Figure 4.

CTs exosomes facilitated survival and inhibited apoptosis in CMECs and enhanced angiogenesis in ischemic-hypoxic conditions in vitro, and CT-miR-21-5p was identified as a key functional molecule. A: The cell viability of CMECs incubated with CT-exos, CT-exos-miR21KO or PBS under normoxia and ischemia-hypoxia. B: Annexin V/PI staining and flow cytometry of CT-exos-, CT-exos-miR21KO-, PBS- or blank-treated CMECs cultured under normoxic and ischemic-hypoxic conditions for 24 h. C: Tube formation assay of CMECs incubated with CT-exos, CT-exos-miR21KO or PBS under normoxic and ischemic-hypoxic conditions for 8 h. The above studies (A-C) were conducted using two animals in at least three repeated experiments for each individual animal. D: The 10 most abundant miRNAs in CT-exos according to RNA sequencing, and miRNA-21-5p is the most prevalent miRNA. E: miR-21-5p expression levels in CMECs incubated with PBS, CT-exos or CT-exos-miR21KO for 24 h. F: (a) Schematics of the transwell assays of CT-exos for CMECs. (b) Flow cytometry analysis for CMECs which was cocultured with no CTs in the upper chamber for 48 h. (c) Flow cytometry analysis for CMECs (lower chamber) cocultured with CTs transfected by the synthesized miR-21-5p-FAM (green) in the upper chamber for 48 h. The green-stained CMCEs in the lower chamber comprised approximately 27%. (d) The green-stained CMCEs were only found in miR-21-5p-FAM (green)-CT treated group. (e) The scattered green dots were found in the miR-21-5p-FAM (green)-CT-treated CMCEs by microscopy. (f) The qPCR assay for transwell treatment confirmed that the miR-21-5p level of CT-treated CMCEs was significantly higher than that of the blank control, while the miR-21-5p level of the miR21KO-CT-treated CMCEs was similar to that of the blank control. *: p < 0.05, **: p < 0.01, #: p < 0.05 compared with the PBS group, &: p < 0.05 compared with the CT-exos-miR21KO group.