Excessive poly(ADP-ribose) Polymerase Activation Research Articles

Poly(ADP-ribose) Polymerase (PARP) and PARP Inhibitors: Mechanisms of Action and Role in Cardiovascular Disorders.

Poly(ADP-ribosyl)ation is an immediate cellular repair response to DNA damage and is catalyzed primarily by poly(ADP-ribose)polymerase-1 (PARP1), which is the most abundant of the 18 different PARP isoforms and accounts for more than 90% of the catalytic activity of PARP in the cell nucleus. Upon detection of a DNA strand break, PARP1 binds to the DNA, cleaves nicotinamide adenine dinucleotide between nicotinamide and ribose and then modifies the DNA nuclear acceptor proteins by formation of a bond between the protein and the ADP-ribose residue. This generates ribosyl-ribosyl linkages that act as a signal for other DNA-repairing enzymes and DNA base repair. Extensive DNA breakage in cells results in excessive activation of PARP with resultant depletion of the cellular stores of nicotinamide adenine dinucleotide (NAD+) which slows the rate of glycolysis, mitochondrial electron transport, and ultimately ATP formation in these cells. This paper focuses on PARP in DNA repair in atherosclerosis, acute myocardial infarction/reperfusion injury, and congestive heart failure and the role of PARP inhibitors in combating the effects of excessive PARP activation in these diseases. Free oxygen radicals and nitrogen radicals in arteries contribute to disruption of the vascular endothelial glycocalyx, which increase the permeability of the endothelium to inflammatory cells and also low-density lipoproteins and the accumulation of lipid in the vascular intima. Mild inflammation and DNA damage within vascular cells promote PARP1 activation and DNA repair. Moderate DNA damage induces caspase-dependent PARP cleavage and vascular cell apoptosis. Severe DNA damage due to vascular inflammation causes excessive activation of PARP1. This causes endothelial cell depletion of NAD+ and ATP, downregulation of atheroprotective SIRT1, necrotic cell death, and ultimately atherosclerotic plaque disruption. Inhibition of PARP decreases vascular endothelial cell adhesion P-selectin and ICAM-1 molecules, inflammatory cells, pro-death caspase-3, and c-Jun N-terminal kinase (JNK) activation and upregulates prosurvival extracellular signal-regulated kinases and AKT, which decrease vascular cell apoptosis and necrosis and limit atherosclerosis and plaque disruption. In myocardial infarction with coronary occlusion then reperfusion, which occurs with coronary angioplasty or thrombolytic therapy, reperfusion injury occurs in as many as 31% of patients and is caused by inflammatory cells, free oxygen and nitrogen radicals, the rapid transcriptional activation of inflammatory cytokines, and the activation of PARP1. Inhibition of PARP attenuates neutrophil infiltration and inflammatory cytokine expression in the reperfused myocardium and preserves myocardial NAD+ and ATP. In addition, PARP inhibition increases the activation of myocyte survival enzymes protein kinase B (Akt) and protein kinase C epsilon (PKCε), and decreases the activity of myocardial ventricular remodeling enzymes PKCα/β, PKCζ/λ, and PKCδ. As a consequence, cardiomyocyte and vascular endothelial cell necrosis is decreased and myocardial contractility is preserved. In heart failure and circulatory shock in animal models, PARP inhibition significantly attenuates decreases in left ventricular systolic pressure, ventricular contractility and relaxation, stroke volume, and increases survival by limiting or preventing upregulation of adhesion molecules, proinflammatory cytokines, myocardial mononuclear cell infiltration, and PKCα/β and PKC λ/ζ. In this manner, PARP inhibition partially restores the myocardial concentrations of NAD+, limits ventricular remodeling and fibrosis, and prevents significant decreases in myocardial contractility. Based primarily on investigations in preclinical models of atherosclerosis, myocardial infarction, and heart failure, PARP inhibition appears to be beneficial in limiting or inhibiting cardiovascular dysfunction. These studies indicate that investigations of acute and chronic PARP inhibition are warranted in patients with atherosclerotic coronary artery disease.

Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations.

Retinitis pigmentosa (RP), an inherited blinding disease, is caused by a variety of different mutations that affect retinal photoreceptor function and survival. So far there is neither effective treatment nor cure. We have previously shown that poly(ADP-ribose)polymerase (PARP) acts as a common and critical denominator of cell death in photoreceptors, qualifying it as a potential target for future therapeutic intervention. A significant fraction of RP-causing mutations affect the genes for the rod photoreceptor phosphodiesterase 6A (PDE6A) subunit, but it is not known whether they all engage the same death pathway. Analysing three homozygous point mutations (Pde6a R562W, D670G, and V685M) and one compound heterozygous Pde6aV685M/R562W mutation in mouse models that match human RP patients, we demonstrate excessive activation of PARP, which correlated in time with the progression of photoreceptor degeneration. The causal involvement of PARP activity in the neurodegenerative process was confirmed in organotypic retinal explant cultures treated with the PARP-selective inhibitor PJ34, using different treatment time-points and durations. Remarkably, the neuroprotective efficacy of PARP inhibition correlated inversely with the strength of the genetically induced insult, with the D670G mutant showing the best treatment effects. Our results highlight PARP as a target for neuroprotective interventions in RP caused by PDE6A mutations and are a first attempt towards personalized, genotype-matched therapy development for RP. In addition, for each of the different mutant situations, our work identifies windows of opportunity for an optimal treatment regimen for further in vivo experimentation and possibly clinical studies.

Effect of cilostazol pretreatment on the PARP/AIF-mediated apoptotic pathway in rat cerebral ischemia-reperfusion models.

The aim of this study was to observe the expression of poly ADP-ribose polymerase (PARP) and apoptosis-inducing factor (AIF) in the CA1 region of the hippocampus and to explore whether cilostazol pretreatment exerts a protective effect on the brain through the PARP/AIF-mediated pathway in a rat model of cerebral ischemia-reperfusion. Rats were randomly divided into three groups: Sham-surgery, ischemia-reperfusion and cilostazol (n=45 rats/group). Rat models of middle cerebral artery occlusion were prepared using a thread occlusion method. Rats in the cilostazol group were administered 30 mg/kg intragastric cilostazol 6 and 2 h before brain ischemia, respectively. Following reperfusion, samples were collected at different time-points (6, 24 and 72 h) and each group was further subdivided into three subgroups (n=15 rats/subgroup). Apoptosis was measured using the terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling method. The protein expression levels of AIF and PARP were detected using western blot analysis and the expression levels of AIF mRNA were determined using the reverse transcription-polymerase chain reaction. AIF nuclear translocation occurred following local cerebral ischemia-reperfusion injury. Apoptosis, levels of AIF and PARP protein expression and levels of AIF mRNA expression were significantly increased in the ischemia-reperfusion group compared with the sham-surgery group (P<0.05). However, apoptosis and the expression levels of AIF protein, PARP protein and AIF mRNA at different time-points were significantly decreased in the cilostazol group compared with the ischemia-reperfusion group (P<0.05). In conclusion, cilostazol has a protective effect on rat cerebral ischemia-reperfusion injury, and acts by inhibiting nerve cell apoptosis by preventing the excessive activation of PARP and AIF nuclear translocation.

Development of a miniaturized assay for the high throughput screening program for poly(ADP-ribose) polymerase-1

Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.

A new poly(ADP-ribose) polymerase inhibitor, FR261529 [2-(4-chlorophenyl)-5-quinoxalinecarboxamide], ameliorates methamphetamine-induced dopaminergic neurotoxicity in mice.

Methamphetamine (METH) administration in mice, results in a chronic dopamine (DA) depletion associated with nerve terminal damage, with DA oxidation and generation of reactive oxygen species (ROS) primarily mediating this neurotoxicity. The oxidative stress induced by METH putatively activates nuclear enzyme poly(ADP-ribose) polymerase (PARP), with excessive PARP activation eventually leading to cell death. In this study, we show that prevention of PARP activation by treatment with FR261529 [2-(4-chlorophenyl)-5-quinoxalinecarboxamide], the compound that was recently identified as a novel PARP inhibitor (IC50 for PARP-1 = 33 nM, IC50 for PARP-2 = 7 nM), protects against both ROS-induced cells injury in vitro and METH-induced dopaminergic neuronal damage in an in vivo Parkinson's disease (PD) model. In PC12 cells, exposure of hydrogen peroxide or METH markedly induced PARP activation, and treatment with FR261529 (1 microM) significantly reduced PARP activation and attenuated cell death. In the mouse METH model, METH (15 mg/kg x 2 i.p., 2 h apart) intoxication accelerated DA metabolism and oxidation in the striatum, with subsequent cell damage in nigrostriatal dopaminergic neurons after 4 days. Oral administration of FR261529 (10 or 32 mg/kg) attenuated the damage of dopaminergic neurons via marked reduction of PARP activity and not via changes in dopamine metabolism or body temperature. These findings indicate that the neuroprotective effects of a novel PARP inhibitor, FR261529, were accompanied by inhibition of METH-induced PARP activation, suggesting that METH induces nigrostriatal dopaminergic neurodegeneration involving PARP activation and also orally active and brain-penetrable PARP inhibitor FR261529 could be a novel attractive therapeutic candidate for neurodegenerative disorders such as PD.

Development of a high-throughput screening-amenable assay for human poly(ADP-ribose) polymerase inhibitors

Introduction: Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. Inhibitors of PARP have been shown to reduce infarct size in animal models of myocardial ischemia. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, head trauma, and neurodegenerative disease, and as an adjunct therapy with chemotherapeutic agents/radiation in cancer therapy. Methods: Assays reported in the literature and commercially available PARP assay kits are labor-intensive, use radioactive reagents, use antibodies, and are not readily amenable to Hadamard transform spectroscopy (HTS). Here we report the development and the validation of a nonradioactive PARP assay suitable for HTS. This is a biotinylated NAD-based colorimetric assay in a 96-well plate format. Results: The assay is sensitive, reproducible, and easy to use. The IC 50 values generated for the known PARP inhibitors are in agreement with those generated using the commercial radioactive kit and those reported in the literature. Discussion: The present study demonstrates a sensitive and reproducible methodology capable of screening human PARP inhibitors in high-throughput format.

Poly(ADP-ribose) Polymerase Activation and Cell Injury in the Course of Rat Heart Heterotopic Transplantation

Free radicals and other reactive species generated during reperfusion of ischemic tissues may cause DNA damage and, consequently, the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). An excessive PARP activation may result in a depletion of intracellular NAD + and ATP, hence cell suffering and, ultimately, cell death. The present study is aimed at clarifying the role of PARP in a heart transplantation procedure and the contribution of myocyte necrosis and/or apoptosis to this process. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (30 or 60 u min). Under these conditions clear signs of oxidative stress, such as lipoperoxidation and DNA strand breaks, were evident. In addition to a marked activation, accompanied by a significant NAD + and ATP depletion, PARP protein levels significantly increased after 60 u min of reperfusion. Ultrastructural analysis showed nuclear clearings, intracellular oedema and plasma membrane discontinuity. Other relevant observations were the absence of typical signs of apoptosis like caspase-3 activation and PARP cleavage, random DNA fragmentation, rise in serum levels of heart damage markers. Our results suggest that during heart transplantation, the activation of PARP, causing energy depletion, results in myocardial cell injury whose dominant feature, at least in our experimental model, is necrosis rather than apoptosis.

A Dinucleotide Repeat Polymorphism at the Poly(ADP-ribose) Polymerase Gene is not Associated with Predisposition to Type 1 Diabetes in French Caucasians

The poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that detects and binds DNA strand breaks. Excessive PARP activation leads to the death of mice islet β-cells by depleting cellular energy reserves. On the other hand, PARP-mutant mice are resistant to streptozotocine-induced diabetes, and in the non-obese diabetic (NOD) mouse model, treatment with nicotinamide, a PARP inhibitor, protects islet cells against cytotoxic actions in vitro and results in a decreased incidence of type 1 diabetes. PARP gene in human is located within a recently identified type 1 diabetes-susceptibility region on chromosome 1q41–42, and contains a polymorphic CA dinucleotide repeat in the promoter region. To consider the putative involvement of PARP polymorphism in predisposition to type 1 diabetes, we performed genotyping for the various alleles of the CA dinucleotide repeat in 158 unrelated French Caucasian patients with type 1 diabetes and 193 ethnically-matched healthy controls. We found no significant difference of PARP alleles distribution between patients and controls, even after stratification of the patients according to HLA class II genotype or to age at disease onset. Our results suggest that this PARP polymorphism does not influence susceptibility to type 1 diabetes in French Caucasians.

Post-treatment with an inhibitor of poly(ADP-ribose) polymerase attenuates cerebral damage in focal ischemia

Poly(ADP-ribose) polymerase (PARP) is thought to play a physiological role in maintaining genomic integrity and in the repair of DNA strand breaks. However, the activation of PARP by free radical-damaged DNA plays a pivotal role in mediating ischemia–reperfusion injury. The excessive activation of PARP causes a rapid depletion of intracellular energy leading to cell death. The present study examined the effect of post-ischemic pharmacological inhibition of PARP in a rat focal cerebral ischemia model. In Long–Evans rats, focal cerebral ischemia was produced by cauterization of the right distal middle cerebral artery (MCA) with bilateral temporary common carotid artery (CCA) occlusion for 90 min. A PARP inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2 H)-isoquinolinone (DPQ; IC 50=1 μM/l) was injected i.p. 30 min after the onset of MCA occlusion (control: 10, 20, 40 and 80 mg/kg; n=7 each). Twenty-four hours later, the total infarct volume was measured. Regional blood flow in the right parietal cortex decreased to approximately 20% of the baseline following MCA occlusion in all groups. PARP inhibition lead to a significant decrease in damaged volume in all treated groups with the largest reduction in the 40 mg/kg group (111.5±24.8 mm 3, mean±SD, p<0.01), compared to the control group (193.5±28.6 mm 3). We also found there was a significant increase of poly(ADP-ribose) immunoreactivity in the ischemic region, as compared to the contralateral side, with DPQ treatment diminishing poly(ADP-ribose) production. These findings indicate that DPQ exerts its neuroprotective effects in vivo by PARP inhibition and that PARP inhibitors may be effective for treating ischemic stroke, even when the treatment is initiated after the onset of ischemia.

Inhibition of poly(ADP-ribose) polymerase: reduction of ischemic injury and attenuation of N-methyl-D-aspartate-induced neurotransmitter dysregulation.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) may play a role in DNA repair. However, in cerebral ischemia, excessive PARP activation may lead to energy depletion and exacerbation of neuronal damage. We examined the effect of inhibiting PARP on (1) the degree of cerebral injury in a rat model of transient focal ischemia and (2) the degree of neurotransmitter dysregulation induced by local cortical perfusion of N-methyl-D-aspartate (NMDA). In experiment 1, rats were subjected to transient ischemia for 90 minutes by occlusion of the middle cerebral artery. After 22.5 hours of reperfusion, lesions were quantified by tetrazolium staining. Untreated rats were compared with those treated with the PARP inhibitor 3-aminobenzamide (10 mg/kg). In experiment 2, rats were implanted with microdialysis probes in the cortex, and 1 mmol/L NMDA was perfused for 2 hours. Extracellular concentrations of neurotransmitter and neuromodulator amino acids were measured. Untreated rats were compared with those given 10 mg/kg 3-aminobenzamide. In experiment 1, PARP inhibition significantly reduced lesion volumes: 204+/-43 mm3 (untreated) versus 90+/-24 mm3 (treated). Neuroprotection was primarily manifested in the cortex. In experiment 2, NMDA perfusion resulted in large elevations of glutamate, taurine, and the lipid component phosphoethanolamine. Levels of the NMDA site modulator D-serine were reduced, and glycine levels appeared unchanged. 3-Aminobenzamide significantly attenuated the elevations in glutamate and phosphoethanolamine but had no effects on D-serine and glycine. Inhibition of PARP reduced injury after transient focal ischemia in rats and attenuated NMDA-induced glutamate efflux and overall neurotransmitter dysregulation. The deleterious effects of excessive PARP activation may be related in part to amplification of excitotoxicity, possibly by cellular energy depletion and additional transmitter release and/or reduced reuptake.

Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia.

Nitric oxide (NO) and peroxynitrite, formed from NO and superoxide anion, have been implicated as mediators of neuronal damage following focal ischemia, but their molecular targets have not been defined. One candidate pathway is DNA damage leading to activation of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP), which catalyzes attachment of ADP ribose units from NAD to nuclear proteins following DNA damage. Excessive activation of PARP can deplete NAD and ATP, which is consumed in regeneration of NAD, leading to cell death by energy depletion. We show that genetic disruption of PARP provides profound protection against glutamate-NO-mediated ischemic insults in vitro and major decreases in infarct volume after reversible middle cerebral artery occlusion. These results provide compelling evidence for a primary involvement of PARP activation in neuronal damage following focal ischemia and suggest that therapies designed towards inhibiting PARP may provide benefit in the treatment of cerebrovascular disease.