Hypoxia–reoxygenation‐induced endothelial barrier failure: role of RhoA, Rac1 and myosin light chain kinase

Abstract

Key points• Hypoxia-reoxygenation induces loss of endothelial barrier function and oedema formation accompanied by a rise in intracellular Ca 2+ , an increase in myosin light chain (MLC) phosphorylation, and RhoA/Rho kinase (Rock) signalling and an inactivation of Rac1.• Neither inhibition of RhoA/Rock signalling nor antagonising Ca 2+ increase could protect against this hypoxia-reoxygenation-induced loss of barrier function.• Inhibition of MLC kinase (MLCK) abrogates hypoxia-reoxygenation-induced MLC phosphorylation and partially protects against hypoxia-reoxygenation-induced endothelial hyperpermeability.• Activation of Rac1 using a cAMP analogue, 8-CPT-O -Me-cAMP, which specifically activates Epac/Rap1 signalling abrogated reoxygenation-induced hyperpermeability. The data help us to better understand the role of Rho GTPases and contractile machinery in the regulation of endothelial barrier function during hypoxia-reoxygenation.Abstract Hypoxia-reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho-GTPases under hypoxia-reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia-reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia-reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE-cadherin from cellular junctions. Inhibition of contractile activation with ML-7 partially protected against hypoxia-reoxygenation-induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia-reoxygenation-induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8-CPT-O -Me-cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE-cadherin at cellular junctions and abrogated reoxygenation-induced hyperpermeability. Similar results were reproduced in isolated saline-perfused rat hearts. These data show that activation of Rac1 but not the inhibition of RhoA preserves endothelial integrity against reoxygenation-induced loss of barrier function.

Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1α in Cofilin Regulation and Cytoplasmic Actin Distribution

“…Interestingly it has been shown in different cell types that hypoxia influences members of the Rho family of GTPases [10]–[14], which are master regulators of the actin cytoskeleton [15], [16]. Besides cell motility the actin cytoskeleton governs many other cellular activities like cytokinesis, endocytosis, cell adhesion and cell shape [17]–[20].…”

The Coronary Circulation as a Target of Cardioprotection

“…65 The endothelial barrier function is made up by the glycocalyx, 66 endothelial cells, and pericytes (particularly at the postcapillary venules 67 ). Endothelial cytoskeletal derangement and hypercontracture induce gap formation, [68][69][70][71] which is enhanced by extracellular adenosine but attenuated by extracellular ATP. 72 Degradation of the glycocalyx also contributes to reduced endothelial barrier function and edema formation 66,73,74 ; tumor necrosis factor α is an important mediator of glycocalyx degradation, 75 and glycocalyx degradation also promotes leukocyte 76 and platelet adherence.…”

Overexpression of Hypoxia-Inducible Factor-1a Exacerbates Endothelial Barrier Dysfunction Induced by Hypoxia

“…Upon activation, HIF-1α regulates a wide range of genes involved in many cellular processes such as angiogenesis, glycolytic energy metabolism, oxygen delivery, cell proliferation, and survival, which collectively lead to the adaptive response of cells and tissues [10,11,12,13,14]. Previous reports have shown that the expression of HIF-1α protein is remarkably increased in endothelial cells exposed to hypoxia [15,30,31]. In accordance with the data from these previous studies, our present study demonstrates that hypoxia treatment induced a significantly increased protein expression of HIF-1α and GLUT-1, an HIF-1 target gene.…”

“…Previous studies have demonstrated that HIF-1α is involved in barrier dysfunction by inducing the expression of HIF-1 target genes such as vascular endothelial growth factor, inducible nitric oxide synthase [38,40,46], aquaporin-4, matrix metalloproteinase-9 [39], macrophage migration inhibitory factor [47], as well as by repressing the expression of vasodilator-stimulated phosphoprotein [48]. However, the activation of MLCK, which in turn leads to an increase of MLC phosphorylation, has been reported to play a critical role in endothelial and intestinal barrier dysfunction induced by hypoxia or inflammation [21,22,23,24,28,29,31,49]. In addition, our previous studies have revealed that HIF-1α-dependent up-regulation of MLCK protein is involved in barrier dysfunction induced by hypoxia or proinflammatory cytokines [15,25,26].…”