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Oxygen-Glucose Deprivation/Reoxygenation Induces Human Brain Microvascular Endothelial Cell Hyperpermeability Via VE-Cadherin Internalization: Roles of RhoA/ROCK2

  • Jie Chen
  • Li Sun
  • Gui-bing Ding
  • Liang Chen
  • Lei Jiang
  • Jun WangEmail author
  • Jin WuEmail author
Article

Abstract

The destruction of the blood-brain barrier (BBB) contributes to a spectrum of neurological diseases such as stroke, and the hyperpermeability of endothelial cells is one of the characters of stroke, which is possibly exacerbated after reperfusion. However, the underlying mechanisms involving hyperpermeability after reperfusion between the endothelial cells remain poorly understood. Therefore, in the present study, the human microvascular endothelial cells (HBMECs) were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) to mimic ischemic stroke condition in vitro with the aim to investigate the potential mechanisms induced by OGD/R. The permeability of cultured HBMECs was measured using FITC-labeled dextran in a Transwell system and transendothelial electrical resistance (TEER), while the RhoA activity was detected by pull-down assay. In addition, the phosphorylation of MYPT1, which reflects the activation of ROCK and the internalization of VE-cadherin, was detected by Western blot. It showed that OGD/R treatment significantly increased the permeability of HBMEC monolayers and facilitated the internalization of VE-cadherin in HBMEC monolayers. Pull-down assay showed that RhoA activation was obviously enhanced after OGD/R treatment, while RhoA and ROCK inhibitor significantly reversed OGD/R-induced HBMEC monolayers hyperpermeability and the internalization of VE-cadherin. Meanwhile, the knockdown assay showed that RhoA small interfering RNA (siRNA) led to similar effects. The inactivation of the downstream effector protein ROCK was also examined. Intriguingly, ROCK2 rather than ROCK1 exerted its adverse effects on HBMEC monolayer integrity, since ROCK2 knockdown markedly reverses the injury of OGD/R in HBMEC monolayers. In conclusion, the present study provides evidence that OGD/R may induce HBMEC monolayer hyperpermeability via RhoA/ROCK2-mediated VE-cadherin internalization, which may provide an impetus for the development of therapeutics targeting BBB damage in ischemic stroke.

Keywords

Oxygen-glucose deprivation and reoxygenation HBMECs permeability VE-cadherin RhoA/ROCK signaling pathway 

Notes

Acknowledgments

We thank Professor Gui bo (Department of Anesthesiology, 1st Affiliated Hospital of Nanjing Medical University, China) for his selfless help in the research process.

Funding

This work was supported by the National Natural Science Foundation of China (81701872) and by a grant from the Nanjing Medical University Science and Technology Development Fund Project (2015NJMUZD010).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Neurologythe Second Affiliated Hospital of Nanjing Medical UniversityNanjingChina
  2. 2.Department of Neurologythe First Affiliated Hospital of Nanjing Medical UniversityNanjingChina
  3. 3.Department of Emergency Medicinethe First Affiliated Hospital of Nanjing Medical UniversityNanjingChina
  4. 4.The Laboratory of Neurotoxicology, School of Public HealthNanjing Medical UniversityNanjingChina

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