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This Article Full Text Full Text (PDF) All Versions of this Article: 295/6/H2417    most recent 00501.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Dossumbekova, A. Articles by Vanden Hoek, T. L. Search for Related Content PubMed PubMed Citation Articles by Dossumbekova, A. Articles by Vanden Hoek, T. L.

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

¹Emergency Resuscitation Center, Department of Medicine, and ²Section of Emergency Medicine, ³Section of Pulmonary and Critical Care, University of Chicago, Chicago, Illinois

Submitted 13 May 2008 ; accepted in final form 14 October 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 µM 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 µM) 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