Introduction Despite advanced therapies for pulmonary arterial hypertension (PAH), the hyperproliferative pulmonary vasculopathy persists. Circulatory microRNAs (miR) offer considerable promise as both a prognostic biomarker, and to identify molecular mechanisms underlying PAH. Previous study from our lab identified whole blood miR-34a as downregulated in patients with PAH.
Objectives To validate changes in whole blood miR-34a levels in patients with PAH and relate them to disease severity and survival, and determine the phenotypic effect on pulmonary artery smooth muscle cells (PASMC).
Methods Whole blood RNA was isolated from 27 treatment-naive patients with PAH, 12 age-matched healthy volunteers (HV) and experimental models of PAH (Monocrotaline-MCT, Sugen5416/hypoxia-SuHx and controls, n = 5/group). Whole blood miR-34a-5p and −3p levels were measured by qPCR. The phenotypic effect of miR-34a-5p and −3p levels was assessed on PASMC in-vitro. Differences between groups were determined by Student’s t-test or ANOVA-Tukey.
Results Whole blood miR-34a-5p was reduced in patients with PAH (p < 0.0001) and experimental models of PAH (MCT p < 0.05, SuHx p < 0.001). Receiver operating characteristic curve identified that miR-34a-5p levels discriminates patients with PAH from HV (AUC = 0.86, p = 0.001). MiR-34a-5p levels were significantly lower in patients with severe PAH, as defined by a cardiac index of <2 vs >2.5 l/min/m2 (p < 0.05) and NT-proBNP > 300 vs <300 ng/l (p < 0.001) and predict survival at 5 years. MiR-34a-5p levels were negatively correlated with pulmonary vascular resistance (r = −0.4, p < 0.05) and pulmonary arterial wedge pressure (r = −0.4, p < 0.05). Preliminary data showed that whole blood miR-34a-3p was reduced in patients with PAH (p = 0.0267) and experimental models of PAH (MCT p < 0.01, SuHx p < 0.01); and delineates patients with PAH from HV (AUC = 0.925, P = 0.01). Transfection of PDGF-stimulated PASMC with miR-34a-5p or −3p inhibitor promote PASMC proliferation (p < 0.001). In contrast, miR-34a-5p and −3p mimic suppress PASMC proliferation (p < 0.05 and p < 0.001 respectively). Additionally, transfection with miR-34a-3p increases caspase-3/7 activities in PASMC (p < 0.0001).
Conclusions Reduced miR-34a-5p levels associate with increased disease severity and poor prognosis in PAH. MiR-34a-5p and −3p levels regulate PASMC proliferative-phenotype in response to PDGF. This research identifies miR-34a-5p and −3p as potential biomarkers, subsequent network analysis may identify novel disease mechanisms. Further experiments in preclinical models are currently underway.