Human umbilical cord mesenchymal stem cell exosomes enhance angiogenesis through the Wnt4/β-catenin pathway

Abstract

Human umbilical cord mesenchymal stem cells (hucMSCs) and their exosomes have been considered as potential therapeutic tools for tissue regeneration; however, the underlying mechanisms are still not well understood. In this study, we isolated and characterized the exosomes from hucMSCs (hucMSC-Ex) and demonstrated that hucMSC-Ex promoted the proliferation, migration, and tube formation of endothelial cells in a dose-dependent manner. Furthermore, we demonstrated that hucMSC-Ex promoted wound healing and angiogenesis in vivo by using a rat skin burn model. We discovered that hucMSC-Ex promoted β-catenin nuclear translocation and induced the increased expression of proliferating cell nuclear antigen, cyclin D3, N-cadherin, and β-catenin and the decreased expression of E-cadherin. The activation of Wnt/β-catenin is critical in the induction of angiogenesis by hucMSC-Ex, which could be reversed by β-catenin inhibitor ICG-001. Wnt4 was delivered by hucMSC-Ex, and the knockdown of Wnt4 in hucMSC-Ex abrogated β-catenin nuclear translocation in endothelial cells. The in vivo proangiogenic effects were also inhibited by interference of Wnt4 expression in hucMSC-Ex. Taken together, these results suggest that hucMSC-Ex-mediated Wnt4 induces β-catenin activation in endothelial cells and exerts proangiogenic effects, which could be an important mechanism for cutaneous wound healing.

Keywords: Angiogenesis; Exosomes; Regenerative medicine; Wnt4; β-Catenin.

Figure 1.

Exosome characterization and internalization. (Ai, Aii): Morphologic analysis of hucMSC-derived exosomes (hucMSC-Ex) by transmission electron microscopy (scale bar = 100 nm). (B): Size distribution measurements under flow conditions by nanoparticle tracking (Bi) with the corresponding video frame (Bii). (C): Phosphate-buffered saline was used as a control (×100) (Ci), and CM-Dil-labeled exosomes were taken up by EA.hy926 cells (×100) (Cii). (Di): SDS-polyacrylamide gel electrophoresis analysis of the protein profiles of cell lysates and exosomes. (Dii): Detection of exosomal marker expression in hucMSC-Ex by Western blot. Abbreviation: hucMSC, human umbilical cord mesenchymal stem cells.

Figure 2.

Exosomes from human umbilical cord mesenchymal stem cells (HucMSC-Ex) prompted the proliferation of endothelial cells. (A): MTT assay for the proliferating ability of EA.hy926 cells with or without hucMSC-Ex treatment. (B): Cell-counting assay for the proliferating ability of hucMSC-Ex-treated EA.hy926 cells. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001. Abbreviations: n.s., not significant; PBS, phosphate-buffered saline.

Figure 3.

HucMSC-Ex prompted the migration of endothelial cells. (A): EA.hy926 cells were treated with different concentrations of exosomes (80, 160 μg/ml) or PBS for 12 hours. Transwell migration assay was performed to analyze the migratory ability of the cells (×100). Cells that migrated to the bottom were counted. ∗∗, p < .01; ∗∗∗, p < .001. (B): EA.hy926 cells treated with different concentrations of exosomes (80, 160 μg/ml) were subjected to a wound-healing assay for 12 hours (×100), and the percentage of closure of the wounded areas was measured. ∗∗, p < .01; ∗∗∗, p < .001. Abbreviations: hucMSC-Ex, exosomes from human umbilical cord mesenchymal stem cells; PBS, phosphate-buffered saline.

Figure 4.

HucMSC-Ex improved the tube-formation ability of endothelial cells and vascular density in cutaneous burn model. (Ai–Aiii): Exosomes increased the tube length in a dose-dependent manner (×40). (Aiv): Measurement of the tube length by ImageJ software. ∗∗, p < .01. (B): Representative micrographs of wound histological images (hematoxylin and eosin staining) at 1 week after treatment with hucMSC-Ex, HFL1-Ex, or PBS (×100). (C): Representative immunofluorescence images of CD31 expression in the wound area after treatment with hucMSC-Ex, HFL1-Ex, or PBS (×200). Abbreviations: HFL-Ex, exosomes from human lung fibroblast; hucMSC-Ex, exosomes from human umbilical cord mesenchymal stem cells; PBS, phosphate-buffered saline.

Figure 5.

HucMSC-Ex prompted angiogenesis by activating Wnt/β-catenin signaling. (A): β-Catenin expression in EA.hy926 cells after exosome treatment (80 or 160 μg/ml) treatment was detected by immunofluorescence staining (×200). ∗∗, p < .01. (B): Cytoplasm and nuclear fractions were prepared from EA.hy926 cells treated with indicated concentrations of hucMSC-Ex or PBS. β-Catenin protein levels were determined using Western blot. (C): EA.hy926 cells were treated with different concentrations of exosomes (80 or 160 μg/ml). The expression of N-cadherin, E-cadherin, PCNA, and cyclin D3 proteins was detected using Western blot. (D): Densitometric analysis of the PCNA Western blot bands (∗∗∗, p < .001) compared with the PBS group (n = 3). (E): Densitometric analysis of the cyclin D3 Western blot bands (∗, p < .05 and ∗∗∗, p < .001) compared with the PBS group (n = 3). (F): The cutaneous-burn rat models were injected with hucMSC-Ex with or without ICG-001 (1 mg per rat). PBS and DMSO were used as controls for hucMSCs and ICG-001, respectively. Representative immunofluorescence images of CD31 expression in the wound area are shown (×200). Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hucMSC-Ex, exosomes from human umbilical cord mesenchymal stem cells; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen.

Figure 6.

HucMSC-Ex-delivered Wnt4 was critical for angiogenesis in vitro and in vivo. (A): HucMSCs were transfected with lentivirus Wnt4-shRNA or control-shRNA. The expression of Wnt4 in hucMSCs and hucMSC-Ex was determined using Western blot. (B): Cytoplasm and nuclear fractions were prepared from EA.hy926 cells treated with exosomes from Wnt4-shRNA or control-shRNA-transfected hucMSCs. β-catenin protein levels were determined using Western blot. (Ci–Ciii): The tube-formation ability of EA.hy926 cells treated with PBS, shWnt4-Ex, or shControl-Ex was determined (×40). (Civ): Measurement of the tube length by Image J software. ∗∗, p < .01; ∗∗∗, p < .001. (D): The rat wound models were treated with PBS, shControl-Ex, or shWnt4-Ex for 1 week. The expression of CD31 in the wound area was determined using immunofluorescence staining (×200). Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hucMSCs, human umbilical cord mesenchymal stem cells; hucMSC-Ex, exosomes from human umbilical cord mesenchymal stem cells; n.s., not significant; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; shControl, control-short hairpin RNA lentiviral expression vector; shControl-Ex, exosomes from control-short hairpin RNA-transfected hucMSCs; shWnt4, Wnt4-short hairpin RNA lentiviral expression vector; shWnt4-Ex, exosomes from Wnt4-short hairpin RNA-transfected hucMSCs.