Over expression of endothelin-1 (ET-1) in lung fibroblasts (LFb) from patients with pulmonary arterial hypertension (PAH), evidence for loss of inhibitory control

Background: ET-1 receptor antagonists are essential elements in the treatment of PAH suggesting a role of ET-1 in PAH pathogenesis. Since LFb are endowed with an endothelinergic system which drives pro-fibrotic processes (Ahmedat et al. Br. J. Pharmacol. (2013) 168: 471), ET-1 expression in LFb from patients with severe PAH was studied. Methods: LFb from patients with PAH obtained after lung transplantation, commercially available LFb from caucasian healthy donors (phLFb) (PromoCell) and MRC-5 cells (human LFb cell line) were cultured for 24 h in absence or presence of cycloheximide (CHX) and/or TGF-s 1 . Expression of ppET-1 mRNA was determined by qPCR. mRNA levels are expressed as n-fold over detection limit (20 PCR cycles over GAPDH; 2 20 -Δ Ct ). Results: ppET-1 mRNA levels in 1 st passage PAH LFb (94.495±27.947, mean±SEM) were markedly higher than in those phLFb (1.174±259) or MRC-5 cells (1.174±259). ppET-1 expression appears to be under inhibitory control of short-living regulatory peptides which can be removed by CHX (Ahmedat et al.). CHX enhanced ppET-1 mRNA in phLFb and MRC-5 cells by 4.284±996 and 2.794±373%, resp., but not in PAH LFb (118±13% of contols). TGF-β 1 (1ng/ml) increased ppET-1 mRNA in phLFb and MRC-5 cells by 1585±603 and 953±184% resp., but not in PAH LFb (107±18% of controls). During passaging of PAH LFb ppET-1 mRNA increased, 5fold in 3 rd passage. Conclusions: Loss of inhibitory control of ET-1 expression in PAH LFb resulting in marked over-expression of ET-1 may be involved in the pathogenesis of PAH. Epigenetic changes which appear to vanish during cell passaging could be an underlying mechanism.