Platelet extracellular vesicles enhance the proangiogenic potential of adipose-derived stem cells in vivo and in vitro

Fig. 1. Identification of PEVs. A SEM showed that PEVs are spherical nanoparticles. B. FACS proved that Annexin V and specific markers CD41a were highly expressed on PEVs surface. C Representative image and analysis data of NTA;

Fig. 2. Identification of ADSCs. A. Representative image of spindle-shaped ADSC in passage 3. B Image of ADSC transfected with eGFP lentivirus in fluorescent field. C Merge image of ADSC transfected with eGFP lentivirus in fluorescent field and bright field. D FACS showed the percentage of CD29, CD44, CD105, CD90, CD34 and CD45 expression in the surface of ADSCs

Fig. 3. Interaction of PEVs with ADSCs. A Confocal laser scanning microscopy of eGFP-ADSC reacted with CM-Dil-labelled PEVs of different concentrations; B confocal laser scanning microscopy of eGFG-ADSC reacted with CM-Dil-labelled PEV at different time points

Fig. 4. PEV preconditioning enhanced viability, migration and attenuated Cocl2-induced hypoxic apoptosis of ADSCs in vitro. After 24 h PEV pre-treatment, Cocl2(700 ng/ml) was used to induce hypoxic apoptosis of ADSCs. Cell apoptosis was evaluated by Live/dead staining and FACS. A Proliferation of ADSCs preconditioned with different concentration of PEVs for different periods of time. B Representative images and quantification of transwell assay. C Calcein-AM/PI staining showed that the dead cells were significantly reduced in the PEV preconditioning groups. The rate of dead cells was expressed as the ratio of dead cells to total cell count. D The apoptosis-related proteins Bax and Bcl2 were evaluated using Western blotting. E FACS showed that Cocl2-induced hypoxic apoptosis rate of ADSC was decreased in PEV preconditioning groups. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01 versus saline; #p < 0.05, ##p < 0.01 versus PEVs (5 µg/ml)

Fig. 5. RT-PCR and Western blot showed the expression of various angiogenic-related genes in P-ADSC were upregulated. A The mRNA level of VEGF, FGF, HGF, PGF, TGF-β, Angpt1,Angpt2 in all groups were assessed using RT-PCR; B Protein levels of VEGF, Angpt1, Angpt2, eNOS, pAkt, pErk1/2, pFAK, pSrc and pPLCγ1 were assessed using Western blot. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01 versus Saline; #p < 0.05, ##p < 0.01 versus PEVs (5 µg/ml)

Fig. 6. Cultured-medium derived from P-ADSCs promoted the proliferation, migration and tube formation of HUVECs. A Cell proliferation was measured using CCK8 assay; B, C representative images and analysis of HUVEC migration treated with CM from ADSC and P-ADSC; D, E representative images and analysis of HUVEC wound healing assay; F, G tube formation assay images and quantification of tube length and network structural complexity. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01 versus saline; #p < 0.05, ##p < 0.01 versus PEVs (5 µg/ml)

Fig. 7. Matrigel plug assay showed proangiogenic properties of PBS, ADSC and P-ADSC in vivo. A Matrigel plugs derived from different groups. Matrigel mixed with PBS, ADSC and P-ADSC was subcutaneously injected into the abdomen of mouse. The plugs were harvested at 14 days after implantation. B, C CD31 and α-SMA immunostaining of Matrigel plug sections and positive staining showed in brown. P-ADSC group showed more blood vessels formation than PBS and ADSC groups; α-SMA, alpha-smooth muscle actin. D Statistical analysis of vessels number in Matrigel plugs. *p < 0.05, **p < 0.01 versus saline, #p < 0.05, ##p < 0.01 versus ADSC. P-ADSC PEV treated ADSCs

Fig. 8. Mouse model showed P-ADSC improved perfusion recovery, attenuated degeneration and strengthened angiogenesis of ischemic hindlimb. A Representative laser perfusion Doppler image (LDPI) of mouse hindlimb before and after surgery. B Blood perfusion recovery in limb ischemia of each group at days 3, 7, 14, 21, post-surgery. C Quantitative analysis of blood recovery measured by LDPI. Blood recovery was represented using the ratio of ischemic hindlimb perfusion to contralateral hindlimb perfusion. D Haematoxylin–eosin (H&E) staining of gastrocnemius muscle of mouse ischemic hindlimb. E, F immunofluorescent staining of CD31 + capillaries in gastrocnemius muscles of ischemic tissue and quantification analysis. *p < 0.05, **p < 0.01 versus Saline, #p < 0.05, ##p < 0.01 versus ADSC. P-ADSC PEV-treated ADSCs