Shock, inflammation and PARP.

The activation of poly(ADP-ribose) polymerase (PARP) is well considered to play an important role in various patho-physiological conditions like inflammation and shock. A vast amount of circumstantial evidence implicates oxygen-derived free radicals (especially, superoxide and hydroxyl radical) and high-energy oxidants (such as peroxynitrite) as mediators of inflammation and shock. ROS (e.g., superoxide, peroxynitrite, hydroxyl radical and hydrogen peroxide) are all potential reactants capable of initiating DNA single strand breakage, with subsequent activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. During the last years, numerous experimental studies have clearly demonstrated the beneficial effects of PARP inhibition in cell cultures through rodent models and more recently in pre-clinical large animal models of acute and chronic inflammation. The aim of this review is to describe recent experimental evidence implicating PARP as a pathophysiological modulator of acute and chronic inflammation.     

Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury

A vast amount of circumstantial evidence implicates oxygen-derived free radicals (especially superoxide and hydroxyl radical) and high-energy oxidants (such as peroxynitrite) as mediators of inflammation, shock, and ischemia/reperfusion injury. The aim of this review is to describe recent developments in the field of oxidative stress research. The first part of the review focuses on the roles of reactive oxygen species (ROS) in shock, inflammation, and ischemia/reperfusion injury. The second part of the review deals with the novel findings using recently identified pharmacological tools (e.g., peroxynitrite decomposition catalysts and selective superoxide dismutase mimetics (SODm) in shock, ischemia/reperfusion, and inflammation. 1) The role of ROS consists of immunohistochemical and biochemical evidence that demonstrates the production of ROS in shock, inflammation, and ischemia/reperfusion injury. ROS can initiate a wide range of toxic oxidative reactions. These include initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane sodium/potassium ATPase activity, inactivation of membrane sodium channels, and other oxidative modifications of proteins. All these toxicities are likely to play a role in the pathophysiology of shock, inflammation, and ischemia/reperfusion. 2) Treatment with either peroxynitrite decomposition catalysts, which selectively inhibit peroxynitrite, or with SODm, which selectively mimic the catalytic activity of the human superoxide dismutase enzymes, have been shown to prevent in vivo the delayed vascular decompensation and the cellular energetic failure associated with shock, inflammation, and ischemia/reperfusion injury. ROS (e.g., superoxide, peroxynitrite, hydroxyl radical, and hydrogen peroxide) are all potential reactants capable of initiating DNA single-strand breakage, with subsequent activation of the nuclear enzyme poly(ADP-ribose) synthetase, leading to eventual severe energy depletion of the cells and necrotic-type cell death. Antioxidant treatment inhibits the activation of poly(ADP-ribose) synthetase and prevents the organ injury associated with shock, inflammation, and ischemia/reperfusion.     

Role of poly(ADP-ribose) glycohydrolase (PARG) in shock, ischemia and reperfusion.

Poly(ADP-ribosyl)ation is regulated by the synthesizing enzyme poly(ADP-ribose) polymerase-1 (PARP-1) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). Homeostasis of poly(ADP-ribosyl)ation has been proposed to be an important regulator for pathogenesis in multi-cellular organisms. Although the role of PARP-1 in tissue damage, inflammation and ischemia has been extensively studied, the function of PARG in various cellular processes is largely unknown. Recent studies using chemical inhibitors of PARG and genetically engineered Drosophila and mouse models that carry a disrupted PARG gene have started to shed new light on the biological function of PARG in vivo. These animal models and cells isolated from them will be useful for further validation of PARG as a potential pharmaceutical target to intervene the pathogenesis induced by acute tissue injury, ischemia and inflammation.     

Potential therapeutic effect of antioxidant therapy in shock and inflammation

Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants. A considerable body of recent evidence suggests that oxidant stress plays a major role in several aspects of acute and chronic inflammation and is the subject of this review. Immunohistochemical and biochemical evidence demonstrate the significant role of reactive oxygen species (ROS) in acute and chronic inflammation. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na+/K+ ATP-ase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of ROS. All these toxicities are likely to play a role in the pathophysiology of shock, inflammation and ischemia and reperfusion. (2) Treatment with either peroxynitrite decomposition catalysts, which selectively inhibit peroxynitrite, or with SODm's, which selectively mimic the catalytic activity of the human superoxide dismutase (SOD) enzymes, have been shown to prevent in vivo the delayed tissue injury and the cellular energetic failure associated with inflammation. ROS (e.g., superoxide, peroxynitrite, hydroxyl radical and hydrogen peroxide) are all potential reactants capable of initiating DNA single strand breakage, with subsequent activation of the nuclear enzyme poly (ADP ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. Antioxidant treatment inhibits the activation of PARS, and prevents the organ injury associated with acute and chronic inflammation.     

Pharmacological actions of melatonin in acute and chronic inflammation

A vast number of experimental and clinical studies implicates oxygen-derived free radicals (especially, superoxide and the hydroxyl radical) and high energy oxidants (such as peroxynitrite) as mediators of acute and chronic inflammation. The purpose of this review is to summarize the pharmacological actions of melatonin in acute and chronic inflammation. Reactive oxygen species can modulate a wide range of toxic oxidative reactions. These include initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane sodium/potassium ATPase activity, inactivation of membrane sodium channels, and other oxidative modifications of proteins. Reactive oxygen species (e.g., superoxide, peroxynitrite, hydrogen peroxide and hydroxyl radical) are all potential reactants capable of initiating DNA single strand breakage, with subsequent activation of the nuclear enzyme poly (ADP ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. These toxic reactions are likely to play a role in the pathophysiology of inflammation. Melatonin has been shown to possess both in vitro and in vivo important antioxidant activities as well as to inhibit the activation of poly (ADP ribose) synthetase. A large number of experimental studies have documented that melatonin exerts important anti-inflammatory actions.     

Role of oxidative-nitrosative stress and downstream pathways in various forms of cardiomyopathy and heart failure

Heart failure is the major cause of hospitalization, morbidity and mortality worldwide. Previous experimental and clinical studies have suggested that there is an increased production of reactive oxygen species (ROS: superoxide, hydrogen peroxide, hydroxyl radical) both in animals and in patients with acute and chronic heart failure. The possible source of increased ROS in the failing myocardium include xanthine and NAD(P)H oxidoreductases, cyclooxygenase, the mitochondrial electron transport chain and activated neutrophils among many others. The excessively produced nitric oxide (NO) derived from NO synthases (NOS) has also been implicated in the pathogenesis of chronic heart failure (CHF). The combination of NO and superoxide yields peroxynitrite, a reactive oxidant, which has been shown to impair cardiac function via multiple mechanisms. Increased oxidative and nitrosative stress also activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP), which importantly contributes to the pathogenesis of cardiac and endothelial dysfunction associated with myocardial infarction, chronic heart failure, diabetes, atherosclerosis, hypertension, aging and various forms of shock. Recent studies have demonstrated that pharmacological inhibition of xanthine oxidase derived superoxide formation, neutralization of peroxynitrite or inhibition of PARP provide significant benefit in various forms of cardiovascular injury. This review discusses the role of oxidative/nitrosative stress and downstream pathways in various forms of cardiomyopathy and heart failure.     

Discovery and structure-activity relationships of modified salicylanilides as cell permeable inhibitors of poly(ADP-ribose) glycohydrolase (PARG)

The metabolism of poly(ADP-ribose) (PAR) in response to DNA strand breaks, which involves the concerted activities of poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG), modulates cell recovery or cell death depending upon the level of DNA damage. While PARP inhibitors show high promise in clinical trials because of their low toxicity and selectivity for BRCA related cancers, evaluation of the therapeutic potential of PARG is limited by the lack of well-validated cell permeable inhibitors. In this study, target-related affinity profiling (TRAP), an alternative to high-throughput screening, was used to identify a number of druglike compounds from several chemical classes that demonstrated PARG inhibition in the low-micromolar range. A number of analogues of one of the most active chemotypes were synthesized to explore the structure-activity relationship (SAR) for that series. This led to the discovery of a putative pharmacophore for PARG inhibition that contains a modified salicylanilide structure. Interestingly, these compounds also inhibit PARP-1, indicating strong homology in the active sites of PARG and PARP-1 and raising a new challenge for development of PARG specific inhibitors. The cellular activity of a lead inhibitor was demonstrated by the inhibition of both PARP and PARG activity in squamous cell carcinoma cells, although preferential inhibition of PARG relative to PARP was observed. The ability of inhibitors to modulate PAR metabolism via simultaneous effects on PARPs and PARG may represent a new approach for therapeutic development.     

A peroxynitrite decomposition catalyst counteracts sensory neuropathy in streptozotocin-diabetic mice

Whereas an important role of free radicals and oxidants in peripheral diabetic neuropathy is well established, the contribution of nitrosative stress and, in particular, of the highly reactive oxidant peroxynitrite, has not been properly explored. Our previous findings implicate peroxynitrite in diabetes-associated motor and sensory nerve conduction deficits and peripheral nerve energy deficiency and poly(ADP-ribose) polymerase activation associated with Type 1 diabetes. In this study the role of nitrosative stress in diabetic sensory neuropathy is evaluated. The peroxynitrite decomposition catalyst Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin (FP15) was administered to control and streptozotocin (STZ)-diabetic mice at the dose of 5 mg kg(-1) day(-1) (FP15), for 3 weeks after initial 3 weeks without treatment. Mice with 6-week duration of diabetes developed clearly manifest thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and approximately 38% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, grey matter of spinal cord, and dorsal root ganglion neurons. FP15 treatment was associated with alleviation of thermal and mechanical hypoalgesia. Tactile response threshold tended to increase in response to peroxynitrite decomposition catalyst treatment, but still remained approximately 59% lower compared with non-diabetic controls. Intraepidermal nerve fiber density was 25% higher in FP15-treated than in untreated diabetic rats, but the difference between two groups did not achieve statistical significance (p=0.054). Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons of peroxynitrite decomposition catalyst-treated diabetic mice were markedly reduced. In conclusion, nitrosative stress plays an important role in sensory neuropathy associated with Type 1 diabetes. The findings provide rationale for further studies of peroxynitrite decomposition catalysts in a long-term diabetic model.     

PARP inhibitors: New tools to protect from inflammation

Poly(ADP-ribosylation) consists in the conversion of β-NAD⁺ into ADP-ribose, which is then bound to acceptor proteins and further used to form polymers of variable length and structure. The correct turnover of poly(ADP-ribose) is ensured by the concerted action of poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) enzymes, which are responsible for polymer synthesis and degradation, respectively. Despite the positive role of poly(ADP-ribosylation) in sensing and repairing DNA damage, generated also by ROS, PARP over-activation could allow NAD depletion and consequent necrosis, thus leading to an inflammatory condition in many diseases. In this respect, inhibition of PARP enzymes could exert a protective role towards a number of pathological conditions; i.e. the combined treatment of tumors with PARP inhibitors/anticancer agents proved to have a beneficial effect in cancer therapy. Thus, pharmacological inactivation of poly(ADP-ribosylation) could represent a novel therapeutic strategy to limit cellular injury and to attenuate the inflammatory processes that characterize many disorders.     

Poly(ADP-ribose)polymerase inhibition - where now?

The poly(ADP-ribose)polymerases (PARPs) catalyse the transfer of ADP-ribose units from the substrate NAD(+) to acceptor proteins, biosynthesising polyanionic poly(ADP-ribose) polymers. A major isoform, PARP-1, has been the target for design of inhibitors for over twenty-five years. Inhibitors of the activity of PARP-1 have been claimed to have applications in the treatment of many disease states, including cancer, haemorrhagic shock, cardiac infarct, stroke, diabetes, inflammation and retroviral infection, but only recently have PARP-1 inhibitors entered clinical trial. Most PARP-1 inhibitors mimic the nicotinamide of NAD(+) and the structure-activity relationships are understood in terms of the structure of the catalytic site. However, five questions remain if PARP-1 inhibitors are to realise their potential in treating human diseases. Firstly, the consensus pharmacophore is a benzamide with N-H conformationally constrained anti to the carbonyl-arene bond but this is also a "pharmacophore" for insolubility in water; can water-solubility be designed into inhibitors without loss of potency? Secondly, some potential clinical applications require tissue-selective PARP-1 inhibition; is this possible through pro-drug approaches? Thirdly, different diseases may require therapeutic PARP-1 inhibition to be either short-term or chronic; are there potential problems associated with chronic inhibition of this DNA-repair process? Fourthly, PARP-1 is one of at least eighteen isoforms; is isoform-selectivity essential, desirable or even possible? Fifthly, PARP activity can be inhibited in cells by inhibition of poly(ADP-ribose)-glycohydrolase (PARG); will this be a viable strategy for future drug design? The answers to these questions will determine the future of disease therapy through inhibition of PARP.     

Unusual increase in lumbar network excitability of the rat spinal cord evoked by the PARP-1 inhibitor PJ-34 through inhibition of glutamate uptake

Overactivity of poly(ADP-ribose) polymerase enzyme 1 (PARP-1) is suggested to be a major contributor to neuronal damage following brain or spinal cord injury, and has led to study the PARP-1 inhibitor 2-(dimethylamino)-N-(5,6-dihydro-6-oxophenanthridin-2yl)acetamide (PJ-34) as a neuroprotective agent. Unexpectedly, electrophysiological recording from the neonatal rat spinal cord in vitro showed that, under control conditions, 1–60 μM PJ-34 per se strongly increased spontaneous network discharges occurring synchronously on ventral roots, persisting for 24 h even after PJ-34 washout. The PARP-1 inhibitor PHE had no similar effect. The action by PJ-34 was reversibly suppressed by glutamate ionotropic receptor blockers and remained after applying strychnine and bicuculline. Fictive locomotion evoked by neurochemicals or by dorsal root stimulation was present 24 h after PJ-34 application. In accordance with this observation, lumbar neurons and glia were undamaged. Neurochemical experiments showed that PJ-34 produced up to 33% inhibition of synaptosomal glutamate uptake with no effect on GABA uptake. In keeping with this result, the glutamate uptake blocker TBOA (5 μM) induced long-lasting synchronous discharges without suppressing the ability to produce fictive locomotion after 24 h. The novel inhibition of glutamate uptake by PJ-34 suggested that this effect may compound tests for its neuroprotective activity which cannot be merely attributed to PARP-1 block. Furthermore, the current data indicate that the neonatal rat spinal cord could withstand a strong, long-lasting rise in network excitability without compromising locomotor pattern generation or circuit structure in contrast with the damage to brain circuits known to be readily produced by persistent seizures.     

Antioxidant properties of Neu2000 on mitochondrial free radicals and oxidative damage

Neu2000 [2-hydroxy-5-(2,3,5,6-tetrafluoro-4 trifluoromethylbenzylamino) benzoic acid] is a dual-acting neuroprotective agent that functions both as a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist and a free radical scavenger. In the present study, we investigated the scavenging activity of Neu2000 on various classes of reactive oxygen species and reactive nitrogen species (ROS/RNS) as well as its efficacy for reducing free radicals and oxidative stress/damage induced in spinal cord mitochondrial preparations. Neu2000 exerted scavenging activity against superoxide, nitric oxide, and hydroxyl radicals, and efficiently scavenged peroxynitrite. In the mitochondrial studies, Neu2000 markedly inhibited ROS/RNS and hydrogen peroxide levels following antimycin treatment. In addition, Neu2000 effectively scavenged hydroxyl radicals generated by iron(III)-ascorbate, reduced protein carbonyl formation mediated by hydroxyl radicals and peroxynitrite, and prevented glutathione oxidation caused by tert-butyl hydroperoxide in isolated mitochondria. Interestingly, incubation of isolated mitochondria with Neu2000 followed by centrifugation and removal of the supernatant also resulted in a concentration-dependent decrease in lipid peroxidation. This observation suggests that Neu2000 enters mitochondria to target free radicals or indirectly affects mitochondrial function in a manner that promotes antioxidant activity. The results of the present study demonstrate that Neu2000 possesses potent in vitro antioxidant activity due, most likely, to its active phenoxy group.     

A dual role for poly(ADP-ribose) polymerase-1 during caspase-dependent apoptosis

2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, catalyzes the generation of reactive oxygen species (ROS) and caspase-dependent apoptosis in human promyelocytic leukemia (HL-60) cells. We now report that TGHQ induces severe DNA damage, as evidenced by DNA ladder formation and H2AX phosphorylation. The subsequent activation of the DNA nick sensor enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), leads to the rapid depletion of ATP and NAD and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). PJ-34 (a PARP-1 inhibitor) completely prevented the formation of PARs, partially attenuated TGHQ-mediated ATP depletion, but had little effect on NAD depletion. Intriguingly, although z-vad-fmk (a pan-caspase inhibitor) attenuated TGHQ-induced apoptosis, cotreatment with PJ-34 led to a further decrease in apoptosis, suggesting that PARP-1 participates in caspase-dependent apoptosis. Indeed, PARP-1 inhibition reduced TGHQ-induced caspase-3, -7, and -9 activation, at least partially by attenuating cytochrome c translocation from mitochondria to the cytoplasm. In contrast, PJ-34 potentiated TGHQ-induced caspase-8 activation, suggesting that PARP-1 plays a dual role in regulating TGHQ-induced apoptosis via opposing effects on the intrinsic (mitochondrial) and extrinsic (death-receptor) pathways. PARP-1 knockdown in HL-60 cells confirmed that PARP-1 participates in effector caspase activation. Finally, PJ-34 also inhibited TGHQ-induced apoptosis-inducing factor (AIF) nuclear translocation, but neither c-jun NH(2)-terminal kinase nor p38 MAPK (p38 mitogen-activated protein kinase) activation was required for AIF translocation. In summary, TGHQ-induced apoptosis of HL-60 cells is accompanied by PARP-1, caspase activation, and AIF nuclear translocation. TGHQ-induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway in a process amenable to PARP inhibition. Residual cell death in the presence of PJ-34 is likely mediated via the extrinsic apoptotic pathway.