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Endothelial Metabolism Driving Angiogenesis: Emerging Concepts and Principles

Cantelmo, Anna Rita PhD; Brajic, Aleksandra MSc; Carmeliet, Peter MD, PhD

doi: 10.1097/PPO.0000000000000133
Reviews: Part I: Tumor Vasculature as a Target: Angiogenesis and Antiangiogenesis
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Angiogenesis has been traditionally studied by focusing on growth factors and other proangiogenic signals, but endothelial cell (EC) metabolism has not received much attention. Nonetheless, glycolysis, one of the major metabolic pathways that converts glucose to pyruvate, is required for the phenotypic switch from quiescent to angiogenic ECs. During vessel sprouting, the glycolytic activator PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3) promotes vessel branching by rendering ECs more competitive to reach the tip of the vessel sprout, whereas fatty acid oxidation selectively regulates proliferation of endothelial stalk cells. These studies show that metabolic pathways in ECs regulate vessel sprouting, more importantly than anticipated. This review discusses the recently discovered role of glycolysis and fatty acid oxidation in vessel sprouting. We also highlight how metabolites can influence EC behavior as signaling molecules by modulating posttranslational modification.

From the Laboratory of Angiogenesis & Neurovascular Link, Vesalius Research Center, VIB, KU Leuven, Belgium.

Conflicts of Interest and Source of Funding: P.C. declares to be named as inventor on patent applications claiming subject matter related to the findings reviewed in this publication. For the remaining authors, none were declared. P.C. is supported by Federal Government Belgium grant (IUAP P7/03), long-term structural funding Methusalem funding by the Flemish Government, FWO grants, AXA Research Fund, ERC Advanced Research Grant, and the Foundation Leducq Transatlantic Artemis Network.

Reprints: Peter Carmeliet, MD, PhD, Laboratory of Angiogenesis & Neurovascular Link, Vesalius Research Center, VIB, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000, Leuven, Belgium. E-mail: peter.carmeliet@vib-kuleuven.be.

The vascular endothelium, a single layer of endothelial cells (ECs) that line the blood vessel lumen, exhibits great flexibility to adapt to environmental cues. For instance, exposure of ECs to laminar shear stress promotes a quiescent state, partly via induction of Kruppel-like factor 2.1 In hypoxic conditions, increased vascular endothelial growth factor (VEGF) levels activate VEGF receptor 2 (VEGFR2) signaling in quiescent ECs, which thereby adopt a migrating “tip” cell behavior at the forefront of the vessel sprout during the angiogenic switch.2 In the elongating sprout, Delta-like 4–mediated activation of Notch signaling promotes proliferation of “stalk” cells that elongate the vascular sprout.3 In addition to numerous genetic and molecular signals regulating this process,4 recent evidence demonstrates that changes in metabolic pathways codetermine the angiogenic switch.

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Glycolysis Regulates Vessel Sprouting

Despite immediate access to oxygen in the blood, sprouting ECs rely primarily on glycolysis to generate energy.5 Upon induction of sprouting by growth factors such as VEGF, quiescent ECs increase glycolysis by up-regulating the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), an activator of phosphofructokinase 1, itself a rate-limiting enzyme of glycolysis.5 Genetic deficiency of PFKFB3 in ECs reduces vascular sprouting by impairing migration of tip cells and proliferation of stalk cells5 (Fig. 1). PFKFB3-driven glycolysis promotes the tip cell phenotype during vessel sprouting, because PFKFB3 overexpression overrules the prostalk activity of Notch signaling. Notably, glycolytic enzymes in ECs associate with F-actin and compartmentalize in lamellipodia and filopodia, where rapid and localized ATP production by glycolysis enables cytoskeleton remodeling during cell migration.5 Together, these findings identify glycolysis as an important regulator of angiogenesis. Glycolysis addiction of ECs makes this pathway a promising target for antiangiogenic therapy.6,7 A paradigm shift in the design of antiglycolytic therapy is that glycolysis in ECs does not need to be completely and permanently blocked, but that transient and partial lowering of glycolysis in ECs suffices to inhibit pathological angiogenesis.6,7 These findings identified antiglycolytic strategy as an alternative antiangiogenic therapy, which might be relevant when considering the intrinsic refractoriness and acquired resistance against current antiangiogenic therapies based on VEGF blockade.

FIGURE 1

FIGURE 1

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Fatty Acid Oxidation: A Novel Role in EC Proliferation

In normal conditions, fatty acid β-oxidation (FAO) contributes to less than 5% of the total amount of ATP in ECs.8 However, in conditions of glucose deprivation, FAO flux is increased in an AMP-activated protein kinase–dependent manner.9 Moreover, VEGF enhances the expression of the fatty acid uptake and trafficking protein FABP4 (fatty acid binding protein 4), which is required for normal EC proliferation.10 Carnitine palmitoyltransferase 1 (CPT1) controls the transfer of long-chain fatty acids into the mitochondria, where they are oxidized, and is thus a rate-limiting step of FAO.9 We recently discovered that endothelial loss of CPT1a causes vascular sprouting defects due to impaired proliferation, not migration of ECs.11 Reduction of FAO in ECs does not cause energy depletion or disturb redox homeostasis, but impairs de novo synthesis of deoxyribonucleotides for DNA replication (Fig. 2). This is most remarkable, because only glucose and glutamine substantially contribute carbons for nucleotide synthesis in (most) cancer cells.12,13 Fatty acid β-oxidation is also critical for vessel sprouting in vivo, as genetic loss of CPT1a in ECs in mice impairs physiological angiogenesis.11 Pharmacological inhibition of CPT1a in mice inhibits pathological ocular angiogenesis, suggesting that targeting FAO in ECs might be a novel strategy for blocking pathological angiogenesis.11 This is especially appealing, given that FAO blockers are already used clinically.14

FIGURE 2

FIGURE 2

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Additional Metabolic Pathways in ECs

Glycolytic intermediates shunted in other metabolic pathways can serve as essential precursors of macromolecules8 (Fig. 3). Glucose-6-phosphate enters the pentose phosphate pathway (PPP), a side pathway of glycolysis that generates nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate, necessary for redox balance and lipid production, and nucleotide synthesis, respectively. The viability and migration of ECs are reduced upon inhibition of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the oxidative PPP branch.15 Inhibition of transketolase, an enzyme involved in the nonoxidative part of the PPP, also reduces EC viability and migration.16 By generating NADPH, which converts oxidized glutathione (GSSG) to its reduced form (GSH), a key cellular antioxidant, the PPP protects ECs against oxidative stress.17

FIGURE 3

FIGURE 3

The hexosamine biosynthesis pathway (HBP) is another side branch of glycolysis that generates N-acetylglucosamine (GlcNAc) for protein O- and N-glycosylation. The rate-limiting HBP enzyme glutamine-fructose-6-phosphate aminotransaminase (GFAT, also known as GFPT1) catalyzes the conversion of the glycolytic intermediate fructose-6-phosphate into glucosamine-6-phosphate (Glc6P), which is in turn metabolized to uridine diphosphate GlcNAc (UDP-GlcNAc). The GlcNAc group of UDP-GlcNAc is used in both O- and N-linked protein glycosylation reactions (Fig. 3).18 Posttranslational glycosylation is important for proper protein function including activity, structure, and localization. The HBP and increased protein glycosylation are implicated in diabetes-induced dysfunction of ECs19,20; however, the exact role of the HBP in sprouting angiogenesis is not well established.

Glutamine is the best studied amino acid in ECs. These cells take up and metabolize glutamine,21 a key carbon and nitrogen source22 that contributes to improved viability of ECs under oxidative stress.23 Inhibition of glutaminase 1, which converts glutamine into glutamate as rate-limiting enzyme of glutaminolysis, causes premature senescence of ECs.24 In addition, ECs rely on glutamine for the synthesis of ornithine, a precursor of proangiogenic polyamines (Fig. 3).25 However, the precise role of glutamine metabolism in vessel sprouting remains to be determined.

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Control of Vessel Sprouting by Metabolite Signaling

Cellular metabolism can control cellular functions via other mechanisms than by regulating energy (ATP) production, redox homeostasis, or biomass production, more in particular through metabolite signaling. For instance, protein acetylation, a common posttranslational modification, requires acetyl-CoA as substrate for lysine acetyltransferases, thus linking metabolism to the regulation of multiple cellular functions.26,27 Intriguingly, many proteins involved in the regulation of EC metabolism are controlled by acetylation. Indeed, peroxisome proliferator–activated receptor γ coactivator 1α, liver kinase B1, and forkhead transcription factor 1 are deacetylated by sirtuin 1 (SIRT1).28–30 Sirtuin 1 is a NAD+-dependent deacetylase, which acts as a sensor of energy and redox state. Sirtuin 1 deficiency causes impaired EC branching and proliferation, leading to reduced blood vessel density.30 Notch is a target of SIRT1 in ECs,31 and by limiting endothelial Notch signaling, SIRT1 modulates tip and stalk behavior.31 Indeed, the Notch1 intracellular domain (NICD), released upon Notch1 receptor activation, is deacetylated by SIRT1, which causes its destabilization and proteasomal degradation. Sirtuin 1–deficient ECs are thus sensitized to Notch signaling and preferentially adopt stalk cell phenotypes31 (Fig. 4A). Notably, SIRT1 can inhibit Notch responses also by epigenetic mechanisms.32 Indeed, SIRT1 associates with the demethylase LSD1 (lysine-specific demethylase 1) in chromatin-associated complexes that repress Notch target gene expression through histone demethylation and deacetylation. Sirtuin 1 also deacetylates endothelial nitric oxide synthase (eNOS), thereby augmenting nitric oxide production and preserving EC function.33

FIGURE 4

FIGURE 4

As mentioned previously, the HBP is involved in glycosylation of angiogenic molecules.21,34,35 Glycosylation of VEGFR2’s extracellular domain leads to interaction with galectin 1, which activates VEGFR2 signaling even in the absence of VEGF36 (Fig. 4B). Glycosylation of other angiomodulatory proteins (eNOS, Akt, Notch1) influences their proangiogenic activity in a context-dependent manner.34,35,37–39 Proangiogenic proteins can be also palmitoylated.40 Reduced palmitoylation of VEGFR2 and eNOS impairs angiogenesis by affecting targeting of VEGFR2 and eNOS to the plasma membrane40,41 (Fig. 4C).

The redox state of the endothelium also controls the activity of angiogenic proteins via oxidation of cysteine residues. Oxidation of C-terminal cysteine residues in VEGFR2 inactivates VEGFR2 signaling, a process impairing angiogenesis.42 Conversely, in response to VEGF, reactive cysteine thiols (Cys-SH) in the cytosolic domain of VEGFR2 and tyrosine-protein kinase Src (c-Src) become oxidized; this promotes the interaction between VEGFR2 and c-Src and results in activation of c-Src, a process that stimulates angiogenesis.43 Elucidation of whether the latter phenomenon promotes angiogenesis in vivo requires further work.

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Conclusions and Future Perspectives

Several antiangiogenic drugs that block VEGF or its receptors have been approved for the treatment of cancer.44 However, the effectiveness of these drugs is limited by intrinsic refractoriness and acquired drug resistance. Recent data showing that the glycolysis regulator PFKFB3 controls vessel sprouting5 and that FAO provides precursors for DNA replication in ECs11 raise the question whether drugs targeting EC metabolism could enhance the response of cancer patients to current antiangiogenic agents or even represent an alternative antiangiogenic strategy. Preclinical animal studies already provided initial proof of concept that PFKFB3 blockade reduces pathological angiogenesis and enhances the antiangiogenic effects of VEGF inhibitors.6 Importantly, the small molecule PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) inhibits EC glycolysis in vivo only transiently and partially, reducing the extra glycolysis associated to pathological angiogenesis, without causing systemic harm.6,7 Also, the pharmacological CPT1 blocker etomoxir reduced pathological ocular neovascularization.11

It will be also interesting to further analyze how metabolite-driven posttranslational modifications influence the activity of angiogenic proteins or metabolic enzymes in ECs. For instance, besides being necessary for the production of membranes and signaling molecules, fatty acids also control cellular functions via activation of lipid-modifying enzymes. Little, however, is known about how lipids affect cellular functions via such protein modifications.45,46 A better understanding of the molecular mechanisms via which metabolite-driven posttranslational protein modifications regulate angiogenic signaling may generate a new perspective in disease diagnosis and treatment.

Finally, there is little known about the metabolism of different endothelial subtypes. Little is also known on how ECs maladapt their metabolism in disease and contribute to EC dysfunction in diabetes or to excessive angiogenesis in cancer. For instance, in diabetes, shunting glycolytic intermediates to side pathways, resulting in oxidative stress, has been documented in ECs exposed to high glucose,47,48 but flux measurements of glycolysis and other metabolic pathways have not been performed.

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ACKNOWLEDGMENT

We apologize to all colleagues whose work was not cited in this review because of space limitations. A.R.C. is a postdoctoral fellow of the Research Foundation-Flanders (FWO); A.B. is PhD student supported by an Erasmus Mundus Western Balkans (ERAWEB) scholarship.

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Keywords:

Angiogenesis; endothelial cells; metabolism

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