Small Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhanced Proangiogenic Potential of Cardiac Fibroblasts via Angiopoietin-Like 4

Figure 1. Characterization of hucMSCs, sEVs, and CFs. (a) The surface markers of hucMSCs measured by flow cytometry. (b) Morphology of hucMSCs. Scale bar: 1000 μm (i) and 500 μm (ii). (c) Morphology of sEVs observed by TEM. Scale bar: 200 nm. (d) Particle size distribution of sEVs measured by NTA. (e) Brownian motion image of hucMSCs. (f) Protein expression of CD9, TSG101, CD81, and calnexin in hucMSCs and sEVs. (g, h) The expression of α-SMA, vimentin, collagen I, periostin, and CD31 on primary CFs was measured by immunofluorescence (g) and western blotting (h). Scale bar: 50 μm.

Figure 2. The sEVs enhanced proangiogenic potential of CFs. Primary CFs were treated with LPS (100 ng/mL) and LPS+sEVs (200 μg/mL) for 24 h, and supernatants were collected. HUVECs were treated with the supernatants derived from pretreated CFs. (a) Proliferation of HUVECs was detected by the CCK-8 assay. (b) The mRNA expression of cyclin D1 was measured by qRT-PCR. (c) Cyclin D1 protein expression was detected by western blotting. (d, f) Migration of HUVECs in different treatment groups was tested by the wound healing assay (d) and Transwell migration assay (f). Scale bar: 500 μm (d) and 100 μm (f). (e) Quantitative analysis of 12 h migration rate in (d). (g) Quantitative analysis of migrated cells in (f). (h–j) Tube formation ability of HUVECs was measured by the tube formation assay (h). Quantitative analysis of the total branching points (i) and tube formation numbers (j). Scale bar: 500 μm. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 3. Angptl4 was increased in CFs treated with sEVs. Primary CFs were treated with LPS (100 ng/mL) and LPS+sEVs (200 μg/mL) for 24 h. mRNA expression of these two groups was compared via RNA-seq analysis. (a) Volcano map of DEGs. (b) Relative fold change of the top 20 angiogenesis-related genes for enrichment analysis. (c) Angptl4 mRNA expression was viewed by qRT-PCR. (d, e) Protein expression of Angptl4 was detected by western blotting. (f, g) Immunofluorescence was used to observe Angptl4 expression. Scale bar: 100 μm. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 4. Inhibition of Angptl4 in primary CFs and sEVs resulted in decreased angiogenesis. (a) Angptl4 protein expression in hucMSCs and sEVs was detected by western blotting. (b, c) hucMSCs (b) and CFs (c) were transfected with Angptl4 siRNA, respectively, and expression of Angptl4 was measured by western blotting. (d) Angptl4 and CD81 expression in sEVs was observed by western blotting after transfection with siRNA. (e) Angptl4 expression in CFs was detected by western blotting after Angptl4 in CFs and in sEVs was knocked down with siRNA. (f) HUVECs were treated with the supernatants derived from pretreated CFs. The proliferation of HUVECs was measured by the CCK-8 assay. (g, h) The migration of HUVECs was detected by the Transwell migration assay (g). Scale bar: 100 μm. Quantitative analysis of the migrated cells (h). (i–k) Tube formation ability of HUVECs was measured by tube formation assay (i). Quantitative analysis of the total branching points (j) and tube formation numbers (k). Scale bar: 500 μm. ∗P < 0.05 and ∗∗P < 0.01. ns: no significant difference between two groups.

Figure 5. Proangiogenic potential of CFs enhanced by sEVs was associated with β-catenin. (a) β-Catenin expression in CFs was detected by western blotting after Angptl4 in CFs and in hucMSCs was knocked down with siRNA. (b) After using the β-catenin inhibitor ICG001 (0, 5, 10, and 15 μM), primary CFs were treated with LPS (100 ng/mL) and LPS+sEVs (200 μg/mL) for 24 h and supernatants were collected to culture HUVECs. The proliferation of HUVECs was measured by the CCK-8 assay. (c) The migration of HUVECs was measured by the Transwell migration assay. Scale bar: 100 μm. (d) Quantitative analysis of migrated cells. (e–g) Tube formation ability of HUVECs was evaluated by the tube formation assay (e). Scale bar: 500 μm. Quantitative analysis of the total branching points (f) and tube formation numbers (g). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.