icatibant and Reperfusion-Injury

Tissue kallikrein is a serine proteinase capable of cleaving kininogen substrate to produce the potent vasodilator kinin peptide. Kinin mediates a complex set of physiological actions through its receptor signaling. Systemic delivery of the kallikrein gene in an adenoviral vector significantly reduced stroke-induced mortality rate, blood pressure elevation, and aortic hypertrophy in hypertensive Dahl-salt sensitive rats fed a high salt diet. Using a focal cerebral ischemic rat model induced by middle cerebral artery occlusion, intravenous or intracerebroventricular kallikrein gene delivery significantly reduced ischemia/repefusion (I/R)-induced neurological deficits, cerebral infarction, neuronal and glial cell apoptosis, and inflammatory cell infiltration, while promoting angiogenesis and neurogenesis in the ischemic brain. A continuous infusion of a sub-depressor dose of tissue kallikrein protein through implanted minipump decreased I/R-induced neurological dysfunction and cerebral infarction, inflammation and oxidative stress independent of kallikrein's blood pressure-lowering effect. Moreover, kallikrein offered neuroprotection even when delivered at one day after the onset of stroke. Kallikrein's protective effects were blocked by the kinin B2 receptor antagonist icatibant. The role of the kinin B2 receptor in mediating the protective effect against ischemic brain injury was further confirmed by increases in mortality rate and post-ischemic brain injury in kinin B2 receptor-deficient mice. Taken together, these results suggest a novel function of kallikrein as an anti-inflammatory and anti-oxidative agent in protecting the brain against ischemic stroke-induced injuries. These findings also raise the possibility that tissue kallikrein may have value in the treatment of acute ischemic stroke.

Topics: Adenoviridae; Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Bradykinin; Brain Ischemia; Cerebral Infarction; Genetic Therapy; Genetic Vectors; Humans; Inflammation; Kinins; Mice; Neovascularization, Pathologic; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Inbred Dahl; Receptors, Peptide; Reperfusion Injury; Salts; Signal Transduction; Stroke; Tissue Kallikreins

Angiotensin-converting enzyme (ACE) inhibitors exert their beneficial effects not only via endocrine mechanisms, but most probably also via interference with autocrine-paracrine actions involving local renin-angiotensin and kallikrein-kinin systems with subsequent autacoid release. Inhibition of ACE (kininase II) results in the reduction of angiotensin II generation and kinin degradation, leading to beneficial cardiovascular effects. Bradykinin and prostacyclin release from isolated rat hearts was increased by local ACE inhibitions with ramiprilat. In different models the bradykinin-mediated effects of ACE inhibition were abolished with the specific B2 kinin-receptor antagonist Hoe 140: The cardioprotective effects of ramiprilat or ramipril such as reduction of postischemic reperfusion injuries in isolated rat hearts or the reduction in infarct size in dogs and rabbits were abolished by coadministration of Hoe 140. Furthermore, left ventricular hypertrophy in rats with aortic banding could be prevented or regression was induced when the ACE inhibitor was given in a non-blood pressure-lowering dose. These beneficial effects were also abolished by Hoe 140. In conclusion, in different experimental models, ACE inhibitors exert cardioprotective effects. An enhancement of endothelial autacoid formation (nitric oxide and prostacyclin) by inhibiting degradation of bradykinin may contribute to these effects.

Topics: Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; Cardiomegaly; Epoprostenol; Heart; Kallikrein-Kinin System; Nitric Oxide; Renin-Angiotensin System; Reperfusion Injury

To examine whether endogenous bradykinin mediates the endothelium-dependent vasomotor dysfunction induced by ischaemia-reperfusion injury, or the protection afforded by remote ischaemic preconditioning in vivo in man.. Randomised double-blind, cross-over study.. Royal Infirmary of Edinburgh, Wellcome Trust Clinical Research Facility.. Twenty healthy male volunteers.. Subjects were randomised to intravenous infusion of the bradykinin B(2) receptor antagonist, HOE-140 (100 μg/kg), or saline placebo in a double-blind, crossover trial. Ischaemia-reperfusion injury was induced in the non-dominant arm by inflating a cuff to 200 mm Hg for 20 min in all subjects. Ischaemia-reperfusion injury was preceded by three cycles of remote ischaemic preconditioning in the dominant arm in 10 subjects.. Bilateral forearm blood flow was assessed using venous occlusion plethysmography during intra-arterial infusion of acetylcholine (5-20 μg/min).. Acetylcholine caused vasodilatation in all studies (p<0.05) that was attenuated by ischaemia-reperfusion injury, both in the presence (p=0.0002) and absence (p=0.04) of HOE-140. Remote ischaemic preconditioning abolished the impairment of endothelium-dependent vasomotor function induced by ischaemia-reperfusion injury. HOE-140 had no effect on the protection afforded by remote ischaemic preconditioning.. These findings do not support a major role for endogenous bradykinin, acting via the B(2) kinin receptor, in the mechanism of ischaemia-reperfusion injury or the protective effects of remote ischaemic preconditioning in man.. NCT00965120 and NCT00965393.

Topics: Acetylcholine; Adrenergic beta-Antagonists; Adult; Bradykinin; Bradykinin Receptor Antagonists; Cross-Over Studies; Double-Blind Method; Endothelium, Vascular; Forearm; Hemodynamics; Humans; Infusions, Intravenous; Injections, Intra-Arterial; Ischemic Preconditioning; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Plethysmography; Prognosis; Reperfusion Injury; Sodium Chloride; Vasodilator Agents

Bradykinin (BK) is used in different tissues. Dose-dependent studies have demonstrated that low doses protect against ischemia/reperfusion (I/R) injury while higher doses lead to adverse effects. Although the beneficial effects of BK infusion were observed in myocardium, its role on the I/R impact in skeletal muscle (SM) has not been fully clarified.. This study was carried out to evaluate the effects of BK, administered in the hindlimbs of rats subjected to I/R.. The study design included three experimental groups: Group 1 control (saline), Group 2 (bradykinin), and Group 3 (HOE 140, a BK2 receptor blocker). In all three groups, rats were subjected to hindlimb ischemia for a total of 2 h followed by continuous 4 h of reperfusion with pharmacological interventions. The methods include analysis of enzymes (lactate dehydrogenase-LDH and creatinine phosphokinase-CPK), cell membrane marker of injury (malondialdeyde-MDA), recruitment of neutrophils (myeloperoxidase-MPO), and apoptosis index (immunohistochemistry TUNEL in situ peroxidase dead end).. Except for the apoptotic index, all parameters studied were shown to be elevated in the reperfusion group intervened with BK. The blocking of BK2 receptors by HOE 140 did not affect the I/R injury.. After 2 h of total ischemia, infusion of bradykinin during 4 h of reperfusion, worsened the I/R injury in the hindlimb skeletal muscle.

Topics: Animals; Apoptosis; Bradykinin; Bradykinin B2 Receptor Antagonists; Creatine Kinase; Hindlimb; L-Lactate Dehydrogenase; Male; Malondialdehyde; Muscle, Skeletal; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Vasodilator Agents

Free radical generation is a key event in cerebral reperfusion injury. Bradykinin (Bk) and interleukin-1β (IL-1β) have both been implicated in edema formation after stroke, although acute Bk application itself results in only a modest permeability increase. We have investigated the molecular mechanism by assessing the permeability of single pial venules in a stroke model. Increased permeability on reperfusion was dependent on the duration of ischemia and was prevented by applying the B(2) receptor antagonist HOE 140. Postreperfusion permeability increases were mimicked by applying Bk (5μM) for 10 min and blocked by coapplying the IL-1 receptor antagonist with Bk. Furthermore, 10 min pretreatment with IL-1β resulted in a 3 orders of magnitude leftward shift of the acutely applied Bk concentration-response curve. The left shift was abolished by scavenging free radicals with superoxide dismutase and catalase. Apocynin coapplied with IL-1β completely blocked the potentiation, implying that NADPH oxidase assembly is the immediate target of IL-1β. In conclusion, this is first demonstration that bradykinin, released during cerebral ischemia, leads to IL-1β release, which in turn activates NADPH oxidase leading to blood-brain barrier breakdown.

Topics: Acetophenones; Animals; Bradykinin; Bradykinin Receptor Antagonists; Brain; Brain Ischemia; Capillary Permeability; Catalase; Enzyme Activation; Female; Free Radical Scavengers; Interleukin-1beta; Male; Microspheres; NADPH Oxidases; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Superoxide Dismutase

Systemic or local delivery of human tissue kallikrein gene (hTK) has been shown to be an effective strategy to alleviate cerebral ischemia/reperfusion (I/R) injury, and tissue kallikrein (TK) administration can suppress glutamate- or acidosis-mediated neurotoxicity in vitro. In the present study, the role of TK in hypoxia/reoxygenation (H/R) induced neuronal cell death was investigated. We found that TK administration could remarkably alleviate H/R-induced neuronal injury by reduction of LDH release and promotion of neuron viability. The protective effects of TK could be counteracted by bradykinin B2 receptor (B2R) antagonist HOE140, which could suppress up-regulation of TK on the ERK signal pathway under H/R condition. These results indicate that TK plays an important role in preventing neurons from H/R damage at least partially through the TK-B2R-ERK1/2 pathway.

Topics: Animals; Bradykinin; Bradykinin Receptor Antagonists; Brain Ischemia; Cells, Cultured; Cerebral Cortex; Cytoprotection; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Rats; Reperfusion Injury; Tissue Kallikreins

PURPOSE. To investigate the effects of an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin II antagonist against retinal ischemia-reperfusion injury in the rat retina. METHODS. Retinal ischemia was induced by increasing intraocular pressure to 130 mm Hg. Rats were treated with an ACE inhibitor (captopril), an angiotensin II type 1 receptor (AT1-R) antagonist (candesartan), an AT2-R antagonist (PD123319), bradykinin, or a bradykinin B2 receptor antagonist (icatibant). At 7 days after the ischemia, retinal damage was evaluated. Immunohistochemistry and image analysis were used to measure changes in the levels of reactive oxygen species (ROS) and the localization of AT1-R. Dark-adapted full-field electroretinography (ERG) was also performed. RESULTS. Pretreatment with captopril or candesartan significantly inhibited the ischemic injury of the inner retina. However, PD123319, bradykinin, or icatibant did not reduce the ischemic damage. In control retinas, retinal vessels were positive for AT1-R. In contrast, 12 hours after ischemia, immunohistochemical analysis detected numerous AT1-R-positive cells in the inner retina in vehicle-treated rats. After ischemia, the production of ROS was detected in retinal cells. However, pretreatment with captopril or candesartan suppressed the production of ROS. On the seventh postoperative day, the amplitudes of the ERG b-waves were significantly lower in the vehicle group than in the groups pretreated with captopril or candesartan. CONCLUSIONS. The present findings demonstrate that ischemic damage promotes the expression of AT1-R in the inner retina. Both the ACE inhibitor and the AT1-R antagonist that were examined can block the stimulation of the AT1-R and attenuate the subsequent ischemic damage in the rat retina.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin II Type 2 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Benzimidazoles; Biphenyl Compounds; Bradykinin; Bradykinin B2 Receptor Antagonists; Captopril; Cell Survival; Electroretinography; Enzyme-Linked Immunosorbent Assay; Female; Imidazoles; Immunoenzyme Techniques; Neuroprotective Agents; Pyridines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptor, Angiotensin, Type 1; Reperfusion Injury; Retina; Retinal Diseases; Retinal Ganglion Cells; Tetrazoles

Angiotensin I-converting enzyme (ACE; kininase II) levels in humans are genetically determined. ACE levels have been linked to risk of myocardial infarction, but the association has been inconsistent, and the causality underlying it remains undocumented. We tested the hypothesis that genetic variation in ACE levels influences myocardial tolerance to ischemia. We studied ischemia-reperfusion injury in mice bearing 1 (ACE1c), 2 (ACE2c, wild type), or 3 (ACE3c) functional copies of the ACE gene and displaying an ACE level range similar to humans. Infarct size in ACE1c was 29% lower than in ACE2c (P<0.05). Pretreatment with a kinin B2 receptor antagonist suppressed this reduction. In ACE3c, infarct size was the same as in ACE2c. But ischemic preconditioning, which reduced infarct size in ACE2c (-63%, P<0.001) and ACE1c (-52%, P<0.05), was not efficient in ACE3c (-2%, NS, P<0.01 vs. ACE2c). In ACE3c, ischemic preconditioning did not decrease myocardial inflammation or cardiomyocyte apoptosis. Pretreatment with a renin inhibitor had no cardioprotective effect in ACE2c, but in ACE3c partially restored (38%) the cardioprotection of ischemic preconditioning. Thus, a modest genetic increase in ACE impairs myocardial tolerance to ischemia. ACE level plays a critical role in cardiac ischemia, through both kinin and angiotensin mediated mechanisms.

Topics: Amides; Angiotensin I; Angiotensin II; Animals; Apoptosis; Blood Pressure; Bradykinin; Bradykinin Receptor Antagonists; Fumarates; Heart; Kinins; Lung; Mice; Mice, Mutant Strains; Myocardial Infarction; Myocardial Ischemia; Myocardium; Peptidyl-Dipeptidase A; Renin; Reperfusion Injury

The bradykinin B2 receptor (B2R) mediates many physiological processes such as hypotension, inflammation and blood-vessel permeability. Hypoxia/reoxygenation (H/R) induces neuronal cell apoptosis. It was found that B2R expression was enhanced in primary cultured cortical neurons after H/R treatment. Addition of bradykinin (BK) alleviated the neuronal damage from H/R. This protective effect of BK was inhibited by the B2R antagonist, HOE140, and the ERK1/2 antagonist, PD98059. The phosphorylation of ERK1/2 was increased under H/R, and the addition of BK enhanced this effect. These results indicate that B2R plays an important role in protecting neurons from damage induced by H/R and this effect may function through the ERK1/2 pathway.

Topics: Animals; Apoptosis; Bradykinin; Bradykinin B2 Receptor Antagonists; Cell Hypoxia; Cells, Cultured; Culture Media; Flavonoids; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Oxygen; Phosphorylation; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptor, Bradykinin B2; Reperfusion Injury

Biomacromolecule like exogenous Kallikrein is difficult to pass through biomembrane and blood brain barrier. So, the use of exogenous Kallikrein for the therapy of nervous system diseases is restricted. We constructed the Protein Transduction Domain-Kallikrein (PTD-Kallikrein), checked its function of penetration and biotoxicity, and observed its influence on neurons and ischemic brain tissues.. PTD-Kallikrein (tissue kallikrein) was prepared by chemical synthesis. After PTD-Kallikrein injected 2.5 hours, rats brains were taken out and contents of Kallikrein were quantitated to observe the function of passing through blood brain barrier. Cell survival rate were measured by XTT methods to determine the peptide's biotoxicity. Apoptosis were inspected by TUNEL. PTD-Kallikrein was administrated immediately after cerebral ischemia. 24h later, infarct volume was determined by TTC stain and IL-1beta, TNF-alpha as well as PGE2 were measured by ELISA.. 1. PTD-Kallikrein can pass through the biomembrane and blood brain barrier; 2. PTD-Kallikrein itself has no obviously biotoxicity. 3. PTD-Kallikrein increases cell survival rate, decreases neurons apoptosis during OGD/recovery; 4. HOE140 inhibits the effects of PTD-Kallikrein. 5. PTD-Kallikrein improves neurological impairment, decreases the infarct volume, and inhibits the release of IL-1beta, TNF-alpha, PGE2. 6. HOE140 inhibits the effects of PTD-Kallikrein on ischemia-reperfusion injury.. 1. PTD-Kallikrein can pass through the biomembrane and BBB efficiently and itself has no obviously biotoxicity. 2. PTD-Kallikrein has neuroprotective effect on neurons and cerebral ischemia injury. 3. PTD-Kallikrein is partially mediated by B2 receptors.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blood-Brain Barrier; Bradykinin; Brain; Brain Ischemia; Cell Membrane Permeability; Cell Survival; Cells, Cultured; Dinoprostone; Interleukin-1beta; Male; Neurons; Rats; Rats, Wistar; Recombinant Fusion Proteins; Reperfusion Injury; Tissue Kallikreins; Tumor Necrosis Factor-alpha

Bradykinin is both a potent vasodilatator and a central inflammatory mediator. Similar to findings in myocardial reperfusion injury, bradykinin might mediate the protective effects of angiotensin-converting enzyme (ACE) inhibition after liver ischemia via increased bradykinin-2-receptor (B-2) stimulation. On the other hand, B-2-inhibition has been shown to reduce liver reperfusion injury. This study was designed to investigate the role of Bradykinin in hepatic reperfusion injury.. Twenty eight rats were allocated randomly to Sham procedure (Sham), 30-min normothermic ischemia (ischemia), ischemia with Ramiprilat (ACE-I), or ischemia with Ramiprilat and B-2-inhibitor HOE 140 (ACE-I+B-2-I). Liver microcirculation and leukocyte adherence were investigated using intravital microscopy 30 min after reperfusion (n = 7 per group). In addition, serum activities of AST and ALT were measured for 7 days (n = 28).. Ischemia was associated with a loss of perfused sinusoids, sinusoidal vasoconstriction, and a reduction in microvascular blood flow. Permanent leukocyte adherence increased both in sinusoids and in postsinusoidal venoles. ACE-I restored sinusoidal perfusion, normalized vasoregulation, maintained sinusoidal blood flow, and inhibited leukocyte adhesion. ACE-I+B-2-I abolished the protective effects linked to ACE-I. Ischemia-induced liver cell injury after 5 h of reperfusion was ameliorated by ACE-I. In the ACE-I+B-2-I group, reduction in liver cell injury was reversed.. After hepatic ischemia, ACE-I reduced reperfusion injury in a B-2-dependent manner. These results suggest a pivotal role for bradykinin in the treatment of reperfusion injury by Ramiprilat, mediating sinusoidal dilation and blunting hepatic inflammation.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Flow Velocity; Bradykinin; Bradykinin B2 Receptor Antagonists; Cell Adhesion; Endothelial Cells; Female; Ischemia; Leukocytes; Liver; Microcirculation; Ramipril; Rats; Rats, Wistar; Receptor, Bradykinin B2; Reperfusion Injury; Vasoconstriction

Ischemia/reperfusion (I/R) in post-arterior post-capillary venules induces an acute inflammatory response, characterized by increased adherence and emigration of leukocytes and vascular permeability, all of which play important roles in cardiovascular disease. The aim of this study was to determine the roles of angiotensin II and AT1 receptor blockade in microvascular I/R injury in rats. Rats were anesthetized and intubated, then the peritoneum was opened and the mesentery was revealed. Small post-capillary venules were examined by in vivo fluorescence microscopy. The flow of erythrocytes and leukocytes was observed under the microscope and video recorded for later dynamic analyses. The superior mesenteric artery (SMA) was ligated with polyethylene tubing and released to induce I/R (20 min of ischemia/60 min of reperfusion). Subsequently, leukocyte adhesion, emigration and albumin leakage were compared with those of non-I/R controls. I/R injury was significantly suppressed by superfusing tissues with the AT1 receptor antagonist losartan (LO; 10 microM). The beneficial effects of LO were inhibited by topical application of either the bradykinin B2 receptor antagonist HOE140 (10 nM) or nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME 10 microM). The effects of LO were lost in the presence of AT2 receptor blocker PD 123319 (PD). In conclusion, LO suppressed and protected against I/R injuries. The possible interaction between AT1 and AT2 receptors was also suggested.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Bradykinin; Capillary Permeability; Cell Adhesion; Leukocyte Rolling; Leukocytes; Losartan; Male; Mesentery; Models, Animal; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Venules; Videotape Recording

Our previous study has shown that human tissue kallikrein protected against ischemia/reperfusion-induced myocardial injury. In the present study, we investigated the protective role of local kallikrein gene delivery in ischemia/reperfusion-induced cardiomyocyte apoptosis and its signaling mechanisms in promoting cardiomyocyte survival. Adenovirus carrying the human tissue kallikrein gene was delivered locally into the heart using a catheter-based technique. Expression and localization of recombinant human kallikrein in rat myocardium after gene transfer were determined immunohistochemically. Kallikrein gene delivery markedly reduced reperfusion-induced cardiomyocyte apoptosis identified by both in situ nick end-labeling and DNA fragmentation. Delivery of the kallikrein gene increased phosphorylation of Src, Akt, glycogen synthase kinase (GSK)-3beta, and Bad(Ser-136) but reduced caspase-3 activation in rat myocardium after reperfusion. The protective effect of kallikrein on apoptosis and its signaling mediators was blocked by icatibant and dominant-negative Akt, indicating a kinin B2 receptor-Akt-mediated event. Similarly, kinin or transduction of kallikrein in cultured cardiomyocytes promoted cell viability and attenuated apoptosis induced by hypoxia/reoxygenation. The effect of kallikrein on cardiomyocyte survival was blocked by dominant-negative Akt and a constitutively active mutant of GSK-3beta, but it was facilitated by constitutively active Akt, catalytically inactive GSK-3beta, lithium, and caspase-3 inhibitor. Moreover, kallikrein promoted Bad.14-3-3 complex formation and inhibited Akt-GSK-3beta-dependent activation of caspase-3, whereas caspase-3 administration caused reduction of the Bad.14-3-3 complex, indicating an interaction between Akt-GSK-caspase-3 and Akt-Bad.14-3-3 signaling pathways. In conclusion, kallikrein/kinin protects against cardiomyocyte apoptosis in vivo and in vitro via Akt-Bad.14-3-3 and Akt-GSK-3beta-caspase-3 signaling pathways.

Topics: 14-3-3 Proteins; Adenoviridae; Animals; Apoptosis; bcl-Associated Death Protein; Blotting, Western; Bradykinin; Carrier Proteins; Caspase 3; Caspases; Cell Survival; DNA; DNA Fragmentation; DNA, Complementary; Gene Transfer Techniques; Genes, Dominant; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hypoxia; Immunohistochemistry; Immunoprecipitation; In Situ Nick-End Labeling; Ischemia; Kallikreins; Kinins; Lithium; Male; Myocardium; Myocytes, Cardiac; Oxygen; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Recombinant Proteins; Reperfusion Injury; Signal Transduction

The administration of bradykinin may attenuate ischemia and reperfusion (I/R) injury by acting on B(2)Rs. Blockade of B(2)R has also been shown to ameliorate lesions associated with I/R injury. In an attempt to explain these contradictory results, the objective of the present work was to investigate the role of and interaction between B(1) and B(2) receptors in a model of intestinal I/R injury in mice. The bradykinin B(2)R antagonist (HOE 140) inhibited reperfusion-induced inflammatory tissue injury and delayed lethality. After I/R, there was an increase in the expression of B(1)R mRNA that was prevented by HOE 140. In mice that were deficient in B(1)Rs (B(1)R(-/-) mice), inflammatory tissue injury was abrogated, and lethality was delayed and partially prevented. Pretreatment with HOE 140 reversed the protective anti-inflammatory and antilethality effects provided by the B(1)R(-/-) phenotype. Thus, B(2)Rs are a major driving force for B(1)R activation and consequent induction of inflammatory injury and lethality. In contrast, activation of B(2)Rs may prevent exacerbated tissue injury and lethality, an effect unmasked in B(1)R(-/-) mice and likely dependent on the vasodilatory actions of B(2)Rs. Blockade of B(1)Rs could be a more effective strategy than B(2) or B(1)/B(2) receptor blockade for the treatment of the inflammatory injuries that follow I/R.

Topics: Animals; Bradykinin; Bradykinin B1 Receptor Antagonists; Bradykinin B2 Receptor Antagonists; Chemokine CCL2; Cytokines; Inflammation Mediators; Interleukin-1; Interleukin-18; Intestinal Mucosa; Intestines; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptor Cross-Talk; Receptor, Bradykinin B1; Receptor, Bradykinin B2; Reperfusion Injury; RNA, Messenger

1. Bradykinin (BK) appears to play an important role in the development and maintenance of inflammation. Here, we assessed the role of the BK B(2) receptor for the injuries that occur after ischemia and reperfusion (I/R) of the territory irrigated by the superior mesenteric artery. 2. Tissue (lung and duodenum) kallikrein activity increased after ischemia with greater enhancement after reperfusion. A selective inhibitor of tissue kallikrein, Phenylacetyl-Phe-Ser-Arg-N-(2,3-dinitrophenyl)-ethylenediamine (TKI, 0.001-10 mg ml(-1)), inhibited kallikrein activity in a concentration-dependent manner in vitro. In vivo, pretreatment with TKI (30 mg kg(-1)) prevented the extravasation of plasma and the recruitment of neutrophils. 3. Similarly, the bradykinin B(2) receptor antagonists, HOE 140 (0.01-1.0 mg kg(-1)) or FR173657 (10.0 mg kg(-1)), inhibited reperfusion-induced increases in vascular permeability and the recruitment of neutrophils in the intestine and lungs. 4. In a model of more severe I/R injury, HOE 140 (1.0 mg kg(-1)) inhibited the increase in vascular permeability, neutrophil recruitment, haemorrhage and tissue pathology. Furthermore, HOE 140 significantly inhibited the elevations of TNF-alpha in tissue and serum and partially prevented lethality. This was associated with an increase in the concentrations of IL-10 in tissue and serum. 5. Thus, our results demonstrate that, following intestinal I/R injury, there is an increase in tissue kallikrein activity and activation of BK B(2) receptors. B(2) receptor activation is essential for the development of inflammatory tissue injury and lethality. These results contrast with those of others showing that BK mostly exerts a protective role during I/R injury.

Topics: Animals; Bradykinin; Bradykinin B2 Receptor Antagonists; Capillary Permeability; Dose-Response Relationship, Drug; Inflammation; Interleukins; Intestinal Mucosa; Intestines; Lung; Male; Mesenteric Artery, Superior; Neutrophil Infiltration; Oligopeptides; Rats; Rats, Wistar; Receptor, Bradykinin B2; Reperfusion Injury; Tissue Kallikreins; Tumor Necrosis Factor-alpha

We sought to determine the mechanisms whereby brief administration of bradykinin (bradykinin preconditioning, BK-PC) before prolonged ischemia followed by reperfusion (I/R) prevents postischemic microvascular dysfunction. Intravital videomicroscopic approaches were used to quantify I/R-induced leukocyte/endothelial cell adhesive interactions and microvascular barrier disruption in single postcapillary venules of the rat mesentery. I/R increased the number of rolling, adherent, and emigrated leukocytes and enhanced venular albumin leakage, effects that were prevented by BK-PC. The anti-inflammatory effects of BK-PC were largely prevented by concomitant administration of a B(2)-receptor antagonist but not by coincident B(1) receptor blockade, nitric oxide (NO) synthase inhibition, or cyclooxygenase blockade. However, NO synthase blockade during reperfusion after prolonged ischemia was effective in attenuating the anti-inflammatory effects of BK-PC. Pan protein kinase C (PKC) inhibition antagonized the beneficial effects of BK-PC but only when administered during prolonged ischemia. In contrast, specific inhibition of the conventional PKC isotypes failed to alter the effectiveness of BK-PC. These results indicate that bradykinin can be used to pharmacologically precondition single mesenteric postcapillary venules to resist I/R-induced leukocyte recruitment and microvascular barrier dysfunction by a mechanism that involves B(2) receptor-dependent activation of nonconventional PKC isotypes and subsequent formation of NO.

Topics: Animals; Anti-Inflammatory Agents; Bradykinin; Bradykinin Receptor Antagonists; Capillaries; Cell Adhesion; Chemotaxis, Leukocyte; Enzyme Inhibitors; Ischemic Preconditioning; Leukocytes; Male; Mesentery; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitrites; omega-N-Methylarginine; Protein Kinase C; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Although a number of recent reports indicate that bradykinin attenuates ischemia- reperfusion (I/R)-induced tissue injury, the mechanisms underlying its protective actions are not fully understood. However, because bradykinin induces endothelial nitric oxide (NO) production and NO donors have been shown to attenuate postischemic leukocyte adhesion, endothelial barrier disruption, and tissue injury, we hypothesized that bradykinin may act to reduce I/R-induced tissue injury by preventing leukocyte recruitment and preserving microvascular barrier function. To address this postulate, we used intravital videomicroscopic approaches to quantify leukocyte-endothelial cell interactions and microvascular barrier function in single postcapillary venules in the rat mesentery. Reperfusion after 20 min of ischemia significantly decreased wall shear rate and leukocyte rolling velocity, increased the number of rolling, adherent, and emigrated leukocytes, and disrupted the microvascular barrier as evidenced by enhanced venular albumin leakage. Superfusion of the mesentery with bradykinin (10 nM) during I/R significantly reduced these deleterious effects of I/R. Although these inhibitory effects of bradykinin were not affected by cyclooxygenase blockade with indomethacin (10 microM), coadministration with NO synthase (N(omega)-nitro-L-arginine methyl ester, 10 microM) or bradykinin B(2)-receptor (HOE-140, 1 microM) antagonists abolished the protective actions of bradykinin. Plasma NO concentration was measured in the mesenteric vein and was significantly decreased after I/R, an effect that was prevented by bradykinin treatment. These results indicate that bradykinin attenuates I/R-induced leukocyte recruitment and microvascular dysfunction by a mechanism that involves bradykinin B(2)-receptor-dependent NO production.

Topics: Animals; Bradykinin; Bradykinin Receptor Antagonists; Capillary Permeability; Chemotaxis, Leukocyte; Male; NG-Nitroarginine Methyl Ester; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Venules

Aminopeptidase P and angiotensin-converting enzyme (ACE) are responsible for the metabolism of exogenously administered bradykinin in the coronary circulation of the rat. It has been shown that ACE inhibitors decrease cytosolic enzyme release from the ischemic rat heart and reduce reperfusion-induced ventricular arrhythmias by increasing endogenous levels of bradykinin. It was hypothesized that the aminopeptidase P inhibitor apstatin could do the same. In an isolated perfused rat heart preparation subjected to global ischemia and reperfusion, both apstatin and ramiprilat (an ACE inhibitor) significantly decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release. The difference between the postischemia and preischemia levels of released CK was reduced 68% by apstatin and 68% by ramiprilat compared with control. The corresponding reductions in LDH release were 74% for apstatin and 81% for ramiprilat. A combination of the inhibitors was not significantly better than either one alone. Apstatin and ramiprilat also significantly reduced the duration of reperfusion-induced ventricular fibrillation by 69 and 61%, respectively. The antiarrhythmic effect of apstatin was reversed by HOE140, a bradykinin B2-receptor antagonist, suggesting that apstatin is acting by potentiating endogenously formed bradykinin. The results demonstrate that the aminopeptidase P inhibitor apstatin is cardioprotective in this model of cardiac ischemia/ reperfusion injury.

Topics: Adrenergic beta-Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Arrhythmias, Cardiac; Bradykinin; Cardiovascular Agents; Creatine Kinase; Drug Interactions; In Vitro Techniques; L-Lactate Dehydrogenase; Male; Peptides; Perfusion; Protease Inhibitors; Ramipril; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ventricular Fibrillation

After transient episodes of ischemia, benefits of thrombolytic or angioplastic therapy may be limited by reperfusion injury. Angiotensin-converting enzyme inhibitors protect the heart against ischemia/reperfusion injury, an effect mediated by kinins. We examined whether the protective effect of the angiotensin-converting enzyme inhibitor ramiprilat on myocardial ischemia/reperfusion is due to kinin stimulation of prostaglandin and/or nitric oxide release. The left anterior descending coronary artery of Lewis inbred rats was occluded for 30 minutes, followed by 120 minutes of reperfusion. Immediately before reperfusion rats were treated with vehicle, ramiprilat, or the angiotensin II type 1 receptor antagonist losartan. We tested whether pretreatment with the kinin receptor antagonist Hoe 140, the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester, or the cyclooxygenase inhibitor indomethacin blocked the effect of ramiprilat on infarct size and reperfusion arrhythmias. In controls, infarct size as a percentage of the area at risk was 79 +/- 3%; ramiprilat reduced this to 49 +/- 4% (P < .001), but losartan had little effect (74 +/- 6%, P = NS). Pretreatment with Hoe 140, NG-nitro-L-arginine methyl ester, or indomethacin abolished the beneficial effect of ramiprilat. Compared with the 30-minute ischemia/120-minute reperfusion group, nonreperfused hearts with 30 minutes of ischemia had significantly smaller infarct size as a percentage of the area at risk, whereas in the 150-minute ischemia group it was significantly larger. This suggests that reperfusion caused a significant part of the myocardial injury, but it also suggests that compared with prolonged ischemia, reperfusion salvaged some of the myocardium. Ventricular arrhythmias mirrored the changes in infarct size. Thus, angiotensin-converting enzyme inhibitors protect the myocardium against ischemia/reperfusion injury and arrhythmias; these beneficial effects are mediated primarily by a kinin-prostaglandin-nitric oxide pathway, not inhibition of angiotensin II formation.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arginine; Arrhythmias, Cardiac; Bradykinin; Bradykinin Receptor Antagonists; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Hemodynamics; Indomethacin; Male; Myocardial Infarction; Myocardial Ischemia; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Ramipril; Rats; Rats, Inbred Lew; Reperfusion Injury

Acute pancreatitis is characterized by two different courses of the disease, edematous and hemorrhagic-necrotizing pancreatitis. The pathogenesis and causes for the progression of pancreatitis are unknown. Ischemia/reperfusion with formation of oxygen free radicals, activation of leukocytes and consecutive failure of the microcirculation has gained attention as a causative factor. Furthermore, the degree of microcirculatory injury correlates with the severity of pancreatitis. The aim of the study was to investigate the influence of long term reperfusion after ischemia of the pancreas for 2 hours on morphological changes and enzyme release of the pancreas in rats. Since the characteristic features of postischemic pancreatic reperfusion injury are kinin-mediated we employed the bradykinin B2-receptor antagonist HOE 140 to inhibit the progression of postischemic changes of the pancreas.. Under ether anesthesia Sprague-Dawley rats (n = 28) were laparotomized, the 4 supplying arteries of the pancreas were isolated (gastroduodenal artery, left gastric artery, splenic artery and caudal pancreaticoduodenal artery) and occluded with microvascular clips for 2 hours. At the end of ischemia the abdomen was closed and the animals were allowed to awake. 15 minutes before end of ischemia an osmotic minipump filled with NaCl (ischemia group NaCl), phosphate buffer (ischemia group phosphate buffer) or HOE 140 dissolved in phosphate buffer (ischemia group HOE 140) was placed intraperitoneally. Control animals underwent sham operation without vessel occlusion; the osmotic minipump was filled with 0.9 % NaCl. Five days after ischemia the animals were sacrificed for histology. Amylase concentration and peripheral leukocyte count were determined at baseline and daily after operation.. After ischemia of 2 hours during reperfusion of 5 days all 14 animals developed histopathological changes as seen in hemorrhagic-necrotizing pancreatitis with a mortality rate of 50 %. These morphological changes were associated with a significant increase (p < 0.05) of pancreas amylase concentration from 1850 +/- 149 U/L before ischemia to a maximum of 3934 +/- 435 U/L at 1st postoperative day and decreased to 1518 +/- 399 U/L at 4th postoperative day. Leukocyte count increased significantly (p < 0.05) from 10 x 10(12)/L to 31 x 10(12)/L. In control animals as well as in animals receiving HOE 140, morphological and enzyme changes typical for acute pancreatitis were absent, leukocyte count increased only slightly.. Ischemia of the pancreas of 2 hours with ensuing reperfusion of 5 days induces morphological and biochemical changes as observed in hemorrhagic-necrotizing pancreatitis. Organ dysfunction after ischemia/ reperfusion can be effectively inhibited by administration of the bradykinin-antagonist HOE 140.

Topics: Amylases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bradykinin; Free Radicals; Ischemia; Leukocyte Count; Necrosis; Pancreas; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury

Ischaemia-reperfusion of the pancreas was performed in 35 anaesthetized Sprague-Dawley rats. The effects of two bradykinin antagonists, HOE-140 (13 micrograms kg-1 intravenous bolus injection, n = 7) and CP-0597 (18 micrograms kg-1 h-1 intravenous infusion, n = 7) on pancreatic microvascular perfusion and leucocyte-endothelium interaction were quantitatively analysed by intravital fluorescence microscopy. Three further groups underwent sham operation (n = 7), ischaemia of 2 h without treatment (n = 7), and ischaemia of 2 h with infusion of phosphate buffer (n = 7). Functional capillary density was significantly greater in animals treated with HOE-140 or CP-0597 than in sham-treated animals, and was decreased to only 60 per cent. Adherence of leucocytes to the endothelium of postcapillary venules was significantly reduced when compared with ischaemia without antagonist. These results demonstrate a positive effect of the two bradykinin antagonists HOE-140 and CP-0597 on microvascular perfusion failure after ischaemia-reperfusion of the pancreas.

Topics: Animals; Bradykinin; Bradykinin Receptor Antagonists; Male; Microcirculation; Pancreas; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Periods of ischemia followed by reperfusion of the ischemic tissue are associated with myocardial damage and ventricular arrhythmia. Angiotensin converting enzyme inhibitors limit the occurrence of these arrhythmias. The protective effects of angiotensin converting enzyme inhibitors may be due to inhibition of bradykinin (BK) degradation, rather than inhibition of angiotensin II formation. Other enzymes which catabolize BK include the endopeptidases EP24.11 and EP24.15. The purpose of this study was to determine if inhibitors of EP24.11 and EP24.15 decrease ischemia/reperfusion injury and if this protection is mediated by BK receptors. Rabbits were anesthetized and prepared for recording of cardiovascular parameters. The chest was opened and a left ventricular artery occluded for 30 min, followed by a 2-hr reperfusion period. Infarct size was determined using triphenyl tetrazolium chloride staining immediately after reperfusion. The enzyme inhibitors, ramiprilat, N-[1-(R,S)-carboxy-3-phenylpropyl]-Phe-pAB, and N[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-pAb, singly and in combinations were administered 3 min before reperfusion. Compared to saline (32.1 +/- 2.1), ramiprilat (18.3 +/- 2.8) and the EP inhibitors (14.4 +/- 1.4 for the combination) significantly decreased infarct size, with the greatest decrease occurring when all three inhibitors were combined (10.6 +/- 1.5). The protective effect of the EP inhibitors was blocked by the BK2 receptor antagonist, HOE 140 (30.1 +/- 2.6). Enzyme assays demonstrated EP24.11 and EP24.15 in the rabbit heart. We conclude that the EP inhibitors decreased ischemia/reperfusion injury by protecting BK from metabolism and that a combination of inhibitors provides superior protection to that given by a single agent.

Topics: Aminobenzoates; Animals; Blood Pressure; Bradykinin; Female; Heart Rate; Kinetics; Male; Myocardial Infarction; Protease Inhibitors; Rabbits; Reperfusion Injury