Abstract
The use of cell secreted factors in clinical settings could be an alternative to conventional cell therapy, with the advantage of limiting concerns generally associated with traditional cell transplantation, such as tumorigenicity, immunoreactivity, and carrying of infections. Based on our published data, we predict a potential role for extracellular vesicles (EVs) in contributing to the proangiogenic activity of human fetal dermal cell secretome. Depletion of nanosized EVs from secretome significantly impaired its ability to induce formation of mesh-like structures in vitro. The isolated EVs were characterized for size and concentration by nanoparticle tracking analysis, and for protein markers (Rab5+, Alix+, CD63+, and calnexin−). The microRNA profile of EVs revealed 87 microRNAs significantly upregulated (≥15-fold increase) in fetal compared to adult dermal cell-derived EVs. Interestingly, these upregulated microRNAs included microRNAs with a validated role in angiogenesis according to literature. Moreover, the DIANA-TarBase v7.0 analysis confirmed enrichment in the KEGG signaling pathways associated with angiogenesis and wound healing, with the identification of putative target genes including thrombospondin 1. To validate the in silico data, EVs were also characterized for total protein contents. When tested in in vitro angiogenesis, fetal dermal cell-derived EVs were more effective than their adult counterpart in inducing formation of complete mesh-like structures. Furthermore, treatment of fibroblasts with fetal dermal-derived EVs determined a 4-fold increase of thrombospondin 1 protein amounts compared with the untreated fibroblasts. Finally, visualization of CSFE-labeled EVs in the cytosol of target cells suggested a successful uptake of these particles at 4-8 hours of incubation. We conclude that EVs are important contributors of the proangiogenic effect of fetal dermal cell secretome. Hence, EVs could also serve as vehicle for a successful delivery of microRNAs or other molecules of therapeutic interest to target cells.
Highlights
The list of clinical conditions related to insufficient angiogenesis is wide, ranging from cardiovascular diseases to impaired wound healing [1]
The nanoparticle tracking analysis (NTA) of pellet preparations obtained by differential ultracentrifugation of conditioned medium (CM) from fetal and adult dermal cells indicated a “mode” diameter size consistent with that of “small extracellular vesicles (EVs)” (
Characterization by Western blot analysis revealed that pellet particles expressed Rab5, Alix, and CD63 proteins, and while were negative for calnexin protein, this latter found in total protein extracts from human fetal dermal cells (Figure 1(c))
Summary
The list of clinical conditions related to insufficient angiogenesis is wide, ranging from cardiovascular diseases to impaired wound healing [1]. There is a great interest in developing clinical strategies ensuring vasculature formation, such as delivery of different cell populations, or administration of proangiogenic growth factors. Among the cell-based therapies, the use of mesenchymal stromal cells (MSCs), which are closely related to pericytes and produce diverse proangiogenic factors, is a promising approach with the potential to stimulate vasculature tissue development [2]. MSC-based therapy applied to regenerative medicine counts hundreds of registered clinical trials with excellent. Despite earlier works ascribing the therapeutic effects of MSCs to their ability to engraft and differentiate to form new permanent tissues, the current consensus view is that MSCs are shortlived after delivery and exert therapeutic benefits through secretion of bioactive factors [3]. In support of MSC paracrine activity, animal studies have shown that administration of MSC-derived soluble factors recapitulates the effects of cell-based therapy [4]. The mixture of growth factors, cytokines, chemokines, and extracellular vesicles (EVs) released by cells is known as secretome and can be collected as cell culture conditioned medium (CM)
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