Dual effects of hyperglycemia on endothelial cells and cardiomyocytes to enhance coronary LPL activity.

In the diabetic heart, there is excessive dependence on fatty acid (FA) utilization to generate ATP. Lipoprotein lipase (LPL)-mediated hydrolysis of circulating triglycerides is suggested to be the predominant source of FA for cardiac utilization during diabetes. In the heart, the majority of LPL is synthesized in cardiomyocytes and secreted onto cell surface heparan sulfate proteoglycan (HSPG), where an endothelial cell (EC)-releasable ß-endoglycosidase, heparanase cleaves the side chains of HSPG to liberate LPL for its onward movement across the EC. EC glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) captures this released enzyme at its basolateral side and shuttles it across to its luminal side. We tested whether the diabetes-induced increase of transforming growth factor-ß (TGF-ß) can influence the myocyte and EC to help transfer LPL to the vascular lumen to generate triglyceride-FA. In response to high glucose and EC heparanase secretion, this endoglycosidase is taken up by the cardiomyocyte (Wang Y, Chiu AP, Neumaier K, Wang F, Zhang D, Hussein B, Lal N, Wan A, Liu G, Vlodavsky I, Rodrigues B. Diabetes 63: 2643-2655, 2014) to stimulate matrix metalloproteinase-9 expression and the conversion of latent to active TGF-ß. In the cardiomyocyte, TGF-ß activation of RhoA enhances actin cytoskeleton rearrangement to promote LPL trafficking and secretion onto cell surface HSPG. In the EC, TGF-ß signaling promotes mesodermal homeobox 2 translocation to the nucleus, which increases the expression of GPIHBP1, which facilitates movement of LPL to the vascular lumen. Collectively, our data suggest that in the diabetic heart, TGF-ß actions on the cardiomyocyte promotes movement of LPL, whereas its action on the EC facilitates LPL shuttling. NEW & NOTEWORTHY Endothelial cells, as first responders to hyperglycemia, release heparanase, whose subsequent uptake by cardiomyocytes amplifies matrix metalloproteinase-9 expression and activation of transforming growth factor-ß. Transforming growth factor-ß increases lipoprotein lipase secretion from cardiomyocytes and promotes mesodermal homeobox 2 to enhance glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1-dependent transfer of lipoprotein lipase across endothelial cells, mechanisms that accelerate fatty acid utilization by the diabetic heart.

Loss of VEGFB and its signaling in the diabetic heart is associated with increased cell death signaling.

Vascular endothelial growth factor B (VEGFB) is highly expressed in metabolically active tissues, such as the heart and skeletal muscle, suggesting a function in maintaining oxidative metabolic and contractile function in these tissues. Multiple models of heart failure have indicated a significant drop in VEGFB. However, whether there is a role for decreased VEGFB in diabetic cardiomyopathy is currently unknown. Of the VEGFB located in cardiomyocytes, there is a substantial and readily releasable pool localized on the cell surface. The immediate response to high glucose and the secretion of endothelial heparanase is the release of this surface-bound VEGFB, which triggers signaling pathways and gene expression to influence endothelial cell (autocrine action) and cardiomyocyte (paracrine effects) survival. Under conditions of hyperglycemia, when VEGFB production is impaired, a robust increase in vascular endothelial growth factor receptor (VEGFR)-1 expression ensues as a possible mechanism to enhance or maintain VEGFB signaling. However, even with an increase in VEGFR1 after diabetes, cardiomyocytes are unable to respond to VEGFB. In addition to the loss of VEGFB production and signaling, evaluation of latent heparanase, the protein responsible for VEGFB release, also showed a significant decline in expression in whole hearts from animals with chronic or acute diabetes. Defects in these numerous VEGFB pathways were associated with an increased cell death signature in our models of diabetes. Through this bidirectional interaction between endothelial cells (which secrete heparanase) and cardiomyocytes (which release VEGFB), this growth factor could provide the diabetic heart protection against cell death and may be a critical tool to delay or prevent cardiomyopathy.NEW & NOTEWORTHY We discovered a bidirectional interaction between endothelial cells (which secrete heparanase) and cardiomyocytes [which release vascular endothelial growth factor B (VEGFB)]. VEGFB promoted cell survival through ERK and cell death gene expression. Loss of VEGFB and its downstream signaling is an early event following hyperglycemia, is sustained with disease progression, and could explain diabetic cardiomyopathy.

Heparanase Overexpression Induces Glucagon Resistance and Protects Animals From Chemically Induced Diabetes.

Heparanase, a protein with enzymatic and nonenzymatic properties, contributes toward disease progression and prevention. In the current study, a fortuitous observation in transgenic mice globally overexpressing heparanase (hep-tg) was the discovery of improved glucose homeostasis. We examined the mechanisms that contribute toward this improved glucose metabolism. Heparanase overexpression was associated with enhanced glucose-stimulated insulin secretion and hyperglucagonemia, in addition to changes in islet composition and structure. Strikingly, the pancreatic islet transcriptome was greatly altered in hep-tg mice, with >2,000 genes differentially expressed versus control. The upregulated genes were enriched for diverse functions including cell death regulation, extracellular matrix component synthesis, and pancreatic hormone production. The downregulated genes were tightly linked to regulation of the cell cycle. In response to multiple low-dose streptozotocin (STZ), hep-tg animals developed less severe hyperglycemia compared with wild-type, an effect likely related to their ß-cells being more functionally efficient. In animals given a single high dose of STZ causing severe and rapid development of hyperglycemia related to the catastrophic loss of insulin, hep-tg mice continued to have significantly lower blood glucose. In these mice, protective pathways were uncovered for managing hyperglycemia and include augmentation of fibroblast growth factor 21 and glucagon-like peptide 1. This study uncovers the opportunity to use properties of heparanase in management of diabetes.

High glucose facilitated endothelial heparanase transfer to the cardiomyocyte modifies its cell death signature.

AIMS: The secretion of enzymatically active heparanase (HepA) has been implicated as an essential metabolic adaptation in the heart following diabetes. However, the regulation and function of the enzymatically inactive heparanase (HepL) remain poorly understood. We hypothesized that in response to high glucose (HG) and secretion of HepL from the endothelial cell (EC), HepL uptake and function can protect the cardiomyocyte by modifying its cell death signature. METHODS AND RESULTS: HG promoted both HepL and HepA secretion from microvascular (rat heart micro vessel endothelial cells, RHMEC) and macrovascular (rat aortic endothelial cells, RAOEC) EC. However, only RAOEC were capable of HepL reuptake. This occurred through a low-density lipoprotein receptor-related protein 1 (LRP1) dependent mechanism, as LRP1 inhibition using small interfering RNA (siRNA), receptor-associated protein, or an LRP1 neutralizing antibody significantly reduced uptake. In cardiomyocytes, which have a negligible amount of heparanase gene expression, LRP1 also participated in the uptake of HepL. Exogenous addition of HepL to rat cardiomyocytes produced a dramatically altered expression of apoptosis-related genes, and protection against HG and H2O2 induced cell death. Cardiomyocytes from acutely diabetic rats demonstrated a robust increase in LRP1 expression and levels of heparanase, a pro-survival gene signature, and limited evidence of cell death, observations that were not apparent following chronic and progressive diabetes. CONCLUSION: Our results highlight EC-to-cardiomyocyte transfer of heparanase to modulate the cardiomyocyte cell death signature. This mechanism was observed in the acutely diabetic heart, and its interruption following chronic diabetes may contribute towards the development of diabetic cardiomyopathy.

Cardiomyocyte-endothelial cell control of lipoprotein lipase.

In people with diabetes, inadequate pharmaceutical management predisposes the patient to heart failure, which is the leading cause of diabetes related death. One instigator for this cardiac dysfunction is change in fuel utilization by the heart. Thus, following diabetes, when cardiac glucose utilization is impaired, the heart undergoes metabolic transformation wherein it switches to using fats as an exclusive source of energy. Although this switching is geared to help the heart initially, in the long term, this has detrimental effects on cardiac function. These include the generation of noxious byproducts, which damage the cardiomyocytes, and ultimately result in increased morbidity and mortality. A key perpetrator that may be responsible for organizing this metabolic disequilibrium is lipoprotein lipase (LPL), the enzyme responsible for providing fat to the hearts. Either exaggeration or reduction in its activity following diabetes could lead to heart dysfunction. Given the disturbing news that diabetes is rampant across the globe, gaining more insight into the mechanism(s) by which cardiac LPL is regulated may assist other researchers in devising new therapeutic strategies to restore metabolic equilibrium, to help prevent or delay heart disease seen during diabetes. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

Cardiomyocyte VEGF Regulates Endothelial Cell GPIHBP1 to Relocate Lipoprotein Lipase to the Coronary Lumen During Diabetes Mellitus.

OBJECTIVE: Lipoprotein lipase (LPL)-mediated triglyceride hydrolysis is the major source of fatty acid for cardiac energy. LPL, synthesized in cardiomyocytes, is translocated across endothelial cells (EC) by its transporter glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1). Previously, we have reported an augmentation in coronary LPL, which was linked to an increased expression of GPIHBP1 following moderate diabetes mellitus. We examined the potential mechanism by which hyperglycemia amplifies GPIHBP1. APPROACH AND RESULTS: Exposure of rat aortic EC to high glucose induced GPIHBP1 expression and amplified LPL shuttling across these cells. This effect coincided with an elevated secretion of heparanase. Incubation of EC with high glucose or latent heparanase resulted in secretion of vascular endothelial growth factor (VEGF). Primary cardiomyocytes, being a rich source of VEGF, when cocultured with EC, restored EC GPIHBP1 that is lost because of cell passaging. Furthermore, recombinant VEGF induced EC GPIHBP1 mRNA and protein expression within 24 hours, an effect that could be prevented by a VEGF neutralizing antibody. This VEGF-induced increase in GPIHBP1 was through Notch signaling that encompassed Delta-like ligand 4 augmentation and nuclear translocation of the Notch intracellular domain. Finally, cardiomyocytes from severely diabetic animals exhibiting attenuation of VEGF were unable to increase EC GPIHBP1 expression and had lower LPL activity at the vascular lumen in perfused hearts. CONCLUSION: EC, as the first responders to hyperglycemia, can release heparanase to liberate myocyte VEGF. This growth factor, by activating EC Notch signaling, is responsible for facilitating GPIHBP1-mediated translocation of LPL across EC and regulating LPL-derived fatty acid delivery to the cardiomyocytes.

Endothelial cell heparanase taken up by cardiomyocytes regulates lipoprotein lipase transfer to the coronary lumen after diabetes.

After diabetes, the heart has a singular reliance on fatty acid (FA) for energy production, which is achieved by increased coronary lipoprotein lipase (LPL) that breaks down circulating triglycerides. Coronary LPL originates from cardiomyocytes, and to translocate to the vascular lumen, the enzyme requires liberation from myocyte surface heparan sulfate proteoglycans (HSPGs), an activity that needs to be sustained after chronic hyperglycemia. We investigated the mechanism by which endothelial cells (EC) and cardiomyocytes operate together to enable continuous translocation of LPL after diabetes. EC were cocultured with myocytes, exposed to high glucose, and uptake of endothelial heparanase into myocytes was determined. Upon uptake, the effect of nuclear entry of heparanase was also investigated. A streptozotocin model of diabetes was used to expand our in vitro observations. In high glucose, EC-derived latent heparanase was taken up by cardiomyocytes by a caveolae-dependent pathway using HSPGs. This latent heparanase was converted into an active form in myocyte lysosomes, entered the nucleus, and upregulated gene expression of matrix metalloproteinase-9. The net effect was increased shedding of HSPGs from the myocyte surface, releasing LPL for its onwards translocation to the coronary lumen. EC-derived heparanase regulates the ability of the cardiomyocyte to send LPL to the coronary lumen. This adaptation, although acutely beneficial, could be catastrophic chronically because excess FA causes lipotoxicity. Inhibiting heparanase function could offer a new strategy for managing cardiomyopathy observed after diabetes.

Hyperglycemia-induced secretion of endothelial heparanase stimulates a vascular endothelial growth factor autocrine network in cardiomyocytes that promotes recruitment of lipoprotein lipase.

OBJECTIVE: During diabetes mellitus, coronary lipoprotein lipase increases to promote the predominant use of fatty acids. We have reported that high glucose stimulates active heparanase secretion from endothelial cells to cleave cardiomyocyte heparan sulfate and release bound lipoprotein lipase for transfer to the vascular lumen. In the current study, we examined whether heparanase also has a function to release cardiomyocyte vascular endothelial growth factor (VEGF), and whether this growth factor influences cardiomyocyte fatty acid delivery in an autocrine manner. APPROACH AND RESULTS: Acute, reversible hyperglycemia was induced in rats, and a modified Langendorff heart perfusion was used to separate the coronary perfusate from the interstitial effluent. Coronary artery endothelial cells were exposed to high glucose to generate conditioned medium, and VEGF release from isolated cardiomyocytes was tested using endothelial cell conditioned medium or purified active and latent heparanase. Autocrine signaling of myocyte-derived VEGF on cardiac metabolism was studied. High glucose promoted latent and active heparanase secretion into endothelial cell conditioned medium, an effective stimulus for releasing cardiomyocyte VEGF. Intriguingly, latent heparanase was more efficient than active heparanase in releasing VEGF from a unique cell surface pool. VEGF augmented cardiomyocyte intracellular calcium and AMP-activated protein kinase phosphorylation and increased heparin-releasable lipoprotein lipase. CONCLUSIONS: Our data suggest that the heparanase-lipoprotein lipase-VEGF axis amplifies fatty acid delivery, a rapid and adaptive mechanism that is geared to overcome the loss of glucose consumption by the diabetic heart. If prolonged, the resultant lipotoxicity could lead to cardiovascular disease in humans.

Endothelial heparanase regulates heart metabolism by stimulating lipoprotein lipase secretion from cardiomyocytes.

OBJECTIVE: After diabetes mellitus, transfer of lipoprotein lipase (LPL) from cardiomyocytes to the coronary lumen increases, and this requires liberation of LPL from the myocyte surface heparan sulfate proteoglycans with subsequent replenishment of this reservoir. At the lumen, LPL breaks down triglyceride to meet the increased demand of the heart for fatty acid. Here, we examined the contribution of coronary endothelial cells (ECs) toward regulation of cardiomyocyte LPL secretion. APPROACH AND RESULTS: Bovine coronary artery ECs were exposed to high glucose, and the conditioned medium was used to treat cardiomyocytes. EC-conditioned medium liberated LPL from the myocyte surface, in addition to facilitating its replenishment. This effect was attributed to the increased heparanase content in EC-conditioned medium. Of the 2 forms of heparanase secreted from EC in response to high glucose, active heparanase released LPL from the myocyte surface, whereas latent heparanase stimulated reloading of LPL from an intracellular pool via heparan sulfate proteoglycan-mediated RhoA activation. CONCLUSIONS: Endothelial heparanase is a participant in facilitating LPL increase at the coronary lumen. These observations provide an insight into the cross-talk between ECs and cardiomyocytes to regulate cardiac metabolism after diabetes mellitus.