Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial cell dysfunction and vascular remodeling. Normally, the endothelium forms an integral cellular barrier to regulate vascular homeostasis. During embryogenesis endothelial cells exhibit substantial plasticity that contribute to cardiac development by undergoing endothelial-to-mesenchymal transition (EndoMT). We determined the presence of EndoMT in the pulmonary vasculature in vivo and the functional effects on pulmonary artery endothelial cells (PAECs) undergoing EndoMT in vitro. Histologic assessment of patients with systemic sclerosis-associated PAH and the hypoxia/SU5416 mouse model identified the presence von Willebrand factor/α-smooth muscle actin-positive endothelial cells in up to 5% of pulmonary vessels. Induced EndoMT in PAECs by inflammatory cytokines IL-1β, tumor necrosis factor α, and transforming growth factor β led to actin cytoskeleton reorganization and the development of a mesenchymal morphology. Induced EndoMT cells exhibited up-regulation of mesenchymal markers, including collagen type I and α-smooth muscle actin, and a reduction in endothelial cell and junctional proteins, including von Willebrand factor, CD31, occludin, and vascular endothelial-cadherin. Induced EndoMT monolayers failed to form viable biological barriers and induced enhanced leak in co-culture with PAECs. Induced EndoMT cells secreted significantly elevated proinflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor α, and supported higher immune transendothelial migration compared with PAECs. These findings suggest that EndoMT may contribute to the development of PAH.

These data describe a novel murine model of PAH, which displays many of the hallmarks of the human disease, thus opening new avenues of investigation to better understand PAH pathophysiology.

Myofibroblasts are the key effector cells responsible for excessive extracellular matrix deposition in multiple fibrotic conditions, including idiopathic pulmonary fibrosis (IPF). The PI3K/Akt/mTOR axis has been implicated in fibrosis, with pan-PI3K/mTOR inhibition currently under clinical evaluation in IPF. Here we demonstrate that rapamycin-insensitive mTORC1 signaling via 4E-BP1 is a critical pathway for TGF-β1 stimulated collagen synthesis in human lung fibroblasts, whereas canonical PI3K/Akt signaling is not required. The importance of mTORC1 signaling was confirmed by CRISPR-Cas9 gene editing in normal and IPF fibroblasts, as well as in lung cancer-associated fibroblasts, dermal fibroblasts and hepatic stellate cells. The inhibitory effect of ATP-competitive mTOR inhibition extended to other matrisome proteins implicated in the development of fibrosis and human disease relevance was demonstrated in live precision-cut IPF lung slices. Our data demonstrate that the mTORC1/4E-BP1 axis represents a critical signaling node during fibrogenesis with potential implications for the development of novel anti-fibrotic strategies.

We report a novel mode of action for imatinib, demonstrating TPH1 down-regulation via inhibition of PDGFR-β signaling. Our data reveal interplay between PDGF and 5-HT pathways within PAH, demonstrating TPH1-dependent imatinib efficacy in collagen-mediated mechanisms of fibrosis.