NBL1 Mediates Endothelial-to-Mesenchymal Transition In Pulmonary Arterial Hypertension Associated With Congenital Heart Disease

Background:Congenital heart disease(CHD)with a large intra-or extracardiac communication,features a left-to-right flow inducing pressure overload of the pulmonary circulation.For unrepaired CHD,constant disturbed blood flow accelerates the progression of pulmonary vascular remodeling,can eventually result in irreversible pulmonary arterial hypertension(PAH).Endothelial-to-mesenchymal transition(EndMT)exerts a critical role in flow-induced vascular remodeling process which is also a common cause of CHD-PAH.Neuroblastoma suppressor of tumorigenicity 1(NBL1)was implicated with CHD-PAH via aggravating phenotype transformation of smooth muscle cells(SMCs).However,the underlying mechanisms regarding the interplay between NBL1 and endothelial cells(ECs)in CHD-PAH remained to be fully elucidated.Aims:To explore the effect of abnormal blood flow on pulmonary vascular injury,four animal models were established.Secondly,Nbl1 knockout rats were constructed to explore the effect of Nbl1 inhibition on the development of shunt-induced PAH.Thirdly,we aimed to identify the potential effect of NBL1 on EndMT in CHD-PAH,and to explore the clinical value of NBL1 in evaluating the function of ECs and the progression of CHDPAH.Methods and Results:To closely reproduce the induced hemodynamic status and proliferative morphological changes observed in CHD-PAH patients,we established a shunt-induced PAH model with Nbl1 knockout rats.The morphological analysis demonstrates that the novel strategy of Nbl1 knockout relieve EndMT and effectively attenuate shunt-induced PAH.Furthermore,the phenotype of EndMT were detected by RNA-seq and further accessed by western blot and immunostaining on pulmonary arteries.Mechanistic experiments were established on human pulmonary artery endothelial cells(hPAECs)to further confirm that EndMT was induced by NBL1 in vitro.After 7 days stimulation with NBL1,the EndMT-related biomarkers,and downstream transcription factors were quantified by RNA-seq,western blot and immunocytochemistry.Both in vivo and in vitro experiments supported the imbalance between increased TGF-β and dysregulation of BMP signaling pathway with NBL1.On the contrary,blocking canonical TGF-β pathway with inhibitors efficiently preserved endothelial phenotype upon NBL1 stimulation.Finally,we detected plasma NBL1 level,EndMT-related biomarkers(ADMA/VCAM-1/IL-6)in CHD-PAH patients with different pulmonary vascular resistance.The correlational analyses results showed that plasma NBL1 level was negatively correlated with pulmonary vascular resistance and EndMT-related biomarkers.Conclusions:NBL1 aggravated shunt-induced PAH through inducing EndMT via TGFβ/BMP signaling pathway.Thus,antagonizing NBL1 and rebalancing TGF-β/BMP signaling pathway may be a viable therapeutic target for CHD-PAH.Furthermore,NBL1 was deeply involved in the severity of EndMT and CHD-PAH,indicating that NBL1 could be a novel marker to evaluate the function of ECs and the progression of CHD-PAH.