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.

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.

Bispyridinium non-oximes: An evaluation of cardiac effects in isolated hearts and smooth muscle relaxing effects in jejunum.

Bispyridinium non-oximes seem to be promising candidates for the generic treatment of nerve agent poisoning as they interact with nicotinic and muscarinic acetylcholine receptors. The lead compound MB327 showed therapeutic effectiveness in vitro and in vivo but was toxic at higher doses. In the present study, the effect of various bispyridinium non-oximes on isolated heart and small intestine function was investigated. Bispyridinium non-oximes and oximes were tested in at least seven different concentrations in rat jejunum preparations pre-treated with carbachol. All bispyridinium non-oximes showed classical dose response curves with MB327 being the most effective (EC50=6.6µM) and MB782 being slightly less effective (EC50=10.4µM). Neither the bispyridinium non-oximes nor the oximes showed cardiotoxic effects in the isolated Langendorff heart. The tested bispyridinum compounds showed no direct cardiac effect but had variable smooth muscle relaxing effects. Further in vivo studies are required to get more insight into potential toxic mechanisms of these promising nerve agent antidotes.

Blaptica dubia as sentinels for exposure to chemical warfare agents - a pilot study.

The increased interest of terrorist groups in toxic chemicals and chemical warfare agents presents a continuing threat to our societies. Early warning and detection is a key component for effective countermeasures against such deadly agents. Presently available and near term solutions have a number of major drawbacks, e.g. lack of automated, remote warning and detection of primarily low volatile chemical warfare agents. An alternative approach is the use of animals as sentinels for exposure to toxic chemicals. To overcome disadvantages of vertebrates the present pilot study was initiated to investigate the suitability of South American cockroaches (Blaptica dubia) as warning system for exposure to chemical warfare nerve and blister agents. Initial in vitro experiments with nerve agents showed an increasing inhibitory potency in the order tabun - cyclosarin - sarin - soman - VX of cockroach cholinesterase. Exposure of cockroaches to chemical warfare agents resulted in clearly visible and reproducible reactions, the onset being dependent on the agent and dose. With nerve agents the onset was related to the volatility of the agents. The blister agent lewisite induced signs largely comparable to those of nerve agents while sulfur mustard exposed animals exhibited a different sequence of events. In conclusion, this first pilot study indicates that Blaptica dubia could serve as a warning system to exposure of chemical warfare agents. A cockroach-based system will not detect or identify a particular chemical warfare agent but could trigger further actions, e.g. specific detection and increased protective status. By designing appropriate boxes with (IR) motion sensors and remote control (IR) camera automated off-site warning systems could be realized.

On-site analysis of acetylcholinesterase and butyrylcholinesterase activity with the ChE check mobile test kit-Determination of reference values and their relevance for diagnosis of exposure to organophosphorus compounds.

Poisoning by organophosphorus compounds (OP) still poses a major medical challenge. Diagnosis of clinical signs of OP poisoning is still the most important parameter for the initiation of specific treatment. However, in case of unspecific signs and of delayed onset of cholinergic crisis a rapid, reliable and on-site analysis of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity would be of great value. Recently the ChE check mobile, a CE-certified ready to use kit for the determination of whole blood AChE and BChE activities, was developed. Here, we evaluated whole blood AChE and BChE reference values with samples taken from 181 male and 61 female volunteers and analyzed them on-site with the ChE check mobile test kit. The analysis of the data revealed a large inter-individual variability (BChE>AChE), only a small sex difference for AChE but a significant difference for BChE activities. The now available normal range values enable an evaluation of determined AChE and BChE activities in case of suspected exposure to OP nerve agents and pesticides. However, the large inter-individual variability of AChE and BChE activities calls for the determination of pre-exposure values in specific subpopulations in order to enable the diagnosis of low-level OP exposure.

Single treatment of VX poisoned guinea pigs with the phosphotriesterase mutant C23AL: Intraosseous versus intravenous injection.

The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge (∼2LD50). C23AL showed a Cmax of 0.63µmolL(-1) after i.o. and i.v. administration of 2mgkg(-1) providing a stable plasma profile up to 180min experimental duration with 0.41 and 0.37µmolL(-1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning.

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.