Abstract
Objectives
Cardiopulmonary bypass has been shown to exert an inflammatory response within the lung, often resulting in postoperative pulmonary dysfunction. Several studies have shown that adenosine A2A receptor (A2AR) activation attenuates lung ischemia-reperfusion injury, however the effect of A2AR activation on cardiopulmonary bypass-induced lung injury has not been studied. We hypothesized that specific A2AR activation by ATL313 would attenuate inflammatory lung injury following cardiopulmonary bypass.
Methods
Adult male Sprague-Dawley rats were randomly divided into three groups: 1) SHAM group (underwent cannulation+heparinization only); 2) CONTROL group (underwent 90-minutes of normothermic cardiopulmonary bypass with normal whole-blood priming solution; 3) ATL group (underwent 90-minutes of normothermic cardiopulmonary bypass with ATL313 added to the normal priming solution).
Results
There was significantly less pulmonary edema and lung injury in the ATL group compared to the CONTROL group. The ATL group had significant reductions in bronchoalveolar lavage interleukin-1, interleukin-6, interferon-γ and myeloperoxidase levels compared to the CONTROL group. Similarly, lung tissue interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly decreased in the ATL group compared to the CONTROL group. There was no significant difference between the SHAM and ATL groups in the amount of pulmonary edema, lung injury, or levels of pro-inflammatory cytokines.
Conclusions
The addition of a potent A2AR agonist to the normal priming solution prior to the initiation of CPB significantly protects the lung from the inflammatory effects of CPB and reduces the amount of lung injury. A2AR agonists could represent a new therapeutic strategy for reducing the potentially devastating consequences of the inflammatory response associated with CPB.
Ultra-mini Abstract
Pharmacologic activation of the adenosine A2A receptor during cardiopulmonary bypass resulted in substantial decreases in pulmonary pro-inflammatory cytokines as well as clinically significant reductions in both postoperative pulmonary edema and lung injury severity in a rat model of cardiopulmonary bypass.
Since its successful introduction by Dr. Gibbon in 19531, cardiopulmonary bypass (CPB) has seen substantial evolution. Despite this, ample evidence exists regarding the massive inflammatory response that is initiated following procedures that employ CPB2–5. The majority of patients undergoing CPB have sufficient reserve in order to overcome this inflammatory insult without clinically observable sequelae. In a smaller cohort of patients however, this inflammatory response can result in system-wide organ dysfunction leading to respiratory failure, renal insufficiency and failure, coagulopathies, neurologic dysfunction, altered hepatic function, and in an even smaller number of patients, acute respiratory distress syndrome and the systemic inflammatory response syndrome2,3. The inflammatory response that is triggered is thought to occur from a highly sensitive set of interactions between the normal circulation and the “foreign” surface of the CPB circuit, which results in an incremental activation of complement, coagulation, fibrinolytic, and inflammatory responses4. Additionally, some argue that end organ ischemia and the subsequent reperfusion injury that results is another powerful player in this inflammatory response2. The ability to reconcile this pathologic and potentially lethal process relies on the ability to attenuate the unyielding inflammatory response that is characteristic of CPB.
The role of adenosine in limiting the inflammatory response to CPB is a logical continuation of the success that has been described with adenosine agonists and their widespread effects on ischemia-reperfusion injury in multiple different organ systems6–9. The adenosine A2A receptor (A2AR) is one of four G-protein coupled receptors that belong to the adenosine receptor family (A1, A2A, A2B, A3). The A2AR is expressed predominantly on inflammatory cells, including neutrophils, macrophages, mast cells, monocytes, and platelets. Once activated, this G-protein coupled receptor leads to an increase in intracellular cyclic adenosine monophosphate (cAMP) which results in a potent inactivation of inflammatory cells, decreased pro-inflammatory cytokine production and release, suppressed neutrophil recruitment and activation, decreased oxygen free radical production, and possibly down-regulation of cellular immunity through impaired CD4+ T-cell activity10,11.
In this study, we examined the effect of A2AR activation on the inflammatory response within the lung caused by CPB by adding the highly selective A2AR agonist, ATL313 [4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}piperidine-1-carboxylic acid methyl ester], to the standard bypass priming solution prior to the initiation of CPB.
Previous research has demonstrated that specific activation of the A2AR subtype has been shown to be have significant anti-inflammatory effects6,9,23,24,26. Furthermore, our institutional research has shown that the specific activation of the A2AR is mediated by ATL3136. We therefore hypothesized that activation of the A2AR in the standard bypass prime prior to the start of CPB would lead to an attenuation in the pulmonary inflammatory response observed following CPB.
Materials and Methods
Animals and Groups
Adult male, Sprague-Dawley rats (Charles River Labs, Wilmington, MA) (350–450 gm) were used for all studies and received humane care in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication No. 85–23, revised 1996). The Animal Care and Use Committee at the University of Virginia reviewed and approved the protocol for this study before experimentation began.
Rats were randomly assigned to one of three groups (n=5 for each group): SHAM group, CPB with normal whole blood priming solution (CONTROL group), and CPB with normal whole blood priming solution plus ATL313 (ATL group). The sham operation consisted of all venous and arterial cannulations as well as full heparinization without cardiopulmonary bypass.
Surgical Procedure
The rat model of CPB was based on the model for extracorporeal circulation in the rat as developed by Grocott and associates12. Rats were anesthetized with 4% isoflurane. After adequate anesthesia was achieved, rats were intubated by direct laryngoscopy with a 14-gauge intravenous catheter (BD Insyte Autoguard, Becton-Dickinson Infusion Therapy Systems Inc., Sandy, UT) and mechanically ventilated by a small-animal ventilator (TOPO Dual Mode Ventilator, Kent Scientific, Torrington, CT) with an air-oxygen mixture (Fi02 = 0.5). During the cannulations, ventilation was adjusted to maintain a partial pressure of carbon dioxide (PaCO2) between 35–42 mmHg. During the surgical procedure, anesthesia was maintained with 2–2.5% Isoflurane, and adjusted as appropriate to achieve adequate depth of anesthesia. All surgeries were performed with standard sterile techniques. The surgical cannulations and CPB circuit are illustrated in Figure 1. Rectal temperature was monitored and regulated at 37.3–37.6°C using a combination of heat lamps, two forced water-heating blankets, and a forced air-warming blanket as needed. Electrocardiography (ECG) and pulse-oximetery were continuously monitored using a small animal portable monitoring system (SurgiVet V3404 Plus, SurgiVet Inc., Waukesha, WI).
Figure 1.
Schematic diagram of the rat cardiopulmonary bypass circuit and surgical cannulations. Ventilator not shown.
The tail artery was identified through a 1 cm ventral tail incision, encircled with 5-0 silk sutures, and cannulated with a modified 22-guage intravenous catheter (BD Insyte, Becton-Dickinson Infusion Therapy Systems Inc., Sandy, UT) to serve as the arterial inflow cannula. Following this, the femoral artery was identified through a 1 cm right groin incision and encircled with 5-0 silk sutures. A 24-guage intravenous catheter (BD Insyte, Becton-Dickinson Infusion Therapy Systems Inc., Sandy, UT) was then inserted for continuous mean arterial pressure (MAP) monitoring as well as intermittent arterial blood gas analysis (Rapidpoint 405 series, Bayer HealthCare LLC, Tarrytown, NY). Next, through a 2 cm midline cervical incision, the right internal jugular vein was identified and encircled with 5-0 silk sutures. A modified 8 Fr multi-orifice pediatric femoral venous cannula (Biomedicus 96830-008, Medtronic Inc., Minneapolis, MN) was inserted into the vein and gently advanced into the right atrium. The position of the venous cannula was confirmed by previous autopsy studies and allowed for complete drainage of the right atrium, bilateral superior vena cava, as well as the inferior vena cava. Upon insertion of the venous cannula, 200 IU of unfractionated heparin were given directly via the venous cannula, allowing for an activated clotting time ≥450 seconds.
Cardiopulmonary Bypass Circuit and Procedure
The CPB circuit (Figure 1) consisted of a sterile 5 mL venous reservoir (Bubble Trap Compliance Chamber, Radnoti Glass Technology Inc., Monrovia, CA). The venous reservoir was connected to a peristaltic roller-pump (Masterflex®, Cole-Parmer Instrument Company, Chicago, IL) through sterile 1.6 mm internal diameter silicone tubing (Tygon®, Cole-Parmer Instrument Company, Chicago IL) connected in series with a custom flow probe (Transonic Flowprobe, Transonic Systems Inc., Ithaca NY) used to continuously monitor blood flow rates during CPB. Additional sterile 1.6 mm internal diameter silicone tubing was used to connect the flow probe to an externally warmed sterile hollow-fiber membrane oxygenator (MiniModule, Membrana, Charlotte, NC) with an active surface area of 0.18 m2. The oxygenator was inverted to serve an additional role as an inline arterial bubble trap and then connected to additional sterile 1.6 mm internal diameter silicone tubing, surrounded by a jacket warmed with circulating water from a separate heat pump, and then to the previously mentioned arterial inflow cannula. Venous drainage was augmented as needed by either adjusting the placement of the venous cannula or changing the height of the venous reservoir relative to the animal in order to increase or decrease gravity drainage of the right atrium and its associated structures.
The CPB circuit was primed with 45 mL of whole blood obtained from 2 to 3 heparinized (250 IU/kg) donor rats phlebotomized under isoflurane anesthesia via direct cardiac puncture. For the ATL group, ATL313 (Adenosine Therapeutics LLC, Charlottesville, VA) was added directly to the whole blood prime at a dosage based on previous studies from our laboratory6. From a 4.6 µmol/L stock solution of ATL313 in normal saline, 1 mL was added to 45 mL of whole blood and gently mixed prior to pump priming. This resulted in a final dosage concentration of 100 nmol/L. For the CONTROL group, an equivalent volume (1 mL) of vehicle (normal saline) was injected into the 45 mL of whole blood prior to priming the CPB circuit. Following the surgical cannulations, heparinization, and adequate pump priming, the animal was connected to the CPB circuit and extracorporeal circulation was slowly initiated to a final flow rate of 160–165 cc/kg/min, which corresponds to 100% of the normal cardiac output in a rat. Once this flow rate was attained, mechanical ventilation was terminated and CPB was carried out for 90-minutes. During CPB, the gas flow to the oxygenator consisted of oxygen, carbon dioxide, and isoflurane. At the conclusion of the 90-minute period, mechanical ventilation was resumed, and all animals were slowly weaned from CPB without the need for inotropes or vasopressors. Once separated from CPB, the rats were decannulated and remained intubated, anesthetized, and mechanically ventilated for an additional 90-minutes.
Physiologic Data and Specimen Collection
Blood pressure, mean arterial pressure, central venous pressure, heart rate, pulse oximetry, temperature and flow rate were monitored continuously during the bypass period and recorded at baseline, 10, 20, 45, 60, and 90-minutes during the bypass procedure. The same variables (excluding flow rate) were monitored following the cessation of CPB and recorded at 30, 60, and 90-minute intervals. In addition, arterial blood gas analysis was performed at the same pre-specified intervals. At the completion of the 90-minute recovery period, the chest was opened and the rat was phlebotomized by direct cardiac puncture. Plasma was collected by centrifugation at 4°C for 20 minutes, and stored at −70°C until cytokine analysis was performed. The left lung was then isolated and removed for subsequent tissue cytokine and wet-to-dry weight ratio analysis. Next, a tracheostomy was performed followed by bronchoalveolar lavage of the entire right lung. After performing the bronchoalveolar lavage, the right lung was removed and fixed by intra-tracheal instillation of 4% paraformaldehyde at 25 cm H2O pressure.
Lung Wet/Dry Weight Ratio
Lung wet/dry weight ratio was used as a measure of pulmonary edema. Samples of the left lower lobe lung tissue were blotted to remove excess blood and weighed immediately after harvest. These samples were then desiccated under vacuum at 55°C until a stable dry weight was achieved.
Bronchoalveolar Lavage
Bronchoalveolar lavage (BAL) was performed on all lungs prior to en-bloc removal and permanent fixation. The right lung was isolated and lavaged 3 times with separate 10 mL aliquots of normal saline. The BAL fluid was centrifuged at 1500g for 10 minutes at 4°C. The supernatant was then snap-frozen for subsequent analysis.
Myeloperoxidase content
Myeloperoxidase (MPO) content in BAL fluid was measured using an MPO enzyme-linked immunosorbent assay (ELISA) kit (Cell Sciences, Canton, MA) and performed according to the manufacturer instructions. MPO content was used as a broad measure of neutrophil activation and sequestration.
Cytokine analysis
The protein levels of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and interferon-γ (IFN-γ) in lung tissue, plasma, and BAL fluid were examined with a Bio-Plex™ multiplex cytokine ELISA system (Bio-Rad Laboratories Inc., Hercules, CA) and performed according to manufacturer instructions. Samples were run in triplicate.
Lung Injury Severity Score
A pathologist, blinded to treatment group, graded each lung sample after appropriate tissue processing and staining (hematoxylin and eosin). Each sample was graded on the presence of the number of macrophages, amount of interstitial infiltrate, and presence of alveolar edema. Each of these three categories was given a score of 0 to 3, resulting in a possible score ranging from 0 for uninjured, normal lungs to 9 for the most severely injured lungs.
Statistics
Values are expressed as the mean ± standard error. All statistical analysis was performed by an independent statistician. Analysis of variance (ANOVA) and the post hoc Bonferroni test were used to determine whether significant differences existed between groups. We considered a p<0.05 to be statistically significant.
Results
Physiologic and arterial blood gas measurements
Physiologic data are detailed in Table 1. The data were similar for all three groups at both the baseline and the post-bypass time points. The addition of ATL313 to the bypass prime did not change any of the physiologic parameters measured when comparing the CONTROL group to the ATL group. Among the three groups, there were no significant differences noted in temperature, arterial oxygen saturation, or hematocrit at any of the time points captured. In the CONTROL and ATL groups, mean arterial pressure decreased significantly while on CPB compared to SHAM (p<0.05 for all time-points). There were no differences noted between the CONTROL group and the ATL group in terms of CPB flow rates, with each at or slightly above the projected goal flow rate of 160–165 cc/kg/min.
Table 1.
Physiologic data
| CPB | Post-CPB | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Baseline | 10 min | 20 min | 45 min | 60 min | 90 min | 30 min | 60 min | 90 min | ||
| MAP (mmHg) | ||||||||||
| Sham | 92 ± 3.6 | 90 ± 2.0a | 92 ± 1.6a | 89 ± 1.6a | 84 ± 1.5a | 85 ± 0.9a | 89 ± 3.5 | 91 ± 3.0 | 90 ± 1.1 | |
| Control | 89 ± 8.9 | 75 ± 5.6 | 73 ± 4.5 | 72 ± 4.4 | 75 ± 4.1 | 71 ± 6.3 | 80 ± 4.5 | 85 ± 4.1 | 88 ± 2.8 | |
| ATL | 88 ± 3.8 | 76 ± 3.9 | 72 ± 5.1 | 76 ± 3.8 | 75 ± 4.1 | 72 ± 4.1 | 82 ± 3.0 | 86 ± 4.6 | 86 ± 4.4 | |
| CPB Flow (ml/kg/min) | ||||||||||
| Sham | - | - | - | - | - | - | - | - | - | |
| Control | - | 161 ± 0.3 | 164 ± 1.1 | 160 ± 0.7 | 165 ± 1.2 | 162 ± 2.3 | - | - | - | |
| ATL | - | 162 ± 0.1 | 161 ± 0.9 | 163 ± 1.2 | 161 ± 1.7 | 164 ± 1.7 | - | - | - | |
| HCT (%) | ||||||||||
| Sham | 37 ± 0.5 | - | 36 ± 0.7 | - | 36 ± 1.1 | - | - | 36 ± 0.4 | - | |
| Control | 39 ± 1.4 | 37 ± 0.6 | 37 ± 0.6 | 38 ± 0.3 | 38 ± 0.6 | 37 ± 1.6 | 38 ± 0.9 | 37 ± 1.3 | 38 ± 1.1 | |
| ATL | 37 ± 0.6 | 36 ± 0.5 | 37 ± 0.9 | 37 ± 0.6 | 38 ± 0.7 | 38 ± 0.2 | 39 ± 0.7 | 39 ± 0.6 | 39 ± 0.5 | |
| Temp (°C) | ||||||||||
| Sham | 37.7 ± .04 | 37.6 ± .07 | 37.5 ± .11 | 37.4 ± .05 | 37.5 ± .03 | 37.4 ± .06 | 37.6 ± .06 | 37.5 ± .07 | 37.6 ± .06 | |
| Control | 37.5 ± .12 | 36.4 ± .09 | 36.9 ± .07 | 37.4 ± .15 | 37.4 ± .07 | 37.4 ± .04 | 37.4 ± .09 | 37.5 ± .08 | 37.5 ± .11 | |
| ATL | 37.4 ± .16 | 36.7 ± .12 | 37.0 ± .08 | 37.4 ± .04 | 37.6 ± .06 | 37.5 ± .08 | 37.5 ± .08 | 37.6 ± .06 | 37.4 ± .07 | |
| SaO2 (%) | ||||||||||
| Sham | 98 ± 0.5 | 99 ± 1.1 | 99 ± 1.0 | 99 ± 0.3 | 98 ± 0.6 | 98 ± 0.5 | 97 ± 0.4 | 99 ± 0.7 | 98 ± 0.2 | |
| Control | 98 ± 0.4 | 99 ± 0.7 | 99 ± 0.7 | 98 ± 0.5 | 98 ± 0.6 | 99 ± 0.7 | 98 ± 0.6 | 97 ± 0.9 | 97 ± 0.2 | |
| ATL | 99 ± 0.2 | 98 ± 0.6 | 98 ± 0.9 | 99 ± 0.7 | 99 ± 0.7 | 98 ± 0.7 | 99 ± 0.3 | 98 ± 0.3 | 98 ± 0.5 | |
Values represent mean ± standard error
Sham (n=5), Control (n=5), ATL (n=5)
MAP = Mean arterial pressure; CPB = Cardiopulmonary bypass; HCT = Hematocrit; Temp = Temperature; SaO2 = Arterial oxygen saturation
p<0.05 versus Control & ATL
Regarding arterial blood gas analysis (Table 2), there were no differences noted between the CONTROL group and the ATL group for pH, PaO2, PaCO2, or HCO3 at any point during the experiment. When comparing the SHAM group to the CONTROL and ATL groups, the SHAM group was noted to have a higher pH and a slightly lower PaO2 at the 20-minute and 60-minute time points (p<0.001). Additionally, the SHAM group was noted to have a slightly lower baseline PaCO2 and HCO3 when compared to the CONTROL and ATL groups (p<0.05).
Table 2.
Arterial blood gas analysis
| CPB | Post-CPB | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Baseline | 10 min | 20 min | 45 min | 60 min | 90 min | 30 min | 60 min | 90 min | ||
| pH | ||||||||||
| Sham | 7.42 ± .01 | - | 7.42 ± .01a | - | 7.45 ± .01a | - | - | 7.45 ± .01a | - | |
| Control | 7.42 ± .02 | 7.39 ± .02 | 7.37 ± .01 | 7.39 ± .02 | 7.39 ± .02 | 7.38 ± .01 | 7.41 ± .01 | 7.38 ± .01 | 7.39 ± .01 | |
| ATL | 7.39 ± .02 | 7.36 ± .03 | 7.36 ± .04 | 7.37 ± .03 | 7.38 ± .03 | 7.39 ± .03 | 7.38 ± .02 | 7.39 ± .01 | 7.38 ± .01 | |
| PaO2 (mmHg) | ||||||||||
| Sham | 283 ± 11 | - | 266 ± 30a | - | 222 ± 13a | - | - | 298 ± 33 | - | |
| Control | 289 ± 20 | 371 ± 53 | 379 ± 46 | 392 ± 53 | 392 ± 49 | 325 ± 58 | 297 ± 31 | 259 ± 24 | 244 ± 15 | |
| ATL | 298 ± 34 | 383 ± 38 | 400 ± 30 | 369 ± 41 | 377 ± 46 | 381 ± 19 | 312 ± 30 | 251 ± 11 | 293 ± 12 | |
| PaCO2 (mmHg) | ||||||||||
| Sham | 36 ± 3b | - | 37 ± 2 | - | 38 ± 3 | - | - | 38 ± 2 | - | |
| Control | 41 ± 4 | 39 ± 1 | 40 ± 1 | 38 ± 2 | 37 ± 3 | 41 ± 2 | 41 ± 2 | 39 ± 2 | 39 ± .4 | |
| ATL | 42 ± 2 | 38 ± 2 | 37 ± 3 | 40 ± 2 | 39 ± 1 | 43 ± 1 | 42 ± 1 | 42 ± 1 | 39 ± 1 | |
| HCO3 (mEq) | ||||||||||
| Sham | 24.4 ± 1.5b | - | 23.9 ± .75 | - | 23.8 ± .63 | - | - | 25.1 ± .94 | - | |
| Control | 26.5 ± 1.4 | 24.9 ± 1.4 | 23.1 ± 1.2 | 23.2 ± 1.2 | 22.9 ± 1.1 | 23.1 ± 1.1 | 23.6 ± 1.7 | 24.6 ± 2.5 | 23.9 ± 1.9 | |
| ATL | 26.1 ± .46 | 25.1 ± .37 | 24.2 ± .43 | 24.1 ± .75 | 23.5 ± .81 | 23.7 ± .87 | 23.8 ± .83 | 24.3 ± .61 | 24.1 ± .44 | |
Values represent mean ± standard error
Sham (n=5), Control (n=5), ATL (n=5)
PaO2 = Partial pressure of oxygen; PaCO2 = Partial pressure of carbon dioxide; HCO3 = bicarbonate
p<0.001 versus Control & ATL
p<0.05 versus Control & ATL
Cytokine analysis
ELISA was used to determine whether the addition of ATL313 had any effect on the quantity of pro-inflammatory cytokines in the lung tissue, BAL fluid, or plasma (Table 3). Within lung tissue, CONTROL animals were found to have marked elevations in IL-1, IL-6, TNF-α, and IFN-γ following the 90-minute recovery period compared to SHAM. In the ATL group, tissue expression of IL-6, TNF-α, and IFN-γ were significantly attenuated. Similar results were observed within the BAL fluid. The CONTROL group was found to have significant elevations in IL-1, IL-6, and IFN-γ compared to SHAM. The addition of ATL313 to the bypass prime (ATL group) significantly reduced the expression of IL-1, IL-6, and IFN-γ back to SHAM levels. There were no differences noted in the levels of TNF-α within the BAL fluid for the three groups. The levels of IL-1, IL-6, TNF-α, and IFN-γ in the plasma were comparatively low and found to be similar between the three groups.
Table 3.
Cytokine analysis
| Lung tissue (pg/mL) | ||||
|---|---|---|---|---|
| IL-1 | IL-6 | TNF-α | IFN-γ | |
| Sham | 10865±1086 | 403±88 | 811±90 | 1870±9 |
| Control | 22839±536a | 7402±371b | 2002±148b | 2530±9c |
| ATL | 21033±694a | 2136±247 | 765±129 | 1770±20 |
| Bronchoalveolar lavage (pg/mL) | ||||
| IL-1 | IL-6 | TNF-α | IFN-γ | |
| Sham | 382±45 | 126±41 | 311±24 | 75±17 |
| Control | 753±45b | 389±70b | 330±10 | 161±17b |
| ATL | 434±45 | 178±12 | 285±43 | 52±7 |
| Plasma (pg/mL) | ||||
| IL-1 | IL-6 | TNF-α | IFN-γ | |
| Sham | 243 ± 13 | 91 ± 5 | 193 ± 14 | 92 ± 10d |
| Control | 249 ± 16 | 102 ± 9 | 169 ± 4 | 124 ± 4 |
| ATL | 225 ± 6 | 89 ± 3 | 165 ± 8 | 124 ± 8 |
Values represent mean ± standard error
Sham (n=5), Control (n=5), ATL (n=5)
p<0.001 versus Sham
p<0.001 versus Sham & ATL
p<0.05 versus Sham & ATL
p<0.05 versus Control & ATL
MPO levels
MPO levels within the BAL fluid were used as an indicator of neutrophil activation and sequestration into alveolar airspace (Figure 2). The CONTROL group was found to have significant elevations in MPO level relative to SHAM. The addition of ATL313 to the standard bypass prime (ATL group) led to a 59% reduction in MPO activity compared to the CONTROL group (p<0.05). There were no significant differences in MPO activity observed between the ATL group and the SHAM group.
Figure 2.
Bronchoalveolar lavage MPO.
Sham (n=5), Control (n=5), ATL (n=5)
*p <0.05 versus Sham & ATL
Wet/Dry weight ratio analysis
Wet-to-dry weight ratios were analyzed as an indicator of pulmonary edema (Figure 3). The CONTROL group was found to have a significantly higher wet-to-dry weight ratio compared to SHAM. The addition of ATL313 to the standard bypass prime (ATL group) resulted in a 63% reduction in wet/dry weight ratio compared to the CONTROL group (p<0.001). There were no significant differences in the wet/dry ratios observed between the ATL group and the SHAM group.
Figure 3.
Wet/dry analysis.
Sham (n=5), Control (n=5), ATL (n=5)
*p<0.001 versus Sham & ATL
Lung Injury Severity
The CONTROL group had considerably more lung injury relative to SHAM. The addition of ATL313 to the bypass prime resulted in significant improvements in lung histology (Figure 4) and lung injury severity score (Table 4) compared to the CONTROL group (p<0.001 for overall lung injury severity score). Lung histology and lung injury severity were similar between the ATL group and the SHAM group.
Figure 4.
Representative hematoxylin-eosin sections of lung tissue. (A) SHAM group, details normal appearing pneumocytes apposed to capillaries without evidence of inflammatory infiltrate, alveolar macrophages, or edema. (B) CONTROL group, shows substantially widened alveolar septa, presence of multiple alveolar macrophages and significant edema. (C) ATL group, alveolar septa are minimally widened by interstitial infiltrate, very few alveolar macrophages are observed, and there is sparse edema.
Table 4.
Lung injury severity scoring
| Group | Interstitial Infiltrate | Macrophages | Alveolar edema | Total Score |
|---|---|---|---|---|
| Sham | 0.2 ± 0.17 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.2 ± 0.17 |
| Control | 0.6 ± 0.21a | 1.6 ± 0.43a | 2.1 ± 0.21a | 4.3 ± 0.41b |
| ATL | 0.3 ± 0.21 | 0.4 ± 0.21 | 0.1 ± 0.14 | 0.8 ± 0.17 |
Values represent mean scores ± standard error
Sham (n=5), Control (n=5), ATL (n=5)
p<0.05 versus Sham & ATL
p<0.001 versus Sham & ATL
Discussion
This study demonstrates that A2AR activation during CPB results in a significant reduction in the pulmonary inflammatory response observed following CPB in a rat model. The addition of ATL313 to the standard bypass prime, prior to the initiation of CPB, resulted in significantly decreased levels of the potent pro-inflammatory cytokines, IL-1, IL-6, TNF-α, and IFN-γ in BAL fluid as well as lung tissue and resulted in decreased neutrophil sequestration and activation (decreased BAL MPO level), decreased pulmonary edema, less lung injury, and preserved lung histology. Presumably these improvements in quantitative inflammatory markers may translate into improved physiology following CPB, although the true effects on lung physiology were not evaluated in this study.
Cardiac surgery using CPB is a well-known trigger of a substantial inflammatory response in both humans following routine CPB and in animal models of CPB2,4,13–16. This inflammatory response can in-turn lead to dysfunction in several organ systems including the cardiovascular, pulmonary, renal, hepatic, gastrointestinal, hematopoetic, and central nervous systems2. This dysfunction can range in severity from mild organ dysfunction to severe life threatening multiorgan failure. Several mechanisms have been shown to contribute to the inflammatory response during CPB including direct surgical trauma, ischemia-reperfusion injury, exposure of blood to the foreign surfaces of the CPB circuit, direct release of endotoxin, and changes in body temperature2,4. We hypothesize that two important mechanisms play a role in the inflammatory response within the lung following CPB in our model: exposure of blood to the foreign CPB circuit and pulmonary ischemia-reperfusion injury.
An important source of inflammatory cell activation in our model is from the interaction of the blood with foreign surfaces of the CPB circuit. Despite improvements in the composition of the internal lining of the circuit, the addition of heparin coated circuits, leukocyte depletion filters, and ultrafiltration techniques, ample evidence exists describing the persistent activation of complement, coagulation, fibrinolytic, and inflammatory responses during CPB2,4. Strong evidence suggests that the end result of these processes is the sequestration and activation of macrophages, eosinophils, neutrophils, mast cells, platelets, endothelial cells and T-lymphocytes.
Another important source of inflammatory activation during CPB may be the result of lung ischemia-reperfusion injury (LIRI). Because perfusion of the lungs during CPB is limited predominantly to flow received from the bronchial arteries, ischemic injury to the lungs during CPB is a likely consequence, despite seemingly adequate systemic perfusion by the CPB pump. Several studies have demonstrated this in various models of CPB. Using a pig model of CPB, Schlensak and collegues17 were able to demonstrate a significant decline in bronchial artery blood flow with the onset of CPB, which led to lung inflammatory activation and subsequent injury. They were able to eloquently show that lung injury and inflammation were markedly reduced with controlled pulmonary artery perfusion during CPB. Similar results have been described by others, in both humans, as well as animal models of CPB18,19. Although we did not directly measure the influence of bronchial artery blood flow in the present study, our results indicate a robust pulmonary inflammatory response following the bypass period with significant elevations in IL-1, IL-6, TNF-α, IFN-γ, MPO, pulmonary edema, and lung parenchymal injury. There was a relative lack of inflammatory activation, as measured by a lack of induction of pro-inflammatory cytokines, in the plasma, which is in contrast to results of other investigators. One possible explanation is that our post-bypass recovery time (90-minutes) was not long enough to demonstrate the propagation of systemic inflammation within the plasma.
Adenosine is a primitive signaling molecule that serves to modulate several physiological responses in the vast majority of mammalian tissues20. More specifically, it has significant anti-inflammatory properties and has been shown to exert a protective role against the development of ischemia induced cell injury6–9, 20–22. Specific activation of the A2AR subtype has been shown to be protective against the development of LIRI6,9,23. Previous institutional research has indicated that the activation of the A2AR is mediated specifically by ATL3136. Whereas the global understanding of A2AR agonists and their influences on lung ischemia-reperfusion injury are largely known, the exact mechanisms by which A2AR activation attenuates the inflammatory response induced by CPB has not been well described. Presumably, the action of ATL313 in limiting the inflammatory response following CPB is similar to the mechanism by which A2AR agonism attenuates ischemia-reperfusion injury in other organ systems. There is extensive, well supported evidence for the role of inflammatory cells in the propagation of the CPB-induced inflammation. A2AR’s have been shown to be present on nearly all inflammatory cells, including macrophages, eosinophils, neutrophils, mast cells, platelets, endothelial cells and T-lymphocytes, and subsequent activation of the A2AR has been shown to be almost uniformly inhibitory in these cell lines20,24. Furthermore, A2AR agonists have been shown to decrease the expression of several adhesion molecules including intercellular adhesion molecule-1, P-selectin, and vascular cell adhesion molecule-1 in myocardial and renal IR models19,21. Many of these same adhesion molecules have been shown to have important effects in the propagation of CPB induced inflammation as well25. Therefore, the observed anti-inflammatory activity of ATL313 in our CPB model could involve two important mechanisms. First, ATL313 could attenuate the inflammatory response generated by the foreign surfaces of the CPB circuit itself, and second, it could reduce the inflammatory response associated with the relative pulmonary ischemia that occurs during CPB.
In conclusion, this report demonstrates that the addition of a potent A2AR agonist to the normal priming solution prior to the initiation of CPB significantly protects the lung from the inflammatory effects of CPB and reduces the amount of lung injury. The ability to adequately protect the lung and other organs from ischemia-reperfusion injury as well as attenuate the activation of an inflammatory response by the CPB circuit itself suggest that A2AR agonists could represent a new therapeutic strategy for reducing the potentially devastating consequences of the inflammatory response associated with CPB.
Acknowledgments
Funding Sources: Cardiovascular Surgery Training Grant (NIH 2 T32 HL007849)
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Disclosures: Dr. Kron is a shareholder in Adenosine Therapeutics, LLC, the corporation that provided the adenosine A2A receptor agonist ATL313.
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