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Reduction of infarct size and postischemic inflammation from ATL-146e, a highly selective adenosine A_2A receptor agonist, in reperfused canine myocardium

Departments of ¹Internal Medicine and ²Chemistry, Experimental Cardiology Laboratory, Cardiovascular Division, University of Virginia Health System, Charlottesville, Virginia

Submitted 14 April 2004 ; accepted in final form 1 December 2004

	ABSTRACT

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Adenosine and adenosine A_2A receptor agonists have been shown to limit myocardial infarct size when given at vasodilatory doses during reperfusion. This beneficial effect is thought to be due, in part, to stimulation of adenosine A_2A receptors on inflammatory cells. The specific aims of this study were to determine whether the anti-inflammatory and cardioprotective properties of a novel adenosine A_2A receptor agonist, ATL-146e (ATL), alone or in combination with the phosphodiesterase IV inhibitor rolipram would occur using very low, nonvasodilating doses. In a canine model of reperfused myocardial infarction, low-dose ATL given alone reduced infarct size by 45% (P < 0.05 vs. control). When ATL was combined with a very low dose of rolipram (0.001 µg·kg^–1·min^–1), a marked reduction in P-selectin expression and neutrophil infiltration (51% lower; P < 0.001 vs. control) was seen and the infarct size reduction (58% lower; P < 0.01 vs. control) was greater than observed with ATL (45% lower; P < 0.05) or rolipram (33% lower; P < 0.05) alone. In conclusion, a low, nonvasodilating dose of ATL, a highly selective adenosine A_2A receptor agonist, reduced infarct size after reperfusion. Furthermore, combining ATL and the phosphodiesterase IV inhibitor rolipram reduced infarct size even more than either agent alone. Such combination therapy may be beneficial clinically by potentiating cardioprotection after coronary reperfusion at doses far below those producing vasodilatation or side effects.

myocardial infarction

Administration of highly selective adenosine A_2A receptor agonists protects against reperfusion injury and markedly reduces postischemic inflammation in a number of experimental models involving various organ systems (11, 16, 23, 27, 29, 36). The potent anti-inflammatory response to these agonists is mediated through stimulation of the G_s protein-coupled A_2A receptor subtype on neutrophils, mast cells, T cells, and platelets, resulting in elevation of intracellular cAMP and activation of protein kinase A, leading to the inhibition of multiple steps in the inflammatory cascade (11, 24, 25, 42, 45). Phosphodiesterase (PDE) type IV inhibitors, such as rolipram, also inhibit inflammation by increasing intracellular cAMP levels (32) and have been shown to act synergistically with adenosine A_2A receptor agonists in reducing neutrophil activation (42) and postischemic tissue injury in the kidney (26).

In the present study, we hypothesized that an adenosine A_2A receptor agonist would afford cardioprotection in reperfused myocardium without the necessity of increasing flow. A secondary hypothesis was that, based on the synergy observed with A_2A adenosine receptor agonists and rolipram in reducing postischemic injury in other tissues and models at our institution (26, 42), the reduction in myocardial infarct size with the combination of ATL-146e + rolipram would be greater than with either drug alone and would be associated with a greater decrease in neutrophil accumulation in the reperfused zone.

The specific aims of this study were to determine whether stimulation of the adenosine A_2A receptor subtype offers cardioprotection from reperfusion injury and whether the anti-inflammatory and infarct size-limiting properties of adenosine A_2A receptor agonists are further enhanced when combined with a PDE type IV inhibitor.

	MATERIALS AND METHODS

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All animal experiments were performed with the approval of the University of Virginia Animal Care and Use Committee and were in compliance with the position of the American Heart Association on the use of research animals.

In Vitro Neutrophil Oxidative Burst Assay

Twenty-five milliliters of venous dog blood were heparinized (10 U/ml), centrifuged (200 g for 8 min), and processed as previously described in detail (42). The effect of ATL-146e (1–1,000 nM) and ATL-146e + rolipram (300 nM, Cedar Knolls, NJ) on neutrophil oxidative burst was assessed as previously described (35). The in vitro samples containing mixed leukocytes were gated for neutrophils by forward and side scatter, and the fluorescence of 10,000 neutrophils was measured in the FL1 channel of a FACScan (Becton-Dickinson) flow cytometer. The results are reported as relative mean fluorescence intensity. Each point is the mean ± SE of nine samples from three different dogs.

In Vivo Canine Experiments

Surgical preparation and experimental protocol. Thirty-two fasted, adult mongrel dogs (mean 23.3 ± 0.6 kg, range 17.3–29.5 kg) were anesthetized with pentobarbital sodium (30 mg/kg iv), tracheally intubated, and mechanically ventilated with room air (Harvard Apparatus). Open-chest surgery and instrumentation were performed as previously described (8, 35).

After a 30-min baseline stabilization period, the left anterior descending coronary artery (LAD) and all visible collaterals were totally occluded for 90 min and reperfused for 120 min. Myocardial surface temperature was monitored and adjusted to match the animal's core temperature. Regional myocardial blood flows were assessed using radiolabeled microspheres during baseline (10 min before occlusion) and at the end of the occlusion and reperfusion periods. Intravenous infusions of ATL-146e (0.01 µg·kg^–1·min^–1; n = 5) and rolipram (0.001 µg·kg^–1·min^–1; n = 5) were tested separately and combined. The separate drug infusions were begun at baseline, 5 min before LAD occlusion, whereas the combination ATL-146e (0.01 or 0.001 µg·kg^–1·min^–1) + rolipram (0.001 µg·kg^–1·min^–1) treatment was begun either at baseline (5 min before LAD occlusion; n = 10) or 30 min before coronary reperfusion (n = 5) and continued throughout the protocol. The maximum dose of ATL-146e that was administered (0.01 µg·kg^–1·min^–1) is below the threshold for producing a vasodilatory response in dogs (8). A control group of seven dogs had no treatment. Myocardial inflammation was assessed with the neutrophil tracer ^99mTc-RP517³⁵ and by postmortem immunohistochemical techniques. Before euthanasia, the LAD was briefly reoccluded and 40 ml of monastral blue dye were rapidly injected into the left atrial catheter to delineate the anatomic risk area.

Postmortem Analysis

Immunohistochemistry. Myocardial biopsies were taken from the normal and infarct areas immediately after excision of the hearts, fixed overnight in 4% paraformaldehyde, and paraffin embedded. Sections were analyzed for P-selectin expression using a rabbit polyclonal anti-P-selectin antibody (10), as previously described (12), and neutrophil staining was performed using naphthol AS-D chloroacetate esterase (18). Sections were observed microscopically, and the images were digitized through an Olympus (BH-2) microprojection system with a Dage-MTI DC-330 color camera (Dage-MTI).

Ex vivo image acquisition and analysis. After excision, the hearts were sliced evenly into four rings from apex to base and placed directly on the collimator of a gamma camera (Siemens Orbiter) for quantitative ex vivo imaging of ^99mTc-RP517 activity, as previously described (35).

Determination of regional myocardial blood flow and ^99mTc-RP517 myocardial activity. The technique used in our laboratory to quantify regional myocardial blood flow by the radioactive microsphere technique has been described previously (8). Seventy-two epicardial, midwall, and endocardial segments and arterial blood samples were counted in a gamma-well counter (MINAXI 5550, Packard Instruments) using standard methods in our laboratory (8, 35). Transmural flow values were calculated as the weighted average of the corresponding epicardial, midwall, and endocardial samples.

^99mTc-RP517 activity in each myocardial segment was expressed as a percentage of the average ^99mTc-RP517 activity in the five normal, left circumflex zone segments from the same animal exhibiting the highest flow during occlusion. ^99mTc-RP517 myocardial uptake was averaged in myocardial segments that had 0–40% of normal flow during occlusion.

Determination of risk area and infarct size. The heart slices were incubated for 10 min at 37°C in a 2% solution of triphenyl tetrazolium chloride to delineate infarct area. Pictures of myocardial slices were obtained with a digital camera (Olympus D-600L), and the risk and infarcted areas were determined with specialized computer software (SigmaScan Pro Image Analysis version 5.0.0, SPSS).

Statistical Analysis

Results are presented as means ±SE. Computations were performed with SYSTAT software (SPSS). Treatment and time effects for regional myocardial blood flow were analyzed using two-way repeated-measures ANOVA. Comparisons between treatment groups for all other parameters were assessed using one-way ANOVA. When appropriate, post hoc testing was performed using Bonferroni's tests. P values of <0.05 were considered significant.

	RESULTS

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In Vitro Neutrophil Oxidative Burst Assay

The in vitro results using canine neutrophils are depicted in Fig. 1. ATL-146e decreased neutrophil oxidative burst in a dose-dependent manner (P < 0.001 by repeated-measures ANOVA), with a 23% inhibition at the 1 µM dose. Combination of ATL-146e with rolipram had a synergistic, inhibitory effect greater than with ATL-146e or alone at all common doses tested (P < 0.01 vs. ATL-146e). Oxidative activity of stimulated neutrophils in the presence of 10 nM ATL-146e + 300 nM rolipram was similar to the basal activity of unstimulated cells.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Flow cytometry analysis of TNF--induced neutrophil oxidative activity with increasing concentrations of ATL-146e in the absence or presence of the phosphodiesterase IV inhibitor rolipram. Note that the TNF--induced neutrophil burst was totally abolished by ATL-146e (10 nM) + rolipram (300 nM). MFI, mean fluorescence intensity.

Hemodynamic parameters. The mean hemodynamic parameters of aortic pressure, heart rate, and first derivative of left ventricular pressure (dP/dt) are presented in Table 1. Despite some minor changes of these parameters within each group over time, no statistical differences between groups in any of the hemodynamic parameters were observed.

Immunohistochemistry. Immunohistochemical analysis of P-selectin and Leder staining of neutrophils from a representative control and ATL-146e + rolipram-treated dog are presented in Fig. 2. P-selectin expression and neutrophil infiltration were visibly reduced in the ATL-146e + rolipram-treated dog. Observation at higher magnification also revealed better myocyte structural integrity with less edema and cellular disruption in the treated group of dogs.

View larger version (146K): [in this window] [in a new window]   Fig. 2. Immunohistochemical and Leder staining of P-selectin (top) and neutrophils (bottom), respectively, in a representative dog from the control (left) and treated (prereperfusion; right) groups. Inset: vessel denoted by arrow at a higher magnification (x1,000). As can be seen, both P-selection expression and neutrophil infiltration were visibly reduced in the treated dog, confirming the inhibition of inflammation by ATL-146e + rolipram treatment.

View larger version (58K): [in this window] [in a new window]   Fig. 3. A: assessment of canine myocardial inflammation with the neutrophil tracer 99mTc-RP517 as measured by gamma well counting of tissue segments. 99mTc-RP517 activity in endocardial and midwall segments with 0–40% of normal flow during coronary occlusion was expressed as a percentage of tracer activity in the normal, nonischemic zone. The ATL-146e + rolipram combination reduced neutrophil infiltration by 51 and 22% when administered at baseline or reperfusion, respectively, and the anti-inflammatory effect was greater than with either agent alone. Base, baseline; Rep, reperfusion. B: representative ex vivo images of 99mTc-RP517 canine myocardial activity after ischemia and reperfusion in a control dog (left) and an animal treated with ATL-146e + rolipram (right). Note that neutrophil infiltration in the previously ischemic area was markedly reduced in the presence of ATL-146e + rolipram.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Myocardial risk area (left) and infarct size (right) in the canine model of myocardial infarction. The combination treatment of ATL-146e + rolipram provided greater protection than with either agent alone. Note that the reduction in infarct size when ATL-146e + rolipram was administered at reperfusion (decrease of 58%) was similar to that when given during baseline (decrease of 50%). LV, left ventricle; RA, risk area.

Rolipram alone. Treatment with rolipram alone beginning at baseline resulted in a 33% reduction in myocardial infarct size to 38.0 ± 6.5% compared with the control group (P < 0.05).

ATL-146e ± rolipram combination. Treatment with ATL-146e + rolipram beginning either at baseline or reperfusion resulted in a marked 58 and 50% decrease in myocardial infarct size, respectively (P < 0.01 vs. control). There was no statistical difference in infarct size reduction when ATL-146e + rolipram was given at baseline vs. given just before reperfusion. The combination treatment resulted in a decrease in myocardial infarct size that was greater than seen with ATL-146e or rolipram alone.

	DISCUSSION

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In canine models of myocardial infarction, low-dose, intravenous administration of the highly selective adenosine A_2A receptor agonist ATL-146e significantly reduced infarct size, both as a pretreatment and when administered just before reperfusion, without causing vasodilatation. Rolipram is a PDE type IV inhibitor that, given alone, resulted in a 33% reduction in infarct size. When both ATL-146e and rolipram were given in combination, infarct size was reduced by 58%. This beneficial effect corresponded with a marked reduction in myocardial inflammation.

Comparison With Previous Experimental Studies

There have been conflicting results using adenosine administered at reperfusion for cardioprotection. In a canine model of myocardial infarction, Pitarys et al. (30) found that adenosine infusion in the early reperfusion period markedly reduced infarct size from 35 to 17% of the risk area, and the protection was sustained over 72 h. In contrast, Vander Heide and colleagues (44) found no reduction in infarct size with intravenous adenosine in a canine model of 90 min of coronary occlusion and reperfusion. Likewise, Goto et al. (9) found that intravenous adenosine infusion during early reperfusion failed to limit myocardial infarct size in rabbits.

The variability in these studies, in part, may be that, in addition to the potent anti-inflammatory effect of A_2A adenosine receptor stimulation, adenosine produces proinflammatory responses mediated by receptors that vary among species; A₃ and A_2B receptors mediate degranulation of rodent and human or canine mast cells, respectively (2, 6, 33). In contrast to the variable results obtained using adenosine, a number of experimental studies have all demonstrated the cardioprotective effects of an adenosine A_2A receptor agonist (CGS-21680) given at the time of reperfusion (11, 16, 23, 36). Infarct size reduction ranged from 40 to 60% when given via the intracoronary route in canine and swine models (11, 16, 36) and 42% when an intermediate dose was administered intravenously in rabbits (23).

Marked infarct size reduction has also been reported in rabbits, dogs, and pigs with AMP579, a mixed adenosine A₁/A₂ adenosine receptor agonist, when given at the time of coronary reperfusion (3, 13, 39, 46). The mechanism for the AMP 579-mediated protection when given at reperfusion has been shown to involve the adenosine A_2A receptor (13). In most of these animal studies, the AMP579 dose given produced hemodynamic changes, including vasodilatation; however, in one study, significant infarct size reduction (50%) was found in a pig model at a low, hemodynamically silent dose (3 µg/kg bolus + 0.3 µg·kg^–1·min^–1 infusion) (39). Unfortunately, the results of a recent clinical trial (ADMIRE) showed no reduction of infarct size with AMP579 treatment (14).

Synergism with ATL-146e and a PDE Type IV Inhibitor

A major finding of the present study was that the reduction in myocardial infarct size and postischemic inflammation was enhanced when the adenosine A_2A receptor agonist ATL-146e was combined with the PDE type IV inhibitor rolipram. ATL-146e alone and rolipram alone (see Fig. 4) resulted in a reduction in ischemic reperfusion injury, but the effect of the combination treatment was greater than observed for either agent alone. The synergism between an adenosine A_2A receptor agonist and rolipram in inhibiting oxidative burst in activated neutrophils was demonstrated in this study using canine neutrophils and has previously been observed when the same in vitro assay was performed on neutrophils obtained from human donors (40). Synergism between rolipram and adenosine or adenosine A_2A receptor agonists has also been shown in vivo in animal models of meningitis (41) and renal ischemia/reperfusion (26).

In the present study, we also found that a very low dose (1 ng·kg^–1·min^–1) of the PDE type IV inhibitor rolipram alone produced a significant reduction in both inflammation and infarct size (reduced 33%). Rolipram was most likely acting in conjunction with the endogenous adenosine produced during the ischemic period to maintain elevated cAMP levels and to reduce inflammation and infarct size. Our results with rolipram alone differ in part from those reported by other laboratories. McVey et al. (22) administered rolipram at the time of reperfusion to see whether it would reduce infarct size in a reperfused rat heart model. In their study, rolipram reduced the inflammatory response as measured by the production of TNF- but did not reduce infarct size. It is possible that any protective effect of rolipram in their study was offset by the marked hypotension that was observed at the dose of rolipram given. Hypotension may have attenuated repefusion flow and exacerbated or prolonged the ischemic period. In another study, Simpson et al. (38) gave rolipram before reperfusion in a canine model. They also found a tendency for a reduction in the level of the neutrophil marker tissue myeloperoxidase (P = 0.0548) with attenuation of the "no-reflow" phenomenon but no reduction in infarct size. One difference between their study and the present study is that we tied off all of the visible collateral vessels during the occlusion period. This would be expected to produce more severe ischemia and may have resulted in a greater production of endogenous adenosine, as well as slowing its washout from the interstitium, during the ischemic period. Zhao et al. (47) reported that endogenous adenosine released from the myocardium during ischemia-reperfusion reduces infarct size by an adenosine receptor-mediated mechanism and that the protection is most pronounced during the early phase of reperfusion. Thus, in our study, a higher level of endogenous adenosine may have been available to act synergistically with rolipram at the time of reperfusion to protect the myocardium.

Reduction of Myocardial Inflammation After Reperfusion

In the previous experimental studies using CGS21680(11, 16, 23, 36), the administered dose of the A_2A agonist was sufficient to produce vasodilatation, especially when administered by the intracoronary route. Although significant cardioprotection was observed in all of these studies, it is impossible to determine whether the observed infarct size reduction was due to a decrease in inflammation, an improvement in tissue perfusion, or a combination of both. In the present study, we gave very low doses of ATL-146e and rolipram that produced no increase in regional myocardial blood flow, yet resulted in a marked reduction in inflammation, as evidenced by inhibition of P-selectin expression and neutrophil infiltration and a 51–58% reduction in myocardial infarct size. Although we chose P-selectin and neutrophils as general markers of the inflammatory response, we cannot rule out an inhibitory effect of the treatment on other inflammatory cell types [e.g., T-lympocytes (4, 15, 17)], tissue cytokines or chemokines, or platelet activation (17, 31).

Clinical Implications

Optimal coronary reperfusion after acute myocardial infarction must result not only in early and sustained patency of the epicardial coronary vessel but maintenance of microvascular flow and tissue perfusion. Reperfusion injury and microvascular no-reflow may attenuate the benefit of reperfusion therapy aimed at reducing infarct size (34).

Myocardial inflammation may be one mechanism contributing to microvascular injury after reperfusion. Intravascular plugging by activated leukocytes may inhibit nutrient blood flow to the myocardium (37). Leukocytes also promote release of oxygen-free radicals and cytokines that induce further injury to the capillary endothelium. Several pharmacological approaches to inhibiting this myocardial inflammatory response consequent to ischemia/reperfusion have been examined in the clinical setting. The AMISTAD trial reported a 33% relative reduction in infarct size with adenosine (20).

Although experimental studies appeared promising (1, 19, 43), a clinical trial using an antibody (Hu23F2G) to block the CD11/CD18 integrin receptor proved negative (5). It should be pointed out that the dose of Hu23F26 used had only saturated 80% of the granulocyte receptors.

In the present experimental study, administration of an adenosine A_2A receptor agonist and a PDE type IV inhibitor alone significantly reduced both infarct zone inflammation after reperfusion and infarct size in a canine model of occlusion and reperfusion. Whether such an impressive outcome would be seen in patients treated with these compounds is yet unknown. Certainly, the highly selective adenosine A_2A receptor agonist may be more effective than adenosine since it is specific for the A_2A receptor eliminating the A_2B effects of hypotension and the A₁ proinflammatory and adverse electrophysiological effects. A clinical trial testing this hypothesis appears warranted.

Study Limitations

One limitation of our study is that we did not test higher doses of ATL-146e and rolipram than those reported. As mentioned above, we selected nonvasodilatory doses to avoid the confounding effect of increased flow on the interpretation of the mechanism for cardioprotection. Higher vasodilating doses of ATL-146e and rolipram might confer greater protection but possibly at the expense of increased side effects.

A second limitation is that, in these acute studies, we were unable to address the question of long-term outcome after short-term treatment. Specifically, does the benefit of infarct size reduction translate into improved cardiac function and enhanced survival in the long term? Further studies in chronic models of myocardial infarction and in humans are needed to address this very important question.

	GRANTS

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This study was supported in part by American Heart Association Grant-In-Aid 0050607N.

	DISCLOSURES

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The adenosine A_2A agonist, ATL-146e, used in this study is owned by Adenosine Therapeutics. Drs. Timothy Macdonald, Joel Linden, George A. Beller, and David K. Glover hold patent rights to this compound and have an equity position in Adenosine Therapeutics.

	ACKNOWLEDGMENTS

 
The authors thank Susan Ramos for expert technical assistance with the immunohistochemical staining. The authors also thank Dr. Sam Green in the Dept. of Cell Biology at the University of Virginia for providing the anti-P-selectin antibody used in these experiments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. K. Glover, Univ. of Virginia/Cardiology Div., PO Box 800500, UVA Health System, Cobb Hall, Rm. 1010, Hospital Dr., Charlottesville, VA 22908-0500 (E-mail: dglover{at}virginia.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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