Extracellular vesicles derived from human umbilical cord mesenchymal stem cells stimulate angiogenesis in myocardial infarction via the microRNA-423-5p/EFNA3 axis

Figure 1. HucMSC-EV identification. A – HucMSCs P3 morphology was observed under a light microscope; B – Oil red O and alizarin red stainings were utilized to detect adipogenic and osteogenic differentiation of hucMSCs, respectively; C – EV morphology was observed by transmission electron microscopy D – Expression of positive and negative characteristic markers of hucMSC-EVs was determined by Western blot; E – EVmiRNA database (http://bioinfo.life.hust.edu.cn/EVmiRNA#!/) was applied to detect miR-423-5p distribution in EVs from diverse sources; F – miR-423-5p content in hucMSC-EVs was measured by RT-qPCR. *P < 0.05 vs hucMSC group

Figure 2. HucMSC-EVs promote in vitro tube formation of HUVECs by delivering miR-423-5p. A – HUVECs were cultured with PKH26-labelled EVs (red) and endocytosis of EVs was detected by immunofluorescence assay; B – miR-423-5p expression in hucMSCs and EVs was tested by RT-qPCR; C – miR-423-5p expression in HUVECs was measured by RT-qPCR; D – VEGF content in cell supernatants was measured by ELISA. E – Angiogenic ability of HUVECs in each group was evaluated by pseudo-tube formation assay. F – Migration ability of HUVECs in each group was detected by Transwell assay.*P < 0.05 vs. PBS group or miR-NC group, #p < 0.05 vs. EV-miR-NC group

Figure 3. Targeting relationship between miR-423-5p and EFNA3. A – Bioinformatics website TargetScan (https://www.targetscan.org/) predicted there were binding sites between EFNA3 and miR-423-5p; B – Binding sites between miR-423-5p and EFNA3; C – Results of dual luciferase reporter gene assay; D – miR-423-5p and EFNA3 mRNA expression levels in HUVECs were tested by RT-qPCR. *P < 0.05 vs. mimic NC group

Figure 4. Interaction effect of miR-423-5p and EFNA3 on in vitro tube formation of HUVECs. A – EFNA3 mRNA expression levels in HUVECs were assessed by RT-qPCR; B – HUVEC migration ability of each group was measured by Transwell assay. C – HUVEC angiogenic ability of each group was evaluated by pseudo-tube formation assay. D – VEGF content in cell supernatants was tested by ELISA. *P < 0.05 vs. the oe-NC + PBS group, #p < 0.05 vs. the oe-NC + EV group

Figure 5. In vivo experiments for observing the effects of EV-miR-423-5p on cardiac function repair in MI rats. A – miR-423-5p in myocardial tissues was measured by RT-qPCR; B – LVEF and LVFS were determined by echocardiography to assess cardiac function. C – Myocardial fibrosis was evaluated by Masson staining. D – Positive capillary CD31 staining in the peri-infarct area was detected by immunohistochemistry. n = 10. E – Positive VEGF staining in the peri-infarct area was determined by immunohistochemistry. n = 10. *P < 0.05 vs the sham group, #p < 0.05 vs. the MI group, &p < 0.05 vs. the MI + EV-miR-NC group