Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

Yuan Zhang,Wenlan Liu,Weiping Li,Ting Wang,Ji Xu,Lijie Ren,Ke Yang

doi:10.3389/fnmol.2016.00079

Abstract

Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1’s downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke.

Highlights

MATERIALS AND METHODSIschemic stroke is a leading cause of disability and mortality in humans
Our data showed that oxygen-glucose deprivation (OGD) induced a significant increase in the protein levels of Enolase-phosphatase 1 (ENOPH1) and its downstream molecule acireductone dioxygenase 1 (ADI1) in cultured brain endothelial cells, which led to increased reactive oxygen species (ROS) generation, endothelial cell apoptosis and increased permeability of endothelial monolayer
To determine whether ENOPH1 plays a role in ischemic blood brain barrier (BBB) injury, we first examined the change of ENOPH1 expression in the BBB, i.e., the cerebral microvessels or capillaries, isolated from the rats that were subjected to 3 h middle cerebral artery occlusion (MCAO) without reperfusion

Summary

Introduction

MATERIALS AND METHODSIschemic stroke is a leading cause of disability and mortality in humans. Western blot analysis showed that ENOPH1 protein levels were significantly increased in bEND3 cells after exposing to OGD for 3 h, but not for 1 h (Figure 2B).

Results

Conclusion