We next interrogated the possibility that let-7b-5p could be transferred from PF exosomes to ECs and the expressional and functional impact of exosomal let-7b-5p uptake by recipient ECs. For these experiments, we adopted a model in which the endogenous expression of miRNAs is reduced. It was previously reported that miRNA biogenesis, and hence intracellular miRNA levels, are decreased after DICER silencing in cultured ECs. Moreover, DICER knockdown (KD) reportedly impairs the angiogenic capacity of cultured ECs.22 We initially confirmed that DICER KD resulted in reduced DICER gene and protein expression (Figures S9A and S9B) and impaired the angiogenic potential of ECs (Figures S9C and S9D). Moreover, as expected, DICER KD reduced let-7b-5p expression (Figure 7A). Next, ECs with either DICER KD or a preserved DICER expression were stimulated with our PF exosomes. Treatment with PF exosomes restored intracellular let-7b-5p expression, which had been compromised by DICER KD (Figure 7A), thus supporting the hypothesis that this proangiogenic miRNA is transferred from the PF exosomes to the recipient ECs. In line with the hypothesis that the let-7b-5p delivered from PF exosomes into ECs is functionally active, ECs treated with PF exosomes responded with a decreased mRNA expression of the miRNA target gene TGFBR1 (Figure 7B). In ECs with DICER KD, treatment with PF exosomes was additionally able to restore DICER expression at protein (Figure S10A), but not mRNA, levels (data not shown). This, together with the aforementioned finding of DICER presence in the exosomes (Figure 4E), further suggests that PF exosomes pass on miRNAs and other components of the RISC machinery to recipient cells. Finally, and in line with their induced let-7b-5p and TGFBR1 expressional changes, PF exosomes restored the angiogenic capacity of DICER KO-ECs (Figures 7C and 7D). To further investigate the transfer of PF exosomal let-7b-5p to ECs and its functional consequence, we preventively suppressed let-7b-5p inside the exosomes. The reduction of exosomal let-7b-5p in PF exosomes transfected with the miRNA inhibitor was confirmed by qPCR (Figure S10B). Moreover, the PF let-7b-5p KD-exosomes could not (1) restore let-7b-5p levels (Figure 7A), (2) decrease TGFBR1 expression (Figure 7B), or (3) improve angiogenesis (Figures 7C and 7D) in recipient DICER-KD ECs. Taken together, these data support the hypothesis that PF exosomes are functionally active and stimulate angiogenesis, at least in part, via the passage of the proangiogenic let-7b-5p to ECs.Figure 7Let-7b-5p Underpins the Angiogenic Action of PF ExosomesECs were transfected with either a sequence that exhibits no homology to the human genome (scramble) or DICER silencing RNA (siRNA) to knock down (KD) DICER for 24 hr, before being treated with either naive PF exosomes or PF exosomes previously depleted of their let-7b-5p content (exosome let-7b-5p KD). (A and B) Columns graphs show the relative expression of (A) let-7b-5p and (B) TGFBR1 in the exosome recipient ECs and untreated cell controls; n = 4. (C) Photomicrographs (the scale bar represents 100 μm) and (D) quantification of Matrigel assays performed using ECs treated as previously indicated; n = 5; 2.5× magnification. All values are mean + SEM. §p ≤ 0.05 and §§§p ≤ 0.001 versus scramble, *p ≤ 0.05 and ***p ≤ 0.001 versus DICER KD, #p ≤ 0.05 and ##p ≤ 0.01 DICER KD treated with naive PF exosomes (one-way ANOVA, Tukey’s post hoc test).