Abstract
This study tests the hypothesis that adenosine A2 receptor activation reduces reperfusion injury by inhibiting neutrophils in a canine model of ischemia and reperfusion. In 16 anesthetized, open-chest dogs, the left anterior descending coronary artery was ligated for 60 min and reperfused for 3 hr. An intracoronary infusion of either the selective adenosine A2 agonist CGS-21680 at 0.2 μg/kg/min (n = 8) or vehicle (n = 8) was started 5 min before reperfusion and discontinued after 60 min. The area at risk was comparable between vehicle-treated and CGS-21680-treated groups (39.6 ± 4.1vs. 37.1 ± 2.5% of left ventricle). Infarction size, determined with triphenyltetrazolium chloride, was smaller in the CGS-21680-treated group than in the vehicle-treated group (15.4 ± 2.9 vs. 29.8 ± 2.3% of area at risk, P < .05 vs. vehicle-treated group). CGS-21680 significantly reduced neutrophil accumulation (myeloperoxidase activity) in the nonnecrotic area at risk tissue, compared with the vehicle-treated group (2.12 ± 0.5 vs. 6.47 ± 0.6 U/g of tissue, P < .05 vs. vehicle-treated group). Inin vitro studies, CGS-21680 reduced platelet-activating factor (PAF)-activated canine neutrophil adherence to the endothelial surface of normal homologous coronary artery segments. Compared with PAF-stimulated neutrophils (188.4 ± 9.4 adhered neutrophils/mm2), CGS-21680 reduced adherence close to base-line levels (46.6 ± 5.8 adhered neutrophils/mm2) at concentrations of 10 μM (65.6 ± 8.2 adhered neutrophils/mm2, P < .05 vs.PAF-stimulated group) and 50 μM (56.6 ± 4.6 adhered neutrophils/mm2, P < .05 vs.PAF-stimulated group). Superoxide anion production (cytochromec reduction) by activated neutrophils was reduced by CGS-21680 from 33.8 ± 5.0 to 8.9 ± 3.6 nmol/5 min/5 × 106 cells (P < .05 vs. PAF-stimulated group). We conclude that specific A2 receptor stimulation with CGS-21680 at reflow reduces reperfusion injury by inhibiting neutrophil-related processes.
Reperfusion, although a necessary event for the preservation of reversibly injured myocardium, is associated with additional injury mediated, in large part, by neutrophils (Dreyer et al., 1991; Lefer et al., 1991). The restoration of blood flow to the ischemic myocardium initiates a cascade of inflammatory-like processes that may contribute to postischemic injury. Within minutes of the initiation of reperfusion, endothelial dysfunction occurs (Tsao and Lefer, 1990). This endothelial dysfunction results from the close proximity of the neutrophil to the coronary endothelium during the adhesion process and the subsequent release of cytotoxic agents into this microenvironment. These agents, including PAF, superoxide anion radical, hydrogen peroxide and elastase, attract and activate more neutrophils to the area (amplification stage) and damage the endothelium. Although the migration of neutrophils across the endothelium into the myocardium occurs much later after reperfusion is initiated (approximately 3 hr after reperfusion), the early neutrophil-endothelial events produce microvascular injury and ultimately myocellular necrosis (Entmanet al., 1991; Lefer et al., 1994; Lefer, 1995).
Adenosine is an endogenous autacoid that has multiple effects on the progression of ischemia and reperfusion injury (Ely and Berne, 1992;Vinten-Johansen et al., 1995a). Adenosine works primarily through two distinct membrane receptors, with A1receptor-mediated effects predominating during ischemia and A2 receptor effects being exerted primarily during reperfusion in models of lethal injury (i.e., infarction) (Zhao et al., 1993). The role of the A3 receptor in cardioprotection has yet to be precisely determined. Activation of adenosine A1 receptors during the ischemic phase has been shown to reduce infarction size and contractile dysfunction, possibly through inhibition of ATP-sensitive K+ channels (Gross and Auchampach, 1992; Toombs et al., 1993) and the subsequent effects of hyperpolarization and reduced calcium overload. Adenosine A2 receptor activation during the reperfusion phase has been shown to reduce infarction size (Norton et al., 1992;Schlack et al., 1993). Selective A2 receptor activation inhibits neutrophil adherence to endothelium and decreases the production of superoxide anion (Cronstein et al., 1992;Zhao et al., 1995) independent of ATP-sensitive K+ channels. These inhibitory effects may be related to A2-mediated reductions in infarction size. Recently, a specific adenosine A2 agonist, CGS-21680, was reported to reduce infarction size in both rabbit and canine models (Nortonet al., 1992; Schlack et al., 1993). However, those studies did not determine whether the mechanism of adenosine A2 receptor-mediated cardioprotection conferred by CGS-21680 was related to the inhibition of neutrophil actions. In the present study, we tested the hypothesis that adenosine A2receptor activation, with the A2-selective agonist CGS-21680 given only during reperfusion, is cardioprotective through inhibition of neutrophil activities.
Methods
All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health Publication 85–23, revised 1985). The protocol was also approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine of Wake Forest University.
Twenty-two mongrel dogs of either gender were initially anesthetized with sodium thiamylal (20 mg/kg i.v.). After endotracheal intubation and cannulation of the right femoral vein, deep anesthesia was maintained by continuous infusion of 0.3 μg/kg/min fentanyl citrate and 0.03 mg/kg/min diazepam. The dog was ventilated with oxygen-enriched room air using a Harvard volume-cycled respirator to maintain arterial pO2 greater than 100 mm Hg. Normal blood gas levels and acid-base status were maintained by adjustment of the rate and volume of respiration or by i.v. administration of sodium bicarbonate, as appropriate. A left thoracotomy was performed at the fifth intercostal space, and the heart was suspended in a pericardial cradle. Millar MPC-500 solid-state pressure transducers (Millar Instruments, Houston, TX) were inserted into the aortic rootvia the right internal mammary artery and into the LV through a puncture in the apex of the heart, to measure MAP and instantaneous LV blood pressure, respectively. An umbilical snare was placed around the inferior vena cava for transient preload reduction. A pair of piezoelectric crystals were placed into the subendocardium of the area of the LV perfused by the LAD in the direction of circumferential fiber shortening, to measure segmental systolic and diastolic length. Anticoagulation was achieved with bolus injections of 300 U of sodium heparin given every 90 min, to maintain activated clotting times of >400 sec. The left carotid artery was cannulated and connected to a large-bore silastic catheter in which a Transonic ultrasonic cannulating-type flow probe (Transonic Systems, Ithaca, NY) was interposed. This circuit was capable of accommodating up to 95-ml/min flow at 100 mm Hg mean pressure. A proximal segment of the LAD was isolated and cannulated with a 14-gauge angiocatheter and immediately perfused via the left carotid artery. Ischemia encountered during the cannulation process lasted no longer than 40 sec and did not alter segmental function, relative to the precannulation period.
Experimental protocol.
Hemodynamic and segmental function data were collected before ischemia for baseline, at the end of ischemia and at 15, 60, 120 and 180 min of reperfusion. After base-line measurements were recorded, 60 min of collateral-deficient ischemia was created by clamping the carotid-LAD perfusion circuit, disconnecting the perfusion circuit from the coronary catheter and venting the collateral blood flow into a beaker. Arteriotomy and collateral diversion reduce the variability in infarction size observed in the canine model of ischemia-reperfusion by reducing the nutritive collateral blood flow (Eng et al., 1987). After 55 min of collateral diverted ischemia, an intracoronary infusion of either CGS-21680 at 0.2 μg/kg/min (n = 8) or vehicle (n = 8) was begun by reconnecting the extracorporeal circuit to the cannula in the LAD, while the circuit remained clamped, and inserting a 25-gauge needle into the silastic tubing just proximal to the cannula and distal to the clamp. At the end of 60 min of ischemia, reperfusion was initiated by removing the clamp, and the intracoronary infusion of CGS-21680 or vehicle was continued for the next 55 min. The AAR was reperfused for a total of 3 hr.
Drug preparation.
CGS-21680 was prepared from a dry powder and dissolved in 0.5 ml of dimethylsulfoxide. When completely dissolved, the drug was diluted with normal saline to achieve a dose of 0.2 μg/kg/min, to be delivered by 60-min intracoronary infusion. Fresh drug was prepared for each experiment. The saline vehicle was prepared with dimethylsulfoxide and saline in the same concentration as used in the CGS-21680-treated group. The dose of drug was calculated to deliver a concentration of drug of about 180 nM, which was a compromise between maximal neutrophil inhibition determined by in vitrotests and systemic vasodilation.
Data collection and analysis.
LV and aortic pressures, segment length data and LAD blood flow measurements were collected in duplicate or triplicate during 12-sec intervals of respiratory apnea. Data were digitized at 200 Hz using a 12-bit analog-to-digital converter (model DT2821; Data Translation Devices, Marlboro, MA) and an IBM-PC computer. Data were stored both on removable media and on the hard disk for archival and subsequent analysis. Two measurements were taken during steady state and three measurements were taken during caval occlusion (for determination of segmental diastolic stiffness). The data were processed using SPECTRUM (Triton Technology, San Diego, CA), a video-graphical interface computer algorithm program developed in our laboratory. The beginning of systole was marked when instantaneous LV dP/dt exceeded 250 mm Hg/sec, and the end-systolic point was marked at peak negativedP/dt. These points were checked visually and adjusted manually as necessary. Segmental shortening was calculated by the formula [(EDSL − ESSL)/EDSL] × 100 and expressed as a percentage. Segmental work was calculated using a point-by-point integration of the pressure-segment length loop over the entire cardiac cycle. Segmental stiffness was calculated by fitting the end-diastolic pressure-segment length data of the variably preloaded pressure-segment length loops obtained during caval occlusion to the equationP ED = α(e β L), whereP ED is the end-diastolic pressure, α and β coefficients are the end-diastolic pressure axis intercept and the degree of curvature, respectively, and L is the EDSL. The β coefficient is a unitless measure of the curvature of the end-diastolic relation (modulus of stiffness).
Determination of AAR and infarction size.
At the end of the experiment, the heart was rapidly arrested with a bolus injection of sodium pentobarbital and excised. The proximal aorta was cannulated, and the heart was stained using a gentian violet infusion deliveredvia the ascending aorta (infusion pressure was maintained at 100 mm Hg). The atria and the right ventricle were removed, and the LV was cut into 3- to 4-mm-thick sections. The unstained region (AAR) was then separated from the blue-stained nonischemic area and incubated for 10 min in a 1% solution of triphenyltetrazolium chloride solution in phosphate buffer, warmed to 37°C. After incubation, all tissues were placed in a 10% formalin solution overnight. The AAR was then subdivided into pale necrotic and brick-red nonnecrotic areas. The AAR and area of necrosis were determined gravimetrically and expressed as a percentage of the total LV mass. The AAR was calculated as 100 × [(mass of necrotic tissue + mass of nonnecrotic ischemic-reperfused tissue)/total mass of LV]. This gravimetric method of infarction sizing compares favorably with the planimetric method (Vinten-Johansenet al., 1992).
Plasma CK activity.
Blood samples for measuring plasma CK activity were taken from the left femoral artery at the same time points at which hemodynamic data were collected. The plasma was analyzed spectrophotometrically for total CK activity (CK-10 kit; Sigma Diagnostics, St. Louis, MO) and protein content. CK levels were expressed as international units per gram of protein.
Cardiac MPO activity.
Tissue samples were taken from the nonischemic zone and the nonnecrotic and necrotic zones of the AAR for spectrophotometric analysis of MPO activity as a measure of neutrophil accumulation in the myocardium, using a method described previously (Mullane et al., 1985; Sato et al., 1995). MPO activity (units per gram of tissue) was calculated as micromoles per minute = ΔA 460/11.3 μmol/(CM × milliliters of reactant ×D)/sample weight, where ΔA 460 is equal to the absorbance change over time at 460 nm, 11.3 is the molar extinction coefficient for H2O2 at 460 nm,CM represents the cuvette path length and D is the volume of supernatant used. One unit of MPO activity is defined as the enzyme activity degrading 1 mmol of H2O2/min at 25°C.
In vitro PMN adherence assay.
Adherence of canine neutrophils to the endothelial cell surface of normal, homologous, coronary arteries (from naive animals not subjected to ischemia and reperfusion) was assessed using neutrophils labeled with Zynaxis PKH26 (Zynaxis Cell Science, Malvern, PA) vital fluorescent dye. One milliliter each of diluent and PKH26 dye (4 μM) was added to a neutrophil suspension of 5 × 106 cells/ml and incubated for 5 min. Two milliliters of PBS containing 10% plasma were subsequently added to stop the labeling reaction, and another 5 ml of PBS were added to the cell suspension. The cells were then centrifuged at 600 × g for 10 min at 4°C. The pellet was resuspended in PBS, and the cells were counted by an automated Coulter counter to determine the population per milliliter of cell suspension. This labeling procedure yields cells with normal function and morphology. In validation studies performed with the labeling procedure, unlabeled and labeled neutrophils demonstrated 95% and 97% viability, respectively, using trypan blue exclusion.
Canine coronary arteries were carefully isolated in cold oxygenated Krebs-Henseleit buffer, so as not to disturb the endothelium. After removal of the fat and connective tissue, the arteries were cut into segments 2 to 3 mm in length. These segments were carefully opened and placed in plastic dishes containing 3 ml of Krebs-Henseleit buffer at 37°C. Labeled neutrophils (4 × 106 cells) were added to the bath alone or in combination with different concentrations of CGS-21680 (1–50 μM). PAF (100 nM) was added to the dishes 5 min after incubation with CGS-21680, and the incubation was continued for an additional 15 min. After incubation, the segments were removed, washed three times with Krebs-Henseleit buffer, placed on glass slides (endothelial side up) and covered with immersion oil and a glass coverslip. Adherence was measured by counting the number of neutrophils adhering to the endothelial surface in six separate microscopic fields under epifluorescence microscopy (490-nm excitation, 504-nm emission).
In vitro superoxide production by PMNs.
Adherence-independent superoxide radical production by activated neutrophils was determined by measuring the SOD-inhibitable reduction of ferricytochrome c to ferrocytochrome c. Neutrophils (5 × 106 cells/ml) were prewarmed to 37°C in the presence of 160 μM cytochrome c and incubated with cytochalasin B (5 μg/ml) and CGS-21680 (1–50 μM) for 5 min. The neutrophils were then stimulated with PAF (100 nM) in a final reaction volume of 0.5 ml. The tests were run in two groups, with and without SOD, to correct for nonspecific activity or color generation. The tubes were centrifuged at 500 × g for 10 min at 4°C. Cytochrome c reduction was measured spectrophotometrically by determining the optical density of the supernatant at 500 nm, using a Vmax kinetic microtiter plate reader (Molecular Devices, Palo Alto, CA). Superoxide radical production was calculated using an extinction coefficient of 21 mM−1 cm−1 for cytochrome c. Results are reported as nanomoles of SOD-inhibitable O2 − produced by a suspension of 5 × 106 neutrophils during the 5-min measurement period.
Statistical analysis.
Data are expressed as mean ± S.E.M. Differences were considered significant when P < .05. Differences in end-point variables between groups were analyzed by Student’s t test. Time-related differences within groups and group differences were analyzed by two-way analysis of variance for repeated measures, with post hoc t tests to determine the points that differed at each time. In vitro data were analyzed with one-way analysis of variance followed by Duncan’s multiple comparisons analysis.
Results
Twenty-two dogs were entered into the study, of which 16 (eight in each group) are represented in the final analysis of the results. Of the six dogs that were excluded from the data analysis, five were from the CGS-21680-treated group and one from the vehicle-treated group. Animals were fully randomized to either group. Two dogs did not finish the protocol (one from each group) because of malignant arrhythmias, three were not reperfused (all from the CGS-21680-treated group and characterized by the absence of reactive hyperemia and the persistence of cyanosis due to thrombus material in the extracorporeal circuit) and one broke protocol (from the CGS-21680-treated group) because of persistent and long-lasting respiratory acidosis.
Hemodynamic Data
The hemodynamic data for both groups are represented in table1. There were no differences between groups for any of the hemodynamic parameters at the control time point. In both groups the heart rate increased over the course of the experiment; however, none of the increases were significant, compared with the previous time point. CGS-21680 treatment after reperfusion showed a statistically significant reduction in heart rate, from that with the vehicle, only at 180 min of reperfusion. The vehicle-treated group displayed a small but significant decrease in LV peak-systolic pressure during ischemia, which was maintained over the remainder of the experiment. The CGS-21680-treated group showed a similar time-related decrease in LV peak-systolic pressure, which did not reach significance. The LV end-diastolic pressure increased slightly in both groups during ischemia but showed no significant group- or time-related differences during the entire experiment. The MAP in the vehicle-treated group remained constant throughout the procedure. However, CGS-21680 treatment at reperfusion caused 13 and 20% decreases in MAP at 15 and 60 min of reperfusion, respectively. There was a group difference in MAP only at 60 min of reperfusion, when the vehicle-treated group displayed a significantly higher pressure than the CGS-21680-treated group. Maximum and minimum dP/dt decreased during ischemia and remained at similar lower levels throughout the procedure in both groups. Coronary blood flow data showed a marked hyperemic response in both groups beginning upon restoration of flow and lasting through the first 1 hr of reperfusion. At 15 and 60 min of reperfusion, the CGS-21680-treated group exhibited a significantly greater blood flow, compared with vehicle. By 120 min of reperfusion, blood flow values returned to base-line levels and were comparable between groups.
Hemodynamic parameters measured throughout the course of the experiment
The calculated PRP for the two groups is summarized in table 1. Even though there were individual group differences at various times for the variables used to calculate the PRP, there were no significant differences in this variable during ischemia and early reperfusion. Although there was a trend toward a lower PRP in the CGS-21680-treated group at the later stages of reperfusion, these differences never reached significance.
Contractile Function Data
Segmental shortening.
The vehicle-treated and CGS-21680-treated groups were comparable with respect to EDSL and ESSL at the control time point (table 2). Both groups exhibited lengthened EDSL and ESSL values during ischemia and throughout the entire reperfusion period. However, there were no group-related differences. During ischemia both groups exhibited a rightward shift in the pressure-length relationship and displayed a significant decline in segmental shortening, changing from active shortening to dyskinesis (fig. 1; table 2). Both groups demonstrated a shift back toward the left in the pressure-segment length relation during the reperfusion phase, but neither group returned to base-line levels of ESSL or segmental shortening. Wall motion remained dyskinetic throughout the entire reperfusion period in the vehicle-treated group, whereas the CGS-21680-treated group showed a slight improvement in shortening during the first 1 hr of reperfusion before becoming dyskinetic for the remainder of the experiment. There were no significant group differences at any of the time points.
Regional segmental length and function for the AAR throughout the experiment
Instantaneous LV pressure-segment length loops for a representative experiment in vehicle-treated (n = 8) (top) and CGS-21680-treated (n = 8) (bottom) groups. Segmental work was calculated as the integral of each pressure-length loop. CNTL, control time point; ISCH, end of ischemia; REP, time of reperfusion.
Segment work.
The calculated segment work for both groups showed a significant decline from control values during ischemia (table2). There was a transient slight recovery of segment work in both groups just after 15 min of reperfusion, which dissipated with time. However, there were no significant differences between the vehicle- and CGS-21680-treated groups at any time during reperfusion.
Segment stiffness.
Segmental stiffness (table 2), calculated as the β-coefficient of the end-diastolic pressure-segment length relationship, was slightly higher in the CGS-21680-treated group at the control time point, compared with the vehicle-treated group (0.36 ± 0.05 vs. 0.21 ± 0.03). Throughout ischemia and reperfusion, the stiffness in both groups increased. However, these changes did not reach significant levels at any other time points.
Infarction Size
The LV weight was significantly greater in the vehicle-treated group, compared with the CGS-21680-treated group (table3). However, the AAR, when expressed as either mass (table 3) or percentage of LV (fig. 2), showed no significant group differences. CGS-21680 treatment at reperfusion significantly reduced the mass of necrotic tissue by 53% of the average vehicle size (table 3). Infarction size was reduced approximately 45% by CGS-21680, compared with vehicle, when expressed either as a percentage of LV or as a percentage of the AAR (fig. 2).
Mass of the areas of the heart (entire LV mass, AAR and area of necrosis) used for the determination of infarction size
Size of the AAR expressed as a percentage of the LV (AAR/LV) and infarction size (area of necrosis) expressed as a percentage of the LV (AN/LV) or AAR (AN/AAR). Vehicle-treated,n = 8; CGS-21680-treated, n = 8. *P < .05 vs. vehicle-treated group.
Plasma CK Activity
Total CK activity was comparable between groups at the control time point (fig. 3). There was a small increase in plasma CK in both vehicle-treated and CGS-21680-treated groups during ischemia. However, after reperfusion, there was a marked increase in CK levels within each group (fig. 3). Starting at 60 min of reperfusion, there was a strong tendency for the CGS-21680-treated group to have a lower plasma CK activity than the vehicle-treated group. However, these differences in total CK activity did not reach significance (P = .21 at 180 min of reperfusion).
Plasma creatine kinase (CK) levels for the two groups, shown for the entire experiment. CK levels are expressed as international units per gram of protein. †P < .05vs. prior time point. Cntl, control; Isch, end of ischemia; R15, reperfusion for 15 min; R60, reperfusion for 60 min; R120, reperfusion for 120 min; R180, reperfusion for 180 min.
Myocardial MPO Activity
MPO activity was similar between the two groups in the normally perfused, nonischemic area of the LV (fig. 4). In the nonnecrotic AAR, the CGS-21680-treated group showed significantly lower levels of MPO activity, compared with the vehicle-treated group. In the necrotic AAR, CGS-21680 treatment decreased the MPO activity by 45%, compared with the vehicle (P = .07). These data suggest that CGS-21680 reduced neutrophil accumulation in the ischemic-reperfused myocardium.
MPO activity in three areas of the myocardium. NI, nonischemic, normally perfused tissue; Isch, ischemic nonnecrotic tissue; Nec, necrotic tissue. MPO activity is expressed as units per gram of protein. *P < .05 vs. vehicle-treated group.
Superoxide Anion Production
As an index of the effect of CGS-21680 on adherence-independent (directly stimulated) neutrophil activity, an in vitro assay of superoxide production by isolated neutrophils was performed. PAF stimulation of neutrophils caused a >5-fold increase in superoxide anion production, compared with unstimulated neutrophils (fig.5). Preincubation with CGS-21680 exhibited a concentration-dependent decrease in superoxide anion production. At a concentration of 50 μM CGS-21680, superoxide radical production was reduced by nearly 75% of PAF-activated levels, which was not different from the unstimulated value. Therefore, CGS-21680 had a direct inhibitory effect on neutrophil superoxide anion generation (fig. 5) triggered by factors other than adherence to the endothelium (fig.6).
Production of superoxide anion by neutrophils, measured spectrophotometrically as the SOD-inhibitable reduction of ferricytochrome c in vitro. The ordinate represents the amount of superoxide produced in 5 min by a suspension of 5 × 106 neutrophils. *P < .05 vs.PAF-treated group.
Adherence of neutrophils to normal coronary artery endothelium. Labeled neutrophils were quantified using epifluorescent microscopy and are represented as the sum of neutrophils adhering to the endothelial surface per square millimeter of endothelium. *P < .05 vs. PAF-treated group. †P < .05vs. PMN-only group.
Neutrophil Adherence to Normal Coronary Endothelium
To further examine the effect of CGS-21680 on neutrophil activity, adherence of activated neutrophils to normal coronary artery endothelium was determined in vitro. PAF stimulation of neutrophils caused a 4-fold increase in the number of neutrophils adhered, compared with that of unstimulated PMNs (fig. 6). Pretreatment of the endothelium and neutrophils with CGS-21680 before activation with PAF significantly reduced adherence, in a dose-dependent manner. At 10 μM, adherence was reduced to approximately one-third of stimulated levels. CGS-21680, we conclude, had a profound inhibitory effect on neutrophil adherence to coronary artery endothelium. CGS-21680 therefore reduced adherence-independent superoxide generation and adherence to the coronary artery endothelium.
Discussion
Reperfusion accounts for a substantial portion of the injury that occurs as the result of reversible coronary occlusion (Forman et al., 1990). This concept of reperfusion injury has been cultivated from numerous studies in which pharmacological interventions were applied only during the reperfusion phase and resulted in a reduction in infarction size. Neutrophils may contribute significantly to the pathogenesis of injury occurring during reperfusion. Dreyer et al. (1991) demonstrated that neutrophils are localized into the previously ischemic myocardium predominantly during the first 1 hr of reperfusion. The present study shows that intracoronary infusion of a specific adenosine A2 agonist, CGS-21680, started during the first 1 hr of reperfusion, reduced infarction size significantly, without marked effects on the determinants of myocardial oxygen demand. The infusion of CGS-21680 was, however, associated with greater LAD blood flow during the period of infusion. In addition, these data support the hypothesis that this reduction in infarction size is associated with reduced neutrophil accumulation (tissue MPO activity) in the ischemic-reperfused myocardium. Additionally, it was shownin vitro that neutrophil-derived superoxide radical production was attenuated by A2 receptor stimulation, as was the adherence of neutrophils to coronary endothelium. These data are consistent with the hypothesis that activation of adenosine A2 receptors during reperfusion reduces myocardial infarction by inhibiting neutrophil activation and associated myocardial injury after reperfusion has been initiated.
The receptor agonist used in this study, CGS-21680, has been used extensively as a specific adenosine A2 agonist (Balwierczaket al., 1991; Norton et al., 1992; Schlacket al., 1993). This compound is more potent at the A2 receptor than at the A1 receptor (EC50 values of 22 nM vs. 760 nM) and is 140-fold more selective for the A2 receptor than the A1 receptor (Hutchison et al., 1989; Webbet al., 1992). CGS-21680 also has well-characterized vasodilator effects that are attributed to its selectivity for the adenosine A2 receptor (Abebe et al., 1994). The half-life of CGS-21680, as measured in rats, is approximately 19 min (Webb et al., 1992). Our hemodynamic data (table 1) indicate a small decrease in MAP during the infusion period. However, the decrease in pressure did not differ from that in the vehicle-treated group after the infusion was discontinued, arguing against a systemic buildup of the drug.
The chronotropic data do not suggest any A1-mediated effects on the heart, although in an in vivo model with intact cardiovascular reflexes these effects may be difficult to observe. The dose of CGS-21680 used in the present study was greater than that used by some other investigators (Bullough et al., 1995). However, as reported by Balwierczak et al. (1991), dogs are much less sensitive to CGS-21680 than other species. Previously, our group showed that adenosine, working through the A1 receptor, had very little effect on infarction size when given specifically during reperfusion (Zhao et al., 1994a). In that study, A1 receptor activity could account for only about 25% of the total protective effect of adenosine, and this protection was exerted during the ischemic phase (Zhao et al., 1994a). Therefore, it is unlikely that any A1receptor activation by the dose of CGS-21680 used in the present study would exert cardioprotection when administered only during reperfusion.
Our results are in apparent contrast to those of Lasley and Mentzer (1992), who concluded that postischemic functional recovery in an isolated rat heart model of myocardial stunning was achieved only by an A1 agonist and not by an A2 agonist. However, in that study the receptor-selective analogs were administered only before the onset of ischemia, followed by reperfusion with drug-free buffer. Therefore, the A2 agonist was not present during the reperfusion phase, when it may have exerted its action. In addition, the buffer solution was free of neutrophils, one of the primary targets of adenosine A2 analog actions. Moreover, neutrophils may play little role in ischemic-reperfusion injury producing myocardial “stunning” in the absence of infarction (Becker, 1991). Therefore, the data reported by Lasley and Mentzer (1992) are not inconsistent with the observations in the present study and are not inconsistent with a role for A2-mediated cardioprotection in models of lethal injury (infarction) where neutrophils are present.
Consistent with the vasodilatory effects of an A2-selective agonist, CGS-21680 exhibited a significant decrease in MAP and an increase in LAD blood flow, relative to the vehicle-treated group, for the duration of its infusion during reperfusion. The decrease in MAP occurred only during reperfusion. Although a correlation has been drawn between reduced afterload (MAP) during ischemia and reduced infarction size, no such correlation has been reported for afterload reduction specifically during reperfusion. In addition, the reduction in aortic blood pressure was counterbalanced by an increase in heart rate, resulting in no change in the PRP as an index of myocardial oxygen demand. The increase in coronary blood flow to the ischemic-reperfused myocardium may have played a role in the cardioprotection elicited by CGS-21680. Stahl et al. (1986) observed an improvement in postischemic function when blood flow was increased to the ischemic-reperfused area in a model of myocardial stunning. Such improvement in contractile performance may have been achieved by improvement of the distribution of blood flow, improving oxygen delivery, or by a local “garden hose” effect produced by vascular turgor. However, studies have demonstrated a marked reduction in infarction size (Vinten-Johansen et al., 1992, 1995b) and an increase in functional recovery (Hori et al., 1991;Vinten-Johansen et al., 1992) when reperfusion was gradually, rather than abruptly, restored. With this gradual reperfusion protocol, coronary blood flow is intentionally limited to approximately 20% of base line during the first 10 min of reflow and is gradually increased to unimpaired levels over 30 min. Therefore, enhanced blood flow during early reperfusion is not clearly associated with a reduction in morphological injury to the myocardium, although a transient improvement in contractile function may be observed, as in the present study. However, we cannot rule out the possibility that a more favorable redistribution of reflow to the subendocardium during reperfusion was involved in infarction reduction with CGS-21680.
Neutrophils are important contributors to in vivoischemic-reperfusion injury models of lethal injury produced by coronary occlusion and reperfusion (Romson et al., 1983;Mehta et al., 1988). Chemotactic stimuli increase adherence and accumulation of neutrophils in the AAR within minutes of the initiation of reperfusion. Adherence of neutrophils to the vascular endothelium is purportedly the initiating factor in the inflammatory-like cascade of neutrophil-mediated damage (Tsao et al., 1990; Kubes, 1993; Sluiter et al., 1993). Neutrophils promote microvascular and myocyte injury by the generation of superoxide anions, hypochlorous acid, proteases and PAF, and by embolization in the microvasculature, possibly contributing to the genesis of the no-reflow phenomenon (Cronstein et al., 1990,1992; Bullough et al., 1995). In addition, activated neutrophils release cytotoxic mediators that may be lethal to myocytes “from a distance,” without emigration into close proximity to the myocardial parenchyma. Within moments of reperfusion, neutrophils adhere to the vascular endothelium and produce measurable loss of endothelial function (Tsao et al., 1990; Lefer et al., 1991), leading to impaired production or release of nitric oxide (Zhao et al., 1994b). Therefore, injury to the endothelium and myocytes may occur early in reperfusion, resulting in interstitial edema, vascular compression, “no-reflow” and necrosis. Adenosine directly inhibits neutrophil-derived, endothelium adherence-independent generation of superoxide anions by receptor-mediated mechanisms (Cronstein et al., 1992; Zhaoet al., 1996). Cronstein et al. (1985) have shown that adenosine also inhibits neutrophil-mediated injury to the vascular endothelium of human umbilical veins by activation of A2receptors. This has recently been extended to the coronary arterial vasculature by Zhao et al. (1996). Furthermore, endogenous adenosine is released by the vascular endothelium in sufficient quantity to inhibit superoxide anion generation by neutrophils, also by an A2-mediated mechanism (Gunther and Herring, 1991). In addition to inhibiting neutrophil adherence and injury to vascular endothelium, Bullough et al. (1995) and Smith et al. (1991) have shown that adenosine retards adherence-dependent injury to myocytes. As shown in the present study, the A2receptor-specific analog CGS-21680 inhibited direct activation of neutrophils (adherence-independent), as well as neutrophil adherence to endothelium. Therefore, adenosine inhibits the early neutrophil actions leading to necrosis and reduces neutrophil-mediated damage to both vascular endothelium and myocytes.
The data from the present study are consistent with the A2-mediated antineutrophil actions of adenosine, because the A2-selective analog CGS-21680 reduced superoxide generation and endothelial cell adherence of PAF-stimulated neutrophilsin vitro. Accordingly, the MPO levels in the myocardium at risk, used as an index of neutrophils accumulated in the tissue, were reduced in the group treated with CGS-21680. Chen et al.(1994) demonstrated a correlation between neutrophil accumulationin vivo, as assessed by histological examination, and tissue MPO activity. In the present study, the neutrophils accumulated in the ischemic-reperfused myocardium may have been either adhering to the vascular endothelium or present as microemboli in the microvasculature. It is unlikely that the accumulated neutrophils had migrated into the parenchyma in proximity to the myocytes, because this process takes several hours to accomplish (Dreyer et al., 1991). MPO levels in the normally perfused myocardium were not altered in the CGS-21680-treated group, arguing against a significant contribution of passively resident neutrophils trapped after tissue sampling to the overall tissue MPO levels and arguing against a systemic antineutrophil effect of CGS-21680. In contrast to the in vitro studies using coincubated neutrophils and endothelium, we cannot conclude that there is a direct cause-and-effect relationship between fewer neutrophils and decreases in infarction size. However, data from the present study, as well as those reported by others (Tsao et al., 1990; Lefer et al., 1991; Ma et al., 1992), strongly suggest that the inhibition of neutrophils reduces consequent injury to vascular endothelium and myocytes. In addition, there is a strong correlation between neutrophil inhibition and infarction reduction (Lucchesi and Mullane, 1986; Lucchesi, 1990; Leferet al., 1993).
In summary, the present study supports the cardioprotective effects of adenosine and adenosine analogs. Specifically, a significant reduction in infarction may by accomplished by selective A2 receptor activation during reperfusion. The period of reperfusion coincides with the initiation of neutrophil activities culminating in vascular and myocyte injury. Reduction of in vitro superoxide generation and adherence to the coronary vascular endothelium, with a concomitant reduction of MPO levels in the AAR, strongly suggest that selective activation of adenosine A2 receptors during reperfusion reduces infarction size by inhibiting neutrophil-mediated damage.
Acknowledgments
We are grateful to Ciba-Geigy Pharmaceuticals (Summit, NJ) for the gift of CGS-21680 and to Sharon Ireland for preparation of the manuscript.
Footnotes
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Send reprint requests to: Jakob Vinten-Johansen, Ph.D., Department of Cardiothoracic Surgery, Cardiothoracic Research Laboratory, Carlyle Fraser Heart Center, Peachtree St., N. E., Atlanta, GA 30365-2225.
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↵1 This study was supported by grants from the National Institutes of Heath (Grant HL46179) and from the American Heart Association, North Carolina Affiliate.
- Abbreviations:
- AAR
- area at risk
- CK
- creatine kinase
- EDSL
- end-diastolic segment length
- ESSL
- end-systolic segment length
- LAD
- left anterior descending coronary artery
- LV
- left ventricle
- MAP
- mean aortic pressure
- MPO
- myeloperoxidase
- PAF
- platelet-activating factor
- PBS
- phosphate-buffered saline
- PMN
- polymorphonuclear leukocyte
- PRP
- pressure-rate product
- SOD
- superoxide dismutase
- Received February 8, 1996.
- Accepted September 25, 1996.
- The American Society for Pharmacology and Experimental Therapeutics