Characterization of large extracellular vesicles (L-EV) derived from human regulatory macrophages (Mreg): novel mediators in wound healing and angiogenesis?

Abstract

Background Large extracellular vesicles (L-EV) with a diameter between 1 and 10 µm are released by various cell types. L-EV contain and transport active molecules which are crucially involved in cell to cell communication. We have shown that secretory products of human regulatory macrophages (Mreg) bear pro-angiogenic potential in-vitro and our recent findings show that Mreg cultures also contain numerous large vesicular structures similar to L-EV with so far unknown characteristics and function. Aim of this study To characterize the nature of Mreg-derived L-EV (L-EVMreg) and to gain insights into their role in wound healing and angiogenesis. Methods Mreg were differentiated using blood monocytes from healthy donors (N = 9) and L-EVMreg were isolated from culture supernatants by differential centrifugation. Characterization of L-EVMreg was performed by cell/vesicle analysis, brightfield/transmission electron microscopy (TEM), flow cytometry and proteome profiling arrays. The impact of L-EVMreg on wound healing and angiogenesis was evaluated by means of scratch and in-vitro tube formation assays. Results Mreg and L-EVMreg show an average diameter of 13.73 ± 1.33 µm (volume: 1.45 ± 0.44 pl) and 7.47 ± 0.75 µm (volume: 0.22 ± 0.06 pl) respectively. Flow cytometry analyses revealed similarities between Mreg and L-EVMreg regarding their surface marker composition. However, compared to Mreg fewer L-EVMreg were positive for CD31 (P < 0.01), CD206 (P < 0.05), CD103 (P < 0.01) and CD45 (P < 0.05). Proteome profiling suggested that L-EVMreg contain abundant amounts of pro-angiogenic proteins (i.e. interleukin-8, platelet factor 4 and serpin E1). From a functional point of view L-EVMreg positively influenced in-vitro wound healing (P < 0.05) and several pro-angiogenic parameters in tube formation assays (all segment associated parameters, P < 0.05; number of meshes, P < 0.05). Conclusion L-EVMreg with regenerative and pro-angiogenic potential can be reproducibly isolated from in-vitro cultured human regulatory macrophages. We propose that L-EVMreg could represent a putative therapeutic option for the treatment of chronic wounds and ischemia-associated diseases.

Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications

“…Prior research conducted by the authors has been dedicated to characterizing Mreg for several years [ 8 , 12 , 16 ], demonstrating their ability to secrete pro-angiogenic factors, particularly under hypoxic conditions [ 8 ]. Building upon these findings, a GMP-compliant protocol for the production of Mreg was developed and a phase I/II study employing Mreg for treating patients with chronic limb threatening ischemia (CLTI) is under preparation.…”

“…Different research groups have demonstrated in recent years that distinct macrophage subtypes can be differentiated in vitro by the administration of various cytokines/ factors (e.g., IL-4, IL-13, IFNγ, LPS), signifying the potential of these in vitro-differentiated cell types in the context of cell therapy [10,11]. We and others have shown that the administration of IFNγ on day 6 of the differentiation period can generate an anti-inflammatory, T-cell suppressive, and potentially pro-angiogenic macrophage subtype known as regulatory macrophages (Mreg) [7,8,[12][13][14][15]. These Mreg cells have already been successfully employed in a clinical study to reduce rejection reactions in kidney transplantations [4,6].…”