* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download 1 - Cardiovascular Research
Survey
Document related concepts
Signal transduction wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Lipid signaling wikipedia , lookup
Paracrine signalling wikipedia , lookup
Biochemical cascade wikipedia , lookup
Basal metabolic rate wikipedia , lookup
Fatty acid metabolism wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Proteases in angiogenesis wikipedia , lookup
Transcript
REVIEW Cardiovascular Research (2016) 111, 172–183 doi:10.1093/cvr/cvw159 Endothelial cell–cardiomyocyte crosstalk in diabetic cardiomyopathy Andrea Wan and Brian Rodrigues* Faculty of Pharmaceutical Sciences, The University of British Columbia, 2405 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 Received 22 January 2016; revised 28 April 2016; accepted 21 May 2016; online publish-ahead-of-print 10 June 2016 Abstract The incidence of diabetes is increasing globally, with cardiovascular disease accounting for a substantial number of diabetes-related deaths. Although atherosclerotic vascular disease is a primary reason for this cardiovascular dysfunction, heart failure in patients with diabetes might also be an outcome of an intrinsic heart muscle malfunction, labelled diabetic cardiomyopathy. Changes in cardiomyocyte metabolism, which encompasses a shift to exclusive fatty acid utilization, are considered a leading stimulus for this cardiomyopathy. In addition to cardiomyocytes, endothelial cells (ECs) make up a significant proportion of the heart, with the majority of ATP generation in these cells provided by glucose. In this review, we will discuss the metabolic machinery that drives energy metabolism in the cardiomyocyte and EC, its breakdown following diabetes, and the research direction necessary to assist in devising novel therapeutic strategies to prevent or delay diabetic heart disease. ----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords Endothelial cell metabolism † Cardiomyocyte metabolism † Heparanase † Lipoprotein lipase † Vascular endothelial growth factor 1. Introduction The incidence of diabetes has reached pandemic proportions, with 347 million people affected globally. The prediction from the World Health Organization is that by 2030, diabetes will be the seventh leading cause of death.1 To manage this chronic disease, patients with diabetes require substantial resources and currently use up a prodigious 15% of national healthcare budgets.1 Treatment of this condition requires appropriate glucose and HbA1c monitoring and subsequent medication management. However, this practice does not exquisitely match the physiological control of glucose homeostasis. As a result, people with diabetes are prone to developing long-term complications including retinopathy, neuropathy, nephropathy, and most importantly cardiovascular disease, which accounts for 50– 80% of diabetes-related deaths.1,2 It should be noted that several large trials assessing HbA1c lowering in patients with diabetes have not confirmed a lower risk of macrovascular events.3 – 5 This implies that a deficiency in carbohydrate metabolism may not be exclusively responsible for the macrovascular events observed following diabetes and that other metabolic disorders may also be contributing factors. Although atherosclerotic vascular disease is a primary reason for this cardiovascular dysfunction, heart failure in patients with diabetes might also be an outcome of an intrinsic heart muscle malfunction (labelled diabetic cardiomyopathy).6 – 8 The aetiology of cardiomyopathy is complex, with a change in cardiomyocyte metabolism being considered a leading stimulus for this dysfunction.9 – 11 However, it should be emphasized that, in addition to cardiomyocytes, endothelial cell (EC) metabolism is also altered following diabetes.12 Furthermore, because the two cell types are interdependent for normal functioning, changes in their respective metabolism will synergistically induce cardiac failure. 2. Diabetic cardiomyopathy Heart disease is a leading reason for death in patients with diabetes, with coronary vessel disease and atherosclerosis being primary causes for the increased incidence of cardiovascular dysfunction.13 In this patient population, the comorbidities of hypertension, abnormal cholesterol, and hypertriglyceridaemia significantly contribute to a more severe cardiovascular pathology and a greater mortality risk.14 However, Type 1 (T1D) and Type 2 (T2D) patients have also been diagnosed with reduced or low-normal diastolic function, left ventricular hypertrophy, and clinically overt congestive heart failure independent of vascular defects and hypertension.15 These observations suggest a specific impairment of heart muscle (termed diabetic cardiomyopathy). Evidence of cardiomyopathy has also been reported in rodent models of T1D16,17 and T2D,18,19 which are comparable with that seen in human diabetic patients. Given that rodents are resistant to atherosclerosis, these models provided strong evidence for the occurrence of diabetic cardiomyopathy. Cardiomyopathy is a complicated disorder, and several factors have been associated with its development. These * Corresponding author. Tel: +(604) 822 4758; fax: +(604) 822 3035, E-mail: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2016. For permissions please email: [email protected]. 173 Cellular crosstalk include (i) an increased stiffness of the left ventricular wall (associated with an accumulation of connective tissue and insoluble collagen),20 (ii) impaired sensitivity to various ligands (e.g. b-agonists and insulin),21,22 (iii) depressed autonomic function (accompanied by alterations in myocardial catecholamine concentrations),23 (iv) compromised endothelium function (related to reduced nitric oxide activity and augmented synthesis of vasoconstrictors),24 (v) abnormalities of various proteins that regulate ion flux (specifically intracellular calcium),25 and (vi) alterations in coronary microcirculation (as a consequence of both structural and functional modifications). 24 The view that diabetic cardiomyopathy could occur as a consequence of early alterations in cardiac metabolism has also been put forward. 3. Conventional cardiomyocyte metabolism With uninterrupted contraction being a unique feature of the heart, the cardiomyocyte has the highest demand for energy. This cell demonstrates substrate promiscuity, enabling it to utilize multiple sources for energy, including fatty acids (FAs), carbohydrates, amino acids, lactate, and ketones.26 Among these, 95% of the energy generated is derived from carbohydrates and FA through mitochondrial metabolism. 3.1 Glucose In a basal setting, glucose and lactate supply 30% of ATP generation, whereas 70% of ATP generation is through FA oxidation (FAO).26 Glucose metabolism requires uptake, glycolysis, and mitochondrial oxidation to yield ATP (Figure 1). Cardiac glucose uptake is dependent on the plasma membrane content of glucose transporters (GLUT1 and GLUT4).27 GLUT1 has predominant plasma membrane localization and accounts for basal glucose uptake, whereas GLUT4 is the dominant transporter in the adult heart. In non-stimulated conditions, a majority of GLUT4 is located in an intracellular pool. However, on stimulation by insulin or contraction-associated activation of AMP-activated protein kinase (AMPK), GLUT4 is redistributed to the plasma membrane to mediate glucose uptake.17,28 The delivered glucose undergoes glycolysis (catalysed by phosphofructokinase-1, PFK-1) to form pyruvate, which then enters the mitochondria for oxidation, a process controlled by the activity of pyruvate dehydrogenase (PDH).8 It should be noted that functions of glucose extend beyond its primary use for energy generation and glycogen storage in the cardiomyocyte. Glucose can also be used for ribose formation or enter into the hexosamine biosynthetic pathway.29 3.2 Fatty acids The cardiomyocyte has a limited capacity to synthesize FA and thus relies on an exogenous and endogenous supply (Figure 1).8 Exogenous FA delivery to the heart involves (i) release from adipose tissue and transport to the heart after complexing with albumin, (ii) lipolysis of circulating triglyceride (TG)-rich lipoproteins (VLDL, chylomicrons) to FA by lipoprotein lipase (LPL) positioned at the EC surface of the coronary lumen,5 and (iii) uptake by sarcolemmal FA transporters including FA translocase (FAT/CD36), FA binding protein (FABPPM), and FA transporter protein (FATP).30 Endogenous FA provision is through the Figure 1 Cardiomyocyte metabolism. In the cardiomyocyte, two of the major substrates that are used for energy generation include glucose and FAs. Glucose uptake into the cardiomyocyte is predominantly a GLUT4 event. Following its entry into the cell, glucose can either be stored as glycogen or undergo glycolytic and oxidative metabolism to generate ATP. FA is the preferred energy substrate of the cardiomyocyte. Its entry is through a number of FA transporters including CD36, FAPBPM, and FATP. FA can also undergo storage in the form of TGs or enter into the mitochondria to be oxidized for ATP. 174 breakdown of endogenous cardiac TG stores. Two essential enzymes involved in TG hydrolysis include adipose TG lipase (ATGL) and hormone-sensitive lipase (HSL).31 Delivered FA can enter the biosynthetic or oxidative pathways.32 In the latter, FAs are oxidized by conversion to fatty acyl-CoA, which is transported into the mitochondria through carnitine palmitoyltransferase (CPT1/ CPT2).33,34 Inside the mitochondria, fatty acyl-CoA undergoes b-oxidation to generate acetyl-CoA, which is oxidized in the tricarboxylic acid cycle to yield ATP. When activated, AMPK, peroxisome proliferator-activated receptor-a (PPAR a ), and malonyl-CoA decarboxylase (MCD), all have substantial roles in modulating FA delivery and oxidation.35 – 37 4. Alterations in cardiomyocyte metabolism following diabetes We and others propose that interruptions in glucose and FA metabolism in the heart are the geneses of diabetic cardiomyopathy.8,26,38,39 Following diabetes, myocardial GLUT4 gene and protein expression are reduced.8,27 However, hyperglycaemia sustains glucose uptake by the diabetic heart, such that glucose influx into the cardiomyocyte remains comparable with control.39 In addition to hyperglycaemia, multiple adaptive mechanisms, at the level of the whole body and intrinsic to the heart, also operate to augment FA supply to the cardiomyocyte. These include adipose tissue lipolysis and plasma lipoprotein-TG,40 vascular LPL activity,41 – 48 and myocyte sarcolemmal FA transporters (e.g. CD36 and FABPPM),9 all of which increase following diabetes. The above changes in cardiac FA are under the supervision of AMPK (early) and PPARs (late). For example, following acute diabetes, rapid activation of AMPK is an adaptation that would ensure adequate cardiac energy production. It does so by increasing FA delivery through its activation of LPL45 in addition to repositioning CD36 to the sarcolemma, an FA transporter that is significantly increased in hearts from STZ-diabetic animals.49 AMPK also participates in the utilization of FA by phosphorylating and inhibiting acetyl-CoA carboxylase, relieving its inhibition on CPT1, hence promoting FAO.8 However, in chronic diabetes, with the addition of augmented plasma and heart lipids, AMPK activation is prevented, and control of FAO is through PPARa.35 Activation of cardiac PPARa has been reported in models of chronic diabetes (STZ-induced diabetic rats,50 ZDF rats,51 and db/db mice50), promoting the expression of genes involved in various steps of FAO. Forkhead box family and subfamily O of transcription factors (FoxOs) are also known to regulate whole-body energy metabolism due to their effect in various tissues such as the liver, adipose tissue, skeletal muscle, and beta cells.52 However, their value in moderating cardiac substrate utilization, especially in conditions like insulin resistance/diabetes, is unknown. On the basis of our results, we believe that unlike AMPK and PPARa, which are time-dependent (early and late phases of diabetes) regulators of cardiac metabolism, FoxO is a key arbitrator of cardiac metabolism, whose activity is enhanced the instant there is a drop in systemic insulin levels.53 We have linked an increase in cardiomyocyte FoxO1 to augmented CD36 in the plasma membrane through actin cytoskeleton rearrangement.54 Collectively, all of the above mechanisms operate to increase FA supply and oxidation. In doing so, augmented FA utilization can significantly reduce cardiac glucose oxidation, and to a lesser extent glycolysis and glucose uptake,29 by increasing (i) citrate (a recognized inhibitor of PFK-1, the rate-limiting enzyme in glycolysis), (ii) pyruvate dehydrogenase kinase 4 (PDK4) expression A. Wan and B. Rodrigues (known to phosphorylate and inhibit PDH), and (iii) acetyl-CoA (which inhibits PDH, either allosterically or through activation of PDK4).8,55 Consequently, intracellular concentrations of glucose and its metabolites accumulate (glucotoxicity) to potentiate O-linked protein glycosylation and interfere with protein functionality.29 When glucose utilization is compromised and surplus FAs are provided to the cardiomyocyte irrespective of its requirements, the damage to the cardiac muscle is severe. In gauging the impact of these excess FAs, two key points to consider are that mitochondrial metabolism of FA requires more oxygen than glucose, and diabetic cardiomyopathy is characterized by decreased capillary density and impaired myocardial perfusion.56 – 58 Additionally, chronic diabetes is associated with a significant decrease in PPARa expression and its associated genes.29 This sponsors a setting where oxygen delivery and augmented FA uptake cannot match its utilization, leading to their storage as TG in cardiomyocytes. Chronically, the reconversion of TG to FA and potentially toxic metabolites, including ceramides, diacylglycerols, and acylcarnitines can provoke cardiomyocyte death (lipotoxicity).9 Interestingly, increasing FA uptake through the overexpression of cardiac human LPL or FA transport protein results in a cardiac phenotype resembling diabetic cardiomyopathy.59,60 Conversely, normalizing cardiac metabolism in diabetic animals reverses the development of cardiomyopathy.8 It should be noted that FAs also play a significant role in promoting cellular insulin resistance.61,62 In addition to the cardiomyocyte, the heart also requires the ECs to perform a myriad of roles, pre-eminently increasing angiogenesis and capillary density, and the secretion of regulatory proteins, obligations that are crucial for sustaining cardiomyocyte function. As angiogenesis63 and the secretion of regulatory proteins64 are largely dependent on glycolytic energy, it becomes pivotal to understand EC metabolism and its projected contribution to diabetic cardiomyopathy, information that is surprisingly sparse and incomplete. 5. Conventional endothelial cell metabolism Prior to defining their metabolism, it is important to appreciate that although ECs from capillaries, large arteries and veins share some common qualities, these cells are exceptionally heterogeneous.65,66 Thus, based on the vessel type (arterial compared with venous and macrocompared with microvascular ECs), anatomic location, and environment, ECs behave differently in their responses to growth and migration stimuli67 and also exhibit characteristic clusters of genes.68 The other caveat is that because the isolation of primary ECs and their long-term maintenance in culture have often proved difficult, EC metabolism is commonly studied in vitro, using EC lines. Hence, the data obtained from one EC may not be universally applicable, nor might it be completely relevant to the intact endothelium in the heart. Unlike the cardiomyocyte, FA generates only 5% of the total amount of ATP in ECs, with the majority being provided by glucose.63 Consequently, glycolytic flux is many folds higher than glucose or FAO, providing the ECs with about 85% of its ATP.63 Interestingly, a recent report suggested that ECs oxidize FAs primarily to feed de novo nucleotide synthesis for DNA replication and EC proliferation (Figure 2).69 In the majority of cells that utilize glucose, the initial step, glycolysis, is followed by oxidation in the mitochondria, the latter being the dominant pathway for ATP generation (Figure 2).70 However, ATP supply in ECs is relatively independent of the mitochondrial oxidative pathway. 175 Cellular crosstalk Figure 2 EC metabolism. In the EC, glucose is the primary substrate used for energy generation. Upon entry using the GLUT1 transporter, glucose undergoes glycolysis—the pre-eminent metabolic pathway—to generate ATP. Under proliferative conditions, the EC shunts pyruvate, a metabolic intermediate of glycolysis, into the mitochondria to generate dNTP that is used for cell growth. Interesting, FAs are not typically used for energy generation in the EC, and instead, contribute to dNTP synthesis. Remarkably, at physiological concentrations, 99% of glucose is metabolized in the glycolytic pathway, with only 1% going into the Krebs cycle in the mitochondria.71 The dependence of ECs on glycolysis has multiple advantages.72 – 74 First, glycolytic enzymes are exquisitely positioned in the cytosol, in close proximity to the actin cytoskeleton. This organization provides an immediate supply of ATP for actin rearrangement to facilitate angiogenesis and vesicular secretion. Second, by utilizing the glycolytic pathway to generate ATP, ECs spare oxygen and FA to fuel the underlying cardiomyocytes. Myocytes have a high demand for oxygen, given that their major substrate for energy production is through the oxidative phosphorylation of FA. Thus, despite being in immediate contact with oxygen in the blood stream, ECs still primarily utilize glycolysis to generate energy. Third, even though the mitochondrial oxidative pathway produces greater amounts of ATP per mole of glucose, the faster rate of glycolysis can match this ATP production. Fourth, ECs are reliant on the glycolytic pathway to generate not only energy but also the required intermediates necessary for cell growth, migration, and angiogenesis. Finally, an additional benefit of such an adaptation is that the EC is protected from mitochondrial electron leakage and generation of chemically reactive molecules [e.g. reactive oxygen species (ROS)] that would otherwise cause EC damage.72 – 74 For glycolysis to occur, glucose enters into the cell with the help of transporters. Of the many different glucose transporters present in ECs, GLUT1 is the major isoform.75,76 This plasma membrane uniporter facilitates not only glucose influx into the ECs, from its luminal side, but also its efflux across the abluminal membrane border.75,77 Intriguingly, there is some evidence suggesting that the GLUT1 distribution is disproportionately skewed towards an abluminal locale.75 However, this information is not always reliable, nor applicable, across different ECs (primary ECs vs. cell lines, microvascular vs. macrovascular ECs, and ECs from diverse tissues). If verified in microvascular ECs, this adaptation will permit faster glucose extrusion than uptake. This will allow the ECs to move glucose to the cardiomyocyte to power the high metabolic requirements of this subjacent cell. Once, inside the ECs, glucose is metabolized by key glycolytic enzymes (e.g. hexokinase II, PFKFB3) to pyruvate, of which ,1% is metabolized by the TCA cycle, whereas the majority is converted to lactate.71 As a result, oxidative pathways account for a minimal amount of ATP generation within the ECs.51 6. Aberrant endothelial cell metabolism in diabetes In ECs, the entry point for glycolysis is glucose uptake by glucose transporters, the predominant one being GLUT1.75,76 Traditionally, GLUT1 was thought of as an insulin-independent transporter, and in response to diabetes, ECs are glucose-blind, with GLUT1 expression remaining unresponsive to hyperglycaemia.78,79 However, this idea has recently been questioned as ECs would conceivably attempt to protect themselves against the damaging effects of excessive glucose influx by reducing GLUT1 expression. One caveat is that, although GLUT1 reduction at the luminal side may be a favourable response, its depletion at the abluminal side will cause inadequate glucose expulsion to the cardiomyocyte. By extension, this will result in an unpredictably high concentration of intracellular glucose, leading to ROS generation and glycolytic inhibition. More recent data suggest that ECs exposed to high glucose (HG) have reduced GLUT1 expression and glucose uptake.80,81 This evidence, together with the reported reduction in enzymatic activity within the glycolytic pathway (e.g. phosphofructokinase), could explain the acknowledged decrease in glycolytic flux during diabetes. 73,82 Stalled glycolytic flux means that glycolytic intermediates accumulate and are shuttled into different metabolic pathways. These include the polyol pathway with the formation of sorbitol and fructose, hexosamine biosynthesis pathway that impedes angiogenesis, methylglyoxal 176 pathway, protein kinase C activation, and defects in mitochondrial biogenesis and fragmentation.73 The net result is excess ROS and reactive nitrogen species production, and advanced glycation end product (AGE) synthesis, mediators of ECs and potentially cardiomyocyte dysfunction.73,83 Mechanisms to explain the altered GLUT1 under conditions of hyperglycaemia include the thioredoxin-interacting protein (TXNIP) system.84 TXNIP, by directly binding to GLUT1, induces its endocytosis and its subsequent breakdown in lysosomes (an acute effect), in addition to reducing the level of GLUT1 mRNA (a chronic outcome).84 TXNIP levels are suppressed in many tumours,85 as cancer cells require high GLUT1 expression to sustain elevated glycolysis.86 Interestingly, TXNIP is an exquisitely glucose-sensitive gene and is induced in response to low insulin or HG.87 Whether diabetes is associated with increased TXNIP expression in ECs derived from hearts of diabetic animals is yet to be determined. An alternate pathway is one where TXNIP can reversibly bind to thioredoxin-1 (TRX1), an interaction that is weakened by ROS, and allows TXNIP to dissociate from oxidized TRX1 to down-regulate GLUT1.88 It is conceivable that with HG and ROS generation, not only is the expression of TXNIP increased but also its dissociation from TRX1, permitting its increased availability for GLUT1 interaction. Finally, a recent study has suggested that cardiomyocyte-derived exosomes, by sending glucose transporters and the associated glycolytic enzymes to the ECs, can modulate endothelial glucose transport and metabolism.89 Whether this process is repressed during hyperglycaemia would be of particular interest as an additional mechanism to explain altered glucose uptake and metabolism in the EC during diabetes. In other cell types like the cardiomyocyte, limited glucose utilization provokes the overconsumption of FA to ensure adequate energy production.8 For this to happen, FA requires uptake and oxidation, processes that are augmented in the cardiomyocyte following diabetes,8 but have yet to be unequivocally substantiated in EC. In culture, ECs have been demonstrated to have reserve oxidative capacity and can increase oxidation under conditions of high metabolic demand or stress.71 Under conditions of glycolytic inhibition following diabetes, there is an expectation that the increased provision of FA would lead to an augmented oxidation of this substrate. However, the verification that FAO increases in the ECs will prompt other more complex questions. For example, is the increase in FAO geared towards ATP generation or nucleotide synthesis? Remarkably, in ECs, acetyl-CoA from FAO is used for DNA synthesis and cell multiplication.69 If excess FA is used for energy production, will its associated undesirable effects in ECs be even more deleterious than that seen in cardiomyocytes? ROS generated by FAO will impede glucose transport and glycolytic enzymes (e.g. GAPDH) and may further reduce glycolysis under HG conditions.12,90,91 To what magnitude are ECs teleologically equipped to handle excess FAO? To explain, the organelle responsible for FAO, mitochondria, make up only 2 – 6% of the ECs compared with their volume in hepatocytes (28%) or cardiomyocytes (32%).92 Thus, excess FA entering the ECs may be associated with a progressively smaller, incremental effect on oxidation—a ceiling effect, such that this substrate will either migrate through these cells or be stored as TG. The latter outcome is especially problematic, given the negative consequences of stored TG in cells other than adipocytes, in addition to the detrimental consequences of excessive FA utilization.10,93 Changes in metabolism and function of EC can participate not only in its own demise but also in cardiomyocyte dysfunction and cell death. A. Wan and B. Rodrigues 7. Endothelial cell: cardiomyocyte crosstalk 7.1 Endothelial cell control of cardiomyocyte metabolism Heparan sulfate proteoglycans (HSPGs) consist of a core protein to which several linear heparan sulfate (HS) side chains are covalently linked and function not only as structural proteins but also as temporary docking sites due to the high content of charged groups in HS.94,95 The latter property is implicitly used to electrostatically bind a number of different proteins including LPL and vascular endothelial growth factor (VEGF).96 Attachment of these bioactive proteins is a clever arrangement, providing the cell with a rapidly accessible auxiliary reservoir, precluding the need for de novo synthesis when the requirement for a particular protein is urgent. The action of EC heparanase on cardiomyocyte HSPG47,64,97 releases LPL48 and VEGF,98 the major players in increasing FA delivery and utilization by the cardiomyocyte to overcome its lack of glucose utilization. ECs have also been implicated in providing functional support to subjacent cardiomyocytes and do so by communicating via soluble paracrine factors. For example, in response to environmental cues like hyperglycaemia, the strategically located ECs act as ‘first responders’99,100 to this cellular disturbance and react by secreting heparanase.64,101 Heparanase is synthesized as an inactive, latent (L-Hep) 65 kDa enzyme that undergoes cellular secretion followed by HSPG-facilitated reuptake.102,103 After undergoing proteolytic cleavage in lysosomes, a 50 kDa polypeptide is formed that is 100-fold more active (A-Hep) than L-Hep.104,105 Within the acidic compartment of lysosomes, A-Hep is stored until mobilized (Figure 3). In the presence of HG, we reported redistribution of lysosomal heparanase from a perinuclear location towards the plasma membrane of ECs, together with elevated secretion into the medium.47 We also determined that ATP release, purinergic receptor activation, cortical actin disassembly, and stress actin formation were essential for HG-induced A-Hep secretion.64 With respect to LPL in the heart, it was suggested that coronary ECs do not synthesize LPL despite its critical function at the vascular lumen.106 However, more recent observations have reported LPL mRNA expression in pure heart EC to amounts that were 25% of those present in pure cardiomyocytes.107 Nevertheless, in the heart, the majority of this enzyme is produced in cardiomyocytes—cardiac tissue that has the highest expression of this enzyme. Following its maturation by lipase maturation factor 1 (LMF1), 108 LPL is subsequently secreted onto HSPG (syndecan 1) binding sites on the myocyte cell surface, where the enzyme is momentarily located.106,108 – 110 From here, LPL is transported across the interstitial space to the luminal surface of ECs where it is bound to HSPG and glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) through an ionic linkage; LPL has an abundant amount of positively charged domains.108 At the lumen, LPL actively metabolizes the TG core of lipoproteins to FA, which are then transported into the heart for numerous metabolic and structural functions (Figure 4). Following diabetes and activation of posttranslational mechanisms,111 the heart rapidly increases LPL at the vascular lumen by transferring this enzyme from the underlying cardiomyocyte.9 We implicated EC heparanase in this process, through a mechanism by which this enzyme detaches LPL from the myocyte surface, for onward movement to the vascular lumen.47,64 The end result Cellular crosstalk 177 Figure 3 Cardiomyocyte– EC crosstalk. In the EC, latent heparanase is first secreted, followed by re-uptake and conversion to active heparanase. The secretion of both latent and active heparanase is dependent on glycolytically produced ATP. Both forms of heparanase are able to liberate myocyte surface-bound proteins including VEGFA and VEGFB. These growth factors have a paracrine influence on the EC, to facilitate angiogenesis through endothelial migration and proliferation. HSPG: heparan sulfate proteoglycan. Figure 4 Synthesis and transport of LPL. Following gene transcription, LPL mRNA is translated to inactive LPL proteins. The inactive monomer undergoes glycosylation, and several other post-translational processes to be dimerized in the endoplasmic reticulum (ER). The fully processed LPL is sorted into vesicles that are targeted towards the cell surface for secretion. This process occurs by the movement along the actin cytoskeleton. Subsequently, it docks with the cell surface and releases LPL onto HSPG-binding sites on the plasma membrane. At the basolateral side of the EC, glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) captures LPL in the interstitial space and transfers it across to the apical side of the EC. GPIHBP1 functions as a platform to enable LPL to hydrolyse lipoprotein-TG and release FAs. More recent data suggest that EC can also synthesize LPL, albeit at a limited capacity. 178 is the increased provision of FA to the diabetic cardiomyocyte through FA transporters like CD36.108 In addition to LPL, VEGF is another myocyte HSPG-bound protein that is released through heparanase action. Of the different VEGF isoforms, VEGFA and VEGFB are notable standouts, abundantly expressed in the cardiomyocyte.112,113 From a single human VEGFA gene, alternative splicing generates a number of isoforms. The predominantly expressed VEGFA165 is the main effector of VEGFA action and is tethered (50– 70%) to the cardiomyocyte extracellular matrix.114 With respect to VEGFB, VEGFB167 constitutes more than 80% of the total VEGFB transcript and is also bound to HSPG. Upon secretion, each has variable affinity for HSPG-binding sites on the myocyte cell surface, an interaction made possible by VEGF heparin-binding domains (amino acids rich in basic residues).96,115 Not only does this liaison protect VEGF against degradation, but it also allows the myocyte matrix to retain a pool of readily releasable growth factors (Figure 3). Following its release by heparanase, VEGFA is competent to provide autocrine signalling. To test the existence of an autocrine pathway for VEGF control of metabolism, we focused on AMPK, a pivotal cellular energy sensor and regulator.98 We reported that recombinant VEGF has a capacity to induce AMPK activation in cardiomyocytes, likely through activation of a calcium-/calmodulin-dependent protein kinase kinase b.98 Interestingly, AMPK activation governs LPL recruitment to the myocyte surface for forward movement to vascular lumen. The mechanisms underlying LPL recruitment embraces p38 mitogenactivated protein kinase activation with subsequent phosphorylation of the heat-shock protein (Hsp) 25. Actin monomers are released from phosphorylated Hsp25 and self-associate to form fibrillar actin. Vesicles containing LPL then move along the actin filament network to bind to HSPG on the cardiomyocyte plasma membrane.98 Strikingly, we observed that VEGF was able to increase LPL translocation to the myocyte cell surface. Our data on the ability of VEGFA to promote LPL movement implicate this growth factor in the cascade of expanding actions that are geared to help the acutely diabetic heart switch its substrate selection to predominately FA. Whether this mechanism persists following chronic diabetes is currently unknown. Animals with longterm STZ-induced diabetes are characterized by reduced cardiac expression of VEGFA and its receptors.56,57 Additionally, other pathways like increased mobilization of fat from adipose tissue mediated by HSL116 and ATGL117 could surpass the efforts of LPL, after chronic diabetes. Following its release from the cardiomyocyte, VEGFB may also have an autocrine signalling action, but related to a mechanism that prevents heart failure rather than modifying cell metabolism.118 VEGFB has been shown to promote cell survival and produce physiological cardiac hypertrophy.119 Indeed, multiple models of heart failure have indicated a significant drop in VEGFB,120 which may be due to decreased VEGFB protein or possibly due to impaired release of VEGFB from HSPG. Whether there is a role for decreased VEGFB in diabetic cardiomyopathy is currently unknown. Overall, under conditions of hyperglycaemia and the associated release of heparanase from the EC, myocyte cellsurface LPL is liberated for onward movement to the vascular lumen to promote FA delivery to the cardiomyocyte. Heparanase-releasable VEGFA aids in the replenishment of HSPG-bound LPL to facilitate the metabolic switching of the cardiomyocyte to FA. As this excessive use of FA could be detrimental to the heart, the VEGFB released ensures cardiomyocyte survival. In addition to the above-mentioned regulatory mechanisms, it should be recalled that EC secretes nitric oxide (NO), which regulates A. Wan and B. Rodrigues vascular tone by relaxing vascular smooth muscle.121 However, NO can also influence the contractile function of cardiomyocytes through beta-adrenergic and muscarinic control.122 Intriguingly, NO has been reported to control cardiac substrate utilization.123,124 Another signalling communicator between EC and cardiomyocytes is neuregulin-1, a growth factor released from EC that has several cardioprotective functions.125,126 In isolated cardiac myocytes, neuregulin-1 has been linked to glucose uptake.127 Finally, given the metabolic flexibility of the cardiomyocyte, and its ability to use a number of substrates for ATP production (high-energy phosphate storage within the cardiomyocyte is minimal),128 it is possible that the products of EC glycolysis, like pyruvate and lactate, can be used by the cardiomyocyte when they become available. In cancer, tumour cells that are highly glycolytic secrete high amounts of lactate, which can be taken up by neighbouring cells and channelled into the TCA cycle.74 Whether this situation is possible in the heart, with cardiomyocytes taking up lactate produced by the EC, has yet to be identified. 7.2 Cardiomyocyte control of endothelial cell metabolism Following its release from the cardiomyocyte in response to heparanase, LPL traverses the interstitial space and binds to its transporter GPIHBP1, a glycoprotein expressed exclusively on capillary EC. GPIHBP1 facilitates LPL relocation from the basolateral to the apical (luminal) side of the EC.129 Out here, it can also act as a platform, binding lipoproteins.130 This allows LPL to actively metabolize the lipoprotein-TG core, thereby liberating FAs that are transported to the cardiomyocytes. We described a novel mechanism in which heparanase-induced VEGFA released from the cardiomyocyte, in a paracrine manner, activated Notch signalling in the EC.131 This resulted in enhanced GPIHBP1 expression, promoted LPL translocation across the EC, and can regulate FA delivery to the cardiomyocytes.131 It is unclear whether cardiomyocyte-VEGFA also assists in FA transport across the EC. VEGFA is known to promote FA-binding protein 4 expression,132 an FA-transporting protein abundantly expressed in microvascular EC in the heart. Similarly, VEGFB released from the cardiomyocyte could also influence EC lipid transport by controlling the expression of vascular EC FATPs, and Vegfb2/2 mice have less uptake and accretion of lipids in the heart.133 Consistent with these observations in mice, VEGFB transgenic rat hearts exhibit increased Fatp4 RNA concentrations, whereas VEGFB knockout rat hearts display reduced amounts of Fatp4 RNA. However, as there was no change in FA uptake between TG, knock-out, and wild-type hearts, the authors concluded that in rats, VEGFB is dispensable for normal cardiac function under unstressed conditions and for high fat diet-induced metabolic changes.119 As FAs traverse the ECs, there is a likelihood that this cell deviates to use this substrate as an alternate energy source during diabetes. However, it is not in the nature of the ECs to increase their consumption of FAs. As such, the ECs can sustain substantial damage when exposed to FAs.134 – 136 As described above, whether there is a role for cardiomyocyte VEGFB in protecting EC against excessive FA use is currently unknown. VEGFB has been shown to decrease gene expression of proteins involved in FAO.119 Increased FA delivery and decreased glucose oxidation in the cardiomyocyte increase FAO. To match the significant increase in FAO, an abundant supply of oxygen to cardiomyocyte is required to efficiently consume these FAs through oxidative phosphorylation. Cardiomyocytes respond by promoting 179 Cellular crosstalk angiogenesis and vasculogenesis to enhance oxygen supply to the myocardium and do so by secreting the necessary paracrine signals that include cardiomyocyte surface-bound VEGFA and VEGFB. VEGFA has a proven role in blood vessel formation, which encompasses angiogenesis, vasculogenesis, and arteriogenesis.115,137 VEGFA can bind to both VEGF receptors 1 and 2 (VEGFR1 and VEGFR2),138 but only by binding VEGFR2 does VEGFA activate downstream signals such as ERK, promoting EC migration and angiogenesis. As a member of the VEGF family, initial studies with VEGFB focused on its role in angiogenesis. Surprisingly, VEGFB does not appear to play a role in angiogenesis under normal conditions or even when overexpressed.139 However, although VEGFB may not directly have a role in angiogenesis, it may play a part in sensitizing ECs to VEGFA-induced angiogenesis.119 It has been suggested that the binding of VEGFB to VEGFR1 leads to less VEGFR1 being available to bind VEGFA, allowing more VEGFA to activate signals via VEGFR2.119,140 In addition to this sensitizing effect, coronary vasculature in VEGFB overexpressing hearts had up to a 5-fold increase in the number of arteries119, whereas adenoviral overexpressing rats had a 2.5-fold increase,141 an effect likely related to EC survival. Overall, this dual action allows VEGFB to indirectly increase angiogenesis to permit tolerable levels of FAO by the cardiomyocyte. Under the condition of diabetes when there is a failure to release VEGFA and VEGFB from the cardiomyocyte, its associated impact on the vasculature will result in an ‘oxygen shortfall’—a situation in which oxygen consumption related to FAO surpasses oxygen delivery, leading to FA storage in the cardiomyocyte and progressive cardiac dysfunction. It should be noted that in addition to this paracrine control of angiogenesis, intrinsic mechanisms within the EC, like FoxO1, have recently been implicated in control of the vascular architecture. 142 FoxO1, a prominent member of the Forkhead box family and subfamily O of transcription factors, has been known to play an important role in cell survival, oxidative stress resistance, energy metabolism, cell cycle arrest, and cell death.143 – 145 FoxO1 is activated in conditions such as fasting, nutrient excess, insulin resistance, diabetes, inflammation, sepsis, and ischaemia.52 Additionally, overexpression of endothelial FoxO1 resulted in restricted vascular expansion.142 Whether the increase in FoxO1 that we observed in diabetic cardiomyocytes is also a feature of EC from diabetic hearts is currently unknown.54 If proved, this could provide an additional mechanism by which the lack of angiogenesis leads to diabetic cardiomyopathy. 8. Significance Inadequate glucose control leads to repeated bouts of hyperglycaemia, which is associated with ineffective glucose uptake (through GLUT1), metabolism, and deficiency in ATP generation within the EC. Additionally, as insulin signalling (via phosphatidylinositol 3-kinase, mitogenactivated protein kinase kinase and endothelial nitric oxide synthase phosphorylation)146,147 stimulates EC insulin transport and capillary recruitment in skeletal muscle, inadequate insulin action in EC is associated with a reduction in insulin delivery, and lower glucose uptake. Should this mechanism be present in the heart, this, together with the incompetence of GLUT4-mediated glucose transport into the cardiomyocyte means that this cell type will begin to use FA exclusively. One way by which the cardiomyocyte handles this increased FA utilization is to deploy LPL to the vascular lumen (with the help of EC heparanase), to break down circulating lipoprotein-TGs. The FA released during this process requires passage through the EC. As FAs traverse the ECs, there is an opportunity for this cell type to utilize FA, but this is not without undesirable outcomes. ECs are both structurally and morphologically geared towards glycolysis, rather than FA utilization [amount of mitochondria in EC is small (,10% of the cytoplasm) compared with cell types that have higher energy demands (e.g. cardiomyocytes, where mitochondria contribute up to 32% of the cytoplasmic volume)].148 Cardiomyocyte-released growth factors (VEGFB) in response to hyperglycaemia could protect EC against FA overload by turning on genes protective against cell death, or supplement the oxygen necessary for cardiomyocyte-mediated FAO by promoting angiogenesis (VEGFA). We propose that under the conditions of diabetes, there is a disruption of the harmonious dialogue between the EC and cardiomyocytes that results in lipotoxicity that can accelerate cardiovascular disease. Gaining more insight into mechanisms that (i) limit EC FA utilization and (ii) ensure adequate oxygen delivery to the cardiomyocyte by accelerating angiogenesis may delay cardiac failure and provide long-term management of this complication during diabetes. 9. Potential therapeutic strategies To restore metabolic equilibrium and curb lipotoxicity, targeting the protein ‘ensemble’, heparanase-LPL-VEGF may help prevent or delay heart dysfunction seen during diabetes. 9.1 Heparanase Heparanase, with a repertoire of functions, can be released from EC in response to HG. It affects myocyte metabolism and does so by interacting with its partners, VEGF and LPL. In the short term (e.g. diabetic patients who have poor control of glucose leading to bouts of hyperglycaemia), it can amplify FA delivery and utilization by the diabetic heart. If these events are prolonged, the resultant lipotoxicity could lead to cardiovascular disease in humans. Globally, inhibitors of heparanase (both active and latent-current strategies to modulate heparanase are aimed exclusively at blocking activity) could be expected to provide critical tools for managing the cardiac complications of diabetes. These include roneparstat (SST0001, a non-anticoagulant chemically modified heparin) and mupafostat (PI88, a mixture of oligosaccharides mimicking HS).149,150 Although we anticipate a delayed or milder cardiomyopathy using these agents, this is not an absolute. Agents that impede heparanase activity may prove to be less effective, especially as HG can also increase secretion of latent inactive heparanase that can fulfil its own functions in releasing LPL.98 9.2 Lipoprotein lipase Cardiac-specific overexpression of LPL causes a severe myopathy characterized by lipid oversupply and deposition, muscle fibre degeneration, excessive dilatation, and impaired left ventricular function in the absence of vascular defects, a situation comparable with diabetic cardiomyopathy.151 Thus, there is a potential benefit to lowering cardiac LPL following diabetes. Angiopoietin-like protein 4 (Angptl-4) is known to convert dimeric active LPL at the vascular lumen into inactive monomers.152 Similarly, apolipoprotein C-III can inhibit LPL by displacement of the enzyme from the lipid droplet.153 The therapeutic advantage of stimulating Angptl-4 or apo C-III may, however, be limited by the possibility to also develop hypertriglyceridaemia. Recent studies have suggested that patients with inactivating mutations in ANGPTL4 have lower levels of TGs and a lower risk of coronary artery disease, linking TG to cardiac disease.154,155 180 A. Wan and B. Rodrigues 9.3 Vascular endothelial growth factor 57 Reduction of myocardial VEGFA, and its association with impaired collateral formation in cardiac tissue,56 is a pivotal event in diabetic cardiomyopathy. Conversely, VEGFA has been implicated in diabetic retinopathy.156 Given the dissimilar global effects of VEGFA, one would anticipate the use of targeted gene therapy to restore VEGFA in the myocardium (local concentrations), rather than an approach to augment systemic levels. Findings on VEGFB in the diabetic heart are lacking. However, given its significant antiapoptotic and survival effects139 and its ability to prepare the EC to VEGFA-induced angiogenesis,119,140 VEGFB has significant therapeutic potential in treating diabetic cardiomyopathy. Acknowledgements We are indebted to Paul Hiebert for his help with the graphics. Conflict of interest: none declared. Funding The present study is supported by an operating grant from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada. References 1. World Health Organization. World Health Organization: Global Health Estimates: Deaths by Cause, Age, Sex, and Country. Geneva: WHO; 2014. 2. Sowers JR, Epstein M, Frohlich ED. Diabetes, hypertension, and cardiovascular disease: an update. Hypertension 2001;37:1053 –1059. 3. Action to Control Cardiovascular Risk in Diabetes Study Group, Gerstein HC, Miller ME, Byington RP, Goff DC Jr, Bigger JT, Buse JB, Cushman WC, Genuth S, Ismail-Beigi F, Grimm RH Jr, Probstfield JL, Simons-Morton DG, Friedewald WT. Effects of intensive glucose lowering in type 2 diabetes. N Engl J Med 2008;358: 2545 –2559. 4. Group AC, Patel A, MacMahon S, Chalmers J, Neal B, Billot L, Woodward M, Marre M, Cooper M, Glasziou P, Grobbee D, Hamet P, Harrap S, Heller S, Liu L, Mancia G, Mogensen CE, Pan C, Poulter N, Rodgers A, Williams B, Bompoint S, de Galan BE, Joshi R, Travert F. Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes. N Engl J Med 2008;358:2560 –2572. 5. Duckworth W, Abraira C, Moritz T, Reda D, Emanuele N, Reaven PD, Zieve FJ, Marks J, Davis SN, Hayward R, Warren SR, Goldman S, McCarren M, Vitek ME, Henderson WG, Huang GD, Investigators V. Glucose control and vascular complications in veterans with type 2 diabetes. N Engl J Med 2009;360:129 –139. 6. Francis G. Diabetic cardiomyopathy: fact or fiction? Heart 2001;85:247 –248. 7. Picano E. Diabetic cardiomyopathy. The importance of being earliest. J Am Coll Cardiol 2003;42:454 –457. 8. An D, Rodrigues B. Role of changes in cardiac metabolism in development of diabetic cardiomyopathy. Am J Physiol Heart Circ Physiol 2006;291:H1489 –H1506. 9. Kim MS, Wang Y, Rodrigues B. Lipoprotein lipase mediated fatty acid delivery and its impact in diabetic cardiomyopathy. Biochim Biophys Acta 2012;1821:800–808. 10. Wende AR, Abel ED. Lipotoxicity in the heart. Biochim Biophys Acta 2010;1801: 311 –319. 11. Rodrigues B, Cam MC, McNeill JH. Myocardial substrate metabolism: implications for diabetic cardiomyopathy. J Mol Cell Cardiol 1995;27:169 – 179. 12. de Zeeuw P, Wong BW, Carmeliet P. Metabolic adaptations in diabetic endothelial cells. Circ J 2015;79:934–941. 13. Luiken JJ, van Nieuwenhoven FA, America G, van der Vusse GJ, Glatz JF. Uptake and metabolism of palmitate by isolated cardiac myocytes from adult rats: involvement of sarcolemmal proteins. J Lipid Res 1997;38:745 – 758. 14. Jung UJ, Choi MS. Obesity and its metabolic complications: the role of adipokines and the relationship between obesity, inflammation, insulin resistance, dyslipidemia and nonalcoholic fatty liver disease. Int J Mol Sci 2014;15:6184 – 6223. 15. Boudina S, Abel ED. Diabetic cardiomyopathy revisited. Circulation 2007;115: 3213 –3223. 16. Ibrahimi A, Bonen A, Blinn WD, Hajri T, Li X, Zhong K, Cameron R, Abumrad NA. Muscle-specific overexpression of FAT/CD36 enhances fatty acid oxidation by contracting muscle, reduces plasma triglycerides and fatty acids, and increases plasma glucose and insulin. J Biol Chem 1999;274:26761 –26766. 17. Luiken JJ, Coort SL, Koonen DP, van der Horst DJ, Bonen A, Zorzano A, Glatz JF. Regulation of cardiac long-chain fatty acid and glucose uptake by translocation of substrate transporters. Pflugers Arch 2004;448:1–15. 18. Aasum E, Belke DD, Severson DL, Riemersma RA, Cooper M, Andreassen M, Larsen TS. Cardiac function and metabolism in type 2 diabetic mice after treatment with BM 17.0744, a novel PPAR-alpha activator. Am J Physiol Heart Circ Physiol 2002; 283:H949 – H957. 19. Belke DD, Larsen TS, Gibbs EM, Severson DL. Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice. Am J Physiol Endocrinol Metab 2000;279:E1104– E1113. 20. Kudo N, Barr AJ, Barr RL, Desai S, Lopaschuk GD. High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5’-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J Biol Chem 1995;270:17513 –17520. 21. Gando S, Hattori Y, Kanno M. Altered cardiac adrenergic neurotransmission in streptozotocin-induced diabetic rats. Br J Pharmacol 1993;109:1276 – 1281. 22. Poornima IG, Parikh P, Shannon RP. Diabetic cardiomyopathy: the search for a unifying hypothesis. Circ Res 2006;98:596 –605. 23. Chiu HC, Kovacs A, Ford DA, Hsu FF, Garcia R, Herrero P, Saffitz JE, Schaffer JE. A novel mouse model of lipotoxic cardiomyopathy. J Clin Invest 2001;107:813 –822. 24. Fang ZY, Prins JB, Marwick TH. Diabetic cardiomyopathy: evidence, mechanisms, and therapeutic implications. Endocr Rev 2004;25:543 –567. 25. Belke DD, Dillmann WH. Altered cardiac calcium handling in diabetes. Curr Hypertens Rep 2004;6:424 –429. 26. Lopaschuk GD, Ussher JR, Folmes CD, Jaswal JS, Stanley WC. Myocardial fatty acid metabolism in health and disease. Physiol Rev 2010;90:207–258. 27. Abel ED. Glucose transport in the heart. Front Biosci 2004;9:201 –215. 28. Glatz JF, Bonen A, Ouwens DM, Luiken JJ. Regulation of sarcolemmal transport of substrates in the healthy and diseased heart. Cardiovasc Drugs Ther 2006;20:471 – 476. 29. Young ME, McNulty P, Taegtmeyer H. Adaptation and maladaptation of the heart in diabetes: Part II: potential mechanisms. Circulation 2002;105:1861 –1870. 30. Schwenk RW, Luiken JJ, Bonen A, Glatz JF. Regulation of sarcolemmal glucose and fatty acid transporters in cardiac disease. Cardiovasc Res 2008;79:249 –258. 31. Haemmerle G, Moustafa T, Woelkart G, Buttner S, Schmidt A, van de Weijer T, Hesselink M, Jaeger D, Kienesberger PC, Zierler K, Schreiber R, Eichmann T, Kolb D, Kotzbeck P, Schweiger M, Kumari M, Eder S, Schoiswohl G, Wongsiriroj N, Pollak NM, Radner FPW, Preiss-Landl K, Kolbe T, Rulicke T, Pieske B, Trauner M, Lass A, Zimmermann R, Hoefler G, Cinti S, Kershaw EE, Schrauwen P, Madeo F, Mayer B, Zechner R. ATGL-mediated fat catabolism regulates cardiac mitochondrial function via PPAR-alpha and PGC-1. Nat Med 2011;17: 1076 –U1082. 32. Young SG, Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis. Genes Dev 2013;27:459 –484. 33. Cuthbert KD, Dyck JR. Malonyl-CoA decarboxylase is a major regulator of myocardial fatty acid oxidation. Curr Hypertens Rep 2005;7:407–411. 34. Jogl G, Hsiao YS, Tong L. Structure and function of carnitine acyltransferases. Ann N Y Acad Sci 2004;1033:17 –29. 35. Kewalramani G, An D, Kim MS, Ghosh S, Qi D, Abrahani A, Pulinilkunnil T, Sharma V, Wambolt RB, Allard MF, Innis SM, Rodrigues B. AMPK control of myocardial fatty acid metabolism fluctuates with the intensity of insulin-deficient diabetes. J Mol Cell Cardiol 2007;42:333–342. 36. Folmes CD, Lopaschuk GD. Role of malonyl-CoA in heart disease and the hypothalamic control of obesity. Cardiovasc Res 2007;73:278 –287. 37. Lefebvre P, Chinetti G, Fruchart JC, Staels B. Sorting out the roles of PPAR alpha in energy metabolism and vascular homeostasis. J Clin Invest 2006;116:571 –580. 38. Stanley WC, Lopaschuk GD, McCormack JG. Regulation of energy substrate metabolism in the diabetic heart. Cardiovasc Res 1997;34:25–33. 39. Bayeva M, Sawicki KT, Ardehali H. Taking diabetes to heart—deregulation of myocardial lipid metabolism in diabetic cardiomyopathy. J Am Heart Assoc 2013;2:e000433. 40. Pulinilkunnil T, Rodrigues B. Cardiac lipoprotein lipase: metabolic basis for diabetic heart disease. Cardiovasc Res 2006;69:329 –340. 41. Rodrigues B, Cam MC, Jian K, Lim F, Sambandam N, Shepherd G. Differential effects of streptozotocin-induced diabetes on cardiac lipoprotein lipase activity. Diabetes 1997; 46:1346 –1353. 42. Sambandam N, Abrahani MA, St Pierre E, Al-Atar O, Cam MC, Rodrigues B. Localization of lipoprotein lipase in the diabetic heart: regulation by acute changes in insulin. Arterioscler Thromb Vasc Biol 1999;19:1526 –1534. 43. Wang Y, Puthanveetil P, Wang F, Kim MS, Abrahani A, Rodrigues B. Severity of diabetes governs vascular lipoprotein lipase by affecting enzyme dimerization and disassembly. Diabetes 2011;60:2041 –2050. 44. Pulinilkunnil T, Abrahani A, Varghese J, Chan N, Tang I, Ghosh S, Kulpa J, Allard M, Brownsey R, Rodrigues B. Evidence for rapid “metabolic switching” through lipoprotein lipase occupation of endothelial-binding sites. J Mol Cell Cardiol 2003;35: 1093 –1103. 45. An D, Pulinilkunnil T, Qi D, Ghosh S, Abrahani A, Rodrigues B. The metabolic “switch” AMPK regulates cardiac heparin-releasable lipoprotein lipase. Am J Physiol Endocrinol Metab 2005;288:E246 –E253. 46. Kim MS, Kewalramani G, Puthanveetil P, Lee V, Kumar U, An D, Abrahani A, Rodrigues B. Acute diabetes moderates trafficking of cardiac lipoprotein lipase through p38 mitogen-activated protein kinase-dependent actin cytoskeleton organization. Diabetes 2008;57:64 –76. Cellular crosstalk 47. Wang F, Kim MS, Puthanveetil P, Kewalramani G, Deppe S, Ghosh S, Abrahani A, Rodrigues B. Endothelial heparanase secretion after acute hypoinsulinemia is regulated by glucose and fatty acid. Am J Physiol Heart Circ Physiol 2009;296:H1108 –H1116. 48. Wang Y, Zhang D, Chiu AP, Wan A, Neumaier K, Vlodavsky I, Rodrigues B. Endothelial heparanase regulates heart metabolism by stimulating lipoprotein lipase secretion from cardiomyocytes. Arterioscler Thromb Vasc Biol 2013;33:894 –902. 49. Luiken JJ, Arumugam Y, Bell RC, Calles-Escandon J, Tandon NN, Glatz JF, Bonen A. Changes in fatty acid transport and transporters are related to the severity of insulin deficiency. Am J Physiol Endocrinol Metab 2002;283:E612 –E621. 50. Buchanan J, Mazumder PK, Hu P, Chakrabarti G, Roberts MW, Yun UJ, Cooksey RC, Litwin SE, Abel ED. Reduced cardiac efficiency and altered substrate metabolism precedes the onset of hyperglycemia and contractile dysfunction in two mouse models of insulin resistance and obesity. Endocrinology 2005;146:5341 –5349. 51. Sharma S, Adrogue JV, Golfman L, Uray I, Lemm J, Youker K, Noon GP, Frazier OH, Taegtmeyer H. Intramyocardial lipid accumulation in the failing human heart resembles the lipotoxic rat heart. FASEB J 2004;18:1692 –1700. 52. Puthanveetil P, Wan A, Rodrigues B. FoxO1 is crucial for sustaining cardiomyocyte metabolism and cell survival. Cardiovasc Res 2013;97:393 – 403. 53. Puthanveetil P, Zhang D, Wang Y, Wang F, Wan A, Abrahani A, Rodrigues B. Diabetes triggers a PARP1 mediated death pathway in the heart through participation of FoxO1. J Mol Cell Cardiol 2012;53:677–686. 54. Puthanveetil P, Wang Y, Zhang D, Wang F, Kim MS, Innis S, Pulinilkunnil T, Abrahani A, Rodrigues B. Cardiac triglyceride accumulation following acute lipid excess occurs through activation of a FoxO1-iNOS-CD36 pathway. Free Radic Biol Med 2011;51: 352 –363. 55. Qi D, Pulinilkunnil T, An D, Ghosh S, Abrahani A, Pospisilik JA, Brownsey R, Wambolt R, Allard M, Rodrigues B. Single-dose dexamethasone induces whole-body insulin resistance and alters both cardiac fatty acid and carbohydrate metabolism. Diabetes 2004;53:1790 –1797. 56. Chou E, Suzuma I, Way KJ, Opland D, Clermont AC, Naruse K, Suzuma K, Bowling NL, Vlahos CJ, Aiello LP, King GL. Decreased cardiac expression of vascular endothelial growth factor and its receptors in insulin-resistant and diabetic States: a possible explanation for impaired collateral formation in cardiac tissue. Circulation 2002;105:373 – 379. 57. Yoon YS, Uchida S, Masuo O, Cejna M, Park JS, Gwon HC, Kirchmair R, Bahlman F, Walter D, Curry C, Hanley A, Isner JM, Losordo DW. Progressive attenuation of myocardial vascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy: restoration of microvascular homeostasis and recovery of cardiac function in diabetic cardiomyopathy after replenishment of local vascular endothelial growth factor. Circulation 2005;111:2073 –2085. 58. Bellomo D, Headrick JP, Silins GU, Paterson CA, Thomas PS, Gartside M, Mould A, Cahill MM, Tonks ID, Grimmond SM, Townson S, Wells C, Little M, Cummings MC, Hayward NK, Kay GF. Mice lacking the vascular endothelial growth factor-B gene (VEGFB) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia. Circ Res 2000;86:E29 –E35. 59. Yagyu H, Chen G, Yokoyama M, Hirata K, Augustus A, Kako Y, Seo T, Hu Y, Lutz EP, Merkel M, Bensadoun A, Homma S, Goldberg IJ. Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy. J Clin Invest 2003;111:419 –426. 60. Yang J, Sambandam N, Han X, Gross RW, Courtois M, Kovacs A, Febbraio M, Finck BN, Kelly DP. CD36 deficiency rescues lipotoxic cardiomyopathy. Circ Res 2007;100:1208 –1217. 61. Zhang L, Keung W, Samokhvalov V, Wang W, Lopaschuk GD. Role of fatty acid uptake and fatty acid beta-oxidation in mediating insulin resistance in heart and skeletal muscle. Biochim Biophys Acta 2010;1801:1 –22. 62. Liu J, Jahn LA, Fowler DE, Barrett EJ, Cao W, Liu Z. Free fatty acids induce insulin resistance in both cardiac and skeletal muscle microvasculature in humans. J Clin Endocrinol Metab 2011;96:438–446. 63. De Bock K, Georgiadou M, Schoors S, Kuchnio A, Wong BW, Cantelmo AR, Quaegebeur A, Ghesquiere B, Cauwenberghs S, Eelen G, Phng LK, 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 RT, Munck S, Daelemans D, Imamura H, Devlieger R, Rider M, Van Veldhoven PP, Schuit F, Bartrons R, Hofkens J, Fraisl P, Telang S, Deberardinis RJ, Schoonjans L, Vinckier S, Chesney J, Gerhardt H, Dewerchin M, Carmeliet P. Role of PFKFB3-driven glycolysis in vessel sprouting. Cell 2013;154:651 – 663. 64. Wang F, Wang Y, Kim MS, Puthanveetil P, Ghosh S, Luciani DS, Johnson JD, Abrahani A, Rodrigues B. Glucose-induced endothelial heparanase secretion requires cortical and stress actin reorganization. Cardiovasc Res 2010;87:127 –136. 65. Aird WC. Endothelial cell heterogeneity. Cold Spring Harb Perspect Med 2012;2: a006429. 66. Jarvis MA, Nelson JA. Human cytomegalovirus tropism for endothelial cells: not all endothelial cells are created equal. J Virol 2007;81:2095 –2101. 67. Zetter BR. The endothelial cells of large and small blood vessels. Diabetes 1981;30: 24 –28. 68. Chi JT, Chang HY, Haraldsen G, Jahnsen FL, Troyanskaya OG, Chang DS, Wang Z, Rockson SG, van de Rijn M, Botstein D, Brown PO. Endothelial cell diversity revealed by global expression profiling. Proc Natl Acad Sci USA 2003;100:10623 –10628. 181 69. Schoors S, Bruning U, Missiaen R, Queiroz KC, Borgers G, Elia I, Zecchin A, Cantelmo AR, Christen S, Goveia J, Heggermont W, Godde L, Vinckier S, Van Veldhoven PP, Eelen G, Schoonjans L, Gerhardt H, Dewerchin M, Baes M, De Bock K, Ghesquiere B, Lunt SY, Fendt SM, Carmeliet P. Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Nature 2015;520:192 –197. 70. Lunt SY, Vander Heiden MG. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu Rev Cell Dev Biol 2011;27:441 –464. 71. Verdegem D, Moens S, Stapor P, Carmeliet P. Endothelial cell metabolism: parallels and divergences with cancer cell metabolism. Cancer Metab 2014;2:19. 72. De Bock K, Georgiadou M, Carmeliet P. Role of endothelial cell metabolism in vessel sprouting. Cell Metab 2013;18:634 – 647. 73. Goveia J, Stapor P, Carmeliet P. Principles of targeting endothelial cell metabolism to treat angiogenesis and endothelial cell dysfunction in disease. EMBO Mol Med 2014;6: 1105–1120. 74. Harjes U, Bensaad K, Harris AL. Endothelial cell metabolism and implications for cancer therapy. Br J Cancer 2012;107:1207 –1212. 75. Gaudreault N, Scriven DR, Moore ED. Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia. Diabetologia 2004;47:2081 –2092. 76. Young CD, Lewis AS, Rudolph MC, Ruehle MD, Jackman MR, Yun UJ, Ilkun O, Pereira R, Abel ED, Anderson SM. Modulation of glucose transporter 1 (GLUT1) expression levels alters mouse mammary tumor cell growth in vitro and in vivo. PLoS One 2011;6:e23205. 77. Cornford EM, Hyman S. Localization of brain endothelial luminal and abluminal transporters with immunogold electron microscopy. NeuroRx 2005;2:27 –43. 78. Fernandes R, Suzuki K, Kumagai AK. Inner blood-retinal barrier GLUT1 in long-term diabetic rats: an immunogold electron microscopic study. Invest Ophthalmol Vis Sci 2003;44:3150 –3154. 79. Hirsch B, Rosen P. Diabetes mellitus induces long lasting changes in the glucose transporter of rat heart endothelial cells. Horm Metab Res 1999;31:645 –652. 80. Rajah TT, Olson AL, Grammas P. Differential glucose uptake in retina- and brainderived endothelial cells. Microvasc Res 2001;62:236 –242. 81. Alpert E, Gruzman A, Riahi Y, Blejter R, Aharoni P, Weisinger G, Eckel J, Kaiser N, Sasson S. Delayed autoregulation of glucose transport in vascular endothelial cells. Diabetologia 2005;48:752–755. 82. Xue W, Cai L, Tan Y, Thistlethwaite P, Kang YJ, Li X, Feng W. Cardiac-specific overexpression of HIF-1{alpha} prevents deterioration of glycolytic pathway and cardiac remodeling in streptozotocin-induced diabetic mice. Am J Pathol 2010;177:97– 105. 83. Eelen G, de Zeeuw P, Simons M, Carmeliet P. Endothelial cell metabolism in normal and diseased vasculature. Circ Res 2015;116:1231 –1244. 84. Wu N, Zheng B, Shaywitz A, Dagon Y, Tower C, Bellinger G, Shen CH, Wen J, Asara J, McGraw TE, Kahn BB, Cantley LC. AMPK-dependent degradation of TXNIP upon energy stress leads to enhanced glucose uptake via GLUT1. Mol Cell 2013;49: 1167–1175. 85. Turturro F, Friday E, Welbourne T. Hyperglycemia regulates thioredoxin-ROS activity through induction of thioredoxin-interacting protein (TXNIP) in metastatic breast cancer-derived cells MDA-MB-231. BMC Cancer 2007;7:96. 86. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer 2004;4:891–899. 87. Dunn LL, Simpson PJ, Prosser HC, Lecce L, Yuen GS, Buckle A, Sieveking DP, Vanags LZ, Lim PR, Chow RW, Lam YT, Clayton Z, Bao S, Davies MJ, Stadler N, Celermajer DS, Stocker R, Bursill CA, Cooke JP, Ng MK. A critical role for thioredoxin-interacting protein in diabetes-related impairment of angiogenesis. Diabetes 2014;63:675 –687. 88. World C, Spindel ON, Berk BC. Thioredoxin-interacting protein mediates TRX1 translocation to the plasma membrane in response to tumor necrosis factor-alpha: a key mechanism for vascular endothelial growth factor receptor-2 transactivation by reactive oxygen species. Arterioscler Thromb Vasc Biol 2011;31:1890 – 1897. 89. Garcia NA, Moncayo-Arlandi J, Sepulveda P, Diez-Juan A. Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes. Cardiovasc Res 2016;109:397 –408. 90. Sawada N, Jiang A, Takizawa F, Safdar A, Manika A, Tesmenitsky Y, Kang KT, Bischoff J, Kalwa H, Sartoretto JL, Kamei Y, Benjamin LE, Watada H, Ogawa Y, Higashikuni Y, Kessinger CW, Jaffer FA, Michel T, Sata M, Croce K, Tanaka R, Arany Z. Endothelial PGC-1alpha mediates vascular dysfunction in diabetes. Cell Metab 2014;19:246–258. 91. Pangare M, Makino A. Mitochondrial function in vascular endothelial cell in diabetes. J Smooth Muscle Res 2012;48:1 –26. 92. Tang X, Luo YX, Chen HZ, Liu DP. Mitochondria, endothelial cell function, and vascular diseases. Front Physiol 2014;5:175. 93. Schulze PC. Myocardial lipid accumulation and lipotoxicity in heart failure. J Lipid Res 2009;50:2137 –2138. 94. Iozzo RV. Heparan sulfate proteoglycans: intricate molecules with intriguing functions. J Clin Invest 2001;108:165 –167. 95. Iozzo RV, San Antonio JD. Heparan sulfate proteoglycans: heavy hitters in the angiogenesis arena. J Clin Invest 2001;108:349–355. 96. Lee TY, Folkman J, Javaherian K. HSPG-binding peptide corresponding to the exon 6a-encoded domain of VEGF inhibits tumor growth by blocking angiogenesis in murine model. PLoS One 2010;5:e9945. 182 97. Bame KJ. Heparanases: endoglycosidases that degrade heparan sulfate proteoglycans. Glycobiology 2001;11:91R –98R. 98. Zhang D, Wan A, Chiu AP, Wang Y, Wang F, Neumaier K, Lal N, Bround MJ, Johnson JD, Vlodavsky I, Rodrigues B. Hyperglycemia-induced secretion of endothelial heparanase stimulates a vascular endothelial growth factor autocrine network in cardiomyocytes that promotes recruitment of lipoprotein lipase. Arterioscler Thromb Vasc Biol 2013;33:2830 –2838. 99. Barrett EJ, Liu Z. The endothelial cell: an “early responder” in the development of insulin resistance. Rev Endocr Metab Disord 2013;14:21–27. 100. Wang F, Zhang D, Wan A, Rodrigues B. Endothelial cell regulation of cardiac metabolism following diabetes. Cardiovasc Hematol Disord Drug Targets 2014;14: 121 –125. 101. Wang Y, Chiu AP, Neumaier K, Wang F, Zhang D, Hussein B, Lal N, Wan A, Liu G, Vlodavsky I, Rodrigues B. Endothelial cell heparanase taken up by cardiomyocytes regulates lipoprotein lipase transfer to the coronary lumen after diabetes. Diabetes 2014; 63:2643 – 2655. 102. Fairbanks MB, Mildner AM, Leone JW, Cavey GS, Mathews WR, Drong RF, Slightom JL, Bienkowski MJ, Smith CW, Bannow CA, Heinrikson RL. Processing of the human heparanase precursor and evidence that the active enzyme is a heterodimer. J Biol Chem 1999;274:29587 –29590. 103. Gingis-Velitski S, Zetser A, Kaplan V, Ben-Zaken O, Cohen E, Levy-Adam F, Bashenko Y, Flugelman MY, Vlodavsky I, Ilan N. Heparanase uptake is mediated by cell membrane heparan sulfate proteoglycans. J Biol Chem 2004;279:44084– 44092. 104. Pikas DS, Li JP, Vlodavsky I, Lindahl U. Substrate specificity of heparanases from human hepatoma and platelets. J Biol Chem 1998;273:18770 –18777. 105. Abboud-Jarrous G, Rangini-Guetta Z, Aingorn H, Atzmon R, Elgavish S, Peretz T, Vlodavsky I. Site-directed mutagenesis, proteolytic cleavage, and activation of human proheparanase. J Biol Chem 2005;280:13568 –13575. 106. Camps L, Reina M, Llobera M, Vilaro S, Olivecrona T. Lipoprotein lipase: cellular origin and functional distribution. Am J Physiol 1990;258:C673 –C681. 107. Coppiello G, Collantes M, Sirerol-Piquer MS, Vandenwijngaert S, Schoors S, Swinnen M, Vandersmissen I, Herijgers P, Topal B, van Loon J, Goffin J, Prosper F, Carmeliet P, Garcia-Verdugo JM, Janssens S, Penuelas I, Aranguren XL, Luttun A. Meox2/Tcf15 heterodimers program the heart capillary endothelium for cardiac fatty acid uptake. Circulation 2015;131:815–826. 108. Dallinga-Thie GM, Franssen R, Mooij HL, Visser ME, Hassing HC, Peelman F, Kastelein JJ, Peterfy M, Nieuwdorp M. The metabolism of triglyceride-rich lipoproteins revisited: new players, new insight. Atherosclerosis 2010;211:1– 8. 109. Eckel RH. Lipoprotein lipase. A multifunctional enzyme relevant to common metabolic diseases. N Engl J Med 1989;320:1060 –1068. 110. Enerback S, Gimble JM. Lipoprotein lipase gene expression: physiological regulators at the transcriptional and post-transcriptional level. Biochim Biophys Acta 1993;1169: 107 –125. 111. Kersten S. Physiological regulation of lipoprotein lipase. Biochim Biophys Acta 2014; 1841:919 –933. 112. Testa U, Pannitteri G, Condorelli GL. Vascular endothelial growth factors in cardiovascular medicine. J Cardiovasc Med (Hagerstown) 2008;9:1190 –1221. 113. Lan L, Wilks A, Morgan TO, Di Nicolantonio R. Vascular endothelial growth factor: tissue distribution and size of multiple mRNA splice forms in SHR and WKY. Clin Exp Pharmacol Physiol Suppl 1995;22:S167 –S168. 114. Krilleke D, DeErkenez A, Schubert W, Giri I, Robinson GS, Ng YS, Shima DT. Molecular mapping and functional characterization of the VEGF164 heparin-binding domain. J Biol Chem 2007;282:28045 –28056. 115. Tammela T, Enholm B, Alitalo K, Paavonen K. The biology of vascular endothelial growth factors. Cardiovasc Res 2005;65:550 –563. 116. Kraemer FB, Shen WJ. Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis. J Lipid Res 2002;43:1585 –1594. 117. Kershaw EE, Hamm JK, Verhagen LA, Peroni O, Katic M, Flier JS. Adipose triglyceride lipase: function, regulation by insulin, and comparison with adiponutrin. Diabetes 2006; 55:148 –157. 118. Dijkstra MH, Pirinen E, Huusko J, Kivela R, Schenkwein D, Alitalo K, Yla-Herttuala S. Lack of cardiac and high-fat diet induced metabolic phenotypes in two independent strains of VEGF-b knockout mice. Sci Rep 2014;4:6238. 119. Kivela R, Bry M, Robciuc MR, Rasanen M, Taavitsainen M, Silvola JM, Saraste A, Hulmi JJ, Anisimov A, Mayranpaa MI, Lindeman JH, Eklund L, Hellberg S, Hlushchuk R, Zhuang ZW, Simons M, Djonov V, Knuuti J, Mervaala E, Alitalo K. VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart. EMBO Mol Med 2014;6:307 –321. 120. Bry M, Kivela R, Leppanen VM, Alitalo K. Vascular endothelial growth factor-B in physiology and disease. Physiol Rev 2014;94:779–794. 121. Bian K, Doursout MF, Murad F. Vascular system: role of nitric oxide in cardiovascular diseases. J Clin Hypertens (Greenwich) 2008;10:304 –310. 122. Hsieh PC, Davis ME, Lisowski LK, Lee RT. Endothelial-cardiomyocyte interactions in cardiac development and repair. Annu Rev Physiol 2006;68:51– 66. 123. Recchia FA, McConnell PI, Loke KE, Xu X, Ochoa M, Hintze TH. Nitric oxide controls cardiac substrate utilization in the conscious dog. Cardiovasc Res 1999;44: 325 –332. A. Wan and B. Rodrigues 124. Recchia FA. Role of nitric oxide in the regulation of substrate metabolism in heart failure. Heart Fail Rev 2002;7:141 –148. 125. Parodi EM, Kuhn B. Signalling between microvascular endothelium and cardiomyocytes through neuregulin. Cardiovasc Res 2014;102:194–204. 126. Odiete O, Hill MF, Sawyer DB. Neuregulin in cardiovascular development and disease. Circ Res 2012;111:1376 –1385. 127. Cote GM, Miller TA, Lebrasseur NK, Kuramochi Y, Sawyer DB. Neuregulin-1alpha and beta isoform expression in cardiac microvascular endothelial cells and function in cardiac myocytes in vitro. Exp Cell Res 2005;311:135 –146. 128. Kolwicz SC Jr, Purohit S, Tian R. Cardiac metabolism and its interactions with contraction, growth, and survival of cardiomyocytes. Circ Res 2013;113:603 –616. 129. Young SG, Davies BS, Voss CV, Gin P, Weinstein MM, Tontonoz P, Reue K, Bensadoun A, Fong LG, Beigneux AP. GPIHBP1, an endothelial cell transporter for lipoprotein lipase. J Lipid Res 2011;52:1869 –1884. 130. Davies BS, Beigneux AP, Barnes RH II, Tu Y, Gin P, Weinstein MM, Nobumori C, Nyren R, Goldberg I, Olivecrona G, Bensadoun A, Young SG, Fong LG. GPIHBP1 is responsible for the entry of lipoprotein lipase into capillaries. Cell Metab 2010;12: 42 –52. 131. Chiu AP, Wan A, Lal N, Zhang D, Wang F, Vlodavsky I, Hussein B, Rodrigues B. Cardiomyocyte VEGF regulates endothelial cell GPIHBP1 to relocate lipoprotein lipase to the coronary lumen during diabetes mellitus. Arterioscler Thromb Vasc Biol 2016;36: 145– 155. 132. Harjes U, Bridges E, McIntyre A, Fielding BA, Harris AL. Fatty acid-binding protein 4, a point of convergence for angiogenic and metabolic signaling pathways in endothelial cells. J Biol Chem 2014;289:23168 –23176. 133. Hagberg CE, Falkevall A, Wang X, Larsson E, Huusko J, Nilsson I, van Meeteren LA, Samen E, Lu L, Vanwildemeersch M, Klar J, Genove G, Pietras K, Stone-Elander S, Claesson-Welsh L, Yla-Herttuala S, Lindahl P, Eriksson U. Vascular endothelial growth factor B controls endothelial fatty acid uptake. Nature 2010;464:917 –921. 134. Khan MJ, Rizwan Alam M, Waldeck-Weiermair M, Karsten F, Groschner L, Riederer M, Hallstrom S, Rockenfeller P, Konya V, Heinemann A, Madeo F, Graier WF, Malli R. Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells. J Biol Chem 2012;287:21110 –21120. 135. Staiger K, Staiger H, Weigert C, Haas C, Haring HU, Kellerer M. Saturated, but not unsaturated, fatty acids induce apoptosis of human coronary artery endothelial cells via nuclear factor-kappaB activation. Diabetes 2006;55:3121 –3126. 136. Symons JD, Abel ED. Lipotoxicity contributes to endothelial dysfunction: a focus on the contribution from ceramide. Rev Endocr Metab Disord 2013;14:59–68. 137. Weis SM, Cheresh DA. Pathophysiological consequences of VEGF-induced vascular permeability. Nature 2005;437:497 – 504. 138. Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem 1994;269:26988– 26995. 139. Li X, Kumar A, Zhang F, Lee C, Tang Z. Complicated life, complicated VEGF-B. Trends Mol Med 2012;18:119 –127. 140. Robciuc MR, Kivela R, Williams IM, de Boer JF, van Dijk TH, Elamaa H, Tigistu-Sahle F, Molotkov D, Leppanen VM, Kakela R, Eklund L, Wasserman DH, Groen AK, Alitalo K. VEGFB/VEGFR1-induced expansion of adipose vasculature counteracts obesity and related metabolic complications. Cell Metab 2016;23:712 –724. 141. Bry M, Kivela R, Holopainen T, Anisimov A, Tammela T, Soronen J, Silvola J, Saraste A, Jeltsch M, Korpisalo P, Carmeliet P, Lemstrom KB, Shibuya M, Yla-Herttuala S, Alhonen L, Mervaala E, Andersson LC, Knuuti J, Alitalo K. Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation. Circulation 2010;122:1725 –1733. 142. Wilhelm K, Happel K, Eelen G, Schoors S, Oellerich MF, Lim R, Zimmermann B, Aspalter IM, Franco CA, Boettger T, Braun T, Fruttiger M, Rajewsky K, Keller C, Bruning JC, Gerhardt H, Carmeliet P, Potente M. FOXO1 couples metabolic activity and growth state in the vascular endothelium. Nature 2016;529:216 –220. 143. Burgering BM, Kops GJ. Cell cycle and death control: long live Forkheads. Trends Biochem Sci 2002;27:352 –360. 144. Hosaka T, Biggs WH III, Tieu D, Boyer AD, Varki NM, Cavenee WK, Arden KC. Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc Natl Acad Sci USA 2004;101:2975 –2980. 145. Essers MA, de Vries-Smits LM, Barker N, Polderman PE, Burgering BM, Korswagen HC. Functional interaction between beta-catenin and FOXO in oxidative stress signaling. Science 2005;308:1181 –1184. 146. Wang H, Wang AX, Liu Z, Barrett EJ. Insulin signaling stimulates insulin transport by bovine aortic endothelial cells. Diabetes 2008;57:540 –547. 147. Kubota T, Kubota N, Kumagai H, Yamaguchi S, Kozono H, Takahashi T, Inoue M, Itoh S, Takamoto I, Sasako T, Kumagai K, Kawai T, Hashimoto S, Kobayashi T, Sato M, Tokuyama K, Nishimura S, Tsunoda M, Ide T, Murakami K, Yamazaki T, Ezaki O, Kawamura K, Masuda H, Moroi M, Sugi K, Oike Y, Shimokawa H, Yanagihara N, Tsutsui M, Terauchi Y, Tobe K, Nagai R, Kamata K, Inoue K, Kodama T, Ueki K, Kadowaki T. Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle. Cell Metab 2011;13:294–307. 148. Kluge MA, Fetterman JL, Vita JA. Mitochondria and endothelial function. Circ Res 2013; 112:1171 –1188. Cellular crosstalk 149. Pala D, Rivara S, Mor M, Milazzo FM, Roscilli G, Pavoni E, Giannini G. Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase. Glycobiology 2016;26:640 –654. 150. Maxhimer JB, Somenek M, Rao G, Pesce CE, Baldwin D Jr, Gattuso P, Schwartz MM, Lewis EJ, Prinz RA, Xu X. Heparanase-1 gene expression and regulation by high glucose in renal epithelial cells: a potential role in the pathogenesis of proteinuria in diabetic patients. Diabetes 2005;54:2172 – 2178. 151. Kim JK, Fillmore JJ, Chen Y, Yu C, Moore IK, Pypaert M, Lutz EP, Kako Y, Velez-Carrasco W, Goldberg IJ, Breslow JL, Shulman GI. Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance. Proc Natl Acad Sci USA 2001;98:7522 –7527. 152. Wang Y, Rodrigues B. Intrinsic and extrinsic regulation of cardiac lipoprotein lipase following diabetes. Biochim Biophys Acta 2015;1851:163 –171. 183 153. Larsson M, Vorrsjo E, Talmud P, Lookene A, Olivecrona G. Apolipoproteins C-I and C-III inhibit lipoprotein lipase activity by displacement of the enzyme from lipid droplets. J Biol Chem 2013;288:33997 –34008. 154. Dewey FE, Gusarova V, O’Dushlaine C, Gottesman O, Trejos J, Hunt C, Van Hout CV, Habegger L, Buckler D, Lai KM, Leader JB, Murray MF, Ritchie MD, Kirchner HL, Ledbetter DH, Penn J, Lopez A, Borecki IB, Overton JD, Reid JG, Carey DJ, Murphy AJ, Yancopoulos GD, Baras A, Gromada J, Shuldiner AR. Inactivating variants in ANGPTL4 and risk of coronary artery disease. N Engl J Med 2016;374: 1123–1133. 155. Myocardial Infarction G, Investigators CAEC. Coding variation in ANGPTL4, LPL, and SVEP1 and the risk of coronary disease. N Engl J Med 2016;374:1134 –1144. 156. Gupta N, Mansoor S, Sharma A, Sapkal A, Sheth J, Falatoonzadeh P, Kuppermann B, Kenney M. Diabetic retinopathy and VEGF. Open Ophthalmol J 2013;7:4–10.