Analysis of Endothelial Fatty Acid Metabolism Using Tracer Metabolomics
Blood vessels are lined by a streamlined monolayer of quiescent endothelial cells (ECs). Although these cells can remain quiescent for years, different stimuli (ischemia, inflammation) and growth factors can activate them and drive a process of new vessel
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troduction Endothelial cells (ECs) are very plastic cell types. They can rapidly switch from periods of quiescence, into intense proliferation during angiogenesis [1]. Until recently, primarily growth factors, receptors, signaling molecules, and related molecules were recognized to drive these processes. However, emerging evidence points out that blood vascular and lymphatic vessel sprouting mechanisms rely on the metabolism of glucose, fatty acids (FAs), and amino acids [2–11]. Over the past years, our group demonstrated that despite close proximity of ECs to oxygen in the blood, ECs mainly
Joanna Kalucka and Bart Ghesquière contributed equally to this work. Angelo D’Alessandro (ed.), High-Throughput Metabolomics: Methods and Protocols, Methods in Molecular Biology, vol. 1978, https://doi.org/10.1007/978-1-4939-9236-2_16, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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rely on glycolysis to produce ATP [2]. Activated ECs are hyperglycolytic and depend largely on glycolysis for angiogenic growth. Hence, anti-glycolytic strategies impair pathological angiogenesis [7, 12]. In addition to glycolysis, other metabolic pathways, including FA metabolism, contribute to the EC differentiation, motility, and growth. FAs play an important role in cells as structural elements of membranes, energy, storage, and signaling molecules. Long-chain FAs (e.g., palmitate) are the products of fatty acid synthesis, which takes place in the cytoplasm, and can be precursors for longer fatty acids and/or substrates for fatty acid ß-oxidation (FAO). FAO progresses in mitochondria in four-step cycles, each time cleaving two carbons that form acetyl-CoA at the end of every cycle. Successively, these acetyl-CoA units can be used for further oxidation in the tricarboxylic acid (TCA) cycle. We recently reported that FAO is critical for vessel sprouting in vivo [3]. Endothelial-selective deletion of carnitine palmitoyltransferase 1A (CPT1A), a mitochondrial FA importer and ratecontrolling enzyme of FAO, causes vessel sprouting defects in vitro and in vivo by impairing EC proliferation, without affecting migration [3]. Moreover, FAO and its key enzyme CPT1A play an important function in the lymphatic development of murine embryos [4]. FAO in lymphatic ECs promotes lymphatic vessel growth and stimulates lymphatic EC proliferation by sustaining the TCA cycle, in conjunction with an anaplerotic substrate, for dNTP synthesis during DNA replication. Recent evidence indicates that FAO in quiescent ECs ensures redox homeostasis by promoting NADPH production, presumably to cope with the high oxygen, and therefore oxidative stress-prone, microenvironment that these cells are exposed to [9]. ECs are also capable of synthesizing FAs, but fatty acid synthetase (FASN), the enzyme synthesizing FAs, also regulates the levels of malonyl-CoA, the substrate of this enzyme. Notably, FASN inhibition elevates cytosolic malonyl-CoA levels, which leads to posttranslational malonylation of mTOR, thereby reducing mTO
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