Heparanase Stimulation of Physiological Cardiac Hypertrophy is Suppressed Following Chronic Diabetes Resulting in Cardiac Remodeling and Dysfunction.

Although endothelial cells control smooth muscle tone in coronary vessels, these cells also influence subjacent cardiomyocyte growth. As heparanase, with exclusive expression in endothelial cells, enables extracellular matrix remodeling, angiogenesis, metabolic reprogramming, and cell survival, it is conceivable that it could also encourage development of cardiac hypertrophy. Global heparanase overexpression resulted in physiological cardiac hypertrophy, likely an outcome of HSPG clustering and activation of hypertrophic signaling. The autocrine effect of heparanase to release neuregulin-1 may have also contributed to this effect. Hyperglycemia induced by streptozotocin-diabetes sensitized the heart to flow-induced release of heparanase and neuregulin-1. Despite this excess secretion, progression of diabetes caused significant gene expression changes related to mitochondrial metabolism and cell death that led to development of pathological hypertrophy and heart dysfunction. Physiological cardiac hypertrophy was also observed in rats with cardiomyocyte-specific VEGFB overexpression. When perfused, hearts from these animals released significantly higher amounts of both heparanase and neuregulin-1. However, subjecting these animals to diabetes triggered robust transcriptome changes related to metabolism, and a transition to pathological hypertrophy. Our data suggest that in the absence of mechanisms that support cardiac energy generation and prevention of cell death, as seen following diabetes, there is a transition from physiological to pathological cardiac hypertrophy and a decline in cardiac function.

Reduction in Insulin Uncovers a Novel Effect of VEGFB on Cardiac Substrate Utilization.

BACKGROUND: The heart relies heavily on external fatty acid (FA) for energy production. VEGFB (vascular endothelial growth factor B) has been shown to promote endothelial FA uptake by upregulating FA transporters. However, its impact on LPL (lipoprotein lipase)-mediated lipolysis of lipoproteins, a major source of FA for cardiac use, is unknown. METHODS: VEGFB transgenic (Tg) rats were generated by using the α-myosin heavy chain promoter to drive cardiomyocyte-specific overexpression. To measure coronary LPL activity, Langendorff hearts were perfused with heparin. In vivo positron emission tomography imaging with [18F]-triglyceride-fluoro-6-thia-heptadecanoic acid and [11C]-palmitate was used to determine cardiac FA uptake. Mitochondrial FA oxidation was evaluated by high-resolution respirometry. Streptozotocin was used to induce diabetes, and cardiac function was monitored using echocardiography. RESULTS: In Tg hearts, the vectorial transfer of LPL to the vascular lumen is obstructed, resulting in LPL buildup within cardiomyocytes, an effect likely due to coronary vascular development with its associated augmentation of insulin action. With insulin insufficiency following fasting, VEGFB acted unimpeded to facilitate LPL movement and increase its activity at the coronary lumen. In vivo PET imaging following fasting confirmed that VEGFB induced a greater FA uptake to the heart from circulating lipoproteins as compared with plasma-free FAs. As this was associated with augmented mitochondrial oxidation, lipid accumulation in the heart was prevented. We further examined whether this property of VEGFB on cardiac metabolism could be useful following diabetes and its associated cardiac dysfunction, with attendant loss of metabolic flexibility. In Tg hearts, diabetes inhibited myocyte VEGFB gene expression and protein secretion together with its downstream receptor signaling, effects that could explain its lack of cardioprotection. CONCLUSIONS: Our study highlights the novel role of VEGFB in LPL-derived FA supply and utilization. In diabetes, loss of VEGFB action may contribute toward metabolic inflexibility, lipotoxicity, and development of diabetic cardiomyopathy.

Flow-Induced Secretion of Endothelial Heparanase Regulates Cardiac Lipoprotein Lipase and Changes Following Diabetes.

Background Lipoprotein lipase (LPL)-derived fatty acid is a major source of energy for cardiac contraction. Synthesized in cardiomyocytes, LPL requires translocation to the vascular lumen for hydrolysis of lipoprotein triglyceride, an action mediated by endothelial cell (EC) release of heparanase. We determined whether flow-mediated biophysical forces can cause ECs to secrete heparanase and thus regulate cardiac metabolism. Methods and Results Isolated hearts were retrogradely perfused. Confluent rat aortic ECs were exposed to laminar flow using an orbital shaker. Cathepsin L activity was determined using gelatin-zymography. Diabetes was induced in rats with streptozotocin. Despite the abundance of enzymatically active heparanase in the heart, it was the enzymatically inactive, latent heparanase that was exceptionally responsive to flow-induced release. EC exposed to orbital rotation exhibited a similar pattern of heparanase secretion, an effect that was reproduced by activation of the mechanosensor, Piezo1. The laminar flow-mediated release of heparanase from EC required activation of both the purinergic receptor and protein kinase D, a kinase that assists in vesicular transport of proteins. Heparanase influenced cardiac metabolism by increasing cardiomyocyte LPL displacement along with subsequent replenishment. The flow-induced heparanase secretion was augmented following diabetes and could explain the increased heparin-releasable pool of LPL at the coronary lumen in these diabetic hearts. Conclusions ECs sense fluid shear-stress and communicate this information to subjacent cardiomyocytes with the help of heparanase. This flow-induced mechanosensing and its dynamic control of cardiac metabolism to generate ATP, using LPL-derived fatty acid, is exquisitely adapted to respond to disease conditions, like diabetes.

Human and mouse muscle transcriptomic analyses identify insulin receptor mRNA downregulation in hyperinsulinemia-associated insulin resistance.

Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have demonstrated that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined, especially on a transcriptome-wide level. Transcriptomic meta-analysis in >450 human samples demonstrated that fasting insulin reliably and negatively correlated with INSR mRNA in skeletal muscle. To establish causality and study the direct effects of prolonged exposure to excess insulin in muscle cells, we incubated C2C12 myotubes with elevated insulin for 16 h, followed by 6 h of serum starvation, and established that acute AKT and ERK signaling were attenuated in this model of in vitro hyperinsulinemia. Global RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin receptor (INSR) signaling, FOXO signaling, and glucose metabolism pathways indicative of 'hyperinsulinemia' and 'starvation' programs. Consistently, we observed that hyperinsulinemia led to a substantial reduction in Insr gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Bioinformatic modeling combined with RNAi identified SIN3A as a negative regulator of Insr mRNA (and JUND, MAX, and MXI as positive regulators of Irs2 mRNA). Together, our analysis identifies mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology and disease progression of type 2 diabetes.

Cardiac-specific VEGFB overexpression reduces lipoprotein lipase activity and improves insulin action in rat heart.

Cardiac muscle uses multiple sources of energy including glucose and fatty acid (FA). The heart cannot synthesize FA and relies on obtaining it from other sources, with lipoprotein lipase (LPL) breakdown of lipoproteins suggested to be a key source of FA for cardiac use. Recent work has indicated that cardiac vascular endothelial growth factor B (VEGFB) overexpression expands the coronary vasculature and facilitates metabolic reprogramming that favors glucose utilization. We wanted to explore whether this influence of VEGFB on cardiac metabolism involves regulation of LPL activity with consequent effects on lipotoxicity and insulin signaling. The transcriptomes of rats with and without cardiomyocyte-specific overexpression of human VEGFB were compared by using RNA sequencing. Isolated perfused hearts or cardiomyocytes incubated with heparin were used to enable measurement of LPL activity. Untargeted metabolomic analysis was performed for quantification of cardiac lipid metabolites. Cardiac insulin sensitivity was evaluated using fast-acting insulin. Isolated heart and cardiomyocytes were used to determine transgene-encoded VEGFB isoform secretion patterns and mitochondrial oxidative capacity using high-resolution respirometry and extracellular flux analysis. In vitro, transgenic cardiomyocytes incubated overnight and thus exposed to abundantly secreted VEGFB isoforms, in the absence of any in vivo confounding regulators of cardiac metabolism, demonstrated higher basal oxygen consumption. In the whole heart, VEGFB overexpression induced an angiogenic response that was accompanied by limited cardiac LPL activity through multiple mechanisms. This was associated with a lowered accumulation of lipid intermediates, diacylglycerols and lysophosphatidylcholine, that are known to influence insulin action. In response to exogenous insulin, transgenic hearts demonstrated increased insulin sensitivity. In conclusion, the interrogation of VEGFB function on cardiac metabolism uncovered an intriguing and previously unappreciated effect to lower LPL activity and prevent lipid metabolite accumulation to improve insulin action. VEGFB could be a potential cardioprotective therapy to treat metabolic disorders, for example, diabetes.NEW & NOTEWORTHY In hearts overexpressing vascular endothelial growth factor B (VEGFB), besides its known angiogenic response, multiple regulatory mechanisms lowered coronary LPL. This was accompanied by limited cardiac lipid metabolite accumulation with an augmentation of cardiac insulin action. Our data for the first time links VEGFB to coronary LPL in regulation of cardiac metabolism. VEGFB may be cardioprotective in metabolic disorders like diabetes.

MK2-Deficient Mice Are Bradycardic and Display Delayed Hypertrophic Remodeling in Response to a Chronic Increase in Afterload.

Background Mitogen-activated protein kinase-activated protein kinase-2 (MK2) is a protein serine/threonine kinase activated by p38α/ß. Herein, we examine the cardiac phenotype of pan MK2-null (MK2-/-) mice. Methods and Results Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P=0.580). At 12 weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, aging was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3, were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+ sensitivity to mitochondrial permeability transition pore opening. Eight weeks of pressure overload increased left ventricular mass in MK2+/+ and MK2-/- mice; however, after 2 weeks the increase was significant in MK2+/+ but not MK2-/- mice. Finally, the pressure overload-induced decrease in systolic function was attenuated in MK2-/- mice 2 weeks, but not 8 weeks, after constriction of the transverse aorta. Conclusions Collectively, these results implicate MK2 in (1) autonomic regulation of heart rate, (2) cardiac mitochondrial function, and (3) the early stages of myocardial remodeling in response to chronic pressure overload.

Loss of function of transcription factor EB remodels lipid metabolism and cell death pathways in the cardiomyocyte.

Glucolipotoxicity following nutrient overload causes cardiomyocyte injury by inhibiting TFEB and suppressing lysosomal function. We ascertained whether in addition to the amount, the type of fatty acids (FAs) and duration of FA exposure regulate TFEB action and dictate cardiomyocyte viability. Saturated FA, palmitate, but not polyunsaturated FAs decreased TFEB content in a concentration- and time-dependent manner in cardiomyocytes. Hearts from high-fat high-sucrose diet-fed mice exhibited a temporal decline in nuclear TFEB content with marked elevation of diacylglycerol and triacylglycerol, suggesting that lipid deposition and TFEB loss are concomitant molecular events. Next, we examined the identity of signaling and metabolic pathways engaged by the loss of TFEB action in the cardiomyocyte. Transcriptome analysis in murine cardiomyocytes with targeted deletion of myocyte TFEB (TFEB-/-) revealed enrichment of differentially expressed genes (DEG) representing pathways of nutrient metabolism, DNA damage and repair, cell death and cardiac function. Strikingly, genes involved in macroautophagy, mitophagy and lysosome function constituted a small portion of DEGs in TFEB-/- cardiomyocytes. In myoblasts and/or myocytes, nutrient overload-induced lipid droplet accumulation and caspase-3 activation were exacerbated by silencing TFEB or attenuated by overexpressing constitutively active TFEB. The effect of TFEB overexpression were persistent in the presence of Atg7 loss-of-function, signifying that the effect of TFEB in the myocyte is independent of changes in the macroautophagy pathway. In the cardiomyocyte, the non-canonical effect of TFEB to reprogram energy metabolism is more evident than the canonical action of TFEB on lysosomal autophagy. Loss of TFEB function perturbs metabolic pathways in the cardiomyocyte and renders the heart prematurely susceptible to nutrient overload-induced injury.

Involvement of Heparanase in Endothelial Cell-Cardiomyocyte Crosstalk.

Traditionally, the management of diabetes has focused mainly on controlling high blood glucose levels. Unfortunately, despite valiant efforts to normalize this blood glucose, poor medication management predisposes these patients to heart failure. Following diabetes, how the heart utilizes different sources of fuel for energy is key to the development of heart failure. The diabetic heart switches from using both glucose and fats, to predominately using fats as an energy resource for maintaining its activities. This transformation to using fats as an exclusive source of energy is helpful in the initial stages of the disease and is tightly controlled. However, over the progression of diabetes, there is a loss of this controlled supply and use of fats, which ultimately has terrible consequences since the uncontrolled use of fats produces toxic by-products which weaken heart function and cause heart disease. Heparanase is a key player that directs how much fats are provided to the heart and does so in association with several partners like LPL and VEGFs. Together, they regulate the amount of fats supplied, and their subsequent breakdown to provide energy. Following diabetes, there is a disruption in this network resulting in fat oversupply and cell death. Understanding how the heparanase-LPL-VEGFs "ensemble" cooperates, and its dysfunction in the diabetic heart would be useful in restoring metabolic equilibrium and limiting diabetes-related cardiac damage.

Diabetes Mellitus Severity and a Switch From Using Lipoprotein Lipase to Adipose-Derived Fatty Acid Results in a Cardiac Metabolic Signature That Embraces Cell Death.

Background Fatty acid (FA) provision to the heart is from cardiomyocyte and adipose depots, plus lipoprotein lipase action. We tested how a graded reduction in insulin impacts the source of FA used by cardiomyocytes and the cardiac adaptations required to process these FA. Methods and Results Rats injected with 55 (D55) or 100 (D100) mg/kg streptozotocin were terminated after 4 days. Although D55 and D100 were equally hyperglycemic, D100 showed markedly lower pancreatic and plasma insulin and loss of lipoprotein lipase, which in D55 hearts had expanded. There was minimal change in plasma FA in D55. However, D100 exhibited a 2- to 3-fold increase in various saturated, monounsaturated, and polyunsaturated FA in the plasma. D100 demonstrated dramatic cardiac transcriptomic changes with 1574 genes differentially expressed compared with only 49 in D55. Augmented mitochondrial and peroxisomal ß-oxidation in D100 was not matched by elevated tricarboxylic acid or oxidative phosphorylation. With increasing FA, although control myocytes responded by augmenting basal respiration, this was minimized in D55 and reversed in D100. Metabolomic profiling identified significant lipid accumulation in D100 hearts, which also exhibited sizeable change in genes related to apoptosis and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells. Conclusions With increasing severity of diabetes mellitus, when the diabetic heart is unable to control its own FA supply using lipoprotein lipase, it undergoes dramatic reprogramming that is linked to handling of excess FA that arise from adipose tissue. This transition results in a cardiac metabolic signature that embraces mitochondrial FA overload, oxidative stress, triglyceride storage, and cell death.

Heparanase protects the heart against chemical or ischemia/reperfusion injury.

Although cancer cells use heparanase for tumor metastasis, favourable effects of heparanase have been reported in the management of Alzheimer's disease and diabetes. Indeed, we previously established a protective function for heparanase in the acutely diabetic heart, where it conferred cardiomyocyte resistance to oxidative stress and apoptosis by provoking changes in gene expression. In this study, we tested if overexpression of heparanase can protect the heart against chemically induced or ischemia/reperfusion (I/R) injury. Transcriptomic analysis of Hep-tg hearts reveal that 240 genes related to the stress response, immune response, cell death, and development were altered in a pro-survival direction encompassing genes promoting the unfolded protein response (UPR) and autophagy, as well as those protecting against oxidative stress. The observed UPR activation was adaptive and not apoptotic, was mediated by activation of ATF6α, and when combined with mTOR inhibition, induced autophagy. Subjecting wild type (WT) mice to increasing concentrations of the ER stress inducer thapsigargin evoked a transition from adaptive to apoptotic UPR, an effect that was attenuated in Hep-tg mouse hearts. Consistent with these observations, when exposed to I/R, the infarct size and markers of apoptosis were significantly lower in the Hep-tg heart compared to WT. Finally, UPR and autophagy inhibitors reduced the protective effects of heparanase overexpression during I/R. Our data suggest that the mechanisms that underlie the role of heparanase in promoting cell survival could be uniquely beneficial to the heart by providing protection against cellular stresses, and could be useful for exploitation as a therapeutic target for the treatment of heart disease.

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.

MK5 haplodeficiency attenuates hypertrophy and preserves diastolic function during remodeling induced by chronic pressure overload in the mouse heart.

MAPK-activated protein kinase-5 (MK5) is a protein serine/threonine kinase that is activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. The physiological function(s) of MK5 remains unknown. Here, we examined the effect of MK5 haplodeficiency on cardiac function and myocardial remodeling. At 12 wk of age, MK5 haplodeficient mice (MK5+/-) were smaller than age-matched wild-type littermates (MK5+/+), with similar diastolic function but reduced systolic function. Transverse aortic constriction (TAC) was used to induce chronic pressure overload in 12-wk-old male MK5+/- and MK5+/+ mice. Two weeks post-TAC, heart weight-to-tibia length ratios were similarly increased in MK5+/- and MK5+/+ hearts, as was the abundance of B-type natriuretic peptide and ß-myosin heavy chain mRNA. Left ventricular ejection fraction was reduced in both MK5+/+ and MK5+/- mice, whereas regional peak systolic tissue velocities were reduced and isovolumetric relaxation time was prolonged in MK5+/+ hearts but not in MK5+/- hearts. The TAC-induced increase in collagen type 1-α1 mRNA observed in MK5+/+ hearts was markedly attenuated in MK5+/- hearts. Eight weeks post-TAC, systolic function was equally impaired in MK5+/+ and MK5+/- mice. In contrast, the increase in E wave deceleration rate and progression of hypertrophy observed in TAC MK5+/+ mice were attenuated in TAC MK5+/- mice. MK5 immunoreactivity was detected in adult fibroblasts but not in myocytes. MK5+/+, MK5+/-, and MK5-/- fibroblasts all expressed α-smooth muscle actin in culture. Hence, reduced MK5 expression in cardiac fibroblasts was associated with the attenuation of both hypertrophy and development of a restrictive filling pattern during myocardial remodeling in response to chronic pressure overload.NEW & NOTEWORTHY MAPK-activated protein kinase-5 (MK5)/p38-regulated/activated protein kinase is a protein serine/threonine kinase activated by p38 MAPK and/or the atypical MAPKs ERK3 and ERK4. MK5 immunoreactivity was detected in adult ventricular fibroblasts but not in myocytes. MK5 haplodeficiency attenuated the progression of hypertrophy, reduced collagen type 1 mRNA, and protected diastolic function in response to chronic pressure overload.

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 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 cells respond to hyperglycemia by increasing the LPL transporter GPIHBP1.

In diabetes, when glucose uptake and oxidation are impaired, the heart is compelled to use fatty acid (FA) almost exclusively for ATP. The vascular content of lipoprotein lipase (LPL), the rate-limiting enzyme that determines circulating triglyceride clearance, is largely responsible for this FA delivery and increases following diabetes. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein [GPIHBP1; a protein expressed abundantly in the heart in endothelial cells (EC)] collects LPL from the interstitial space and transfers it across ECs onto the luminal binding sites of these cells, where the enzyme is functional. We tested whether ECs respond to hyperglycemia by increasing GPIHBP1. Streptozotocin diabetes increased cardiac LPL activity and GPIHBP1 gene and protein expression. The increased LPL and GPIHBP1 were located at the capillary lumen. In vitro, passaging EC caused a loss of GPIHBP1, which could be induced on exposure to increasing concentrations of glucose. The high-glucose-induced GPIHBP1 increased LPL shuttling across EC monolayers. GPIHBP1 expression was linked to the EC content of heparanase. Moreover, active heparanase increased GPIHBP1 gene and protein expression. Both ECs and myocyte heparan sulfate proteoglycan-bound platelet-derived growth factor (PDGF) released by heparanase caused augmentation of GPIHBP1. Overall, our data suggest that this protein "ensemble" (heparanase-PDGF-GPIHBP1) cooperates in the diabetic heart to regulate FA delivery and utilization by the cardiomyocytes. Interrupting this axis may be a novel therapeutic strategy to restore metabolic equilibrium, curb lipotoxicity, and help prevent or delay heart dysfunction that is characteristic of diabetes.

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.

G1/S transcription factor orthologues Swi4p and Swi6p are important but not essential for cell proliferation and influence hyphal development in the fungal pathogen Candida albicans.

The G(1)/S transition is a critical control point for cell proliferation and involves essential transcription complexes termed SBF and MBF in Saccharomyces cerevisiae or MBF in Schizosaccharomyces pombe. In the fungal pathogen Candida albicans, G(1)/S regulation is not clear. To gain more insight into the G(1)/S circuitry, we characterized Swi6p, Swi4p and Mbp1p, the closest orthologues of SBF (Swi6p and Swi4p) and MBF (Swi6p and Mbp1p) components in S. cerevisiae. The mbp1Δ/Δ cells showed minor growth defects, whereas swi4Δ/Δ and swi6Δ/Δ yeast cells dramatically increased in size, suggesting a G(1) phase delay. Gene set enrichment analysis (GSEA) of transcription profiles revealed that genes associated with G(1)/S phase were significantly enriched in cells lacking Swi4p and Swi6p. These expression patterns suggested that Swi4p and Swi6p have repressing as well as activating activity. Intriguingly, swi4Δ/Δ swi6Δ/Δ and swi4Δ/Δ mbp1Δ/Δ strains were viable, in contrast to the situation in S. cerevisiae, and showed pleiotropic phenotypes that included multibudded yeast, pseudohyphae, and intriguingly, true hyphae. Consistently, GSEA identified strong enrichment of genes that are normally modulated during C. albicans-host cell interactions. Since Swi4p and Swi6p influence G(1) phase progression and SBF binding sites are lacking in the C. albicans genome, these factors may contribute to MBF activity. Overall, the data suggest that the putative G(1)/S regulatory machinery of C. albicans contains novel features and underscore the existence of a relationship between G(1) phase and morphogenetic switching, including hyphal development, in the pathogen.