Adenosine A₂A agonist improves lung function during ex vivo lung perfusion.

Abstract

Ex vivo lung perfusion (EVLP) is a novel technique than can be used to assess and potentially repair marginal lungs that may otherwise be rejected for transplantation. Adenosine has been shown to protect against pulmonary ischemia-reperfusion (IR) injury through its A(2A) receptor. We hypothesized that combining EVLP with adenosine A(2A) receptor agonist treatment would enhance lung functional quality and increase donor lung use.
Eight bilateral pig lungs were harvested and flushed with cold Perfadex (Vitrolife, Englewood, CO). After 14 hours of storage at 4°C, EVLP was performed for 5 hours on 2 explanted lung groups: (1) control group lungs (n = 4) were perfused with Steen Solution (Vitrolife) and dimethyl sulfoxide and (2) treated group lungs (n = 4) received 10 μM CGS21680, a selective A(2A) receptor agonist, in a Steen solution-primed circuit. Lung histologic features, tissue cytokines, gas analysis, and pulmonary function were compared between groups.
Treated lungs demonstrated significantly less edema as reflected by wet-dry weight ratio (6.6 versus 5.2; p < 0.03) and confirmed by histologic examination. In addition, treated lung demonstrated significantly lower levels of interferon-γ (IFN- γ) (45.1 versus 88.5; p < 0.05). Other measured tissue cytokine levels (interleukin [IL]-1β, IL-6, and IL-8) were lower in the treatment group, but values failed to reach statistical significance. The oxygenation index was improved in the treated group (1.5 versus 2.3; p < 0.01) as was mean airway pressure (10.3 versus 13; p < 0.009).
Combined use of adenosine A(2A) agonist and EVLP significantly attenuates the inflammatory response in acutely injured lungs after IR and enhances pulmonary function. This combination may improve donor lung quality and could increase the donor lung pool for transplantation.

Full text

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Adenosine A2AAgonist Improves Lung Function
During Ex Vivo Lung Perfusion
Abbas Emaminia, MD,* Damien J. LaPar, MD, MS,* Yunge Zhao, MD, PhD,
John F. Steidle, MS, David A. Harris, BS, Victor E. Laubach, PhD, Joel Linden, PhD,
Irving L. Kron, MD, and Christine L. Lau, MD
Department of Thoracic and Cardiovascular Surgery, University of Virginia, Charlottesville, Virginia
Background. Ex vivo lung perfusion (EVLP) is a novel
technique than can be used to assess and potentially
repair marginal lungs that may otherwise be rejected for
transplantation. Adenosine has been shown to protect
against pulmonary ischemia-reperfusion (IR) injury
through its A2Areceptor. We hypothesized that combin-
ing EVLP with adenosine A2Areceptor agonist treatment
would enhance lung functional quality and increase
donor lung use.
Methods. Eight bilateral pig lungs were harvested and
flushed with cold Perfadex (Vitrolife, Englewood, CO).
After 14 hours of storage at 4°C, EVLP was performed for
5 hours on 2 explanted lung groups: (1) control group
lungs (n ? 4) were perfused with Steen Solution (Vit-
rolife) and dimethyl sulfoxide and (2) treated group
lungs (n ? 4) received 10 ?M CGS21680, a selective A2A
receptor agonist, in a Steen solution–primed circuit. Lung
histologic features, tissue cytokines, gas analysis, and
pulmonary function were compared between groups.
Results. Treated lungs demonstrated significantly less
edema as reflected by wet-dry weight ratio (6.6 versus 5.2;
p < 0.03) and confirmed by histologic examination. In
addition, treated lung demonstrated significantly lower
levels of interferon-? (IFN- ?) (45.1 versus 88.5; p < 0.05).
Other measured tissue cytokine levels (interleukin [IL]-
1?, IL-6, and IL-8) were lower in the treatment group, but
values failed to reach statistical significance. The oxygen-
ation index was improved in the treated group (1.5 versus
2.3; p < 0.01) as was mean airway pressure (10.3 versus 13;
p < 0.009).
Conclusions. Combined use of adenosine A2Aagonist
and EVLP significantly attenuates the inflammatory re-
sponse in acutely injured lungs after IR and enhances
pulmonary function. This combination may improve
donor lung quality and could increase the donor lung
pool for transplantation.
(Ann Thorac Surg 2011;92:1840–6)
© 2011 by The Society of Thoracic Surgeons
L
eases. However there are still major obstacles in the way
of successful lung transplantation that include (1) a
shortage of suitable donors, (2) ischemia-reperfusion (IR)
injury, (3) allograft rejection, and (4) the development of
bronchiolitis obliterans and chronic graft rejection [1].
Despite breakthroughs in donor management and or-
gan preservation, fewer than 30% of lungs are considered
transplantable by currently accepted criteria [2], and the
majority of lungs are removed from the donor pool
because of their marginal status. Donor lung injury may
be a result of the mechanism of death, excessive fluid
resuscitation, aspiration, or neurogenic edema [3, 4].
Shortages in suitable lungs for transplantation contribute
to delays in lung transplantation and increase patient
morbidity and mortality. Many lung transplant centers
ung transplantation remains a widely accepted treat-
ment for the majority of end-stage pulmonary dis-
use extended criteria or marginal lungs with acceptable
short-term and long-term results. Using organs from
non–heart beating donors (NHBDs) provides an addi-
tional alternative to increase the number of available
donor lungs. Several groups have successfully trans-
planted lungs from NHBDs [5–7]. However proper as-
sessment of such organs before transplantation is crucial.
Steen and associates [8] addressed this problem by
developing an ex vivo perfusion circuit in which lungs
are perfused and ventilated in physiologic condition.
Although the primary use of ex vivo perfusion is to assess
lung quality, recent studies have demonstrated its use in
the preservation and salvage of previously unacceptable
organs [9].
Adenosine is an endogenous compound released in
response to inflammation, exerting both proinflamma-
tory and antiinflammatory effects through binding to 4
adenosine receptors (A1, A2A, A2B, and A3). Based on our
previous experience examining acute pulmonary injury
and the effects of adenosine, we hypothesized that ex vivo
perfusion of lungs with adenosine A2Aagonist after signif-
icant ischemic injury would attenuate the acute inflamma-
tory response to IR injury and prove beneficial in maintain-
ing pulmonary function and mitigating edema formation.
Accepted for publication June 21, 2011.
*Drs Emaminia and LaPar contributed equally to this article.
Presented at the Poster Session of the Forty-seventh Annual Meeting of
The Society of Thoracic Surgeons, San Diego, CA, Jan 31–Feb 2, 2011.
Address correspondence to Dr Lau, Division of Thoracic and Cardiovas-
cular Surgery, University of Virginia School of Medicine, PO Box 800679,
Charlottesville, VA22903; e-mail: cll2y@virginia.edu.
© 2011 by The Society of Thoracic Surgeons
Published by Elsevier Inc
0003-4975/$36.00
doi:10.1016/j.athoracsur.2011.06.062
GENERAL THORACIC

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Material and Methods
Animal Care
All animals received humane care in accordance with the
Guide for Care and Use of Laboratory animals (pub-
lished by the National Institute of Health, National Insti-
tutes of Health publication no. 86–23, revised 1996). The
study protocol was approved by the University of Vir-
ginia Animal Care and Use Committee before
experimentation.
Study Design
Domestic pigs (n ? 8) with a weight range of 30 to 40 kg
were randomly assigned to 2 groups. The control group
underwent 14 hours of ischemia followed by 5 hours of
EVLP with Steen Solution (Vitrolife, Englewood, CO) and
dimethyl sulfoxide. The treatment group underwent the
same procedure but received CGS-21680 (Sigma-Aldrich,
St. Louis, MO), a selective A2Areceptor agonist. A2A
receptor agonist dosing was determined based on our
previous experiments in cell culture and murine mod-
els of IR injury. A dose of 10 ?M of the compound was
added to the prime at the beginning of perfusion and at
3 hours based on studies of the compound reported
elsewhere [10].
Donor Lung Retrieval and Preservation
The donor lung harvest procedure was identical for both
groups and is described in detail elsewhere [11]. Briefly,
pigs were anesthetized with an intramuscular injection of
ketamine (50 mg/kg) and xylazine (5 mg/kg). Endotra-
cheal intubation was performed and lungs were venti-
lated with an inspired oxygen fraction (FiO2) of 1.0, a tidal
volume of 8.0 mL/kg, a respiratory rate of 18 to 20
breath/min, and positive end-expiratory pressure of 5.0
cm H2O. Anesthesia was maintained with 2.0% isoflurane
throughout the procedure. After administration of 200
U/kg heparin sodium, a median sternotomy was per-
formed and the pericardium and bilateral pleura were
opened. After pulmonary artery (PA) cannulation with a
cardioplegia cannula (Terumo Cardiovascular Systems,
Ann Arbor, MI) prostaglandin E1 (PGE1; 10 mg/kg) was
injected directly into the main PA. Lungs were then
flushed with 1 L of cold Perfadex (Vitrolife) through the
PA cannula while both lungs were cooled using saline
slush. The heart and bilateral lungs were resected en
bloc. Lungs were inflated with 100% oxygen to tidal
volume, and the trachea was clamped. The heart and
lung bloc was dissected ex vivo, and the heart was
excised leaving a generous left atrial (LA) cuff in place. A
funnel-shaped plastic cannula (Vitrolife) was then sewn
to the atrial cuff using polypropylene suture. An addi-
tional 1-L retrograde flush of cold Perfadex was then
performed through the newly sewn cannula. Lungs were
immersed in cold Perfadex and kept at 4°C for 14 hours
before ex vivo perfusion. This ischemic time was chosen
based on previous studies done on EVLP with extended
cold ischemic time [12, 13].
Priming the Ex Vivo Circuit and Preparation of Lungs
for Perfusion
The ex vivo perfusion circuit consisted of a bypass
centrifugal pump (Bio-Medicus, Medtronic, Minneapolis,
MN), cardiopulmonary bypass membrane oxygenator,
and venous reservoir (Sorin Group USA, Arvada, CO), a
heat exchanger (Dideco), and polyethylene tubes. The
circuit was primed with 1 L of Steen Solution (Vitrolife),
sodium heparin 10,000 U (APP Pharmaceutical, Schaum-
burg, IL), cefazolin 500 mg (Hikma Farmaceutica [Portu-
gal SA], Terrugem, Sintra, Portugal) and methylpred-
nisolone 500 mg (Pfizer, New York, NY).
After 14 hours of cold ischemia, the trachea and PA
were cannulated. Specially designed cannulas with
built-in pressure catheters (Vitrolife) were used to mon-
itor PA and LA pressures throughout the experiment. In
addition, 0.5 mL/min of mixed gases (nitrogen 86%,
carbon dioxide 8%, and oxygen 6%) was supplied to the
oxygenator to deoxygenate the perfusate returning from
the lungs to a pco2of 35 to 45 mm Hg.
EVLP
The experimental lungs were transferred to an XVIVO
chamber (Vitrolife) (Fig 1). The LA cannula was con-
nected first and retrograde flow with Steen Solution was
initiated. This step was performed to remove air and
remaining Perfadex from the pulmonary vasculature and
cannulas. Once the flow ascended the PA cannula, the
outflow cannula was connected to the PA cannula and
antegrade flow was initiated. Perfusion began with low
flow (0.1 L/min) at 25°C for 20 minutes. When the
perfusate temperature reached 32°C, ventilation was ini-
tiated (Harvard Apparatus model 607 respiration pump,
Harvard Apparatus, Holliston, MA) with Fio2 of 0.2,
ventilatory rate of 8 breaths/min, tidal volume of 8.0
Fig 1. Lungs are placed in the XVIVO evaluation box and are con-
nected to ventilator and perfusion system. Blood flow is from left
atrium into a reservoir by gravity. It passes a centrifugal pump, is
deoxygenated in an oxygenator, and is pumped back into the lung
through the pulmonary artery. Pulmonary artery and left atrial
pressures are monitored by specially designed cannulas with built-in
pressure lines.
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mL/kg, and a positive end-expiratory pressure of 5.0 cm
H2O. Within the first hour, antegrade PA flow was
gradually increased to a maximum flow of 40% of cardiac
output (calculated as 100 mL/kg of animal weight) or less
if PA pressure was more than 15 mm Hg. Similarly, LA
pressure was maintained at a range of 0 to 5 mm Hg by
adjusting the height of the venous reservoir (Table 1).
Assessment
Pulmonary artery flow, PA pressure (PAP), and LA pres-
sure (LAP) were recorded continuously during EVLP.
Inflow and outflow perfusate samples were collected
hourly to analyze oxygenation and calculate pH. A re-
cruitment maneuver was performed 10 minutes before
each gas analysis to a maximum PA wedge pressure of 20
cm H2O. A small tissue sample was obtained from the left
lower lobe at 3 hours for wet-dry weight ratio assessment
and histologic examination. The oxygenation index was
calculated as (Fio2[%] ? mean airway pressure [mm
Hg]/Po2[mm Hg]) and pulmonary vascular resistance ?
PAP – LAP/PA flow (Wood units). At the end of EVLP,
pieces of tissue from all lobes were collected, weighed,
and desiccated under vacuum at 55°C until a stable dry
weight was achieved. The lung wet-dry weight ratio was
used to assess the degree of pulmonary edema after
EVLP. A tissue sample from the left upper lobe was
excised, fixed in 4% formalin, and stained with hematox-
ylin and eosin for histologic examination. In addition, 100
mL of Steen Solution was replaced at 2- and 4-hour time
points to ensure freshness of perfusate components and
to maintain consistent glucose and bicarbonate levels.
Lung Tissue Collection and Protein Extraction
Lung tissues from left and right lower lobes were col-
lected after EVLP was finished (5 hours). Tissues were
weighed, minced, and homogenized in 10? phosphate
buffered saline (gram-milliliter ? 1:10) with FastPrep
FP120 (Thermo Fisher Scientific, Waltham, MA). Samples
were then centrifuged at 8,000 rpm for 10 minutes at 4°C
and were stored at ?80°C for future enzyme-linked
immunosorbent assay (ELISA) analysis.
ELISA Analysis
Porcine enzyme linked immunosorbant assay (ELISA kits
for interleukin (IL)-1?, IL-6, IL-8, and interferon gamma
(IFN-?) were purchased from RayBiotech, Inc (Norcross,
GA). Cytokines were measured in porcine lung extrac-
tion according to the manufacturer’s instructions. Briefly,
the lung extraction samples (100 ?L/well) were added to
96 well plates, which were precoated with specific anti-
bodies separately and incubated overnight at 4°C. One
hundred microliters of biotin antibodies were added to
each well and incubated 1 hour at room temperature
followed by the addition of 100 ?L Streptavidin solution
(RayBiotech Inc, Norcross, GA) and incubation at room
temperature for 45 minutes. A 100-?L TMB One-Step
Substrate Reagent (RayBiotech Inc) was then added to
each well and incubated 30 minutes at room temperature.
Finally, 50 ?L Stop Solution (RayBiotech Inc) was added
to each well and read at 450 nm immediately. Each
sample was analyzed in triplicate.
Statistical Analysis
Values are expressed as mean ? standard error of the
mean. For comparison of variables between the 2 groups,
a 2-tailed Student t test was used. A p value less than 0.05
was considered statistically significant.
Results
Swine weight and perioperative physiologic parameters
(cardiac output, arterial Pao2,time from median sternot-
omy to cold flush, and cannula sewing time) were not
significantly different between the experimental groups.
All lungs underwent 5 hours of continuous EVLP. There
was no difference between the perfusate pH of control
and treatment groups.
The oxygenation index was significantly improved in
the treated group compared with the control lungs (1.5 ?
0.17 versus 2.3 ? 0.07; p ? 0.01) (Fig 2). Although the
lungs in the treated group had better oxygenation re-
flected as higher pulmonary vein Pao2, this improvement
failed to reach statistical significance.
Significant functional differences existed between ex-
perimental lung groups. After treatment with A2Aago-
nist, mean airway pressures were significantly decreased
compared with those of the control lungs (10.3 ? 0.6
versus 13 ? 0.06; p ? 0.009) (Fig 3). Similarly, expiratory
minute volume was improved in the treated group com-
pared with the control lungs (7.7 ? 0.5 mL versus 7.0 ?
0.8 mL; p ? 0.04). Adenosine A2Atreatment also im-
proved both inspiratory (403 ? 61.5 mL versus 332 ? 52.7
mL; p ? 0.1) and expiratory (428 ? 7.7 mL versus 319 ?
62.3 mL, p ? 0.07) tidal volumes compared with controls;
however these improvements failed to reach statistical
significance. Pulmonary vascular resistance, although
Table 1. Summary of Circuit Characteristics and Lung
Injury After Ex Vivo Lung Perfusion
Variable
Control
Group
(n ? 4)
Treatment
Group
(n ? 4)
p
Value
Pulmonary artery flow
(L/min)
Pulmonary artery
pressure (mm Hg)
Left atrial pressure
(mm Hg)
Pao2/Fio2(mm Hg)
Oxygenation index
PVR (dine.cm/sec5)
Interleukin 1? (pg/mL)
Interleukin-6 (pg/mL)
Interleukin-8 (pg/mL)
Interferon-? (pg/mL)
0.5 ? 0.10.7 ? 0.090.1
15.4 ? 4.414.1 ? 2.90.6
1.5 ? 0.41.2 ? 0.1 0.1
429.5 ? 36.5
1.5 ? 0.17
1228 ? 307
2695 ? 34
6455 ? 152
3029 ? 66
88 ? 12
439.9 ? 55.5
2.3 ? 0.07
1177 ? 337
2307 ? 90
5214 ? 224
2869 ? 37
45 ? 10
0.8
? 0.01
0.4
0.4
0.3
0.2
0.03
Values are presented as mean ? standard deviation.
PVR ? pulmonary vascular resistance.
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lower in the treated group, was not significantly different
compared with the controls (p ? 0.4).
Significant differences in pulmonary edema and pro-
inflammatory cytokine expression were observed among
experimental lungs after EVLP. W/D weight ratio of lung
samples collected at the 5-hour time point was signifi-
cantly lower for the treated group compared with control
lungs (5.2 ? 0.1 versus 6.6 ? 0.3; p ? 0.03) (Fig 4). In
addition to more pronounced intraalveolar fluid accumu-
lation, control lung samples showed thicker interalveolar
septa with more inflammatory cell infiltration into alve-
olar walls by 5 hours compared with compound-exposed
lungs. With respect to cytokine expression, the lung
tissue IFN-? level was significantly lower in the treatment
group than in the control group (45.1 ? 10.0 versus 88.5 ?
12.2 pg/mL; p ? 0.05) (Fig 5). Similarly, tissue levels of
IL-1?, IL-6, and IL-8 were also lower among adenosine
A2A-agonist treated lungs; however these trends failed to
reach statistical significance.
Comment
Herein we have reported the novel use of ex vivo lung
perfusion with the concomitant administration of an
adenosine A2Aagonist to attenuate acute IR injury in
porcine lungs. Specifically, we have demonstrated that
treatment of experimental lungs with adenosine A2A
agonist during EVLP results in an improved oxygenation
index, decreased mean airway pressure, and improved
expiratory minute volume compared with control lungs.
In addition, adenosine A2Aagonist–perfused lungs in-
curred significantly decreased pulmonary edema and
tissue IFN-?, and displayed histologic evidence of re-
duced acute pulmonary injury. These results corroborate
the previously established benefits of adenosine A2A
receptor agonism on the attenuation of acute IR lung
injury and further extend the application of ex vivo lung
Fig 2. Oxygenation index was significantly improved after 5 hours
of ex vivo lung perfusion with Steen Solution and A2Aagonist com-
pared with lungs perfused with Steen Solution only; *p ? 0.01.
(EVLP ? ex vivo lung perfusion; A2AR ? A2Areceptor.)
Fig 3. Lungs after 5 hours of ex vivo lung perfusion (EVLP) with
A2Aagonist revealed significantly lower mean airway pressure com-
pared with control lungs; *p ? 0.009. (A2AR ? A2Areceptor.)
Fig 4. Comparison of wet-dry (W/D) weight ratio between the treat-
ment and control groups. Low edema formation rates were observed
in lungs exposed to the A2Aagonist; *p ? 0.03. (EVLP ? ex vivo
lung perfusion; A2AR ? A2Areceptor.)
Fig 5. Tissue level of interferon gamma (IFN-?) was significantly
lower in treatment lungs after 5 hours of ex vivo lung perfusion
(EVLP) and exposure to adenosine A2Aagonist; *p ? 0.05. (A2AR ?
A2Areceptor.)
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perfusion to the successful reconditioning of explanted
lungs in preparation for transplantation.
Although lung transplantation is the accepted defini-
tive treatment for many end-stage lung conditions, avail-
ability of acceptable donor organs remains a major ob-
stacle. In 2007, only 30% of available lungs from deceased
donors were used for transplantation [2]. EVLP is a
technique that allows visual evaluation of donor lungs,
monitoring of ventilator and perfusion capacities of the
explanted organ, and gas analysis [14]. In addition EVLP
provides an environment to restore and reexpand atelec-
tatic lungs more efficiently, to decrease the presence of
hypoxic vasoconstriction, and to remove small clots from
the pulmonary microvasculature through a retrograde
flush. Thus use of EVLP could translate into fewer
postoperative pulmonary complications in lung trans-
plant patients. In fact EVLP has been used for prolonged
periods to assess, preserve, and treat organs [8, 9, 14].
Steen and colleagues [15] demonstrated that lung isch-
emic time after explantation may be extended up to 20
hours.
The use of treatment modalities in concert with EVLP
has been a recent focus of investigation. Although soli-
tary perfusion of lungs with Steen Solution has been
shown to be superior to cold preservation of the organ
[13], the use of various treatment strategies during EVLP
has recently been explored for both NHBDs and brain-
dead donors with marginal lungs. Inci and colleagues
[16] demonstrated that the addition of a fibrinolytic
agent, urokinase, to the perfusate in a pig EVLP model of
NHBD lung improved graft function by reporting a
reduction in pulmonary vascular resistance and an in-
crease in oxygenation at the end of perfusion. Cypel and
colleagues [13] used an adenoviral vector to transfer IL10,
an antiinflammatory cytokine gene, into airways and
alveoli at the beginning of EVLP. Kakishita and cowork-
ers [17] showed no significant improvement in pulmo-
nary function after neutralization of proinflammatory
cytokines tumor necrosis factor-? and IL-8 from the
perfusate using an adsorbent membrane.
In this study we tested the impact of an adenosine A2A
agonist on the pulmonary function of acutely injured
experimental lungs during normothermic EVLP. Adeno-
sine is a purine nucleoside produced in response to
ischemia that acts through its interaction with 4 different
adenosine receptors (A1, A2A, A2B, and A3) [18]. Adeno-
sine A2Areceptors are highly expressed on bone mar-
row–derived cells, including T lymphocytes, neutrophils,
monocytes, macrophages, platelets, and mast cells [1].
Activation of the A2Areceptor on bone marrow–derived
cells is the primary mechanism for the protective effects
of A2Areceptor agonists in acute pulmonary injury [19].
The cellular responses appear to be mediated mostly
through cyclic adenosine monophosphate [19] and result
in inhibition of oxidative burst in neutrophils [20], re-
duced cytokine release [21], and, inhibition of leukocyte
activation [22]. We previously reported the inhibitory
effects of A2Areceptor agonism on acute lung IR injury in
several animal models [23–25]. A2Aagonists have been
shown to attenuate pulmonary dysfunction, edema, neu-
trophil infiltration, proinflammatory cytokine produc-
tion, and microvascular leak after lung transplantation
[25]. As a result we sought to further investigate the
potential protective effect of an adenosine A2Aagonist
during EVLP to salvage acutely injured lungs. Although
the antiinflammatory role of A2Aagonists after reperfu-
sion has been extensively studied in the past, the recon-
ditioning of injured lungs, as we have described, before
transplantation has not yet been investigated. In our
EVLP model, the addition of a selective adenosine A2A
agonist to the perfusate resulted in significant reduction
of edema and a better oxygenation index. Functionally,
treated lungs showed improved mean airway pressure
and expiratory minute volume. Interestingly we found
that the most optimal recorded gas measurements oc-
curred during the first and second hours of EVLP, an
observation that may be secondary to the compound’s
short half-life (25 to 30 minutes) [10].
Acute lung IR injury is modulated by specific bone
marrow–derived cells and proinflammatory cytokines.
IFN-? has been shown to be involved in migration and
cytokine production of macrophages [26–28]. It has also
been demonstrated to be an important mediator in the
development of inflammatory response in lung injury
[29]. Eppinger and associates [30] showed that vascular
leakage of serum albumin was increased in a rat model of
lung IR injury. They noticed an increased level of cyto-
kines released from macrophages, including IFN-?,
shortly after reperfusion started. Our group and others
have previously shown that alveolar macrophages have a
pivotal role in lung IR injury by releasing proinflamma-
tory cytokines and chemokines, such as IFN-?, in re-
sponse to oxidative stress [31–33]. Because the lung
possesses resident macrophages as a defense against
environmental pathogens [30], it seems likely that these
cells were targets of the antiinflammatory effect of aden-
osine A2Aagonist in our study. One of the pulmonary
protective mechanisms of adenosine A2Aagonist is
through activation of receptors on CD4?, T lymphocytes,
and neutrophils. These cells would then attenuate down-
stream intercellular signaling cascade events involving
other cell types such as alveolar macrophages and epi-
thelial and endothelial cells [34].
We have previously shown that adenosine A2Arecep-
tor activation results in the greatest protection against
lung IR injury when given before ischemia and during
reperfusion [24]. With the experience gained through this
experiment, it can be proposed that treating lungs with
adenosine A2Aagonist ex vivo, in addition to recipient
treatment, would result in even better outcomes. Future
experiments combining the superior impact of adenosine
A2Aon donor lung and recipient are warranted. We are
currently planning a series of experiments using treated
EVLP lungs for subsequent lung transplantation to con-
tinue our assessment of the impact of A2Aadministration
and the benefits of EVLP. These studies will help to
further determine the potential clinical use of A2Arecep-
tor agonists.
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Limitations
Select limitations deserve discussion in this study. In this
experiment, lungs were not transplanted after EVLP was
finished and organs were removed from the circuit. We
are working on further experiments in which transplan-
tation of perfused lungs will be considered. In addition,
the admittedly small sample size in each experimental
group limited the ability to detect statistically significant
differences between observed trends in various measures
of lung injury between control and treatment groups.
Baseline Po2, which is recorded before explantation of
lungs, serves as a valuable measure for comparison with
final oxygen content and was not recorded in this study.
The most significant differences between the control and
treatment groups occurred during the first 2 hours of
EVLP. Although not surprising because adenosine A2A
receptor treatment typically acts very early in the inflam-
matory process after reperfusion, it is unclear whether
prolonged treatment will derive any additional benefits
during the EVLP process. Regarding cytokine expression,
1 potential reason inflammatory tissue cytokines were
not significantly decreased after 5 hours could be a
function of the short half-life of the A2Aagonist itself.
Future studies are planned with slow infusion of the
agent throughout the EVLP to further assess this possi-
bility. Finally, the acellular nature of perfusate in this
experiment likely excluded important sources of inflam-
matory cytokine production.
Conclusions
We conclude that the combined use of adenosine A2A
agonist and EVLP significantly attenuates the inflamma-
tory response in acutely injured lungs after IR to enhance
pulmonary function. This combined treatment modality
offers a novel and clinically translatable approach to
enhance donor lung quality and a potential to increase
the donor lung pool for transplantation.
This study was supported by Award Number T32HL007849
(DJL) from the National Heart, Lung, and Blood Institute. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Heart,
Lung, and Blood Institute or the National Institutes of Health.
Dr Lau is supported by a grant sponsored by the National
Heart, Lung, and Blood Institute (1K08HL094704-01), the
CVRC Partner’s Grant, and was the AATS John W. Kirklin
Research Fellow.
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