Dermal Fibroblasts Uptake Exosomes From Endothelial Cells and Promote Migration

Objectives: This study examines if exosomes from microvascular endothelial cells influence wound resolution by targeting fibroblasts, one of the essential players in skin regeneration. Our objectives are to isolate and characterize exosomes from human dermal microvascular endothelial cells, to examine the uptake of these exosomes by human dermal fibroblasts (FBs), and to observe the effect of endothelial exosome uptake on fibroblast migration.
Methods: Endothelial cells were cultured until 80% confluence, then incubated in medium containing exosome free fetal bovine serum for 48-hours. Exosomes were collected and purified from the culture supernatant of the Endothelial cells (EC-exos). Nanoparticle tracking was used to determine the quantity and size distribution of the exosomes. Green fluorescently labeled EC-exos were placed on plated FBs for 24 hours to assess uptake using fluorescent microscopy. A transwell migration assay that employed 0.1% crystal violet staining was used to assess fibroblast migration. To assess the effects of EC-exos on FB function, a scratch assay was used to measure migration. Fibroblasts were seeded in 3-well culture inserts (Ibidi) creating two artificial gaps on a confluent monolayer. Mitomycin C (1μg/mL) was applied to the monolayer for one hour after the inserts were removed to preclude proliferation and scratch closure was assessed at 0, 12, 24, and 48 hours.
Results: Following exosome purification, nanoparticle analysis showed a mean particle size of 148.1 nm, which is characteristic of exosomes. Fluorescent microscopy and confocal microscopy showed uptake of exosomes from HDMVECs by the HDFs at 15% concentration. FBs treated with EC-exos exhibited enhanced migration in the transwell migration assay and accelerated scratch closure.
Conclusions: ECs produce functional exosomes that can be successfully isolated and taken up by FBs causing changes in migration patterns, suggesting that endothelial cells can influence fibroblast function in wounds via exosomes.