The morphological hallmark of pulmonary hypertension is thickening of the vessel wall and narrowing or occlusion of the lumen. A defining feature of pulmonary vascular remodeling is the increased presence of a-smooth muscle actin (a-SMA)-positive cells within the vessel wall. The cellular origin and mechanisms governing the accumulation of these cells are fundamental issues in pulmonary hypertension and the focus of ongoing research. Over the years, different cell types have been identified and proposed as precursors of a-SMA-positive cells in remodeled vessels. These include circulating bone marrow-derived cells (1, 2) and lung resident cells, ranging from pericytes (3, 4) to vascular smooth muscle cells (SMCs)(5), mesenchymal stem and progenitor cells (6, 7), and endothelial cells (ECs)(8-10). A direct contribution of ECs to the a-SMA-positive cell pool is a particularly intriguing process because it would involve transdifferentiation of one cell type into another, with a concomitant change in cell-type marker expression and functional behavior. In the setting of the pulmonary circulation, endothelial-to-mesenchymal transition (EndMT) is associated with downregulation or loss of typical EC markers such as CD31, von Willebrand factor, and junctional protein VE-cadherin, and the acquisition of a smooth muscle cell-like phenotype with expression of a-SMA and collagen 1. Acquisition of mesenchymal markers is accompanied with higher migratory and invasive capacities of transdifferentiated ECs (11, 12). Thus, under certain conditions and with disease progression, transdifferentiated ECs with an SMC-like phenotype could then directly contribute to the a-SMA-positive pool of cells in remodeled vessels. In this issue of the Journal, Mammoto and colleagues (pp. 194-207) demonstrate that overexpression of Twist1 can lead to EndMT (13). Together with Snail and ZEB, Twist1 belongs to the family of transcription factors that orchestrate the acquisition of mesenchymal markers and changes in cellular behavior that enable increased cell motility and survival (14). The current study provides a missing piece of the puzzle by explaining how increased expression of Twist1 in lung samples from patients with pulmonary arterial hypertension, as previously reported (10), could lead to EndMT (Figure 1). Through multiple in vitro experiments using human pulmonary arterial ECs (HPAECs), the authors clearly show that Twist1 enhances transforming growth factor b receptor 2 (TGF-bR2) expression and SMAD2 phosphorylation, and decreases VE-cadherin levels. Loss of EC markers, such as VE-cadherin and CD31, corresponds to loosened cell-cell contact, higher migratory properties, and possibly a phenotypic switch indicated by enhanced expression of a-SMA. Indeed, overexpression of Twist1 led to higher levels of a-SMA in HPAECs. The central role of Twist1, or rather its activation, in the cell transition was substantiated by experiments with the mutant construct (Twist1S42A), where the transition was not observed. Further, the authors verified the necessity of Twist1 Ser42 phosphorylation for EndMT in ex vivo studies by using implanted fibril gels containing fluorescently labeled HPAECs in mouse lungs. To further interrogate their findings, the authors applied an EC-specific knockout of Twist1 in a hypoxic mouse model (Twist1fl/flTie2-cre mice), and observed that these animals developed less vascular remodeling and pulmonary hypertension than control littermates. To date, most studies investigating EndMT have relied exclusively on in vitro analysis of cells cultured on plastic. The authors of the current study went beyond in vitro analysis and extending their …