity of p21 Rho family proteins. In the present study we have analyzed the effect of Rho-directed toxins on the actin cytoskeleton and monolayer integrity of endothelial cells. We report here that Escherichia colicytotoxic necrotizing factor 1 (CNF1) activates Rho in human umbilical vein endothelial cells (HUVEC). In confluent monolayers, CNF1 treatment induces prominent stress fiber formation without significantly modifying peripheral localization of VE-cadherin, a specific marker of vascular endothelial cell adherens junctions. Further, Rho activation with CNF1 blocks thrombin-induced redistribution of VE-cadherin staining and gap formation in HUVEC monolayers. Inhibition of Rho by prolonged treatment of cells with C3 exoenzyme (Clostridium botulinum) eliminates actin stress fibers without disrupting the continuity of VE-cadherin staining, indicating that Rho-dependent stress fibers are not required for maintaining this adhesion receptor at sites of intercellular contact. Lethal toxin (Clostridium sordellii), an inhibitor of Rac as well as Ras and Rap, potently disrupts the actin microfilament system and monolayer integrity in HUVEC cultures.

Modification of epithelial cell barrier permeability and intercellular junctions by Clostridium sordellii lethal toxins

“…The exact pathway between Rac and cortical actin polymerization which is inhibited by LTs remains to be defined. It is noteworthy that LTs also induce a disorganization of AJs in endothelial cells which involves a Rac‐dependent actin depolymerization and a Rac pathway independent of actin dissociation (Vouret‐Craviari et al ., 1999; Waschke et al ., 2004; Waschke et al ., 2005).…”

Endothelial Cell-to-Cell Junctions: Molecular Organization and Role in Vascular Homeostasis

“…An important issue is how permeability-increasing agents modify junctional architecture and eventually create intercellular gaps. Cell retraction is certainly an intuitive mechanism, which has been observed in cultured cells upon exposure to thrombin, histamine, plasmin, and other permeability-increasing agents (9,68,84,92,130,288,298,338) or in vivo in inflamed vessels (26). More subtle changes, such as phosphorylation of junctional proteins and their dissociation from the actin cytoskeleton, may result in increased permeability without frank appearance of intercellular gaps (9).…”

“…Although apparently dispensable for the formation and maintenance of VE-cadherin at AJ in the endothelium, small GTPases play an important role in the action of permeability-increasing agents. Thrombin strongly increases RhoA and Rac activation (329,330,338,351), and this causes endothelial cell rounding and retraction, as well as increase in permeability (92,130,338). However, Rho activation alone is not sufficient to increase endothelial permeability.…”

Clostridium sordellii Lethal Toxin Kills Mice by Inducing a Major Increase in Lung Vascular Permeability

“…50 Actin cytoskeleton is in tight interaction with specific structures at the cell periphery involved in cell-cell contacts such as tight junctions and adherens junctions. The involvement of the Rho proteins in the regulation of cell-cell junctions in endothelial cells has been investigated either in ex vivo endothelial cells from human umbilical vein and porcine pulmonary arteria (human umbilical vein endothelial cells and porcine aortic endothelial cells) [51][52][53][54] or in immortalized endothelial cell lines from human or murine myocardial vessels. 55,56 Rho and Rac are the main regulators of the cell Figure 8.…”