Role of Endothelial Cell Metabolism in Vessel Sprouting
2013; Cell Press; Volume: 18; Issue: 5 Linguagem: Inglês
10.1016/j.cmet.2013.08.001
ISSN1932-7420
AutoresKatrien De Bock, Μαρία Γεωργιάδου, Peter Carmeliet,
Tópico(s)Adipose Tissue and Metabolism
ResumoEndothelial cells (ECs) are quiescent for years but can plastically switch to angiogenesis. Vascular sprouting relies on the coordinated activity of migrating tip cells at the forefront and proliferating stalk cells that elongate the sprout. Past studies have identified genetic signals that control vascular branching. Prominent are VEGF, activating tip cells, and Notch, which stimulates stalk cells. After the branch is formed and perfused, ECs become quiescent phalanx cells. Now, emerging evidence has accumulated indicating that ECs not only adapt their metabolism when switching from quiescence to sprouting but also that metabolism regulates vascular sprouting in parallel to the control by genetic signals. Endothelial cells (ECs) are quiescent for years but can plastically switch to angiogenesis. Vascular sprouting relies on the coordinated activity of migrating tip cells at the forefront and proliferating stalk cells that elongate the sprout. Past studies have identified genetic signals that control vascular branching. Prominent are VEGF, activating tip cells, and Notch, which stimulates stalk cells. After the branch is formed and perfused, ECs become quiescent phalanx cells. Now, emerging evidence has accumulated indicating that ECs not only adapt their metabolism when switching from quiescence to sprouting but also that metabolism regulates vascular sprouting in parallel to the control by genetic signals. Blood vessels arose in evolution for various reasons. First and foremost, they supply oxygen, nutrients, and growth factors to tissues while draining toxic metabolic waste. They also ensure immune surveillance, thus allowing immune cells to patrol the organism for foreign antigens or invaders. Interestingly, vessels are evolutionarily closely associated with organismal metabolism. Indeed, in primitive invertebrates, blood vessels were initially hollow matrix tubes that were not lined by endothelial cells (ECs), thus allowing only slow, sluggish, turbulent blood flow and limited tissue perfusion (Muñoz-Chápuli et al., 2005Muñoz-Chápuli R. Carmona R. Guadix J.A. Macías D. Pérez-Pomares J.M. The origin of the endothelial cells: an evo-devo approach for the invertebrate/vertebrate transition of the circulatory system.Evol. Dev. 2005; 7: 351-358Crossref PubMed Scopus (30) Google Scholar). Only when organisms required a more rapid metabolism (for instance, to predate) did vessels become lined by ECs in order to establish faster laminar blood flow and more efficient perfusion (Muñoz-Chápuli et al., 2005Muñoz-Chápuli R. Carmona R. Guadix J.A. Macías D. Pérez-Pomares J.M. The origin of the endothelial cells: an evo-devo approach for the invertebrate/vertebrate transition of the circulatory system.Evol. Dev. 2005; 7: 351-358Crossref PubMed Scopus (30) Google Scholar). However, how ECs rewire their own metabolism when switching from quiescence to vascular branching and whether such metabolic adaptations affect vascular branching remain much less studied. ECs are highly plastic cells and can rapidly switch from a long-term quiescent state to active growth upon stimulation by hypoxia or growth factors. According to the prevalent model of vascular sprouting (Potente et al., 2011Potente M. Gerhardt H. Carmeliet P. Basic and therapeutic aspects of angiogenesis.Cell. 2011; 146: 873-887Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), an endothelial tip cell takes the lead by navigating at the vascular forefront. Following the tip cell, endothelial stalk cells elongate the branch by proliferating, whereas endothelial phalanx cells line quiescent perfused vessels. The process of tip and stalk cell differentiation is under the tight control of VEGF and Notch signaling and other genetic signals (Potente et al., 2011Potente M. Gerhardt H. Carmeliet P. Basic and therapeutic aspects of angiogenesis.Cell. 2011; 146: 873-887Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). VEGF promotes tip cell induction and filopodia formation and induces the expression of the Notch ligand Delta-like 4 (DLL4), which activates Notch signaling in neighboring cells and thereby suppresses VEGF receptor 2 (VEGFR-2) expression and tip cell behavior (Figure 1A). Tip and stalk cells do not exhibit permanently fixed cell fates but dynamically switch between tip and stalk cell phenotypes (Jakobsson et al., 2010Jakobsson L. Franco C.A. Bentley K. Collins R.T. Ponsioen B. Aspalter I.M. Rosewell I. Busse M. Thurston G. Medvinsky A. et al.Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting.Nat. Cell Biol. 2010; 12: 943-953Crossref PubMed Scopus (179) Google Scholar). In a matter of hours, a tip cell that lacks the fitness to compete for the leading position can be overtaken by a stalk cell, which then acquires a tip position. This mechanism may ensure that vessel branching relies on the fittest cells. However, little is known about the different metabolic characteristics and requirements of these various EC subtypes and whether Notch controls metabolism in ECs. First, we will overview our current understanding of the various metabolic pathways in ECs, and then we will discuss how these pathways regulate vessel sprouting, illustrating a major role for glycolysis in this process. Glucose delivery to peripheral organs occurs via paracellular transport as well as a transcellular route. In fact, only a small fraction of the glucose that is taken up by ECs is phosphorylated for further internal metabolization. ECs take up glucose through facilitated diffusion, an energy-independent process facilitated by glucose transporters (GLUT), mainly by GLUT-1. VEGF increases GLUT-1 expression in ECs through the activation of PI3K-AKT signaling (Yeh et al., 2008Yeh W.L. Lin C.J. Fu W.M. Enhancement of glucose transporter expression of brain endothelial cells by vascular endothelial growth factor derived from glioma exposed to hypoxia.Mol. Pharmacol. 2008; 73: 170-177Crossref PubMed Scopus (30) Google Scholar). Reduced GLUT-1 levels in ECs decrease glucose uptake in peripheral organs (Huang et al., 2012Huang Y. Lei L. Liu D. Jovin I. Russell R. Johnson R.S. Di Lorenzo A. Giordano F.J. Normal glucose uptake in the brain and heart requires an endothelial cell-specific HIF-1α-dependent function.Proc. Natl. Acad. Sci. USA. 2012; 109: 17478-17483Crossref PubMed Scopus (9) Google Scholar). In humans, impaired glucose transport across the blood-brain barrier due to GLUT-1 mutations causes the glucose transporter protein syndrome, which is characterized by infantile seizures, developmental delay, and microcephaly (Klepper et al., 1999Klepper J. 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In ECs of intact coronary arteries, glucose is taken up at the periphery of the cell and accumulates close to cell-to-cell junctions, where the majority of glucose transporters are anchored. This compartmentalization of glucose produces a concentration gradient between the cytosol and the interstitial space that might facilitate transcellular transport of glucose (Gaudreault et al., 2008Gaudreault N. Scriven D.R. Laher I. Moore E.D. Subcellular characterization of glucose uptake in coronary endothelial cells.Microvasc. Res. 2008; 75: 73-82Crossref PubMed Scopus (16) Google Scholar). Divergent effects of insulin on glucose uptake and metabolism in ECs have been reported (Artwohl et al., 2007Artwohl M. Brunmair B. Fürnsinn C. Hölzenbein T. Rainer G. Freudenthaler A. Porod E.M. Huttary N. Baumgartner-Parzer S.M. Insulin does not regulate glucose transport and metabolism in human endothelium.Eur. J. Clin. 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Nonetheless, most studies report that ECs do not rely on oxidative metabolism but are highly glycolytic, generating more than 80% of their ATP in this pathway (Culic et al., 1997Culic O. Gruwel M.L. Schrader J. Energy turnover of vascular endothelial cells.Am. J. Physiol. 1997; 273: C205-C213PubMed Google Scholar, De Bock et al., 2013De Bock K. Georgiadou M. Schoors S. Kuchnio A. Wong B.W. Cantelmo A.R. Quaegebeur A. Ghesquière B. Cauwenberghs S. Eelen G. et al.Role of PFKFB3-driven glycolysis in vessel sprouting.Cell. 2013; 154: 651-663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, Krützfeldt et al., 1990Krützfeldt A. Spahr R. Mertens S. Siegmund B. Piper H.M. Metabolism of exogenous substrates by coronary endothelial cells in culture.J. Mol. Cell. Cardiol. 1990; 22: 1393-1404Abstract Full Text PDF PubMed Scopus (42) Google Scholar). In the presence of physiological glucose concentrations, only <1% of pyruvate generated in glycolysis is oxidized in the tricarboxylic acid (TCA) cycle (De Bock et al., 2013De Bock K. Georgiadou M. Schoors S. Kuchnio A. Wong B.W. Cantelmo A.R. Quaegebeur A. Ghesquière B. Cauwenberghs S. Eelen G. et al.Role of PFKFB3-driven glycolysis in vessel sprouting.Cell. 2013; 154: 651-663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). However, when glucose and glycolysis levels drop, the oxidation of glucose (as well as of palmitate and amino acids) is enhanced, indicating that ECs switch to oxidative metabolism when anaerobic glycolysis is impaired (known as the Crabtree effect) (Krützfeldt et al., 1990Krützfeldt A. Spahr R. Mertens S. Siegmund B. Piper H.M. Metabolism of exogenous substrates by coronary endothelial cells in culture.J. Mol. Cell. Cardiol. 1990; 22: 1393-1404Abstract Full Text PDF PubMed Scopus (42) Google Scholar). ECs increase their glycolytic flux when switching from quiescence to proliferation and migration (De Bock et al., 2013De Bock K. Georgiadou M. Schoors S. Kuchnio A. Wong B.W. Cantelmo A.R. Quaegebeur A. Ghesquière B. Cauwenberghs S. Eelen G. et al.Role of PFKFB3-driven glycolysis in vessel sprouting.Cell. 2013; 154: 651-663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In pathological conditions, such as pulmonary hypertension or latent infection with Kaposi’s sarcoma-associated herpesvirus, glycolysis is increased while oxygen consumption is reduced in ECs (Delgado et al., 2010Delgado T. Carroll P.A. Punjabi A.S. Margineantu D. Hockenbery D.M. Lagunoff M. Induction of the Warburg effect by Kaposi’s sarcoma herpesvirus is required for the maintenance of latently infected endothelial cells.Proc. Natl. Acad. Sci. USA. 2010; 107: 10696-10701Crossref PubMed Scopus (39) Google Scholar, Fijalkowska et al., 2010Fijalkowska I. Xu W. Comhair S.A. Janocha A.J. 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Eelen G. et al.Role of PFKFB3-driven glycolysis in vessel sprouting.Cell. 2013; 154: 651-663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Also, the genetic deficiency of PFKFB3 in ECs causes vascular hypobranching in mice (De Bock et al., 2013De Bock K. Georgiadou M. Schoors S. Kuchnio A. Wong B.W. Cantelmo A.R. Quaegebeur A. Ghesquière B. Cauwenberghs S. Eelen G. et al.Role of PFKFB3-driven glycolysis in vessel sprouting.Cell. 2013; 154: 651-663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Similar to fibroblasts (Lemons et al., 2010Lemons J.M. Feng X.J. Bennett B.D. Legesse-Miller A. Johnson E.L. Raitman I. Pollina E.A. Rabitz H.A. Rabinowitz J.D. Coller H.A. Quiescent fibroblasts exhibit high metabolic activity.PLoS Biol. 2010; 8: e1000514Crossref PubMed Scopus (76) Google Scholar, Valcourt et al., 2012Valcourt J.R. Lemons J.M. Haley E.M. Kojima M. Demuren O.O. Coller H.A. Staying alive: metabolic adaptations to quiescence.Cell Cycle. 2012; 11: 1680-1696Crossref PubMed Scopus (17) Google Scholar), ECs have substantial baseline glycolysis levels when they are quiescent and only double their glycolysis flux when they are activated to divide and migrate (De Bock et al., 2013De Bock K. Georgiadou M. Schoors S. Kuchnio A. Wong B.W. Cantelmo A.R. Quaegebeur A. Ghesquière B. Cauwenberghs S. Eelen G. et al.Role of PFKFB3-driven glycolysis in vessel sprouting.Cell. 2013; 154: 651-663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Accordingly, when vessel sprouting is stimulated in hypoxic conditions, ECs enhance glycolysis by no more than 50% (Dobrina and Rossi, 1983Dobrina A. Rossi F. Metabolic properties of freshly isolated bovine endothelial cells.Biochim. Biophys. Acta. 1983; 762: 295-301Crossref PubMed Scopus (32) Google Scholar). 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Quiescent fibroblasts exhibit high metabolic activity.PLoS Biol. 2010; 8: e1000514Crossref PubMed Scopus (76) Google Scholar, Valcourt et al., 2012Valcourt J.R. Lemons J.M. Haley E.M. Kojima M. Demuren O.O. Coller H.A. Staying alive: metabolic adaptations to quiescence.Cell Cycle. 2012; 11: 1680-1696Crossref PubMed Scopus (17) Google Scholar), ECs need a high baseline glycolysis level for homeostatic maintenance, and blocking glycolysis by 80% by 2-deoxy-D-glucose is toxic for these cells (Merchan et al., 2010Merchan J.R. Kovács K. Railsback J.W. Kurtoglu M. Jing Y. Piña Y. Gao N. Murray T.G. Lehrman M.A. Lampidis T.J. Antiangiogenic activity of 2-deoxy-D-glucose.PLoS ONE. 2010; 5: e13699Crossref PubMed Scopus (24) Google Scholar, Wang et al., 2011aWang Q. Liang B. Shirwany N.A. 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Alvarez D.F. Al-Mehdi A.B. Alexeyev M. Stevens T. Critical role for lactate dehydrogenase A in aerobic glycolysis that sustains pulmonary microvascular endothelial cell proliferation.Am. J. Physiol. Lung Cell. Mol. Physiol. 2010; 299: L513-L522Crossref PubMed Scopus (27) Google Scholar). Hemodynamic forces such as blood flow also stimulate glycolysis through shear forces acting on the EC glycocalyx (Suárez and Rubio, 1991Suárez J. Rubio R. Regulation of glycolytic flux by coronary flow in guinea pig heart. Role of vascular endothelial cell glycocalyx.Am. J. Physiol. 1991; 261: H1994-H2000PubMed Google Scholar). The proangiogenic molecules VEGF and FGF2 increase PFKFB3-driven glycolysis, whereas DLL4, which activates Notch signaling and decreases branching, reduces glycolysis in ECs (De Bock et al., 2013De Bock K. Georgiadou M. Schoors S. Kuchnio A. Wong B.W. Cantelmo A.R. Quaegebeur A. Ghesquière B. Cauwenberghs S. Eelen G. et al.Role of PFKFB3-driven glycolysis in vessel sprouting.Cell. 2013; 154: 651-663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). At first sight, it may seem paradoxical that quiescent ECs rely on glycolysis, given that they could take advantage of the available oxygen in their immediate environment in the blood to more efficiently generate ATP via oxidative phosphorylation. Indeed, per glucose molecule, glycolysis produces a net total of only two molecules of ATP, whereas glucose oxidation yields up to 36 molecules of ATP. Nonetheless, one of the prime tasks of ECs is to vascularize avascular tissues through sprouting. If they relied primarily (or solely) on oxidative metabolism, then ECs would be unable to generate ATP in oxygen-depleted areas. In fact, given that interstitial oxygen levels drop faster than glucose levels over a distance away from a blood vessel, ECs can continue to rely on anaerobic glycolysis in such conditions (Buchwald, 2011Buchwald P. A local glucose-and oxygen concentration-based insulin secretion model for pancreatic islets.Theor. Biol. Med. Model. 2011; 8: 20Crossref PubMed Scopus (12) Google Scholar, Gatenby and Gillies, 2004Gatenby R.A. Gillies R.J. Why do cancers have high aerobic glycolysis?.Nat. Rev. Cancer. 2004; 4: 891-899Crossref PubMed Scopus (1502) Google Scholar). Indeed, ECs are resistant to hypoxia as long as glucose is available but become sensitive to oxygen deprivation when glucose is limiting (Mertens et al., 1990Mertens S. Noll T. Spahr R. Krützfeldt A. Piper H.M. Energetic response of coronary endothelial cells to hypoxia.Am. J. Physiol. 1990; 258: H689-H694PubMed Google Scholar). Another reason is that glycolysis rapidly generates ATP, which ECs need in order to form highly motile and rapidly moving lamellipodia and filopodia. Moreover, as long as glucose is not limiting in the extracellular milieu, glycolysis can generate similar amounts of ATP as glucose oxidation (Locasale and Cantley, 2011Locasale J.W. Cantley L.C. Metabolic flux and the regulation of mammalian cell growth.Cell Metab. 2011; 14: 443-451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Another advantage of glycolytic metabolism is that glycolysis and its side pathways generate the necessary precursors for macromolecules needed in order for ECs to grow, divide, and migrate (see below). Also, a low-oxidative metabolism generates fewer reactive oxygen species (ROS) and less oxidative stress in the high-oxygen environment that quiescent ECs are exposed to. Finally, by consuming less oxygen, they can transfer more oxygen to perivascular cells, thereby improving tissue oxygenation. ECs also store intracellular glucose reserves as glycogen (Amemiya, 1983Amemiya T. Glycogen metabolism in the capillary endothelium. Electron histochemical study of glycogen synthetase and phosphorylase in the pecten capillary of the chick.Acta Histochem. 1983; 73: 93-96Crossref PubMed Google Scholar, Numano et al., 1974Numano F. Takahashi T. Kuroiwa T. Shimamoto T. Glycogen in endothelial cells. Electronmicroscopic studies of polyglucose synthesized by phosphorylase in endothelial cells of aorta and heart muscle of rabbits.Exp. Mol. Pathol. 1974; 20: 168-174Crossref PubMed Scopus (3) Google Scholar, Vizán et al., 2009Vizán P. Sánchez-Tena S. Alcarraz-Vizán G. Soler M. Messeguer R. Pujol M.D. Lee W.N. Cascante M. 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Characterization of the metabolic changes underlying growth factor angiogenic activation: identification of new potential therapeutic targets.Carcinogenesis. 2009; 30: 946-952Crossref PubMed Scopus (20) Google Scholar). In cerebral microvascular ECs, norepinephrine induces glycogenolysis, whereas 5-hydroxytryptamine stimulates glycogenesis (Spatz et al., 1986Spatz M. Mrsulja B.B. Wroblewska B. Merkel N. Bembry J. Modulation of glycogen metabolism in cerebromicrovascular smooth muscle and endothelial cultures.Biochem. Biophys. Res. Commun. 1986; 134: 484-491Crossref PubMed Scopus (4) Google Scholar). Overall, little is known about the role and importance of glycogen in ECs, but the inhibition of glycogen phosphorylase impairs EC viability and migration (Vizán et al., 2009Vizán P. Sánchez-Tena S. Alcarraz-Vizán G. Soler M. Messeguer R. Pujol M.D. Lee W.N. Cascante M. Characterization of the metabolic changes underlying growth factor angiogenic activation: identification of new potential therapeutic targets.Carcinogenesis. 2009; 30: 946-952Crossref PubMed Scopus (20) Google Scholar). This raises the question of whether ECs use this endogenous glucose storage to sprout into avascular glucose-deprived areas. The pentose phosphate pathway (PPP) is a side branch of glycolysis that cells use for divergent purposes (Figure 2). In this pathway, glucose-6-phosphate is oxidized to pentose sugars and reduces equivalents in two phases. The irreversible oxidative branch (oxPPP) generates NADPH and ribose-5-phosphate (R5P), whereas the reversible nonoxidative arm (non-oxPPP) produces only R5P. The latter is used for the synthesis of nucleotides, whereas NADPH is used for the reductive biosynthesis of lipids, production of NO, or reconversion of oxidized glutathione (GSSG) to reduced glutathione (GSH), a major cellular redox buffer. Depending on the cellular needs and context, the PPP can serve to promote cellular growth and division by increasing the biosynthesis of macromolecules (Cairns et al., 2011Cairns R.A. Harris I.S. Mak T.W. Regulation of cancer cell metabolism.Nat. Rev. Cancer. 2011; 11: 85-95Crossref PubMed Scopus (641) Google Scholar, Lunt and Vander Heiden, 2011Lunt S.Y. Vander Heiden M.G. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation.Annu. Rev. Cell Dev. Biol. 2011; 27: 441-464Crossref PubMed Scopus (231) Google Scholar, Vander Heiden et al., 2009Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science. 2009; 324: 1029-1033Crossref PubMed Scopus (2149) Google Scholar), or the oxPPP can ensure redox homeostasis (Anastasiou et al., 2011Anastasiou D. Poulogiannis G. Asara J.M. Boxer M.B. Jiang J.K. Shen M. Bellinger G. Sasaki A.T. Locasale J.W. 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