Paper Title
YAP-Mediated Endothelial to Mesenchymal Transition Contributes to Pulmonary Arterial Hypertension

Abstract
Pulmonary arterial hypertension (PAH) is a severe and progressive disease, featured by pulmonary vascular remodeling causing medial hypertrophy and muscularization, adventitial thickening, occlusive neointima, and complex plexiform lesions leading to obliteration of precapillary pulmonary arteries and sustained elevation of pulmonary arterial pressure. Cumulative data from preclinical animal models demonstrate that disturbed pulmonary blood flow preceded by endothelial injury is a prerequisite for the development of vascular remodeling including neointimal and plexiform lesions and medial thickening. Histological exams of lungs from PAH patients and animal models revealed that those vascular lesions are mainly seen at the bifurcation of intra-acinar arteries where there is disturbed blood flow. Neointimal and complex plexiform lesions demonstrate a central zone of proliferative tissue that express both endothelial markers and smooth muscle α-actin. The nature and origin of these cells remain controversial, but they are believed to be endothelial lineage. Recently, it has been shown that endothelial to mesenchymal transition (EndoMT) can contribute to the accumulation of smooth muscle-like cells in plexiform lesions. The stimuli that trigger EndoMT in PAH and the mechanisms through which it occurs remain unknown. YAP (yes-associated protein)/TAZ (transcriptional coactivator with PDZ-binding motif) serves as downstream effectors of the Hippo-YAP as well as transcriptional co-activator in the Hippo-YAP pathway to regulate tissue growth by controlling the expression of many genes, such as connective tissue growth factor (CTGF), that are involved in promoting cell survival, proliferation and differentiation. In the present study, we explored the role of YAP and disturbed blood flow in EndoMT of lung endothelial cells (ECs) isolated from PAH patients or healthy donors. We found that lung ECs from PAH patients expressed higher levels of EndoMT markers, α-SMA, TCF-8and Snail-1 under disturbed flow in comparison to that underlaminar flow. The degree of EndoMT marker expression under disturbed flow was significantlyhigher in PAH lung ECs compared to that of healthy donor’s lung ECs. Lung ECs from PAH patients, exposed to disturbed flow, exhibited a higher proliferation rates and were more resistant to staurosporin-induced apoptosis than cellsexposed to laminar flow. Nevertheless,lung ECs from healthy donors, exposed to disturbed flow, had lower proliferation rates and were more sensitive to staurosporin-induced apoptosis than cells exposed to laminar flow. Moreover, disturbed flow increased expression of transcription factor YAP and CTGF. The level of increase was significantly higher in disturbed flow-exposed PAH ECs compared to that in donor’s ECs exposed to disturbedflow. Inhibition of YAPwith verteporfin or knock-down of YAP or CTGF using siRNA prevented disturbed flow-induced EndoMT and proliferation of PAH lung ECs. More importantly, YAP inhibitor verteporfin prevented pulmonary hypertension, vascular remodeling, plexiform lesion formation and EndoMT in PAH rat model. These results indicate that lung ECs from PAH patients undergo YAP-mediatedEndoMT and hyper-proliferation that contribute to neointimal and plexiform lesions in PAH. Thus, YAPpathway might be a noveltherapeutic target in PAH treatment.