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(Circulation. 1995;91:393-402.)
© 1995 American Heart Association, Inc.

Articles

Cardioprotective Effects of a C1 Esterase Inhibitor in Myocardial Ischemia and Reperfusion

Michael Buerke, MD; Toyoaki Murohara, MD, PhD; Allan M. Lefer, PhD

From the Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa.

Correspondence to Dr Allan M. Lefer, Department of Physiology, Jefferson Medical College, 1020 Locust St, Philadelphia, PA 19107.

	Abstract

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Background Myocardial injury after ischemia and reperfusion can be attributed largely to the effects of polymorphonuclear leukocytes (PMN). The complement system plays an important role as a chemotactic agent, affecting adhesion molecule expression and neutrophil accumulation.

Methods and Results In the present study, the cardioprotective effects of C1 esterase inhibitor (C1 INH) were examined in a feline model of myocardial ischemia and reperfusion (90 minutes of ischemia followed by 270 minutes of reperfusion). C1 INH (15 mg/kg) administered 10 minutes before reperfusion significantly attenuated myocardial necrosis compared with vehicle (10±2% and 29±2% necrosis as a proportion of area at risk, respectively; P<.01). Myocardial preservation was also related to reduced plasma accumulation of creatine kinase activity. C1 INH treatment resulted in improved recovery of cardiac contractility and preservation of coronary vascular endothelial function, as assessed by relaxation in response to acetylcholine, compared with contractility and preservation of endothelial function in vehicle-treated animals (69±6% and 20±4% relaxation, respectively; P<.01). In addition, cardiac myeloperoxidase activity (an index of PMN accumulation) in the ischemic area was significantly reduced after C1 INH treatment. Furthermore, immunohistochemical analysis of ischemic-reperfused myocardial tissue demonstrated deposition of the first component of the classic complement pathway, C1q, on cardiac myocytes and coronary vessels.

Conclusions Blocking of the classic complement pathway by C1 INH appears to be an effective means of preserving ischemic myocardium from reperfusion injury. The mechanism of this cardioprotective effect appears to be inhibition of PMN-endothelium interaction; this inhibition leads to preservation of normal endothelial function, which results in reduced cardiac necrosis.

Key Words: proteins • esterases • endothelium • leukocytes • reperfusion

	Introduction

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Prolonged myocardial ischemia without reperfusion inevitably results in myocardial cell death. Although early reperfusion is a goal of therapy, reperfusion itself contributes to additional injury.^{1 2} This reperfusion injury is preceded by endothelial dysfunction³ and a disturbed homeostatic balance between circulating neutrophils and the coronary vasculature. A decrease in the release of basal nitric oxide initiates a series of events, including neutrophil adhesion to the coronary endothelial cell surface within 20 minutes after the onset of reperfusion and neutrophil accumulation in the myocardium 3 hours after reperfusion.^{4 5} These events lead to enhanced myocardial cell injury and increased myocardial necrosis.⁶

The complement system is thought to play a major role in initiating some of the inflammatory events occurring in ischemia and reperfusion.^{7 8} The classic complement pathway can be activated by certain sensitizing antibodies, cardiac mitochondrial particles, cardiolipin, or the fibrinolytic system.^{8 9} C3a and C5a, anaphylatoxins of the complement cascade, are potent leukocyte chemotactic agents, and C5a induces the synthesis and release of cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor– in macrophages. Infusion of C5a into the coronary arteries of pigs also results in reduced cardiac contractile function.¹⁰ Additional components of the complement cascade C5b-9, known as the terminal membrane attack complex (MAC), stimulate the synthesis of reactive oxygen metabolites and leukotriene B₄ in neutrophils.^{11 12} The complement system also activates the adhesion of neutrophils to the endothelium, because the MAC induces rapid translocation of P-selectin from Weibel-Palade bodies to the endothelial surface.¹³ Similarly, CD11b/CD18, the ß₂ integrin leukocyte adhesion complex, functions as complement receptor 3.¹⁴

The adhesion process that follows reperfusion of the ischemic myocardium starts with neutrophil rolling along the endothelial surface, largely mediated by P-selectin expressed on the endothelial surface and by constitutively expressed L-selectin on the neutrophil surface.¹⁵ The major ligands for selectin-mediated adherence are Lewis^x-containing carbohydrates as well as other glycolipids or glycoproteins.¹⁶ The rolling process tethers the neutrophils to the endothelial cell surface, leading to platelet activating factor–mediated activation of polymorphonuclear leukocytes (PMN) (ie, shape change, shedding of L-selectin, and conformational changes in CD11b/CD18, components of a complement cascade).^{17 18} This activation leads to tight adhesion mediated by the interaction of CD11b/CD18 with intercellular adhesion molecule–1 (ICAM-1), and this tight adhesion can result in transmigration of the neutrophils into the extravascular space. Monoclonal antibodies directed against P-selectin, L-selectin, or a sialyl Lewis^x-containing oligosaccharide prevent neutrophils from adhering to the coronary endothelium, preserve coronary endothelial function, and attenuate myocardial necrosis after myocardial ischemia and reperfusion.^{19 20 21}

Inhibition of the complement cascade at the receptor level has been shown to be cardioprotective in different in vitro²² and in vivo²³ models of myocardial ischemia and reperfusion. However, few data are available on the effect on reperfusion injury of complement system blockade at an early step in the complement cascade. Therefore, the major purposes of this study were to determine the effects of a C1 esterase inhibitor (C1 INH) on myocardial tissue injury, cardiac contractility, adherence of neutrophils to the coronary vascular endothelium, and coronary endothelial integrity in a well-established model of myocardial ischemia and reperfusion.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Determination of C1 INH Activity
To determine the ability of C1 INH to block the classic complement pathway, we used an erythrocyte hemolytic assay. Two hundred microliters of sensitized sheep erythrocytes (1.5x10⁸ cells/mL) (Sigma) were incubated with the first component of the classic complement pathway, C1q (0.01 to 0.25 µg/mL) and either 10 µL C1q-depleted human serum (Sigma) or 10 µL normal human serum. The volume was then adjusted to 550 µL with gelatin veronal buffer (Sigma) and the tubes were placed in a shaker bath at 37°C for 15 minutes; then 1 mL ice-cold gelatin veronal buffer was added to each tube to stop the reaction. The unlysed cells were removed by centrifugation at 800g at 4°C for 10 minutes. The absorbance of the supernatant was determined spectrophotometrically at 412 nm. Absorbance in the presence of normal serum was considered 100% of complement activity. The complement activity of the other tubes was calculated by dividing the absorbance of each tube by the absorbance of the normal serum.

To determine the effect of C1 INH on C1q-induced hemolysis, we incubated sensitized sheep erythrocytes with C1q-depleted human serum in the presence of 0.1 µg/mL C1q with and without different concentrations of C1 INH and determined hemolytic activity as described above.

Experimental Protocol
Adult male cats (2.7 to 3.8 kg) were anesthetized with sodium pentobarbital (30 mg/kg IV). An intratracheal cannula was inserted through a midline incision in each cat, and the animals were placed on intermittent positive-pressure ventilation (Harvard small-animal respirator). A polyethylene catheter was inserted into the right external jugular vein for additional pentobarbital infusion to maintain a surgical plane of anesthesia and for administration of C1 INH or its vehicle. A polyethylene catheter was inserted through the left femoral artery and positioned in the abdominal aorta for the measurement of mean arterial blood pressure (MABP) by use of a pressure transducer (Cobe Instruments). After a midsternal thoracotomy, the anterior pericardium was incised and a 3-0 silk suture was placed around the left anterior descending (LAD) coronary artery 8 to 10 mm from its origin. A high-fidelity catheter tip pressure transducer (model MPC 500, Millar Instruments, Inc) was introduced into the left ventricle through the apical dimple. The catheter was positioned during observation of left ventricular pressure (LVP) and dP/dt waveform (an index of myocardial contractility) and was secured in place by a silk suture. Standard lead II of the scalar ECG was used to determine heart rate and ST segment elevation. ST segment elevation was determined by analysis of the ECG recording at 50 mm/s every 20 minutes. The ECG, MABP, LVP, and dP/dt were continuously monitored on a Hewlett Packard 78304 A oscilloscope and recorded on a Gould 2400 S oscillographic recorder every 20 minutes. The pressure-rate index (PRI), an approximation of myocardial oxygen demand, was calculated as the product of MABP and heart rate divided by 1000.

After all surgical procedures were completed, the cats were allowed to stabilize for 30 minutes, at which time baseline readings of ECG, MABP, LVP, and dP/dt were recorded. Myocardial ischemia (MI) was induced by tightening the initially placed reversible ligature around the LAD so that the vessel was completely occluded. The time at which this was done was designated time point 0. Eighty minutes after coronary occlusion (10 minutes before reperfusion), 15 mg/kg C1 INH (Behring) or its vehicle (PBS) was given intravenously as a bolus. Ten minutes later (ie, after a total of 90 minutes of ischemia), the LAD ligature was untied, and the ischemic myocardium was reperfused for 4.5 hours. The cats were randomly divided into two major groups. Six cats undergoing myocardial infarction plus reperfusion received PBS (1 mL/kg) and six cats undergoing the same received C1 INH (15 mg/kg in PBS). Preliminary studies indicated that 7 mg/kg C1 INH exerted only a moderate degree of cardioprotection in this model.

Determination of Myocardial Necrosis
At the end of the 4.5-hour reperfusion period, the ligature around the LAD was again tightened. Twenty milliliters of 0.5% Evans blue was rapidly injected into the left ventricle to stain the area of myocardium perfused by the patent coronary arteries (ie, the area not at risk). The area at risk was identified as the area that did not stain. Immediately after this injection, the heart was rapidly excised and placed in warmed, oxygenated Krebs-Henseleit solution. The left circumflex (LCx) and LAD coronary arteries were isolated and removed for study of coronary ring vasoactivity and PMN adherence. The right ventricle, the great vessels, and fat tissue were carefully removed, and the left ventricle was sliced parallel to the atrioventricular groove in 3-mm-thick sections. The unstained portion of the myocardium was separated from the Evans blue–stained portion of the myocardium. The area at risk was sectioned into 1-mm³ cubes and incubated in 0.1% nitroblue tetrazolium in phosphate solution at pH 7.4 and 37°C for 15 minutes. The tetrazolium dye forms a blue formazan complex in the presence of myocardial cells containing active dehydrogenases and their cofactors. The irreversibly injured or necrotic portion of the myocardium at risk, which did not stain, was separated from the stained (ischemic but nonnecrotic) portion of the myocardium. The three portions of the myocardium (nonischemic, ischemic nonnecrotic, and ischemic necrotic) were weighed. Results are expressed as area at risk as a proportion of the total left ventricular mass, the area of necrotic tissue as a proportion of the area at risk, and the area of necrotic tissue as a proportion of the total left ventricular mass.

In three additional cats receiving vehicle, the above-described procedures were performed except the area at risk was evenly divided in two before nitroblue tetrazolium staining. One portion was incubated with 0.5 mg/mL C1 INH and the other with an equal volume of PBS to determine whether C1 INH altered the staining properties of the nitroblue tetrazolium. The area of necrotic tissue was 32±5% of the area at risk in the control samples and 34±4% of the area at risk in the samples incubated with C1 INH (difference not significant). Thus, C1 INH had no effect on nitroblue tetrazolium staining properties and therefore could not artifactually alter the determination of myocardial necrosis.

Plasma Creatine Kinase Analysis
Arterial blood samples (2 mL) were drawn immediately before ligation and hourly thereafter. The blood was collected in polyethylene tubes containing 200 IU heparin sodium. Samples were centrifuged at 2000g at 4°C for 20 minutes, and the plasma was decanted for biochemical analysis. Plasma protein concentration was assayed using the biuret method of Gornall.²⁴ Plasma creatine kinase (CK) activity was measured using the method of Rosalki²⁵ and expressed as IU/µg protein. All assays were measured without knowledge of each cat's experimental group. In three cats receiving vehicle, the above-described procedures were performed except that aliquots of the final plasma samples were incubated with 0.5 mg/mL C1 INH or an equal volume of PBS to determine whether C1 INH altered the CK assay. CK activities were 21.4±1.5 IU/µg protein in PBS-treated samples and 22.5±1.6 IU/µg in samples incubated with C1 INH. These values were not significantly different, indicating that C1 INH had no direct effect on the CK assay.

Determination of Myocardial Myeloperoxidase Activity
The myocardial activity of myeloperoxidase (MPO), an enzyme occurring virtually exclusively in neutrophils, was determined by the method of Bradley et al²⁶ as modified by Mullane et al.²⁷ The myocardium was homogenized in 0.5% hexadecyltrimethylammonium bromide (Sigma) and dissolved in 50 mmol/L potassium phosphate buffer at pH 6.0 using a Polytron (PCU-2) homogenizer (Brinkmann Instruments). Homogenates were centrifuged at 12 500g at 2°C for 30 minutes. The supernatants were then collected and reacted with 0.167 mg/mL o-dianisidine dihydrochloride (Sigma) and 0.0005% hydrogen peroxide in 50 mmol/L phosphate buffer at pH 6.0. The change in absorbance was measured spectrophotometrically at 460 nm. One unit of MPO is defined as that quantity of enzyme hydrolyzing peroxide at a rate of 1 mmol/min at 25°C. In three cats receiving vehicle, the above-described procedures were performed except that one half of the necrotic tissue was incubated with 0.5 mg/mL C1 INH or an equal volume of PBS to determine whether C1 INH altered the MPO assay. The MPO activities were 1.02±0.4 U/100 mg tissue in the Krebs-Henseleit solution–treated samples and 0.96±0.4 U/100 mg tissue in the samples incubated with C1 INH. These values were not significantly different, indicating that C1 INH had no direct effect on the MPO assay.

PMN Isolation and Labeling
Peripheral blood (20 mL) was collected from the femoral artery at the beginning of the surgical procedure, and PMNs were isolated by a procedure modified from Lafrado and Olsen²⁸ and described in detail previously.^{19 20} After centrifugation to remove platelets, the remaining blood was mixed with 6% dextran (average molecular weight 60 000 to 90 000; Sigma) and PBS to allow erythrocytes to settle for 40 to 60 minutes. The leukocyte-enriched fraction was layered onto Percoll gradient of 80%, 62%, and 50% (Sigma). The gradient was then centrifuged to separate the different cell populations. PMNs were collected from the 62% to 80% interface and washed twice with PBS before being assayed for viability using trypan blue exclusion. PMN preparations obtained by this method were in general >95% pure and >95% viable.

Isolated autologous PMNs were then labeled with a fluorescent dye (Sigma) according to the method of Yuan and Fleming.²⁹ One milliliter of the diluent was added to a loose cell pellet containing about 10 million cells. One milliliter of PKH2-GL dye (4 mmol/L) was added to the cell suspension, mixed, and then incubated for 5 minutes. Two milliliters of PBS containing 10% PPP was added to stop the labeling reaction, and another 5 mL of PBS was added to the suspension. Cells were then centrifuged at 400g for 10 minutes at room temperature. The supernatants were removed, and the cells were resuspended in PBS and recounted. This labeling procedure does not affect the normal morphology and function of cat PMNs.²⁹

PMN Adherence to Ischemic-Reperfused Coronary Artery Endothelium
The ischemic-reperfused LAD and the nonischemic LCx coronary artery segments were isolated. The artery segments were placed into warmed Krebs-Henseleit solution consisting of (mmol/L) NaCl 118, KCl 4.75, CaCl₂ · 2H₂O 2.54, KH₂PO₄ 1.19, MgSO₄ · 7H₂O 1.19, NaHCO₃ 12.5, and glucose 10.0, with a pH of 7.4. Coronary artery segments were carefully cleaned of fat and connective tissue and cut into rings 2 to 3 mm in length. These rings were opened and placed into 5-mL round cell culture dishes containing 3 mL Krebs-Henseleit solution. Unstimulated PMNs (400 000/mL) were added to the culture dishes and incubated in a shaker bath (120 agitations per minute) for 20 minutes at 37°C. Coronary artery segments were then removed from the culture dishes and dipped three to four times into fresh Krebs-Henseleit solution to wash off loose PMNs. The coronary rings were placed face up on glass slides. The number of adhering PMNs on the endothelium was assessed by fluorescence microscopy (Nikon) and expressed as PMNs/mm².

Vasorelaxation of Isolated Coronary Rings
Both LAD and LCx coronary segments were isolated from the heart and placed into warmed Krebs-Henseleit solution as described above. Coronary vessels were cleaned of connective tissue and cut into rings 2 to 3 mm in length. The rings were then mounted on stainless steel hooks, transferred to 10-mL tissue baths, and connected to FT-03 force displacement transducers (Grass Instrument Co) for recording of changes in force on a Grass model 7 oscillographic recorder. The baths were filled with 10 mL Krebs-Henseleit solution and gassed with 95% O₂ and 5% CO₂ at 37°C. Coronary rings were initially stretched to give a preload of 0.5 g of force and equilibrated for 90 minutes. During this period, the Krebs-Henseleit solution in the tissue baths was replaced every 15 minutes. After equilibration, the rings were stimulated with 100 nmol/L U-46619 (9,11-epoxymethano-PGH₂, Biomol Research Laboratories), a thromboxane A₂ mimetic, to generate about 0.5 g of developed force. Once the contraction reached a stable plateau, acetylcholine, an endothelium-dependent vasodilator, was added to the bath in cumulative concentrations of 0.1, 1, 10, and 100 nmol/L. After the response stabilized, the rings were washed three times and allowed to equilibrate for 20 minutes to reach baseline once again. The procedure, including addition of U-46619, was repeated with another endothelium-dependent vasodilator, A-23187 (1, 10, 100, and 1000 nmol/L), and an endothelium-independent vasodilator, acidified NaNO₂ (0.1, 1, 10, and 100 µmol/L), that was titrated to pH 2. Addition of equal volumes of Krebs-Henseleit solution titrated to pH 2 produced no detectable vasorelaxation in cat coronary artery rings. Vasorelaxation was calculated as percent relaxation from the peak U-46619–induced contraction.

Immunohistochemistry
For immunohistochemical analysis, 8 additional cats were subjected to no ischemia, 90 minutes of ischemia, 90 minutes of ischemia plus 20 minutes of reperfusion, or 90 minutes of ischemia plus 60 minutes of reperfusion. At the end of the reperfusion period, the hearts were removed and immediately cannulated through the aorta. The hearts were perfused at 50 mm Hg with Krebs-Henseleit solution for 2 minutes until the heart was cleared of blood. Perfusion was then switched to 4% paraformaldehyde in PBS (pH 7.4, 4°C) for 5 minutes to perfusion-fix the hearts. Full-thickness slices of the ischemic and nonischemic left ventricular wall (1 mm thick and 5 mm wide) were fixed for 1.5 hours at 4°C in 4% paraformaldehyde. After 1.5 hours, the ventricular slices were dehydrated in a graded series of acetone solutions (50%, 70%, 90%, and 100%) at 4°C. After dehydration, the sections were infiltrated with methacrylate (Immunobed; Polysciences Inc) at room temperature for 24 hours and subsequently embedded in methacrylate at 4°C for 12 hours. Glass knives were used to cut 4-µm-thick tissue sections, which were then placed on Vectabond-coated slides (Vector Laboratories).

Immunohistochemical procedures on plastic sections were performed by methods described previously by Beckstead et al³⁰ and modified by Weyrich et al,²⁰ using the avidin-biotin immunoperoxidase technique (Vectastain ABC reagent; Vector Laboratories). Incubation of the primary anti-C1q monoclonal antibody (MAb), 57 mg/mL (Calbiochem), with the cardiac tissue samples was carried out overnight at room temperature at dilutions of 1:10, 1:50, and 1:100 of the anti-C1q MAb. The 1:50 dilution gave the greatest degree of immunolocalization with the least amount of nonspecific background staining. The sections were lightly counterstained with the hematoxylin solution Gill No. 3 and examined with a Zeiss Axioplan light microscope. Tissue sections from each heart were analyzed in duplicate on separate days for each primary antibody.

Statistical Analysis
All values in the text, table, and figures are presented as mean±SEM derived from independent experiments. All data on infarct size, endothelial function, cardiac MPO, and PMN adherence were subjected to ANOVA followed by Fisher's t test. All data on CK, PRI, and dP/dt_max were analyzed by ANOVA incorporating repeated measurements. Values of P<.05 were considered statistically significant.

	Results

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Inhibitory Effects of C1 INH on Complement-Mediated Hemolysis
Incubation of sensitized sheep erythrocytes with C1q in C1q-depleted serum resulted in a concentration-dependent C1q-induced hemolysis of the cells (ie, activation of the classic complement pathway). Ten microliters of C1q hemolyzed 90% of the erythrocytes, whereas C1q-depleted serum by itself hemolyzed only 32% (Fig 1). Coincubation of 10 µL C1q (0.1 µg/mL) with C1 INH (0.05 to 1 mg/mL) resulted in a concentration-dependent inhibition of the hemolytic activity that was almost complete at 1 mg/mL. These results clearly demonstrate the efficacy of C1 INH in inhibiting activation of the classic complement pathway (Fig 1).

View larger version (37K): [in this window] [in a new window]   Figure 1. Bar graph of hemolytic activity of C1q, the first component of the classic complement pathway, and the inhibition of sensitized sheep red cell hemolysis by C1 esterase inhibitor (C1 INH), expressed as percent hemolysis. C1q (0.1 µg/mL) hemolyzed 90% of the cells. The inhibition of hemolysis by C1 INH was concentration-dependent over the range of 0.05 to 1 mg/mL. Height of bars are means; brackets represent SEM for five cats. **P<.05; ***P<.01.

Cardiac Electrophysiological and Hemodynamic Changes
Before coronary occlusion, there were no significant differences in any of the cardiovascular variables measured in the two groups of cats (vehicle and C1 INH) that underwent myocardial infarction and reperfusion. Several minutes after LAD occlusion, the ST segment of the ECG became significantly elevated, peaking by 1 hour. There was no significant difference in peak ST segment elevation between the two groups (0.18±0.02 mV in the vehicle group and 0.17±0.02 mV in the C1 INH group), indicating that the ischemic insult was similar in both groups. After reperfusion, the ST segment decreased to nearly control values in all cats, indicating that coronary perfusion had been effective. During reperfusion, there was a noticeable increase in the incidence of premature ventricular contractions in all cats. One cat in each group developed ventricular fibrillation, which was successfully converted to a normal sinus rhythm by a single application of cardiac defibrillation (DC electronic defibrillator; Sanborn Co). In both groups, PRI decreased significantly 1 hour after coronary occlusion (P<.05) and gradually returned to baseline values after reperfusion. There were no significant differences between the two groups at any of the hourly PRI readings, suggesting that C1 INH did not alter myocardial oxygen demand (Fig 2).

View larger version (16K): [in this window] [in a new window]   Figure 2. Pressure-rate index expressed as (mean arterial blood pressure [in mm Hg]xbeats per minute)/1000 during the 6-hour observation of myocardial infarction followed by reperfusion (R) in two groups of 6 cats each (one group [] was administered C1 esterase inhibitor [C1 INH] and the other [] was administered vehicle [V]). All values are mean±SEM.The pressure-rate index decreased significantly (P<.05) in both groups 1 hour after coronary occlusion (O). There were no significant differences between the two groups at any time after addition of C1 INH or vehicle.

Effect of C1 INH on Myocardial Injury After Reperfusion
To ascertain the effects of C1 INH on the degree of myocardial salvage of ischemic tissue after reperfusion, we measured the amount of necrotic cardiac tissue as a percentage of either the area at risk or of total left ventricular mass. There was no significant difference in the wet weights of the areas at risk expressed as a percentage of total left ventricular mass (Fig 3), indicating that a comparable region of myocardial ischemia occurred in both groups. About 30% of the jeopardized myocardium became necrotic in the vehicle group. In contrast, the necrotic area, expressed as either a percent of the area at risk or a percent of the total left ventricular mass, was significantly greater (P<.01) in cats treated with C1 INH. Therefore, C1 INH (15 mg/kg) significantly protected against necrotic injury in the cats that underwent ischemia and reperfusion (Fig 3).

View larger version (29K): [in this window] [in a new window]   Figure 3. Tissue wet weights of area at risk as a percentage of the total left ventricular wet weight (left), necrotic tissue as a percentage of area at risk (middle), and necrotic tissue as a percentage of the total left ventricular wet weight (right) for two groups of 6 cats each that underwent myocardial infarction plus reperfusion. Solid bars indicate data from cats administered vehicle; shaded bars indicate data from cats administered C1 esterase inhibitor. Height of bars are means; brackets represent SEM.

To further evaluate the preservation of ischemic tissue, we measured plasma CK activity, a biochemical marker of myocardial injury. In the two groups that underwent ischemia and reperfusion, plasma CK activity increased slightly during the period of myocardial ischemia. However, a washout of CK into the circulating blood occurred within the first hour of reperfusion (Fig 4). This increase in circulating CK progressed markedly during the remaining 4 hours of reperfusion in cats receiving only vehicle. In contrast, cats treated with C1 INH had significantly lower plasma CK activities compared with their counterparts that received only vehicle. The effect was sustained over the entire reperfusion period, suggesting that C1 INH significantly attenuated myocardial reperfusion injury (Fig 4).

View larger version (17K): [in this window] [in a new window]   Figure 4. Plasma creatine kinase (CK) activity expressed as international units (IU)/µg protein, measured hourly throughout the experiment for two groups of 6 cats each (C1 INH [] receiving C1 esterase inhibitor [C1 INH] and vehicle [] receiving vehicle [V]) that underwent myocardial infarction followed by reperfusion (R). All values are mean±SEM. O indicates occlusion. *P<.05 compared with the group administered C1 INH.

Effect of C1 INH on Cardiac Function
Left ventricular pressure and its first derivative (dP/dt), an index of myocardial contractility, were measured by a catheter tip manometer in the left ventricular cavity. Initial values were comparable in both groups that underwent ischemia and reperfusion. However, maximal left ventricular pressure and myocardial contractility (measured as positive dP/dt_max) decreased upon occlusion of the LAD to about 70% of control in both groups. In cats given only vehicle, contractility decreased further during the first 15 minutes of reperfusion and recovered only very slowly thereafter. In contrast, dP/dt_max in C1 INH–treated cats recovered more rapidly. After 4.5 hours of reperfusion, the dP/dt_max of the cats receiving C1 INH was significantly higher than that of cats given only vehicle (P<.05) (Fig 5). These results suggest that C1 INH not only prevents myocardial necrosis after reperfusion of the ischemic myocardium but also plays a role in myocardial salvage, as indicated by the preservation of mechanical performance of the heart.

View larger version (17K): [in this window] [in a new window]   Figure 5. Effects on dP/dtmax (a measure of coronary contractility) of administration of 15 mg/kg C1 esterase inhibitor (C1 INH) () or its vehicle (V) () in two groups of 6 cats each during 90 minutes of ischemia and 270 minutes of reperfusion. Data are expressed as percentage of initial values. All values are means; brackets indicate SEM. O indicates occlusion. *P<.01 compared with vehicle.

Effect of C1 INH on Coronary Endothelial Function
Because endothelial dysfunction is an early and critical event in reperfusion injury, we assessed endothelial function by comparing the vasorelaxant activity of isolated coronary artery rings in response to the endothelium-dependent vasodilators acetylcholine and A-23187 and to the endothelium-independent vasodilator NaNO₂. In the rings isolated from cats that underwent ischemia and reperfusion who received only vehicle, the acetylcholine-induced and A-23187–induced relaxations were significantly less than those in C1 INH–treated animals, whereas the NaNO₂-induced relaxation was similar in the two groups. Fig 6 summarizes the vasorelaxant responses to acetylcholine, A-23187, and NaNO₂ in isolated cat coronary artery rings. C1 INH significantly protected against the loss of endothelium-dependent relaxation observed in coronary artery rings after myocardial ischemia and reperfusion.

View larger version (48K): [in this window] [in a new window]   Figure 6. Summary of vasorelaxant responses of ischemia-reperfused left anterior descending (LAD) coronary artery rings (left) and nonischemic left circumflex (LCX) coronary artery rings (right) to 100 nmol/L acetylcholine, 1 µmol/L A-23187, and 100 µmol/L NaNO2. Solid bars indicate data from cats administered vehicle; shaded bars indicate data from cats administered C1 esterase inhibitor. Bar heights are means; brackets indicate SEM for 10 to 12 rings. **P<.01 compared with vehicle.

Neutrophil Accumulation in the Ischemic-Reperfused Area
Accumulation of neutrophils in the ischemic region during reperfusion has been thought to be one of the major mechanisms responsible for reperfusion injury. We therefore measured MPO activity in the nonischemic, ischemic, and necrotic portions of the myocardium as a marker for neutrophil accumulation. Fig 7 summarizes these data. In the nonischemic myocardium (ie, the area not at risk), MPO activity was very low in both groups that underwent myocardial infarction, and there was no significant difference between them. However, the cats that received only vehicle exhibited a marked increase in MPO activity in both the ischemic and necrotic regions. In contrast, the C1 INH–treated cats exhibited a significantly lower MPO activity in both ischemic nonnecrotic myocardial tissue (25% of that in the cats receiving vehicle, P<.05) and necrotic myocardial tissue (40% of that in the cats receiving vehicle, P<.01). These results indicate that C1 INH significantly retarded neutrophil accumulation in the myocardium of ischemic-reperfused cats.

View larger version (21K): [in this window] [in a new window]   Figure 7. Cardiac myeloperoxidase activity in area not at risk, area at risk, and necrotic area expressed as units (U)/100 mg tissue wet weight for the two groups of 6 cats each that underwent myocardial infarction followed by reperfusion. Solid bars indicate data from cats administered vehicle; shaded bars indicate data from cats administered C1 esterase inhibitor. Heights of bars are means; brackets represent SEM.

Effects of C1 INH on Circulating White Blood Cells
To determine whether C1 INH exerted any leukopenic effects that could contribute to its cardioprotective effects or its reduced MPO activity in the ischemic-reperfused myocardium, we counted the number of circulating white blood cells (WBCs) over the experimental period. Peripheral WBCs were counted 5 minutes before coronary occlusion, 5 minutes before reperfusion, and 30, 150, and 270 minutes after reperfusion. WBC counts did not change significantly over the course of the experiment in either the vehicle-treated or the C1 INH–treated group, and there were no significant differences in total WBC counts between the two groups at any time (Table). Differential counts were made to determine the percentage of the leukocytes that were neutrophils. In all analyzed samples, PMNs were 55% to 65% of total WBCs. These results clearly indicate that administration of C1 INH did not produce leukopenia in the cats. Thus, we cannot attribute the observed cardioprotective effects of C1 INH to changes in the number of circulating WBCs.

View this table: [in this window] [in a new window]   Table 1. Circulating Leukocyte Counts in Cats Receiving C1 Esterase Inhibitor (C1 INH) or Vehicle During Myocardial Ischemia and Reperfusion

Effect of C1 INH Administration In Vivo on PMN Adherence to Ischemia-Reperfused Coronary Endothelium Ex Vivo
An initial step in neutrophil-mediated reperfusion injury is the increased adhesion of neutrophils to the vascular endothelium. When unstimulated autologous PMNs were added alone to nonischemic-reperfused control LCx coronary arteries for 20 minutes, very few neutrophils adhered to the endothelial surface. However, in cats receiving vehicle, unstimulated PMNs added to LAD coronary arteries after ischemia and 270 minutes of reperfusion resulted in a dramatic increase in the number of PMNs adhering to the coronary endothelium (Fig 8). When autologous unstimulated PMNs were incubated with the LAD coronary arteries isolated from the cats treated with C1 INH, the number of PMNs adhering to the coronary endothelium after the same protocol was significantly less (P<.001)(Fig 8). Thus, C1 INH treatment prevented adherence of PMNs to the coronary endothelium after myocardial ischemia and reperfusion.

View larger version (22K): [in this window] [in a new window]   Figure 8. Effects of administration of C1 esterase inhibitor (C1 INH) (shaded bars) or vehicle (solid bars) in vivo on unstimulated neutrophil adherence to nonischemic-reperfused left circumflex (LCX) artery endothelium and ischemic-reperfused left anterior descending (LAD) coronary endothelium. Data are expressed as numbers of polymorphonuclear leukocytes (PMNs) adhering to each square millimeter of coronary endothelium. Bar heights are means, brackets indicate SEM, and numbers at the bottoms of the bars are numbers of coronary rings studied.

Immunohistochemical Localization of C1 After Myocardial Ischemia and Reperfusion
The presence of C1q in the myocardium after ischemia and reperfusion was detected by an anti-C1q MAB assay using an avidin-biotin immunoperoxidase procedure. Nonischemic sections of heart tissue (ie, those taken from myocardium perfused by the LCx coronary artery or by the LAD coronary artery after 0 minutes of ischemia) did not demonstrate any immunostaining. Similarly, no labeling of myocardial or endothelial cells was observed in immunohistological preparations in which either the primary antibody (anti-C1q MAb) or the biotinylated secondary antibody (mouse IgG) was replaced with nonimmune serum.

Although it was not seen in the nonischemic sections, C1q was evident in sections from all groups that had undergone ischemia followed by reperfusion for periods of 0 to 60 minutes. Intense immunolocalization of anti-C1q MAb was evident at 60 minutes after reperfusion (Fig 9). Significant immunolocalization of anti-C1q MAb was also seen at 0 and 20 minutes of reperfusion (Fig 9), although the staining reaction tended to be patchier. Immunolocalization of C1q was prevalent on cardiac myocytes and in the coronary vasculature, particularly the endothelium. These results indicate that reperfusion of the ischemic myocardium results in deposition of C1q in cardiac tissue.

View larger version (0K): [in this window] [in a new window]   Figure 9. Photomicrographs of heart tissue incubated with monoclonal antibody to C1q, the first component of the classic pathway, and labeled with peroxidase substrate solution. Brown reaction product is present at sites of antigen localization (arrows). A, Myocardium exposed to 0 minutes of ischemia; B, myocardium exposed to 90 minutes of ischemia followed by 0 minutes of reperfusion; C, cross section of a positively stained coronary vessel after 90 minutes of ischemia and 20 minutes of reperfusion; D, ischemic-reperfused myocardium negative control with omission of the primary antibody. The four panels are all the same magnification. A indicates coronary artery; V, coronary venule.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our data clearly demonstrate cardioprotective properties of C1 INH in myocardial ischemia and reperfusion. The cardioprotection exerted by C1 INH in this study was characterized by a 65% reduction in the amount of necrotic myocardium and a markedly attenuated plasma CK activity after reperfusion compared with what was seen in cats given vehicle. The reduction in necrotic tissue caused by C1 INH administration cannot be attributed to differences in the severity of ischemia because both groups that underwent ischemia and reperfusion developed equivalent anatomic areas of myocardium at risk and similar ST segment elevations. In addition, the two groups' comparable PRIs rule out the possibility that the cardioprotective effect of the C1 INH was related to reductions in myocardial oxygen demand. Because cats have a very low coronary collateral blood flow (about 7%),^{31 32 33} it is also unlikely that the protective effect of C1 INH could be explained by variations in collateral flow to the ischemic myocardium (ie, alterations in oxygen supply).

The cardioprotective effects of C1 INH also include improved cardiac function. This is the first study to demonstrate that blocking the classic complement pathway in vivo preserves cardiac contractility after ischemia and reperfusion. Specifically, cardiac contractility (dP/dt_max) recovered rapidly during the first few hours of reperfusion. Our results coincide with those of Shandelya et al,²² who used a soluble complement receptor 1 (sCR1) in vitro and observed improved cardiac contractile function and coronary flow in postischemic rat hearts. The sCR1 exerted these effects by inhibition of the neutrophil-derived oxidative burst. Diminished contractile function after reperfusion is known as myocardial stunning³⁴ and is most likely related to free radical release at the onset of reperfusion.³⁵ Because C1 INH exerted no direct hemodynamic effects, it is possible that C1 INH preserves contractile function by inhibiting PMN-endothelium interaction rather than having a direct cardiotonic effect.

C1 INH treatment not only provided significant myocardial protection but also preserved endothelial function. Endothelial dysfunction occurs shortly after the onset of reperfusion (within 5 minutes) and is characterized by a reduction in basal release of nitric oxide.^{3 36} Moreover, this loss of basal nitric oxide clearly leads to increased PMN adherence to the coronary vascular endothelium 20 minutes after reperfusion.^{4 5} Adhered and activated neutrophils release a variety of cytotoxic mediators (hydrogen peroxide, superoxide anion, hydroxyl radical, and elastase) that lead to increased tissue injury.^{6 37 38 39} These mediators further aggravate endothelial dysfunction, resulting in increased PMN adhesion to the vascular endothelium and subsequent myocardial necrosis 2 to 4 hours after reperfusion. However, in the present study, administration of C1 INH significantly preserved endothelial function as measured by coronary vascular relaxation in response to both acetylcholine and A-23187. In addition, administration of C1 INH in vivo resulted in diminished PMN adherence to the coronary endothelium 4.5 hours after reperfusion. These protective effects of C1 INH on the endothelium might be explained by inhibition of the PMN-endothelium interaction; this inhibition reduces firm PMN adherence, subsequent PMN mediator release, and ultimate myocardial tissue injury.

One important component of the myocardial salvage afforded by C1 INH is very likely due to its ability to diminish neutrophil adherence to the endothelium, presumably retarding complement activation and deposition. Clearly, neutrophils are involved in feline myocardial ischemia and reperfusion, because we observed significant increases in MPO activity in vehicle-treated ischemic myocardial tissue. This effect is probably due to the binding of C1q to the DNA of injured cells, leading to C1r and C1s activation and further formation of complement products.^{40 41} Moreover, an anti-CD18 MAb was protective to a comparable degree in this model of ischemia followed by reperfusion.³¹ In contrast, C1 INH treatment resulted in reduced MPO activity in the reperfused myocardium. The effects of C1 INH cannot be attributed to changes in circulating WBC counts because both groups had comparable and normal WBC counts throughout the myocardial ischemia and reperfusion. These data eliminate the possibility that C1 INH administration in vivo exerted leukopenic effects, a phenomenon known to be cardioprotective in myocardial ischemia–reperfusion injury.⁴² The reduced PMN accumulation after C1 INH administration observed in our study is in agreement with findings in other experiments involving ischemia and reperfusion, in which complement depletion with cobra venom factor resulted in significant inhibition of myocardial injury and reduced PMN infiltration into the ischemic myocardium.^{7 8}

Accumulation of C1q has been demonstrated in the ischemic reperfused myocardium by Rossen et al^{40 41} and has been related to increased neutrophil accumulation in this area. Their results further support our immunohistochemical findings of deposition of C1q in ischemic myocardium (after 90 minutes of ischemia) and in reperfused myocardium (after 90 minutes of ischemia plus 20 minutes of reperfusion and 90 minutes of ischemia plus 60 minutes of reperfusion). C1q could be observed on cardiac myocytes as well as on the coronary vascular endothelium. The ischemic myocardium releases membrane particles, pieces of mitochondria, and other subcellular components that bind C1q and activate the complement cascade.^{8 9} In addition, chemotactic and PMN-activating activity of postischemic cardiac lymph was found within the first 4 hours of reperfusion and correlated well with the appearance of C1q and C5a in cardiac lymph.^{43 44} The C1 activation results in generation of the bimolecular complex C4b,C2a, which is referred to as C3 convertase and forms C3a and C3b. This results in the splitting of C5 into C5a and C5b, with subsequent creation of the MAC C5b-9.^{45 46} Immunohistochemical analysis of autopsy material from patients with myocardial infarction has identified C5b-9 deposits in myocardial tissue.⁴⁷ Furthermore, Weisman et al²³ detected C5b-9 deposition on capillaries and venules in their murine model of myocardial ischemia and reperfusion.

Different complement factors exert a variety of inflammatory effects. C3a and C5a are potent leukocyte chemotactic agents. Blocking of C3b by sCR1 results in cardioprotective effects both in vitro²² and in vivo.²³ C5a induces synthesis and release of cytokines including interleukin-1, interleukin-6, and tumor necrosis factor– in macrophages. These cytokines induce the expression of the immunoglobulin superfamily adhesion molecules such as ICAM-1, which serves as a major counterreceptor for CD11b/CD18 on neutrophils. Blocking of either ICAM-1 or CD18 significantly reduces myocardial injury in ischemic-reperfused cats.^{31 48} Further infusion of C5a into coronary arteries of pigs results in reduced contractile function.¹⁰ The terminal MAC C5b-9 has been shown to stimulate the synthesis of reactive oxygen metabolites¹² and leukotriene B₄ neutrophils.¹¹ These mediators lead to the accumulation and activation of neutrophils and promote the conversion of reversibly injured myocytes to irreversibly injured myocytes (ie, they promote reperfusion injury). In addition, the complement system stimulates neutrophil-endothelium adhesion,¹⁴ because the MAC C5b-9 induces rapid translocation of P-selectin from Weibel-Palade bodies to the endothelial surface.¹³ Furthermore, complement-induced generation of oxygen free radicals might be an important stimulus for endothelial P-selectin expression.⁴⁹ Rapid expression of P-selectin is an important trigger for neutrophil rolling, which precedes activation and tight adherence of the neutrophils.^{15 17 18 49} Blocking P-selectin either with an MAb or a soluble sialyl Lewis^x–containing oligosaccharide reduces myocardial reperfusion injury in cats.^{20 21}

The complement-mediated myocardial injury after ischemia and reperfusion can be attributed to direct pathophysiological actions of complement⁵⁰ and can be indirectly augmented by complement-activated neutrophils.⁵¹ The cardioprotective effects of the C1 INH observed in the present study are quite dramatic, because we blocked the complement cascade in its first step and thereby prevented all the subsequent steps of the cascade (particularly C3). Blocking the classic complement pathway by administration of C1 INH significantly attenuates many key events, including release of chemotactic agents, PMN accumulation, endothelial activation, and PMN-endothelium interaction in this model of myocardial ischemia and reperfusion.

In conclusion, we have demonstrated that in vivo administration of C1 INH attenuates myocardial necrosis, preserves endothelial function, and sustains normal cardiac performance after myocardial ischemia and reperfusion. These protective effects could be attributed in large part to reduced PMN accumulation after C1 INH administration in the reperfused myocardium. Furthermore, these in vivo results demonstrate the important role of the classic complement pathway in inflammatory states that follow myocardial ischemia and reperfusion.

	Acknowledgments

 
This study was supported in part by grant GM-45434 from the National Institutes of Health. Dr Buerke is a postdoctoral fellow supported by Deutsche Forschungsgemeinschaft, and Dr Murohara is a postdoctoral fellow supported by the Japan Heart Foundation. The authors gratefully acknowledge Robert Craig for his excellent technical assistance in the biochemical analysis of creatine kinase activity. The authors wish to thank Dr H. Isliker, Lausanne, Switzerland, for his critical comments and Dr B. Eisele of Behringwerke AG, Marburg, Germany, for the supply of the complement inhibitor Berinert.

Received July 7, 1994; accepted August 9, 1994.

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				R. Bauernschmitt, C. Schreiber, and R. Lange C1 esterase inhibitor concentrate during surgery with cardiopulmonary bypass: Is there an indication beyond substitution therapy in patients with hereditary angioneurotic edema? J. Thorac. Cardiovasc. Surg., August 1, 2000; 120(2): 427 - 428. [Full Text]

				A. Agah, M. C. Montalto, C. L. Kiesecker, M. Morrissey, M. Grover, K. L. Whoolery, R. P. Rother, and G. L. Stahl Isolation, Characterization, and Cloning of Porcine Complement Component C7 J. Immunol., July 15, 2000; 165(2): 1059 - 1065. [Abstract] [Full Text] [PDF]

				H.-J. Rupprecht, J. v. Dahl, W. Terres, K. M. Seyfarth, G. Richardt, H.-P. Schulthei{beta}, M. Buerke, F. H. Sheehan, and H. Drexler Cardioprotective Effects of the Na+/H+ Exchange Inhibitor Cariporide in Patients With Acute Anterior Myocardial Infarction Undergoing Direct PTCA Circulation, June 27, 2000; 101(25): 2902 - 2908. [Abstract] [Full Text] [PDF]

				C. D. Collard, A. Vakeva, M. A. Morrissey, A. Agah, S. A. Rollins, W. R. Reenstra, J. A. Buras, S. Meri, and G. L. Stahl Complement Activation after Oxidative Stress : Role of the Lectin Complement Pathway Am. J. Pathol., May 1, 2000; 156(5): 1549 - 1556. [Abstract] [Full Text] [PDF]

				C. Caliezi, W. A. Wuillemin, S. Zeerleder, M. Redondo, B. Eisele, and C. E. Hack C1-Esterase Inhibitor: An Anti-Inflammatory Agent and Its Potential Use in the Treatment of Diseases Other Than Hereditary Angioedema Pharmacol. Rev., March 1, 2000; 52(1): 91 - 112. [Abstract] [Full Text] [PDF]

				J. E. Jordan, Z.-Q. Zhao, and J. Vinten-Johansen The role of neutrophils in myocardial ischemia-reperfusion injury Cardiovasc Res, September 1, 1999; 43(4): 860 - 878. [Abstract] [Full Text] [PDF]

				W. K. Lagrand, C. A. Visser, W. T. Hermens, H. W. M. Niessen, F. W. A. Verheugt, G.-J. Wolbink, and C. E. Hack C-Reactive Protein as a Cardiovascular Risk Factor : More Than an Epiphenomenon? Circulation, July 6, 1999; 100(1): 96 - 102. [Abstract] [Full Text] [PDF]

				H.W.M. Niessen, W.K. Lagrand, C.A. Visser, C.J.L.M. Meijer, and C.E. Hack Upregulation of ICAM-1 on cardiomyocytes in jeopardized human myocardium during infarction Cardiovasc Res, March 1, 1999; 41(3): 603 - 610. [Abstract] [Full Text] [PDF]

				K. Yasojima, C. Schwab, E. G. McGeer, and P. L. McGeer Human Heart Generates Complement Proteins That Are Upregulated and Activated After Myocardial Infarction Circ. Res., October 19, 1998; 83(8): 860 - 869. [Abstract] [Full Text] [PDF]

				M. Buerke, D. Prüfer, M. Dahm, H. Oelert, J. Meyer, and H. Darius Blocking of Classical Complement Pathway Inhibits Endothelial Adhesion Molecule Expression and Preserves Ischemic Myocardium from Reperfusion Injury J. Pharmacol. Exp. Ther., July 1, 1998; 286(1): 429 - 438. [Abstract] [Full Text]

				K. Yasojima, K. S. Kilgore, R. A. Washington, B. R. Lucchesi, and P. L. McGeer Complement Gene Expression by Rabbit Heart : Upregulation by Ischemia and Reperfusion Circ. Res., June 15, 1998; 82(11): 1224 - 1230. [Abstract] [Full Text] [PDF]

				A. P. Vakeva, A. Agah, S. A. Rollins, L. A. Matis, L. Li, and G. L. Stahl Myocardial Infarction and Apoptosis After Myocardial Ischemia and Reperfusion : Role of the Terminal Complement Components and Inhibition by Anti-C5 Therapy Circulation, June 9, 1998; 97(22): 2259 - 2267. [Abstract] [Full Text] [PDF]

				S. C. Makrides Therapeutic Inhibition of the Complement System Pharmacol. Rev., March 1, 1998; 50(1): 59 - 88. [Abstract] [Full Text] [PDF]

				J. Sonntag, M. H Wagner, E. Strauss, and M. Obladen Complement and contact activation in term neonates after fetal acidosis Arch. Dis. Child. Fetal Neonatal Ed., March 1, 1998; 78(2): 125F - 128. [Abstract] [Full Text]

				C. D. Collard, A. Vakeva, C. Bukusoglu, G. Zund, C. J. Sperati, S. P. Colgan, and G. L. Stahl Reoxygenation of Hypoxic Human Umbilical Vein Endothelial Cells Activates the Classic Complement Pathway Circulation, July 1, 1997; 96(1): 326 - 333. [Abstract] [Full Text]

				G. Horstick, A. Heimann, O. Gotze, G. Hafner, O. Berg, P. Boehmer, P. Becker, H. Darius, H.-J. Rupprecht, M. Loos, et al. Intracoronary Application of C1 Esterase Inhibitor Improves Cardiac Function and Reduces Myocardial Necrosis in an Experimental Model of Ischemia and Reperfusion Circulation, February 4, 1997; 95(3): 701 - 708. [Abstract] [Full Text]

				W. K. Lagrand, H. W.M. Niessen, G.-J. Wolbink, L. H. Jaspars, C. A. Visser, F. W.A. Verheugt, C. J.L.M. Meijer, and C. E. Hack C-Reactive Protein Colocalizes With Complement in Human Hearts During Acute Myocardial Infarction Circulation, January 7, 1997; 95(1): 97 - 103. [Abstract] [Full Text]

				G. L. Schaer, L. J. Spaccavento, K. F. Browne, K. A. Krueger, D. Krichbaum, J. M. Phelan, W. O. Fletcher, C. L. Grines, S. Edwards, M. K. Jolly, et al. Beneficial Effects of RheothRx Injection in Patients Receiving Thrombolytic Therapy for Acute Myocardial Infarction: Results of a Randomized, Double-Blind, Placebo-Controlled Trial Circulation, August 1, 1996; 94(3): 298 - 307. [Abstract] [Full Text]

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