Amniotic extracellular vesicles' effect on equine endometrial cells challenged with LPS: a proteomic analysis

Aim: A balanced uterine environment is crucial for the positive outcome of a pregnancy. Any alteration of its physiological status could affect embryo-maternal crosstalk and impair the mare fertility, as occurs in endometrial inflammatory conditions. The use of regenerative medicine strategies, such as extracellular vesicles (EVs), is emerging as promising therapeutic alternatives. This study aimed to identify the proteins involved in the anti-inflammatory action of equine amniotic EVs on endometrial cells (ECs) challenged by lipopolysaccharide (LPS). Methods: An in vitro model of endometrial inflammation was set up by stressing ECs with 10 ng/mL LPS for 3 h. Subsequently, LPS-treated cells were incubated with 400 × 10⁶ EVs/mL for 24 h. Results: The nano-liquid chromatography-high-resolution mass spectrometry (nLC-HRMS) approach identified 794 differentially regulated proteins with P < 0.05 and |Log2FC| > 1. Treatment with amniotic EVs entailed an overexpression of several proteins involved in the inflammatory response, including annexins, extracellular matrix (ECM) proteins, antioxidant enzymes and mediators, and transcription factors and regulators. Conversely, other pro-inflammatory mediators were also underexpressed, including cytokines, oxidative factors and matrix metalloproteinase inhibitors. Conclusion: These data suggest that amniotic EVs may exert an anti-inflammatory action on LPS-stressed ECs by multiple mechanisms, including cytokine regulation, antioxidant protection, transcriptional regulation, ECM remodeling and alteration of other key signaling mediators involved in the inflammatory response. These findings could provide useful information on the proteins involved in EVs effect during the initial 24 h of their incorporation into ECs.