(WHS-P2.02) ENDOTHELIAL CELL-DERIVED EXTRACELLULAR VESICLES AND THEIR MICRORNAS DOWNREGULATE FIBROTIC PATHWAYS IN FIBROBLASTS
Friday, May 17, 2024
10:30 AM – 11:30 AM East Coast USA Time
Background: An adequate wound healing response requires the coordination of intercellular signals between multiple cell types. During the proliferative and remodeling phases of healing, endothelial cells and fibroblasts are likely to communicate. We hypothesize that this communication occurs through the release of endothelial cell-derived extracellular vesicles (ECEVs), which contain microRNAs that transcriptionally regulate fibrotic gene signatures and influence fibroblast behavior.
Methods: ECEVs were collected from primary human dermal microvascular endothelial cells using Exoquick-TC and validated using Nanoparticle Tracking Analysis (NTA), Western blot (WB), and transmission electron microscopy (TEM). Bulk RNA sequencing was performed on human dermal fibroblasts treated with ECEVs. ECEVs were also sequenced to determine microRNA content. The relative expression of select gene targets and ECEV microRNAs was validated with qRT-PCR of fibroblasts treated with ECEVs for 24 hours. Fibroblast function was assessed following transfection of a microRNA candidate that had increased expression in ECEV-treated fibroblasts.
Results: Ingenuity Pathway Analysis (IPA) was used to identify top Canonical Pathways affected in fibroblasts treated with ECEVs. The microRNA target filter in IPA was then applied to determine the pathway-specific transcriptional targets of the top 20 microRNAs found in ECEVs. The analysis identified Pulmonary Fibrosis Idiopathic, Hepatic Fibrosis, and Wound Healing as the top 3 downregulated pathways in ECEV-treated fibroblasts, suggesting that ECEVs exert anti-fibrotic effects on fibroblasts. Among these three pathways, 11 common ECEV microRNAs were predicted to target genes related to fibroblast proliferation, differentiation into myofibroblasts, contraction, and formation of collagen. Five genes (ACTA2, MAP2K6, PDGFA, TGFBR3, COL1A2) demonstrated reduced relative expression in ECEV-treated fibroblasts when validated using qRT-PCR (p < 0.05). MiR-126-3p was the most highly expressed microRNA in ECEVs, comprising over 20% of total microRNA content. Intracellular levels of miR-126-3p in fibroblasts were also elevated (p < 0.05) following ECEV treatment, and transfection of fibroblasts with mimics of miR-126-3p resulted in impaired migration and proliferation (p < 0.05).
Conclusions: ECEVs contain microRNAs that are predicted to repress pro-fibrotic gene signatures in fibroblasts. Preliminary in vitro validation suggests that the transfer of miR-126-3p from ECEVs to fibroblasts is one mediator of this anti-fibrotic effect. In the context of wound healing, this endothelial cell-fibroblast communication may be a means of precluding early ECM deposition or contraction, allowing proper vascular remodeling in the wound bed.