A MOLECULAR SIGHT AT THE CARGO OF EQUINE AMNIOTIC MESENCHYMAL STROMAL CELL-DERIVED EXTRACELLULAR VESICLES.

Most cells comprising mesenchymal stem cells (MSCs), which are adult multipotent cells, can release extracellular vesicles (EVs), spherical nanoparticles containing a cargo of genetic materials, lipids and proteins by which they ensure cell-to-cell communication. Extracellular vesicles represent a new cell- free strategy in tissue function and regeneration, and recently, those released by equine amniotic mesenchymal stromal cells (eAMCs) have been employed to restore fertility of mares affected by chronic endometritis (CE). Since the content of EVs varies in relation to the releasing cells, this study sheds light on certain proteins of the eAMC-EVs with anti-inflammatory capacity or involved in embryo-maternal communication. In addition, the glycosylation pattern of the glycodelin contained in eAMC-EVs was also investigated, as glycosylation determines the biological activities of the glycodelin and varies depending on the glycodelin glycoforms.Western blotting and primary antibodies against L-4, L-1O, L1Ra, FN-y, glycodelin A, TGFi31, ADAM 10 and a-actinin-4 were employed for studying the protein EV content, while a panel of seven FITC-labelled lectins (ConA, PHA-E, RCA120, SNA, AAL, LTA, UEA I) was chosen for evaluating the N-glycosylation and fucose presence in eAMC- EV glycodelin.The results showed positive hybridization signals of the expected molecular size for all proteins analyzed, as well as the presence of N-linked glycans containing fucosylated and sialylated residues in glycodelin. The clinical efficacy of the treatment based on eAMC-EVs to restore the fertility of mares affected by CE is upported by the results of this study showing that the anti-inflammatory protein cargo may have a potential role in reducing uterine inflammation. Moreover, proteins such as ADAM10 with a known role in notching signalling, TGFi31, a shuttle molecule in the crosstalk between the conceptus and the mother, glycodelin, a glycoprotein involved in cell-mediated cell adhesion and a-actinin-4, an actin-cross linking protein, may have a role in preparing the uterine environment to receive the embryo.This study highlights the role of EVs in regenerative veterinary medicine.