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J Proteome Res. 2017 Aug 4;16(8):2836-2847. doi: 10.1021/acs.jproteome.7b00144. Epub 2017 Jul 6.

Hypoxia-Induced Changes in the Fibroblast Secretome, Exosome, and Whole-Cell Proteome Using Cultured, Cardiac-Derived Cells Isolated from Neonatal Mice.

Author information

1
Department of Physiology and Translational Biology and Engineering Program (TBEP), University of Toronto , Toronto, Ontario M5G 1M1, Canada.
2
The Donnelly Centre for Cellular and Biomolecular Research, Ted Rogers Centre for Heart Research, University of Toronto , Toronto, Ontario M5S 3E1, Canada.

Abstract

Cardiac fibroblasts (CFs) represent a major subpopulation of cells in the developing and adult heart. Cardiomyocyte (CM) and CF intercellular communication occurs through paracrine interactions and modulate myocyte development and stress response. Detailed proteomic analysis of the CF secretome in normal and stressed conditions may offer insights into the role of CF in heart development and disease. Primary neonatal mouse CFs were isolated and cultured for 24 h in 21% (normoxic) or 2% (hypoxic) O2. Conditioned medium was separated to obtain exosomes (EXO) and EXO-depleted secretome fractions. Multidimensional protein identification technology was performed on secreted fractions. Whole cell lysate data were also generated to provide subcellular context to the secretome. Proteomic analysis identified 6163 unique proteins in total. Statistical (QSpec) analysis identified 494 proteins differentially expressed between fractions and oxygen conditions. Gene Ontology enrichment analysis revealed hypoxic conditions selectively increase expression of proteins with extracellular matrix and signaling annotations. Finally, we subjected CM pretreated with CF secreted factors to hypoxia/reoxygenation. Viability assays suggested altered viability due to CF-derived factors. CF secretome proteomics revealed differential expression based on mode of secretion and oxygen levels in vitro.

KEYWORDS:

cardiac fibroblast; cardiovascular; exosome; paracrine factors; secretome

PMID:
28641008
DOI:
10.1021/acs.jproteome.7b00144
[Indexed for MEDLINE]

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