Postischemic poly (ADP-ribose) polymerase (PARP) inhibition reduces ischemia reperfusion injury in a hind-limb ischemia model

doi:10.1016/j.surg.2009.12.006

Surgery

Volume 148, Issue 1, July 2010, Pages 110-118

https://doi.org/10.1016/j.surg.2009.12.006 Get rights and content

Background

Several experiments were designed to determine whether the systemic, postischemic administration of PJ34,which is a poly-adenosine diphosphate (ADP)-ribose polymerase inhibitor, decreased tissue injury and inflammation after hind-limb ischemia reperfusion (I/R).

Methods

C57BL6 mouse limbs were subjected to 1.5 h ischemia followed by 24-h reperfusion. The treatment group (PJ) received intraperitoneal PJ34 (30 mg/kg) immediately before reperfusion, as well as 15 min and 2 h into reperfusion. The control group (CG) received lactated Ringer's alone at the same time intervals as PJ34 administration. The skeletal muscle levels of adenosine triphosphate (ATP), macrophage inflammatory protein-2 (MIP-2), keratinocyte derived chemokine (KC), and myeloperoxidase (MPO) were measured. Quantitative measurement of skeletal muscle tissue injury was assessed by microscopic analysis of fiber injury.

Results

ATP levels were higher in limbs of PJ versus CG mice (absolute ATP: 4.7 ± 0.35 vs 2.3 ± 0.15-ng/mg tissue, P = .002). The levels of MIP-2, KC, and MPO were lower in PJ versus CG mice (MIP-2: 1.4 ± 0.34 vs 3.67 ± 0.67-pg/mg protein, P = .014; KC: 4.97 ± 0.97 vs 12.65 ± 3.05-pg/mg protein, P = .037; MPO: 46.27 ± 10.53 vs 107.34 ± 13.58-ng/mg protein, P = .008). Muscle fiber injury was markedly reduced in PJ versus CG mice (4.25 ± 1.9% vs 22.68 ± 3.0% total fibers, P = .0004).

Conclusion

Systemic postischemic administration of PJ34 preserved skeletal muscle energy levels, decreased inflammatory markers, and preserved tissue viability post-I/R. These results support PARP inhibition as a viable treatment for skeletal muscle I/R in a clinically relevant post hoc scenario.

Section snippets

Animal protocol

All experimental procedures were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee in accordance with the “Principles of Laboratory Animal Care” (Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publication No. 86-23, Revised 1996). C57BL6 mice (male, 20–25 g; Jackson Laboratory, Bar Harbor, ME) were anesthetized using intraperitoneal administration of 60 mg/kg of pentobarbital. C57BL6 mice were subjected to 1.5 h of

Histologic analysis

The percentage of injured fibers in PJ34- versus LR-treated hind limbs was assessed using defined histologic criteria for injury. The percent of injured fibers was significantly lower in mice treated with PJ34 versus animals treated with LR (4.25 ± 1.9% vs 22.68 ± 3.0%; P = .0004; Fig 1). Representative photomicrographs of skeletal muscle for treated and untreated mice are shown in Fig 2. Treated mice had a few scattered clusters of injured cells among predominantly normal polygonal skeletal

Discussion

These experiments demonstrate that postischemic treatment with PJ34 results in decreased skeletal muscle fiber injury, preservation of tissue ATP (ie, metabolic rescue), and decreased inflammation (levels of tissue cytokines and leukocyte activation) in a model of skeletal muscle I/R. These results were obtained in a postischemic treatment model in which PJ34 was administered systemically (via intraperitoneal injections) starting immediately before reperfusion.

Previously, our laboratory

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Poly(ADP-ribose) Polymerase (PARP) is Critically Involved in Liver Ischemia/reperfusion-injury
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Poly(ADP-ribose) polymerase (PARP) is a DNA-repairing enzyme activated by extreme genomic stress, and therefore is potently activated in the remnant liver suffering from ischemia after surgical resection. However, the impact of PARP on post-ischemic liver injury has not been elucidated yet.
We investigated the impact of PARP on murine hepatocyte/liver injury induced by hypoxia/ischemia, respectively.
PJ34, a specific inhibitor of PARP, markedly protected against hypoxia/reoxygenation (H/R)-induced cell death, though z-VAD-fmk, a pan-caspase inhibitor similarly showed the protective effect. PJ34 did not affect H/R-induced caspase activity or caspase-mediated cell death. z-VAD-fmk also did not affect the production of PAR (i.e., PARP activity). Therefore, PARP- and caspase-mediated cell death occurred in a mechanism independent of each other in H/R. H/R immediately induced activation of PARP and cell death afterwards, both of which were suppressed by PJ34 or Trolox, an antioxidant. This suggests that H/R-induced cell death occurred redox-dependently through PARP activation. H/R and OS induced nuclear translocation of apoptosis inducing factor (AIF, a marker of parthanatos) and RIP1-RIP3 interaction (a marker of necroptosis), both of which were suppressed by PJ34. H/R induced PARP-mediated parthanatos and necroptosis redox-dependently. In mouse experiments, PJ34 significantly reduced serum levels of AST, ALT & LDH and areas of hepatic necrosis after liver ischemia/reperfusion, similar to z-VAD-fmk or Trolox.
PARP, activated by ischemic damage and/or oxidative stress, may play a critical role in post-ischemic liver injury by inducing programmed necrosis (parthanatos and necroptosis). PARP inhibition may be one of the promising strategies against post-ischemic liver injury.
Avoid the trap: Targeting PARP1 beyond human malignancy
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PARP1 is a poly(ADP-ribose) polymerase (PARP) enzyme that plays a critical role in regulating DNA damage response. The main enzymatic function of PARP1 is to catalyze a protein post-translational modification known as poly(ADP-ribosyl)ation (PARylation). Human cancers with homologous recombination deficiency are highly sensitive to PARP1 inhibitors. PARP1 is aberrantly activated in many non-oncological diseases, leading to the excessive NAD⁺ depletion and PAR formation, thus causing cell death and tissue damage. PARP1 deletion offers a profound protective effect in the relevant animal models. However, many of the current PARP1 inhibitors also induce PARP1 trapping, which drives subsequent DNA damage, innate immune response and cytotoxicity. This minireview provides an overview of the basic biology of PARP1 trapping, and its implications in disease. Furthermore, we also discuss the recent development of PARP1 PROTAC compounds, and their utility as “non-trapping” PARP1 degraders for the potential amelioration of non-oncological diseases driven by aberrant PARP1 activation.
Hind limb ischemia-reperfusion injury in diet-induced obese mice
2014, Journal of Surgical Research
Obesity is a major risk factor for the development of diabetes. Limb ischemia–reperfusion injury (IR) is a common clinical problem in diabetics who have compromised lower extremity perfusion. This study compared the histologic, metabolic, and functional outcomes after hind limb IR in diet-induced obese (DIO) and non-diabetic (ND) mice during the acute and the regenerative phases of IR.
DIO and ND mice were subjected to 1.5 h unilateral hind limb ischemia followed by 1- or 28-d IR. Muscle morphology, metabolic, and genomic stress were evaluated at days 1 and 28 IR; Acute inflammation and thrombosis were only measured at day-1 IR. At day 28, IR, skeletal muscle contractility, and maturation were also assessed.
At day-1 IR, similar levels of acute muscle fiber necrosis were seen in both groups. DIO mice demonstrated substantially greater inflammatory, prothrombotic, and genomic stress responses, which were also associated with a greater reduction in energy substrates and Akt phosphorylation. At 28d, there was no difference in the peak forces generated in the hind limbs for the two groups. DIO mice had reduced fatigue resistance compared with ND and larger areas of fat accumulation although there was no significant difference in muscle fiber maturation.
DIO mice had an exacerbated acute response to IR with enhanced metabolic deficit, fat accumulation, and defective functional recovery during the regenerative phase of IR. These changes in fatigue resistance reflect compromised functional recovery after IR injury and have relevance for the functional recovery of patients with metabolic syndrome and insulin resistance.
Reduced hind limb ischemia-reperfusion injury in Toll-like receptor-4 mutant mice is associated with decreased neutrophil extracellular traps
2013, Journal of Vascular Surgery
Citation Excerpt :
Further evidence to suggest the specificity of decreased cellular stress in the TLR4m mice subjected to hind limb IR was the observed decrease in PARP activation. A previous report from our laboratory using pharmacologic interventions in a clinically relevant post hoc scenario showed only a transient decrease in PARP activity at 7 hours of reperfusion,34 whereas in this report, a significant decrease in PARP activity was observed even at 48 hours of reperfusion. TLR4m mice had substantially decreased systemic levels of the proinflammatory cytokine, MCP-1, but not KC.
Ischemia-reperfusion (IR) injury is a significant problem in the management of patients with acute limb ischemia. Despite rapid restoration of blood flow after technically successful open and endovascular revascularization, complications secondary to IR injury continue to occur and limit clinical success. Our aim was to create a murine model of hind limb IR injury to examine the role of Toll-like receptor-4 (TLR4) and to determine whether inactive TLR4 led to a decrease in the detection of neutrophil extracellular traps (NETs), which are known to be highly thrombogenic and may mediate microvascular injury.
A calibrated tension tourniquet was applied to unilateral hind limb of wild-type (WT) and TLR4 receptor mutant (TLR4m) mice for 1.5 hours to induce ischemia and then removed to initiate reperfusion. At the end of 48 hours of reperfusion, mice were euthanized and hind limb tissue and serum specimens were collected for analysis. Hematoxylin and eosin-stained sections of hind limb skeletal muscle tissue were examined for fiber injury. For immunohistochemistry, mouse monoclonal antihistone H2A/H2B/DNA complex antibody to detect NETs and rabbit polyclonal antimyeloperoxidase antibody were used to identify infiltrating cells containing myeloperoxidase. Muscle adenosine triphosphate levels, nuclear factor (NF)-κB activity, the α-subunit of inhibitor of NF-κB light polypeptide gene enhancer, poly (adenosine diphosphate-ribose) polymerase activity, and inducible nitric oxide synthase expression were measured. Systemic levels of keratinocyte-derived chemokine, monocyte chemotactic protein-1, and vascular endothelial growth factor in the serum samples were also examined.
IR injury in the hind limb of WT mice demonstrated significant levels of muscle fiber injury, decreased energy substrates, increased NF-κB activation, decreased levels of α-subunit of inhibitor of NF-κB light polypeptide gene enhancer, increased inducible nitric oxide synthase expression, and increased poly (adenosine diphosphate-ribose) polymerase activity levels compared with the TLR4m samples. Additionally, there was marked decrease in the level of neutrophil and monocyte infiltration in the TLR4m mice, which corresponded to similar levels of decreased NET detection in the interstitial space and in microvascular thrombi. In situ nuclease treatment of WT tissue sections significantly diminished the level of NET immunostaining, demonstrating the specificity of the antibody to detect NETs and suggesting a potential role for nuclease treatment in IR injury.
These results suggest a pivotal role for TLR4 in mediating hind limb IR injury and suggest that NETs may contribute to muscle fiber injury.
Unusual increase in lumbar network excitability of the rat spinal cord evoked by the PARP-1 inhibitor PJ-34 through inhibition of glutamate uptake
2012, Neuropharmacology
Citation Excerpt :
It is difficult to relate the present in vitro findings to the in vivo administration of PJ-34 since, to the best of our knowledge, the actual plasma concentration of this drug and its final redistribution to the brain tissue remain unclear. Since the dose of PJ-34 injected into experimental animal is 3-30 mg/kg (Abdelkarim et al., 2001; Virág and Szabó, 2002; Kauppinen et al., 2009; Crawford et al., 2010), assuming uniform drug distribution throughout body compartments of an adult rat (and ignoring any bound fraction that might lower the free drug concentration), one might estimate a plasma concentration of approximately 37 μM after 10 mg/kg, that is in the range of the concentrations tested in vitro in the present report. Even if the actual concentration of free PJ-34 at neuronal membrane level is likely to be lower, it seems feasible that it would still be compatible with those tested in the present study.
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.
Adverse effects of bilateral lower limb ischemia-reperfusion on inducing kidney injuries in rats could be ameliorated by platonin
2012, Acta Anaesthesiologica Taiwanica
Citation Excerpt :
Reperfusion of the acutely ischemic limb is a typical ischemia–reperfusion (I/R) injury that may cause injuries to vital organ, including the lungs and kidneys.1 Abundant data indicated that overproduction of reactive oxygen species (ROS) and proinflammatory molecules, and the subsequent inflammatory response induced by I/R is one of the most crucial underlying mechanisms.1–4 This concept is supported by previous data that the I/R-induced organ injuries could be mitigated by therapies aiming at decreasing the oxidative stress and/or inflammatory response.2,5–7
Bilateral lower limb ischemia–reperfusion (I/R) could cause significant oxidative stress, elicit inflammatory response, and subsequently induce kidney injury in animals. We tested the effects of platonin, a potent antioxidant, on mitigating the kidney injury induced by lower limb I/R in rats.
Adult male rats were allocated to receive I/R or I/R plus platonin (100 μg/kg intravenous injection immediately after reperfusion), and denoted as the I/R or the I/R-P group, respectively (n = 10 in each group). Sham groups were run simultaneously. Bilateral lower limb I/R was achieved by applying rubber-band tourniquets high around each thigh for 3 hours, followed by reperfusion for 6 hours. After sacrifice, the level of kidney injury was assayed.
I/R significantly increased the plasma concentrations of blood urea nitrogen (BUN) and creatinine (Cr). However, this effect could be mitigated by platonin, as the plasma concentrations of BUN and Cr of the I/R-P group were significantly lower than those of the I/R group. Moreover, histological findings revealed moderate injury in kidney tissues of the I/R group and mild injury in those of the I/R-P group. In addition, the leukocyte infiltration and myeloperoxidase activity in kidney tissues as well as the renal concentrations of inflammatory molecules (i.e., cyclooxygenase-2/prostaglandin E₂, interleukin-6, and macrophage inflammatory protein-2) and malondialdehyde (i.e., the index of lipid peroxidation) of the I/R group were significantly higher than those of the I/R-P group.
Platonin attenuates kidney injury induced by bilateral lower limb I/R in rats.

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: Supported by the Pacific Vascular Research Foundation, the American Diabetes Association, the Geneen Fund at the Massachusetts General Hospital, and Grant 1R01AR055843 from the National Institutes of Health.

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Original CommunicationPostischemic poly (ADP-ribose) polymerase (PARP) inhibition reduces ischemia reperfusion injury in a hind-limb ischemia model

Background

Methods

Results

Conclusion

Section snippets

Animal protocol

Histologic analysis

Discussion

J Thorac Cardiovasc Surg

J Surg Res

J Am Coll Surg

Clinics

J Vasc Surg

Surgery

J Surg Res

J Surg Res

J Vasc Surg

J Thorac Cardiovasc Surg

Long-term results of percutaneous aspiration embolectomy

Cardiovasc Intervent Radiol

Management of acute leg ischaemia in the elderly

Br J Surg

Acute limb ischemia due to arterial embolism or thrombosis: influence of limb ischemia versus pre-existing cardiac disease on postoperative mortality rate

J Cardiovasc Surg

Neutrophil mediated remote organ injury after lower torso ischemia and reperfusion is selectin and complement dependent

J Trauma

Role of neutrophils in ischemia-reperfusion-induced microvascular injury

Am J Physiol

S-nitroso-N-acetylcysteine protects skeletal muscle against reperfusion injury

Microsurgery

Role of heparin in reducing skeletal muscle infarction in ischemia-reperfusion

Microcirc Endothelium Lymphatics

Polyadenosine diphosphate-ribose polymerase inhibition modulates skeletal muscle injury following ischemia reperfusion

Arch Surg

Inhibition of iNOS with 1400W improves contractile function and alters nos gene and protein expression in reperfused skeletal muscle

Microsurgery

Original Communication
Postischemic poly (ADP-ribose) polymerase (PARP) inhibition reduces ischemia reperfusion injury in a hind-limb ischemia model