Background/Purpose: Extracellular vesicles (EV) are a diverse assortment of lipid bilayer-bound vesicles of various sizes and origin. Exosomes are a subset of EV arising from multivesicular bodies released into the extracellular space and into the circulation that contain various macromolecules including numerous microRNA (miRNA) and proteins. EV can mediate intercellular communication by fusing and releasing their macromolecular contents into target cells. The mechanism of the establishment and progression of a profibrotic phenotype in Systemic Sclerosis (SSc) is not currently well understood. Microvascular damage is considered an early event in SSc pathogenesis and EV produced by damaged microvascular endothelial cells may represent an important component in the initiation and progression of SSc.  Here, we characterized the miRNA content of EV isolated from cultured normal human lung microvascular endothelial cells (HLMVEC) treated with TGF-β1, TGF-β2, or TGF-β3 to determine the specific influence of the TGF-β isoforms on the RNA content of HLMVEC.

Methods: Commercially obtained normal HLMVEC were treated with 10 ng/ml of TGF-β1, TGF-β2, or TGF-β3 in FBS-free media. Culture media was isolated from duplicate wells after 72h and EV were isolated by resin-based purification. Total EV RNA was isolated and RNA sequencing (RNA-seq) was performed by 50bp paired-end RNA-seq at 10-20 million reads per sample and aligned to the reference genome (hg19). Differential analysis of RNA content was performed by comparing each treatment group to untreated cells using OASIS 2.0. Gene set enrichment and pathway analysis of miRNA targets was performed employing the MirPath v3.0. Differential expression of selected EV RNA was verified by qPCR.

Results: Treatment of cultured normal HLMVEC with TGF-β isoforms altered the RNA contents of EV isolated from these cells. In addition to changes in miRNA content, marked differences in the amounts and identity of other classes of small RNA such as piRNA were induced upon treatment with the TGF-β isoforms. Analysis of EV miRNA targets indicated extracellular matrix (ECM)-receptor interactions, proteoglycans, ECM components and TGF-β signaling were among the most significantly targeted pathways.

Conclusion: Despite similarities in structure and sequence, TGF-β protein isoforms result in distinct populations of HLMVEC-derived EV. The pattern of upregulated and downregulated EV miRNA from TGF-β1- and TGF-β3-treated HLMVEC show considerable overlap compared with TGF-β2-treated HLMVEC. However, each TGF-β isoform mediated distinct patterns and levels of EV small RNA species. Pathway analysis revealed that numerous pathways are regulated by all three isoforms although the individual genes affected and the magnitude of the changes produced differed for each specific TGF-β isoform. The pathways targeted indicate that EV miRNA could affect target cells by altering the pathways involved in extracellular matrix synthesis, potentially inducing a profibrotic phenotype in target cells. This mechanism may explain the extension of SSc-associated pathological alterations from afftected to normal tissues.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tgf-%ce%b2-isoforms-modulate-the-rna-cargo-of-extracellular-vesicles-exosomes-isolated-from-cultured-normal-human-lung-microvascular-endothelial-cells-a-mechanistic-link-between-endothelial-cell-dy/