Ewa K. Zuba‐Surma

Emerging evidence suggests that stem cell‐derived extracellular vesicles (EVs) may induce pro‐regenerative effects in ischemic tissues by delivering bioactive molecules, including microRNAs. Recent studies have also shown pro‐regenerative benefits of EVs derived from induced pluripotent stem (iPS) cells. However, the underlying mechanisms of EV benefits and the role of their transferred regulatory molecules remain incompletely understood. Accordingly, we investigated the effects of human iPS‐derived EVs (iPS‐EVs) enriched in proangiogenic miR‐126 (iPS‐miR‐126‐EVs) on functional properties of human endothelial cells (ECs) in vitro. We also examined the outcomes following EV injection in a murine model of limb ischemia in vivo. EVs were isolated from conditioned media from cultures of unmodified and genetically modified human iPS cells overexpressing miR‐126. The iPS‐miR‐126‐EVs were enriched in miR‐126 when compared with control iPS‐EVs and effectively transferred miR‐126 along with other miRNAs to recipient ECs improving their functional properties essential for ischemic tissue repair, including proliferation, metabolic activity, cell survival, migration, and angiogenic potential. Injection of iPS‐miR‐126‐EVs in vivo in a murine model of acute limb ischemia promoted angiogenesis, increased perfusion, and enhanced functional recovery. These observations corresponded with elevated expression of genes for several proangiogenic factors in ischemic tissues following iPS‐miR‐126‐EV transplantation. These results indicate that innate pro‐regenerative properties of iPS‐EVs may be further enhanced by altering their molecular composition via controlled genetic modifications. Such iPS‐EVs overexpressing selected microRNAs, including miR‐126, may represent a novel acellular tool for therapy of ischemic tissues in vivo.

Human immune cells possess the ability to react complexly and effectively after contact with microbial virulence factors, including those transported in cell-derived structures of nanometer sizes termed extracellular vesicles (EVs). EVs are produced by organisms of all kingdoms, including fungi pathogenic to humans. In this work, the immunomodulatory properties of EVs produced under oxidative stress conditions or at host concentrations of CO2 by the fungal pathogen Candida albicans were investigated. The interaction of EVs with human pro-monocytes of the U-937 cell line was established, and the most notable effect was attributed to oxidative stress-related EVs. The immunomodulatory potential of tested EVs against human THP-1 macrophages was verified using cytotoxicity assay, ROS-production assay, and the measurement of cytokine production. All fungal EVs tested did not show a significant cytotoxic effect on THP-1 cells, although a slight pro-oxidative impact was indicated for EVs released by C. albicans cells grown under oxidative stress. Furthermore, for all tested types of EVs, the pro-inflammatory properties related to increased IL-8 and TNF-α production and decreased IL-10 secretion were demonstrated, with the most significant effect observed for EVs released under oxidative stress conditions.

It has been repeatedly reported that the cells of organisms in all kingdoms of life produce nanometer-sized lipid membrane-enveloped extracellular vesicles (EVs), transporting and protecting various substances of cellular origin. While the composition of EVs produced by human pathogenic fungi has been studied in recent decades, another important challenge is the analysis of their functionality. Thus far, fungal EVs have been shown to play significant roles in intercellular communication, biofilm production, and modulation of host immune cell responses. In this study, we verified the involvement of biofilm-derived EVs produced by two different strains of Candida albicans—C. albicans SC5314 and 3147 (ATCC 10231)—in various aspects of biofilm function by examining its thickness, stability, metabolic activity, and cell viability in the presence of EVs and the antifungal drug caspofungin. Furthermore, the proteolytic activity against the kininogen-derived antimicrobial peptide NAT26 was confirmed by HPLC analysis for C. albicans EVs that are known to carry, among others, particular members of the secreted aspartic proteinases (Saps) family. In conclusion, EVs derived from C. albicans biofilms were shown to be involved in biofilm tolerance to caspofungin, biofilm detachment, and fungal proteolytic activity.