The fibrin-derived peptide Bbeta15-42 protects the myocardium against ischemia-reperfusion injury.

In the event of a myocardial infarction, current interventions aim to reopen the occluded vessel to reduce myocardial damage and injury. Although reperfusion is essential for tissue salvage, it can cause further damage and the onset of inflammation. We show a novel anti-inflammatory effect of a fibrin-derived peptide, Bbeta15-42. This peptide competes with the fibrin fragment N-terminal disulfide knot-II (an analog of the fibrin E1 fragment) for binding to vascular endothelial (VE)-cadherin, thereby preventing transmigration of leukocytes across endothelial cell monolayers. In acute or chronic rat models of myocardial ischemia-reperfusion injury, Bbeta15-42 substantially reduces leukocyte infiltration, infarct size and subsequent scar formation. The pathogenic role of fibrinogen products is further confirmed in fibrinogen knockout mice, in which infarct size was substantially smaller than in wild-type animals. Our findings conclude that the interplay of fibrin fragments, leukocytes and VE-cadherin contribute to the pathogenesis of myocardial damage and reperfusion injury. The naturally occurring peptide Bbeta15-42 represents a potential candidate for reperfusion therapy in humans.

Preconditioning by toll-like receptor 2 agonist Pam3CSK4 reduces CXCL1-dependent leukocyte recruitment in murine myocardial ischemia/reperfusion injury.

OBJECTIVE: To test whether preconditioning with a toll-like receptor (TLR) 2 agonist protects against myocardial ischemia and reperfusion by interfering with chemokine CXCL1 release from cardiomyocytes. DESIGN: C3H mice were challenged with vehicle or synthetic TLR2 agonist Pam3Cys-Ser-Lys4 (Pam3CSK4; 1 mg/kg) 24 hrs before myocardial ischemia (20 mins) and reperfusion (2 hrs or 24 hrs). Infarct size, troponin T release, and leukocyte recruitment were quantified. In murine cardiomyocytes (HL-1), we studied the expression/activation profile of TLR2 in response to stimulation with Pam3CSK4 (0.01-1 mg/mL). Furthermore, we studied the chemokine ligand 1 (CXCL1) response to Pam3CSK4 and ischemia/reperfusion in vivo and in vitro. SETTING: University hospital research laboratory. SUBJECTS: Anesthetized male mice and murine cardiomyocytes. MEASUREMENTS AND MAIN RESULTS: Preconditioning by Pam3CSK4 reduced infarct size and troponin T release. This was accompanied by a decreased recruitment of leukocytes into the ischemic area and an improved cardiac function. In HL-1 cells, TLR2 activation amplified the expression of the receptor in a time-dependent manner and led to CXCL1 release in a concentration-dependent manner. Preconditioning by Pam3CSK4 impaired CXCL1 release in response to a second inflammatory stimulus in vivo and in vitro. CONCLUSIONS: Preconditioning by TLR2 agonist Pam3CSK4 reduces myocardial infarct size after myocardial ischemia/reperfusion. One of the mechanisms involved is a diminished chemokine release from cardiomyocytes, which subsequently limits leukocyte infiltration.

Rosiglitazone is cardioprotective in a murine model of myocardial I/R.

The peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a regulator of anti-inflammatory genes. One of its agonists, rosiglitazone-widely used in the treatment of type 2 diabetes mellitus-has recently been reported to increase the risk for myocardial infarction. In contrast, various studies provide evidence for a rosiglitazone-induced cardioprotection in different models of acute myocardial I/R. Here, we report that this protection can still be observed after 28 days of reperfusion in a murine model even when treatment commenced after the period of ischemia (reperfusion therapy). In vitro, cells from the rat cardiomyoblast cell line H9c2(2-1) are protected against oxidative stress by incubation with rosiglitazone, which can be abrogated by dexamethasone or cycloheximide. The antioxidant enzyme heme oxygenase 1 is up-regulated in these cells after rosiglitazone treatment. Our data provide further evidence that rosiglitazone exerts protective effects during myocardial I/R and might contribute to the reevaluation of the approved drug rosiglitazone.

Caspase inhibitor zVAD.fmk reduces infarct size after myocardial ischaemia and reperfusion in rats but not in mice.

OBJECTIVE: Apoptosis of cardiomyocytes has been suggested to contribute to outcome following myocardial ischaemia and reperfusion (MI/R). Caspase inhibitors were developed as potential therapeutics for MI/R. However, various reports using the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) in the latter setting present conflicting results. Therefore, it is still unclear whether inhibition of apoptosis by caspase inhibitors promotes cardioprotection. MATERIALS AND METHODS: This study evaluated whether zVAD.fmk or novel caspase inhibitor quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh) reduce myocardial infarct size in mice. Secondly, we tested zVAD.fmk's potential infarct-sparing effects in rats and whether these are accompanied by improved left ventricular function. RESULTS: In mice neither zVAD.fmk nor Q-VD-OPh reduced infarct size. In rats, however, zVAD.fmk reduced infarct size following ischaemia (25min) and reperfusion (7 days) by approximately 53%. This was, however, accompanied by an increase in left ventricular end-diastolic pressure. CONCLUSION: This study provides further evidence that abrogation of apoptosis via caspase inhibition might not be sufficient to effectively limit infarct size following MI/R.

Lipoteichoic acid induces delayed myocardial protection in isolated rat hearts: a comparison with endotoxin.

OBJECTIVE: Preconditioning with bacterial wall fragments lipopolysaccharide (LPS) or lipoteichoic acid (LTA) reduce myocardial infarct size after ischaemia and reperfusion (I/R) in rats. Preconditioning with LTA reduces neutrophil accumulation during reperfusion and thereby ameliorates one of myocardial reperfusion injury's most important mechanisms. In this study, we use an ex vivo model of regional myocardial I/R to investigate LTA versus LPS induced preconditioning in a system devoid of leukocytes. METHODS: Rats were subjected to LTA or LPS with or without dexamethasone pre-treatment. Twenty-four hours after LTA or LPS challenge, hearts were removed and retrogradely perfused in a Langendorff set-up. Hearts underwent 20 min of regional ischaemia followed by 2 h of reperfusion. Ischaemic preconditioning (IPC) was performed as a positive control. Myocardial infarct size was determined as the primary end-point. RESULTS: LTA and LPS preconditioning both lead to a marked reduction in infarct size similar to IPC, however, no significant differences were found between LTA and LPS. The reduction in infarct size was abrogated by dexamethasone pre-treatment. CONCLUSION: We conclude that preconditioning with LTA and likewise with LPS confers myocardial protection in an ex vivo setting devoid of leukocytes. Dexamethasone inhibits preconditioning, suggesting that the underlying mechanism is dependent upon induction of a systemic inflammatory response to a LTA or LPS stimulus.

Toll-like receptor 9 expression in murine and human adrenal glands and possible implications during inflammation.

CONTEXT: Sepsis is a leading cause of death in the Western world and can be associated with failure of the hypothalamic-pituitary-adrenal axis. A coordinated response of the adrenal and immune system is of vital importance for survival during sepsis. Within the immune response, Toll-like receptors (TLRs) play a crucial role by recognizing pathogen-associated molecules such as bacterial DNA. TLR-9 can detect motifs of unmethylated cytosine-phosphate-guanine (CpG) dinucleotides (CpG-DNA) being present in bacterial DNA. OBJECTIVE: We investigated whether TLR-9 is expressed in human and murine adrenal glands and whether its activation is associated with an adrenal response. DESIGN: Human fetal and adult adrenal glands; wild-type, C57BL/6 and TLR-9 deficient (TLR-9-/-) mice; and in vitro cell line models were used in the study. SETTING: The study took place at a university hospital. RESULTS: TLR-9 is expressed in human and murine adrenal glands, as well as in in vitro cell lines (Y-1 and NCI-H295R cells). CpG-oligodeoxynucleotide challenge caused a 3-fold increase in plasma levels of corticosterone in wild-type mice. This effect was not observed in TLR-9-/- mice. Furthermore, CpG-oligodeoxynucleotide challenge resulted in a strong release of several inflammatory cytokines, such as TNF-alpha, and IL-1beta, -6, -10, and -12 in vivo as well as in vitro. Again, this effect was not present in TLR-9-/- mice. CONCLUSIONS: TLR-9 is present in both murine and human adrenal glands. TLR-9 stimulation led to a corticosterone and inflammatory cytokine response. TLR-9 may play a role in the regulation of the hypothalamic-pituitary-adrenal axis during conditions in which bacterial DNA is present.

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.

Thrombelastography for the monitoring of lipopolysaccharide induced activation of coagulation.

During Gram-negative sepsis, lipopolysaccharide (LPS) activates toll-like receptor (TLR) 4 and induces complex responses of immune system and haemostasis. In the present study we investigated whether thrombelastography is suitable to monitor the LPS-induced activation of coagulation. Whole blood samples from healthy volunteers were incubated with LPS for various incubation periods (0-5 hrs), thereafter rotation thrombelastography was performed. Incubation of whole blood (>or=3 h) with LPS markedly reduced clotting time; after 5 hrs the variable was reduced from 459+/-39 sec to 80+/-20 sec while the other thrombelastography variables (angle alpha, clot formation time, maximal clot formation) remained unaltered. EC(50) of the LPS effect on whole blood clotting time was 18 microg/ml. In isolated leukocytes, diluted in platelet poor plasma, far lower LPS-concentrations were effective: 10 ng/ml LPS reduced clotting time from 439+/-68 sec to 200+/-56 sec. Experiments with the protein synthesis inhibitor cycloheximide and active site-inhibited factor VIIa revealed that LPS exerts its effects via the synthesis of tissue factor. Addition of tissue factor to whole blood samples revealed that a concentration of 100 fmol/l can be detected using thrombelastography. In whole blood samples the tissue factor concentration induced by LPS amounted up to 12 pmol/l. In summary, thrombelastography proved to be a sensitive and reliable tool for the determination of LPS-induced tissue factor mediated activation of haemostasis in whole blood samples.

NSAIDs increase GM-CSF release by human synoviocytes: comparison with nitric oxide-donating derivatives.

Non-steroidal anti-inflammatory drugs (NSAIDs) are used to treat the condition of rheumatoid arthritis, where levels of prostaglandin E2 (PGE2) and granulocyte macrophage-colony stimulating factor (GM-CSF) are elevated in the synovial fluid. NO-NSAIDs are a new class of cyclooxygenase (COX)-inhibitors developed by coupling a nitric oxide (NO)-donating moiety to conventional NSAIDs. We show that, in cytokine-treated synoviocytes (from non-rheumatic patients), NO-naproxen and NO-flurbiprofen like their parent compounds concentration-dependently reduce the levels of PGE2 (an index of COX-2 activity), with a corresponding rise in the release of GM-CSF. Unlike acetylsalicylic acid (ASA), NO-ASA reduces the levels of PGE2, without increasing GM-CSF release, although cell viability is reduced at the highest concentration (1 mM). The effects of NSAIDs and NO-NSAIDs on GM-CSF release were attributable to the PGE2 mediated cyclic (c) AMP pathway because PGE2 reversed the effects of COX blockade. Second, phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-201724 (both of which elevate cAMP levels) decreased GM-CSF release, in the presence of PGE2. Finally, neither sodium nitroprusside nor zaprinast (both of which elevate cGMP levels) affected GM-CSF or PGE2 release. Our findings demonstrate that GM-CSF is regulated by NSAIDs and NO-NSAIDs via inhibition of COX and appears to be mediated via the cAMP pathway. NO-ASA is the exception, because it does not increase GM-CSF release, although at millimolar concentrations cell viability is reduced.

The effects and metabolic fate of nitroflurbiprofen in healthy volunteers.

OBJECTIVE: Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are a new class of cyclooxygenase (COX) inhibitors. To investigate whether these drugs actually release nitric oxide (NO), we labeled the nitroxy group of nitroflurbiprofen with nitrogen 15 to determine the metabolic fate of this compound in humans. METHOD: Six healthy volunteers who fasted were given an oral dose of 15 N-nitroflurbiprofen (100 mg). Samples of blood, urine, and gastric headspace gas were taken over a 24-hour period to determine the levels of nitroflurbiprofen, flurbiprofen, total nitrate/nitrite, 15 N-nitrate/nitrite, COX activity, and gastric NO. In a crossover study (1 week apart), a further 6 healthy volunteers who fasted were given an oral dose of nitroflurbiprofen (100 mg) or flurbiprofen (65 mg) and levels of gastric NO were determined. RESULTS: Nitroflurbiprofen was undetectable in the systemic circulation. Levels of 15 N-nitrate/nitrite (5.2% +/- 1.5% enrichment) and flurbiprofen (2.4 +/- 0.7 microg/mL) peaked at 4 hours in the plasma and gradually decreased thereafter. In unstimulated blood, the plasma levels of thromboxane B 2 (COX-1 activity) were 2 to 3 ng/mL, and after calcium ionophore stimulation, large amounts of thromboxane B 2 were produced (112 +/- 31 ng/mL). Prostaglandin E 2 was undetectable in unstimulated blood. After lipopolysaccharide stimulation, the plasma levels of prostaglandin E 2 increased to 15 +/- 4 ng/mL. The metabolite flurbiprofen inhibited plasma COX-1 activity for the duration of the study period (maximum inhibition at 4 hours), whereas COX-2 activity recovered after 6 hours. In the crossover study, levels of gastric NO were higher in subjects given nitroflurbiprofen, when compared with those given flurbiprofen. (The area under the curve for gastric NO was 435 +/- 107 ppm . h versus 305 +/- 94 ppm . h [95% confidence interval of the difference, 89-172 ppm . h; P < .001]). CONCLUSION: Nitroflurbiprofen was undetectable in the systemic circulation, suggesting metabolism to 15 N-nitrate/nitrite and flurbiprofen in the presystemic circulation. Levels of gastric NO were significantly higher after ingestion of nitroflurbiprofen than flurbiprofen.

Innate immunity, coagulation and surgery.

Inflammation is the host's defense mechanism to infection or trauma including surgical procedures. In the clinic, non-infectious inflammation plays an important part in cardiology (e.g. Percutaneous transluminal coronary angioplasty, PTCA), intensive care medicine (e.g. polytrauma), cardiac (e.g. extracorporeal circulation) and vascular surgery (e.g. reperfusion injury). An imbalance of the inflammatory response can cause an acute condition like sepsis or long-term Cardiovascular disease (CVD), both of which are leading killers in the Western world. Alterations in coagulation, innate immunity and endothelial function represent key aspects in the mechanism of inflammation and are the link between the pathogenesis of these two diseases. Studying inflammatory pathways or targeting specific mediators during inflammation may help to develop strategies to improve the clinical outcome of patients undergoing major surgery, where postoperative inflammation plays a crucial role.

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.

Toll-like receptor 4 plays a crucial role in the immune-adrenal response to systemic inflammatory response syndrome.

Sepsis and septic shock are leading killers in the noncoronary intensive care unit, and they remain worldwide health concerns. The initial host defense against bacterial infections involves Toll-like receptors (TLRs), which detect and respond to microbial ligands. In addition, a coordinated response of the adrenal and immune systems is crucial for survival during severe inflammation. Previously, we demonstrated a link between the innate immune system and the endocrine stress response involving TLR-2. Like TLR-2, TLR-4 is also expressed in human and mouse adrenals. In the present work, by using a low dose of LPS to mimic systemic inflammatory response syndrome, we have revealed marked cellular alterations in adrenocortical tissue and an impaired adrenal corticosterone response in TLR-4-/- mice. Our findings demonstrate that TLR-4 is a key mediator in the crosstalks between the innate immune system and the endocrine stress response. Furthermore, TLR polymorphisms could contribute to the underlying mechanisms of impaired adrenal stress response in patients with bacterial sepsis.

Impaired adrenal stress response in Toll-like receptor 2-deficient mice.

Septicemia is one of the major health concerns worldwide, and rapid activation of adrenal steroid release is a key event in the organism's first line of defense during this form of severe illness. The family of Toll-like receptors (TLRs) is critical in the early immune response upon bacterial infection, and TLR polymorphisms are frequent in humans. Here, we demonstrate that TLR-2 deficiency in mice is associated with reduced plasma corticosterone levels and marked cellular alterations in adrenocortical tissue. TLR-2-deficient mice have an impaired adrenal corticosterone release after inflammatory stress induced by bacterial cell wall compounds. This defect appears to be mediated by a decrease in systemic and intraadrenal cytokine expression, including IL-1, tumor necrosis factor alpha, and IL-6. Our data demonstrate a link between the innate immune system and the endocrine stress response. The critical role of TLR-2 in adrenal glucocorticoid regulation needs to be considered in patients with inflammatory disease.