Laminar Shear Stress Inhibits Endothelial Cell Metabolism via KLF2-Mediated Repression of PFKFB3

Abstract

E ndothelial cells form the inner lining of all blood vessels and not only regulate transport of nutrients to the underlying 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, relatively 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 determine the quiescent state of endothelial cells.3 This biomechanical 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 antiinflammatory and antithrombotic proteins, whereas proinflammatory 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. 5,6See accompanying editorial on page 13Recent 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. 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 © 2014 American Heart Association, Inc. 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 ma...

Endothelial transcription factor KLF2 negatively regulates liver regeneration via induction of activin A

“…To obtain insight into the mechanisms of endothelial KLF2 control of hepatocyte proliferation, we determined whether endothelial cells communicate in a paracrine manner with hepatocytes. Therefore, we overexpressed KLF2 by using lentiviral vectors (18) in ECs in vitro (Fig. 4A) and determined whether supernatants (SNs) of endothelial cells overexpressing KLF2 regulated hepatocyte proliferation.…”

Endothelium

“…Exposure of endothelial cells to laminar flow impairs their capacity to uptake glucose and their mitochondrial content in a Kruppel-like factor (KLF)-2 dependent manner. 40 This effect results from the inhibition caused by shear stress of key glycolytic enzymes, and in particular, repression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3) promoter. All together these findings demonstrate that shear stress repression of endothelial metabolism is mediated by KLF2 and that PFKFB3 seems as an underestimated regulator of endothelial cell phenotype.…”

Manipulating Angiogenesis by Targeting Endothelial Metabolism: Hitting the Engine Rather than the Drivers—A New Perspective?

“…Moreover, upon establishment of blood flow in the newly formed vessel network, ECs experience shear stress. This further promotes EC quiescence also through reduction of glycolytic metabolism, via krueppel-like factor 2 dependent transcriptional suppression of PFKFB3, hexokinase 2, and PFK-1 (Galie et al, 2014;Doddaballapur et al, 2015). These data illustrate the pivotal role of PFKFB3-driven glycolysis during active sprouting angiogenesis and its regulation at different levels (transcription, mRNA stability, and cellular compartmentalization).…”