defibrotide and Reperfusion-Injury

Defibrotide is a single-stranded DNA fraction obtained from mammalian lung and is able to increase prostacyclin production by endothelial cells. It has profibrinolytic and antithrombotic properties, and we have successfully used it as an anti-ischaemic drug in many in-vivo experimental models. In fact, we showed that defibrotide treatment significantly protected rat heart, kidney and liver from ischaemia and postischaemic reperfusion injury. In rats treated with defibrotide, the functionality and metabolic activity of ischaemic organs were significantly protected from impairment as compared to controls treated only with the vehicle of the drug. In the present work we evaluated all our results, together with those of others, in order to hypothesize the mechanism of action of the drug. We postulated that the prominent function of defibrotide is to inhibit platelet and leukocyte adhesion to endothelial cells: this may depend on reduced cell activation possibly following drug interaction with adenosine receptors. Defibrotide could also favour endothelial-cell function through binding with haemoglobin: such binding permits oxygen release and preservation of endothelium-derived relaxing factor. Moreover, prostacyclin production by endothelial cells is responsible for many drug activities and also limits superoxide radical generation.

Topics: Adenine Nucleotides; Animals; Cell Adhesion; Endothelium; Epoprostenol; Heart Transplantation; Ischemia; Kidney; Kidney Transplantation; Leukocytes; Myocardial Reperfusion Injury; NAD; Platelet Adhesiveness; Polydeoxyribonucleotides; Rats; Rats, Wistar; Receptors, Purinergic P1; Reperfusion Injury

During some surgical interventions, temporary occlusion of the hepatic blood supply may cause ischaemia-reperfusion (I/R) injury and hepatic dysfunction. In this study the protective effect of defibrotide (DEF) was evaluated in a rat model of liver I/R injury. Four groups of rats were subjected to the following protocols: saline infusion without ischaemia, DEF infusion without ischaemia, DEF infusion with hepatic I/R, and saline infusion with hepatic I/R. After a midline laporatomy, liver ischaemia was induced by 45 min of portal occlusion. DEF 175 mg/kg(-1) was infused before ischaemia in 10 ml of saline. The same volume of saline was infused into the control animals. At the end of the 45-min reperfusion interval, the animals were sacrified. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzyme activities were determined in haemolysates, and malondialdehyde (MDA) level in the liver tissue was measured. Tissue MDA levels were significantly higher in the I/R plus saline group compared to the sham operation control groups (p < 0.01 and p < 0.05, respectively). Tissue MDA levels decreased in the DEF plus I/R group compared to the I/R plus saline group (p < 0.05), but DEF could not reduce tissue lipid peroxidation to the levels of the control sham operation groups. SOD and GSH-Px enzyme activities were significantly higher in DEF-treated animals than in the other groups (p < 0.05). These results suggest that DEF protects liver against I/R injury by increasing the antioxidant enzyme levels.

Topics: Animals; Glutathione Peroxidase; Liver; Male; Malondialdehyde; Polydeoxyribonucleotides; Rats; Reperfusion Injury; Superoxide Dismutase

1. The purpose of this study was to investigate the protective effects of defibrotide, a single-stranded polydeoxyribonucleotide, on ischaemia-reperfusion injury to the liver using a rat model. 2. Ischaemia of the left and median lobes was created by total inflow occlusion for 30 min followed by 60 min of reperfusion. Hepatic injury was assessed by the release of liver enzymes (alanine transferase, ALT and lactic dehydrogenase, LDH). Hepatic oxidant stress was measured by superoxide production, lipid peroxidation and nitrite/nitrate formation. Leukocyte-endothelium interaction and Kupffer cell mobilization were quantified by measuring hepatic myeloperoxidase (MPO), polymorphonuclear leukocyte adherence to superior mesenteric artery (SMA) and immunostaining of Kupffer cell. 3. Defibrotide treatment resulted in a significant inhibition of postreperfusion superoxide generation, lipid peroxidation, serum ALT activity, serum LDH activity, MPO activity, serum nitrite/nitrate level, leukocyte adherence to SMA, and Kupffer cell mobilization, indicating a significant attenuation of hepatic dysfunction. 4. A significant correlation existed between liver ischaemia/reperfusion and hepatic injury, suggesting that liver ischaemia/reperfusion injury is mediated predominantly by generation of oxygen free radicals and mobilization of Kupffer cells. 5. We conclude that defibrotide significantly protects the liver against liver ischaemia/reperfusion injury by interfering with Kupffer cell mobilization and formation of oxygen free radicals. This study provides strong evidence that defibrotide has important beneficial effects on acute inflammatory tissue injury such as that occurring in the reperfusion of the ischaemic liver.

Topics: Animals; Free Radicals; Ischemia; Kupffer Cells; Liver; Male; Polydeoxyribonucleotides; Rats; Rats, Sprague-Dawley; Reperfusion Injury

In the present study, in vitro effects of Defibrotide (D) on perfusion-induced changes in the morphology of endothelium were investigated by scanning (SEM) and transmission (TEM) electron microscope. Human umbilical cord veins were incubated or perfused with platelet-rich plasma alone (PRP) or platelet-rich plasma with Defibrotide (PRP+D) at 3ml/min or 14ml/min and the changes observed were compared. SEM examination of luminal surfaces demonstrated that perfusion with high flow rates may damage endothelial cells and lead to morphological changes which may be prevented by the presence of Defibrotide in the perfusate. Also, the marked reduction in the number of adhered platelets on luminal surface of veins incubated or perfused with Defibrotide compared to veins treated with platelet-rich plasma only revealed that Defibrotide has anti-thrombotic effects. TEM examination of ruthenium red (RR) stained thin sections of veins demonstrated that perfusion disrupts the glycosaminoglcan (GAG) coat on endothelial cells. But the presence of D in the perfusate preserves the integrity of GAG, indicating further cytoprotective effects of the drug on endothelium.

Topics: Endothelium, Vascular; Fibrinolytic Agents; Glycosaminoglycans; Humans; Microscopy, Electron; Microscopy, Electron, Scanning; Platelet Aggregation Inhibitors; Polydeoxyribonucleotides; Reperfusion Injury; Umbilical Veins

The authors previously demonstrated the protective activity of defibrotide (a profibrinolytic and antithrombotic drug) on endothelial cells. In the present work they examine the efficacy of defibrotide in protecting rat kidney from ischaemic/reperfusion injury by studying the modifications of intrarenal adenine nucleotide levels. Right renal ischaemia of 60 min and reperfusion of 30 min were induced in adult male Wistar rats. Defibrotide was administered as a bolus through a catheter inserted into the left femoral vein 5 min before the beginning of ischaemia at the dose of 32 mg/kg and continuously infused during ischaemia/reperfusion through the same vein at the final dose of 32 mg/kg in 5 ml of saline at the rate of 3 ml/h. Rats treated with vehicle of the drug were used as controls. At the end of postischaemic reperfusion, the ischaemic and left kidneys were rapidly removed and frozen in liquid nitrogen. Tissue extracts were prepared, and their ATP, ADP, AMP, cAMP, NAD+, and NADH contents were determined by using luminescence methods. In controls, ATP intrarenal levels were significantly higher in the left kidney than in the ischaemic organ of the same rat (3405 +/- 320 vs. 378 +/- 36 nmol/g fresh tissue and mean +/- s.e.m. of 10 experiments). Defibrotide treatment significantly protected ischaemic kidneys from the drop in ATP intrarenal content (1465 +/- 147 vs. 3124 +/- 303 nmol/g fresh tissue measured in the left kidney).

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Disease Models, Animal; Fibrinolytic Agents; Ischemia; Kidney; Male; Polydeoxyribonucleotides; Rats; Rats, Wistar; Reperfusion Injury

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Energy Metabolism; Kidney Transplantation; Liver; Male; NAD; Organ Preservation; Polydeoxyribonucleotides; Rats; Rats, Wistar; Reperfusion Injury; Transplantation, Isogeneic

Topics: Adenine Nucleotides; Animals; Creatinine; Cyclic AMP; Fibrinolytic Agents; Kidney Transplantation; Male; NAD; Oxidation-Reduction; Polydeoxyribonucleotides; Rats; Rats, Inbred Strains; Reperfusion Injury; Urea

Topics: Adenosine Triphosphate; Animals; Fibrinolytic Agents; Ischemia; Kidney; Liver; Male; Myocardial Reperfusion Injury; Polydeoxyribonucleotides; Rats; Rats, Inbred Strains; Reperfusion Injury

Defibrotide, a polydeoxyribonucleotide obtained from mammalian lungs, has been demonstrated to exert profibrinolytic and antithrombotic activity through stimulation of vascular prostacyclin (PGI2) production. We studied the effect of defibrotide administration in protecting liver and heart from ischaemic and postischaemic reperfusion damage. Defibrotide was administered as an i.v. bolus (30 mg/kg) at the beginning of liver ischaemia and at the same dose continuously during 60 min of postischaemic reperfusion. ATP levels were significantly improved in livers of defibrotide-treated rats as compared to those obtained in livers of rats treated with vehicle of the drug. Intrahepatic cytoplasmic and mitochondrial NAD+/NADH ratios were higher in defibrotide-treated than in vehicle-treated animals. The hearts, isolated from rats according to the transplantation procedure, were subjected to different times of warm + cold ischaemia. During ischaemia, the hearts were perfused continuously with 60 mg/kg of defibrotide or vehicle of the drug. The loss of creatine phosphokinase and lactate dehydrogenase activities due to an increased ischaemia time was limited in defibrotide-perfused hearts. Intracardiac ATP and ADP levels were significantly higher in defibrotide-treated organs than in controls. Our results demonstrate the efficacy of defibrotide in protecting liver and heart from ischaemia.

Topics: Animals; Coronary Disease; Cytoplasm; Epoprostenol; Ischemia; Liver; Male; Mitochondria, Liver; Myocardium; Nucleotides; Polydeoxyribonucleotides; Rats; Rats, Inbred Strains; Reperfusion Injury

1. Defibrotide, a single-stranded polydeoxyribonucleotide obtained from bovine lungs, has significant anti-thrombotic, pro-fibrinolytic and prostacyclin-stimulating properties. 2. The present study was designed to evaluate the effects of defibrotide on infarct size and regional myocardial blood flow in a rabbit model of myocardial ischaemia and reperfusion. 3. Defibrotide (32 mg kg-1 bolus + 32 mg kg-1 h-1, i.v.) either with or without co-administration of indomethacin (5 mg kg-1 x 2, i.v.) was administered 5 min after occlusion of the left anterior-lateral coronary artery and continued during the 60 min occlusion and subsequent 3 h reperfusion periods. 4. Defibrotide significantly attenuated the ischaemia-induced ST-segment elevation and abolished the reperfusion-related changes (R-wave reduction and Q-wave development) in the electrocardiogram. In addition, defibrotide significantly improved myocardial blood flow in normal and in ischaemic, but not in infarcted sections of the heart. The improvement in blood flow in normal perfused myocardium, but not in the ischaemic area was prevented by indomethacin. 5. Although the area at risk was similar in all animal groups studied, defibrotide treatment resulted in a 51% reduction of infarct size. Indomethacin treatment abolished the reduction of infarct size seen with defibrotide alone. 6. The data demonstrate a considerable cardioprotective effect of defibrotide in the reperfused ischaemic rabbit myocardium. This effect may be related, at least in part, to a stimulation of endogenous prostaglandin formation. Other possible mechanisms are discussed.

Topics: Animals; Blood Pressure; Coronary Circulation; Coronary Disease; Electrocardiography; Heart Rate; Hemodynamics; Indomethacin; Male; Microspheres; Myocardial Infarction; Polydeoxyribonucleotides; Rabbits; Reperfusion Injury