Akt activates NOS3 and separately restores barrier integrity in H(2)O(2)-stressed human cardiac microvascular endothelium

作者：Dossumbekova Anar; Berdyshev Evgeny V; Gorshkova Irina; Shao Zuohui; Li Changqing; Long Phillip; Joshi Atul; Natarajan Viswanathan; Hoek Terry L Vanden^*

来源：American Journal of Physiology - Heart and Circulatory Physiology, 2008, 295(6): H2417-H2426.

DOI：10.1152/ajpheart.00501.2008

• oxidative stress
• nitric oxide synthase 3
• Akt/protein kinase B
• endothelial cell barrier function
• hydrogen peroxide

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 H(2)O(2)-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 mu M H(2)O(2) 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. H(2)O(2) also induced NO generation that was associated with NOS3 Ser-1177 site phosphorylation and Thr-495 dephosphorylation, with Ser-1177 effects attenuated by LY294002 and an Akt inhibitor, Akt/PKB signaling inhibitor-2 (API-2). H(2)O(2) induced significant barrier disruption in HCMVEC within minutes, but recovery started within 30 min and normalized over hours. The NOS inhibitor N(omega)-nitro-L-arginine methyl ester (200 mu M) blocked NO generation but had no effect on H(2)O(2)-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 H(2)O(2)-affected barrier function in HCMVEC. Independent of NOS3 regulation, Akt proves to be critical for the restoration of barrier integrity in HCMVEC.

• 出版日期2008-12