Skip to main content

Advertisement

SpringerLink
Log in

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

  • Published:
Molecular Neurobiology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

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

References

  1. Kesari KK, Kumar S, Behari J (2012) Pathophysiology of microwave radiation: effect on rat brain. Appl Biochem Biotechnol 166:379–388

    Article  CAS  PubMed  Google Scholar 

  2. Mortazavi SM, Mahbudi A, Atefi M, Bagheri S, Bahaedini N et al (2011) An old issue and a new look: electromagnetic hypersensitivity caused by radiations emitted by GSM mobile phones. Technol Health Care 19:435–443

    CAS  PubMed  Google Scholar 

  3. Del Vecchio G, Giuliani A, Fernandez M, Mesirca P, Bersani F et al (2009) Effect of radiofrequency electromagnetic field exposure on in vitro models of neurodegenerative disease. Bioelectromagnetics 30:564–572

    Article  PubMed  Google Scholar 

  4. Kuo YC, Lu CH (2011) Effect of human astrocytes on the characteristics of human brain-microvascular endothelial cells in the blood-brain barrier. Colloids Surf B Biointerfaces 86:225–231

    Article  CAS  PubMed  Google Scholar 

  5. Thal SC, Luh C, Schaible EV, Timaru-Kast R, Hedrich J et al (2012) Volatile anesthetics influence blood-brain barrier integrity by modulation of tight junction protein expression in traumatic brain injury. PLoS One 7:e50752

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Liebner S, Kniesel U, Kalbacher H, Wolburg H (2000) Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells. Eur J Cell Biol 79:707–717

    Article  CAS  PubMed  Google Scholar 

  7. Hawkins BT, Davis TP (2005) The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57:173–185

    Article  CAS  PubMed  Google Scholar 

  8. Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 38:323–337

    Article  CAS  PubMed  Google Scholar 

  9. Balbuena P, Li W, Ehrich M (2011) Assessments of tight junction proteins occludin, claudin 5 and scaffold proteins ZO1 and ZO2 in endothelial cells of the rat blood-brain barrier: cellular responses to neurotoxicants malathion and lead acetate. Neurotoxicology 32:58–67

    Article  CAS  PubMed  Google Scholar 

  10. Lafuente JV, Argandona EG, Mitre B (2006) VEGFR-2 expression in brain injury: its distribution related to brain-blood barrier markers. J Neural Transm 113:487–496

    Article  CAS  PubMed  Google Scholar 

  11. Do B (2010) Vascular endothelial growth factors and vascular permeability. Cardiovasc Res 87:262–271

    Article  Google Scholar 

  12. Wang WDW, Borchardt RT (2001) VEGF increases BMEC monolayer permeability by affecting occludin expression and tight junction assembly. Am J Physiol Heart Circ Physiol 280:H434–H440

    CAS  PubMed  Google Scholar 

  13. Kaya D, Gursoy-Ozdemir Y, Yemisci M, Tuncer N, Aktan S et al (2005) VEGF protects brain against focal ischemia without increasing blood-brain permeability when administered intracerebroventricularly. J Cereb Blood Flow Metab 25:1111–1118

    Article  CAS  PubMed  Google Scholar 

  14. Storkebaum E, Lambrechts D, Carmeliet P (2004) VEGF: once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays 26:943–954

    Article  CAS  PubMed  Google Scholar 

  15. Wachtel M, Frei K, Ehler E, Bauer C, Gassmann M et al (2002) Extracellular signal-regulated protein kinase activation during reoxygenation is required to restore ischaemia-induced endothelial barrier failure. Biochem J 367:873–879

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gonzalez-Mariscal L, Tapia R, Chamorro D (2008) Crosstalk of tight junction components with signaling pathways. Biochim Biophys Acta Biomembr 1778:729–756

    Article  CAS  Google Scholar 

  17. Ning L, Kunnimalaiyaan M, Chen H (2008) Regulation of cell-cell contact molecules and the metastatic phenotype of medullary thyroid carcinoma by the Raf-1/MEK/ERK pathway. Surgery 144:920–924, discussion 924–925

    Article  PubMed  PubMed Central  Google Scholar 

  18. Salford LG, Brun A, Sturesson K, Eberhardt JL, Persson BR (1994) Permeability of the blood-brain barrier induced by 915 MHz electromagnetic radiation, continuous wave and modulated at 8, 16, 50, and 200 Hz. Microsc Res Tech 27:535–542

    Article  CAS  PubMed  Google Scholar 

  19. Schirmacher A, Winters S, Fischer S, Goeke J, Galla HJ et al (2000) Electromagnetic fields (1.8 GHz) increase the permeability to sucrose of the blood-brain barrier in vitro. Bioelectromagnetics 21:338–345

    Article  CAS  PubMed  Google Scholar 

  20. Williams WM, Del Cerro M, Michaelson SM (1984) Effect of 2450 MHz microwave energy on the blood-brain barrier to hydrophilic molecules. B. Effect on the permeability to HRP. Brain Res 319:171–181

    Article  CAS  PubMed  Google Scholar 

  21. Zhang YM, Zhou Y, Qiu LB, Ding GR, Pang XF (2012) Altered expression of matrix metalloproteinases and tight junction proteins in rats following PEMF-induced BBB permeability change. Biomed Environ Sci 25:197–202

    PubMed  Google Scholar 

  22. Vadim Zinchuka OG-Z (2009) Recent advances in quantitative colocalization analysis: focus on neuroscience. Prog Histochem Cytochem 44:125–172

    Article  Google Scholar 

  23. Ding GR, Qiu LB, Wang XW, Li KC, Zhou YC et al (2010) EMP-induced alterations of tight junction protein expression and disruption of the blood-brain barrier. Toxicol Lett 196:154–160

    Article  CAS  PubMed  Google Scholar 

  24. Nittby H, Brun A, Eberhardt J, Malmgren L, Persson BR et al (2009) Increased blood-brain barrier permeability in mammalian brain 7 days after exposure to the radiation from a GSM-900 mobile phone. Pathophysiology 16:103–112

    Article  CAS  PubMed  Google Scholar 

  25. Franke H, Streckert J, Bitz A, Goeke J, Hansen V et al (2005) Effects of Universal Mobile Telecommunications System (UMTS) electromagnetic fields on the blood-brain barrier in vitro. Radiat Res 164:258–269

    Article  CAS  PubMed  Google Scholar 

  26. Franke H, Ringelstein EB, Stogbauer F (2005) Electromagnetic fields (GSM 1800) do not alter blood-brain barrier permeability to sucrose in models in vitro with high barrier tightness. Bioelectromagnetics 26:529–535

    Article  PubMed  Google Scholar 

  27. Kuribayashi M, Wang J, Fujiwara O, Doi Y, Nabae K et al (2005) Lack of effects of 1439 MHz electromagnetic near field exposure on the blood-brain barrier in immature and young rats. Bioelectromagnetics 26:578–588

    Article  PubMed  Google Scholar 

  28. McQuade JM, Merritt JH, Miller SA, Scholin T, Cook MC et al (2009) Radiofrequency-radiation exposure does not induce detectable leakage of albumin across the blood-brain barrier. Radiat Res 171:615–621

    Article  CAS  PubMed  Google Scholar 

  29. Zhou JX, Ding GR, Zhang J, Zhou YC, Zhang YJ et al (2013) Detrimental effect of electromagnetic pulse exposure on permeability of in vitro blood-brain-barrier model. Biomed Environ Sci 26:128–137

    CAS  PubMed  Google Scholar 

  30. Kuo YC, Lu CH (2012) Modulation of efflux proteins by electromagnetic field for delivering azidothymidine and saquinavir into the brain. Colloids Surf B Biointerfaces 91:291–295

    Article  CAS  PubMed  Google Scholar 

  31. Stam R (2010) Electromagnetic fields and the blood-brain barrier. Brain Res Rev 65:80–97

    Article  PubMed  Google Scholar 

  32. Murakami T, Felinski EA, Antonetti DA (2009) Occludin phosphorylation and ubiquitination regulate tight junction trafficking and vascular endothelial growth factor-induced permeability. J Biol Chem 284:21036–21046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Rao R (2009) Occludin phosphorylation in regulation of epithelial tight junctions. Ann N Y Acad Sci 1165:62–68

    Article  CAS  PubMed  Google Scholar 

  34. Aschner M, Fitsanakis VA, dos Santos AP, Olivi L, Bressler JP (2006) Blood-brain barrier and cell-cell interactions: methods for establishing in vitro models of the blood-brain barrier and transport measurements. Methods Mol Biol 341:1–15

    CAS  PubMed  Google Scholar 

  35. Shah KK, Yang L, Abbruscato TJ (2012) In vitro models of the blood-brain barrier. Methods Mol Biol 814:431–449

    Article  CAS  PubMed  Google Scholar 

  36. Wilhelm I, Fazakas C, Krizbai IA (2011) In vitro models of the blood-brain barrier. Acta Neurobiol Exp (Wars) 71:113–128

    Google Scholar 

  37. Musch MW, Walsh-Reitz MM, Chang EB (2006) Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption. Am J Physiol Gastrointest Liver Physiol 290:G222–G231

    Article  CAS  PubMed  Google Scholar 

  38. Helms HC, Waagepetersen HS, Nielsen CU, Brodin B (2010) Paracellular tightness and claudin-5 expression is increased in the BCEC/astrocyte blood-brain barrier model by increasing media buffer capacity during growth. AAPS J 12:759–770

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liebner S, Fischmann A, Rascher G, Duffner F, Grote EH et al (2000) Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme. Acta Neuropathol 100:323–331

    Article  CAS  PubMed  Google Scholar 

  40. Wuest DM, Wing AM, Lee KH (2013) Membrane configuration optimization for a murine in vitro blood-brain barrier model. J Neurosci Methods 212:211–221

    Article  CAS  PubMed  Google Scholar 

  41. Bohara M, Kambe Y, Nagayama T, Tokimura H, Arita K et al (2014) C-type natriuretic peptide modulates permeability of the blood-brain barrier. J Cereb Blood Flow Metab

  42. Cioni C, Turlizzi E, Zanelli U, Oliveri G, Annunziata P (2012) Expression of tight junction and drug efflux transporter proteins in an in vitro model of human blood-brain barrier. Front Psychiatry 3:47

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zehendner CM, Librizzi L, Hedrich J, Bauer NM, Angamo EA et al (2013) Moderate hypoxia followed by reoxygenation results in blood-brain barrier breakdown via oxidative stress-dependent tight-junction protein disruption. PLoS One 8:e82823

    Article  PubMed  PubMed Central  Google Scholar 

  44. Zhu H, Wang Z, Xing Y, Gao Y, Ma T et al (2012) Baicalin reduces the permeability of the blood-brain barrier during hypoxia in vitro by increasing the expression of tight junction proteins in brain microvascular endothelial cells. J Ethnopharmacol 141:714–720

    Article  CAS  PubMed  Google Scholar 

  45. Qiu LB, Ding GR, Zhang YM, Zhou Y, Wang XW et al (2009) Effects of electromagnetic pulse on blood-brain barrier permeability and tight junction proteins in rats. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 27:539–543

    CAS  PubMed  Google Scholar 

  46. Li X, Peng R-Y, Hu X, Wang S, Gao Y, Wang L et al (2011) Changes and significance of occludin expression in rats with blood-brain barrier injury induced by microwave radiation. Med J Chin People’s Liberation Army 36:765–769

    CAS  Google Scholar 

  47. DeMaio L, Chang YS, Gardner TW, Tarbell JM, Antonetti DA (2001) Shear stress regulates occludin content and phosphorylation. Am J Physiol Heart Circ Physiol 281:H105–H113

    CAS  PubMed  Google Scholar 

  48. Salmeri M, Motta C, Anfuso CD, Amodeo A, Scalia M et al (2013) VEGF receptor-1 involvement in pericyte loss induced by Escherichia coli in an in vitro model of blood brain barrier. Cell Microbiol 15:1367–1384

    Article  CAS  PubMed  Google Scholar 

  49. Dejana E, Orsenigo F, Lampugnani MG (2008) The role of adherens junctions and VE-cadherin in the control of vascular permeability. J Cell Sci 121:2115–2122

    Article  CAS  PubMed  Google Scholar 

  50. Davis B, Tang J, Zhang L, Mu D, Jiang X et al (2010) Role of vasodilator stimulated phosphoprotein in VEGF induced blood-brain barrier permeability in endothelial cell monolayers. Int J Dev Neurosci 28:423–428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Mendonca MC, Soares ES, Stavale LM, Raposo C, Coope A et al (2013) Expression of VEGF and Flk-1 and Flt-1 receptors during blood-brain barrier (BBB) Impairment following Phoneutria nigriventer spider venom exposure. Toxins (Basel) 5:2572–2588

    Article  Google Scholar 

  52. Li X, Peng R-Y, Hu XJ, Wang SM, Gao Y, Wang L, Zhao L, Dong J et al (2010) Effects of microwave exposure on VEGF and FLK-1 of hippocampus in the rats. Chin J Stereology Image Anal 4:017

    Google Scholar 

  53. Basuroy S, Seth A, Elias B, Naren AP, Rao R (2006) MAPK interacts with occludin and mediates EGF-induced prevention of tight junction disruption by hydrogen peroxide. Biochem J 393:69–77

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Laboratory of Experimental Pathology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing, 100850, China

    Li-Feng Wang, Ya-Bing Gao, Shui-Ming Wang, Li Zhao, Ji Dong, Bin-Wei Yao, Xin-Ping Xu, Gong-Min Chang, Xiang-Jun Hu & Rui-Yun Peng

  2. Human Resource Department, Beijing Chuiyangliu Hospital, 2 Chuiyangliu South Street, Beijing, 100022, China

    Xiang Li

  3. Radiation Protection, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing, 100850, China

    Hong-Mei Zhou

Authors
  1. Li-Feng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ya-Bing Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shui-Ming Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Li Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ji Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bin-Wei Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xin-Ping Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gong-Min Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hong-Mei Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Xiang-Jun Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Rui-Yun Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

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

Additional information

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12035-014-8848-9

Keywords

Search

Navigation