Abstract 149: Endothelial NADPH Oxidase 4 Worsens Ischemic Stroke by Suppressing eNOS Activity
NADPH oxidase (Nox) family proteins are a major source of reactive oxygen species (ROS) and play various important roles in immune systems, arteriosclerosis, angiogenesis and aging. We have previously demonstrated that among the Nox proteins, Nox4 is abundantly expressed and is a major source of ROS in endothelial cells; however, its roles in endothelial cells are not fully understood. In the present study, we investigated the roles of endothelial Nox4 in brain ischemia, using mice with endothelial cell-specific Nox4 overexpression (Tg-EC-Nox4) and cultured human brain microvascular endothelial cells (HMEC) overexpressing Nox4.
Nox4 was abundantly expressed and was significantly upregulated in response to hypoxia at O2 1% (2.0-fold increase, p<0.001) in HMEC. Immunofluorescent double staining demonstrated that in a middle cerebral occlusion model (MCAO) of wild type mice, Nox4 was markedly upregulated in endothelial cells in peri-infarct area. We subjected Tg-EC-Nox4 mice and their littermates to either transient (ischemia 90 min and reperfusion) or permanent MCAO. Triphenyltetrazolium chloride staining demonstrated that infarct volume at 1 day after MCAO was significantly larger in Tg-EC-Nox4 than wild-type mice in both the models (transient MCAO: 151 %, permanent MCAO: 132 %, p<0.05). We overexpressed Nox4 by adenovirus in HMEC. In the endothelial cells overexpressing Nox4, angiopoietin-2 (Ang-2) was drastically upregulated (30-fold increase, p<0.0001) at mRNA levels and the phosphorylation of Akt at Ser-473 (47 %, p<0.01) and endothelial nitric oxide synthase (eNOS) at Ser-1177 (43 %, p<0.01) were significantly attenuated. Treatment with Ang-2 suppressed the phosphorylation of Akt and eNOS induced by angiopoietin-1 in a dose-dependent manner in HMEC. Consistently, Ang-2 mRNA was significantly increased (2.0-fold, p<0.05), and the phosphorylation of Akt (37 %, p<0.05) and eNOS (41 %, p=0.12) were suppressed in the ischemic hemisphere of Tg-EC-Nox4, compared to those of wild-type mice. In conclusion, the overexpression of endothelial Nox4 may increase infarct volume by suppressing eNOS activity through the upregulation of Ang-2 in brain ischemia.
- © 2012 by American Heart Association, Inc.