Next, we questioned whether exosomal miRNAs contributed to the angiogenic responses elicited by the PF exosomes. To answer this, we focused on let-7b-5p, which was chosen because it was highly expressed in the PF exosomes (Figure 3A), including as conjugated with AGO-2 (Figure 4D), and because other members of the Let-7 family were known to stimulate angiogenesis.18, 19 Expressional data were validated using standard curves. In fact, we found that in comparison with plasma, the whole PF contained higher let-7b-5p and lower miR-122 copy numbers (Figures S7A and S7B). Moreover, we confirmed the presence of let-7b-5p in PF exosomes (Figure S7C). By transfecting ECs with a let-7b-5p mimic, a let-7b-5p inhibitor, or their respective controls (see Figure 6A for validation of the impact of transfection on let-7b-5p in ECs), we demonstrated that let-7b-5p promotes angiogenesis in vitro. In fact, increased let-7b-5p improved capillary-like tube formation on Matrigel (Figure 6B, top), while let-7b-5p inhibition produced the opposite effect (Figure 6B, bottom), and decreased EC proliferation (Figure 6D, right). By contrast, let-7b-5p did not affect the survival of cultured ECs (Figure 6C). We also determined whether the expression of three previously validated direct target genes of let-7b-5p was affected by forcing let-7b-5p expressional changes in ECs. Increased let-7b-5p reduced the mRNA expression of the antiangiogenic TGFBR1 only20, 21 (Figure 6E), leaving unchanged the other two potential target genes, LOX-1 and CASPASE 3 (Figures S8A and S8B). Therefore, the latter were not taken forward for further analyses.Figure 6The PF-Enriched let-7b-5p Is a Proangiogenic MicroRNA(A) ECs were transfected with either a let-7b-5p mimic (right) or a let-7b-5p inhibitor (left). The random sequence anti-miR miRNA inhibitor (scramble) was used as control. Efficiency of let-7b-5p transfection was assessed by qPCR using U6 as a normalizer. (B) Matrigel assay photomicrograph (the scale bar represents 100 μm) and quantification (total length of tube-like structures), 5× magnification. (C and D) Caspase activity (C) and BrdU incorporation (D) of ECs transfected as previously described. (E) Relative expression of the let-7b-5p direct target gene TGFBR1 (measured by PCR using ubiquitin C [UBC] as normalizer). All values are mean + SEM. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus the respective scramble control (unpaired Student’s t test); n = 4.