Isolation and characterization of endothelial-to-mesenchymal transition-cells in pulmonary arterial hypertension

Endothelial-to-mesenchymal transition (EndMT) is a process in which endothelial cells lose polarity and cell-to-cell contacts, and undergo a dramatic remodeling of the cytoskeleton. It has been implicated in initiation and progression of pulmonary arterial hypertension (PAH). However, the characteristics of cells which have undergone EndMT-cells in vivo have not been reported and so remain unclear. To study this, sugen5416 and hypoxia (SuHx)-induced PAH was established in Cdh5-Cre / Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J double-transgenic mice, in which GFP was stably expressed in pan-endothelial cells. After 3 weeks of SuHx, flow cytometry and immunohistochemistry demonstrated CD144-negative and GFP-positive cells (complete EndMT-cells) possessed higher proliferative and migratory activity compared to other mesenchymal cells. While CD144-positive and α-SMA-positive cells (partial EndMT-cells) continued to express endothelial progenitor cell markers, complete EndMT-cells were Sca-1-rich mesenchymal cells with high proliferative and migratory ability. When transferred in fibronectin-coated chamber slides containing smooth muscle media, α-SMA robustly expressed in these cells compared to cEndMT-cells that were grown in maintenance media. Demonstrating additional paracrine effects, conditioned medium from isolated complete EndMT-cells induced enhanced mesenchymal proliferation and migration, and increased angiogenesis as compared to conditioned medium from resident mesenchymal cells. Overall, these findings show that EndMT-cells could contribute to the pathogenesis of PAH both directly by transformation into smooth muscle-like cells with higher proliferative and migratory potency, as well as indirectly, through paracrine effects on vascular intimal and medial proliferation.