04 February 2019 by Jianhui Lin

Reduced circulatory miR-34a levels and its role in EndoMT in Pulmonary Arterial Hypertension

Rationale:

Data suggest that endothelial mesenchymal transition (EndoMT) is implicated in endothelial dysfunction and accumulation of α-smooth muscle actin (α-SMA) expressing cells in the pathogenesis of pulmonary arterial hypertension (PAH). microRNAs (miRs) offer considerable promise as both prognostic biomarkers, and to identify molecular mechanisms underlying PAH. We previously reported that circulatory miR-34a expression levels as downregulated in two independent cohorts of patients with PAH and regulates genes associated with hyperproliferative vasculopathy.

Objective:

To investigate the role of miR-34a in regulation of EndoMT in pulmonary artery endothelial cells (PAEC).

Methods:

Human PAEC (Lonza) were grown in basal media (EGM-2 with 2% fetal calf serum, FCS; Lonza) and growth factor bullet kit (Lonza). EndoMT was induced by the addition of 10 ng/mL TNF-α, 10 ng/mL TGF-β, and/or 10 ng/mL IL- 1β (PeproTech) to PAEC growth media, with conditioned media replaced every 48 hours for 7 days. EndoMT was assessed in PAEC following transfection of either miR-34a-5p and miR-34a-3p mimics or antagonists, or scrambled miR using western immunoblotting.

Results:

Transfection of PAEC with miR-34a-5p or -3p mimics suppress EndoMT induced by combinatorial stimulation of TGF-β1, IL-1β and TNF-α, assessed by reduced EndoMT markers (α-SMA, Vimentin and Zeb1) and increased endothelial marker (VWF and CD31). BMP9 treatment at 10 ng/mL, also suppressed EndoMT and increased endothelial markers (VWF and CD31) compared to non-BMP9 treated groups. Moreover, miR-34a-5p and miR-34a-3p expression levels were significantly elevated in PAEC that exposed to BMP9 treatment compared non-BMP9 treated controls.

Conclusion:

This research identifies miR-34a as a key miR in the regulation of pulmonary vascular cell phenotype associated with EndoMT and a potential novel BMP9-miR34a axis in the pathobiology of PAH. Further experiments in preclinical models are currently underway to demonstrate a causal link between miR-34a levels and EndoMT in the pathogenesis of PAH.

 

About the author


profile picture of Jianhui  Lin

Jianhui Lin

PhD Student

University of Sheffield

United Kingdom

Key Contributors

Jianhui Lin, James Iremonger, Josephine A. Pickworth, Alexander M.K. Rothman, Helen L. Casbolt, Nadine D. Arnold, Charles Elliot, Robin Condliffe, David G. Kiely, Zhi-Cheng Jing, Allan Lawrie


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