Elevated plasma angiopoietin-2 levels and primary graft dysfunction after lung transplantation.
ABSTRACT Primary graft dysfunction (PGD) is a significant contributor to early morbidity and mortality after lung transplantation. Increased vascular permeability in the allograft has been identified as a possible mechanism leading to PGD. Angiopoietin-2 serves as a partial antagonist to the Tie-2 receptor and induces increased endothelial permeability. We hypothesized that elevated Ang2 levels would be associated with development of PGD.
We performed a case-control study, nested within the multi-center Lung Transplant Outcomes Group cohort. Plasma angiopoietin-2 levels were measured pre-transplant and 6 and 24 hours post-reperfusion. The primary outcome was development of grade 3 PGD in the first 72 hours. The association of angiopoietin-2 plasma levels and PGD was evaluated using generalized estimating equations (GEE).
There were 40 PGD subjects and 79 non-PGD subjects included for analysis. Twenty-four PGD subjects (40%) and 47 non-PGD subjects (59%) received a transplant for the diagnosis of idiopathic pulmonary fibrosis (IPF). Among all subjects, GEE modeling identified a significant change in angiopoietin-2 level over time in cases compared to controls (p = 0.03). The association between change in angiopoietin-2 level over the perioperative time period was most significant in patients with a pre-operative diagnosis of IPF (p = 0.02); there was no statistically significant correlation between angiopoietin-2 plasma levels and the development of PGD in the subset of patients transplanted for chronic obstructive pulmonary disease (COPD) (p = 0.9).
Angiopoietin-2 levels were significantly associated with the development of PGD after lung transplantation. Further studies examining the regulation of endothelial cell permeability in the pathogenesis of PGD are indicated.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Tight regulation of vascular permeability is necessary for normal development and deregulated vascular barrier function contributes to the pathogenesis of various diseases, including acute respiratory distress syndrome, cancer and inflammation. The angiopoietin (Ang)-Tie2 pathway is known to control vascular permeability. However, the mechanism by which the expression of Tie2 is regulated to control vascular permeability has not been fully elucidated. Here we show that transcription factor Twist1 modulates pulmonary vascular leakage by altering the expression of Tie2 in a context-dependent way. Twist1 knockdown in cultured human lung microvascular endothelial cells decreases Tie2 expression and phosphorylation and increases RhoA activity, which disrupts cell-cell junctional integrity and increases vascular permeability in vitro. In physiological conditions, where Ang1 is dominant, pulmonary vascular permeability is elevated in the Tie2-specific Twist1 knockout mice. However, depletion of Twist1 and resultant suppression of Tie2 expression prevent increase in vascular permeability in an endotoxin-induced lung injury model, where the balance of Angs shifts toward Ang2. These results suggest that Twist1-Tie2-Angs signaling is important for controlling vascular permeability and modulation of this mechanism may lead to the development of new therapeutic approaches for pulmonary edema and other diseases caused by abnormal vascular permeability.PLoS ONE 09/2013; 8(9):e73407. · 3.53 Impact Factor
- Annals of the American Thoracic Society. 05/2014; 11(4):598-9.
- [Show abstract] [Hide abstract]
ABSTRACT: Objectives: Angiopoietin-1 and angiopoietin-2 are important factors in regulating endothelial vascular permeability. This study evaluated perioperative changes in serum levels of angiopoietin-1 and -2 in patients undergoing cardiac surgery. Design: Measurement of serum levels of angiopoietin-1 and angiopoietin-2 in samples collected during a previously conducted prospective multicenter observational study. Setting: Three university hospitals. Participants: 84 adult patients undergoing cardiac surgery. Intervention: Serum levels of angiopoietins were measured at baseline, immediately after surgery (post-op), and the day after surgery (POD-1). Measurements and Main Results: Serum levels of angiopoietin-2 were elevated by POD-1 (median 3.3 ng/mL, interquartile range [IQR] 2.5–4.6 ng/mL) compared with baseline (median 1.6 ng/mL, IQR 1.3–2.1 ng/mL, p < 0.0001), and angiopoietin-1 levels were decreased immediately after surgery (baseline median 23.2 ng/mL, IQR 10.2–32.8 ng/mL; post-op median 8.0 ng/mL, IQR 1.5–13.2 ng/mL, p < 0.0001). Angiopoietin-2 levels on POD-1 in patients undergoing off-pump coronary artery bypass grafting were significantly lower than those in patients undergoing aortic surgery (p = 0.0009) and valve surgery (p = 0.008). Angiopoietin-2 levels on POD-1 had a predictive performance of area under the curve (AUC) of the receiver operating characteristic curve 0.74 for mechanical ventilation > 3 days. Angiopoietin-1 levels and the angiopoietin-2/angiopoietin-1 ratio showed lower predictive performance (AUC values 0.58 and 0.68, respectively). Conclusions: Angiopoietin-2 serum levels were elevated after cardiac surgery. Elevated angiopoietin-2 had a good predictive performance for respiratory failure after cardiac surgery, perhaps reflecting the severity of lung dysfunction related to postoperative increases in vascular permeability.Journal of Cardiothoracic and Vascular Anesthesia 03/2014; · 1.48 Impact Factor
Elevated Plasma Angiopoietin-2 Levels and Primary Graft
Dysfunction after Lung Transplantation
Joshua M. Diamond1*., Mary K. Porteous2., Edward Cantu3, Nuala J. Meyer1, Rupal J. Shah1,
David J. Lederer4, Steven M. Kawut1,5,6, James Lee1, Scarlett L. Bellamy1, Scott M. Palmer7,
Vibha N. Lama8, Sangeeta M. Bhorade9, Maria Crespo10, Ejigayehu Demissie1,5, Keith Wille11,
Jonathan Orens12, Pali D. Shah12, Ann Weinacker13, David Weill13, Selim Arcasoy4, David S. Wilkes14,
Lorraine B. Ware15", Jason D. Christie1,5"for the Lung Transplant Outcomes Group
1Pulmonary, Allergy, and Critical Care Division, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America,
2Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 3Division of Cardiovascular Surgery,
University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 4Division of Pulmonary, Allergy, and Critical Care Medicine, Columbia
University College of Physicians and Surgeons, New York, New York, United States of America, 5Center for Clinical Epidemiology and Biostatistics, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 6Penn Cardiovascular Institute, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania, United States of America, 7Division of Pulmonary, Allergy, and Critical Care Medicine, Duke University, Raleigh-Durham, North Carolina, United
States of America, 8Division of Pulmonary, Allergy, and Critical Care Medicine, University of Michigan, Ann Arbor, Michigan, United States of America, 9Division of
Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, United States of America, 10Division of Pulmonary, Allergy, and Critical Care, University of
Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 11Division of Pulmonary and Critical Care Medicine, University of Alabama at Birmingham, Birmingham,
Alabama, United States of America, 12Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Johns Hopkins University Hospital, Baltimore,
Maryland, United States of America, 13Division of Pulmonary and Critical Care Medicine, Stanford University, Palo Alto, California, United States of America, 14Division of
Pulmonary, Allergy, Critical Care, and Occupational Medicine, Indiana University School of Medicine and Indiana University Health Lung Transplant Program, Indianapolis,
Indiana, United States of America, 15Departments of Medicine and Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee, United States
Introduction: Primary graft dysfunction (PGD) is a significant contributor to early morbidity and mortality after lung
transplantation. Increased vascular permeability in the allograft has been identified as a possible mechanism leading to
PGD. Angiopoietin-2 serves as a partial antagonist to the Tie-2 receptor and induces increased endothelial permeability. We
hypothesized that elevated Ang2 levels would be associated with development of PGD.
Methods: We performed a case-control study, nested within the multi-center Lung Transplant Outcomes Group cohort.
Plasma angiopoietin-2 levels were measured pre-transplant and 6 and 24 hours post-reperfusion. The primary outcome was
development of grade 3 PGD in the first 72 hours. The association of angiopoietin-2 plasma levels and PGD was evaluated
using generalized estimating equations (GEE).
Results: There were 40 PGD subjects and 79 non-PGD subjects included for analysis. Twenty-four PGD subjects (40%) and 47
non-PGD subjects (59%) received a transplant for the diagnosis of idiopathic pulmonary fibrosis (IPF). Among all subjects,
GEE modeling identified a significant change in angiopoietin-2 level over time in cases compared to controls (p=0.03). The
association between change in angiopoietin-2 level over the perioperative time period was most significant in patients with
a pre-operative diagnosis of IPF (p=0.02); there was no statistically significant correlation between angiopoietin-2 plasma
levels and the development of PGD in the subset of patients transplanted for chronic obstructive pulmonary disease (COPD)
Conclusions: Angiopoietin-2 levels were significantly associated with the development of PGD after lung transplantation.
Further studies examining the regulation of endothelial cell permeability in the pathogenesis of PGD are indicated.
Citation: Diamond JM, Porteous MK, Cantu E, Meyer NJ, Shah RJ, et al. (2012) Elevated Plasma Angiopoietin-2 Levels and Primary Graft Dysfunction after Lung
Transplantation. PLoS ONE 7(12): e51932. doi:10.1371/journal.pone.0051932
Editor: Samir M. Parikh, Beth Israel Deaconess Medical Center, United States of America
Received July 16, 2012; Accepted November 14, 2012; Published December 19, 2012
Copyright: ? 2012 Diamond et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by National Institutes of Health grants R01 HL087115, R01 HL081619, K24 HL103844, and K12 HL090021. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
" These authors a.
PLOS ONE | www.plosone.org1 December 2012 | Volume 7 | Issue 12 | e51932
re equal senior authors
Primary graft dysfunction (PGD), a form of acute lung injury,
represents a major cause of early morbidity and mortality after
lung transplantation. Thought to be the result of ischemia-
reperfusion injury (IRI), PGD affects between 10–30% of lung
transplant recipients [1–8]. IRI-induced PGD manifests with
epithelial cell injury, impairment of fibrinolytic and coagulation
cascades, cytokine and chemokine dysregulation, and alteration of
vascular permeability .
Altered pulmonary vascular permeability is the result of
a complex interplay between various regulatory cytokines and
growth factors. Previous studies have focused on the relationship
between PGD and cytokines known to be involved in regulating
vascular permeability. Higher pre-transplant levels of plasma
VEGF predicted the development of severe PGD . Elevated
gene expression and protein production of endothelin-1, a medi-
ator of vascular permeability, were associated with an increased
incidence of PGD [11–12]. The detection of these markers of
activated endothelium in the setting of PGD suggests that
pulmonary vascular disruption may be a critical factor in the
development of the syndrome.
The angiopoietins are a group of vascular growth factors
involved in angiogenesis, endothelial cell permeability, and
regulation of inflammatory cascades . Angiopoietin 1 (Ang1)
serves as a constitutive Tie-2 agonist promoting vessel stability
and preventing vascular leak. In contrast, Ang2 is abundant in
the resting state, but when released by activated endothelium,
endothelial responses to inflammatory stimuli, including tumor
necrosis factor-alpha . Ang2 production is induced by
VEGF, hypoxia, and hyperoxia and competes with Ang1,
functioning as a Tie-2 receptor antagonist in most contexts .
Several studies demonstrate that patients with acute lung injury
(ALI) and acute respiratory distress syndrome (ARDS) have
higher levels of both plasma and bronchoalveolar lavage Ang2
and that elevated plasma Ang2 levels are associated with
increased mortality [15–21].
Given the strong association of Ang2 with all-cause ARDS, we
hypothesized that lung transplant recipients who developed PGD
would have higher post-transplant plasma Ang2 levels compared
to those without PGD.
Materials and Methods
Approval from the Institutional Review Board of each site
(University of Pennsylvania, Columbia University, University of
Alabama-Birmingham, Johns Hopkins University, Duke Univer-
sity, Indiana University, University of Michigan, University of
Pittsburgh, University of Chicago, Vanderbilt University, and
Stanford University) and informed consent from each subject were
obtained. Consent from all participants was written and the
consent form utilized for enrollment was approved by each of the
participating center’s Institutional Review Boards listed above. A
signed consent form was a requirement for enrollment in the
cohort and inclusion in this study. None of the research performed
as part of this study was performed outside of the United States.
A multicenter, nested case control study was performed on
study subjects selected from eight of the eleven participating
centers within the ongoing Lung Transplant Outcomes group
(LTOG) cohort. Subjects were eligible for inclusion if they
underwent lung transplantation between July 2002 and September
2009 for an indication of either IPF or COPD.
Cases were defined by development of any grade 3 PGD within
72 hours of allograft reperfusion. Controls were defined as subjects
who did not meet the criteria specified for cases. Cases and
controls were frequency matched for predisposing diagnosis
leading to transplant. Study subjects were limited to those with
IPF or COPD, the two most common indications for lung
transplantation, in order to ensure power for a within diagnosis
analysis. This study population has been used previously to
evaluate the association of leptin and long pentraxin-3 with PGD
after lung transplantation [6,7].
PGD Grade Determination
The primary case definition for this study was the development
of any grade 3 PGD within the first 72 hours after allograft
reperfusion. PGD grades were determined according to the
International Society for Heart and Lung Transplantation
(ISHLT) . Two trained physicians independently assessed
chest x-rays from immediately post-transplantation and from 24,
48, and 72 hours after transplantation and assigned PGD grades
for each x-ray (subject-level classification kappa=0.95). Following
exclusion of secondary causes, grade 3 PGD was defined by the
presence of diffuse alveolar infiltrates and a ratio of the partial
pressure of arterial oxygen to the fraction of inspired oxygen
(PaO2/FiO2) of less than 200.
Measurement of Ang2 Concentration
Blood samples were collected from the participants preopera-
tively and 6 and 24 hours after reperfusion. Plasma samples were
processed within 30 minutes and then stored at 280uC. Ang2
plasma level was measured in duplicate utilizing a commercially
available ELISA assay (R & D Systems, Minneapolis MN). The
mean coefficient of variation for the assay was 2.3%. All study
personnel were blinded to the PGD status of the study subjects.
Study subject characteristics were compared using parametric
and non-parametric tests as appropriate. Data were missing from
0–19% of the patient characteristics; mean pulmonary artery
pressure was the only covariate with more than 10% missingness.
Simple imputation was used to account for missing covariate data.
The primary analysis utilized generalized estimating equations
(GEE) to identify differences in Ang2 levels over time and across
study subjects. Based on previous studies identifying differences in
biomarker levels across predisposing diagnoses, we a priori defined
diagnosis leading to transplantation as a possible effect modifier
and performed diagnosis specific analyses [5,6]. Potential con-
founding by recipient and donor age, sex, and race/ethnicity,
cardiopulmonary bypass use, transplant surgical type, ischemic
time, intra-operative mean pulmonary artery pressure, and packed
red blood cell (PRBC) transfusion volumes was assessed in-
dividually using multivariable GEE modeling. A change in the
b coefficient, after inclusion of a covariate, of greater than 20%
was used to identify confounding . A sensitivity analysis was
performed using the presence of grade 3 PGD at 72 hours after
reperfusion as a more severe phenotype. The secondary analysis
was a comparison of median Ang2 levels prior to transplant and 6
and 24 hours after transplant using Wilcoxon rank sum tests across
groups. A p,0.05 was pre-defined for statistical significance for all
tests. All statistical analyses were performed using Stata 11.2
software (STATA Corp., College Station, TX).
Elevated Ang2 Associated with PGD
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Forty cases and 79 controls were included for analysis. Donor,
recipient and intra-operative characteristics are presented in
Table 1. Cases and controls demonstrated similar donor and
recipient characteristics. There was a higher incidence of
cardiopulmonary bypass use among cases compared with controls
(54% vs. 30%, p=0.01). PGD cases received a larger volume of
packed red blood cells intraoperatively than controls (1063 ml vs.
696 ml, p=0.04). There were 16 cases with COPD and 24 cases
GEE models were used to assess the association between change
in Ang2 level over time and the risk of PGD (Figure 1). While
Ang2 levels increased from pre-transplant to post-transplant
measurements in all subjects, there were significant differences in
the slope of the change in Ang2 level over time in cases compared
to controls (p=0.03). Among subjects limited to a diagnosis of
COPD, there were no significant differences in longitudinal Ang2
levels between cases and controls (p=0.9). In those subjects
transplanted for IPF, GEE longitudinal modeling identified
a significant difference in the trend of Ang2 levels across all three
time points between cases and controls, with cases having
Table 1. Subject characteristics stratified as PGD cases and non-PGD controls.
CharacteristicsPGD (n=40) Non-PGD (n=79)p
Age, yr 55 (52, 58)56 (53, 58) 0.7
Female Gender 30%46% 0.1
African American13% 5%
Age, yr 34 (29, 38) 33 (30, 36)0.7
Female Gender48% 43% 0.6
Cause of death0.9
Blunt Trauma 3%4%
Head Trauma 30%35%
COPD 40% 41%
Transplant Type, single 38%38%0.9
Use of Cardiopulmonary Bypass 54% 30%0.01
Time on Bypass, min235 (201, 270)210 (184, 236)0.2
Ischemic Time, min 308 (281, 334)282 (262, 302)0.1
Mean Pulmonary Artery Pressure, mmHg 47 (40, 54)41 (36, 46)0.3
Packed Red Blood Cells, ml 1063 (675, 1450)696 (538, 854)0.04
Fresh Frozen Plasma, ml893 (702, 1084) 1062 (769, 1354)0.3
Platelets, ml421 (99, 743)228 (46, 411)0.3
PGD is defined as any grade 3 PGD during first 72 hours.
Continuous variables are expressed as means with 95% confidence intervals, while dichotomous and categorical variables are expressed as percentages, which may not
exactly total 100% because of rounding.
Elevated Ang2 Associated with PGD
PLOS ONE | www.plosone.org3 December 2012 | Volume 7 | Issue 12 | e51932
significantly larger increases in Ang2 plasma levels compared with
In the overall study population, there was no significant
difference in median Ang2 concentrations between cases and
controls prior to transplantation (1909 pg/ml vs. 2064 pg/ml;
p=0.9) or 24 hours after allograft reperfusion (6221 pg/ml vs.
4869 pg/ml; p=0.1), with borderline significance at 6 hours
(3707 pg/ml vs. 3188 pg/ml; p=0.05). Pre-transplant levels of
Ang2 were significantly higher in patients with IPF vs. COPD
(3330 pg/ml vs. 2051 pg/ml, p=0.005). Among patients with
COPD, there was no significant difference in Ang2 level between
cases and controls pre-transplant (1895 pg/ml vs. 1824 pg/ml), 6
hours after transplant (3247 pg/ml vs. 2843 pg/ml) or 24 hours
after transplant (4944 pg/ml vs. 4412 pg/ml) (p.0.6 for all time
points). Among patients with IPF, Ang2 concentrations at 6 hours
were significantly higher in cases compared to controls (4578 pg/
ml vs. 3218 pg/ml, p=0.01) with borderline significance at 24
hours (7115 pg/ml vs. 5046 pg/ml, p=0.06). There was no
significant difference in Ang2 level prior to transplant in the IPF
cases and controls (2538 pg/ml vs. 2318 pg/ml, p=0.6).
Figure 1. Longitudinal median Angiopoietin 2 level across the pre-transplant, 6-h post-transplant, and 24-h post-transplant time
points. A) all study subjects, B) subjects with COPD, and C) subjects with IPF. Solid line represents PGD cases and dashed line represents PGD-free
controls. Error bars represent the 95% CI. All p-values reported are from GEE modeling.
Elevated Ang2 Associated with PGD
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The effects of potential confounders on Ang2 levels and the
development of PGD were assessed using multivariable GEE
modeling (Table 2). The use of cardiopulmonary bypass and mean
pulmonary artery pressure confounded the association between
longitudinal Ang2 level and PGD, resulting in loss of statistical
significance on adjustment. There was no evidence of confounding
of the relationship between PGD and Ang2 level by any donor or
recipient characteristics or by total ischemic time, blood product
transfusion volume, or transplant type (Table 2).
A sensitivity analysis performed defining cases as those subjects
with grade 3 PGD at 72 hours produced similar results. GEE
modeling among all study subjects identified a significant differ-
ence in the change in Ang2 level over time in cases compared to
controls (p=0.04). Among COPD subjects, there was no
significant association between Ang2 trend and the development
of PGD (p=0.9). Among IPF subjects, there was a significant
difference in the change in Ang2 level across the three time points
between cases and controls (p=0.02).
The purpose of this study was to define the association of plasma
Ang2 concentrations with the development of PGD after lung
transplantation. We found that post-transplant Ang2 plasma levels
were significantly associated with the development of PGD. There
was also a significant difference in the change in Ang2
concentration over time between patients with IPF who developed
PGD compared with those without PGD.
Our identification of an association of elevated Ang2 levels and
PGD is consistent with evidence for a central role of Ang2 in the
development of syndromes with similar mechanisms to PGD.
Variation in the ANGPT2 gene, which encodes Ang2, was
associated with risk of trauma-associated ALI and with a higher
proportion of full-length Ang2 plasma isoforms . Circulating
levels of Ang2 are also significantly higher in trauma patients with
ALI compared to matched ALI-free controls . Ang2 levels
were also significantly higher in patients with severe sepsis
compared to those with sepsis alone or to those without sepsis or
systemic inflammatory response syndrome . Higher Ang2
plasma levels were associated with increased mortality in a surgical
population with ALI and in the NHLBI ARDS network
population. Mechanistically, both exogenous Ang2 or plasma
from patients with ALI induces permeability in vitro, and normal
barrier function can be restored by adding Ang1 . Experi-
mental models to augment Ang1 with either cell-based or
mesenchymal stem cell therapy resulted in mitigated parameters
of lung injury [25,26]. Given the shared pathology between ALI
and PGD, our findings are consonant with studies of Ang2’s role in
ALI. We now extend the relevance of Ang2 to the lung transplant
The results of this study provide further evidence for a significant
role of endothelial barrier disruption in the development of PGD.
Krenn et al. have demonstrated that VEGF levels are higher in
patients with cystic fibrosis (CF) compared to those with COPD
and that CF patients are at higher risk of PGD compared to those
with COPD. Furthermore, CF patients had higher lung water
content post-reperfusion compared to COPD subjects . In
a general population of lung transplant recipients, pre-transplant
VEGF serum concentrations were higher in patients ultimately
developing PGD compared to those without PGD . The
consistent demonstration of an association between upregulated
Ang2 production across related syndromes, combined with the
strong association between endothelial disruption and the de-
velopment of PGD, adds validity to our identification of an
association between post-transplant Ang2 plasma levels and PGD.
There was evidence of confounding of the relationship between
plasma Ang2 level and the development of PGD by cardiopul-
monary bypass and pulmonary arterial pressures. Elevated
pulmonary pressures and bypass are both independent risk factors
for PGD [28–30]. Bypass use has been associated with elevations
in Ang2 plasma levels and increased vascular permeability [31,32].
Pulmonary arterial hypertension has also been associated with
elevated plasma Ang2 levels with increased Ang2 gene expression
in plexiform lesions from lung tissue samples . Abnormalities
in the Ang2/Tie2 pathway may in part explain the association of
bypass and elevated pulmonary pressures with PGD; that is, longer
Table 2. Generalized estimating equation (GEE) analysis of association between PGD and Ang2 plasma level.
GEE b-coefficient for PGD, 61025
Unadjusted Base Model (n=119)5.6 (0.5, 10.8)0.03
COPD only (n=48)
21.0 (213, 11)0.9
IPF only (n=71) 7.6 (1.4, 13.8) 0.02
Base Model Adjusted for (n=119):
Cardiopulmonary Bypass 3.5 (21.9, 8.8)0.2
Transplant Type 5.8 (0.5, 11.1) 0.03
Recipient Age 5.6 (0.4, 10.7)0.03
Recipient Sex 6.4 (1.0, 11.9)0.02
Donor Age5.7 (0.5, 10.9) 0.03
Donor Sex 5.5 (0.4, 10.7)0.04
Total ischemic time4.5 (20.7, 9.6) 0.09
Mean Pulmonary Artery Pressure 4.2 (21.4, 9.7)0.1
Packed Red Blood Cells 4.7 (20.6, 10.0) 0.08
b-coefficient and p-value are generated from GEE models.
COPD=Chronic Obstructive Pulmonary Disease.
IPF=Idiopathic Pulmonary Fibrosis.
Elevated Ang2 Associated with PGD
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cardiopulmonary bypass time may induce Ang2 release from
endothelium, triggering vascular barrier breakdown and fluid
extravasation, therefore contributing pathogenically to PGD.
A subgroup analysis stratified by pre-operative diagnosis
identified a significant association between plasma Ang2 levels
and PGD in the IPF subgroup, but not the COPD subgroup.
Furthermore, the association between change in Ang2 level over
the peri-operative time period and PGD was only significant in the
IPF subgroup, suggesting that the role of Ang2 in the development
of PGD is diagnosis-specific. We have identified significant
differences in the association of plasma levels of long pentraxin-
3, an innate immune mediator, and clara cell secretory protein,
a marker of epithelial cell disruption, and PGD when comparing
patients with fibrotic and non-fibrotic lung disease [5,6]. Ang2 is
overexpressed and Ang1 expression decreased in patients with IPF
compared to those with connective tissue disease associated
pulmonary fibrosis, indicating an altered angiogenic profile in
patients with IPF . Likewise, prior studies have demonstrated
that serum VEGF levels were significantly higher in patients with
CF compared to COPD, both before transplant and immediately
after allograft reperfusion . The diagnosis-specific association
of Ang2 plasma levels with PGD provides further evidence that
PGD may have differential involvement of cellular pathways
depending on the pre-operative pulmonary diagnosis.
There are several limitations to this study. While we hypothesize
that the association of elevated Ang2 plasma levels and PGD may
indicate disruption of pulmonary endothelial cells with leakage
into the systemic circulation, we cannot be sure of the source of the
Ang2 release into plasma. While Ang2 is generally believed to be
predominantly an endothelial protein resident in Weibel-Palade
bodies, it has been demonstrated in alveolar epithelial cells as well
. In the absence of lung biopsy samples with staining for Ang2
localization, we cannot determine the cellular source of the Ang2
detected in plasma. Additionally, systemic endothelium may be the
source of the Ang2. The functional implications of the association
between PGD and elevated plasma Ang2 levels are an area for
future study. We were unable to evaluate the association between
donor Ang2 levels at the time of organ procurement and the
development of PGD in the recipient. With the increased
utilization of ex vivo lung perfusion (EVLP) to improve allograft
function, changes in Ang2 levels from the allograft prior to
implantation may also correlate with PGD risk. EVLP may be
a powerful tool to analyze the role of the Ang2 pathway in the
pathogenesis of PGD. There is also the potential for PGD
misclassification. Evaluation of chest x-rays for the development of
PGD was performed by two independent physicians and grade
was assigned using a validated scoring system [22,35]. Sensitivity
analysis using a more severe phenotype of PGD, grade 3 at 72
hours after reperfusion, confirmed our findings. Our evaluation of
the Tie-2/Ang2 pathway is limited to plasma Ang2 levels. Recent
studies have demonstrated utility in the Ang2/Ang1 ratio for
predicting poor outcomes associated with ALI, and genetically-
determined splice variants have been identified by our group
[16,36]. Plasma samples for the patients included in this study
have been exhausted, limiting our ability to further analyze the
role of Ang2/Ang1 ratio, different isoforms, or the role of VEGF
plasma level in PGD and will be the focus of future investigations.
While the Ang-2/Ang-1 ratio may be considered a measure of
relative antagonism and agonism of the Tie-2 receptor as
hypothesized by Ong et al., it is not clear that the ratio adds to
the association demonstrated between Ang2 alone and PGD .
Tie2 phosphorylation studies are beyond the scope of this study as
no biopsy samples are taken as part of the LTOG protocol, either
prior to organ procurement or after allograft implantation and
reperfusion. Finally, as with previous biomarker evaluations of
PGD, given the lack of significant pre-transplant differences in
Ang2 plasma levels between cases and controls, it is not possible to
define a causal relationship between Ang2 production and the
development of PGD post-transplant. Ang2 measurements were in
some cases made concurrently with the identification of PGD. In
some cases, chest radiographs and oxygenation parameters
demonstrated a patient had PGD prior to the 6 and 24 hour
collection times used in this study. Therefore, Ang2 plasma level
does not serve as a useful diagnostic tool for PGD. However, we
can conclude a likely mechanistic role for endothelial disruption
and the development of PGD, and that the Tie2/Ang2 pathway
may be an ideal target for pharmacologic intervention.
In summary, we identified elevated Ang2 plasma levels to be
strongly associated with the development of PGD after lung
transplantation for patients with IPF. This provides increasing
support for a central role for endothelial cell disruption in the
development of PGD. Evaluating the role for potential therapeu-
tics targeting aberrant vascular permeability, including EVLP is an
important area of future study.
The participating centers and investigators in the Lung Transplant
Outcomes Group were:
University of Pennsylvania (Coordinating site):
Jason Christie, MD, MS (PI)
Steven M. Kawut, MD, MS
Alberto Pocchetino, MD
Y. Joseph Woo, MD
Ejigayehu Demissie, MSN
Robert M. Kotloff, MD
Vivek N. Ahya, MD
James Lee, MD, MS
Denis Hadjiliadis, MD, MHS
Melanie Rushefski, BS
Richard Aplenc, MD
Clifford Deutschman, MD, MS
Benjamin Kohl, MD
Edward Cantu, MD
Joshua M. Diamond, MD, MS
Rupal J. Shah, MD
David Lederer, MD, MS (PI)
Selim Arcasoy, MD
Joshua Sonett, MD
Jessie Wilt, MD
Frank D’Ovidio, MD
Lori Shah, MD
Hilary Robbins, MD
Matthew Bacchetta, MD
Nilani Ravichandran, NP
Genevieve Reilly, NP
Jeffrey Okun, MD
Debbie Rybak, BA
Michael Koeckert, BA
Robert Sorabella, BA
Nisha Ann Philip, MBBS
Nadine Al-Naamani, MD
Matthew LaVelle, BS
Megan Larkin, MPH
Shefali Sanyal, BS
Lorraine Ware, MD (PI)
Aaron Milstone, MD (PI)
Jean Barnes, RN
Stephanie Logan, RN
Carla Ramsey, RN
Elevated Ang2 Associated with PGD
PLOS ONE | www.plosone.org6December 2012 | Volume 7 | Issue 12 | e51932
Shaquita Claybrooks, RN
Ann Weinacker, MD (PI)
Susan Spencer Jacobs, MSN
Val Scott, MSN
Tal Alfasi, MS
University of Alabama, Birmingham:
Keith Wille, MD (PI)
Necole Harris, RN
Johns Hopkins University:
Jonathan Orens, MD (PI)
Ashish Shah, MD
John McDyer, MD
Christian Merlo, MD, MPH
Matthew Pipeling, MD
Reda Girgis, MD
Karen Oakjones, RN
University of Michigan:
Vibha Lama, MD, MS (PI)
Fernando Martinez, MD, MS
Douglas R. Armstrong R.N, MS
Mary Maliarik, BS
Scott M. Palmer, MD, MHS (PI)
David Zaas, MD, MBA
R. Duane Davis, MD
Ashley Finlen-Copeland, MSW
William A. Davis
University of Chicago:
Sangeeta Bhorade, MD (PI)
Mark Lockwood, RN,MSN
University of Pittsburgh:
Maria Crespo, MD (PI)
Dr. Joseph Pilewski
Dr. Christian Bermudez
David S. Wilkes, MD
David Wilson Roe, MD
Thomas Wozniak, MD
Ronda L. McNamee, RN
Kim A. Fox, RN
Danyel F. Gooch, RN
Tonya Isaacs, RN
Conceived and designed the experiments: MD MKP NJM SMK RJS EC
LBW SLB JDC. Performed the experiments: LBW. Analyzed the data:
JMD MKP NJM SMK SLB SMP RJS LBW EC JDC. Contributed
reagents/materials/analysis tools: JMD MKP DJL JL EC VNL SMB MC
SLB ED KW JO AW DW SA PDS DSW LBW SMP JDC. Wrote the
paper: Revised the manuscript for important intellectual content and
provided final approval for the version to be submitted: JMD MKP NJM
SMK DJL JL EC RJS SLB VNL SMB MC ED KW JO AW DW SA PDS
DSW LBW SMP JDC.
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