In horses, endotoxemia, such as with strangulating and inflammatory lesions of the small intestines and large colon, is a leading cause of morbidity and death.1,2 Ischemia-reperfusion injury and severe inflammation lead to intestinal mucosal injury with increased absorption of endotoxins and subsequent systemic inflammatory response syndrome that can lead to multiple organ failure. Methods to decrease intestinal IRI and inflammation and the effects of endotoxins on systemic inflammation may improve the prognosis for affected horses.
Poly(ADP-ribose) polymerase-1, which is the most abundant, ubiquitous, and studied member of the large family of poly(ADP-ribose) polymerase enzymes,3 is involved in the IRI process. When activated in response to DNA damage from oxidative, nitrosative, genotoxic, oncogenic, thermal, inflammatory, or metabolic stress, PARP1 induces synthesis of poly(ADP-ribose) polymers, with the oxidized form of nicotinamide adenine dinucleotide serving as the donor of ADP-ribose units. The poly(ADP-ribose) polymers and other acceptor proteins (eg, histones, DNA repair proteins, transcription factors, and chromatin modulators) then attach to the polymerase,3–5 resulting in rapid depletion of the intracellular pools of ATP and oxidized nicotinamide adenine dinucleotide, which in turn leads to decreased rates of glycolysis and mitochondrial respiration and potentially culminates in energy crisis–induced cell necrosis.6 Poly(ADP-ribose)-dependent cell death may also be caused by cellular translocation of the apoptosis-inducing factor from the mitochondrion to the nucleus, which occurs in a poly(ADP)-ribosylation–dependent manner and leads to DNA fragmentation and cell death. In addition, PARP1 activation plays a central role in inflammation through different pathways.7–9 For instance, PARP1 induces release of high mobility group box 1, a proinflammatory mediator that can induce macrophage activation and TNF-α production.10 Activation of PARP1 is also involved in inflammatory responses mediated by nuclear factor κ-light-chain-enhancer of activated B cells and in responses with secretion of other proinflammatory mediators (eg, TNF-α, interleukin-1β, P-selectin, intracellular adhesion molecule, and inducible nitric oxide synthetase).11 As expected, considering the involvement of PARP1 in the inflammatory cascade and cell death, activation of PARP1 is involved in processes such as IRI, endotoxemia, and hemorrhagic shock. Therefore, PARP1 inhibition may be a strategy for decreasing IRI, inflammation, and the effects of endotoxin on systemic inflammation.
Numerous studies12–16 show that the absence or pharmacologic inhibition of PARP1 is responsible for several protective effects on gut epithelium, such as improved mucosal barrier function, decreased inflammatory cell infiltration, lowered proinflammatory cytokines production, and reduced tissue damage. Because PARP1 inhibition may afford partial protection against endotoxemia in horses by decreasing capillary leakage and permeability, use of PARP1 along with current treatments for IRI and endotoxemia in affected horses may be beneficial. Species-specific research, however, is needed because the extent of endotoxin response varies among species and individuals.17 Thus, research investigating the role of PARP1 inhibition on LPS-stimulated PBMCs of horses is desirable.
We hypothesized that for horses, TFN-α production would be lower in IFN-γ– and LPS-stimulated PBMCs exposed to versus not exposed to PARP1 inhibitors and that efficacy differed among PARP1 inhibitors. The aim of the present study was to evaluate effects, including potential protective effects, of PARP1 inhibitors on the production of TNF-α by IFN-γ– and LPS-stimulated PBMCs as an in vitro model of inflammation in horses.
This study was performed at the New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pa, and the School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pa.
This manuscript represents a portion of a thesis submitted by Dr. Cacciolatti to the Università degli Studi di Torino, Italy, as partial fulfillment of the requirements for the degree of Doctor in Veterinary Medicine.
Supported in part by the Raymond Firestone Research Foundation; New Bolton Center, School of Veterinary Medicine, University of Pennsylvania; and Narkovet Consulting LLC.
The authors declare that there were no conflicts of interest.
Presented in abstract form at the 24th Annual Scientific Meeting of the European College of Veterinary Surgeons, Berlin, July 2015.
The authors thank Jessica Bader and Jesse Vanderhoeff for technical support, Dr. Mary Robinson for the use of laboratory space, and Dr. Samantha Hart for assistance with research grant preparation.
Peripheral blood mononuclear cell
Tumor necrosis factor-α
Gibco DPBS with no calcium and no magnesium, Mediatech Inc, Manassas, Va.
Ficoll-Hypaque, GE Healthcare Bio-Sciences AB, Uppsala, Sweden.
Centrifuge 5810R, Eppendorf AG, Hauppage, NY.
Gibco HBSS with no calcium, magnesium, or phenol red, Thermo Fisher Scientific Inc, Waltham, Mass.
Hyclone fetal bovine serum, SH30070, Thermo Fisher Scientific Inc, Waltham, Mass.
Gibco penicillin-streptomycin (10,000 U/mL), Thermo Fisher Scientific Inc, Waltham, Mass.
Gibco trypan blue solution, 0.4%, Thermo Fisher Scientific Inc, Waltham, Mass.
Costar clear TC-treated 12-well plates, Corning Inc, Corning, NJ.
Human fibronectin, No. 365008, BD Biosciences, San Jose, Calif.
Heraeus HERAcell 150, DJB Labcare Ltd, Newport Pagnell, Buckinghamshire, England.
Recombinant human INF-γ, No. HC2030, Hycult Biotech Inc, Wayne, Pa.
LPS from Escherichia coli O55:B5, No. L2880, Millipore Sigma, St Louis, Mo.
Equine TNFα ELISA Reagent Kit, ESS0017, Thermo Scientific, Rockford, Ill.
Benchmark Plus Microplate Spectrophotometer System, Bio-Rad Laboratories Inc, Hercules, Calif.
PJ-34, Enzo Life Sciences Inc, Farmingdale, NY.
Veliparib, Enzo Life Sciences Inc, Farmingdale, NY.
Olaparib, Selleck Chemicals, Houston, Tex.
1. Mair TS, Smith LJ. Survival and complication rates in 300 horses undergoing surgical treatment of colic. Part 1: short-term survival following a single laparotomy. Equine Vet J 2005;37:296–302.
3. D'Amours D, Desnoyers S, D'Silva I, et al. Poly(ADP-ribosyl) ation reactions in the regulation of nuclear functions. Biochem J 1999;342:249–268.
5. Meyer-Ficca ML, Meyer RG, Jacobson EL, et al. Poly(ADP-ribose) polymerases: managing genome stability. Int J Biochem Cell Biol 2005;37:920–926.
6. Ha HC, Snyder SH. Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc Natl Acad Sci U S A 1999;96:13978–13982.
7. Hong SJ, Dawson TM, Dawson VL. Nuclear and mitochondrial conversations in cell death: PARP-1 and AIF signaling. Trends Pharmacol Sci 2004;25:259–264.
9. Cregan SP, Dawson VL, Slack RS. Role of AIF in caspase-dependent and caspase-independent cell death. Oncogene 2004;23:2785–2796.
10. Ditsworth D, Zong WX, Thompson CB. Activation of poly(ADP)-ribose polymerase (PARP-1) induces release of the pro-inflammatory mediator HMGB1 from the nucleus. J Biol Chem 2007;282:17845–17854.
11. Bai P, Virag L. Role of poly(ADP-ribose) polymerases in the regulation of inflammatory processes. FEBS Lett 2012;586:3771–3777.
12. Di Paola R, Genovese T, Caputi AP, et al. Beneficial effects of 5-aminoisoquinolinone, a novel, potent, water-soluble, inhibitor of poly (ADP-ribose) polymerase, in a rat model of splanchnic artery occlusion and reperfusion. Eur J Pharmacol 2004;492:203–210.
13. Di Paola R, Mazzon E, Xu W, et al. Treatment with PARP-1 inhibitors, GPI 15427 or GPI 16539, ameliorates intestinal damage in rat models of colitis and shock. Eur J Pharmacol 2005;527:163–171.
14. Taner AS, Cinel I, Ozer L, et al. Poly(ADP-ribose) synthetase inhibition reduces bacterial translocation in rats after endotoxin challenge. Shock 2001;16:159–162.
15. Jijon HB, Churchill T, Malfair D, et al. Inhibition of poly(ADP-ribose) polymerase attenuates inflammation in a model of chronic colitis. Am J Physiol Gastrointest Liver Physiol 2000;279:G641–G651.
16. Oliver FJ, Ménissier-de Murcia J, Nacci C, et al. Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP-ribose) polymerase-1 deficient mice. EMBO J 1999;18:4446–4454.
18. Fidalgo-Carvalho I, Craigo JK, Barnes S, et al. Characterization of an equine macrophage cell line: application to studies of EIAV infection. Vet Microbiol 2009;136:8–19.
19. Morris DD, Moore JN, Fischer K, et al. Endotoxin-induced tumor necrosis factor activity production by equine peritoneal macrophages. Circ Shock 1990;30:229–236.
20. Douglas HF, Southwood LL, Meyer-Ficca ML, et al. The activity and inhibition of poly(ADP-ribose) polymerase-1 in equine peripheral blood mononuclear cells in vitro. J Vet Emerg Crit Care (San Antonio) 2015;25:528–537.
21. Sosna J, Voigt S, Mathieu S, et al. TNF-induced necroptosis and PARP-1-mediated necrosis represent distinct routes to programmed necrotic cell death. Cell Mol Life Sci 2014;71:331–348.
22. Madison DL, Stauffer D, Lundblad JR. The PARP inhibitor PJ34 causes a PARP1-independent, p21 dependent mitotic arrest. DNA Repair (Amst) 2011;10:1003–1013.
23. Antolín AA, Jalencas X, Yélamos J, et al. Identification of pim kinases as novel targets for PJ34 with confounding effects in PARP biology. ACS Chem Biol 2012;7:1962–1967.
24. Jagtap P, Soriano FG, Virág L, et al. Novel phenanthridinone inhibitors of poly (adenosine 5′-diphosphate-ribose) synthetase: potent cytoprotective and antishock agents. Crit Care Med 2002;30:1071–1082.
25. Iványi Z, Hauser B, Pittner A, et al. Systemic and hepatosplanchnic hemodynamic and metabolic effects of the PARP inhibitor PJ34 during hyperdynamic porcine endotoxemia. Shock 2003;19:415–421.
26. Goldfarb RD, Marton A, Szabó E, et al. Protective effect of a novel, potent inhibitor of poly(adenosine 5′-diphosphateribose) synthetase in a porcine model of severe bacterial sepsis. Crit Care Med 2002;30:974–980.
27. Veres B, Gallyas, Jr. F, Varbiro G, et al. Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock. Biochem Pharmacol 2003;65:1373–1382.
28. Veres B, Radnai B, Gallyas F Jr, et al. Regulation of kinase cascades and transcription factors by a poly(ADP-ribose) polymerase-1 inhibitor, 4-hydroxyquinazoline, in lipopolysaccharide-induced inflammation in mice. J Pharmacol Exp Ther 2004;310:247–255.
29. Pacher P, Cziráki A, Mabley JG, et al. Role of poly(ADP-ribose) polymerase activation in endotoxin-induced cardiac collapse in rodents. Biochem Pharmacol 2002;64:1785–1791.
30. Tasatargil A, Dalaklioglu S, Sadan G. Inhibition of poly(ADP-ribose) polymerase prevents vascular hyporesponsiveness induced by lipopolysaccharide in isolated rat aorta. Pharmacol Res 2005;51:581–586.
31. Yilmaz B, Sahin P, Ordueri E, et al. Poly(ADP-ribose) polymerase inhibition improves endothelin-1-induced endothelial dysfunction in rat thoracic aorta. Ups J Med Sci 2014;119:215–222.
32. Lobo SM, Orrico SR, Queiroz MM, et al. Pneumonia-induced sepsis and gut injury: effects of a poly-(ADP-ribose) polymerase inhibitor. J Surg Res 2005;129:292–297.
33. Li X, Ling Y, Cao Z, et al. Targeting intestinal epithelial cell-programmed necrosis alleviates tissue injury after intestinal ischemia/reperfusion in rats. J Surg Res 2018;225:108–117.