Growth differentiation factor 6 (GDF6): a novel regulator of pulmonary vascular remodeling in pulmonary hypertension secondary to left heart disease

Introduction: Pulmonary hypertension due to left heart disease (PH-LHD) is the most common type of pulmonary hypertension (PH) and freguently aggravated by pulmonary arterial (PA) remodeling. Altered pulmonary hemodynamics are primarily sensed by vascular endothelial cells (ECs) which in turn undergo remodeling, yet underlying molecular mechanisms remain to be characterized.

Methods and results: We identified that in PH-LHD, increased proliferation and migration of PA ECs is associated with reduced expression of bone morphogenetic protein receptor type II (BMPR2) and decreased activation of the downstream effectors SMAD1/5/8 of the bone morphogenetic protein (BMP) signaling pathway. Bulk RNA sequencing revealed upregulation of a BMPR2 ligand – growth differentiation factor 6 (GDF6) – in PA samples of PH-LHD patients and in an experimental PH-LHD rat model induced by surgical aortic-banding, as compared to healthy donors and sham controls, respectively. Increased levels of GDF6 protein in PA biosamples and plasma of PH-LHD patients, isolated human pulmonary arterial smooth muscle cells and ECs, and lungs of AoB rats was confirmed by western blotting. The functional role of GDF6 in ECs was further addressed in gain- and loss-of-function experiments, which showed that treatment of healthy ECs with exogenous GDF6 promoted cell proliferation and migration, whereas knockdown of GDF6 in PH-LHD ECs reduced these cellular functions. We further demonstrated that in healthy ECs GDF6 could activate not only BMPR2, but also transforming growth factor beta (TGF-β) and vascular endothelial growth factor receptor (VEGFR) signaling pathways, as identified by increased phosphorylation of SMAD2/3 and ERK1 respectively. Yet, in PH-LHD ECs exogenous GDF6 failed to activate the BMPR2 pathway, while activation of the TGF-β and VEGFR pathways was further increased, , indicating a dysbalance of signaling pathways towards pro-proliferative signaling. Pharmacological inhibition of either the TGF-β receptor (TGFβR) or VEGFR2 abolished the effects of exogenous GDF6 on SMAD2/3 and ERK1 phosphorylation in healthy ECs, and attenuated the effect of GDF6 on EC proliferation and migration.

Conclusions: GDF6 expression is increased in PAs from PH-LHD patients and AoB rats. In PH-LHD, GDF6 promotes the proliferation and migration of PA ECs by activating TGF-β and ERK signaling pathways and may as such contribute critically to PA remodeling, These findings highlight the need for further in-depth study of GDF6´s functional role in vivo.