Adipose Mesenchymal Extracellular Vesicles as Alpha-1-Antitrypsin Physiological Delivery Systems for Lung Regeneration

Abstract

Accumulating evidence shows that Mesenchymal Stem/Stromal Cells (MSCs) exert their therapeutic effects by the release of secretome, made of both soluble proteins and nano/microstructured extracellular vesicles (EVs). In this work, for the first time, we proved by a proteomic investigation that adipose-derived (AD)-MSC-secretome contains alpha-1-antitrypsin (AAT), the main elastase inhibitor in the lung, 72 other proteins involved in protease/antiprotease balance, and 46 proteins involved in the response to bacteria. By secretome fractionation, we proved that AAT is present both in the soluble fraction of secretome and aggregated and/or adsorbed on the surface of EVs, that can act as natural carriers promoting AAT in vivo stability and activity. To modulate secretome composition, AD-MSCs were cultured in different stimulating conditions, such as serum starvation or chemicals (IL-1β and/or dexamethasone) and the expression of the gene encoding for AAT was increased. By testing in vitro the anti-elastase activity of MSC-secretome, a dose-dependent effect was observed; chemical stimulation of AD-MSCs did not increase their secretome anti-elastase activity. Finally, MSC-secretome showed anti-bacterial activity on Gram-negative bacteria, especially for Klebsiellapneumoniae. These preliminary results, in addition to the already demonstrated immunomodulation, pave the way for the use of MSC-secretome in the treatment of AAT-deficiency lung diseases.

Keywords: alpha-1-antitrypsin; anti-elastase; lung diseases; mesenchymal exosomes; mesenchymal extracellular vesicles; mesenchymal microvesicles; mesenchymal secretome.

Figure 1

(A) Proteins secreted by human Adipose-Derived Mesenchymal Stem/Stromal Cells (AD-MSCs) and grouped in four functional categories including protease/peptidase activity, protease/peptidase inhibitor activity, response to bacteria, and acute-phase response (see Table S1); in addition, proteins belonging to a functional category were further split, where possible, by protein families. (B) Topological evaluation of the network (290 nodes and 3836 interactions) reconstructed by combining proteins secreted by human AD-MSCs and Homo sapiens protein-protein interaction (PPI) data [7]. Proteins in the graph showed betweenness, centroid, and degree values above the average calculated on the whole network, thus are considered hubs; the complete list (290 proteins) and the corresponding topological parameters (betweenness, centroid, and degree) are shown in Table S2. (C) Proteins secreted by human AD-MSCs experimentally identified [7] and annotated as SERPINA1 interactors.

Figure 2

Total protein and lipid content of freeze-dried secretome collected between 0–24 and 24–48 h of serum starvation; mean values ± standard deviation, n = 3.

Figure 3

Total proteins and lipids dosed into MSC-secretome fractions. Mean values ± standard deviation, n = 3. Different letters (a, b, c) indicate significant differences between the means (p < 0.05), whereas the same letter indicates no significant difference (p > 0.05).

Figure 4

(A) Total proteins and lipids dosed into freeze-dried secretome collected after AD-MSC stimulation with different conditions (serum starvation (ST) and/or IL-1β, dexamethasone (DEX) and IL-1β + DEX). Mean values ± standard deviation, n = 3. Different letters (a, b, c) indicate significant differences between the means (p < 0.05), whereas the same letter indicates no significant difference (p > 0.05). (B) SERPINA1 relative expression as a function of the stimulating conditions. Multifactor ANOVA, mean values ± least significant difference (LSD), n = 3.

Figure 5

(A) Anti-elastase activities of freeze-dried MSC-secretome fractions; mean values ± standard deviation, n = 3. (B) Results of average elastase inhibition % as a function of the secretome fraction. Multifactor ANOVA, mean values ± LSD, n = 3.

Figure 6

(A) Anti-elastase activities of freeze-dried MSC-secretome obtained stimulating AD-MSCs with IL-1β and/or DEX; mean values ± standard deviation, n = 3. (B) Results of average elastase inhibition % as a function of the different stimulating conditions. Multifactor ANOVA, mean values ± LSD, n = 3.

Figure 7

(A) Microbicidal effect (ME) of lyo-secretome on the different bacterial strains at 2 and 6 mg/mL after 5 and 24 h of contact time. Mean values ± standard deviation, n = 3. (B) Lyo-secretome microbicidal effect as a function of the bacterial strain. Multifactor ANOVA, mean values ± LSD, n = 3.