Kenneth S. Kilgore

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.

Effects of heparin and N-acetyl heparin on ischemia/reperfusion-induced alterations in myocardial function in the rabbit isolated heart.

Evidence is presented that heparin pretreatment produces protective effects on myocardial tissue distinct from its anticoagulant activity. The present study examines the ability of heparin sulfate and N-acetyl heparin (a derivative of heparin devoid of anticoagulant effects) to protect the heart from injury associated with global ischemia and reperfusion. Male New Zealand White rabbits were administered either heparin sulfate (n = 7, 300 U/kg i.v.), N-acetyl heparin (n = 6, 1.73 mg/kg i.v.), or vehicle (n = 6). Two hours after treatment, the hearts were removed, perfused on a Langendorff apparatus, and subjected to 30 minutes of global ischemia, followed by 45 minutes of reperfusion. During reperfusion, creatine kinase concentrations in the coronary sinus effluent were greater in hearts from vehicle-treated rabbits compared with hearts from N-acetyl heparin-treated and heparin-treated rabbits. Left ventricular end-diastolic pressure after 45 minutes of reperfusion in the vehicle-treated group was 64 +/- 15 mm Hg compared with 17 +/- 4 and 10 +/- 3 mm Hg in the heparin-pretreated and N-acetyl heparin-pretreated groups, respectively. Heparin, but not N-acetyl heparin, increased the activated partial thromboplastin time, consistent with its known anticoagulant action. Heparin and N-acetyl heparin inhibited complement-mediated erythrocyte lysis in a concentration-dependent manner. The glycosaminoglycans, in contrast to r-hirudin, reduced complement activation-induced injury in the rabbit isolated heart. The results demonstrate that heparin or N-acetyl heparin, administered to the intact rabbit, protects the isolated heart from subsequent myocardial dysfunction secondary to ischemia/reperfusion. The cardioprotective effects of heparin and N-acetyl heparin are independent of an antithrombin mechanism.

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.

The use of hibernation induction triggers for cardiac transplant preservation.

Cardiac transplant is hindered by donor shortage and preservation time. Extended extracorporeal preservation could increase the number and distribution of hearts for transplantation. Interestingly, mammalian hibernation biology closely parallels the altered cardiac cellular physiology noted with hypothermic organ storage. The present study undertook to test whether treatment with hibernation induction triggers could improve myocardial functional recovery following prolonged ischemic storage in a nonhibernating mammalian model. To study this hypothesis, isolated rabbit hearts had baseline functional and metabolic parameters recorded and then received either hypothermic storage only or standard cardioplegia, or cardioplegia containing 1 mg/kg D-Ala2-Leu5-enkaphalin (DADLE), which mimics natural hibernation, or preperfusion with DADLE, administered for 15 min at 2 mmol, 25 min prior to cardioplegic ischemia. Hearts were then subjected to 18 hr of global ischemic storage at 4 degrees C. Isovolumic developed pressure, coronary flows, and myocardial oxygen consumption were significantly improved with DADLE pretreatment vs. all groups after storage and reflow. Furthermore, DADLE hearts demonstrated better histological ultrastructure preservation following prolonged storage ischemia. This study demonstrates that hibernation protection with DADLE is beneficial for prolonged cardiac storage. The use of hibernation induction triggers is promising for organ preservation and deserve further mechanistic study.

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.

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.

Reduction of myocardial infarct size after ischemia and reperfusion by the glycosaminoglycan pentosan polysulfate.

Activation of the complement system contributes to the tissue destruction associated with myocardial ischemia/reperfusion. Pentosan polysulfate (PPS), a negatively charged sulfated glycosaminoglycan (GAG) and an effective inhibitor of complement activation, was studied for its potential to decrease infarct size in an experimental model of myocardial ischemia/reperfusion injury. Open-chest rabbits were subjected to 30-min occlusion of the left coronary artery followed by 5 h of reperfusion. Vehicle (saline) or PPS (30 mg/kg/h) was administered intravenously immediately before the onset of reperfusion and every hour during the reperfusion period. Treatment with PPS significantly (p < 0.05) reduced infarct size as compared with vehicle-treated animals (27.5+/-2.9% vs. 13.34+/-2.6%). Analysis of tissue demonstrated decreased deposition of membrane-attack complex and neutrophil accumulation in the area at risk. The results indicate that, like heparin and related GAGs, PPS possesses the ability to decrease infarct size after an acute period of myocardial ischemia and reperfusion. The observations are consistent with the suggestion that PPS may mediate its cytoprotective effect through modulation of the complement cascade.

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.

Preconditioning reduces tissue complement gene expression in the rabbit isolated heart

Both preconditioning and inhibition of complement activation have been shown to ameliorate myocardial ischemia-reperfusion injury. The recent demonstration that myocardial tissue expresses complement components led us to investigate whether preconditioning affects complement expression in the isolated heart. Hearts from New Zealand White rabbits were exposed to either two rounds of 5 min global ischemia followed by 10 min reperfusion (ischemic preconditioning) or 10 μM of the ATP-dependent K+(KATP) channel opener pinacidil for 30 min (chemical preconditioning) before induction of 30 min global ischemia followed by 60 min of reperfusion. Both ischemic and chemical preconditioning significantly ( P < 0.05) reduced myocardial C1q, C1r, C3, C8, and C9 mRNA levels. Western blot and immunohistochemistry demonstrated a similar reduction in C3 and membrane attack complex protein expression. The KATPchannel blocker glyburide (10 μM) reversed the depression of C1q, C1r, C3, C8, and C9 mRNA expression observed in the pinacidil-treated hearts. The results suggest that reduction of local tissue complement production may be one means by which preconditioning protects the ischemic myocardium.Both preconditioning and inhibition of complement activation have been shown to ameliorate myocardial ischemia-reperfusion injury. The recent demonstration that myocardial tissue expresses complement components led us to investigate whether preconditioning affects complement expression in the isolated heart. Hearts from New Zealand White rabbits were exposed to either two rounds of 5 min global ischemia followed by 10 min reperfusion (ischemic preconditioning) or 10 microM of the ATP-dependent K+ (KATP) channel opener pinacidil for 30 min (chemical preconditioning) before induction of 30 min global ischemia followed by 60 min of reperfusion. Both ischemic and chemical preconditioning significantly (P < 0.05) reduced myocardial C1q, C1r, C3, C8, and C9 mRNA levels. Western blot and immunohistochemistry demonstrated a similar reduction in C3 and membrane attack complex protein expression. The K(ATP) channel blocker glyburide (10 microM) reversed the depression of C1q, C1r, C3, C8, and C9 mRNA expression observed in the pinacidil-treated hearts. The results suggest that reduction of local tissue complement production may be one means by which preconditioning protects the ischemic myocardium.

N-Acetylheparin Pretreatment Reduces Infarct Size in the Rabbit

The ability of the heparin derivative, N-acetylheparin (NHEP) to protect the heart from regional ischemia/reperfusion injury was examined in vivo. NHEP (2 mg/kg i.v.) or vehicle was administered 2 h before occlusion of the left circumflex coronary (LCX) artery. Open-chest, anesthetized rabbits were subjected to 30 min of regional myocardial ischemia followed by 5 h of reperfusion. Myocardial myeloperoxidase activity, membrane attack complex (MAC) deposition and IL-8 generation were assessed in supernatant samples from the area at risk. Infarct size in rabbits pretreated with NHEP (32.5 ± 3.8%, n = 10) decreased by 41% compared to infarct size in rabbits that received vehicle (55.3 ± 4.9%, n = 10; p = 0.002). Accumulation of neutrophils within the ischemic region, as assessed by myeloperoxidase activity, declined by 45% (p < 0.05) in AAR from NHEP-treated animals compared to AAR from vehicle-treated animals. Levels of MAC and IL-8 obtained from AAR were less in NHEP-pretreated animals compared to controls. These results suggest that NHEP may protect the myocardium by inhibiting complement activation and subsequent neutrophil infiltration.