The role of p-rex2 protein in the dysfunction of the endothelial barrier

Abstract

The endothelial barrier is formed by tightly integrated endothelial cells (ECs), which loosen their interaction and then recover again in a controlled way to open paracellular gaps for the transport of leukocytes and macromolecules across the endothelium. Endothelial barrier dysfunction occurs when this reversible mechanism is disrupted, and it leads to various pathological conditions due to the accumulation of cells and molecules within the surrounding tissues. The endothelial barrier is stabilized by Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of the Rho family of GTPases. The Rac1 also regulates enzyme complex nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, uncontrolled activation of which leads to over-production of reactive oxygen species (ROS) and thus dysfunction of the endothelial barrier. The guanine nucleotide exchange factors (GEFs) such as phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-Rex1), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 2 (P-Rex2), and T-lymphoma invasion and metastasis-inducing protein 1 (Tiam1) control the activity of Rac1 in ECs. The present study investigated the effects of inflammatory conditions on Rac1 activation to reveal the molecular pathway directing Rac1 towards its role in endothelial barrier dysfunction. Our study demonstrated that P-Rex2 overexpression reduced the integrity of the endothelial barrier by downregulating the assembly of vascular endothelial cadherin (VE-cadherin) Adherens junctions (AJs) and actin at the cell-cell junctions, and TNF-α dramatically enhanced the effects of P-Rex2 on the EC barrier. Consistently, P-Rex2 overexpression resulted in increased barrier permeability. Furthermore, our findings showed that P-Rex2-mediated barrier disruption in response to TNF-α was mediated via Rac1 activation. Under basal conditions, the pharmacological and biological intervention of Rac1 activation using NSC23766 and dominant-negative Rac1 (Rac1-DN) resulted in gap formation, dramatically increased stress fibers, and failed to protect endothelial barrier against TNF-α. In contrast to that, P-Rex2 overexpression directed Rac1 to an opposite role. Inhibition of Rac1 activation in P-Rex2 overexpressing cells recovered endothelial barrier via reassembly of AJs and peripheral actin. Moreover, it was protective against TNF-α induced barrier disruption. Similarly, expression of constitutively active Rac1 (Rac1-CA) was protective for the barrier under basal conditions, but it dramatically enhanced actin stress fibers in P-Rex2 overexpressing ECs, supporting the role of Rac1 in the P-Rex2-mediated EC barrier dysfunction. In accordance with these findings, P-Rex2 overexpression induced EC migration while triggering in vitro sprouting in ECs. Chemical inhibition of Rac1 attenuated EC migration only in the P-Rex2 overexpressing cells, indicating that P-Rex2 mediated EC migration is Rac1-dependent. Additionally, P-Rex2 overexpression significantly upregulated intracellular ROS production in ECs. Inhibition of ROS using ROS scavenger N-Acetyl-L-cysteine (NAC) protected the EC barrier against P-Rex2 mediated loss of VE-cadherin from cell-cell junctions and increased localization of actin at the cell periphery. Interestingly, the NADPH oxidase inhibitor VAS2870 prevented barrier loss caused by P-Rex2 overexpression in a concentration-dependent manner both with and without TNF-α stimuli, indicating that P-Rex2 leads to TNF-α-induced NADPH oxidase-dependent ROS production in ECs. In conclusion, the data of the presented study demonstrates that P-Rex2 regulates endothelial barrier dysfunction downstream of TNF- α signaling via activating Rac1. As a result of Rac1 activation, NADPH oxidase-dependent excessive ROS production causes rearrangement of AJs and actin dynamics leading to gap formation between adjacent ECs, which in turn increases barrier permeability, endothelial migration, and sprouting. P-Rex2 may offer an important target to prevent inflammation-induced ROS production and endothelial dysfunction. Manipulation of P-Rex2 activity might be a therapeutic approach to maintain barrier stabilization.