Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Interplay of Liver-Heart Inflammatory Axis and Cannabinoid 2 Receptor Signaling in an Experimental Model of Hepatic Cardiomyopathy.

BACKGROUND AND AIMS: Hepatic cardiomyopathy, a special type of heart failure, develops in up to 50% of patients with cirrhosis and is a major determinant of survival. However, there is no reliable model of hepatic cardiomyopathy in mice. We aimed to characterize the detailed hemodynamics of mice with bile duct ligation (BDL)-induced liver fibrosis, by monitoring echocardiography and intracardiac pressure-volume relationships and myocardial structural alterations. Treatment of mice with a selective cannabinoid-2 receptor (CB2 -R) agonist, known to attenuate inflammation and fibrosis, was used to explore the impact of liver inflammation and fibrosis on cardiac function. APPROACH AND RESULTS: BDL induced massive inflammation (increased leukocyte infiltration, inflammatory cytokines, and chemokines), oxidative stress, microvascular dysfunction, and fibrosis in the liver. These pathological changes were accompanied by impaired diastolic, systolic, and macrovascular functions; cardiac inflammation (increased macrophage inflammatory protein 1, interleukin-1, P-selectin, cluster of differentiation 45-positive cells); and oxidative stress (increased malondialdehyde, 3-nitrotyrosine, and nicotinamide adenine dinucleotide phosphate oxidases). CB2 -R up-regulation was observed in both livers and hearts of mice exposed to BDL. CB2 -R activation markedly improved hepatic inflammation, impaired microcirculation, and fibrosis. CB2 -R activation also decreased serum tumor necrosis factor-alpha levels and improved cardiac dysfunction, myocardial inflammation, and oxidative stress, underlining the importance of inflammatory mediators in the pathology of hepatic cardiomyopathy. CONCLUSIONS: We propose BDL-induced cardiomyopathy in mice as a model for hepatic/cirrhotic cardiomyopathy. This cardiomyopathy, similar to cirrhotic cardiomyopathy in humans, is characterized by systemic hypotension and impaired macrovascular and microvascular function accompanied by both systolic and diastolic dysfunction. Our results indicate that the liver-heart inflammatory axis has a pivotal pathophysiological role in the development of hepatic cardiomyopathy. Thus, controlling liver and/or myocardial inflammation (e.g., with selective CB2 -R agonists) may delay or prevent the development of cardiomyopathy in severe liver disease.

Decorin Protects Cardiac Myocytes against Simulated Ischemia/Reperfusion Injury.

Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.

Disruption of Renal Arginine Metabolism Promotes Kidney Injury in Hepatorenal Syndrome in Mice.

Tubular dysfunction is an important feature of renal injury in hepatorenal syndrome (HRS) in patients with end-stage liver disease. The pathogenesis of kidney injury in HRS is elusive, and there are no clinically relevant rodent models of HRS. We investigated the renal consequences of bile duct ligation (BDL)-induced hepatic and renal injury in mice in vivo by using biochemical assays, real-time polymerase chain reaction (PCR), Western blot, mass spectrometry, histology, and electron microscopy. BDL resulted in time-dependent hepatic injury and hyperammonemia which were paralleled by tubular dilation and tubulointerstitial nephritis with marked upregulation of lipocalin-2, kidney injury molecule 1 (KIM-1) and osteopontin. Renal injury was associated with dramatically impaired microvascular flow and decreased endothelial nitric oxide synthase (eNOS) activity. Gene expression analyses signified proximal tubular epithelial injury, tissue hypoxia, inflammation, and activation of the fibrotic gene program. Marked changes in renal arginine metabolism (upregulation of arginase-2 and downregulation of argininosuccinate synthase 1), resulted in decreased circulating arginine levels. Arginase-2 knockout mice were partially protected from BDL-induced renal injury and had less impairment in microvascular function. In human-cultured proximal tubular epithelial cells hyperammonemia per se induced upregulation of arginase-2 and markers of tubular cell injury. CONCLUSION: We propose that hyperammonemia may contribute to impaired renal arginine metabolism, leading to decreased eNOS activity, impaired microcirculation, tubular cell death, tubulointerstitial nephritis and fibrosis. Genetic deletion of arginase-2 partially restores microcirculation and thereby alleviates tubular injury. We also demonstrate that BDL in mice is an excellent, clinically relevant model to study the renal consequences of HRS. (Hepatology 2018; 00:000-000).

Cannabinoid-1 receptor deletion in podocytes mitigates both glomerular and tubular dysfunction in a mouse model of diabetic nephropathy.

AIMS: To determine the specific role of podocyte-expressed cannabinoid-1 receptor (CB1 R) in the development of diabetic nephropathy (DN), relative to CB1 R in other renal cell types. MATERIAL AND METHODS: We developed a mouse model with a podocyte-specific deletion of CB1 R (pCB1Rko) and challenged this model with streptozotocin (STZ)-induced type-1 DN. We also assessed the podocyte response to high glucose in vitro and its effects on CB1 R activation. RESULTS: High glucose exposure for 48 hours led to an increase in CB1 R gene expression (CNR1) and endocannabinoid production in cultured human podocytes. This was associated with podocyte injury, reflected by decreased podocin and nephrin expression. These changes could be prevented by Cnr1-silencing, thus identifying CB1R as a key player in podocyte injury. After 12 weeks of chronic hyperglycaemia, STZ-treated pCB1Rko mice showed elevated blood glucose similar to that of their wild-type littermates. However, they displayed less albuminuria and less podocyte loss than STZ-treated wild-type mice. Unexpectedly, pCB1Rko mice also have milder tubular dysfunction, fibrosis and reduction of cortical microcirculation compared to wild-type controls, which is mediated, in part, by podocyte-derived endocannabinoids acting via CB1 R on proximal tubular cells. CONCLUSIONS: Activation of CB1 R in podocytes contributes to both glomerular and tubular dysfunction in type-1 DN, which highlights the therapeutic potential of peripheral CB1 R blockade.

PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). METHODS: We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. RESULTS: PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1-/- animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. CONCLUSION: Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer. LAY SUMMARY: Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.

Chronic plus binge ethanol feeding induces myocardial oxidative stress, mitochondrial and cardiovascular dysfunction, and steatosis.

Alcoholic cardiomyopathy in humans develops in response to chronic excessive alcohol consumption; however, good models of alcohol-induced cardiomyopathy in mice are lacking. Herein we describe mouse models of alcoholic cardiomyopathies induced by chronic and binge ethanol (EtOH) feeding and characterize detailed hemodynamic alterations, mitochondrial function, and redox signaling in these models. Mice were fed a liquid diet containing 5% EtOH for 10, 20, and 40 days (d) combined with single or multiple EtOH binges (5 g/kg body wt). Isocalorically pair-fed mice served as controls. Left ventricular (LV) function and morphology were assessed by invasive pressure-volume conductance approach and by echocardiography. Mitochondrial complex (I, II, IV) activities, 3-nitrotyrosine (3-NT) levels, gene expression of markers of oxidative stress (gp91phox, p47phox), mitochondrial biogenesis (PGC1α, peroxisome proliferator-activated receptor α), and fibrosis were examined. Cardiac steatosis and fibrosis were investigated by histological/immunohistochemical methods. Chronic and binge EtOH feeding (already in 10 days EtOH plus single binge group) was characterized by contractile dysfunction (decreased slope of end-systolic pressure-volume relationship and preload recruitable stroke work), impaired relaxation (decreased time constant of LV pressure decay and maximal slope of systolic pressure decrement), and vascular dysfunction (impaired arterial elastance and lower total peripheral resistance). This was accompanied by enhanced myocardial oxidative/nitrative stress (3-NT; gp91phox; p47phox; angiotensin II receptor, type 1a) and deterioration of mitochondrial complex I, II, IV activities and mitochondrial biogenesis, excessive cardiac steatosis, and higher mortality. Collectively, chronic plus binge EtOH feeding in mice leads to alcohol-induced cardiomyopathies (National Institute on Alcohol Abuse and Alcoholism models) characterized by increased myocardial oxidative/nitrative stress, impaired mitochondrial function and biogenesis, and enhanced cardiac steatosis.

Novel, selective EPO receptor ligands lacking erythropoietic activity reduce infarct size in acute myocardial infarction in rats.

Erythropoietin (EPO) has been shown to protect the heart against acute myocardial infarction in pre-clinical studies, however, EPO failed to reduce infarct size in clinical trials and showed significant safety problems. Here, we investigated cardioprotective effects of two selective non-erythropoietic EPO receptor ligand dimeric peptides (AF41676 and AF43136) lacking erythropoietic activity, EPO, and the prolonged half-life EPO analogue, darbepoetin in acute myocardial infarction (AMI) in rats. In a pilot study, EPO at 100U/mL significantly decreased cell death compared to vehicle (33.8±2.3% vs. 40.3±1.5%, p<0.05) in rat neonatal cardiomyocytes subjected to simulated ischemia/reperfusion. In further studies (studies 1-4), in vivo AMI was induced by 30min coronary occlusion and 120min reperfusion in male Wistar rats. Test compounds and positive controls for model validation (B-type natriuretic peptide, BNP or cyclosporine A, CsA) were administered iv. before the onset of reperfusion. Infarct size (IS) was measured by standard TTC staining. In study 1, 5000U/kg EPO reduced infarct size significantly compared to vehicle (45.3±4.8% vs. 59.8±4.5%, p<0.05). In study 2, darbepoetin showed a U-shaped dose-response curve with maximal infarct size-reducing effect at 5µg/kg compared to the vehicle (44.4±5.7% vs. 65.9±2.7%, p<0.01). In study 3, AF41676 showed a U-shaped dose-response curve, where 3mg/kg was the most effective dose compared to the vehicle (24.1±3.9% vs. 44.3±2.5%, p<0.001). The positive control BNP significantly decreased infarct size in studies 1-3 by approximately 35%. In study 4, AF43136 at 10mg/kg decreased infarct size, similarly to the positive control CsA compared to the appropriate vehicle (39.4±5.9% vs. 58.1±5.4% and 45.9±2.4% vs. 63.8±4.1%, p<0.05, respectively). This is the first demonstration that selective, non-erythropoietic EPO receptor ligand dimeric peptides AF41676 and AF43136 administered before reperfusion are able to reduce infarct size in a rat model of AMI. Therefore, non-erythropoietic EPO receptor peptide ligands may be promising cardioprotective agents.

Renin-Angiotensin-Aldosterone Signaling Inhibitors-Losartan, Enalapril, and Cardosten-Prevent Infarction-induced Heart Failure Development in Rats.

CONTEXT: The activation of the renin-angiotensin-aldosterone system (RAAS) plays an important role in the pathophysiology of congestive heart failure, which is the reason that angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin 2 receptor blockers (ARBs) have become established therapies for heart failure. However, it is still not known whether preventive treatment with losartan or enalapril can reduce symptoms of infarction-induced heart failure. Ultra-low dose (ULD) drug therapy is thought to exert specific activity, with a lower chance of side effects. OBJECTIVES • The research team had hypothesized that preventive treatment with inhibitors of RAAS signaling-losartan, enalapril, and a preparation of a ULD antibody (ie, cardosten), which target the angiotensin type 1 (AT1) receptor-might alleviate pathological hypertrophy and/or functional decline in infarction-induced heart failure. METHODS: The research team treated male Wistar rats orally for 30 d with 20 mg/kg of losartan, 10 mg/kg enalapril, 5 or 7.5 mL/kg of cardosten, or a control solution, started 1 d prior to permanent coronary occlusion. A sham-operated group functioned as a second control group. SETTINGS: The study was conducted at the Department of Biochemistry of the Faculty of Medicine at the University of Szeged in Szeged, Hungary, in cooperation with the Pharmahungary Group, also in Szeged, Hungary, and with OOO "NPF" Materia Medica Holding Ltd in Moscow, Russia. OUTCOME MEASURES: To determine cardiac functional parameters in vivo, the research team inserted a catheter into the left ventricle of the rats and measured the parameters of ventricular pressure, and cardiac output was determined by thermodilution. Morphological parameters were measured after heart isolation in transverse sections by a digital caliper. RESULTS: A total of 30 d after permanent coronary ligation, both losartan and enalapril, significantly decreased mean arterial blood pressure (MABP), attenuated the development of the left-ventricular anterior-wall and septum hypertrophy, and reduced scar thickness compared with the vehicle control group. The deterioration of cardiac output and the increase in total peripheral resistance (TPR) due to coronary ligation were significantly inhibited by both losartan and enalapril. The effects of cardosten were comparable with those of losartan and enalapril on cardiac morphology, left ventricular function, and TPR; however, it did not influence MABP. Moreover, in contrast to losartan and enalapril, cardosten did not decrease the rate of survival. CONCLUSIONS: The study was the first to have demonstrated that preventive treatment with losartan, enalapril, or cardosten can attenuate pathological hypertrophy in infarction-induced heart failure in rats.

Activation of PPARß/δ protects cardiac myocytes from oxidative stress-induced apoptosis by suppressing generation of reactive oxygen/nitrogen species and expression of matrix metalloproteinases.

Heart failure still remains one of the leading causes of morbidity and mortality worldwide. A major contributing factor is reactive oxygen/nitrogen species (RONS) overproduction which is associated with cardiac remodeling partly through cardiomyocyte apoptosis. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily and have been implicated in cardioprotection. However, the molecular mechanisms are largely unexplored. In this study we sought to investigate the potential beneficial effects evoked by activation of PPARß/δ under the setting of oxidative stress induced by H2O2 in adult rat cardiac myocytes. The selective PPARß/δ agonist GW0742 inhibited the H2O2-induced apoptosis and increased cell viability. In addition, generation of RONS was attenuated in cardiac myocytes in the presence of PPARß/δ agonist. These effects were abolished in the presence of the PPARß/δ antagonist indicating that the effect was through PPARß/δ receptor activation. Treatment with PPARß/δ agonist was also associated with attenuation of caspase-3 and PARP cleavage, upregulation of anti-apoptotic Bcl-2 and concomitant downregulation of pro-apoptotic Bax. In addition, activation of PPARß/δ inhibited the oxidative-stress-induced MMP-2 and MMP-9 mRNA upregulation. It is concluded that PPARß/δ activation exerts a cytoprotective effect in adult rat cardiac myocytes subjected to oxidative stress via inhibition of oxidative stress, MMP expression, and apoptosis. Our data suggest that the novel connection between PPAR signaling and MMP down-regulation in cardiac myocytes might represent a new target for the management of oxidative stress-induced cardiac dysfunction.