Bioanalytical and proteomic approaches in the study of pathologic ECs dysfunctionality, oxidative stress and the effect of PFKFB3 modulators

Abstract

The vascular endothelium is one of the major targets of oxidative stress, playing an important role in the development of vascular-related disorders, such as atherosclerotic disease1. Oxidative stress increases the formation of reactive species (ROS), such as peroxides and hydroxyl radicals. ROS are signalling molecules and are able to induce leukocyte adhesion and promote the vascular endothelial permeability, that leads to the dysfunction of endothelium- related signal transduction pathways as well as redox-regulated transcription factors2. ROS cause reorganization of the actin filament, formation of an intracellular gap and changes in the cell shape. The endothelium contains adherents and tight junctions that are responsible for maintaining the restrictive barrier. Figure A1 shows the subcellular endothelial sites regulated by oxidative stress. In the normal structure of vascular endothelium, the lateral cell border contains occluding protein in association with zonula occludens-1 (ZO-1); VE-cadherin in association with a-, b-, and g-catenins; other proteins such as platelet endothelial cell adhesion molecule 1 (PECAM-1). Cortical actin filaments are directly linked with ZO-1, vinculin and α-actinin, that connect catenin to VE-cadherin in actin filament network. Intercellular adhesion molecule-1 (ICAM-1) expressed on the endothelial luminal surface might also involve in cortical actin filaments. ECs continuously express surface adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), ICAM-1, ICAM-2 and P-selectin (stored in Weible-Palade bodies). However, oxidative stress causes phosphorylation and reorganization of occludin and PECAM-1, reduces levels of vascular endothelium catenins and actin-binding proteins. In the actin filament network, oxidant stress causes the increased formation of stress fibers and decreased cortical actin band, resulting in disruption of the tight and adherens junctions. ROS promote cell contraction and stress fiber formation due to the increased phosphorylation of myosin light chain (MLC). Additionally, oxidative stress functionally upregulates adhesion molecules