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-EV_Mreg) 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-EV_Mreg were isolated from culture supernatants by differential centrifugation. Characterization of L-EV_Mreg was performed by cell/vesicle analysis, brightfield/transmission electron microscopy (TEM), flow cytometry and proteome profiling arrays. The impact of L-EV_Mreg on wound healing and angiogenesis was evaluated by means of scratch and in-vitro tube formation assays.

Results: Mreg and L-EV_Mreg 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-EV_Mreg regarding their surface marker composition. However, compared to Mreg fewer L-EV_Mreg 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-EV_Mreg contain abundant amounts of pro-angiogenic proteins (i.e. interleukin-8, platelet factor 4 and serpin E1). From a functional point of view L-EV_Mreg 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-EV_Mreg with regenerative and pro-angiogenic potential can be reproducibly isolated from in-vitro cultured human regulatory macrophages. We propose that L-EV_Mreg could represent a putative therapeutic option for the treatment of chronic wounds and ischemia-associated diseases.

Keywords: Angiogenesis; Large extracellular vesicles; Macrophages; Wound healing.

Fig. 1

Schematic representation of in-vitro differentiation of human monocytes to Mreg and L-EV_Mreg (left) and differential centrifugation steps leading to L-EV_Mreg enriched pellets (right). SN, supernatant; EV, extracellular vesicles

Fig. 2

Vesicle and cell analysis. A. Representative analysis of a sample containing Mreg and L-EV_Mreg using the MOXI counter. B. Representative analysis of a sample containing Mreg and L-EV_Mreg using the Nucleocounter. Note that nucleus lacking vesicles are not detected using the Nucleocounter method. C. Quantitative results from vesicle and cell analysis

Fig. 3

Morphology of Mreg derived L-EV (L-EV_Mreg). A. Brighfield microscopy after harvest on day 7. B. Transmission electron microscopy after harvest on day 7. Note that L-EV_Mreg share several morphological features of Mreg cells such as numerous of vesicles/vacuoles and pseudopodia-like extensions. N nucleus, V vesicle/vacuole, P pseudopodia

Fig. 4

Cell surface marker characterization of Mreg derived L-EV (L-EV_Mreg). A. Representative scatter plot (FCS vs. SSC) showing L-EV_Mreg and Mreg populations at day 7. B. Flow cytometric analysis of surface marker characteristics of L-EV_Mreg and Mreg. FSC, forward scatter; SSC, sideward scatter; *, P < 0.05; **, P < 0.01

Fig. 5

Characterization of angiogenic proteins in Mreg derived L-EV (L-EV_Mreg). A. Representative proteome profiling array membrane incubated with sonicated L-EV_Mreg showing the relative abundance of 55 proteins involved in angiogenesis. Note that each protein is represented by duplicate spots on the respective membrane. B. Three-dimensional heatmap analysis of A. C. Arrangement of protein spots on the membrane. Proteins with the highest signal intensities are depicted in bold

Fig. 6

Effects of Mreg derived L-EV (L-EV_Mreg) on in-vitro wound healing and angiogenesis. A. Photomicrographs of a representative wound healing assay employing human endothelial cells (HUVEC) with addition of 10⁶ L-EV_Mreg/ml and without addition of L-EV_Mreg. Note the presence of numerous L-EV in the treatment group (arrows). Scale bars denote 250 µm. B. Statistical analysis of the effect of L-EV_Mreg addition on relative wound closure (control = 1) after 8 h. The column denotes the mean ± SD of 3 independent experiments; *, P < 0.05 (one sample student t-test vs. 1). C. Representative images of tube formation analysis employing HUVEC cell cultures with and without the addition of L-EV_Mreg. Scale bars denote 500 µm. D. The influence of L-EV_Mreg addition on various tube formation parameters. Horizontal columns denote the mean ± SD of 3 independent experiments. *, P < 0.05; **, P < 0.01 (one sample student t-test vs. 0). Green color, branches related parameters; yellow color, segments related parameters; cyan color, meshes related parameters; blue color, nodes related parameters; red color, junctions related parameters. For parameter definitions and details on the angiogenesis analyzer tool please refer to: http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ&lang=en