Effect of hypoxia and reoxygenation on the isolated rabbit heart determined by monoclonal antimyosin antibody uptake.

OBJECTIVE: The aim was to examine the effect of hypoxia and reoxygenation upon the isolated rabbit heart, and to determine whether or not irreversible tissue injury develops in association with the reintroduction of molecular oxygen to the previously hypoxic heart. METHODS: Isolated rabbit hearts suspended on a Langendorff apparatus and perfused with a modified Krebs-Henseleit buffer were subjected to either 5 or 30 min hypoxia and, when applicable, followed by 45 min reoxygenation. The effect of hypoxia and reoxygenation upon the isolated heart was determined with a 125labelled monoclonal antibody to the intracellular protein myosin. Determination of tissue creatine kinase release and morphological analysis, using lanthanum chloride as a marker of vessel damage, were also performed to document the uptake of antimyosin with myocardial tissue injury. RESULTS: Hearts subjected to 30 min hypoxia followed by 45 min reoxygenation showed a significant increase in antimyosin uptake when compared to hearts exposed to 30 min hypoxia. Creatine kinase release and morphological analysis showed an increase in intracellular damage in hearts receiving 30 min hypoxia and 45 min reoxygenation when compared to hearts receiving 30 min hypoxia without subsequent reoxygenation. Hearts subjected to 5 min hypoxia followed by reoxygenation did not show a significant increase in antimyosin uptake as compared to continuously oxygenated control hearts or hearts made hypoxic for 5 min without subsequent reoxygenation. CONCLUSIONS: Antimyosin antibody binding increased in hearts subjected to hypoxia and reoxygenation compared to hearts subjected to hypoxia without reoxygenation. The data provide compelling evidence that reoxygenation of hypoxic tissue exacerbates the extension of cellular damage. The ability of superoxide dismutase and catalase to decrease antimyosin uptake suggests that reactive oxygen species play a role in reoxygenation induced myocardial damage. This study also provides evidence that the labelled antimyosin antibody provides a convenient approach to quantitate the extent of damage induced by hypoxia with and without subsequent reoxygenation.

Cardioprotective effects of heparin or N-acetylheparin in an in vivo model of myocardial ischaemic and reperfusion injury.

OBJECTIVE: The aim was to determine if either heparin or N-acetylheparin could reduce the extent of myocardial injury resulting from 90 min of coronary artery occlusion and 6 h of reperfusion in the anaesthetised dog. METHODS: Heparin or N-acetylheparin was given in three repeated intravenous doses of 2 mg.kg-1. Drug or vehicle (0.9% saline) was given 75 min after onset of ischaemia and 90 and 180 min after reperfusion. To ensure an equal degree of myocardial ischaemia induced by left circumflex coronary artery occlusion among the three groups of animals studied, only animals with ischaemic zone blood flow of < or = 0.16 ml.min-1.g-1 were included in the final analysis. RESULTS: Ischaemic zone blood flow was 0.068(SEM 0.0016) ml.min-1.g-1 in control animals (n = 13), 0.083(0.017) ml.min-1.g-1 in heparin treated animals (n = 10), and 0.094(0.010) ml.min-1.g-1 in N-acetylheparin treated animals (n = 10). Baseline haemodynamic variables did not differ among the three groups studied. Heparin treatment alone significantly increased bleeding time and activated partial thromboplastin time. Electrocardiographic ST segment elevation, an indicator of regional ischaemia at the onset of coronary occlusion, was not different among control, heparin, or N-acetylheparin groups. The area of the left ventricle at risk of infarct was 39.8(1.5)%, 38.6(0.7)%, and 37.3(2.0)% in control, heparin, and N-acetylheparin treated groups, respectively. Myocardial infarct size, as a percentage of area at risk, was 43.0(3.7)%, 30.7(3.9)%, and 24.5(3.7)% in control, heparin, and N-acetylheparin treated animals, respectively (P < 0.05, control v heparin and N-acetylheparin). CONCLUSIONS: The glycosaminoglycans, heparin or N-acetylheparin, can reduce the extent of myocardial injury associated with regional ischaemia and reperfusion in the canine heart. The mechanism of cytoprotection is unrelated to alterations in the coagulation cascade and may involve inhibition of complement activation in response to tissue injury.

Platelet GPIIb/IIIa receptor inhibition by SC-49992 prevents thrombosis and rethrombosis in the canine carotid artery.

OBJECTIVE: Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptors represent the final common pathway for aggregation. GPIIb/IIIa inhibition with antibodies or RGD peptides prevents arterial thrombosis. The present study examined the ability of SC-49992 (SC), a GPIIb/IIIa receptor antagonist, to prevent thrombosis in a canine model of carotid artery thrombosis. METHODS: Both carotid arteries of anaesthetised dogs were instrumented with Doppler probes. A 300 microA current was applied to the intimal surface of the right carotid artery via an intraluminal electrode; time to occlusive thrombus formation was noted. SC (30 and 60 micrograms/.kg-1 x min-1, intravenously) or saline was infused for 240 min. The procedure for thrombus formation was repeated after 60 min of drug infusion for the left carotid artery. RESULTS: SC did not alter heart rate or blood pressure. Frequency of occlusive thrombus formation was reduced or prevented in a dose dependent manner (control = 100%, n = 12; SC 30 micrograms = 50%, n = 6; SC 60 micrograms = 0%, n = 6; p < 0.05). SC resulted in a reduction in thrombus weight (p < 0.05) v control. Ex vivo platelet aggregation to ADP and arachidonic acid was inhibited. Platelet reactivity remained inhibited 60 min after cessation of SC infusion. In a second group of animals, a carotid artery thrombus was formed and lysed with administration of anisoylated plasminogen streptokinase activator complex (0.05 U.kg-1). SC (60 micrograms.kg-1 x min-1, intravenously, n = 6) or saline (n = 6) was infused for 240 min. In dogs receiving saline there was an 83% rate of rethrombosis; none of the SC treated animals had reocclusion after recanalisation (p < 0.05). CONCLUSIONS: SC-49992 inhibits ex vivo platelet aggregation, prevents occlusive thrombus formation in a canine model of arterial thrombosis, and prevents rethrombosis after thrombolysis. The data are consistent with results obtained with murine monoclonal antibodies directed against the platelet GPIIb/IIIa receptor.

Cardioprotective effects of ranolazine (RS-43285) in the isolated perfused rabbit heart.

OBJECTIVE: The aim was to examine the putative cardioprotective effects of the novel antianginal agent, ranolazine, using an isolated rabbit heart model of ischaemia and reperfusion. METHODS: Hearts from male New Zealand White rabbits were perfused in the Langendorff mode with a recirculating Krebs buffer at a constant flow of 20-25 ml.min-1. After equilibration, hearts were treated with ranolazine (10 or 20 microM) or vehicle control for 10 min before exposure to a 30 min period of global ischaemia and 60 min reperfusion; a normoxic control group was also studied. Haemodynamic variables (left ventricular pressure), myocardial creatine kinase, and potassium release were measured at baseline (preischaemic) and at selected points during reperfusion; tissue calcium and ATP content were also measured and electron microscopy was performed. RESULTS: Left ventricular developed pressure during reperfusion was improved (p < 0.05) in a concentration dependent manner by 10 and 20 microM ranolazine (the baseline value was unaffected) with the latter dose resulting in a return to preischaemic values. The release of creatine kinase and potassium was reduced in the ranolazine groups (p < 0.05). A 2.5-fold increase in tissue calcium content in vehicle treated hearts at the end of reperfusion (compared to normoxic time control) was reduced by 10 microM ranolazine (p < 0.05) and completely prevented by 20 microM ranolazine. Similarly, the decrease in tissue ATP was largely inhibited by ranolazine in a concentration dependent manner. Electron microscopy showed that 20 microM ranolazine prevented the occurrence of many indications of reperfusion injury observed in vehicle treated control hearts, for example, blurring of myofibrillar Z bands, derangement of myofibrillar architecture, disruption of mitochondrial cristae and matrices, and the appearance of electron-dense bodies within them. The deposition of lanthanum chloride, a marker of blood vessel integrity, is also modified in the ranolazine treated hearts. CONCLUSIONS: Ranolazine has impressive cardioprotective properties in an isolated rabbit heart model of ischaemia and reperfusion, suggesting that the drug warrants further research into its precise mechanism of action.

Postischemic function and protein kinase C signal transduction.

BACKGROUND: The protective effects of myocardial preconditioning may occur by way of multiple mechanisms, with G-protein-mediated protein kinase C (PKC) translocation as a final common pathway. In this study we investigate the pharmacologic induction of preconditioning, by PKC translocation, using PKC agonists/antagonists to reveal its effects on contractile function after myocardial ischemia. METHODS: Langendorff-perfused rabbit hearts received: (1) control; (2) dimethyl sulfoxide (vehicle); (3) acetylcholine (0.55 mmol/L; PKC agonist); (4) 1,2-s,n-dioctanoylglycerol (DOG; 22 mmol/L; PKC agonist); (5) chelerythrine (0.8 mmol/L; PKC antagonist); or (6) DOG-chelerythrine followed by a 2-hour ischemic period, using modified St. Thomas cardioplegia and a 45-minute reperfusion period. The period of ischemia was chosen so as to allow for improvement by appropriate agonists. To observe metabolic changes, tissue nucleotides and nucleosides were measured. Membrane and cytosolic fractions of PKC were determined by an anti-PKC antibody directed against the PKC delta isozyme. Lactate levels and myocardial pH were measured. RESULTS: The PKC agonists DOG and acetylcholine showed the greatest recovery of developed pressure (68% +/- 2%, 60% +/- 9%, respectively). Although pH, lactate, and nucleotide levels were similar between groups at all times, myocyte PKC translocation demonstrated 25% of PKC delta isoforms on cell membrane sites during baseline, which shifted to 67% delta 17% with unprotected ischemia. DOG mimicked this shift with 58% delta 12% of PKC delta isoforms on membranes, which was also blocked by chelerythrine to 35% +/- 7%. CONCLUSIONS: These data demonstrate that PKC translocation results in improved postischemic function, not by alteration of energetics or metabolism, and deserves further investigation.

Use of "natural" hibernation induction triggers for myocardial protection.

BACKGROUND: Hypothermic cardioplegia provides adequate myocellular protection, yet stunning and dysfunction remain significant problems. Interestingly, the subcellular changes of hibernation parallel the altered biology of induced cardiac ischemia, but are well tolerated by hibernating mammalian myocardium. Hibernation induction trigger (HIT) from winter-hibernating animal serum induces hibernation in active animals. Hibernation induction trigger is opiate in nature and is similar to the delta 2 opioids. METHODS: To determine whether HIT could improve myocardial recovery following global ischemia, we gave 37 isolated rabbit hearts either standard cardioplegia or cardioplegia containing summer-active woodchuck, hibernating woodchuck, or black bear HIT serum or a delta 2 opioid, D-Ala2-Leu5-enkephalin, before 2 hours of global ischemia. RESULTS: Hibernation induction trigger appeared not to have an active mechanism during ischemia, as all hearts had equal recovery. In contrast, when examining for a preischemia mechanism, 23 additional rabbits received 3 days pretreatment with summer-active woodchuck or HIT hibernating woodchuck or black bear serum, or were preperfused with D-Ala2-Leu5-enkephalin or D-pen2,5-enkephalin, a-delta 1 opioid, again before 2 hours of global ischemia. Postischemic ventricular function, coronary flows, myocardial oxygen consumption, and ultrastructural preservation were all significantly improved with HIT and D-Ala2-Leu5-enkephalin pretreatment. CONCLUSION: "Natural" HIT protection is superior to standard cardioplegia alone and may have clinical application.

Delta opioid receptors and low temperature myocardial protection.

BACKGROUND: Cardiac surgery continues to be limited by an inability to achieve complete myocardial protection. This may result from the use of hypothermic cardioplegia. Interestingly, the subcellular changes of animal hibernation parallel the altered biology of induced hypothermic myocardial ischemia, but are well tolerated by hibernated mammalian myocardium. Evidence indicates this protection is mediated by activation of the delta opioid receptor, which elicits profound metabolic effects at the whole animal, organ, and cell level. In this study, we sought to determine if pentazocine, with agonist activity at the delta opioid receptor, could improve myocardial recovery following global ischemia over a wide range of temperatures. METHODS: Isolated rabbit hearts received either standard cardioplegia or were pretreated with racemic, d or 1 isomer pentazocine. Hearts were then subjected to 2 hours at 34 degrees C, or 3.5 hours at 20 degrees C, or 4 hours at 10 degrees C of cardioplegic ischemia and reperfused. Functional recovery was compared to controls. RESULTS: Isovolumic developed pressure, coronary flow, oxygen consumption, and ultrastructural preservation were enhanced with pentazocine delta opioid mediated protection, which appears to be additive to standard cardioplegia, even at low temperatures. CONCLUSIONS: Teleologically, delta opioid protection parallels animal hibernation, which occurs from 34 degrees down to 0 degrees C. The use of delta opioid receptor agonists may have important clinical implications for cardiac surgery and deserves further study.

Reperfusion injury after myocardial infarction: the role of free radicals and the inflammatory response.

Development of thrombolytic therapy as a treatment for myocardial infarction has focused attention on the events that occur upon reperfusion of ischemic myocardial tissue. Although it is well documented that salvage of the ischemic myocardium is dependent upon timely reperfusion, it is likely that the very events critical for survival may, in fact, lead to further tissue injury. A widely recognized source of reperfusion injury is the generation of oxygen-derived free radicals. These reactive oxygen species, which are formed within the first moments of reperfusion, are known to be cytotoxic to surrounding cells. In addition, strong support exists for the involvement of the inflammatory system in mediating tissue damage upon reperfusion. Coincident with the recruitment of neutrophils and activation of the complement system is an increase in the loss of viable cells. Although a number of mechanisms are likely to be involved in reperfusion injury, this discussion focuses on the roles that oxygen-derived free radicals and the inflammatory system play in mediating reperfusion injury.

Neutrophil adhesion to human endothelial cells is induced by the membrane attack complex: the roles of P-selectin and platelet activating factor.

A variety of inflammatory diseases are accompanied by activation of the complement system. We examined the role of the membrane attack complex (MAC) in mediating neutrophil adhesion to endothelial cells. To assemble the MAC in endothelial cell monolayers, a C5b-like molecule was created through the treatment of purified C5 with the oxidizing agent chloramine-T, followed by addition of the remaining components (C6-C9) that constitute the MAC. Use of this method abrogated potentially confounding effects mediated by other complement components (e.g., C5a). MAC assembly resulted in a rapid (30 min), concentration-dependent increase in neutrophil adherence. A monoclonal antibody directed against P-selectin inhibited MAC-mediated neutrophil adhesion. A whole cell EIA confirmed P-selectin expression after formation of the MAC. Incubation of neutrophils with the platelet-activating factor receptor antagonist, CF 3988, also significantly decreased adhesion, indicating that PAF plays a role in MAC-mediated adhesion. These results suggest that the MAC can promote neutrophil adhesion through P-selectin and PAF-mediated mechanisms.

Modulation of LPS-induced pulmonary neutrophil infiltration and cytokine production by the selective PPARbeta/delta ligand GW0742.

OBJECTIVE: To define the anti-inflammatory effects of PPARbeta/delta activation by use of the selective PPARbeta/delta ligand (GW0742) in a model of lipopolysaccharide (LPS)-induced pulmonary inflammation. METHODS: Male BALB/c mice were pretreated for three days with the PPARbeta/delta agonist, GW0742, prior to induction of LPS-mediated pulmonary inflammation. Bronchial alveolar lavage fluid (BALF) was analyzed for inflammatory cell influx and for levels of pro-inflammatory mediators. BALF-derived inflammatory cells were also collected for mRNA analysis. RESULTS: Pretreatment with GW0742 resulted in a significant decrease in leukocyte recruitment into the pulmonary space. Protein and mRNA levels of the pro-inflammatory cytokines IL-6, IL-1beta and TNFalpha in BALF were found to be significantly decreased in GW0742-treated animals (30 mg/kg). A significant decrease in granulocyte macrophage-colony stimulating factor (GM-CSF), a major regulator of neutrophil chemotaxis (via its downstream actions on TNFalpha and other cytokines/chemokines), activation and survival, was also noted in the BALF levels of GW0742-treated animals. CONCLUSIONS: The present study demonstrates that activation of PPARbeta/delta attenuates the degree of inflammation in a model of LPS-induced pulmonary inflammation and may therefore represent a novel therapeutic approach for the treatment of inflammation-mediated pathologies.

Surgically induced osteoarthritis in the rat results in the development of both osteoarthritis-like joint pain and secondary hyperalgesia.

OBJECTIVE: In the present study, we sought to develop/characterize the pain profile of a rat model of surgically induced osteoarthritis (OA). METHODS: OA was surgically induced in male Lewis rats (200-225 g) by transection of the medial collateral ligament and medial meniscus of the femoro-tibial joint. In order to characterize the pain profile, animals were assessed for a change in hind paw weight distribution (HPWD), development of mechanical allodynia, and the presence of thermal and mechanical hyperalgesia. Rofecoxib and gabapentin were examined for their ability to decrease change in weight distribution and tactile allodynia. RESULTS: Transection of the medial collateral ligament and medial meniscus of male Lewis rats resulted in rapid (<3 days) changes in hind paw weight bearing and the development of tactile allodynia (secondary hyperalgesia). There was, however, no appreciable effect on thermal hyperalgesia or mechanical hyperalgesia. Treatment with a single dose of rofecoxib (10 mg/kg, PO, day 21 post surgery) or gabapentin (100mg/kg, PO, day 21 post surgery) significantly attenuated the change in HPWD, however, only gabapentin significantly decreased tactile allodynia. CONCLUSION: The rat medial meniscal tear (MMT) model mimics both nociceptive and neuropathic OA pain and is responsive to both a selective cylooxygenase-2 (COX-2) inhibitor commonly utilized for OA pain (rofecoxib) and a widely prescribed drug for neuropathic pain (gabapentin). The rat MMT model may therefore represent a predictive tool for the development of pharmacologic interventions for the treatment of the symptoms associated with OA.

Complement inhibitors in myocardial ischemia/reperfusion injury.

Myocardial ischemia/reperfusion injury is accompanied by an inflammatory response contributing to reversible and irreversible changes in tissue viability and organ function. Endothelial and leukocyte responses are involved in tissue injury, orchestrated primarily by the complement cascade. Anaphylatoxins, and assembly of the membrane attack complex contribute directly and indirectly to further tissue damage. Tissue salvage can be achieved by depletion of complement components, thus making evident a contributory role for the complement cascade in ischemia/reperfusion injury. The complexity of the complement cascade provides numerous sites as potential targets for therapeutic interventions designed to modulate the complement response to injury. The latter is exemplified by the ability of a soluble form of complement receptor 1 (sCR1) to decrease infarct size in in vivo models of ischemia/reperfusion injury as well as prevent myocyte and vascular injury and organ dysfunction by interdicting assembly of the membrane attack complex. Effective inhibitors of complement are not limited to newly developed compounds or solubilized forms of endogenous regulators of complement activation. Therapeutic agents in common use, such as heparin and related non-anticoagulant glycosaminoglycans, are known to inhibit the complement activation in vitro as well as in vivo and may prove useful as cytoprotective agents.

Monoclonal antibody [7E3 F(ab')2] prevents arterial but not venous rethrombosis.

The role of the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor and the efficacy of GPIIb/IIIa receptor antagonists on reocclusion after arterial versus venous thrombolysis is unknown. We used a canine model of simultaneous carotid artery and jugular vein thrombosis to evaluate the effect of the murine monoclonal antibody 7E3 [7E3 F(ab')2] on intravascular thrombotic reocclusion after thrombolysis in both vessels. The left carotid artery and right jugular vein were instrumented with flow probes, a critical stenosis, and an electrode through which an anodal current was applied to induce formation of an occlusive thrombus. After persistent occlusion of both vessels, 7E3 (0.8 mg/kg, n = 7) or saline (n = 10) was administered intravenously (i.v.) immediately after the local administration of anisolylated plasminogen streptokinase activator complex (APSAC, 0.1 U/kg). Ex vivo platelet aggregation in response to ADP or arachidonic acid was inhibited completely, and bleeding time was increased threefold by 7E3. The administration of 7E3 did not affect the activated partial thromboplastin time as compared with control values. The time to reperfusion of either vessel was not altered significantly in the presence of 7E3. Arterial or venous thrombolysis after APSAC was achieved in 10 of 10 and 8 of 10 control animals, respectively, but rethrombosis occurred in both the carotid artery and jugular vein in each of the saline-treated animals. Treatment with 7E3 prevented cyclic flow variations and reocclusion in the carotid artery (p < 0.05) in all treated animals (n = 7). In contrast, 7E3 did not suppress cyclic flow variations in the jugular vein, and rethrombosis occurred in five of six vessels despite the presence of platelet inhibition as assessed ex vivo. Residual arterial thrombus weights were reduced by pretreatment with 7E3 (saline = 24 +/- 4 mg, 7E3 = 11 +/- 3 mg; p < 0.05), whereas residual venous thrombus weights were not affected (saline 25 +/- 5 mg and 7E3 26 +/- 11 mg). The results indicate that inhibition of the platelet GPIIb/IIIa receptor does not prevent jugular vein rethrombosis despite its ability to prevent rethrombosis in the carotid artery. Different thrombotic mechanisms are involved in forming arterial and venous thrombi. Interventions that modulate arterial thrombotic events need not affect occlusive thrombotic activity in the venous circulation.

Sulfhydryl compounds, captopril, and MPG inhibit complement-mediated myocardial injury.

Factors including complement activation, neutrophil infiltration, and oxygen-derived free radicals have been implicated in the pathogenesis of myocardial tissue injury during ischemia and reperfusion. Certain sulfhydryl-containing compounds have been shown to inhibit complement activation. The sulfhydryl compounds captopril and N-(2-mercaptopropionyl)-glycine (MPG) are antioxidant compounds that previously have been shown to protect the myocardium from ischemia and reperfusion-induced damage. In this study, captopril (an angiotensin-converting-enzyme inhibitor; ACEI) and MPG, and the non-sulfhydryl compound enalaprilat (also an ACEI) were tested for their ability to protect the isolated perfused rabbit heart against complement-induced injury. Both captopril and MPG protected hearts against complement-mediated increases in left ventricular end-diastolic pressure and increases in coronary arterial perfusion pressure in a concentration-dependent manner, whereas enalaprilat was not protective. The ability of these compounds to inhibit complement activation also was tested using an in vitro complement-mediated red blood cell hemolysis assay. These findings offer additional insight as to the mechanism whereby captopril, MPG, and possibly other sulfhydryl compounds, may be acting to provide cytoprotection during myocardial ischemia and reperfusion.

Complement gene expression by rabbit heart: upregulation by ischemia and reperfusion.

Activation of the complement system has been implicated in the pathogenesis of myocardial ischemia/reperfusion injury. It has always been assumed that liver is the primary source of complement components. In the present study, we used the reverse-transcriptase polymerase chain reaction technique to establish that the mRNAs for complement proteins C3 and C9 are expressed in rabbit heart. Rabbit liver, brain, spleen, and kidney were also shown to express C3 and C9 mRNAs. We used Western blotting to establish that these mRNAs in heart are translated into the corresponding proteins. We further established that dramatic upregulation of the mRNAs occurred in Langendorff-perfused isolated hearts subjected to ischemia and reperfusion. C3 mRNA was always expressed at higher levels than was C9 mRNA, but C9 mRNA showed greater upregulation under stress. Compared with levels in control hearts subjected to 5 minutes of normoxic perfusion, hearts subjected to 0.5 hours of ischemia followed by 1 hour of reperfusion had a 4.72-fold increase in C3 mRNA and a 19.5-fold increase in C9 mRNA. By contrast, C3 mRNA in hearts subjected to 3.5 hours of normoxic perfusion showed no change, and those subjected to 3.5 hours of ischemia showed only a 1.72-fold increase, whereas C9 mRNA levels increased by 5.17-fold after 3.5 hours of normoxic perfusion and 12.5-fold after 3.5 hours of ischemia. The results of this study demonstrate for the first time that heart tissue is capable of expressing genes and proteins of the complement system, although it is not yet known which cell types are responsible. They further demonstrate that ischemia and reperfusion of the heart promotes a rapid upregulation of the mRNAs encoding the complement proteins C3 and C9 and that these abnormal levels considerably exceed those of normal liver. These observations are consistent with the hypothesis that local production of complement proteins may contribute significantly to the degree of ischemic injury to the myocardium and that complement expression is augmented by reperfusion.

Ex vivo reversal of heparin-mediated cardioprotection by heparinase after ischemia and reperfusion.

Glycosaminoglycans, including heparin, have been demonstrated both in vitro and in vivo to protect the ischemic myocardium against reperfusion injury. In the present study, we sought to determine whether the cardioprotective effects of heparin administration could be reversed by the heparin-degrading enzyme heparinase. New Zealand white rabbits were pretreated with heparin (300 U/kg i.v.) or vehicle (saline). Two hours after treatment, hearts were removed, perfused on a Langendorff apparatus, and subjected to 25 min of global ischemia, followed by 45 min of reperfusion. Hemodynamic variables were obtained before ischemia (baseline) and every 10 min throughout the reperfusion period. Compared with vehicle-treated rabbits, the left ventricular end-diastolic and left ventricular developed pressures were improved significantly (p <.05) in the heparin-treated group. Ex vivo administration of heparinase (5 U/ml) immediately before the onset of global ischemia was associated with a reversal of the heparin-mediated cardioprotection. The uptake of a radiolabeled antibody to the intracellular protein myosin and creatine kinase release were used to determine membrane integrity and discriminate between viable and nonviable myocardial tissue. The uptake of radiolabeled antimyosin antibody and release of creatine kinase after reperfusion were increased in heparin-pretreated hearts exposed to heparinase, indicating a loss of membrane integrity and increased myocyte injury. These results demonstrate that neutralization of heparin by heparinase promotes increased myocardial injury after reperfusion of the ischemic myocardium.

Antibodies to neutrophil cytoplasmic antigens induce monocyte chemoattractant protein-1 secretion from human monocytes.

Antibodies to neutrophil cytoplasmic antigens (ANCA) have been found in the serum samples of patients with a number of vasculitides (e.g., Wegener's granulomatosis, small vessel vasculitis, and idiopathic necrotizing and cresentic glomerulonephritis). Although detection of ANCA in serum samples has proven to be useful diagnostically and in selected activity of disease monitoring situations, the pathogenetic role of ANCA in vasculitis remains ill-defined. We sought to determine whether purified ANCA promotes the secretion of monocyte chemoattractant protein-1 (MCP-1) from isolated human peripheral blood monocytes. P (perinuclear)- and C (cytoplasmic)- ANCA were purified from the serum samples of patients with either Wegener's granulomatosis, small vessel vasculitis, or idiopathic necrotizing and cresentic glomerulonephritis. Human peripheral blood monocytes from healthy subjects were incubated with either C-ANCA immunoglobulin G (IgG), P-ANCA IgG, or nonspecific IgG, and the conditioned media were analyzed for MCP-1 activity. A monocyte chemotaxis assay was utilized to functionally quantify secreted chemotactic activity. Secretion of monocyte chemotactic activity was found to be antibody concentration-dependent and time-dependent, with maximal chemotaxis measured in media collected 24 hours after the addition of either C- or P-ANCA IgG. A specific antibody directed against human MCP-1 largely inhibited monocyte chemotaxis, indicating that MCP-1 is the predominant monocyte chemotactic mediator present in the conditioned medium. An MCP-1 enzyme-linked immunosorbent assay further supported the conclusion that P- and C-ANCA IgG can trigger MCP-1 secretion by monocytes. These data indicate that incubation of monocytes with ANCA promotes the dose-dependent release of the chemotactic beta-chemokine MCP-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble complement receptor type 1 prevents human complement-mediated damage of the rabbit isolated heart.

The purpose of this study was to determine if recombinant human soluble CR1 (sCR1) could prevent tissue damage associated with the activation of human complement. Directly mediated human complement-dependent myocardial injury was induced in the rabbit isolated heart perfused with a Krebs-Henseleit buffer containing 6% human plasma. There were three study groups: 1) 6% heat-inactivated human plasma (control); 2) 6% normal human plasma (NHP); or 3) 6% normal human plasma + 20 nM sCR1 (NHP + sCR1). Recorded functional parameters of the control group remained stable throughout the duration of the 70-min protocol. Complement activation in hearts perfused with 6% NHP increased the diastolic pressure; decreased developed pressure; and increased coronary perfusion pressure. These alterations were accompanied by a decrease in the maximum positive and negative dP/dt. Complement activation also increased cardiac muscle lymphatic fluid flow rate. The changes were greatest between 20 and 40 min, but persisted for the duration of the protocol. sCR1 (20 nM) in the perfusate containing 6% NHP prevented the complement-mediated alterations in the systolic, developed, and coronary perfusion pressures. sCR1 prevented the decrement in the positive and negative dP/dt, and the increase in the lymphatic fluid flow rate. Values for each of these parameters in hearts perfused with 6% NHP + sCR1 were not altered from those of controls at any time point in the protocol. Ultrastructural changes were present in tissues perfused with 6% NHP along with immunohistochemical evidence for presence of the terminal C5b-9 complex. sCR1 prevented the ultrastructural changes and the formation of the terminal complex. sCR1 offers significant protection against the cytolytic effects resulting from activation of the human complement system.