1 E ndothelial cells form the inner lining of all blood vessels and not only regulate transport of nutrients to the under- lying tissue but also coordinate the formation of new blood vessels, a process termed angiogenesis. Therefore, endothelial cells are highly plastic cells that are capable of switching from a resting quiescent state in normal conduit blood vessels to a highly proliferative and migratory state when angiogenesis takes place. Resting quiescent endothelial cells are termed phalanx cells, 1 whereas migratory angiogenic endothelial cells are referred to as tip cells, which are followed by proliferating so-called stalk cells. 2 Although the mechanisms regulating tip and stalk cell behavior have been extensively studied, rela- tively little is known about the control of the phalanx state. Shear stress, the force that laminar blood flow exerts on endothelial cells, is thought to be one of the factors that deter- mine the quiescent state of endothelial cells. 3 This biomechan- ical stimulus induces the expression of the transcription factor Krüppel-like factor 2 (KLF2), which orchestrates a network of genes that elicit a quiescent endothelial cell phenotype. 4,5 Among the factors that are upregulated by KLF2 are anti- inflammatory and antithrombotic proteins, whereas proin- flammatory and prothrombotic factors are downregulated by KLF2. 4 Although not all effects of shear stress on endothelial cells are mediated by KLF2, KLF2 coordinates approximately half of the gene expression programs evoked by shear stress. 5,6 Recent studies have highlighted the importance of cellular metabolism for the control of endothelial cell phenotype. 7,8 Particularly, it was shown that angiogenic endothelial cells rely heavily on glycolysis for migration and proliferation. 9 The enzyme PFKFB3 is a key regulator of glycolysis in endothelial cells that has been shown to promote angiogenic sprouting. 9–11 However, how resting endothelial cells control their metabolic activity and whether this affects the functional properties of the phalanx phenotype is unclear. © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.114.304277 Objective—Cellular metabolism was recently shown to regulate endothelial cell phenotype profoundly. Whether the atheroprotective biomechanical stimulus elicited by laminar shear stress modulates endothelial cell metabolism is not known. Approach and Results—Here, we show that laminar flow exposure reduced glucose uptake and mitochondrial content in endothelium. Shear stress–mediated reduction of endothelial metabolism was reversed by silencing the flow-sensitive transcription factor Krüppel-like factor 2 (KLF2). Endothelial-specific deletion of KLF2 in mice induced glucose uptake in endothelial cells of perfused hearts. KLF2 overexpression recapitulates the inhibitory effects on endothelial glycolysis elicited by laminar flow, as measured by Seahorse flux analysis and glucose uptake measurements. RNA sequencing showed that shear stress reduced the expression of key glycolytic enzymes, such as 6-phosphofructo-2-kinase/fructose- 2,6-biphosphatase-3 (PFKFB3), phosphofructokinase-1, and hexokinase 2 in a KLF2-dependent manner. Moreover, KLF2 represses PFKFB3 promoter activity. PFKFB3 knockdown reduced glycolysis, and overexpression increased glycolysis and partially reversed the KLF2-mediated reduction in glycolysis. Furthermore, PFKFB3 overexpression reversed KLF2- mediated reduction in angiogenic sprouting and network formation. Conclusions—Our data demonstrate that shear stress–mediated repression of endothelial cell metabolism via KLF2 and PFKFB3 controls endothelial cell phenotype. (Arterioscler Thromb Vasc Biol. 2015;35:00-00.) Key Words: endothelium ◼ glycolysis ◼ metabolism ◼ shear stress down regulated gene-1 protein, human Received on: July 7, 2014; final version accepted on: October 16, 2014. From the Institute for Cardiovascular Regeneration, Center of Molecular Medicine, Goethe University, Frankfurt am Main, Germany (A.D., K.M.M., Y.M., T.L., S.D., R.A.B.); The Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, The Netherlands (R.H.H.); The Max- Delbrück-Center, Berlin, Germany (X.Y., W.C.); Department of Cardiology, Internal Medicine III, University of Frankfurt, Frankfurt, Germany (A.M.Z.); Angiogenesis and Metabolism Laboratory, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (M.P.); and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt am Main, Germany (A.M.Z., S.D.). The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304277/-/DC1. Correspondence to Reinier A. Boon, Institute for Cardiovascular Regeneration, Center of Molecular Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. E-mail boon@med.uni-frankfurt.de Laminar Shear Stress Inhibits Endothelial Cell Metabolism via Krüppel-Like Factor 2–Mediated Repression of 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase-3 Anuradha Doddaballapur, Katharina M. Michalik, Yosif Manavski, Tina Lucas, Riekelt H. Houtkooper, Xintian You, Wei Chen, Andreas M. Zeiher, Michael Potente, Stefanie Dimmeler, Reinier A. 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