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Proteomics profile of mesenchymal stromal cells and extracellular vesicles in normoxic and hypoxic conditions

  • Author Footnotes
    ⁎ These authors contributed equally to this work.
    Cássia Lisboa Braga
    Footnotes
    ⁎ These authors contributed equally to this work.
    Affiliations
    Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
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  • Luana Rocha da Silva
    Affiliations
    Toxicology Laboratory, Oswaldo Cruz Institute, Rio de Janeiro, Brazil

    Center for Technological Development in Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
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  • Renata Trabach Santos
    Affiliations
    Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
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  • Luiza Rachel Pinheiro de Carvalho
    Affiliations
    Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
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  • Samuel Coelho Mandacaru
    Affiliations
    Toxicology Laboratory, Oswaldo Cruz Institute, Rio de Janeiro, Brazil

    Center for Technological Development in Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
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  • Monique Ramos de Oliveira Trugilho
    Affiliations
    Toxicology Laboratory, Oswaldo Cruz Institute, Rio de Janeiro, Brazil

    Center for Technological Development in Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
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  • Patricia Rieken Macedo Rocco
    Affiliations
    Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

    National Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, Brazil

    Rio de Janeiro Innovation Network in Nanosystems for Health–NanoSaúde, Research Support Foundation of the State of Rio de Janeiro, Rio de Janeiro, Brazil
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  • Fernanda Ferreira Cruz
    Affiliations
    Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

    National Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, Brazil

    Rio de Janeiro Innovation Network in Nanosystems for Health–NanoSaúde, Research Support Foundation of the State of Rio de Janeiro, Rio de Janeiro, Brazil
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  • Pedro Leme Silva
    Correspondence
    Correspondence: Pedro Leme Silva, PhD, Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Avenida Carlos Chagas Filho, s/n, Bloco G-014, Ilha do Fundão, Rio de Janeiro 21941-902, Brazil.
    Affiliations
    Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

    National Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, Brazil

    Rio de Janeiro Innovation Network in Nanosystems for Health–NanoSaúde, Research Support Foundation of the State of Rio de Janeiro, Rio de Janeiro, Brazil
    Search for articles by this author
  • Author Footnotes
    ⁎ These authors contributed equally to this work.
Published:September 30, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.08.009

      Abstract

      Background aims

      Although bone marrow-derived mesenchymal stromal cells (MSCs) have demonstrated success in pre-clinical studies, they have shown only mild therapeutic effects in clinical trials. Hypoxia pre-conditioning may optimize the performance of bone marrow-derived MSCs because it better reflects the physiological conditions of their origin. It is not known whether changes in the protein profile caused by hypoxia in MSCs can be extended to the extracellular vesicles (EVs) released from them. The aim of this study was to evaluate the proteomics profile of MSCs and their EVs under normoxic and hypoxic conditions.

      Methods

      Bone marrow-derived MSCs were isolated from six healthy male Wistar rats. After achieving 80% confluence, MSCs were subjected to normoxia (MSC-Norm) (21% oxygen, 5% carbon dioxide, 74% nitrogen) or hypoxia (MSC-Hyp) (1% oxygen, 5% carbon dioxide, 94% nitrogen) for 48 h. Cell viability and oxygen consumption rate were assessed. EVs were extracted from MSCs for each condition (EV-Norm and EV-Hyp) by ultracentrifugation. Total proteins were isolated from MSCs and EVs and prepared for mass spectrometry. EVs were characterized by nanoparticle tracking analysis. Proteomics data were analyzed by PatternLab 4.0, Search Tool for the Retrieval of Interacting Genes/Proteins, Gene Ontology, MetaboAnalyst and Reactome software.

      Results

      Cell viability was higher in MSC-Hyp than MSC-Norm (P = 0.007). Basal respiration (P = 0.001), proton leak (P = 0.004) and maximal respiration (P = 0.014) were lower in MSC-Hyp than MSC-Norm, and no changes in adenosine triphosphate-linked and residual respiration were observed. The authors detected 2177 proteins in MSC-Hyp and MSC-Norm, of which 147 were identified in only MSC-Hyp and 512 were identified in only MSC-Norm. Furthermore, 718 proteins were identified in EV-Hyp and EV-Norm, of which 293 were detected in only EV-Hyp and 30 were detected in only EV-Norm. Both MSC-Hyp and EV-Hyp showed enrichment of pathways and biological processes related to glycolysis, the immune system and extracellular matrix organization.

      Conclusions

      MSCs subjected to hypoxia showed changes in their survival and metabolic activity. In addition, MSCs under hypoxia released more EVs, and their content was related to expression of regulatory proteins of the immune system and extracellular matrix organization. Because of the upregulation of proteins involved in glycolysis, gluconeogenesis and glucose uptake during hypoxia, production of reactive oxygen species and expression of immunosuppressive properties may be affected.

      Key Words

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