Katalin Erdélyi

Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation

Hydrogen sulfide (H2S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H2S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H2S and NO exert their effects via separate pathways, the results of the current study show that H2S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H2S increases intracellular cyclic guanosine 5′-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H2S-stimulated angiogenic response, and attenuated H2S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H2S signaling. Conversely, silencing of the H2S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H2S in the vascular activity of NO. The actions of H2S and NO converge at cGMP; though H2S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H2S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H2S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H2S are mutually required for the physiological control of vascular function.

Intramitochondrial hydrogen sulfide production by 3-mercaptopyruvate sulfurtransferase maintains mitochondrial electron flow and supports cellular bioenergetics

It is well established that exposure of mammalian cells to hydrogen sulfide (H2S) suppresses mitochondrial function by inhibiting cytochrome‐c oxidase (CcOX; complex IV). However, recent experimental data show that administration of H2S to mammalian cells can serve as an electron donor and inorganic source of energy. The aim of our study was to investigate the role of endogenously produced H2S in the regulation of mitochondrial electron transport and oxidative phosphorylation in isolated liver mitochondria and in the cultured murine hepatoma cell line Hepa1c1c7. Low concentrations of H2S (0.1–1 μM) elicited a significant increase in mitochondrial function, while higher concentrations of H2S (3–30 μM) were inhibitory. The positive bioenergetic effect of H2S required a basal activity of the Krebs cycle and was most pronounced at intermediate concentrations of succinate. 3‐mercaptopyruvate (3‐MP), the substrate of the mitochondrial enzyme 3‐mercaptopyruvate sulfurtransferase (3‐MST) stimulated mitochondrial H2S production and enhanced mitochondrial electron transport and cellular bioenergetics at low concentrations (10–100 nM), while at higher concentrations, it inhibited cellular bioenergetics. SiRNA silencing of 3‐MST reduced basal bioenergetic parameters and prevented the stimulating effect of 3‐MP on mitochondrial bioenergetics. Silencing of sulfide quinone oxidoreductase (SQR) also reduced basal and 3‐MP‐stimulated bioenergetic parameters. We conclude that an endogenous intramitochondrial H2S‐producing pathway, governed by 3‐MST, complements and balances the bioenergetic role of Krebs cycle‐derived electron donors. This pathway may serve a physiological role in the maintenance of mitochondrial electron transport and cellular bioenergetics.—Módis, K., Coletta, C., Erdélyi, K., Papapetropoulos, A., Szabo, C. Intramitochondrial hydrogen sulfide production by 3‐mercaptopyruvate sulfurtransferase maintains mitochondrial electron flow and supports cellular bioenergetics. FASEB J. 27, 601–611 (2013). www.fasebj.org

Dual role of poly(ADP-ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells

Activation of poly(ADP‐ribose) polymerase‐1 (PARP1) has been shown to mediate cell death induced by genotoxic stimuli. The role of poly(ADP‐ribose) glycohydrolase (PARG), the enzyme responsible for polymer degradation, has been largely unexplored in the regulation of cell death. Using lentiviral gene silencing we generated A549 lung adenocarcinoma cell lines with stably suppressed PARG and PARP1 expression (shPARG and shPARP1 cell lines, respectively) and determined parameters of apoptotic and necrotic cell death following hydrogen peroxide exposure. shPARG cells accumulated large amounts of poly(ADP‐ribosyl)ated proteins and exhibited reduced PARP activation. Hydrogen peroxide‐induced cell death is regulated by PARG in a dual fashion. Whereas the shPARG cell line (similarly to shPARP1 cells) was resistant to the necrotic effect of high concentrations of hydrogen peroxide, these cells exhibited stronger apoptotic response. Both shPARP1 and especially shPARG cells displayed a delayed repair of DNA breaks and exhibited reduced clonogenic survival following hydrogen peroxide treatment. Translocation of apoptosis‐inducing factor could not be observed, but cells could be saved by methyl pyruvate and α‐ketoglutarate, indicating that energy failure may mediate cytotoxicity in our model. These data indicate that PARG is a survival factor at mild oxidative damage but contributes to the apoptosis‐necrosis switch in severely damaged cells.—Erdélyi, K., Bai, P., Kovács, I., Szabó, E., Mocsar, G., Kakuk, A., Szabó, C., Gergely, P., Virag, L. Dual role of poly(ADP‐ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells. FASEB J. 23, 3553–3563 (2009). www.fasebj.org

Monoacylglycerol Lipase Controls Endocannabinoid and Eicosanoid Signaling and Hepatic Injury in Mice

BACKGROUND & AIMS The endocannabinoid and eicosanoid lipid signaling pathways have important roles in inflammatory syndromes. Monoacylglycerol lipase (MAGL) links these pathways, hydrolyzing the endocannabinoid 2-arachidonoylglycerol to generate the arachidonic acid precursor pool for prostaglandin production. We investigated whether blocking MAGL protects against inflammation and damage from hepatic ischemia/reperfusion (I/R) and other insults. METHODS We analyzed the effects of hepatic I/R in mice given the selective MAGL inhibitor JZL184, in Mgll(-/-) mice, fatty acid amide hydrolase(-/-) mice, and in cannabinoid receptor type 1(-/-) (CB1-/-) and cannabinoid receptor type 2(-/-) (CB2-/-). Liver tissues were collected and analyzed, along with cultured hepatocytes and Kupffer cells. We measured endocannabinoids, eicosanoids, and markers of inflammation, oxidative stress, and cell death using molecular biology, biochemistry, and mass spectrometry analyses. RESULTS Wild-type mice given JZL184 and Mgll(-/-) mice were protected from hepatic I/R injury by a mechanism that involved increased endocannabinoid signaling via CB2 and reduced production of eicosanoids in the liver. JZL184 suppressed the inflammation and oxidative stress that mediate hepatic I/R injury. Hepatocytes were the major source of hepatic MAGL activity and endocannabinoid and eicosanoid production. JZL184 also protected from induction of liver injury by D-(+)-galactosamine and lipopolysaccharides or CCl4. CONCLUSIONS MAGL modulates hepatic injury via endocannabinoid and eicosanoid signaling; blockade of this pathway protects mice from liver injury. MAGL inhibitors might be developed to treat conditions that expose the liver to oxidative stress and inflammatory damage.

Poly (ADP-ribose) polymerase-1 is a key mediator of liver inflammation and fibrosis.

Poly (ADP‐ribose) polymerase 1 (PARP‐1) is a constitutive enzyme, the major isoform of the PARP family, which is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. Pharmacological inhibitors of PARP provide significant therapeutic benefits in various preclinical disease models associated with tissue injury and inflammation. However, our understanding the role of PARP activation in the pathophysiology of liver inflammation and fibrosis is limited. In this study we investigated the role of PARP‐1 in liver inflammation and fibrosis using acute and chronic models of carbon tetrachloride (CCl4)‐induced liver injury and fibrosis, a model of bile duct ligation (BDL)‐induced hepatic fibrosis in vivo, and isolated liver‐derived cells ex vivo. Pharmacological inhibition of PARP with structurally distinct inhibitors or genetic deletion of PARP‐1 markedly attenuated CCl4‐induced hepatocyte death, inflammation, and fibrosis. Interestingly, the chronic CCl4‐induced liver injury was also characterized by mitochondrial dysfunction and dysregulation of numerous genes involved in metabolism. Most of these pathological changes were attenuated by PARP inhibitors. PARP inhibition not only prevented CCl4‐induced chronic liver inflammation and fibrosis, but was also able to reverse these pathological processes. PARP inhibitors also attenuated the development of BDL‐induced hepatic fibrosis in mice. In liver biopsies of subjects with alcoholic or hepatitis B‐induced cirrhosis, increased nitrative stress and PARP activation was noted. Conclusion: The reactive oxygen/nitrogen species‐PARP pathway plays a pathogenetic role in the development of liver inflammation, metabolism, and fibrosis. PARP inhibitors are currently in clinical trials for oncological indications, and the current results indicate that liver inflammation and liver fibrosis may be additional clinical indications where PARP inhibition may be of translational potential. (Hepatology 2014;59:1998–2009)

Cellular bioenergetics is regulated by PARP1 under resting conditions and during oxidative stress

PURPOSE The goal of the current studies was to elucidate the role of the principal poly(ADP-ribose)polymerase isoform, PARP1 in the regulation of cellular energetics in endothelial cells under resting conditions and during oxidative stress. METHODS We utilized bEnd.3 endothelial cells and A549 human transformed epithelial cells. PARP1 was inhibited either by pharmacological inhibitors or by siRNA silencing. The Seahorse XF24 Extracellular Flux Analyzer was used to measure indices of mitochondrial respiration (oxygen consumption rate) and of glycolysis (extracellular acidification rate). Cell viability, cellular and mitochondrial NAD(+) levels and mitochondrial biogenesis were also measured. RESULTS Silencing of PARP1 increased basal cellular parameters of oxidative phosphorylation, providing direct evidence that PARP1 is a regulator of mitochondrial function in resting cells. Pharmacological inhibitors of PARP1 and siRNA silencing of PARP1 protected against the development of mitochondrial dysfunction and elevated the respiratory reserve capacity in endothelial and epithelial cells exposed to oxidative stress. The observed effects were unrelated to an effect on mitochondrial biogenesis. Isolated mitochondria of A549 human transformed epithelial cells exhibited an improved resting bioenergetic status after stable lentiviral silencing of PARP1; these effects were associated with elevated resting mitochondrial NAD+ levels in PARP1 silenced cells. CONCLUSIONS PARP1 is a regulator of basal cellular energetics in resting endothelial and epithelial cells. Furthermore, endothelial cells respond with a decrease in their mitochondrial reserve capacity during low-level oxidative stress, an effect, which is attenuated by PARP1 inhibition. While PARP1 is a regulator of oxidative phosphorylation in resting and oxidatively stressed cells, it only exerts a minor effect on glycolysis.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX’s cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly(ADP)-ribose polymerase 1 (PARP)-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing

Extracellular ATP binds to and signals through P2X7 receptors (P2X7Rs) to modulate immune function in both inflammasome‐dependent and ‐independent manners. In this study, P2X7‐/‐ mice, the pharmacological agonists ATP‐magnesium salt (Mg‐ATP; 100 mg/kg, EC50 ≈ 1.32 mM) and benzoylbenzoyl‐ATP (Bz‐ATP; 10 mg/kg, EC50 ≈ 285 μM), and antagonist oxidized ATP (oxi‐ATP; 40 mg/kg, IC50 ≈ 100 μM) were used to show that P2X7R activation is crucial for the control of mortality, bacterial dissemination, and inflammation in cecal ligation and puncture‐induced polymicrobial sepsis in mice. Our results with P2X7‐/‐ bone marrow chimeric mice, adoptive transfer of peritoneal macrophages, and myeloid‐specific P2X7‐/‐ mice indicate that P2X7R signaling on macrophages is essential for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, which is independent of the inflammasome. By using the connexin (Cx) channel inhibitor Gap27 (0.1 mg/kg, IC50 ≈ 0.25 μM) and pannexin channel inhibitor probenecid (10 mg/kg, IC50 ≈ 11.7 μM), we showed that ATP release through Cx is important for inhibiting inflammation and bacterial burden. In summary, targeting P2X7Rs provides a new opportunity for harnessing an endogenous protective immune mechanism in the treatment of sepsis.—Csóka, B., Németh, Z. H., Törő, G., Idzko, M., Zech, A., Koscsó, B., Spolarics, Z., Antonioli, L., Cseri, K., Erdélyi, K., Pacher, P., Haskó, G. Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing. FASEB J. 29, 3626‐3637 (2015). www.fasebj.org

Cytoprotective effect of gallotannin in oxidatively stressed HaCaT keratinocytes: The role of poly(ADP-ribose) metabolism

Abstract:  Oxidative stress‐induced cytotoxicity is mediated in part by accelerated poly‐ADP ribosylation. Peroxynitrite and hydrogen peroxide cause DNA breakage triggering the activation of the DNA nick sensor enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1). Overactivation of PARP‐1 leads to cell dysfunction and cell death mainly due to depletion of NAD+ (the substrate of PARP‐1) and ATP. PARP‐1 attaches most ADP‐ribose residues onto itself, leading to downregulation of enzyme activity. Here, we have investigated the role of poly(ADP‐ribose) glycohydrolase (PARG), the poly(ADP‐ribose)‐catabolyzing enzyme in oxidative stress‐induced cytotoxicity in HaCaT cells. We have found that inhibition of PARG by gallotannin (GT) (50 µM) provided significant cytoprotection to peroxynitrite‐ or hydrogen peroxide‐treated HaCaT cells, as assessed by lactate dehydrogenase release and propidium iodide uptake (parameters of necrotic cell death) as well as caspase activation (apoptotic parameter). GT pretreatment has also inhibited the depletion of cellular NAD+ pools in hydrogen peroxide‐ or peroxynitrite‐treated HaCaT cells. GT caused the accumulation of poly(ADP‐ribose) and concomitant inhibition in cellular PARP activity in oxidatively stressed cells. Therefore, PARG is likely to contribute to maintaining the active state of PARP‐1 by continuously removing inhibitory ADP‐ribose residues from PARP‐1.

Hydrogen peroxide-induced poly(ADP-ribosyl)ation regulates osteogenic differentiation-associated cell death.

We set out to investigate the role of poly(ADP-ribosylation), the attachment of NAD(+)-derived (ADP-ribose)(n) polymers to proteins, in the regulation of osteogenic differentiation of SAOS-2 cells and mesenchymal stem cells. In osteogenic differentiation medium, SAOS-2 cells showed mineralization and expressed alkaline phosphatase and osteoblastic marker genes such as Runx2, osterix, BMP2, and osteopontin. The cells also released hydrogen peroxide, displayed poly(ADP-ribose) polymerase (PARP) activation, and showed commitment to cell death (apoptosis and necrosis). Scavenging reactive oxygen species by glutathione or decomposing hydrogen peroxide by the addition of catalase reduced differentiation, PARP activation, and cell death. We silenced the expression of the main PAR-synthesizing enzyme PARP-1 and the PAR-degrading enzyme poly(ADP-ribose) glycohydrolase (PARG) in SAOS-2 osteosarcoma cells (shPARP-1 and shPARG, respectively). Both shPARP-1- and shPARG-silenced cells exhibited altered differentiation, with the most notable change being increased osteopontin expression but decreased alkaline phosphatase activity. PARP-1 silencing suppressed both apoptotic and necrotic cell death, but the PARP inhibitor PJ34 sensitized cells to cell death, indicating that the effects of PARP-1 silencing are not related to the activity of the enzyme. PARG silencing resulted in more apoptosis and, in the last days of differentiation, a shift from apoptosis toward necrosis. In conclusion our data prove that hydrogen peroxide-induced poly(ADP-ribose) signaling regulates cell death and osteodifferentiation.