Homeostatic effects of exosomal KLF2 microRNAs: implications for treatment of pulmonary hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a severe disorder leading to right heart failure. Recently identified disabling mutations in shear stress-activated transcription factor Krüppel-like factor 2 (KLF2) in hereditary PAH suggest its potential role in the disease. I studied the role of KLF2-induced exosomal microRNAs (miRNAs) in PAH. Methods. Changes in miRNA profile were studied in exosomes purified from control and KLF2-overexpressing human pulmonary artery endothelial cells (HPAECs). Expression of KLF2-induced miRNAs and their target genes was studied in flow-stimulated HPAECs and pulmonary hypertensive mice using quantitative PCR and RNAscope in situ hybridization. The effect of KLF2 miRNAs on HPAECs function and pulmonary vascular remodeling and haemodynamics in Sugen/hypoxia mice were studied. Results. KLF2 induced differential expression of 86 exosomal miRNAs in HPAECs. KLF2 exosomes attenuated apoptosis, reduced TNF-α- and hypoxia-induced activation of inflammatory transcription factor NFκB in pulmonary endothelial cells in vitro, mimicking the actions of KLF2. Eight miRNAs, increased by KLF2 and reduced in PAH, were transfected into HPAECs. Of these, miR-181a-5p and miR-324-5p synergistically prevented apoptosis and inflammatory responses in endothelial cells. RNA sequencing of miR-181a- 5p and miR-324-5p-overexpressing HPAECs confirmed reduced expression of predicted miRNA target genes implicated in vascular remodelling, including ETS-1, NOTCH4, TNF-Α, IL-1, MMP10, MAPK and NFATC2. Physiological flow increased expression of KLF2, miR-181a-5p and miR-324-5p and reduced expression of their target genes in HPAECs. miRNA treatment also attenuated pulmonary vascular remodelling, improved pulmonary haemodynamics and reduced expression of Notch4 and ETS-1 in Sugen/hypoxia preclinical model of PAH. Conclusions: The results show dysregulation of KLF2 signalling in the pulmonary endothelium in PAH and highlight the potential therapeutic role of endothelial KLF-2- induced miRNAs in PAH