Comprehensive Proteomic Analysis of Mesenchymal Stem Cell Exosomes Reveals Modulation of Angiogenesis via Nuclear Factor-KappaB Signaling

Abstract

Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.

Proteomic characterisation reveals active Wnt-signalling by human multipotent stromal cells as a key regulator of beta cell survival and proliferation

“…MSCs exert their therapeutic effects primarily through the secretion of trophic signals within damaged organs, or impact tissue repair from distant sites by secretion of regenerative effectors into the circulation within exosomes or microvesicles [7]. Exosomes harvested from MSCs have been shown to contain proangiogenic proteins and promote the healing of ischaemic tissue [8,9]. MSCs also secrete a wide variety of immunomodulatory molecules that dampen autoimmunity via modulation of immune cell functions [10,11].…”

Intranasal MSC-derived A1-exosomes ease inflammation, and prevent abnormal neurogenesis and memory dysfunction after status epilepticus

“…Moreover, several studies suggest that therapeutic benefits of MSC administration are largely explained by paracrine effects mediated by soluble factors or EVs secreted by MSCs (21)(22)(23)(24). Furthermore, EVs can cross the blood-brain barrier and deliver various therapeutic factors to the brain (25,26).…”

Extracellular vesicles as regulators of tumor fate: crosstalk among cancer stem cells, tumor cells and mesenchymal stem cells

“…More importantly in the context of this review, MSC-EVs contain and transfer other proteins, the functions of which can be associated-among other options-with tumor physiology: TIMP-1, TIMP-2, MMP-9, MMP-2 (related to tumor growth and matrix remodeling) and VEGF and PDGF (related to angiogenesis) (92). Proteomic analysis of MSCs-derived EVs revealed a wide spectrum of proteins with biological functions associated with regulation of the cell cycle, proliferation, cell migration and angiogenesis (97,98). Collino et al (99) analyzed the composition of MSCEVs, showing differential protein enrichment among the subpopulations of EVs related to inflammation, interleukin, FGF and TGF-β pathways, which have implications for tumor evolution (100).…”