TNF-α alters the release and transfer of microparticle-encapsulated miRNAs from endothelial cells

Abstract

MicroRNAs (miRNAs) encapsulated within microparticles (MPs) are likely to have a role in cell-to-cell signaling in a variety of diseases, including atherosclerosis. However, little is known about the mechanisms by which different cell types release and transfer miRNAs. Here, we examined TNF-α-induced release of MP-encapsulated miR-126, miR-21, and miR-155 from human aortic endothelial cells (ECs) and their transfer to recipient cells. ECs were treated with TNF-α (100 ng/ml) in the presence or absence of inhibitors that target different MP production pathways. MPs released in response to TNF-α were characterized by: 1) 70-80% decrease in miRNA/MP levels for miR-126 and -21 but a significant increase in pre-miR-155 and miR-155 (P < 0.05), 2) 50% reduction in uptake by recipient cells (P < 0.05), and 3) diminished ability to transfer miRNA to recipient cells. Cotreatment of donor ECs with TNF-α and caspase inhibitor (Q-VD-OPH, 10 μM) produced MPs that had: 1) 1.5- to 2-fold increase in miRNA/MP loading, 2) enhanced uptake by recipient cells (2-fold), and 3) increased ability to transfer miR-155. Cotreatment of ECs with TNF-α and Rho-associated kinase (ROCK) inhibitor (10 μM) produced MPs with features similar to those produced by TNF-α treatment alone. Our data indicate that TNF-α induced the production of distinct MP populations: ROCK-dependent, miRNA-rich MPs that effectively transferred their cargo and were antiapoptotic, and caspase-dependent, miRNA-poor MPs that were proapoptotic. These data provide insight into the relationship between MP production and extracellular release of miRNA, as well as the potential of encapsulated miRNA for cell-to-cell communication.

Keywords: TNF-α; atherosclerosis; endothelial cells; extracellular RNA; miRNA release and transfer; microparticles.

Fig. 1.

Precursor and mature microRNA (miR)-126 (A), miR-21 (B), and miR-155 (C) content of microparticles (MPs) produced by endothelial cells (ECs) subjected to TNF-α (100 μg/ml) for 24 h. MP-encapsulated microRNA (miRNA) was assessed by the ratio of miRNA level in MP fraction divided by the number of MPs. Values expressed relative to control MPs. *P < 0.05, n = 10.

Fig. 2.

Effect of TNF-α (100 ng/ml) plus caspase inhibitor on precursor and mature miR-126, -21, and -155 levels in ECs (A) and MPs (B). Relative levels of intracellular miRNA were normalized to U6 and expressed relative to TNF-α treatment alone (no significant changes, n = 6). MP-encapsulated miRNA was assessed by the ratio of miRNA level in MP fraction divided by the number of MPs. Values expressed relative to TNF-α MPs. caspase-I, caspase inhibitor. *P < 0.05, n = 6.

Fig. 3.

Effect of TNF-α (100 ng/ml) plus Rho-activated kinase (ROCK) inhibition (Y-27632, 10 µM) on precursor and mature miR-126, -21, and -155 levels in ECs (A) and MPs (B). Relative levels of intracellular miRNA were normalized to U6 and expressed relative to TNF-α treatment alone (*P < 0.05, n = 5). MP-encapsulated miRNA was assessed by the ratio of miRNA level in MP fraction divided by the number of MPs. Values expressed relative to TNF-α MPs. ROCK-I, ROCK inhibitor *P < 0.05, n = 5.

Fig. 4.

MPs from TNF-α- and TNF-α plus ROCK inhibitor-treated donor ECs (B) are not taken up by recipient cells as avidly as MPs from untreated donor cells (A) or donor cells treated with TNF-α plus caspase inhibitor (C). Red, phalloidin stain for F-actin; blue, DAPI for cell nucleus; green (arrows), calcein from labeled donor MPs that had been taken up by recipient cells. Micrographs are representative of 3 separate experiments. MPs were isolated from different donor EC treatment groups, labeled with calcein-AM, and subsequently incubated with recipient ECs for 24 h. D: grouped mean green fluorescence values in recipient ECs after 24 h incubation with MPs from different donor ECs. x-Axis indicates different donor cell treatments. *P < 0.05 vs. control; #P < 0.05 between groups, n = 6.

Fig. 5.

MPs from donor ECs treated with TNF-α or TNF-α plus ROCK inhibitor have diminished capacity to transfer miR-155 to recipient ECs, which is abrogated by caspase inhibitor. MPs were isolated from donor ECs that had been untreated (control) or treated with TNF-α with and without caspase and ROCK inhibitor for 24 h. Different groups of donor MPs were incubated with recipient ECs for 2 h. MiRNA-155 levels are expressed relative to recipient ECs incubated with MPs from control donor cells. x-Axis indicates different donor cell treatments. NS, not significant. *P < 0.05 vs. control; #P < 0.05 between groups, n = 3.

Fig. 6.

The effect of MPs from different donor EC treatment groups on recipient cell apoptosis. Recipient ECs were incubated with MPs from untreated donor cells (control), donor cells treated with TNF-α alone, or donor cells treated with TNF-α plus inhibitor (i.e., caspase-I, ROCK-I) for 24 h, and caspase-3 activity was measured in the recipient cells. Results are expressed relative to caspase-3 activity in recipient ECs incubated with MPs from control donor cells. *P < 0.05 vs. control; n = 5.