Endothelial-to-mesenchymal transition (EndMT) is a dynamic biological process
present during development and involved in a variety of pathological vascular
remodelling scenarios. However, despite our growing understanding of the key
cellular alterations required, the precise molecular determinants governing this
phenotypical transition remain elusive. With long non-coding RNAs (lncRNA) now
emerging as powerful regulators of gene expression we sought to understand their role
in the process of EndMT.
To replicate EndMT in vitro and characterise its molecular signature, human umbilical
vein endothelial cells (HUVEC) and human pulmonary artery endothelial cells
(HPAEC) were exposed to a continuous co-treatment of transforming growth factor-beta 2 (TGF-β2) and interleukin 1 beta (IL-1β) for a total of 7 days. Using high-throughput RNA-sequencing analysis of these cells, a total of 103 differential
expressed lncRNAs were identified. Of these, the downregulation of the lncRNA
MIR503HG was found to be a prevalent feature present in multiple human primary
EC types undergoing EndMT in vitro. Further analysis revealed that depletion of
MIR503HG was sufficient to elicit a robust EndMT phenotype, with a significant
increase in the expression of SNAI2, ACTA2 and COL1A1, accompanied by
repression of CD31. Conversely, ectopic expression of a single MIR503HG transcript
suppressed these hallmark EndMT-associated changes despite TGF-β2 and IL-1β co-treatment. Accompanying RNA-sequencing of these cells showed that the
overexpression of MIR503HG alone was able to inhibit over 25% of the EndMT
transcriptional profile. Crucially, these changes were found to be independent of the
functional regulation of miR-503 and miR-424, found within the MIR503HG locus. Our findings were then confirmed in vivo using a sugen/hypoxia-induced model of
pulmonary hypertension (PH) established in endothelial lineage-tracing mice. Here,
the expression of the MIR503HG mouse homolog (Gm28730) was significantly
downregulated in association with an EndMT profile in the lung. Conversely, targeted
up-regulation of MIR503HG in the mouse lung significantly suppressed the
appearance of mesenchymal markers in CD31+ cells during PH. Notably, MIR503HG
availability was also found to be decreased in lung tissue sections from patients with
idiopathic pulmonary arterial hypertension (IPAH) and cultured blood outgrowth ECs
isolated from patients with heritable pulmonary arterial hypertension (HPAH).
Collectively, our studies identify MIR503HG as essential in maintaining EC
phenotypical commitment and preventing EndMT both in vitro and during disease
