Anderson et al. (2016) sought to characterize the mesenchymal stem cell (MSC) proteome under ischemic culture conditions to determine factors that aid angiogenesis.1 Using high-resolution isoelectric focusing in conjunction with liquid chromatography–tandem mass spectrometry (HRIEF-LC-MS/MS), the researchers elucidated the role of MSC exosomal proteins, including nuclear factor kappa B (NFκB), in tissue repair following hypoxic damage.
Certain disease states, including peripheral artery disease, reduce oxygen tension in tissues, causing damage due to ischemia. Tissue repair is difficult without establishing new blood supply. MSCs help heal ischemic tissues primarily by promoting angiogenesis, or the formation of new blood-carrying capacity. Anderson et al. used a mass spectrometric workflow for deep proteome coverage of cellular lysates to characterize factors important in this therapeutic role.
The researchers obtained MSCs from bone marrow aspirates harvested from three healthy, nonsmoking male donors. Once the team established stable cultures, they exposed the cells to low-oxygen conditions to simulate tissue ischemia, and then harvested both MSCs and exosomes independently. Exosome harvesting took place following 40 hours’ conditioning. They confirmed exosome isolation by identifying characteristic morphologies visually with scanning electron microscopy.
Following protein extraction and digestion with trypsin, Anderson et al. labeled the peptides using tandem mass tagging (TMT) isobaric labeling reagents. They used TMT10plex reagents for the cell preparation protein quantitation and TMTsixplex reagants for the exosome digests (both Thermo Scientific). Following the labeling step, the researchers examined the peptides with the HRIEF-LC-MS/MS workflow. Mass spectrometric evaluation took place on an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) operated in data-dependent acquisition mode, coupled with nanoLC fractionation.
Overall, MSCs in culture produced 6,342 proteins, of which 1,927 identified as exosomal and 580 as membrane-associated. The exosomal proteome contained 457 proteins not identified in the MSC cell preparations.
Comparing cells cultured under standard nonhypoxic conditions, ischemia induced differential expression of 843 proteins with both high- and low-abundance analytes affected. Network, pathway and gene ontology analysis showed upregulation of pathways involving platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor. Interactome analysis revealed clustering of signaling molecules interacting with the PDGF receptor, the EGF receptor and NFκB nodes.
Ischemic conditions also stimulated exosome release into the culture medium, accompanied by an increase in proteins associated with cholesterol and lipid biosynthesis. Gene ontology analysis highlighted vascular and endothelial proteins, with network analysis indicating NFκB involvement. For this reason, the research team focused on NFκB and its role in angiogenesis, using primary human umbilical vascular endothelial cell (HUVEC) cultures for further characterization.
Under ischemic conditions, NFκB treatment induced angiogenesis in the HUVEC cultures, as shown by tubule formation. Anderson et al. could repeat this by treating the HUVECs with ischemia-stimulated MSC exosomes. The research team could abolish both responses with NFκB inhibitor pyrrolidine dithiocarbamate co-treatment, suggesting that the NFκB is an important pathway in MSC exosomal angiogenesis induction.
From these data, the researchers suggest that further characterization of the NFκB and other pathways is valid for exploring the therapeutic potential of MSCs in treatment of ischemic tissue damage.
1. Anderson, J.D., et al. (2016) “Comprehensive proteomic analysis of mesenchymal stem cell exosomes reveals modulation of angiogenesis via nuclear factor-KappaB signaling,” Stem Cells, 34(3) (pp. 601–613), doi: 10.1002/stem.2298.