Akt activates NOS3 and separately restores barrier integrity in H₂O₂-stressed human cardiac microvascular endothelium

Abstract

Dossumbekova A, Berdyshev EV, Gorshkova I, Shao Z, Li C, Long P, Joshi A, Natarajan V, Vanden Hoek TL. Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium. Am J Physiol Heart Circ Physiol 295: H2417-H2426, 2008. First published October 17, 2008 doi:10.1152/ajpheart.00501.2008.-The integrity of microvascular endothelium is an important regulator of myocardial contractility. Microvascular barrier integrity could be altered by increased reactive oxygen species (ROS) stress seen within minutes after cardiac arrest resuscitation. Akt and its downstream target nitric oxide (NO) synthase (NOS)3 can protect barrier integrity during ROS stress, but little work has studied these oxidant stress responses in human cardiac microvascular endothelial cells (HCMVEC). We, therefore, studied how ROS affects barrier function and NO generation via Akt and its downstream target NOS3 in HCMVEC. HCMVEC exposed to 500 M H2O2 had increased Akt phosphorylation within 10 min at both Ser-473 and Thr-308 sites, an effect blocked by the phosphatidylinositol 3-kinase inhibitor LY-294002. H2O2 also induced NO generation that was associated with NOS3 Ser-1177 site phosphorylation and Thr-495 dephosphorylation, with Ser-1177 effects attenuated by LY-294002 and an Akt inhibitor, Akt/PKB signaling inhibitor-2 (API-2). H2O2 induced significant barrier disruption in HCMVEC within minutes, but recovery started within 30 min and normalized over hours. The NOS inhibitor N -nitro-L-arginine methyl ester (200 M) blocked NO generation but had no effect on H2O2-induced barrier permeability or the recovery of barrier integrity. By contrast, the Akt inhibitor API-2 abrogated HCMVEC barrier restoration. These results suggest that oxidant stress in HCMVEC activates NOS3 via Akt. NOS3/NO are not involved in the regulation of H2O2-affected barrier function in HCMVEC. Independent of NOS3 regulation, Akt proves to be critical for the restoration of barrier integrity in HCMVEC. oxidative stress; nitric oxide synthase 3; Akt/protein kinase B; endothelial cell barrier function; hydrogen peroxide MICROCIRCULATORY DYSFUNCTION is an important etiological component of ischemia-reperfusion injury. A significant percentage of victims of cardiac arrest, despite being initially resuscitated, die of heart-related causes within hours due to recurrent arrhythmias and cardiovascular collapse (4,23,28,47). Recent work suggests that these cardiac arrest events are temporally associated with interruptions in microvascular blood flow to the heart that begin within minutes of cardiopulmonary resuscitation (CPR) and may affect the return of spontaneous circulation (ROSC) (17). Furthermore, serious microvascular dysfunction in critical organs such as the heart and brain is seen within hours of ROSC and is associated with critical organ dysfunction (1, 8).The mechanism of microvascular endothelial dysfunction in the post-cardiac arrest heart is not well known. Our studies in a swine model of cardiac arrest suggest that o...

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