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J Proteomics. 2016 Feb 5;133:100-112. doi: 10.1016/j.jprot.2015.12.011. Epub 2015 Dec 21.

Characterization of secretomes from a human blood brain barrier endothelial cells in-vitro model after ischemia by stable isotope labeling with aminoacids in cell culture (SILAC).

Author information

1
Neurovascular Research Laboratory, Vall d'Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: victor.llombart.sebastia@vhir.org.
2
Neurovascular Research Laboratory, Vall d'Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: teresa.garcib@gmail.com.
3
Proteomics Laboratory, Vall d'Hebron Institute of Oncology (VHIO), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: joanjosepbech@gmail.com.
4
Neurovascular Research Laboratory, Vall d'Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: alba.simats@vhir.org.
5
Neurovascular Research Laboratory, Vall d'Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: alebustamanterangel@gmail.com.
6
Neurovascular Research Laboratory, Vall d'Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: dolors1985@gmail.com.
7
Proteomics Laboratory, Vall d'Hebron Institute of Oncology (VHIO), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: greverter@imim.es.
8
Proteomics Laboratory, Vall d'Hebron Institute of Oncology (VHIO), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: fcanals@vhio.net.
9
Neurovascular Research Laboratory, Vall d'Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: marguillamon@gmail.com.
10
Neurovascular Research Laboratory, Vall d'Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain. Electronic address: 31862jmv@comb.es.

Abstract

The human immortalized brain endothelial cell line hCMEC/D3 is considered a simple in-vitro model of the blood-brain-barrier. Our aim was to characterize changes in the secretome of hCMEC/D3 subjected to oxygen and glucose deprivation (OGD) to identify new proteins altered after ischemia and that might trigger blood-brain-barrier disruption and test their potential as blood biomarkers for ischemic stroke. Using a quantitative proteomic approach based on SILAC, 19 proteins were found differentially secreted between OGD and normoxia/normoglycemia conditions. Among the OGD-secreted proteins, protein folding was the main molecular function identified and for the main canonical pathways there was an enrichment in epithelial adherens junctions and aldosterone signaling. Western blot was used to verify the MS results in a set of 9 differentially secreted proteins and 5 of these were analyzed in serum samples of 38 ischemic stroke patients, 18 stroke-mimicking conditions and 18 healthy controls.

SIGNIFICANCE:

"We characterized changes in the secretome of hCMEC/D3 cells after an ischemic insult by SILAC and identified proteins associated with ischemia that might be involved in the disruption of the blood-brain barrier. Besides we analyzed the putative potential of the candidate proteins to become biomarkers for the diagnosis of ischemic stroke.

KEYWORDS:

Biomarkers; Blood–brain-barrier; Cerebral ischemia; Endothelial cells; Ischemic stroke; Secretome; Silac

PMID:
26718731
DOI:
10.1016/j.jprot.2015.12.011
[Indexed for MEDLINE]
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