© 2014 Nature America, Inc. All rights reserved. ARTICLES NATURE MEDICINE ADVANCE ONLINE PUBLICATION 1 PH is a severe and debilitating disease characterized by remodeling of the pulmonary vessels, leading to a progressive increase in pulmo- nary vascular resistance, increased afterload on the right ventricle (RV) and, ultimately, right-heart failure 1 . Variants of PH may affect up to 100 million people worldwide 2 . Although marked progress has been made in the past decade in the treatment of group I PH, pul- monary arterial hypertension (PAH) 3–5 , current approaches provide mainly symptomatic relief. Disease progression is inevitable in most patients with PAH, and mortality remains unacceptably high 6 . Thus, the identification of new molecular or signaling pathways that drive maladaptive inward remodeling of pulmonary arteries that may serve as therapeutic targets is urgently needed. The vascular wall of pulmonary arteries is characterized as a pro- proliferative and anti-apoptotic microenvironment 7,8 . Accordingly, the upregulation of growth factors 9–11 , as well as metabolic and mitochondrial abnormalities 12–14 that are reminiscent of cancer, are present in PAH, and anti-neoplastic drugs have beneficial effects in preclinical and clinical PAH 9,15,16 . These abnormali- ties may involve immune or inflammatory cell recruitment 17–19 and increased cytokine or chemokine expression in remodeled pulmonary vessels 20,21 . Thus, multiple stimuli mediate vascular remodeling 22–24 and may complicate therapeutic approaches. We hypothesized that targeting of a central downstream effector on which various pro-proliferative and inflammatory stimuli con- verge, such as the FoxO transcription factors, might provide a new solution for such multifactorial etiology. So far, this strategy has not been explored in the treatment of PAH. FoxO transcription factors belong to a family of transcriptional regulators characterized by a conserved DNA-binding domain termed the forkhead box 25 . FoxOs, when present in the nucleus and bound to promoters that contain the FoxO consensus motif, can act as tran- scriptional activators and repressors. In mammals, four FoxO isoforms have been identified: FoxO1, FoxO3, FoxO4 and FoxO6 (refs. 26,27). Although there is some functional redundancy among these isoforms, genetic loss of individual FoxO isoforms results in specific phenotypes, suggesting differing roles for each of the isoforms 26 . FoxOs control various cellular responses 28,29 and have been implicated in vascular structural maintenance 30–33 . FoxO activity is tightly controlled by post-translational modifications, such as phosphorylation and ubiqui- tination. These modifications alter FoxO transcriptional activity, DNA binding, subcellular localization and protein stability 29,34 . For example, phosphorylation of FoxO1 at Thr24 and Ser256 creates binding motifs for the 14-3-3 scaffolding proteins, thereby causing the translocation of FoxO from the nucleus and its retention in the cytoplasm. Here we present evidence that among FoxO isoforms, FoxO1 is cen- trally involved in the hyperproliferative and apoptosis-resistant phe- notype of PASMCs, the hallmark of PH. We present a new therapeutic concept focusing on this transcriptional regulator, which we show to be highly effective in three preclinical models of severe PAH, as well as in ex vivo–cultured PASMCs isolated from human PAH lungs. 1 Max-Planck-Institute for Heart and Lung Research, Department of Lung Development and Remodeling, member of the German Center for Lung Research (DZL), Bad Nauheim, Germany. 2 Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), member of the DZL, Justus-Liebig University, Giessen, Germany. 3 Department of Molecular Cardiology, University Clinic of Giessen and Marburg, Giessen, Germany. 4 Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany. 5 Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA. Correspondence should be addressed to S.S.P. (soni.pullamsetti@mpi-bn.mpg.de). Received 11 June 2013; accepted 18 August 2014; published online 26 October 2014; doi:10.1038/nm.3695 Pro-proliferative and inflammatory signaling converge on FoxO1 transcription factor in pulmonary hypertension Rajkumar Savai 1,2 , Hamza M Al-Tamari 1 , Daniel Sedding 3,4 , Baktybek Kojonazarov 2 , Christian Muecke 1 , Rebecca Teske 3 , Mario R Capecchi 5 , Norbert Weissmann 2 , Friedrich Grimminger 2 , Werner Seeger 1,2 , Ralph Theo Schermuly 2 & Soni Savai Pullamsetti 1,2 Pulmonary hypertension (PH) is characterized by increased proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Forkhead box O (FoxO) transcription factors are key regulators of cellular proliferation. Here we show that in pulmonary vessels and PASMCs of human and experimental PH lungs, FoxO1 expression is downregulated and FoxO1 is inactivated via phosphorylation and nuclear exclusion. These findings could be reproduced using ex vivo exposure of PASMCs to growth factors and inflammatory cytokines. Pharmacological inhibition and genetic ablation of FoxO1 in smooth muscle cells reproduced PH features in vitro and in vivo. Either pharmacological reconstitution of FoxO1 activity using intravenous or inhaled paclitaxel, or reconstitution of the transcriptional activity of FoxO1 by gene therapy, restored the physiologically quiescent PASMC phenotype in vitro, linked to changes in cell cycle control and bone morphogenic protein receptor type 2 (BMPR2) signaling, and reversed vascular remodeling and right-heart hypertrophy in vivo. Thus, PASMC FoxO1 is a critical integrator of multiple signaling pathways driving PH, and reconstitution of FoxO1 activity offers a potential therapeutic option for PH.