Activation of VEGF/Flk-1-ERK Pathway Induced Blood–Brain Barrier Injury After Microwave Exposure

Abstract

Microwaves have been suggested to induce neuronal injury and increase permeability of the blood–brain barrier (BBB), but the mechanism remains unknown. The role of the vascular endothelial growth factor (VEGF)/Flk-1-Raf/MAPK kinase (MEK)/extracellular-regulated protein kinase (ERK) pathway in structural and functional injury of the blood–brain barrier (BBB) following microwave exposure was examined. An in vitro BBB model composed of the ECV304 cell line and primary rat cerebral astrocytes was exposed to microwave radiation (50 mW/cm2, 5 min). The structure was observed by scanning electron microscopy (SEM) and the permeability was assessed by measuring transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) transmission. Activity and expression of VEGF/Flk-1-ERK pathway components and occludin also were examined. Our results showed that microwave radiation caused intercellular tight junctions to broaden and fracture with decreased TEER values and increased HRP permeability. After microwave exposure, activation of the VEGF/Flk-1-ERK pathway and Tyr phosphorylation of occludin were observed, along with down-regulated expression and interaction of occludin with zonula occludens-1 (ZO-1). After Flk-1 (SU5416) and MEK1/2 (U0126) inhibitors were used, the structure and function of the BBB were recovered. The increase in expression of ERK signal transduction molecules was muted, while the expression and the activity of occludin were accelerated, as well as the interactions of occludin with p-ERK and ZO-1 following microwave radiation. Thus, microwave radiation may induce BBB damage by activating the VEGF/Flk-1-ERK pathway, enhancing Tyr phosphorylation of occludin, while partially inhibiting expression and interaction of occludin with ZO-1.

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Abbreviations

BBB:

Blood–brain barrier

EMP:

Electromagnetic pulse

ERK:

Extracellular-regulated protein kinase

GFAP:

Glial fibrillary acidic protein

HRP:

Horseradish peroxidase

IM:

Inverted microscope

LSCM:

Laser scanning confocal microscope

MEK:

MAPK kinase

MMP:

Matrix metalloproteinase

qRT-PCR:

Quantitative real-time PCR

SEM:

Scanning electron microscope

TEER:

Transendothelial electrical resistance

TJs:

Tight junctions

ZO:

Zonula occludens

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (81172620 and 81372926) and Beijing Natural Science Foundation (7122127). We are grateful to Sa Zhang and Kai Wang of the National Center of Biomedical Analysis for their kind help with electron microscopy and laser scanning confocal microscopy, Associate Professor Yue-Feng Yang and Guang-Xing Bian of Beijing Institute of Radiation Medicine for in vitro BBB models.

Authors’ Contributions

The work presented here was carried out in collaboration between all authors. Rui-Yun Peng and Xiang-Jun Hu conceived the project. Li-Feng Wang and Xiang Li performed the experiments. Shui-Ming Wang and Ya-Bing Gao conceived the study and participated in the design of the study. Ji Dong, Li Zhao, and Bin-Wei Yao participated in sample collection. Xin-Ping Xu performed the quantitative analysis. Gong-Min Chang helped to analyze the data and performed the statistical analysis. Hong-Mei Zhou was responsible for microwave radiation.

Conflict of Interest

The author(s) declare that they have no competing interests.

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Correspondence to Xiang-Jun Hu or Rui-Yun Peng.

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Li-Feng Wang and Xiang Li are co-first authors.

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Wang, LF., Li, X., Gao, YB. et al. Activation of VEGF/Flk-1-ERK Pathway Induced Blood–Brain Barrier Injury After Microwave Exposure. Mol Neurobiol 52, 478–491 (2015). https://doi.org/10.1007/s12035-014-8848-9

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Keywords

  • Microwave
  • In vitro blood–brain barrier model
  • VEGF
  • ERK
  • Occludin