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PACAP Enhances Barrier Properties of Cerebral Microvessels

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Abstract

Cerebral microvascular endothelial cells—coming in contact with pericytes and astrocytes—constitute the structural basis of the blood-brain barrier (BBB). The continuous belt of interendothelial tight junctions (TJs) and the presence of specific transport systems, enzymes, and receptors in the brain endothelium regulate the molecular and cellular traffic into the central nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having several cellular protective effects. However, little is known about the effects of PACAP on the cerebral endothelium and BBB functions. Here, we show that PACAP has no significant pro-survival role in cerebral microvascular endothelial cells; however, it improves the barrier properties of the brain endothelium. PACAP induces an increase in the transendothelial electrical resistance, which is the most important marker of the tightness of the TJs. Moreover, PACAP has a protective role against glucose deprivation- and oxidative stress-induced junctional damage in microvascular brain endothelial cells.

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Acknowledgments

This work was supported by grants from the Hungarian Research Fund (OTKA PD-100958, K-100807, K-104984), the National Development Agency (Hungary-Romania Cross-Border Co-operation Program 2007-2013: HURO/1101/173/2.2.1, the TÁMOP-4.2.2.A-11/1/KONV-2012-0024, and 4.2.2.A-11/1/KONV-2012-0052 projects), the PTE-MTA “Lendület” Program, NAP, and the Arimura Foundation. I. Wilhelm was supported by the János Bolyai Research Fellowship of the Hungarian Academy of Sciences (BO/00320/12/8). The research of C. Fazakas and A. Tamás was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/2-11/1-2012-0001 “National Excellence Program.”

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Authors and Affiliations

  1. Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary

    Imola Wilhelm, Csilla Fazakas & István A. Krizbai

  2. Department of Anatomy, PTE-MTA “Lendület” PACAP Research Team, University of Pécs, Pécs, Hungary

    Andrea Tamás & Dóra Reglődi

  3. Department of Medical Chemistry, University of Szeged, Szeged, Hungary

    Gábor Tóth

Authors
  1. Imola Wilhelm
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  2. Csilla Fazakas
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  3. Andrea Tamás
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  4. Gábor Tóth
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  5. Dóra Reglődi
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  6. István A. Krizbai
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Corresponding author

Correspondence to István A. Krizbai.

Additional information

Imola Wilhelm and Csilla Fazakas contributed equally to this work.

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Supplementary Fig. 1

Effect of PACAP on the survival of cerebral endothelial cells. D3 cells were exposed to glucose-deprivation and DMNQ-induced oxidative stress in the absence or presence of 100 nM PACAP. A: Phase contrast images showing the morphological changes. B: Densitometric analysis of pro-caspase 3 and the pro-apoptotic proteins Bad, Bax and cleaved caspase 3 from the apoptosis array presented on Fig. 2b. (JPEG 88 kb)

High resolution image (TIFF 18790 kb)

Supplementary Fig. 2

Effect of PACAP on the localization of tight junction proteins. RBECs were exposed to glucose-deprivation and/or DMNQ-induced oxidative stress in the absence or presence of 100 nM PACAP. Immunofluorescence stainings were performed using anti-occludin or anti-claudin-5 antibodies. Junctional disruption (indicated by arrows) is represented by the discontinuity or disappearance of the membrane staining. One representative of three independent experiments is presented. (JPEG 67 kb)

High resolution image (TIFF 18790 kb)

Supplementary Fig. 3

Effect of PACAP on the expression of tight and adherens junction proteins. RBECs or D3 cells were exposed to glucose-deprivation and DMNQ-induced oxidative stress in the absence or presence of 100 nM PACAP. Western-blot experiments were performed using the RIPA-soluble fractions. Densitometric analysis of three independent experiments is presented. **: p < 0.05, *: p < 0.01 compared to control, ##: p < 0.05 compared to G-free + DMNQ, as assessed by ANOVA and Bonferroni’s post hoc test. (JPEG 7 kb)

High resolution image (TIFF 2196 kb)

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Wilhelm, I., Fazakas, C., Tamás, A. et al. PACAP Enhances Barrier Properties of Cerebral Microvessels. J Mol Neurosci 54, 469–476 (2014). https://doi.org/10.1007/s12031-014-0260-4

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  • DOI: https://doi.org/10.1007/s12031-014-0260-4

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