Targeting endothelial cell metabolism: new therapeutic prospects?

Abstract

Endothelial cells (ECs) line blood vessels and function as a vital conduit for oxygen and nutrients, but can also form vascular niches for various types of stem cells. While mostly quiescent throughout adult life, ECs can rapidly switch to a highly active state, and start to sprout in order to form new blood vessels. ECs can also become dysfunctional, as occurs in diabetes and atherosclerosis. Recent studies have demonstrated a key role for EC metabolism in the regulation of angiogenesis, and showed that EC metabolism is even capable of overriding genetic signals. In this review, we will review the basic principles of EC metabolism and focus on the metabolic alterations that accompany EC dysfunction in diabetes and vessel overgrowth in cancer. We will also highlight how EC metabolism influences EC behavior by modulating post-translational modification and epigenetic changes, and illustrate how dietary supplementation of metabolites can change EC responses. Finally, we will discuss the potential of targeting EC metabolism as a novel therapeutic strategy.

Keywords

angiogenesis / metabolism / endothelial cell dysfunction / anti-angiogenic therapy

The basics of endothelial cell biology

In healthy adults, ECs can remain quiescent for years. Originally considered only as an inert lining, solely meant to conduct the blood, ECs perform many more functions, among which the supply of oxygen and nutrients, the removal of metabolic waste, the transport of white blood cells for immune surveillance, hemostasis, regulation of the vascular tone ( Davignon and Ganz, 2004) and the control of transendothelial transport of macromolecules ( Pober et al., 2009). Moreover, ECs rapidly respond to pro-angiogenic stimuli that orchestrate vascularization of tissues deprived of oxygen and nutrients. Vascular endothelial growth factor (VEGF) is a key player in the tightly coordinated process of angiogenesis. Through VEGF/VEGF receptor 2 (VEGFR2) signaling, quiescent ECs adopt a migrating “tip” cell behavior at the forefront of the vessel sprout ( Potente et al., 2011). In the elongating sprout, Delta-like 4 (DLL4)-mediated activation of Notch signaling promotes the phenotype of trailing “stalk” cells that proliferate behind the tip cell and extend the vascular sprout ( Phng and Gerhardt, 2009). ECs compete for the leading position in a highly dynamic process of repositioning of tip and stalk cells, with non-competitive tip cells being overtaken by fitter stalk cells ( Jakobsson et al., 2010). In addition to numerous genetic signals regulating this process ( Eelen et al., 2013), a metabolic switch in ECs has been recently postulated to control vessel sprouting as well ( De Bock et al., 2013b).

In the adult, new vessels can grow via sprouting (angiogenesis), but bone marrow-derived endothelial progenitors can also contribute to new vessel growth (vasculogenesis), though their relevance remains debated ( Moschetta et al., 2014). ECs can also arise from resident stem cells in the vessel wall ( Kawabe and Hasebe, 2014), or from endothelial colony-forming cells in the circulation ( Pelosi et al., 2014). ECs also establish a vascular niche for stem cells in the brain, hematopoietic system in the bone marrow and cancers ( Gómez-Gaviro et al., 2012; Takakura, 2012; Mendelson and Frenette, 2014; Tabe and Konopleva, 2014). Since the role of EC metabolism in vessel sprouting using genetic models has only recently being studied, and nearly nothing is known about the metabolism of EC progenitors or vascular niche ECs, we will restrict our review to overviewing the metabolic changes of differentiated ECs during vessel sprouting in health and disease.

Glycolysis fuels vessel sprouting

Despite immediate access to oxygen in the blood, sprouting ECs rely primarily on glycolysis for energy production ( De Bock et al., 2013b). VEGF stimulation doubles the glycolytic flux of ECs and increases the expression of the glycolysis regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), an activator of phosphofructokinase 1, itself a rate-limiting enzyme in glycolysis ( De Bock et al., 2013b). Genetic or pharmacologic inhibition of PFKFB3 impairs sprouting of ECs in vitro, and reduces vessel branching and outgrowth in vivo ( De Bock et al., 2013b). Importantly, glycolysis can overrule genetic signals that regulate EC specification during vessel sprouting. Indeed, overexpression of PFKFB3 converts genetically-programmed (by Notch) stalk cells into tip cells, while PFKFB3 knockdown diminishes tip cell behavior ( De Bock et al., 2013b). Notably, glycolytic enzymes in ECs associate with F-actin and compartmentalize in lamellipodia and filopodia, where rapid and localized ATP production enables cytoskeleton remodeling during cell migration ( De Bock et al., 2013b).

In sprouting ECs, glycolysis accounts for the production of 85% of the total cellular ATP content ( De Bock et al., 2013b). The role of mitochondria in ECs remains more debated. Certain studies show that mitochondria in ECs are predominantly involved in the production of reactive oxygen species (ROS) for signaling purposes ( Quintero et al., 2006; Groschner et al., 2012), while another study documented a role for mitochondrial respiration in the production of ATP in proliferating ECs ( Coutelle et al., 2014).

Additional metabolic pathways in ECs

Glycolytic products and intermediates shunted in other metabolic pathways can serve as essential precursors of macromolecules ( De Bock et al., 2013a). Glucose-6-phosphate enters the pentose phosphate pathway (PPP), a side pathway of glycolysis that generates NADPH and ribose-5-phosphate, necessary for redox balance and nucleotide synthesis, respectively. The viability and migration of ECs are reduced upon inhibition of both glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the oxidative PPP branch, and transketolase (TKT), a rate-limiting enzyme of the non-oxidative PPP branch ( Zhang et al., 2000; Vizán et al., 2009). In addition, the PPP serves as a protector against oxidative stress through NADPH-dependent conversion of oxidized glutathione (GSSG) to its reduced form (GSH), a key cellular antioxidant ( Leopold et al., 2003).

The hexosamine biosynthetic pathway (HBP) is another side branch of glycolysis that generates N-acetylglucosamine for protein O- and N-glycosylation. In particular, fructose-6-phospate is shunted in the HBP and converted to glucosamine-6-phospate (GlucN6P), which is in turn metabolized to uridine diphosphate N-acetylglucosamine (UDPGlcNAc), a substrate for glycosylation. As discussed in more detail below, the HBP may serve as a nutrient-sensing pathway that is responsible for glycosylation of proteins that influence angiogenesis ( Merchan et al., 2010).

Amino acid metabolism can be used for energy production, redox homeostasis and biosynthesis of macromolecules, though its role in ECs remains largely enigmatic. L-arginine is used by endothelial nitric oxide synthase (eNOS) to produce citrulline and nitric oxide (NO) ( Tousoulis et al., 2012). In addition, glutamine metabolism is critical for ECs, since inhibition of glutaminase-1, which converts glutamine into glutamate as rate-limiting enzyme of glutaminolysis, causes premature senescence of ECs ( Unterluggauer et al., 2008). In addition, ECs rely on glutamine for the synthesis of ornithine, a precursor of pro-angiogenic polyamines ( Wu et al., 2000). Nevertheless, the precise role of glutamine metabolism in vessel sprouting remains to be determined.

The role of fatty acid β-oxidation (FAO) in ECs has not been clearly defined as well. Knockdown of the fatty acid binding protein 4 (FABP4), which mediates intracellular transport and metabolism of fatty acids, decreases VEGF-dependent proliferation in ECs ( Elmasri et al., 2009). FABP4 gene expression is upregulated by Notch ( Harjes et al., 2014). In certain conditions, FAO might represent a fuel source for ECs, such as during glucose deprivation, when ECs increase the FAO flux ( Dagher et al., 2001). However, in normal conditions, FAO contributes to<5% of the total amount of ATP in ECs ( De Bock et al., 2013a). Whether FAO regulates redox homeostasis in ECs, as it does in stressed cancer cells ( Jeon et al., 2012; Carracedo et al., 2013), remains unknown.

Metabolic features of ECs in disease

In cancer, inflammation and ocular disorders, angiogenesis is excessive and deregulated. Conversely, in diabetes, atherosclerosis and other cardiovascular disorders, the vascular endothelium is dysfunctional, though vessel overgrowth also occurs in a context-dependent manner ( Rajendran et al., 2013). In the following sections, we will focus on the metabolic features of ECs in diabetes and cancer as examples of, respectively, EC dysfunction and vessel overgrowth. As a general consideration, metabolism of ECs in disease has been mostly studied in vitro by mimicking disease-like conditions, for instance by exposing healthy ECs to high glucose, when studying diabetes. It should be mentioned however that high glucose cannot adequately and comprehensively reflect the more complex, multifactorial situation of diabetes in vivo and the different mechanisms in diabetes type I and type II. Another consideration is that the relative contribution of different metabolic pathways in ECs may vary between species, EC subtypes or the experimental conditions ( Oldendorf et al., 1977; Parra-Bonilla et al., 2010). Nonetheless, despite these limitations, exciting insights have been obtained from the available studies.

EC dysfunction in diabetes

Experimental and clinical evidence indicates that hyperglycemia is a major risk factor for EC dysfunction in diabetes ( Funk et al., 2012). Hyperglycemia contributes to maladaptive changes of EC metabolism, resulting in macro- and microvascular complications in diabetes. It is widely accepted that the functional perturbation of the endothelium under hyperglycemic conditions is to a large part attributable to the generation of reactive oxygen species (ROS) and oxidative stress, a finding that has been confirmed in diabetic mice and patients, in vivo or ex vivo after isolation of ECs (Fig. 1) ( Wu and Meininger, 1995; Meininger et al., 2000; Meininger et al., 2004; Kizhakekuttu et al., 2012; Tian et al., 2012; Cho et al., 2013; Hernandez-Mijares et al., 2013; Mackenzie et al., 2013; Sudhahar et al., 2013; Venkatesan et al., 2013). So far, various metabolic changes in ECs have been suggested ( Brownlee, 2001; Rask-Madsen and King, 2013). First, high glucose levels cause mitochondrial dysfunction, leading to increased production of ROS via protein kinase C-dependent activation of NADPH-dependent oxidases ( Brandes et al., 2014).The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is then indirectly inactivated by ROS ( Du et al., 2003) and, in parallel, high glucose levels inhibit G6PD, a rate limiting PPP enzyme, thus decreasing the production of NADPH for glutathione reduction ( Zhang et al., 2000). The stalling of the PPP and glycolysis in turn shunts more glucose to the polyol pathway, where it is converted to sorbitol by aldolase reductase (ALR2), further depleting NADPH stores and increasing ROS levels ( Lorenzi, 2007).

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), an important vasodilator that also inhibits platelet aggregation ( Forstermann and Sessa, 2012). The activity of eNOS is dependent on the availability of the cofactors tetrahydrobiopterin (BH4) and coenzyme Q (CoQ10) ( Gorren et al., 2000; Crabtree et al., 2009; Mugoni et al., 2013). Thus, eNOS becomes “uncoupled” when BH4 is limiting, resulting in the production of excess ROS instead of NO ( Stroes et al., 1997; Mugoni et al., 2013). High glucose levels uncouple eNOS, because of reduced BH4 availability and elevated levels of oxidizing peroxynitrite, and favor O-glycosylation of eNOS, further impairing its activity and hence increasing ROS levels ( Du et al., 2001; Zou et al., 2002; Cai et al., 2005; Beleznai and Bagi, 2012). eNOS is also uncoupled in atherosclerosis ( Takaya et al., 2007). ROS also impair angiogenesis via ligand-independent phosphorylation of VEGFR2’s cytosolic domain by Src family kinases, leading to its reduced availability at the cell surface ( Warren et al., 2014).

The highly reactive 3-deoxyglucosone derived from the sorbitol pathway promotes the formation of advanced glycation end products (AGE) ( Wautier and Schmidt, 2004). By binding to receptors of AGEs (RAGE), these modified proteins in turn further enhance vascular inflammation, leakage and ROS production ( Manigrasso et al., 2014). AGE levels are increased when the AGE precursor methylglyoxal (which is elevated by high glucose levels ( Liu et al., 2012)) is formed from the glycolytic metabolites dihydroxyacetone phosphate (DHAP) and glyceraldeyde-3-phosphate (G3P). In diabetic patients, elevated plasma levels of methylglyoxal have been associated with hyperglycemia, and aggravate vascular complications by increasing oxidative stress ( Matafome et al., 2013; van Eupen et al., 2013). Methylglyoxal elevates oxidative stress by uncoupling eNOS ( Su et al., 2013) and by inhibiting NADPH-generating enzymes ( Morgan et al., 2014).

In addition to the aforementioned maladaptive changes of glycolysis and its side pathways, risk factors such as hyperglycemia and hyperlipidemia cause mitochondria damage and subsequent EC dysfunction, which leads to inflammation, oxidative stress and EC death (apoptosis) that in turn result in impaired vasodilation, vascular remodeling, vascular leakage and subsequent vascular diseases ( Sena et al., 2013; Tang et al., 2014). High glucose causes mitochondrial dysfunction and fragmentation, defective mitochondrial biogenesis, disturbed mitochondrial autophagy (which results in the accumulation of damaged mitochondria, such as irreversibly depolarized or leaky mitochondria), and mitochondrial ROS production ( Makino et al., 2010; Pangare and Makino, 2012; Mishiro et al., 2014; Santos et al., 2014). Atherosclerosis inducers, including oxidized low-density lipoprotein (oxLDL), triglycerides, fatty acids and hyperglycemia, also upregulate mitochondrial production of ROS and inhibit the endothelial ROS buffering system, which causes damage of mitochondrial DNA and other mitochondrial components, important for normal EC function ( Tang et al., 2014). Normalization of mitochondrial ROS levels restored the activity of GAPDH ( Du et al., 2000), prevented the high glucose-induced activation of the polyol and hexosamine pathway and the formation of AGEs ( Nishikawa et al., 2000; Brownlee, 2005).

For a long time, it remained unknown why vascular damage still occurs in diabetes patients even when glycemia levels are under control. Hyperglycemic memory may explain why glucose control has failed to improve cardiovascular outcomes in subjects with diabetes. Indeed, hyperglycemia promotes EC dysfunction even after normalization of glucose levels. Emerging evidence support the notion that ROS-driven hyperglycemic stress is remembered in ECs. The mitochondrial adaptor protein p66Shc is involved in the hyperglycemic memory of ECs, since continued p66Shc activation and mitochondrial translocation are associated with persistent ROS production, decreased NO levels, and EC apoptosis ( Tang et al., 2014). In the future, it will be relevant to assess the relative importance of each of these maladapted metabolic pathways described above.

While ECs become dysfunctional in many organs in diabetic patients, in the retina, elevated ROS levels cause apoptosis of pericytes (mural cells covering microvessels), leading to vessel disintegration, bleeding and aneurysms ( Rask-Madsen and King, 2013). This impairs perfusion, causing retinal tissue ischemia, which in turn is a strong stimulus for neovascularization, overall leading to overgrowth of leaky vessels (diabetic retinopathy) ( Antonetti et al., 2012).

Vessel overgrowth in cancer

The vasculature of solid tumors is morphologically and functionally aberrant, and characterized by highly permeable and fragile vessels, intense vessel sprouting and loss of hierarchical architecture of the vascular network ( Potente et al., 2011; Jain, 2013). To date, the metabolic features of tumor ECs have been poorly characterized. Nevertheless, the switch from a quiescent to an angiogenic phenotype (as in cancer) is metabolically taxing, and tumor ECs are highly activated (Fig. 2). Indeed, VEGF signaling increases glycolytic flux by inducing glucose uptake through elevated expression of the glucose transporter GLUT1 ( Yeh et al., 2008). VEGF also upregulates the expression of PFKFB3, which promotes EC specification to sprout-forming tip cells ( De Bock et al., 2013b). Elevated intratumoral lactate concentrations are a good indicator of the glycolytic adaptation of tumors and correlate with poor clinical outcome in a variety of human cancers ( Hirschhaeuser et al., 2011). Notably, tumor cell-derived lactate can act as bona fide signaling molecule for ECs. Once taken up by tumor-associated ECs through the monocarboxylate transporter 1 (MCT1), lactate induces activation of hypoxia-inducible factor-1α (HIF-1α) ( Sonveaux et al., 2012). Indeed, lactate inhibits the oxygen-sensing prolyl hydroxylase-2 (PHD2), a negative regulator of HIF-1α, thereby stabilizing HIF-1α levels ( Végran et al., 2011). In turn, HIF-1α activation increases the expression of pro-angiogenic factors, such as VEGFR2, basic fibroblast growth factor (bFGF) and nuclear factor kappa B (NFκB), thereby increasing angiogenesis ( Végran et al., 2011; Sonveaux et al., 2012).

Besides functioning as a signaling molecule in tumor ECs, lactate can also be converted to pyruvate to fuel the tricarboxylic acid (TCA) cycle. The interconversion of lactate and pyruvate is mediated by lactate dehydrogenase (LDH), a tetrameric enzyme composed of different subunits, which give rise to different isoenzymes depending on the tetramer composition. LDH with a high LDH-A subunit content converts pyruvate into lactate, and is abundant in glycolytic, proliferative pulmonary microvascular ECs ( Parra-Bonilla et al., 2010), while expression of LDH-B, the subunit converting lactate to pyruvate, is upregulated in tumor endothelium ( van Beijnum et al., 2006), possibly to promote the use of lactate into oxidative metabolism.

Control of post-translational modifications by metabolism in ECs

Post-translational modifications (PTMs) regulate the biological activity of multiple proteins, also of angiogenic proteins. Protein acetylation, a common PTM, requires acetyl-CoA as substrate for lysine acetyltransferases, thus linking metabolism to the regulation of multiple cellular functions ( Friedmann and Marmorstein, 2013; Choudhary et al., 2014). Not surprisingly, acetylation of several angio-modulatory proteins in ECs influences angiogenesis. This has been demonstrated for Notch ( Guarani et al., 2011), HIF-1α ( Lim et al., 2010), cortactin ( Kaluza et al., 2011), VEGFR2 ( Zecchin et al., 2014) and the forkhead transcription factor 1(Foxo1) ( Potente et al., 2007). Interestingly, the NAD⁺-dependent deacetylase SIRT1, which acts as a sensor of energy and redox state, reverses the acetylation status of many of these proteins (Fig. 3A). Inactivation of SIRT1 favors acetylation of the signaling-active Notch intracellular domain (NICD), thereby preventing ubiquitination and proteasomal degradation of NICD; the resultant increase in Notch signaling promotes differentiation to an endothelial stalk cell phenotype, which impairs vessel sprouting ( Guarani et al., 2011). SIRT1 also deacetylates and inactivates Foxo1, a negative regulator of angiogenesis ( Potente et al., 2007). It also deacetylates HIF-1α, thereby suppressing its transcriptional activity and impairing tumor growth and angiogenesis ( Lim et al., 2010). Furthermore, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is a target of SIRT1, and deacetylation of PGC-1α modulates its activity ( Rodgers et al., 2005). In normal conditions, PGC-1α stimulates the formation of blood vessels ( Arany et al., 2008; Saint-Geniez et al., 2013), while in hyperglycemic conditions, PGC-1α induces EC dysfunction and blunts angiogenesis ( Sawada et al., 2014). Whether SIRT1-dependent deacetylation of PGC-1α has a role in diabetes-induced EC dysfunction is unknown.

The link between metabolism and epigenetics is also becoming increasingly evident, as recent reports indicate that ATP citrate lyase (ACLY) and the pyruvate dehydrogenase complex (PDC), two enzymes that generate acetyl-CoA for metabolic purposes, have been shown to exert also non-metabolic functions by influencing the rate of histone acetylation in the nucleus in response to nutrients and growth factors, thereby modulating the expression of target genes ( Wellen et al., 2009; Lee et al., 2014; Sutendra et al., 2014). It will be interesting to explore whether such mechanisms are also operational in ECs and regulate angiogenesis.

As mentioned above, the HBP is involved in glycosylation of angiogenic molecules ( Takahashi and Shibuya, 1997; Du et al., 2000; Benedito et al., 2009). For instance, glycosylation of VEGFR2’s extracellular domain facilitates an interaction with galectin-1, which activates VEGFR2 signaling, even in the absence of VEGF (Fig. 3B). This finding might help to explain part of the tumor refractoriness to anti-VEGF treatment and clarify why blood vessels can still grow in the presence of anti-VEGF therapies ( Croci et al., 2014). Glycosylation of other angio-modulatory proteins (eNOS, Akt, Notch1) influences their pro-angiogenic activity in a context-dependent manner ( Takahashi and Shibuya, 1997; Du et al., 2001; Federici et al., 2002; Luo et al., 2008; Benedito et al., 2009).

In addition to de novo synthesis of lipids, fatty acid synthase (FASN) generates palmitate, which can be used for palmitoylation of the pro-angiogenic protein eNOS in ECs ( Wei et al., 2011) (Fig. 3C). Hence, FASN deficiency impedes angiogenesis by impairing the targeting of the pro-angiogenic VEGFR2 and eNOS to the plasma membrane through reduced palmitoylation ( Browne et al., 2006; Wei et al., 2011). Of note, insulin induces palmitoylation of eNOS, and palmitoylation of eNOS is decreased in insulin-deficient and insulin-resistant mouse models ( Wei et al., 2011).

The redox state of the endothelium can also control the activity of angiogenic proteins via oxidation of cysteine residues. For instance, oxidation of C-terminal cysteine residues in VEGFR2 inactivates VEGFR2 signaling, a process that impairs angiogenesis ( Kang et al., 2011) (Fig. 3D). The antioxidant enzyme peroxiredoxin II protects VEGFR2 against such oxidative inactivation, explaining why peroxiredoxin II deficiency suppresses tumor angiogenesis ( Kang et al., 2011). However, the effects of oxidation are contextual, since in response to VEGF, (presumably other) reactive cysteine thiols (Cys-SH) in the cytosolic domain of VEGFR2 and 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 ( Lee et al., 2011). Whether the latter phenomenon indeed promotes angiogenesis in vivo and how these seemingly opposite findings can be reconciled and whether they occur in different conditions requires further work.

Role of epigenetic DNA methylation in ECs

DNA methylation is an epigenetic modification that regulates various cellular processes and affects progression of several diseases such as cancer and diabetes ( Dawson and Kouzarides, 2012; Yang et al., 2012; Dayeh et al., 2014; Nilsson et al., 2014; Stefan et al., 2014). The transfer of the methyl group is mediated by DNA methyltransferases (DNMT), which use as universal methyl donor S-adenosylmethionine (SAM), an intermediate of one-carbon metabolism ( Struck et al., 2012; Locasale, 2013). Aberrant epigenetic modifications can also contribute to EC dysfunction – this has been best documented in atherosclerosis ( Mitra et al., 2011; Kim et al., 2012; Kumar et al., 2013). DNA methylation patterns in atherosclerotic plaques of human patients and apolipoprotein E-deficient (apoE^-/- ) mice are different from those in healthy tissues ( Hiltunen et al., 2002; Castillo-Díaz et al., 2010; Nazarenko et al., 2011; Nazarenko et al., 2013). ApoE^-/- mice already display global changes in DNA methylation preceding atherosclerotic lesion formation ( Lund et al., 2004). Also, the finding that inhibiting DNA methyltransferase 1 (DNMT1), the enzyme responsible for de novo DNA methylation, with 5-aza-2′-deoxycytidine reduces atherosclerotic lesion burden in apoE^-/- mice further supports a role for DNA methylation in atherogenesis ( Dunn et al., 2014). There is increasing evidence that part of these epigenetic changes in atherosclerosis take place in ECs.

Indeed, exposure of ECs to atherogenic lipoproteins upregulates the expression of p66shc by inducing DNA hypomethylation of the p66shc promoter; since this gene promotes oxidative stress in response to a high fat diet ( Napoli et al., 2003), these lipoproteins cause EC dysfunction ( Kim et al., 2012). Further, treatment of ECs with low-density lipoproteins (LDL) upregulates the expression of DNMT1. This leads to hypermethylation of the promoter of Kruppel-like factor 2 (KLF2), and thereby a decrease in its expression. Since KLF2 mediates vascular homeostasis and has anti-inflammatory and anti-atherosclerotic activity, LDLs render ECs dysfunctional ( Kumar et al., 2013). Disturbed blood flow, another risk factor for atherosclerosis, also induces genome-wide changes in the DNA methylation pattern in ECs, resulting in global changes of gene expression ( Dunn et al., 2014). In particular, disturbed blood flow causes hypermethylation of the promoter and thus suppression of the expression of KLF4, another atheroprotective transcription factor in ECs, thus promoting progression of the disease ( Jiang et al., 2014).

It is tempting to speculate that aberrant epigenetic DNA modifications may induce maladaptive changes of EC metabolism, which could further contribute to atherosclerosis, but such a link has only recently emerged. Indeed, a recent study demonstrated that KLF2 lowers glycolysis in ECs by downregulating the expression of PFKFB3, an activator of glycolysis ( Doddaballapur et al., 2015). However, what precisely the consequences are for glycolysis in ECs in atherosclerotic plaques and how such metabolic changes might modify the cellular activities remains to be explored.

Metabolites controling EC function?

By virtue of their localization, ECs are exposed directly to metabolites and nutrients in the blood, such as glucose, lipids and amino acids. Emerging evidence indicates that a change in the availability of blood metabolites can affect the cellular activities of ECs. One example is how lipids affect ECs. In addition to shunting fatty acids from the circulation to surrounding tissues ( Hagberg et al., 2010; Hagberg et al., 2012), the endothelium itself also responds to lipids in the circulation. Indeed, cholesterol influences the formation of membrane lipid rafts in ECs, a process that facilitates membrane localization and signaling of the pro-angiogenic VEGFR2 ( Fang et al., 2013). Hence, cholesterol efflux from ECs to high-density lipoprotein (HDL) reduces vessel sprouting ( Fang et al., 2013). Another example is how circulating apolipoprotein B (apoB) negatively affects angiogenesis by upregulating the expression of VEGFR1 ( Avraham-Davidi et al., 2012). While only a few studies to date have documented such a relationship, they open exciting future research avenues on how dietary nutrients or metabolites (or proteins interacting with them) might influence EC dysfunction in diabetes and atherosclerosis, or modulate vessel overgrowth in cancer and ocular disease.

Dietary metabolite supplementation to protect or inhibit ECs?

Given that metabolite scan alter EC functions, the question arises whether dietary supplementation of metabolites might represent a strategy to ameliorate vascular disease. We provide here two examples.

Dietary supplementation of L-arginine to protect ECs in atherosclerosis

In the folate cycle, methyl-tetrahydrofolate (mTHF) donates a methyl group to homocysteine (hCys) to generate methionine ( Locasale, 2013). Methionine itself is an important methyl donor for the methylation of lysine and arginine residues in target proteins ( Leiper and Nandi, 2011). Due to the turnover of methylated proteins, asymmetric dimethylated arginine (ADMA) is released. This metabolic by-product functions as an endogenous inhibitor of eNOS, leading to uncoupling of this enzyme, a process that results in reduced NO production but increased ROS formation ( Dhillon et al., 2003; Leiper and Nandi, 2011). In patients with atherosclerosis who have elevated ADMA plasma levels ( Boger, 2009), dietary supplementation of L-arginine increases NO production by directly competing with ADMA for the binding to eNOS ( Bode-Böger et al., 2007; Leiper and Nandi, 2011). These elevated NO levels improve endothelium-dependent vasodilation and reverse the hyperadhesive phenotype of monocytes and T-lymphocytes to the endothelium in atherosclerotic patients, processes that counteract disease progression ( Chan et al., 2000). Especially atherosclerotic patients with a low ratio of L-arginine/ADMA in the plasma could benefit from L-arginine administration ( Bode-Böger et al., 2007).

Dietary supplementation of n-3 PUFAs to reduce pathological angiogenesis

Several studies have reported an anti-angiogenic effect of long-chain n-3, polyunsaturated fatty acids (PUFAs), also known as omega-3 (ω-3) fatty acids, in addition to their anti-thrombotic and anti-inflammatory properties ( Calder, 2014; Wang et al., 2014). n-3 PUFAs inhibit EC functions directly, in part by reducing the expression of VEGFR2, matrix metalloproteinase (MMP)-2 and MMP-9, which dampen EC migration and capillary tube formation ( Yang et al., 1998; Tsuji et al., 2003; Tsuzuki et al., 2007). Indirect anti-angiogenic effects related to a decrease in VEGF levels have also been ascribed to n-3 PUFAs. All this can explain why an omega-3-enriched diet reduces tumor growth and microvessel density ( Rose and Connolly, 1999; Mukutmoni-Norris et al., 2000; Tevar et al., 2002; Calviello et al., 2004; Zhang et al., 2013). n-3 PUFAs also attenuate diabetic retinopathy, a pathological neovascularization in the eye that causes blindness ( Connor et al., 2007; Yanai et al., 2014). However, not all studies report an anti-angiogenic effect of n-3 PUFAs ( Hu et al., 2014), suggesting that their angio-modulatory activity is context-dependent.

Targeting EC metabolism in vessel overgrowth and EC dysfunction

Vessel overgrowth in cancer

Several agents that block metabolic pathways have been demonstrated to inhibit pathological angiogenesis. Glycolytic blockers, for instance, inhibit vessel overgrowth. The aforementioned findings that glycolysis is essential and necessary for vessel sprouting render this metabolic pathway an attractive therapeutic target. Initial studies showed that the non-metabolizable glucose analog 2-deoxy-D-glucose (2DG), which blocks all cellular glucose metabolism, induces EC cytotoxicity and impairs EC proliferation, migration and vessel formation in vitro ( Chung et al., 2004; Nef et al., 2008; Merchan et al., 2010). However, adverse systemic effects due to complete and permanent blockade of glucose metabolism by 2DG limit its therapeutic potential ( Zhang et al., 2014). More recent studies provided a paradigm shift in the design of anti-glycolytic therapy, utilizing a PFKFB3-blocker to inhibit glycolysis only transiently and partially. Importantly, this strategy reduced only the “hyper-glycolysis,” i.e. the extra amount of glycolysis that was increased and necessary for ECs to switch from quiescence to sprouting. This transient and partial reduction in glycolytic flux was sufficient to reduce pathological angiogenesis in the eye, inflamed skin and gut ( Schoors et al., 2014a, 2014b; Xu et al., 2014), but since it did not eliminate baseline glycolysis entirely, this strategy did not affect quiescent healthy vessels, nor did it induce systemic effects ( Schoors et al., 2014b).

EC dysfunction in atherosclerosis and diabetes

By inhibiting HMG-CoA reductase and thereby cholesterol synthesis, statins have been approved for the prevention of cardiovascular events in dyslipidemia patients ( Wilkinson et al., 2014). In addition to lipid-lowering, anti-inflammatory and other effects, statins also decrease EC dysfunction by restoring the activity of eNOS ( Rolfe et al., 2005; Biasucci et al., 2010; Antoniades et al., 2011). This effect relies on a non-lipid lowering mechanism. Indeed, eNOS mRNA stability is reduced by RhoA signaling; for RhoA to become activated, it must be anchored in the plasma membrane, a process that requires prenylation of RhoA. Prenylation itself requires the substrate geranylgeranyl pyrophosphate (GGPP), an intermediate of the mevalonate pathway, which is inhibited by statins. Hence, by inhibiting RhoA activation in ECs through a decreased availability of geranylgeranyl, statins restore eNOS expression ( Rolfe et al., 2005).

The anti-diabetic drug metformin is another example of a drug that affects EC functions by acting on metabolism and critical regulators of cellular metabolism. Metformin inhibits complex I-mediated mitochondrial respiration and, secondary to the resultant drop in energy charge, activates AMP-activated protein kinase (AMPK), a critical energy sensor of cellular energy homeostasis that integrates multiple signaling networks to coordinate a wide array of compensatory, protective and energy-sparing responses ( Sena et al., 2011; Foretz et al., 2014; Pernicova and Korbonits, 2014). In the setting of atherosclerosis and diabetes, metformin reduces EC dysfunction and improves vascular homeostasis ( Foretz et al., 2014). This effect of metformin is in line with its known effects on stimulating vessel sprouting in vitro and angiogenesis/ischemic tissue revascularization in vivo ( Takahashi et al., 2014; Venna et al., 2014). This protective effect of metformin on ECs relies in part on the reduction of oxidative and endoplasmic reticulum stress (induced by high glucose and lipid levels), inhibition of NAD(P)H oxidases ( Batchuluun et al., 2014), activation of eNOS ( Davis et al., 2006; Eriksson and Nyström, 2014; Valente et al., 2014), upregulation of VEGF ( Cittadini et al., 2012; Martin et al., 2012), decreased expression of RAGEs and on abrogation of the suppression of SIRT1 expression ( Arunachalam et al., 2014).

However, in the tumor setting, metformin mostly inhibits tumor growth and in some cases also tumor angiogenesis ( Algire et al., 2011; Dowling et al., 2012; Akinyeke et al., 2013; Mohammed et al., 2013; Hadad et al., 2014; Orecchioni et al., 2014; Qu and Yang, 2014). This is at least in part due to direct inhibitory effects on ECs, though indirect effects on cancer cells have been also documented, albeit at high metformin concentrations ( Foretz et al., 2014). Indeed, metformin inhibits proliferation and migration of ECs, and induces endothelial progenitor apoptosis in vitro ( Tan et al., 2009; Esfahanian et al., 2012; Dallaglio et al., 2014; Orecchioni et al., 2014). It remains however to be explored whether metformin treatment of ECs induces the same switch from anabolic to catabolic metabolism as occurs in other cell types ( Foretz et al., 2014), and whether these changes in EC metabolism also contribute to its observed contextual effects.

As discussed above, hyperglycemic ECs redirect excess glucose-derived metabolites into the ALR2-dependent polyol pathway, leading to increased production of AGEs and ROS. Ocular neovascularization is a common complication in diabetes. Preclinical findings indicate that inhibition of ALR2 reduces the progression of early diabetic retinopathy ( Obrosova and Kador, 2011). In human diabetics, the ALR2 inhibitor epalrestat delays the progression of retinopathy ( Hotta et al., 2012). Overall, these examples illustrate the therapeutic potential of targeting EC metabolism for the treatment of vascular disorders.

Conclusions and future perspectives

Current strategies to inhibit pathological angiogenesis rely primarily on the inhibition of growth factor (VEGF) signaling ( Singh and Ferrara, 2012). However, refractoriness and drug resistance limit the efficacy of these treatments. There is thus a need for anti-angiogenic therapies that act via complementary mechanisms. It will be interesting to explore whether drugs targeting EC metabolism could enhance the response of cancer patients to current anti-angiogenic agents. Preclinical animal studies have already provided initial proof of concept that glycolytic PFKFB3 blockade enhances the anti-angiogenic effects of VEGF inhibitors ( Schoors et al., 2014a).

But metabolic inhibitors may also improve anti-angiogenic therapy in other ways. For instance, patients suffering wet age-related macular degeneration (AMD), the most frequent blinding disease in the elderly population, receive frequent intra-ocular injections of anti-VEGF therapies, causing local adverse effects and personal discomfort from the needle injections in the eye. An alternative to avoid recurrent intra-ocular injections would rely on the use of a drug that can be intravenously administered without causing systemic adverse effects. Recent studies revealed that systemic administration of the glycolytic PFKFB3 blocker 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) caused a partial and transient reduction of glycolysis in ECs, which sufficed to inhibit choroidal neovascularization in a preclinical mouse model of wet AMD, without a need for intra-ocular injections ( Schoors et al., 2014b).

It will be also interesting to characterize how current anti-cancer and anti-angiogenic drugs affect EC metabolism, since recent studies documented substantial changes of cancer cell metabolism upon treatment with anti-angiogenic drugs ( Keunen et al., 2011; Sounni et al., 2014; Curtarello et al., 2015). In tumor models, anti-VEGF therapy induced a selection of highly glycolytic and tumorigenic cancer cells, which showed reduced responsiveness to VEGF blockade ( Curtarello et al., 2015). Also, withdrawal of VEGF receptor tyrosine kinase inhibitors induced a metabolic shift toward lipid metabolism in cancer cells, suggesting that inhibition of lipid metabolism might represent a promising strategy to overcome the tumor aggressiveness induced by the withdrawal of anti-angiogenic agents ( Sounni et al., 2014). Understanding the effects of anti-cancer treatments on EC metabolism promises to improve overall anti-cancer treatment options.

Finally, there is nearly nothing known about the metabolism of the different types of endothelial progenitors or vascular niche cells, neither it is known how these cells adapt their metabolism when they proliferate and differentiate to ECs, or maladapt their metabolism in disease. It is also not known how quiescent vascular niche ECs harboring dormant disseminated breast cancer cells adapt their metabolism when these niche ECs start to sprout to promote micrometastatic outgrowth ( Ghajar et al., 2013). EC metabolism is a young field with multiple opportunities to address novel questions, and offering unexplored translational opportunities for improving anti-angiogenic or EC-protective therapy.

References

[1] Akinyeke T, Matsumura S, Wang X, Wu Y, Schalfer E D, Saxena A, Yan W, Logan S K, Li X (2013). Metformin targets c-MYC oncogene to prevent prostate cancer. Carcinogenesis, 34(12): 2823–2832

[2] Algire C, Amrein L, Bazile M, David S, Zakikhani M, Pollak M (2011). Diet and tumor LKB1 expression interact to determine sensitivity to anti-neoplastic effects of metformin in vivo. Oncogene, 30(10): 1174–1182

[3] Antonetti D A, Klein R, Gardner T W (2012). Diabetic retinopathy. N Engl J Med, 366(13): 1227–1239

[4] Antoniades C, Bakogiannis C, Leeson P, Guzik T J, Zhang M H, Tousoulis D, Antonopoulos A S, Demosthenous M, Marinou K, Hale A, Paschalis A, Psarros C, Triantafyllou C, Bendall J, Casadei B, Stefanadis C, Channon K M (2011). Rapid, direct effects of statin treatment on arterial redox state and nitric oxide bioavailability in human atherosclerosis via tetrahydrobiopterin-mediated endothelial nitric oxide synthase coupling. Circulation, 124(3): 335–345

[5] Arany Z, Foo S Y, Ma Y, Ruas J L, Bommi-Reddy A, Girnun G, Cooper M, Laznik D, Chinsomboon J, Rangwala S M, Baek K H, Rosenzweig A, Spiegelman B M (2008). HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha. Nature, 451(7181): 1008–1012

[6] Arunachalam G, Samuel S M, Marei I, Ding H, Triggle C R (2014). Metformin modulates hyperglycaemia-induced endothelial senescence and apoptosis through SIRT1. Br J Pharmacol, 171(2): 523–535

[7] Avraham-Davidi I, Ely Y, Pham V N, Castranova D, Grunspan M, Malkinson G, Gibbs-Bar L, Mayseless O, Allmog G, Lo B, Warren C M, Chen T T, Ungos J, Kidd K, Shaw K, Rogachev I, Wan W, Murphy P M, Farber S A, Carmel L, Shelness G S, Iruela-Arispe M L, Weinstein B M, Yaniv K (2012). ApoB-containing lipoproteins regulate angiogenesis by modulating expression of VEGF receptor 1. Nat Med, 18(6): 967–973

[8] Batchuluun B, Inoguchi T, Sonoda N, Sasaki S, Inoue T, Fujimura Y, Miura D, Takayanagi R (2014). Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells. Atherosclerosis, 232(1): 156–164

[9] Beleznai T, Bagi Z (2012). Activation of hexosamine pathway impairs nitric oxide (NO)-dependent arteriolar dilations by increased protein O-GlcNAcylation. Vascul Pharmacol, 56(3-4): 115–121

[10] Benedito R, Roca C, Sörensen I, Adams S, Gossler A, Fruttiger M, Adams R H (2009). The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell, 137(6): 1124–1135

[11] Biasucci L M, Biasillo G, Stefanelli A (2010). Inflammatory markers, cholesterol and statins: pathophysiological role and clinical importance. Clin Chem Lab Med, 48: 1685–1691

[12] Bode-Böger S M, Scalera F, Ignarro L J (2007). The L-arginine paradox: Importance of the L-arginine/asymmetrical dimethylarginine ratio. Pharmacol Ther, 114(3): 295–306

[13] Boger R H (2009). Asymmetric dimethylarginine: understanding the physiology, genetics, and clinical relevance of this novel biomarker. Proceedings of the 4th International Symposium on ADMA. Pharmacol Res, 60: 447

[14] Brandes R P, Weissmann N, Schröder K (2014). Redox-mediated signal transduction by cardiovascular Nox NADPH oxidases. J Mol Cell Cardiol, 73: 70–79

[15] Browne C D, Hindmarsh E J, Smith J W (2006). Inhibition of endothelial cell proliferation and angiogenesis by orlistat, a fatty acid synthase inhibitor. FASEB J, 20: 2027–2035

[16] Brownlee M (2001). Biochemistry and molecular cell biology of diabetic complications. Nature, 414(6865): 813–820

[17] Brownlee M (2005). The pathobiology of diabetic complications: a unifying mechanism. Diabetes, 54(6): 1615–1625

[18] Cai S, Khoo J, Channon K M (2005). Augmented BH4 by gene transfer restores nitric oxide synthase function in hyperglycemic human endothelial cells. Cardiovasc Res, 65(4): 823–831

[19] Calder P C (2014). Marine omega-3 fatty acids and inflammatory processes: Effects, mechanisms and clinical relevance. Biochim Biophys Acta

[20] Calviello G, Di Nicuolo F, Gragnoli S, Piccioni E, Serini S, Maggiano N, Tringali G, Navarra P, Ranelletti F O, Palozza P (2004). n-3 PUFAs reduce VEGF expression in human colon cancer cells modulating the COX-2/PGE2 induced ERK-1 and-2 and HIF-1alpha induction pathway. Carcinogenesis, 25(12): 2303–2310

[21] Carracedo A, Cantley L C, Pandolfi P P (2013). Cancer metabolism: fatty acid oxidation in the limelight. Nat Rev Cancer, 13(4): 227–232

[22] Castillo-Díaz S A, Garay-Sevilla M E, Hernández-González M A, Solís-Martínez M O, Zaina S (2010). Extensive demethylation of normally hypermethylated CpG islands occurs in human atherosclerotic arteries. Int J Mol Med, 26(5): 691–700

[23] Chan J R, Böger R H, Bode-Böger S M, Tangphao O, Tsao P S, Blaschke T F, Cooke J P (2000). Asymmetric dimethylarginine increases mononuclear cell adhesiveness in hypercholesterolemic humans. Arterioscler Thromb Vasc Biol, 20(4): 1040–1046

[24] Cho Y E, Basu A, Dai A, Heldak M, Makino A (2013). Coronary endothelial dysfunction and mitochondrial reactive oxygen species in type 2 diabetic mice. Am J Physiol Cell Physiol, 305(10): C1033–C1040

[25] Choudhary C, Weinert B T, Nishida Y, Verdin E, Mann M (2014). The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol, 15(8): 536–550

[26] Chung S J, Lee S H, Lee Y J, Park H S, Bönger R, Kang Y H (2004). Pyruvate protection against endothelial cytotoxicity induced by blockade of glucose uptake. J Biochem Mol Biol, 37(2): 239–245

[27] Cittadini A, Napoli R, Monti M G, Rea D, Longobardi S, Netti P A, Walser M, Samà M, Aimaretti G, Isgaard J, Saccà L (2012). Metformin prevents the development of chronic heart failure in the SHHF rat model. Diabetes, 61(4): 944–953

[28] Connor K M, SanGiovanni J P, Lofqvist C, Aderman C M, Chen J, Higuchi A, Hong S, Pravda E A, Majchrzak S, Carper D, Hellstrom A, Kang J X, Chew E Y, Salem N Jr, Serhan C N, Smith L E (2007). Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nat Med, 13(7): 868–873

[29] Coutelle O, Hornig-Do H T, Witt A, Andree M, Schiffmann L M, Piekarek M, Brinkmann K, Seeger J M, Liwschitz M, Miwa S, Hallek M, Krönke M, Trifunovic A, Eming S A, Wiesner R J, Hacker U T, Kashkar H (2014). Embelin inhibits endothelial mitochondrial respiration and impairs neoangiogenesis during tumor growth and wound healing. EMBO Mol Med, 6(5): 624–639

[30] Crabtree M J, Tatham A L, Al-Wakeel Y, Warrick N, Hale A B, Cai S, Channon K M, Alp N J (2009). Quantitative regulation of intracellular endothelial nitric-oxide synthase (eNOS) coupling by both tetrahydrobiopterin-eNOS stoichiometry and biopterin redox status: insights from cells with tet-regulated GTP cyclohydrolase I expression. J Biol Chem, 284(2): 1136–1144

[31] Croci D O, Cerliani J P, Dalotto-Moreno T, Méndez-Huergo S P, Mascanfroni I D, Dergan-Dylon S, Toscano M A, Caramelo J J, García-Vallejo J J, Ouyang J, Mesri E A, Junttila M R, Bais C, Shipp M A, Salatino M, Rabinovich G A (2014). Glycosylation-dependent lectin-receptor interactions preserve angiogenesis in anti-VEGF refractory tumors. Cell, 156(4): 744–758

[32] Curtarello M, Zulato E, Nardo G, Valtorta S, Guzzo G, Rossi E, Esposito G, Msaki A, Pastò A, Rasola A, Persano L, Ciccarese F, Bertorelle R, Todde S, Plebani M, Schroer H, Walenta S, Mueller-Klieser W, Amadori A, Moresco R M, Indraccolo S (2015). VEGF-targeted therapy stably modulates the glycolytic phenotype of tumor cells. Cancer Res, 75(1): 120–133

[33] Dagher Z, Ruderman N, Tornheim K, Ido Y (2001). Acute regulation of fatty acid oxidation and amp-activated protein kinase in human umbilical vein endothelial cells. Circ Res, 88(12): 1276–1282

[34] Dallaglio K, Bruno A, Cantelmo A R, Esposito A I, Ruggiero L, Orecchioni S, Calleri A, Bertolini F, Pfeffer U, Noonan D M, Albini A (2014). Paradoxic effects of metformin on endothelial cells and angiogenesis. Carcinogenesis, 35(5): 1055–1066

[35] Davignon J, Ganz P (2004). Role of endothelial dysfunction in atherosclerosis. Circulation, 109(23 Suppl 1): III27–III32

[36] Davis B J, Xie Z, Viollet B, Zou M H (2006). Activation of the AMP-activated kinase by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo by promoting the association of heat shock protein 90 and endothelial nitric oxide synthase. Diabetes, 55(2): 496–505

[37] Dawson M A, Kouzarides T (2012). Cancer epigenetics: from mechanism to therapy. Cell, 150(1): 12–27

[38] Dayeh T, Volkov P, Salö S, Hall E, Nilsson E, Olsson A H, Kirkpatrick C L, Wollheim C B, Eliasson L, Rönn T, Bacos K, Ling C (2014). Genome-wide DNA methylation analysis of human pancreatic islets from type 2 diabetic and non-diabetic donors identifies candidate genes that influence insulin secretion. PLoS Genet, 10(3): e1004160

[39] De Bock K, Georgiadou M, Carmeliet P (2013a). Role of endothelial cell metabolism in vessel sprouting. Cell Metab, 18(5): 634–647

[40] De Bock K, Georgiadou M, Schoors S, Kuchnio A, Wong B W, Cantelmo A R, Quaegebeur A, Ghesquière B, Cauwenberghs S, Eelen G, Phng L K, Betz I, Tembuyser B, Brepoels K, Welti J, Geudens I, Segura I, Cruys B, Bifari F, Decimo I, Blanco R, Wyns S, Vangindertael J, Rocha S, Collins R T, Munck S, Daelemans D, Imamura H, Devlieger R, Rider M, Van Veldhoven P P, Schuit F, Bartrons R, Hofkens J, Fraisl P, Telang S, Deberardinis R J, Schoonjans L, Vinckier S, Chesney J, Gerhardt H, Dewerchin M, Carmeliet P (2013b). Role of PFKFB3-driven glycolysis in vessel sprouting. Cell, 154(3): 651–663

[41] Dhillon B, Badiwala M V, Maitland A, Rao V, Li S H, Verma S (2003). Tetrahydrobiopterin attenuates homocysteine induced endothelial dysfunction. Mol Cell Biochem, 247(1-2): 223–227

[42] Doddaballapur A, Michalik K M, Manavski Y, Lucas T, Houtkooper R H, You X, Chen W, Zeiher A M, Potente M, Dimmeler S, Boon R A (2015). Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3. Arterioscler Thromb Vasc Biol, 35(1): 137–145

[43] Dowling R J O, Niraula S, Stambolic V, Goodwin P J (2012). Metformin in cancer: translational challenges. J Mol Endocrinol, 48(3): R31–R43

[44] Du X, Matsumura T, Edelstein D, Rossetti L, Zsengellér Z, Szabó C, Brownlee M (2003). Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells. J Clin Invest, 112(7): 1049–1057

[45] Du X L, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M (2001). Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest, 108(9): 1341–1348

[46] Du X L, Edelstein D, Rossetti L, Fantus I G, Goldberg H, Ziyadeh F, Wu J, Brownlee M (2000). Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci USA, 97(22): 12222–12226

[47] Dunn J, Qiu H, Kim S, Jjingo D, Hoffman R, Kim C W, Jang I, Son D J, Kim D, Pan C, Fan Y, Jordan I K, Jo H (2014). Flow-dependent epigenetic DNA methylation regulates endothelial gene expression and atherosclerosis. J Clin Invest, 124(7): 3187–3199

[48] Eelen G, Cruys B, Welti J, De Bock K, Carmeliet P (2013). Control of vessel sprouting by genetic and metabolic determinants. Trends Endocrinol Metab, 24(12): 589–596

[49] Elmasri H, Karaaslan C, Teper Y, Ghelfi E, Weng M, Ince T A, Kozakewich H, Bischoff J, Cataltepe S (2009), Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells. FASEB J, 23: 3865–3873

[50] Eriksson L, Nyström T (2014). Activation of AMP-activated protein kinase by metformin protects human coronary artery endothelial cells against diabetic lipoapoptosis. Cardiovasc Diabetol, 13(1): 152

[51] Esfahanian N, Shakiba Y, Nikbin B, Soraya H, Maleki-Dizaji N, Ghazi-Khansari M, Garjani A (2012). Effect of metformin on the proliferation, migration, and MMP-2 and -9 expression of human umbilical vein endothelial cells. Mol Med Rep, 5: 1068–1074

[52] Fang L, Choi S H, Baek J S, Liu C, Almazan F, Ulrich F, Wiesner P, Taleb A, Deer E, Pattison J, Torres-Vázquez J, Li A C, Miller Y I (2013). Control of angiogenesis by AIBP-mediated cholesterol efflux. Nature, 498(7452): 118–122

[53] Federici M, Menghini R, Mauriello A, Hribal M L, Ferrelli F, Lauro D, Sbraccia P, Spagnoli L G, Sesti G, Lauro R (2002). Insulin-dependent activation of endothelial nitric oxide synthase is impaired by O-linked glycosylation modification of signaling proteins in human coronary endothelial cells. Circulation, 106(4): 466–472

[54] Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B (2014). Metformin: from mechanisms of action to therapies. Cell Metab, 20(6): 953–966

[55] Forstermann U, Sessa W C (2012). Nitric oxide synthases: regulation and function. Eur Heart J, 33: 829–837, 837a–837d

[56] Friedmann D R, Marmorstein R (2013). Structure and mechanism of non-histone protein acetyltransferase enzymes. FEBS J, 280: 5570–5581

[57] Funk S D, Yurdagul A Jr, Orr A W (2012). Hyperglycemia and endothelial dysfunction in atherosclerosis: lessons from type 1 diabetes. Int J Vasc Med, 2012: 569654

[58] Ghajar C M, Peinado H, Mori H, Matei I R, Evason K J, Brazier H, Almeida D, Koller A, Hajjar K A, Stainier D Y, Chen E I, Lyden D, Bissell M J (2013). The perivascular niche regulates breast tumour dormancy. Nat Cell Biol, 15(7): 807–817

[59] Gómez-Gaviro M V, Lovell-Badge R, Fernández-Avilés F, Lara-Pezzi E (2012). The vascular stem cell niche. J Cardiovasc Transl Res, 5(5): 618–630

[60] Gorren A C, Bec N, Schrammel A, Werner E R, Lange R, Mayer B (2000). Low-temperature optical absorption spectra suggest a redox role for tetrahydrobiopterin in both steps of nitric oxide synthase catalysis. Biochemistry, 39(38): 11763–11770

[61] Groschner L N, Waldeck-Weiermair M, Malli R, Graier W F (2012). Endothelial mitochondria–less respiration, more integration. Pflugers Arch, 464: 63–76

[62] Guarani V, Deflorian G, Franco C A, Krüger M, Phng L K, Bentley K, Toussaint L, Dequiedt F, Mostoslavsky R, Schmidt M H, Zimmermann B, Brandes R P, Mione M, Westphal C H, Braun T, Zeiher A M, Gerhardt H, Dimmeler S, Potente M (2011). Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase. Nature, 473(7346): 234–238

[63] Hadad S M, Hardie D G, Appleyard V, Thompson A M (2014). Effects of metformin on breast cancer cell proliferation, the AMPK pathway and the cell cycle. Clin Transl Oncol, 16(8): 746–752

[64] Hagberg C E, Falkevall A, Wang X, Larsson E, Huusko J, Nilsson I, van Meeteren L A, Samen E, Lu L, Vanwildemeersch M, Klar J, Genove G, Pietras K, Stone-Elander S, Claesson-Welsh L, Ylä-Herttuala S, Lindahl P, Eriksson U (2010). Vascular endothelial growth factor B controls endothelial fatty acid uptake. Nature, 464(7290): 917–921

[65] Hagberg C E, Mehlem A, Falkevall A, Muhl L, Fam B C, Ortsäter H, Scotney P, Nyqvist D, Samén E, Lu L, Stone-Elander S, Proietto J, Andrikopoulos S, Sjöholm A, Nash A, Eriksson U (2012). Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes. Nature, 490(7420): 426–430

[66] Harjes U, Bridges E, McIntyre A, Fielding B A, Harris A L (2014). Fatty acid-binding protein 4, a point of convergence for angiogenic and metabolic signaling pathways in endothelial cells. J Biol Chem, 289(33): 23168–23176

[67] Hernandez-Mijares A, Rocha M, Rovira-Llopis S, Bañuls C, Bellod L, de Pablo C, Alvarez A, Roldan-Torres I, Sola-Izquierdo E, Victor V M (2013). Human leukocyte/endothelial cell interactions and mitochondrial dysfunction in type 2 diabetic patients and their association with silent myocardial ischemia. Diabetes Care, 36(6): 1695–1702

[68] Hiltunen M O, Turunen M P, Häkkinen T P, Rutanen J, Hedman M, Mäkinen K, Turunen A M, Aalto-Setälä K, Ylä-Herttuala S (2002). DNA hypomethylation and methyltransferase expression in atherosclerotic lesions. Vasc Med, 7(1): 5–11

[69] Hirschhaeuser F, Sattler U G, Mueller-Klieser W (2011). Lactate: a metabolic key player in cancer. Cancer Res, 71(22): 6921–6925

[70] Hotta N, Kawamori R, Fukuda M, Shigeta Y, Aldose Reductase Inhibitor-Diabetes Complications Trial Study G (2012). Long-term clinical effects of epalrestat, an aldose reductase inhibitor, on progression of diabetic neuropathy and other microvascular complications: multivariate epidemiological analysis based on patient background factors and severity of diabetic neuropathy. Diabet Med, 29: 1529–1533

[71] Hu J, Popp R, Frömel T, Ehling M, Awwad K, Adams R H, Hammes H P, Fleming I (2014). Müller glia cells regulate Notch signaling and retinal angiogenesis via the generation of 19,20-dihydroxydocosapentaenoic acid. J Exp Med, 211(2): 281–295

[72] Jain R K (2013). Normalizing tumor microenvironment to treat cancer: bench to bedside to biomarkers. J Clin Oncol, 31(17): 2205–2218

[73] Jakobsson L, Franco C A, Bentley K, Collins R T, Ponsioen B, Aspalter I M, Rosewell I, Busse M, Thurston G, Medvinsky A, Schulte-Merker S, Gerhardt H (2010). Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat Cell Biol, 12(10): 943–953

[74] Jeon S M, Chandel N S, Hay N (2012). AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress. Nature, 485(7400): 661–665

[75] Jiang Y Z, Jiménez J M, Ou K, McCormick M E, Zhang L D, Davies P F (2014). Hemodynamic disturbed flow induces differential DNA methylation of endothelial Kruppel-Like Factor 4 promoter in vitro and in vivo. Circ Res, 115(1): 32–43

[76] Kaluza D, Kroll J, Gesierich S, Yao T P, Boon R A, Hergenreider E, Tjwa M, Rössig L, Seto E, Augustin H G, Zeiher A M, Dimmeler S, Urbich C (2011). Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin. EMBO J, 30(20): 4142–4156

[77] Kang D H, Lee D J, Lee K W, Park Y S, Lee J Y, Lee S H, Koh Y J, Koh G Y, Choi C, Yu D Y, Kim J, Kang S W (2011). Peroxiredoxin II is an essential antioxidant enzyme that prevents the oxidative inactivation of VEGF receptor-2 in vascular endothelial cells. Mol Cell, 44(4): 545–558

[78] Kawabe J, Hasebe N (2014). Role of the vasa vasorum and vascular resident stem cells in atherosclerosis. Biomed Res Int, 2014: 701571

[79] Keunen O, Johansson M, Oudin A, Sanzey M, Rahim S A, Fack F, Thorsen F, Taxt T, Bartos M, Jirik R, Miletic H, Wang J, Stieber D, Stuhr L, Moen I, Rygh C B, Bjerkvig R, Niclou S P (2011). Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. Proc Natl Acad Sci USA, 108(9): 3749–3754

[80] Kim Y R, Kim C S, Naqvi A, Kumar A, Kumar S, Hoffman T A, Irani K (2012). Epigenetic upregulation of p66shc mediates low-density lipoprotein cholesterol-induced endothelial cell dysfunction. Am J Physiol Heart Circ Physiol, 303(2): H189–H196

[81] Kizhakekuttu T J, Wang J, Dharmashankar K, Ying R, Gutterman D D, Vita J A, Widlansky M E (2012). Adverse alterations in mitochondrial function contribute to type 2 diabetes mellitus-related endothelial dysfunction in humans. Arterioscler Thromb Vasc Biol, 32(10): 2531–2539

[82] Kumar A, Kumar S, Vikram A, Hoffman T A, Naqvi A, Lewarchik C M, Kim Y R, Irani K (2013). Histone and DNA methylation-mediated epigenetic downregulation of endothelial Kruppel-like factor 2 by low-density lipoprotein cholesterol. Arterioscler Thromb Vasc Biol, 33(8): 1936–1942

[83] Lee J V, Carrer A, Shah S, Snyder N W, Wei S, Venneti S, Worth A J, Yuan Z F, Lim H W, Liu S, Jackson E, Aiello N M, Haas N B, Rebbeck T R, Judkins A, Won K J, Chodosh L A, Garcia B A, Stanger B Z, Feldman M D, Blair I A, Wellen K E (2014). Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation. Cell Metab, 20(2): 306–319

[84] Lee M, Choy W C, Abid M R (2011). Direct sensing of endothelial oxidants by vascular endothelial growth factor receptor-2 and c-Src. PLoS ONE, 6(12): e28454

[85] Leiper J, Nandi M (2011). The therapeutic potential of targeting endogenous inhibitors of nitric oxide synthesis. Nat Rev Drug Discov, 10(4): 277–291

[86] Leopold J A, Zhang Y Y, Scribner A W, Stanton R C, Loscalzo J (2003). Glucose-6-phosphate dehydrogenase overexpression decreases endothelial cell oxidant stress and increases bioavailable nitric oxide. Arterioscler Thromb Vasc Biol, 23(3): 411–417

[87] Lim J H, Lee Y M, Chun Y S, Chen J, Kim J E, Park J W (2010). Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypoxia-inducible factor 1alpha. Mol Cell, 38(6): 864–878

[88] Liu H, Yu S, Zhang H, Xu J (2012). Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2. PLoS ONE, 7(10): e46720

[89] Locasale J W (2013). Serine, glycine and one-carbon units: cancer metabolism in full circle. Nat Rev Cancer, 13(8): 572–583

[90] Lorenzi M (2007). The polyol pathway as a mechanism for diabetic retinopathy: attractive, elusive, and resilient. Exp Diabetes Res, 2007: 61038

[91] Lund G, Andersson L, Lauria M, Lindholm M, Fraga M F, Villar-Garea A, Ballestar E, Esteller M, Zaina S (2004). DNA methylation polymorphisms precede any histological sign of atherosclerosis in mice lacking apolipoprotein E. J Biol Chem, 279(28): 29147–29154

[92] Luo B, Soesanto Y, McClain D A (2008). Protein modification by O-linked GlcNAc reduces angiogenesis by inhibiting Akt activity in endothelial cells. Arterioscler Thromb Vasc Biol, 28(4): 651–657

[93] Mackenzie R M, Salt I P, Miller W H, Logan A, Ibrahim H A, Degasperi A, Dymott J A, Hamilton C A, Murphy M P, Delles C, Dominiczak A F (2013). Mitochondrial reactive oxygen species enhance AMP-activated protein kinase activation in the endothelium of patients with coronary artery disease and diabetes. Clin Sci (Lond), 124(6): 403–411

[94] Makino A, Scott B T, Dillmann W H (2010). Mitochondrial fragmentation and superoxide anion production in coronary endothelial cells from a mouse model of type 1 diabetes. Diabetologia, 53(8): 1783–1794

[95] Manigrasso M B, Juranek J, Ramasamy R, Schmidt A M (2014). Unlocking the biology of RAGE in diabetic microvascular complications. Trends Endocrinol Metab, 25(1): 15–22

[96] Martin M J, Hayward R, Viros A, Marais R (2012). Metformin accelerates the growth of BRAF V600E-driven melanoma by upregulating VEGF-A. Cancer Discov, 2(4): 344–355

[97] Matafome P, Sena C, Seiça R (2013). Methylglyoxal, obesity, and diabetes. Endocrine, 43(3): 472–484

[98] Meininger C J, Cai S, Parker J L, Channon K M, Kelly K A, Becker E J, Wood M K, Wade L A, Wu G (2004). GTP cyclohydrolase I gene transfer reverses tetrahydrobiopterin deficiency and increases nitric oxide synthesis in endothelial cells and isolated vessels from diabetic rats. FASEB J, 18: 1900–1902

[99] Meininger C J, Marinos R S, Hatakeyama K, Martinez-Zaguilan R, Rojas J D, Kelly K A, Wu G (2000). Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency. Biochem J, 349(Pt 1): 353–356

[100] Mendelson A, Frenette P S (2014). Hematopoietic stem cell niche maintenance during homeostasis and regeneration. Nat Med, 20(8): 833–846

[101] Merchan J R, Kovács K, Railsback J W, Kurtoglu M, Jing Y, Piña Y, Gao N, Murray T G, Lehrman M A, Lampidis T J (2010). Antiangiogenic activity of 2-deoxy-D-glucose. PLoS ONE, 5(10): e13699

[102] Mishiro K, Imai T, Sugitani S, Kitashoji A, Suzuki Y, Takagi T, Chen H, Oumi Y, Tsuruma K, Shimazawa M, Hara H (2014). Diabetes mellitus aggravates hemorrhagic transformation after ischemic stroke via mitochondrial defects leading to endothelial apoptosis. PLoS ONE, 9(8): e103818

[103] Mitra S, Khaidakov M, Lu J, Ayyadevara S, Szwedo J, Wang X W, Chen C, Khaidakov S, Kasula S R, Stone A, Pogribny I, Mehta J L (2011). Prior exposure to oxidized low-density lipoprotein limits apoptosis in subsequent generations of endothelial cells by altering promoter methylation. Am J Physiol Heart Circ Physiol, 301(2): H506–H513

[104] Mohammed A, Janakiram N B, Brewer M, Ritchie R L, Marya A, Lightfoot S, Steele V E, Rao C V (2013). Antidiabetic drug metformin prevents progression of pancreatic cancer by targeting in part cancer stem cells and mTOR signaling. Transl Oncol, 6(6): 649–659

[105] Morgan P E, Sheahan P J, Davies M J (2014). Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal. PLoS ONE, 9(1): e86564

[106] Moschetta M, Mishima Y, Sahin I, Manier S, Glavey S, Vacca A, Roccaro A M, Ghobrial I M (2014). Role of endothelial progenitor cells in cancer progression. Biochim Biophys Acta, 1846(1): 26–39

[107] Mugoni V, Postel R, Catanzaro V, De Luca E, Turco E, Digilio G, Silengo L, Murphy M P, Medana C, Stainier D Y, Bakkers J, Santoro M M (2013). Ubiad1 is an antioxidant enzyme that regulates eNOS activity by CoQ10 synthesis. Cell, 152(3): 504–518

[108] Mukutmoni-Norris M, Hubbard N E, Erickson K L (2000). Modulation of murine mammary tumor vasculature by dietary n-3 fatty acids in fish oil. Cancer Lett, 150(1): 101–109

[109] Napoli C, Martin-Padura I, de Nigris F, Giorgio M, Mansueto G, Somma P, Condorelli M, Sica G, De Rosa G, Pelicci P (2003). Deletion of the p66Shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet. Proc Natl Acad Sci USA, 100(4): 2112–2116

[110] Nazarenko M S, Markov A V, Lebedev I N, Sleptsov A A, Frolov A V, Barbash O L, Puzyrev V P (2013). DNA methylation profiling of the vascular tissues in the setting of atherosclerosis. Mol Biol (Mosk), 47(3): 398–404

[111] Nazarenko M S, Puzyrev V P, Lebedev I N, Frolov A V, Barbarash O L, Barbarash L S (2011). Methylation profiling of human atherosclerotic plaques. Mol Biol (Mosk), 45(4): 610–616

[112] Nef H M, Möllmann H, Joseph A, Troidl C, Voss S, Vogt A, Weber M, Hamm C W, Elsässer A (2008). Effects of 2-deoxy-D-glucose on proliferation of vascular smooth muscle cells and endothelial cells. J Int Med Res, 36(5): 986–991

[113] Nilsson E, Jansson P A, Perfilyev A, Volkov P, Pedersen M, Svensson M K, Poulsen P, Ribel-Madsen R, Pedersen N L, Almgren P, Fadista J, Rönn T, Klarlund Pedersen B, Scheele C, Vaag A, Ling C (2014). Altered DNA methylation and differential expression of genes influencing metabolism and inflammation in adipose tissue from subjects with type 2 diabetes. Diabetes, 63(9): 2962–2976

[114] Nishikawa T, Edelstein D, Du X L, Yamagishi S, Matsumura T, Kaneda Y, Yorek M A, Beebe D, Oates P J, Hammes H P, Giardino I, Brownlee M (2000). Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature, 404(6779): 787–790

[115] Obrosova I G, Kador P F (2011). Aldose reductase / polyol inhibitors for diabetic retinopathy. Curr Pharm Biotechnol, 12(3): 373–385

[116] Oldendorf W H, Cornford M E, Brown W J (1977). The large apparent work capability of the blood-brain barrier: a study of the mitochondrial content of capillary endothelial cells in brain and other tissues of the rat. Ann Neurol, 1(5): 409–417

[117] Orecchioni S, Reggiani F, Talarico G, Mancuso P, Calleri A, Gregato G, Labanca V, Noonan D M, Dallaglio K, Albini A, Bertolini F (2014). The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells. Int J Cancer

[118] Pangare M, Makino A (2012). Mitochondrial function in vascular endothelial cell in diabetes. J Smooth Muscle Res, 48(1): 1–26

[119] Parra-Bonilla G, Alvarez D F, Al-Mehdi A B, Alexeyev M, Stevens T (2010). Critical role for lactate dehydrogenase A in aerobic glycolysis that sustains pulmonary microvascular endothelial cell proliferation. Am J Physiol Lung Cell Mol Physiol, 299(4): L513–L522

[120] Pelosi E, Castelli G, Testa U (2014). Endothelial progenitors. Blood Cells Mol Dis, 52(4): 186–194

[121] Pernicova I, Korbonits M (2014). Metformin—mode of action and clinical implications for diabetes and cancer. Nat Rev Endocrinol, 10(3): 143–156

[122] Phng L K, Gerhardt H (2009). Angiogenesis: a team effort coordinated by notch. Dev Cell, 16(2): 196–208

[123] Pober J S, Min W, Bradley J R (2009). Mechanisms of endothelial dysfunction, injury, and death. Annu Rev Pathol, 4(1): 71–95

[124] Potente M, Gerhardt H, Carmeliet P (2011). Basic and therapeutic aspects of angiogenesis. Cell, 146(6): 873–887

[125] Potente M, Ghaeni L, Baldessari D, Mostoslavsky R, Rossig L, Dequiedt F, Haendeler J, Mione M, Dejana E, Alt F W, Zeiher A M, Dimmeler S (2007). SIRT1 controls endothelial angiogenic functions during vascular growth. Genes Dev, 21(20): 2644–2658

[126] Qu H, Yang X (2014). Metformin inhibits angiogenesis induced by interaction of hepatocellular carcinoma with hepatic stellate cells. Cell Biochem Biophys

[127] Quintero M, Colombo S L, Godfrey A, Moncada S (2006). Mitochondria as signaling organelles in the vascular endothelium. Proc Natl Acad Sci USA, 103(14): 5379–5384

[128] Rajendran P, Rengarajan T, Thangavel J, Nishigaki Y, Sakthisekaran D, Sethi G, Nishigaki I (2013). The vascular endothelium and human diseases. Int J Biol Sci, 9(10): 1057–1069

[129] Rask-Madsen C, King G L (2013). Vascular complications of diabetes: mechanisms of injury and protective factors. Cell Metab, 17(1): 20–33

[130] Rodgers J T, Lerin C, Haas W, Gygi S P, Spiegelman B M, Puigserver P (2005). Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature, 434(7029): 113–118

[131] Rolfe B E, Worth N F, World C J, Campbell J H, Campbell G R (2005). Rho and vascular disease. Atherosclerosis, 183(1): 1–16

[132] Rose D P, Connolly J M (1999). Antiangiogenicity of docosahexaenoic acid and its role in the suppression of breast cancer cell growth in nude mice. Int J Oncol, 15(5): 1011–1015

[133] Saint-Geniez M, Jiang A, Abend S, Liu L, Sweigard H, Connor K M, Arany Z (2013). PGC-1α regulates normal and pathological angiogenesis in the retina. Am J Pathol, 182(1): 255–265

[134] Santos J M, Mishra M, Kowluru R A (2014). Posttranslational modification of mitochondrial transcription factor A in impaired mitochondria biogenesis: implications in diabetic retinopathy and metabolic memory phenomenon. Exp Eye Res, 121: 168–177

[135] Sawada N, Jiang A, Takizawa F, Safdar A, Manika A, Tesmenitsky Y, Kang K T, Bischoff J, Kalwa H, Sartoretto J L, Kamei Y, Benjamin L E, Watada H, Ogawa Y, Higashikuni Y, Kessinger C W, Jaffer F A, Michel T, Sata M, Croce K, Tanaka R, Arany Z (2014). Endothelial PGC-1α mediates vascular dysfunction in diabetes. Cell Metab, 19(2): 246–258

[136] Schoors S, Cantelmo A R, Georgiadou M, Stapor P, Wang X, Quaegebeur A, Cauwenberghs S, Wong B W, Bifari F, Decimo I, Schoonjans L, De Bock K, Dewerchin M, Carmeliet P (2014a). Incomplete and transitory decrease of glycolysis: a new paradigm for anti-angiogenic therapy? Cell Cycle, 13(1): 16–22

[137] Schoors S, De Bock K, Cantelmo A R, Georgiadou M, Ghesquière B, Cauwenberghs S, Kuchnio A, Wong B W, Quaegebeur A, Goveia J, Bifari F, Wang X, Blanco R, Tembuyser B, Cornelissen I, Bouché A, Vinckier S, Diaz-Moralli S, Gerhardt H, Telang S, Cascante M, Chesney J, Dewerchin M, Carmeliet P (2014b). Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis. Cell Metab, 19(1): 37–48

[138] Sena C M, Matafome P, Louro T, Nunes E, Fernandes R, Seiça R M (2011). Metformin restores endothelial function in aorta of diabetic rats. Br J Pharmacol, 163(2): 424–437

[139] Sena C M, Pereira A M, Seiça R (2013). Endothelial dysfunction- a major mediator of diabetic vascular disease. Biochim Biophys Acta, 1832(12): 2216–2231

[140] Singh M, Ferrara N (2012). Modeling and predicting clinical efficacy for drugs targeting the tumor milieu. Nat Biotechnol, 30(7): 648–657

[141] Sonveaux P, Copetti T, De Saedeleer C J, Végran F, Verrax J, Kennedy K M, Moon E J, Dhup S, Danhier P, Frérart F, Gallez B, Ribeiro A, Michiels C, Dewhirst M W, Feron O (2012). Targeting the lactate transporter MCT1 in endothelial cells inhibits lactate-induced HIF-1 activation and tumor angiogenesis. PLoS ONE, 7(3): e33418

[142] Sounni N E, Cimino J, Blacher S, Primac I, Truong A, Mazzucchelli G, Paye A, Calligaris D, Debois D, De Tullio P, Mari B, De Pauw E, Noel A (2014). Blocking lipid synthesis overcomes tumor regrowth and metastasis after antiangiogenic therapy withdrawal. Cell Metab, 20(2): 280–294

[143] Stefan M, Zhang W, Concepcion E, Yi Z, Tomer Y (2014). DNA methylation profiles in type 1 diabetes twins point to strong epigenetic effects on etiology. J Autoimmun, 50: 33–37

[144] Stroes E, Kastelein J, Cosentino F, Erkelens W, Wever R, Koomans H, Lüscher T, Rabelink T (1997). Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest, 99(1): 41–46

[145] Struck A W, Thompson M L, Wong L S, Micklefield J (2012). S-adenosyl-methionine-dependent methyltransferases: highly versatile enzymes in biocatalysis, biosynthesis and other biotechnological applications. ChemBioChem, 13(18): 2642–2655

[146] Su Y, Qadri S M, Wu L, Liu L (2013). Methylglyoxal modulates endothelial nitric oxide synthase-associated functions in EA.hy926 endothelial cells. Cardiovasc Diabetol, 12(1): 134

[147] Sudhahar V, Urao N, Oshikawa J, McKinney R D, Llanos R M, Mercer J F, Ushio-Fukai M, Fukai T (2013). Copper transporter ATP7A protects against endothelial dysfunction in type 1 diabetic mice by regulating extracellular superoxide dismutase. Diabetes, 62(11): 3839–3850

[148] Sutendra G, Kinnaird A, Dromparis P, Paulin R, Stenson T H, Haromy A, Hashimoto K, Zhang N, Flaim E, Michelakis E D (2014). A nuclear pyruvate dehydrogenase complex is important for the generation of acetyl-CoA and histone acetylation. Cell, 158(1): 84–97

[149] Tabe Y, Konopleva M (2014). Advances in understanding the leukaemia microenvironment. Br J Haematol, 164(6): 767–778

[150] Takahashi N, Shibata R, Ouchi N, Sugimoto M, Murohara T, Komori K (2014). Metformin stimulates ischemia-induced revascularization through an eNOS dependent pathway in the ischemic hindlimb mice model. J Vasc Surg

[151] Takahashi T, Shibuya M (1997). The 230 kDa mature form of KDR/Flk-1 (VEGF receptor-2) activates the PLC-gamma pathway and partially induces mitotic signals in NIH3T3 fibroblasts. Oncogene, 14(17): 2079–2089

[152] Takakura N (2012). Formation and regulation of the cancer stem cell niche. Cancer Sci, 103(7): 1177–1181

[153] Takaya T, Hirata K, Yamashita T, Shinohara M, Sasaki N, Inoue N, Yada T, Goto M, Fukatsu A, Hayashi T, Alp N J, Channon K M, Yokoyama M, Kawashima S (2007). A specific role for eNOS-derived reactive oxygen species in atherosclerosis progression. Arterioscler Thromb Vasc Biol, 27(7): 1632–1637

[154] Tan B K, Adya R, Chen J, Farhatullah S, Heutling D, Mitchell D, Lehnert H, Randeva H S (2009). Metformin decreases angiogenesis via NF-kappaB and Erk1/2/Erk5 pathways by increasing the antiangiogenic thrombospondin-1. Cardiovasc Res, 83(3): 566–574

[155] Tang X, Luo Y X, Chen H Z, Liu D P (2014). Mitochondria, endothelial cell function, and vascular diseases. Front Physiol, 5: 175

[156] Tevar R, Jho D H, Babcock T, Helton W S, Espat N J (2002). Omega-3 fatty acid supplementation reduces tumor growth and vascular endothelial growth factor expression in a model of progressive non-metastasizing malignancy. JPEN J Parenter Enteral Nutr, 26(5): 285–289

[157] Tian X Y, Wong W T, Xu A, Lu Y, Zhang Y, Wang L, Cheang W S, Wang Y, Yao X, Huang Y (2012). Uncoupling protein-2 protects endothelial function in diet-induced obese mice. Circ Res, 110(9): 1211–1216

[158] Tousoulis D, Kampoli A M, Tentolouris C, Papageorgiou N, Stefanadis C (2012). The role of nitric oxide on endothelial function. Curr Vasc Pharmacol, 10(1): 4–18

[159] Tsuji M, Murota S I, Morita I (2003). Docosapentaenoic acid (22:5, n-3) suppressed tube-forming activity in endothelial cells induced by vascular endothelial growth factor. Prostaglandins Leukot Essent Fatty Acids, 68(5): 337–342

[160] Tsuzuki T, Shibata A, Kawakami Y, Nakagawa K, Miyazawa T (2007). Conjugated eicosapentaenoic acid inhibits vascular endothelial growth factor-induced angiogenesis by suppressing the migration of human umbilical vein endothelial cells. J Nutr, 137(3): 641–646

[161] Unterluggauer H, Mazurek S, Lener B, Hütter E, Eigenbrodt E, Zwerschke W, Jansen-Dürr P (2008). Premature senescence of human endothelial cells induced by inhibition of glutaminase. Biogerontology, 9(4): 247–259

[162] Valente A J, Irimpen A M, Siebenlist U, Chandrasekar B (2014). OxLDL induces endothelial dysfunction and death via TRAF3IP2: inhibition by HDL3 and AMPK activators. Free Radic Biol Med, 70: 117–128

[163] van Beijnum J R, Dings R P, van der Linden E, Zwaans B M, Ramaekers F C, Mayo K H, Griffioen A W (2006). Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature. Blood, 108(7): 2339–2348

[164] van Eupen M G, Schram M T, Colhoun H M, Hanssen N M, Niessen H W, Tarnow L, Parving H H, Rossing P, Stehouwer C D, Schalkwijk C G (2013). The methylglyoxal-derived AGE tetrahydropyrimidine is increased in plasma of individuals with type 1 diabetes mellitus and in atherosclerotic lesions and is associated with sVCAM-1. Diabetologia, 56(8): 1845–1855

[165] Végran F, Boidot R, Michiels C, Sonveaux P, Feron O (2011). Lactate influx through the endothelial cell monocarboxylate transporter MCT1 supports an NF-κB/IL-8 pathway that drives tumor angiogenesis. Cancer Res, 71(7): 2550–2560

[166] Venkatesan B, Valente A J, Das N A, Carpenter A J, Yoshida T, Delafontaine J L, Siebenlist U, Chandrasekar B (2013). CIKS (Act1 or TRAF3IP2) mediates high glucose-induced endothelial dysfunction. Cell Signal, 25(1): 359–371

[167] Venna V R, Li J, Hammond M D, Mancini N S, McCullough L D (2014). Chronic metformin treatment improves post-stroke angiogenesis and recovery after experimental stroke. Eur J Neurosci, 39(12): 2129–2138

[168] Vizán P, Sánchez-Tena S, Alcarraz-Vizán G, Soler M, Messeguer R, Pujol M D, Lee W N, Cascante M (2009). Characterization of the metabolic changes underlying growth factor angiogenic activation: identification of new potential therapeutic targets. Carcinogenesis, 30(6): 946–952

[169] Wang W, Zhu J, Lyu F, Panigrahy D, Ferrara K W, Hammock B, Zhang G (2014). ω-3 polyunsaturated fatty acids-derived lipid metabolites on angiogenesis, inflammation and cancer. Prostaglandins Other Lipid Mediat, 113-115: 13–20

[170] Warren C M, Ziyad S, Briot A, Der A, Iruela-Arispe M L (2014). A ligand-independent VEGFR2 signaling pathway limits angiogenic responses in diabetes. Sci Signal, 7(307): ra1

[171] Wautier J L, Schmidt A M (2004). Protein glycation: a firm link to endothelial cell dysfunction. Circ Res, 95(3): 233–238

[172] Wei X, Schneider J G, Shenouda S M, Lee A, Towler D A, Chakravarthy M V, Vita J A, Semenkovich C F (2011). De novo lipogenesis maintains vascular homeostasis through endothelial nitric-oxide synthase (eNOS) palmitoylation. J Biol Chem, 286(4): 2933–2945

[173] Wellen K E, Hatzivassiliou G, Sachdeva U M, Bui T V, Cross J R, Thompson C B (2009). ATP-citrate lyase links cellular metabolism to histone acetylation. Science, 324(5930): 1076–1080

[174] Wilkinson M J, Laffin L J, Davidson M H (2014). Overcoming toxicity and side-effects of lipid-lowering therapies. Best Pract Res Clin Endocrinol Metab, 28(3): 439–452

[175] Wu G, Haynes T E, Li H, Meininger C J (2000). Glutamine metabolism in endothelial cells: ornithine synthesis from glutamine via pyrroline-5-carboxylate synthase. Comp Biochem Physiol A Mol Integr Physiol, 126(1): 115–123

[176] Wu G, Meininger C J (1995). Impaired arginine metabolism and NO synthesis in coronary endothelial cells of the spontaneously diabetic BB rat. Am J Physiol, 269(4 Pt 2): H1312–H1318

[177] Xu Y, An X, Guo X, Habtetsion T G, Wang Y, Xu X, Kandala S, Li Q, Li H, Zhang C, Caldwell R B, Fulton D J, Su Y, Hoda M N, Zhou G, Wu C, Huo Y (2014). Endothelial PFKFB3 plays a critical role in angiogenesis. Arterioscler Thromb Vasc Biol, 34(6): 1231–1239

[178] Yanai R, Mulki L, Hasegawa E, Takeuchi K, Sweigard H, Suzuki J, Gaissert P, Vavvas D G, Sonoda K H, Rothe M, Schunck W H, Miller J W, Connor K M (2014). Cytochrome P450-generated metabolites derived from ω-3 fatty acids attenuate neovascularization. Proc Natl Acad Sci USA, 111(26): 9603–9608

[179] Yang B T, Dayeh T A, Volkov P A, Kirkpatrick C L, Malmgren S, Jing X, Renström E, Wollheim C B, Nitert M D, Ling C (2012). Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes. Mol Endocrinol, 26(7): 1203–1212

[180] Yang S P, Morita I, Murota S I (1998). Eicosapentaenoic acid attenuates vascular endothelial growth factor-induced proliferation via inhibiting Flk-1 receptor expression in bovine carotid artery endothelial cells. J Cell Physiol, 176(2): 342–349

[181] Yeh W L, Lin C J, Fu W M (2008). Enhancement of glucose transporter expression of brain endothelial cells by vascular endothelial growth factor derived from glioma exposed to hypoxia. Mol Pharmacol, 73(1): 170–177

[182] Zecchin A, Pattarini L, Gutierrez M I, Mano M, Mai A, Valente S, Myers M P, Pantano S, Giacca M (2014). Reversible acetylation regulates vascular endothelial growth factor receptor-2 activity. J Mol Cell Biol, 6(2): 116–127

[183] Zhang D, Li J, Wang F, Hu J, Wang S, Sun Y (2014). 2-Deoxy-D-glucose targeting of glucose metabolism in cancer cells as a potential therapy. Cancer Lett, 355(2): 176–183

[184] Zhang G, Panigrahy D, Mahakian L M, Yang J, Liu J Y, Stephen Lee K S, Wettersten H I, Ulu A, Hu X, Tam S, Hwang S H, Ingham E S, Kieran M W, Weiss R H, Ferrara K W, Hammock B D (2013). Epoxy metabolites of docosahexaenoic acid (DHA) inhibit angiogenesis, tumor growth, and metastasis. Proc Natl Acad Sci USA, 110(16): 6530–6535

[185] Zhang Z, Apse K, Pang J, Stanton R C (2000). High glucose inhibits glucose-6-phosphate dehydrogenase via cAMP in aortic endothelial cells. J Biol Chem, 275(51): 40042–40047

[186] Zou M H, Shi C, Cohen R A (2002). Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest, 109(6): 817–826

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg