International Journal of Molecular Sciences Article N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV–Cell Interaction and Functional Activation of Endothelial Cells Marta Clos-Sansalvador 1,2 , Sergio G. Garcia 1,2 , Miriam Morón-Font 1 , Charles Williams 3,4 , Niels-Christian Reichardt 3,5 , Juan M. Falcón-Pérez 4,6,7 , Antoni Bayes-Genis 8,9,10,11 , Santiago Roura 8,10,12 , Marcella Franquesa 1 , Marta Monguió-Tortajada 1,8,9, * and Francesc E. Borràs 1,13, * 1 REMAR-IGTP Group, Germans Trias i Pujol Research Institute (IGTP) & Nephrology Department, University Hospital Germans Trias i Pujol (HUGTiP), Can Ruti Campus, 08916 Badalona, Spain 2 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain 3 CIC biomaGUNE, Glycotechnology Group, Basque Research and Technology Alliance (BRTA), 20014 San Sebastian, Spain 4 Exosomes Laboratory, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), 20850 Derio, Spain 5 Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 20014 San Sebastian, Spain 6 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, 41092 Madrid, Spain 7 Basque Foundation for Science (IKERBASQUE), 48009 Bilbao, Spain Citation: Clos-Sansalvador, M.; 8 ICREC Research Program, Health Science Research Institute Germans Trias i Pujol, Can Ruti Campus, Garcia, S.G.; Morón-Font, M.; 08916 Badalona, Spain Williams, C.; Reichardt, N.-C.; 9 iCor, Cardiology Service, Germans Trias i Pujol University Hospital, 08916 Badalona, Spain Falcón-Pérez, J.M.; Bayes-Genis, A.; 10 CIBERCV, Instituto de Salud Carlos III, 41092 Madrid, Spain 11 Department of Medicine, Universitat Autònoma de Barcelona (UAB), 08193 Barcelona, Spain Roura, S.; Franquesa, M.; 12 Faculty of Medicine, University of Vic-Central University of Catalonia (UVic-UCC), 08500 Vic, Spain Monguió-Tortajada, M.; et al. 13 Department of Cell Biology, Physiology and Immunology, Universitat de Barcelona (UB), 08007 Barcelona, Spain N-Glycans in Immortalized * Correspondence: mmonguio@igtp.cat (M.M.-T.); feborras@igtp.cat (F.E.B.); Tel.: +34-930-330-526 (F.E.B.) Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for Abstract: Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered EV–Cell Interaction and Functional as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and Activation of Endothelial Cells. Int. J. Mol. Sci. 2022, 23, 9539. https:// regenerative properties. However, the interaction mechanisms between EV and target cells are not doi.org/10.3390/ijms23179539 fully understood. The surface glycans could be key players in EV–cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of Academic Editor: Denis N. Silachev N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells’ interaction for subsequent Received: 25 July 2022 EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Accepted: 13 August 2022 Wharton’s Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and Published: 23 August 2022 treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled Publisher’s Note: MDPI stays neutral iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based with regard to jurisdictional claims in tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV published maps and institutional affil- is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their iations. tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and Copyright: © 2022 by the authors. fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV Licensee MDPI, Basel, Switzerland. to permit EV–cell interactions and associated functions. This article is an open access article distributed under the terms and Keywords: mesenchymal stem/stromal cells; extracellular vesicles; exosomes; glycans; glycosylation; conditions of the Creative Commons Attribution (CC BY) license (https:// lectins creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2022, 23, 9539. https://doi.org/10.3390/ijms23179539 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 9539 2 of 16 1. Introduction Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) have already demonstrated their efficient modulation of inflammation and the exertion of regenera- tive properties in a number of both preclinical models [1] and clinical trials [2]. However, the specific mechanism of action of MSC-EV remains unclear. In this context, it is widely accepted that EV cargo (lipids, RNA and proteins) can be shuttled to the target cells by endocytosis (clathrin-dependent or caveolae-dependent) [3–6], phagocytosis [7], macropinocytosis [8] or by plasma/endosomal membrane fusion in acidic pH conditions, when EV and cells’ plasma membrane display the same fluidity [9]. More- over, it has also been established that this transfer of vesicular content plays a crucial role in the intercellular communication in several biological processes, including angiogen- esis [10,11], cell migration [12], inflammation regulation [13,14], bone formation [15] or interneuronal communication [16]. The transcriptomic, proteomic and lipidomic profiles of EV have been widely studied to find the molecules implicated in EV–cell interaction [17]. However, the specific mechanisms of EV recognition and their subsequent capture by target cells are still not fully elucidated. The implication of glycans in important cellular mechanisms in non-EV-related studies, such as cell adhesion [18,19], self- and non-self-recognition [20,21], molecular trafficking and receptor activation, has turned glycans into a new focus of molecules in which to find the possible mechanisms of EV function and targeting. In this context, a few studies (reviewed in [22,23]) have already reported EV glycan profiling analysis or specific receptor– ligand interactions for EV uptake in a glycan-dependent manner. Most of them referred to cancer-derived EV, urine EV or plasma EV. Specifically, heparan sulfate proteoglycans (HSPGs) were implicated in the uptake of tumor-derived EV by CHO cells [4], and in the paracrine intercommunication of the hepatic stellate cells [24]. The sialic acid residues have also been involved in EV-uptake in vitro [25,26] and in vivo [27], and the depletion of these sialic acid residues has also been shown to modify the EV biodistribution in mice [28], highlighting the importance of protein and lipid glycosylation in EV targeting. Thus, in the present study, we aimed to evaluate the implication of MSC-EV N- glycosylation in EV–endothelial cell interaction, EV-uptake and also, in specific MSC-EV described functions, such as endothelial cell recruitment and tube-like formation stimula- tion. Our results showed that the removal of N-glycosylation by PNGase-F treatment on MSC-EV affected both capture and functionality on HUVEC. 2. Results 2.1. Isolation and Characterization of iWJ-MSC-EV The isolated iWJ-MSC-EV were screened for the MSC marker CD90, and the EV markers CD63 and CD9. The SEC elution profile of iWJ-MSC-EV, treated or not with PNGase-F (Figure 1A), showed the EV elution indistinctly from the EV treatment. The EV were detected in fractions 4–6, nicely separated from the protein, which eluted later, confirming the previously published results [29]. Nevertheless, we observed a decrease in the MFI in the expression of all of the three markers after the addition of PNGase-F, especially affecting CD90 (CD9: 33%, p < 0.05; CD63: 24% n.s; CD90: 75%, p < 0.0001; n = 3; Figure 1B), which could be indicative of N-glycosylation sensitive epitopes. The fractions containing iWJ-MSC-EV were pooled together and analyzed by cryo-electron microscopy, which confirmed that both iWJ-MSC-EV and iWJ-MSC-EV PNGase-F had a double membrane, nanosize and round shape (Figure 1C). The iWJ-MSC-EV and iWJ-MSC- EV PNGase-F were also screened for calnexin and ezrin markers. The calnexin dotblot revealed no important reticulum endoplasmic contamination in the EV samples, and ezrin was positive for both of the iWJ-MSC-EV types (Figure 1D). 1B), which could be indicative of N-glycosylation sensitive epitopes. The fractions con- taining iWJ-MSC-EV were pooled together and analyzed by cryo-electron microscopy, which confirmed that both iWJ-MSC-EV and iWJ-MSC-EV PNGase-F had a double mem- brane, nanosize and round shape (Figure 1C). The iWJ-MSC-EV and iWJ-MSC-EV Int. J. Mol. Sci. 2022, 23, 9539 PNGase-F were also screened for calnexin and ezrin markers. The calnexin dotblot 3 of re- 16 vealed no important reticulum endoplasmic contamination in the EV samples, and ezrin was positive for both of the iWJ-MSC-EV types (Figure1D). Figure1.1.EV Figure EVwere wereisolated isolatedfrom fromiWJ-MSC iWJ-MSCconditioned conditioned medium medium byby SEC SEC and and characterized. characterized. (A)(A) Rep- Repre- resentative SEC elution profile of iWJ-MSC-EV, treated or not with PNGase-F, obtained by bead- sentative SEC elution profile of iWJ-MSC-EV, treated or not with PNGase-F, obtained by bead-based based flow cytometry and positive for the EV and MSC markers CD63, CD9 and CD90. Protein flow cytometry and positive for the EV and MSC markers CD63, CD9 and CD90. Protein elution elution was measured at 280nm absorbance and occurred later; (B) Comparative analysis of MSC- was EV measured at 280nm markers between absorbance untreated andand occurred later; iWJ-MSC-EV. PNGase-F-treated (B) Comparative Eachanalysis of MSC-EVtomark- dot corresponds inde- ers between pendent EVuntreated and PNGase-F-treated characterizations. iWJ-MSC-EV. The statistic differences Each dotfor are indicated corresponds to independent * p < 0.05 and **** p < 0.0001 EV by characterizations. two-way ANOVA The withstatistic differences a Šídák’s are indicatedtest; multiple comparisons p