Pulmonary arterial hypertension (PAH) is a severe disorder characterised by progressive pulmonary vascular remodeling, leading to right heart failure. Endothelial apoptosis and inflammation contribute to the pathogenesis of the disease. Shear stress- activated transcription factor Krueppel-like factor 2 (KLF2) maintains vascular homeostasis and exosomes from KLF2-overexpressing cells have atheroprotective effects. We hypothesized that KLF2-induced exosomal microRNAs may attenuate endothelial dysfunction in pulmonary hypertension.
Methods and Results
KLF2 overexpression in human pulmonary artery endothelial cells (HPAECs) was induced by adenoviral gene transfer. Incubation of HPAECs with exosomes purified from KLF2-overexpressing cells attenuated starvation-induced apoptosis and reduced TNF-α- and hypoxia-induced activation of NFkB. The analysis of microRNA profile in exosomes isolated from control and KLF2-overexpressing HPAECs identified 86 differentially expressed miRs. Five miRs known to be reduced in plasma of PAH patients but elevated in KLF2-overexpressing cells, were transfected into HPAECs and their effects on endothelial apoptosis and NFkB activity were studied. Only miR-181a and miR-324a mimics had endothelium-protective effects in all experimental conditions and their combined actions were stronger compared with single treatments. Inhibitors of miR-181a-5p and miR324-5p reversed protective effects of KLF2 exosomes, suggesting that these two miRs mediate homeostatic effects of KLF2. The levels of miR-181a-5p and miR-324-5p were significantly reduced in blood-derived endothelial progenitor cells (BOECs) from PAH patients (n=7).
RNA sequencing followed by pathway analysis in HPAECs transfected with miR-181a-5p and miR 324-5p (single and combined treatments), revealed a number of gene targets involved in vascular remodelling, including ETS-1, Notch4, TNF-α, IL-1, MMP10 (miR-181a) and ETS1, MAPK, SOCS1, NFATC2 (miR-324). Vascular delivery of miR181a and miR324 mimics in Sugen/Hypoxia mouse model of PAH attenuated pulmonary vascular remodelling and reduced expression of miR targets, Notch4 and ETS-1 in the lung.
Therapeutic supplementation of KLF-2-induced miRs may help design new treatment strategies in pulmonary hypertension.