Bß(15-42) attenuates the effect of ischemia-reperfusion injury in renal transplantation.

Renal ischemia-reperfusion contributes to reduced renal allograft survival. The peptide Bß(15-42), a breakdown product of fibrin, attenuates inflammation induced by ischemia-reperfusion in the heart by competitively blocking the binding of leukocytes to endothelial VE-cadherin, but whether it could improve outcomes in renal transplantation is unknown. Here, we tested the ability of Bß(15-42) to ameliorate the effects of renal ischemic injury during allogenic kidney transplantation in mice. In our renal transplantation model (C57BL/6 into BALB/c mice), treatment with Bß(15-42) at the time of allograft reperfusion resulted in significantly improved survival of recipients during the 28-day follow-up (60% versus 10%). Bß(15-42) treatment decreased leukocyte infiltration, expression of endothelial adhesion molecules, and proinflammatory cytokines. Treatment significantly attenuated allogenic T cell activation and reduced cellular rejection. Moreover, Bß(15-42) significantly reduced tubular epithelial damage and apoptosis, which we reproduced in vitro. These data suggest that Bß(15-42) may have therapeutic potential in transplant surgery by protecting grafts from ischemia-reperfusion injury.

Bbeta(15-42) protects against acid-induced acute lung injury and secondary pseudomonas pneumonia in vivo.

RATIONALE: Acute lung injury (ALI) is a serious condition in critically ill patients that predisposes to secondary bacterial pneumonia. Vascular leak is a hallmark in the pathogenesis of ALI. The fibrin-derived peptide Bbeta(15-42) was shown to preserve endothelial barriers, thereby reducing vascular leak. The potential therapeutic role of Bbeta(15-42) in ALI has not been addressed so far. OBJECTIVES: To investigate the therapeutic potential of Bbeta(15-42) in ALI and secondary pneumonia induced by Pseudomonas aeruginosa. METHODS: The effect of the fibrin-derived peptide Bbeta(15-42) was studied in models of ALI, induced either by pulmonary administration of LPS or hydrochloric acid. Lung inflammation was analyzed by quantifying cell influx, cytokine levels, and oxidized lipids. Vascular leak was determined by Evans Blue extravasations and alveolar protein content. In subsequent two-hit studies, mice were infected with P. aeruginosa 24 hours after induction of aspiration pneumonitis and effects of Bbeta(15-42) on inflammation, bacterial clearance, and survival were evaluated. MEASUREMENTS AND MAIN RESULTS: After LPS or acid inhalation, proinflammatory cytokine levels, neutrophil influx, and vascular leak were found diminished in mice treated with Bbeta(15-42). Acid aspiration impaired macrophage functions and rendered mice more susceptible to subsequent P. aeruginosa infection, whereas mice that received Bbeta(15-42) during acid aspiration and were subsequently challenged with bacteria displayed reduced inflammation, enhanced bacterial clearance, and ultimately improved survival. CONCLUSIONS: The fibrin-derived peptide Bbeta(15-42) exerted protective effects during ALI, resulting in diminished lung injury and preserved antibacterial properties of macrophages, which improved outcome during subsequent P. aeruginosa pneumonia.

The effects of the fibrin-derived peptide Bbeta(15-42) in acute and chronic rodent models of myocardial ischemia-reperfusion.

Many compounds have been shown to prevent reperfusion injury in various animal models, although to date, translation into clinic has revealed several obstacles. Therefore, the National Heart, Lung, and Blood Institute convened a working group to discuss reasons for such failure. As a result, the concept of adequately powered, blinded, randomized studies for preclinical development of a compound has been urged. We investigated the effects of a fibrin-derived peptide Bbeta(15-42) in acute and chronic rodent models of ischemia-reperfusion at three different study centers (Universities of Dusseldorf and Vienna, TNO Biomedical Research). A total of 187 animals were used, and the peptide was compared with the free radical scavenger Tempol, CD18 antibody, alpha-C5 antibody, and the golden standard, ischemic preconditioning. We show that Bbeta(15-42) robustly and reproducibly reduced infarct size in all models of ischemia-reperfusion. Moreover, the peptide significantly reduced plasma levels of the cytokines interleukin 1beta, tumor necrosis factor alpha, and interleukin 6. In rodents, Bbeta(15-42) inhibits proinflammatory cytokine release and is cardioprotective during ischemia-reperfusion injury.

Fibrin(ogen) and its fragments in the pathophysiology and treatment of myocardial infarction.

The occlusion of a coronary artery leads to ischemia of the myocardium, while permanent occlusion results in cell death and myocardial dysfunction. Early restoration of blood flow is the only means to reduce or prevent myocardial necrosis, but-paradoxically-reperfusion itself contributes to injury of the heart. In animal models, this phenomenon is well described, and there are many different unrelated approaches to reduce reperfusion injury. In humans, however, pharmacological interventions have so far failed to reduce myocardial reperfusion injury. We summarize the pathogenesis of reperfusion injury, detailing the role of fibrin(ogen) and its derivatives. Moreover, we introduce a new concept for fibrin derivatives as potential targets for reperfusion therapy.

Peptide Bbeta(15-42) preserves endothelial barrier function in shock.

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bbeta15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bbeta15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bbeta15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bbeta15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bbeta15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bbeta15-42 is confirmed in Fyn(-/-) mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bbeta15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.

Inducible nitric oxide synthase and heme oxygenase-1 in the lung during lipopolysaccharide tolerance and cross tolerance.

OBJECTIVE: Pretreatment with low-dose lipopolysaccharide protects cells/organs against a subsequent lethal Gram-negative (lipopolysaccharide tolerance) or Gram-positive (cross tolerance) stimulus. We determined whether this occurs in the rat lung. The involvement of inducible nitric oxide synthase and heme oxygenase-1 was evaluated. DESIGN: Laboratory study. SETTING: University hospital laboratory. SUBJECTS: Anesthetized male Wistar rats. INTERVENTIONS: To test the hypothesis, rats received saline or lipopolysaccharide (1 mg/kg). At 2, 4, 8, 16, or 24 hrs later, blood samples and lung tissue were taken to determine messenger RNA, protein concentration, and activity of inducible nitric oxide synthase and heme oxygenase-1. In additional experiments, rats were challenged with lipopolysaccharide (1 mg/kg) and subjected to Gram-negative (lipopolysaccharide) or Gram-positive (lipoteichoic acid and peptidoglycan) shock 24 hrs later. These studies were carried out in the presence and absence of inducible nitric oxide synthase or heme oxygenase-1 inhibitors (1400W or tin protoporphyrin IX). Following 6 hrs of shock, lung tissue was taken to determine lung damage and heme oxygenase-1 concentration and activity. MEASUREMENTS AND MAIN RESULTS: In the rat lung, lipopolysaccharide (1 mg/kg) induced a significant increase in inducible nitric oxide synthase protein at 8 hrs with a corresponding increase in plasma nitrate/nitrite at 8-16 hrs. Simultaneously, heme oxygenase-1 messenger RNA transcripts were observed at 8-16 hrs, and maximal expression of the protein followed (24 hrs). Pretreatment with low-dose lipopolysaccharide reduced myeloperoxidase activity (neutrophil infiltration) and wet-dry ratio (pulmonary edema) in the lungs of animals subjected to Gram-negative or Gram-positive shock, demonstrating tolerance. Pretreatment with low-dose lipopolysaccharide and the selective inducible nitric oxide synthase inhibitor 1400W reduced heme oxygenase-1 protein expression, and lung protection was abolished. Tin protoporphyrin IX did not affect heme oxygenase-1 expression, but heme oxygenase activity and lung protection were significantly reduced. CONCLUSIONS: We propose that nitric oxide (most likely inducible nitric oxide synthase derived) regulates the induction of heme oxygenase-1 in the lung, which in turn plays an important part in pulmonary protection during lipopolysaccharide tolerance and cross tolerance.

The fibrin-derived peptide Bbeta15-42 is cardioprotective in a pig model of myocardial ischemia-reperfusion injury.

OBJECTIVE: The fibrin-derived peptide Bbeta15-42 has been shown to reduce infarct size in rodent models of ischemia-reperfusion injury. To increase its potential for translation into the clinic, we studied the effects of Bbeta15-42 in pigs, whose coronary anatomy is similar to that of humans. In addition, we evaluated the pharmacokinetics and safety of Bbeta15-42 in several species, including humans. DESIGN: Animal study and phase I trial. SETTING: University hospital and contract research laboratories. SUBJECTS: Pigs/healthy volunteers. INTERVENTIONS: Male farm-bred Landrace pigs were subjected to 1 hr of left anterior descending coronary artery occlusion followed by 3 hrs of reperfusion. At the time of reperfusion, Bbeta15-42 (2.4 mg/kg, n = 6) or random peptide (control; 2.4 mg/kg, n = 6) was administered as an intravenous bolus. As a positive control, pigs were subjected to ischemic preconditioning (n = 6). Cardiac damage and hemodynamics were recorded. Biodistribution and pharmacokinetics of Bbeta15-42 were determined in rats and dogs. In a phase I trial involving 30 male healthy volunteers, pharmacokinetics and safety were tested in a randomized, double-blinded, placebo-controlled, parallel-group, single ascending dose study. MEASUREMENTS AND MAIN RESULTS: Bbeta15-42 and ischemic preconditioning significantly reduced myocardial infarct size and troponin I levels. Bbeta15-42 also reduces interleukin-6 levels, underlining its anti-inflammatory properties. Furthermore, in humans, the pharmacokinetics of the peptide Bbeta15-42 were comparable to those of animals, and no serious adverse effects were observed. CONCLUSIONS: Bbeta15-42 elicits cardioprotection in pigs and is clinically safe in phase I testing of humans. This study confirms the new concept of a pathogenic role of fibrin derivatives in myocardial reperfusion injury, which can be inhibited by peptide Bbeta15-42.