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.