The Angiogenic Potential of Adipose Mesenchymal Stem Cell-derived Extracellular Vesicles is modulated by Basic Fibroblast Growth Factor

Abstract

Objective: The aim of the present study was to investigate the effects of basic fibroblast growth factor (bFGF) on the angiogenic properties of extracellular vesicles (EVs) secreted by adipose-derived mesenchymal stem cells (ASCs) in culture. Methods: We isolated EVs from ASC (ASC-EVs) cultured with bFGF (FGF-EVs) or without bFGF (b-EVs). We compared the EV angiogenic action on human microvascular endothelial cells (HMEC) in vitro by capillary- like structure formation assay and in vivo by subcutaneous injection of HMEC-containing Matrigel in SCID mice. We analysed micro-RNA composition of EVs by PCR array and we selected two mi-RNAs changed after bFGF stimulation: miR-223 and miR-21. We transfected HMEC with short antisense anti-miR-223 or with mimic miR- 21 to compensate the action of EVs. Results: In vitro, the total length and the number of branches of vessel-like structures formed by FGF-EV- stimulated HMEC were significantly reduced in respect to b-EV stimulation and the structures were significantly larger than after b-EV stimulation. In vivo, vessels formed by FGF-EV-stimulated HMEC were significantly lower in number in respect to those formed by b-EV-stimulated HMEC, but they were significantly larger in size and contained cells positive for mouse smooth muscle actin. We found that bFGF led to molecular changes in ASC- EVs characterized by decreased expression of angiogenic factors. Moreover, bFGF stimulation up-regulated the expression of the anti-angiogenic miR-223 and decreased the level of pro-angiogenic miR-21. The effects of FGF- EVs were antagonized by the inhibition of miR-233 and by the miR-21 mimic Conclusions: The results indicate that culture conditions may modify the pro-angiogenic activity of ASC- derived EVs, changing their protein and RNA contents. In particular, bFGF induces production of EVs that stimulate vessel stabilization rather than an increase in vessel number.