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Cav-1 Protein Levels in Serum and Infarcted Brain Correlate with Hemorrhagic Volume in a Mouse Model of Thromboembolic Stroke, Independently of rt-PA Administration

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Abstract

Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for acute ischemic stroke. However, its administration is still limited due to the associated increased risk of hemorrhagic transformation (HT). rt-PA may exacerbate blood-brain barrier (BBB) injury by several mechanisms that have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae, has been linked to the endothelial barrier function. The effects of rt-PA on Cav-1 expression remain largely unknown. Here, Cav-1 protein expression after ischemic conditions, with or without rt-PA administration, was analyzed in a murine thromboembolic middle cerebral artery occlusion (MCAO) and in brain microvascular endothelial bEnd.3 cells subjected to oxygen/glucose deprivation (OGD). Our results show that Cav-1 is overexpressed in endothelial cells of infarcted area and in bEnd.3 cell line after ischemia but there is disagreement regarding rt-PA effects on Cav-1 expression between both experimental models. Delayed rt-PA administration significantly reduced Cav-1 total levels from 24 to 72 h after reoxygenation and increased pCav-1/Cav-1 at 72 h in the bEnd.3 cells while it did not modify Cav-1 immunoreactivity in the infarcted area at 24 h post-MCAO. Importantly, tissue Cav-1 positively correlated with Cav-1 serum levels at 24 h post-MCAO and negatively correlated with the volume of hemorrhage after infarction, the latter supporting a protective role of Cav-1 in cerebral ischemia. In addition, the negative association between baseline serum Cav-1 levels and hemorrhagic volume points to a potential usefulness of baseline serum Cav-1 levels to predict hemorrhagic volume, independently of rt-PA administration.

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Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

BBB:

Blood-brain barrier

bEnd.3:

Immortalized mouse brain endothelial cell line

BSA:

Bovine serum albumin

Cav-1:

Caveolin-1

CTR:

Control

DAB:

Diaminobenzidine

FITC-BSA:

Fluorescein isothiocyanate labelled bovine serum albumin

HT:

Hemorrhagic transformation

MCAO:

Middle cerebral artery occlusion

MTT:

3-(4,5-Dimethylthiazol)-2,5-diphenyl tetrazolium bromide

OGD:

Oxygen/glucose deprivation

pCav-1:

Phosphorylated caveolin-1

rt-PA:

Recombinant tissue plasminogen activator

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Acknowledgements

The authors thank Dr. Maria Buxó for her support in the statistical data analysis.

Funding

This work was supported by grants from Instituto de Salud Carlos III and co-financed by the European Development Regional Fund “A Way to Achieve Europe” Health Strategic Action Program PI13/02258 and PI17/02123 (MC), PI20/00535 (IL), and Spanish Stroke Research Network RETICS RD12/0014/0010 (MC), and RD16/0019/0003 (JS), RD16/0019/0004 (MC), and RD16/0019/0009 (IL); from Regional Madrid Government B2017/BMD- 3688 (IL); from Spanish Ministry of Science and Innovation PID2019-106581RB-I00 (MAM); from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MAM); and from Fundación La Caixa HR17_00527 (MAM). P. Comajoan was a recipient of a predoctoral fellowship from the University of Girona (IF-UdG 2015).

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Authors and Affiliations

  1. Cerebrovascular Pathology Research Group, Department of Neurology, Girona Biomedical Research Institute (IDIBGI), Parc Hospitalari Martí i Julià, C/Dr. Castany s/n, M2 Building, 17190, Salt, Girona, Spain

    Carme Gubern-Mérida, Pau Comajoan, Gemma Huguet, Juan Manuel Sánchez, Joaquín Serena & Elisabet Kádár

  2. Cellular and Molecular Neurobiology Research Group, Department of Biology, University of Girona (UdG), Aulari Comú building, C/Maria Aurèlia Capmany 40, 17003, Girona, Spain

    Carme Gubern-Mérida, Pau Comajoan, Gemma Huguet, Irene Puig-Parnau, Joaquín Serena & Elisabet Kádár

  3. Neurovascular Research Unit, Department of Pharmacology and Toxicology and Instituto Universitario de Investigación en Neuroquímica (IUIN), Instituto de Investigación Hospital 12 de Octubre (i+12), Complutense University of Madrid (UCM), Pza. Ramón y Cajal s/n, 28040, Madrid, Spain

    Isaac García-Yebenes & Ignacio Lizasoain

  4. Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain

    María Angeles Moro

  5. Analytical and Environmental Chemistry Research Group, Department of Chemistry, University of Girona (UdG), C/Maria Aurèlia Capmany 69, 17003, Girona, Spain

    Juan Manuel Sánchez

  6. Department of Neurology, A Coruña University Hospital/A Coruña Biomedical Research Institute, Xubias de Arriba 84, 15006A, Coruña, Spain

    Mar Castellanos

Author notes

  1. Carme Gubern-Mérida and Pau Comajoan are joint first authors.

    • Elisabet Kádár and Mar Castellanos are joint last authors.

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      1. Carme Gubern-Mérida
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      Contributions

      CG, PC, and EK performed in vitro procedures and Western blot, ELISA, and immunofluorescence analysis. IP helped with immunohistochemistry analysis. IG performed the mouse thromboembolic stroke model. CG, PC, EK, and GH designed the experiments and interpreted data. IL, MAM, JS, and MC contributed to data interpretation and, joint to JMS, critically revised the manuscript. CG, PC, and EK were the major contributors in writing the manuscript. All the authors read and approved the final manuscript and agreed to be accountable in ensuring appropriate answer to questions related to the accuracy and integrity of any part of the work.

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      Correspondence to Elisabet Kádár or Mar Castellanos.

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      All procedures were performed in accordance with the European Communities Council Directive (86/609/EEC) and approved by the Ethics Committee on Animal Welfare of University Complutense (PROEX No. 016/18) and are reported according to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

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      Gubern-Mérida, C., Comajoan, P., Huguet, G. et al. Cav-1 Protein Levels in Serum and Infarcted Brain Correlate with Hemorrhagic Volume in a Mouse Model of Thromboembolic Stroke, Independently of rt-PA Administration. Mol Neurobiol 59, 1320–1332 (2022). https://doi.org/10.1007/s12035-021-02644-y

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