Joseph C. Fantone

The Role of the Neutrophil and Free Radicals in Ischemic Myocardial Injury

The association of coronary artery thrombosis with the onset of acute myocardial infarction has provided the rationale for the development of pharmacologic and/or physical methods for the restoration of coronary artery blood flow. The institution of pharmacologic methods for myocardial reperfusion has resulted in a reduction in mortality leading to acceptance of thrombolytic therapy as the standard approach to the management of patients with an evolving acute myocardial infarction [I]. It is a well established fact that prolonged myocardial ischemia leads to a time-dependent loss in the viability of myocardial cells in the jeopardized region of the heart and that the restoration of coronary artery blood flow is fundamental in order to arrest the progression of cell death and for the restoration of myocardial function. The restoration of blood flow is essential for repair of the reversible changes induced by ischemia and for the continued survival of the myocardial cells at risk of permanent damage. The reintroduction of oxygen at the time of reperfusion, however, may be detrimental to the reoxygenated myocyte as well as being beneficial. A number of recent reviews have been devoted to an examination of the question of whether reactive species of oxygen or oxygen radicals can contribute to the development of irreversible myocardial cell injury during the period of reperfusion [2, 3, 4, 51. The purpose of this editorial is to focus attention on the potential role of the polymorphonuclear leukocyte (PMN) as a determinant of the ultimate extent of irreversible myocardial injury after ischemia and reperfusion, and to call attention to those factors which modulate the inflammatory response to myocardial cell injury.

Polymorphonuclear leukocyte-mediated cell and tissue injury: Oxygen metabolites and their relations to human disease

Reactive oxygen metabolic products derived from an activated NADPH oxidase present in the cell membrane of PMNs and mononuclear phagocytic cells play a critical role in the hosts defense against bacterial infection. Recent studies have also demonstrated the ability of these toxic products to initiate eukaryotic cell injury and promote the development of the acute inflammatory responses. Experimental studies suggest that neutrophil-derived oxygen metabolites contribute to the development of the tissue injury associated with a variety of disease states, including emphysema, myocardial infarction, adult respiratory distress syndrome, immune complex-mediated vasculitis, and rheumatoid arthritis. Future studies to define further the mechanisms by which reactive oxygen-derived metabolic products mediate tissue injury will provide insight into the development of new therapeutic strategies for the modulation of disease states that are mediated by the recruitment and activation of PMNs.

Iloprost inhibits neutrophil function in vitro and in vivo and limits experimental infarct size in canine heart.

The prostacyclin analogue iloprost (ZK 36374) inhibits neutrophil activation in vitro, reduces neutrophil accumulation in inflammatory skin lesions, and reduces ultimate infarct size in an anesthetized open-chest canine model of regional ischemia and reperfusion. Doprost (0.1–100 μM) inhibited the in vitro production of superoxide anion by canine neutrophils in a concentration-dependent manner. Doprost (100 ng/kg/min i.v.) inhibited C5a-induced neutrophil migration into inflammatory skin lesions as assessed by the neutrophil-specific enzyme marker, myeloperoxidase. The myeloperoxidase activity determined 2 hours after the intradermal administration of C5a in each of the groups was control 13.3 ± 1.8 units/g tissue (n = 12) and iloprost 6.5 ± 0.9 units/g (n = 12), p <0.01. Iloprost was administered to anesthetized open-chest dogs (100 ng/kg/min) 10 minutes after left circumflex coronary artery (LCCA) occlusion and continued during the 90-minute occlusion period and the first 2 hours of reperfusion. Regional myocardial blood flow was similar between treatment groups at baseline, 5 minutes and 80 minutes after LCCA occlusion, and after 1 hour of reperfusion. Infarct size, assessed 6 hours after reperfusion, was reduced by iloprost treatment: 22.4 ± 3.1 % of the area at risk (n = 15) compared with 42.4 ± 3.3% of control (n = 13), p <0.01. Doprost treatment reduced the accumulation of neutrophils (measured by myeloperoxidase activity) in the ischemic myocardium at the Interface between infarcted and noninfarcted tissue: control (n = 9) 9.0 ± 1.8 units/g tissue, iloprost (n = 6) 2.0 ± 0.4 units/g, p <0.01. The ability of iloprost to reduce Infarct size may be related both to a reduction in arterial blood pressure and to a modulation of neutrophil infiltration and activation at the site of tissue injury.

Prostaglandin E1 and prostaglandin I2 modulation of superoxide production by human neutrophils

15(S)-15-methyl-prostaglandin E1 and prostaglandin I2 rapidly and reversibly inhibit formyl-methionyl-leucyl-phenylalanine induced superoxide production by human neutrophils. In contrast, 15(S)-15-methyl-prostaglandin E1 and prostaglandin I2 did not alter the rate or the total amount of superoxide production by human neutrophils stimulated with either phorbol myristate acetate or arachidonic acid. These data suggest that the production of superoxide anion by human neutrophils may be mediated by at least two mechanisms, one regulated by prostaglandins and intracellular cyclic adenosine monophosphate levels and a second independent of prostaglandin modulation.

Reduction of the size of infarction by allopurinol in the ischemic-reperfused canine heart.

This study was performed to assess the effect of allopurinol in a canine preparation of myocardial infarction. Dogs underwent occlusion of the left circumflex coronary artery for 90 min, followed by reperfusion for 6 hr. Three groups were studied: (1) control, (2) dogs receiving 25 mg/kg allopurinol 18 hr before occlusion and 50 mg/kg 5 min before occlusion, and (3) dogs receiving allopurinol as above plus 5 mg/kg superoxide dismutase over 1 hr beginning 15 min before reperfusion. Infarct size expressed as a percentage of the area at risk was 40 +/- 4 in the control group, 22 +/- 5 in the allopurinol group (p less than .05 vs control), and 17 +/- 4 in the allopurinol plus superoxide dismutase group (p less than .05 vs control). The differences in infarct size were not due to differences in myocardial oxygen supply or demand. Neutrophil superoxide anion production was not altered by allopurinol treatment. The results suggest that myocardial xanthine oxidase may generate oxygen radicals that play a role in myocardial injury due to ischemia and reperfusion.

Prostacyclin protects ischemic reperfused myocardium in the dog by inhibition of neutrophil activation.

Prostacyclin (PGI2) and the stable PGI2 analogue SC39902 (6,9 alpha-epoxy,5S-fluoro-11 alpha, 15S-dehydroxyprosta-6,13E-dien-1-oic acid, sodium salt) were studied in anesthetized open-chest dogs subjected to 90 minutes of left circumflex coronary artery (LCCA) occlusion and 6 hours of reperfusion. PGI2 (50 ng/kg/min, infused into the left atrium) reduced infarct mass by 59% compared to control, but SC39902 (1.5 micrograms/kg/min) failed to produce a significant reduction in infarct size. Both PGI2 and SC39902 reduced mean arterial blood pressure, heart rate, and rate-pressure product to the same extent. Regional myocardial blood flow measured with radiolabelled tracer microspheres did not demonstrate an increase in regional blood flow to the ischemic myocardium during the 90 minutes of LCCA occlusion in the PGI2 and control treatment groups. Canine neutrophils were isolated from whole blood and activated with opsonized zymosan. PGI2 produced a concentration-dependent inhibition of neutrophil activation as measured by superoxide production in vitro, whereas SC39902 failed to effectively inhibit neutrophil activation. Neutrophil migration into inflammatory skin lesions was effectively attenuated when dogs were pretreated with PGI2 (50 ng/kg/min, intravenously). Therefore, it is suggested that the cytoprotective effect of PGI2 during myocardial ischemia and reperfusion is related to an inhibition of neutrophil migration and the production of cytotoxic activated oxygen species.

Protective effects of N-2-mercaptopropionyl glycine against myocardial reperfusion injury after neutrophil depletion in the dog: evidence for the role of intracellular-derived free radicals.

Reperfusion of the previously ischemic myocardium is associated with the production of oxygen free radicals and their metabolites, which contribute to the ultimate extent of irreversible myocardial injury. The relative importance of polymorphonuclear leukocytes vs intracellular-derived oxygen metabolites has remained uncertain. We evaluated the effectiveness of a free-radical scavenger, N-2-mercaptopropionyl glycine (MPG), in limiting infarct size after ischemia/reperfusion in dogs that were depleted of neutrophils with specific antisera. Twenty-four urethane-anesthetized open-chest dogs were subjected to 90 min of ischemia by occlusion of the left circumflex coronary artery followed by 6 hr of reperfusion. Dogs were randomly assigned to receive nonimmune serum, neutrophil antiserum, or neutrophil antiserum plus MPG (20 mg/kg intra-atrially 15 min before reperfusion was initiated and for 45 min after reperfusion). Infarct size, as a percent of the area at risk, was reduced by 33% in the neutrophil antiserum group as compared with the nonimmune group (30.7 +/- 2.7% vs 45.6 +/- 3.7%, p less than .01). The combined administration of neutrophil antiserum plus MPG reduced the size of infarction by 63% of the area at risk compared with that in the nonimmune group (17.0 +/- 2.7% vs 45.6 +/- 3.7%, p less than .01). The reduction in infarct size with neutrophil antiserum plus MPG was significantly greater than that with the neutrophil antiserum alone (p less than .01). The areas at risk did not differ among the groups. Myocardial protection could not be explained on the basis of hemodynamic differences. The observation that MPG enhances the protective effects of neutrophil depletion suggests that both extramyocardial- and intramyocardial-derived oxygen free radicals contribute significantly to reperfusion-induced myocardial injury.

Canine myocardial reperfusion injury: protection by a free radical scavenger, N-2-mercaptopropionyl glycine.

Summary: Oxygen-derived free radicals and their metabolites may contribute to the extension of irreversible cellular injury, which occurs on reperfusion of the previously ischemic myocardium. Therefore, therapy directed against the toxic effects of reactive oxygen species may provide protection to the ischemic myocardium, which undergoes subsequent reperfusion. We evaluated the effectiveness of N-2-mercaptopropionyl glycine (MPG), a free radical scavenger, to limit the extent of irreversible injury resulting from 90 min of ischemia followed by 6 h of reperfusion in a canine model of myocardial infarction. In three groups of dogs, MPG (20 mg/kg) was administered as a constant infusion into the left atrium. Group I received MPG for 2 h, starting 15 min before occlusion of the left circumflex coronary artery and ending 15 min after reperfusion. Group II received MPG for 1 h, starting 15 min before reperfusion. Group III received MPG for 1 h beginning 45 min after reperfusion. Each group was compared with its respective saline control group. Infarct size was reduced by 35% in Group I (32.2 ± 5.1% vs. 47.7 ± 3.4% of the area at risk, p < 0.05) and Group II (31.4 ± 3.6% vs. 47.5 ± 5.1% of the area at risk, p < 0.025) in comparison with the saline treated control animals. In contrast, in Group III infarct size did not differ significantly from the saline-treated control group (45.9 ± 3.3% vs. 47.7 ± 3.5% of the area at risk). The percent of left ventricle at risk did not differ among the groups. The beneficial effects of MPG could not be explained on the basis of hemodynamic differences. In addition, MPG did not influence regional myocardial blood flow. In vitro studies indicated that MPG effectively scavanges O2- generated by the hypoxanthine-xanthine oxidase reaction, as well as by PMA-activated polymorphonuclear leukocytes. Based on these observations, we propose that MPG exerts its beneficial effects by protecting against free radical-mediated damage during the early phase of reperfusion.

Autopsy findings in eight patients with fatal H1N1 influenza.

A novel H1N1 influenza A virus emerged in April 2009, and rapidly reached pandemic proportions. We report a retrospective observational case study of pathologic findings in 8 patients with fatal novel H1N1 infection at the University of Michigan Health Systems (Ann Arbor) compared with 8 age-, sex-, body mass index-, and treatment-matched control subjects. Diffuse alveolar damage (DAD) in acute and organizing phases affected all patients with influenza and was accompanied by acute bronchopneumonia in 6 patients. Organizing DAD with established fibrosis was present in 1 patient with preexisting granulomatous lung disease. Only 50% of control subjects had DAD. Peripheral pulmonary vascular thrombosis occurred in 5 of 8 patients with influenza and 3 of 8 control subjects. Cytophagocytosis was seen in all influenza-related cases. The autopsy findings in our patients with novel H1N1 influenza resemble other influenza virus infections with the exception of prominent thrombosis and hemophagocytosis. The possibility of hemophagocytic syndrome should be investigated in severely ill patients with H1N1 infection.

A qualitative exploration of how the conflict between the formal and informal curriculum influences student values and behaviors.

Purpose The third-year students at one medical school told the authors that values core to patient-centered care were impossible to practice in clerkships, in a culture where supervisors role modeled behaviors in direct conflict with patient-centered care. As they developed a new medical student curriculum, the authors designed the Family Centered Experience (FCE) to help students achieve developmental goals and understand the importance of and provide a foundation for patient-centered care. Method The authors solicited members of the first cohort to complete the FCE (the class of 2007) to participate in this focus-group-based study halfway through the third year. They explored the influence of the FCE on students’ experiences in the third-year clerkships, and how conflicts between the two learning experiences shaped these students’ values and behaviors. Results Students reported that during clerkships they experienced strong feelings of powerlessness and conflict between what they had learned about patient-centered care in the first two years and what they saw role modeled in the third year. Based on students’ comments, the authors categorized students into one of three groups: those whose patient-centered values were maintained, compromised, or transformed. Conclusions Students revealed that their conflict was connected to feelings of powerlessness, along with exacerbating factors including limited time, concerns about expectations for their behavior, and pessimism about change. Role modeling had a significant influence on consequences related to students’ patient-centered values.