The role of the primary cilium in pulmonary arterial hypertension

Abstract

Introduction Vascular remodeling in pulmonary arterial hypertension (PAH) is characterized by endothelial dysfunction and smooth muscle cell proliferation and hypertrophy ultimately causing right heart failure and death. Up to now, the molecular mechanisms of PAH pathogenesis have not been adequately resolved, preventing the development of novel therapies specifically targeting vascular remodeling in PAH. Based on increased expression levels of PDGF-BB and TGF-β in both patients and animal models with PAH, these signaling pathways have been proposed to play a critical role as drivers of lung vascular remodeling. Among multiple other effects, PDGF-BB and TGF-β cause shortening of the primary cilium – an antenna-like organelle functioning as a flow-sensor and signaling hub on most eukaryotic cells. Loss or shortening of cilia is characteristically associated with cell proliferation and promotes PDGF signaling, thus potentially establishing a positive feedback loop. We thus hypothesized that PAH is associated with a loss or shortening of primary cilia in pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs) which in turn drives cell proliferation and thus, remodeling in the pulmonary arterial wall. Methods Pulmonary artery tissue from PH-patients and non-PH donors was fixed and stained for acetylated α-tubulin to determine the number of primary cilia. PAECs and PASMCs from PAH patients and non-PAH donors were exposed in vitro to three different characteristic stimuli or mediators, respectively, of PAH, namely PDGF-BB, TGF-β1, or hypoxia (1%). Cells were fixed and stained for measurement of primary cilia length. In parallel, donor and PAH-SMCs were compared functionally in migration and proliferation assays. IFT88 - an essential structural protein of the primary cilium - was knocked down by siRNA in PAECs and PASMCs to assess the effect of cilium loss on migration and proliferation. Results In pulmonary arteries of PAH patients, the number of primary cilia per area was reduced by 80% as compared to donor lungs. PAH-SMCs were found to have shorter cilia and similar to IFT88 knock-down ECs and SMCs showed increased migration and proliferation as compared to healthy control cells. Additionally, primary cilia of PAECs, PASMCs, and PAH-SMCs stimulated with PDGF-BB, TGF-β1, or hypoxia were shorter as compared to controls. Lithium, which has been shown to elongate cilia in other cell types, was able to rescue cilia length and reduced migration and proliferation in PAH-SMCs. Conclusions Here, we demonstrate that PAH is associated with a loss of cilia in vivo and reduced cilium length in vitro. Primary cilium loss or shortening may promote progression of vascular remodeling as indicated by the fact that cilium loss causes proliferation/migration in PAH-SMCs, and their parallel reversal by lithium. Cilium loss may as such be an important propagator of vascular remodeling in PAH and may present a target for novel therapeutic interventions such as lithium.