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

Key points • Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema formation accompanied by a rise in intracellular Ca2+, 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 Ca2+ 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. 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 activate