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CHEST Original Research

COPD

Altered Surfactant Protein-A Expression


in Type II Pneumocytes in COPD
Eleni M. Vlachaki, MD; Anastassios V. Koutsopoulos, MD, PhD; Nikolaos Tzanakis, MD,
PhD; Eirini Neofytou, MSc; Marianna Siganaki, MD; Ioannis Drositis, MD;
Andreas Moniakis, MD; Sophia Schiza, MD, PhD; Nikolaos M. Siafakas, MD, PhD, FCCP;
and Eleni G. Tzortzaki, MD, PhD, FCCP

Background: Pulmonary surfactant protein A (SP-A) is a lectin, with multiple functions that con-
tribute to innate host defense and the regulation of the inflammatory process in the lung. In
normal conditions, SP-A seems to protect against the effects of smoking. However, studies in smokers
with or without COPD are limited.
Methods: Western blots on lung tissue specimens from 60 male subjects (32 patients with COPD,
18 smokers without COPD, and 10 control nonsmokers) for SP-A and the housekeeping protein
actin were carried out. Additionally, the SP-A expression pattern was evaluated by immuno-
histochemistry in formalin-fixed, paraffin-embedded lung tissue sections from the same subjects.
Results: Western blots revealed significantly higher SP-A levels in control nonsmokers (4.8 6 0.05)
when compared with patients with COPD (0.6 6 0.7) and smokers without COPD (2.4 6 0.9),
(P , .05). However, differences that were not statistically significant were observed in SP-A levels
among the patients with COPD and the smokers without COPD (P 5 .12). The immunohistochem-
ical examinations showed an increase in the overall number of type II pneumocytes per high-
power field in patients with COPD, but a decreased ratio of SP-A positive type II pneumocytes to
total type II pneumocytes, compared with smokers without COPD (P 5 .001). This ratio was also
correlated with FEV1 (percent predicted [% pred]), (r 5 0.490, P 5 .001). The overall number of
alveolar macrophages per high-power field was significantly higher in patients with COPD com-
pared with smokers without COPD (P 5 .001). The ratio of SP-A positive alveolar macrophages
was increased in patients with COPD when compared with smokers without COPD (P 5 .002),
while this was correlated with airway obstruction (FEV1, % pred) (r 5 0.281, P 5 .04).
Conclusions: Our results indicate that altered SP-A expression could be another link to COPD
pathogenesis and highlights the need for further studies on surfactant markers in COPD.
CHEST 2010; 137(1):3745

Abbreviations: (% pred) 5 percent predicted; SP 5 surfactant protein; TTF 5 thyroid transcription factor-1

Pulmonary surfactant is a complex mixture of lipids


and proteins synthesized mainly by type II pneu-
behavior of the lung. However, the newly established
immunomodulatory properties of lectins SP-A and
mocytes.1 Four surfactant proteins (SPs) have been SP-D elucidated their important role in a number of
defined: SP-A, SP-B, SP-C, and SP-D.1,2 SPs were lung diseases.1 Although lectins have the potential to
initially thought to play a key role in the biophysical
Funding/Support: The present study was supported by a Grant
Manuscript received May 6, 2009; revision accepted July 14, 2009. of Hellenic Thoracic Society.
Affiliations: From the Department of Thoracic Medicine (Drs Correspondence to: Eleni Tzortzaki, MD, PhD, FCCP, Depart-
Vlachaki, Tzanakis, Neofytou, Schiza, Siafakas, and Tzortzaki, and ment of Thoracic Medicine, University Hospital of Heraklion,
Ms Siganaki), the Department of Pathology (Dr Koutsopoulos), Medical School, University of Crete, 71110 Heraklion, Crete, Greece;
and the Department of Thoracic Surgery (Drs Drositis and e-mail: tzortzaki@med.uoc.gr
Moniakis), University Hospital of Heraklion, Medical School, 2010 American College of Chest Physicians. Reproduction
University of Crete, Greece. of this article is prohibited without written permission from the
Part of this article has been presented in abstract form (Vlachaki American College of Chest Physicians (www.chestjournal.org/
E, Tzortzaki EG, Koutsopoulos A, et al. Proc Am Thorac Soc. site/misc/reprints.xhtml).
2006:A624). DOI: 10.1378/chest.09-1029

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contribute to the pathogenesis of COPD, little work Materials and Methods
has been done in this field.2
COPD is a slowly progressive disease character- Subjects
ized by reduced maximum expiratory flow and slow The study population comprised 60 subjects who underwent
forced emptying of the lungs, features that are not lung resection for a solitary peripheral nodule of malignant car-
fully reversible.3 Abnormal inflammatory responses cinoma. Spirometric evaluation was performed according to
to several noxious agents (mainly smoke from ciga- standard protocols 1 week before lobectomy. Patients who had
a severe COPD exacerbation or lower respiratory tract infec-
rettes), the involvement of a variety of inflammatory
tion within 4 weeks before their entrance in the study were
cells and cytokines, and the persistent destructive excluded.
oxidative process, accused of being the pathogenesis Three groups were defined: 32 smokers with COPD, 18 smok-
of COPD, may affect the composition and the func- ers without COPD and without lung function impairment, and 10
tion of surfactant.4 It has been also shown that smoking, control nonsmokers. The American Thoracic Society/European
Respiratory Society Consensus Statement13 was used for the diag-
the major cause of COPD, might affect surfactant
nosis and assessment of severity for the patients with COPD.
homeostasis and function.5 Along this line, Otto- Informed consent was obtained from all subjects, and the proto-
Verberne and colleagues4 reported a protective col was approved by the medical ethics committee of the Univer-
effect of pulmonary surfactant on elastase-induced sity Hospital of Heraklion in Crete.
emphysema in mice. Moreover, Honda et al6 stud-
ied the levels of immunomodulatory SP-A and Human Tissue Preparation
SP-D in BAL and reported decreased levels of SP-A Human lung tissue samples (two or three blocks) were
and SP-D in smokers compared with nonsmokers. obtained from all subjects, derived from the subpleural parenchyma
These results have recently been confirmed by at least 5 cm away from the solitary nodule. During surgery, all
Betsuyaku et al,7 whereas Guo et al8 reported that patients received general anesthesia under controlled mechani-
cal ventilation. Tissue samples for Western blots were immedi-
certain SP-A alleles may increase susceptibility
ately frozen in liquid nitrogen and stored at 280C until use. Tis-
to COPD. sue samples for immunohistochemistry were immediately fixed
Nevertheless, current concepts suggest that the in 10% buffered formalin for at least 24 h. After fixation, each
various surfactant proteins differ both in their local- tissue block was embedded in paraffin. Sections 5 mm thick were
ization and biologic action; for example, the lectins cut following routine procedures. To overcome possible tissue
volume differences between different groups when counting
SP-A and SP-D have been related to the host
various cells (eg, pneumocytes and alveolar macrophages), we
defense of the lung.9-11 In the present study, we spe- evaluated the ratios of SP-A positive pneumocytes to total pneu-
cifically focused on the immunomodulatory protein mocytes and the ratios of SP-A-positive macrophages to total
SP-A, considering its important role in the pulmo- macrophages.
nary host defense.1,2 In normal conditions, SP-A
is generally beneficial in protecting the lungs from Western Blots
oxidants and inflammatory and infectious stress.1,2 Western blot detection of SP-A and actin was performed using
However, SP-A immunity functions seem to be standard procedures. In detail, lung tissue specimens from all
impaired in chronic smokers,5-7 which may play a cru- subjects were homogenized to obtain the corresponding pro-
cial role in the development of COPD. Furthermore, tein extracts. The protein lysate was added to one-third volume
we have previously reported microsatellite DNA of sodium dodecyl sulfate-preparation buffer (NuPAGE LDS
4 3 LDS Sample Buffer; Invitrogen Corp; Carlsbad, CA). Sample
instability in patients with COPD in G29802 marker.12 preparations of each lung protein sample (50 ng) were separated
Microsatellite marker G29802 is located on the using 12.5% sodium dodecyl sulfate-polyacrylamide gel electro-
10q22 chromosomal region next to SP-A1 gene. We phoresis under reducing conditions. The gels were then trans-
hypothesized that microsatellite DNA instability in ferred using electrophoresis to a nitrocellulose membrane. Immuno-
G29802 marker in patients with COPD could be detection was done using a primary mouse anti-SP-A antibody
(PE-10; Dako, Ltd; Hellas, Greece), an exposure time of 1 min,
an indication of a deficient mismatch repair in the and enhanced chemiluminescence. The protein presence was
region and perhaps a disruption of the adjacent assessed using the mouse antiactin antibody (MAB 1501; Chemi-
SP-A gene. con; Temecula, CA). The lanes of the blotted membranes were
On this basis, we attempted to explore (1) the quantified using Photoshop Image Analysis Software (CS2, Adobe
SP-A protein levels, using Western blots and Systems Inc; San Jose, CA).
immunohistochemistry in smokers with or without
Immunohistochemistry
COPD and in control nonsmokers, and (2) the
relationship between the SP-A expression levels Immunostaining of SP-A and thyroid transcription factor-1
and the degree of airway obstruction. To the best (TTF-1) in formalin-fixed, paraffin-embedded lung tissue sec-
of our knowledge, this is the first study to evaluate tions was carried out using the biotin-based kit Link-Label
(IHC Detection Systems; Biogenix; San Ramon, CA) according
the SP-A expression in lung biopsies from patients to manufacturer instructions. The monoclonal mouse antihu-
with COPD, smokers without COPD, and control man SP-A, clone PE-10 antibody (Dako Ltd) was used in a 1:50
nonsmokers. dilution at room temperature for 1 h. The monoclonal mouse

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anti-TTF-1 antibody (Santa Cruz Biotechnology, Inc) was used Immunostaining Images
on adjacent serial sections to count the total type II pneumo-
cytes and SP-A positive type 2 cells. Sections were counter- Figure 2 shows representative SP-A immuno-
stained with Mayers hematoxylin. Negative control experi- stained images of lung tissue in a patient with COPD
ments for nonspecific binding were performed with preimmune (Fig 2A), in a smoker without COPD (Fig 2B), and in
serum.
a control nonsmoker (Fig 2C). Figure 3 shows positive
Quantification of the Immunohistochemical Reaction
TTF-1 immunostained images of type II pneumo-
cytes in a patient with COPD (Fig 3A) and in a smoker
The evaluation of the total type II pneumocytes (columnar without COPD (Fig 3B). Alveolar macrophages were
alveolar lining cells), alveolar macrophages (irregularly distributed TTF-1 negative.
in the alveoli with foamy cytoplasm and indented nuclei), and
SP-A-positive cells stained in purple (type II pneumocytes and
macrophages) was performed using a grid with dimensions 10 3 10 Type II Pneumocytes and SP-A Expression
for light microscopy (Olympus; Olympus America Inc.; Center
Valley, PA), at 340 magnification (manual counting). Two observers
Figures 2A and 2B show representative SP-A
(E.M.V., A.V.K.) specialized in lung pathology examined the immunostaining of type II pneumocytes in lung tissue
sections independently, without prior knowledge of the subjects sections from a patient with COPD and a smoker
clinical data. The interobserver variability of measurements was without COPD, respectively. The total number of
expressed as the % coefficient of variation. The interobserver type II pneumocytes was higher in patients with
coefficient of variation was , 10%.
Twenty microscopic fields of each slide, in a cross configura-
COPD in comparison with smokers without COPD,
tion, were evaluated for all subjects. The results were expressed in as shown in Figure 4 (P 5 .002). The ratio of SP-A-
cells per millimeter squared. positive type II pneumocytes (SP-A-positive type II
pneumocytes/total type II pneumocytes, expressed
Statistical Analysis as %) was higher in smokers without COPD com-
Clinical characteristics are presented as mean 6 SD and cell
pared with patients with COPD (P 5 .001) (Fig 4B).
characteristics as median (range) unless stated otherwise. Differ-
ences between the groups of subjects were tested using the Mann- Alveolar Macrophages and SP-A Expression
Whitney U test for non-normal distributed variables and the Stu-
dent t test for normal variables for independent groups. Pearsons Figures 2A and 2B show SP-A positive alveolar
for normal or Spearmans for non-normal correlation coefficients macrophages from a patient with COPD and a smoker
were used to evaluate the associations between variables. The without COPD. The total number of macrophages
statistical software SPSS Version 15 for Windows (SPSS Inc.; per millimeter squared was significantly higher in
Chicago, IL) was used for the entire analysis. A P value of , 0.05
was considered statistically significant.
patients with COPD compared with smokers without
COPD (P 5 .001) ( Fig 5A).
The ratio of SP-A-positive alveolar macrophages
Results (SP-A-positive alveolar macrophages/total alveolar
macrophages, expressed as a percentage) was sig-
The anthropometric characteristics and spirometric
nificantly higher (P 5 .002) in patients with COPD
values of patients with COPD, smokers without COPD,
compared with smokers without COPD (Fig 5B). In
and control nonsmokers are shown in Table 1. Western
addition, the scatter of the values of the ratio in patients
blot analysis showed SP-A levels were decreased,
with COPD was smaller (Fig 5B).
although not statistically significant, in patients with
COPD (0.6 6 0.7) in comparison with smokers with- Correlation of SP-A Expression
out COPD (2.4 6 0.9), (P 5 .12). SP-A levels in control
nonsmokers were significantly higher (4.8 6 0.05), The ratio of SP-A-positive type II pneumocytes to
(P , .05) in comparison with smokers with or without total type II pneumocytes was significantly corre-
COPD (Fig 1). lated with FEV1 values (r 5 0.490, P 5 .001) (Fig 6A).

Table 1Anthropometric Characteristics and Spirometric Values of Study Subjects

Smokers With COPD Smokers Without COPD Control Nonsmokers P Valuea


Sex, M/F 32/0 18/0 10/0
Age, y 64 6 6.5 56.5 6 10.2 57.6 6 16.7 .03
Smoking, P-Y 59 6 20.4 49.1 6 26.7 NS
FEV1, % pred 63.6 6 15.5 94.7 6 12.4 101.2 6 14 .0001
FVC, % pred 79.3 6 17.6 91 6 12.5 95.8 6 15 .02
FEV1/FVC, % 62.3 6 6.2 81.4 6 4.7 83.4 6 2 .0001
M/F 5 male/female; NS 5 not significant; P-Y 5 pack-years of smoking.
aComparison between smokers with COPD and smokers without COPD.

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Figure 1. Representative Western blots of SP-A and actin in
human lung tissues from two patients with COPD, one smoker
without COPD, and one control nonsmoker. Immunodetection
was done using primary mouse anti-SP-A antibody (A). Quantita-
tive analysis (mean 6 SD) for SP-A normalized to actin showed
decreased but not statistically significant levels in patients with
COPD in comparison with smokers without COPD, whereas in
control nonsmokers, the SP-A levels were significantly higher than
in smokers with or without COPD (B). NS 5 not significant differ-
ence (P 5 .12) between patients with COPD and smokers without
COPD; SP-A 5 surfactant protein A. ** 5 significant difference
(P , .05) between patients with COPD and control nonsmokers.

In Figure 6A, we illustrated that the decreased SP-A


ratio was related with a higher degree of obstruction
(lower FEV1 percent predicted [% pred]). The ratio
of SP-A-positive alveolar macrophages to total alve-
olar macrophages was significantly (r 5 0.281,
P 5 .04) correlated with FEV1 (Fig 6B). No signifi-
cant correlation between the total number of SP-A-
positive cells and age was found in any of the
subjects.

Discussion
Figure 2. Immunostaining of SP-A in human lung tissue (origi-
To our knowledge, this study is the first to evalu- nal magnification 3400). A positive SP-A hyperplastic type II
pneumocyte [PN II (+)] stained in pink, negative hyperplastic
ate protein levels and tissue distribution of SP-A in SP-A type II pneumocytes [PN II (-)] stained in blue, and posi-
human lung biopsies from patients with COPD, tive alveolar macrophages [MACR (+)] stained in purple, from a
smokers without COPD, and control nonsmokers representative patient with COPD (A). A hyperplastic positive
SP-A type II pneumocyte [PN II (+)] cell stained in purple, a
using an adequate sample size and two different negative SP-A type I pneumocyte [PN I (-)] stained in blue, and
experimental approaches for each patient. SP-A positive alveolar macrophages [MACR (+)] stained in purple,
levels, as revealed by Western blots, reflected the from a representative smoker without COPD (B). Positive SP-A
pneumocytes [PN (+)] stained in purple, from a representative
overall protein expression from the homogenized control nonsmoker (C). See Figure 1 legend for expansion of
lung extracts (including SP-A from pneumocytes other abbreviations.

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Figure 3. Positive TTF-1 immunostaining in type II pneumocytes [PN II (+)] in a patient with COPD
(A) and in a smoker without COPD (B). In both A and B, the alveolar macrophages [MACR (-)] were
TTF-1 negative (original magnification 3400). TTF-1 5 thyroid transcription factor-1.

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Figure 4. Total number of pneumocytes II per millimeter squared in patients with COPD and in smok-
ers without COPD (A). SP-A positive pneumocytes II (expressed as a percentage of the total pneumo-
cytes II) in patients with COPD and in smokers without COPD (B). Lines represent median values. 5
patients with COPD; 5 smokers without COPD (control subjects). See Figure 1 legend for expansion
of other abbreviations.

and alveolar macrophages), while immunohistochem- COPD compared with smokers without COPD
ical examinations further identified the specific cell (Fig 5A), whereas the ratio of SP-A-positive mac-
subtypes expressing SP-A protein in the human lung. rophages to the total macrophages was higher in
Western blots revealed significantly higher SP-A patients with COPD. This was also correlated with
levels in control nonsmokers compared with patients airway obstruction (FEV1 % pred) (Fig 6B).
with COPD and smokers without COPD (P , .05). The pathogenetic role of surfactant was first
However, differences that were not statistically sig- described in infant respiratory distress syndrome,
nificant were observed in SP-A levels among patients in which surfactant deficiency induced the disease.14,15
with COPD and smokers without COPD (P 5 .12), Later, surfactant biochemical, functional, and immuno-
( Fig 1). Interestingly, although immunostaining modulatory abnormalities were reported in certain
revealed increased overall numbers of type II pneumo- other lung diseases, including COPD.14,16
cytes in patients with COPD in comparison with
smokers without COPD (Fig 4A), the ratio of SP-A- Increased Type II Pneumocytes in COPD
positive type II pneumocytes to total type II pneu-
mocytes was decreased in COPD patients (Fig 4B). Our results revealed an increased number of type
In addition, the decreased SP-A ratio in patients with II pneumocytes in patients with COPD compared
COPD was related to lower FEV1 (% pred), (Fig 6A). with smokers without COPD. This result was expected
Furthermore, the overall number of alveolar mac- because of the known extensive tissue damage in
rophages was significantly higher in patients with COPD and is also in agreement with the mechanical

Figure 5. Total number of macrophages per millimeter squared in patients with COPD and in smokers
without COPD (A). SP-A positive macrophages (expressed as % of the total number of macrophages) in
patients with COPD and in smokers without COPD (B). Lines represent median values. 5 patients
with COPD; 5 smokers without COPD. See Figure 1 legend for expansion of abbreviation.

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Figure 6. The relationship between SP-A-positive pneumocytes II expressed as % of the total pneumo-
cytes II and FEV1 (% pred) in patients with COPD and in smokers without COPD (A). The relationship
between SP-A-positive alveolar macrophages expressed as % of total alveolar macrophages and FEV1 (%
pred) in all subjects (B). 5 patients with COPD; 5 smokers without COPD. See Figure 1 legend for
expansion of other abbreviations.

injury hypothesis.10,11 Increased numbers of type smokers, suggesting that this may contribute to the
II pneumocytes have been reported in previous increased prevalence of respiratory infections,6
studies, especially in rats after induced lung impaired efferocytosis (removal of apoptotic bodies),
injury.5,17 The alveolar epithelium, when injured by and abnormal tissue remodeling.20 Two recent studies
cigarette smoke, initiates repair responses of dif- by Hu et al23,24 reported decreased SP-A levels in the
ferentiation and proliferation of cells to cover the lung tissue and lavage fluid of rats after chronic ciga-
defect from the injury.18,19 Type II pneumocytes rette smoke exposure, concluding that cigarette smoke
proliferate during lung injury and chronic inflam- might have important effects on SP-A metabolism
matory states,19 such as COPD, to produce type 1 cells. and host defense functions.
Furthermore, type II pneumocytes produce spe- The association between cigarette smoke, innate
cific molecules, enzymes, and proteins, such as SP-A, immunity, lung inflammation, and COPD pathogen-
which participate in lung defense against noxious esis has been extensively studied, and several theories
agents.20,21 have been proposed.25-28 In the susceptible smoker,
continuous exposure initially affects the lung epithe-
Decreased SP-A Expression in Type II lial barrier system and, in particular, its cellular com-
Pneumocytes in COPD Was Related With ponent via repeated oxidative stress, which may result
the Degree of Airway Obstruction in oxidative DNA damage and acquired somatic
mutations of these cells.27-30 In previous studies, we
Interestingly, we have found a decreased ratio of have reported acquired genetic instability in the G29802
SP-A-positive type II pneumocytes (SP-A-positive marker in patients with COPD,12,31, hypothesizing
type II pneumocytes/total type II pneumocytes) in that this could be an indication of a deficient DNA
patients with COPD compared with smokers without mismatch repair in the region and perhaps a disrup-
COPD (Fig 4B), and this was related to a higher tion of the adjacent SP-A gene. The decreased SP-A
degree of obstruction (Fig 6A). One explanation could expression in type II pneumocytes in patients with
be that SP-A levels are lower in patients with COPD COPD, as revealed by this study, could support our
as a result of disease-related epithelial destruction initial hypothesis.
that depletes the type II pneumocytes and clara
cells responsible for SP-A production. In support of SP-A Expression in Alveolar Macrophages
this scenario, the degree of airway obstruction was
inversely correlated with the SP-A levels. In normal In accordance with previous studies, we found
conditions, SP-A is generally beneficial in protecting increased numbers of macrophages in the lung paren-
the lungs from oxidants and inflammatory and infec- chyma of patients with COPD.32 We further reported
tious stress.21 It is also involved in the clearance of higher numbers of SP-A-positive alveolar macrophages
apoptotic and necrotic cells.20 However, studies have in patients with COPD than in smokers without
shown that host defense functions of surfactant may COPD. Moreover, the ratio of SP-A-positive alveolar
be impaired in the chronic smoker.7,22 Decreased lev- macrophages to total alveolar macrophages was sig-
els of SP-A were reported in BAL fluid from chronic nificantly correlated with FEV1(Fig 6B).

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A possible explanation for the increased SP-A obstruction. The reported relationship, although not
amounts found in the alveolar macrophages of direct cause and effect, may suggest either an abnor-
patients with COPD could come from SP-A fractions mal production of SP-A in type II pneumocytes or
scavenged from the alveolar space. This could reflect an augmented SP-A turnover in the lungs of patients
either an impaired mechanism of SP-A production or with COPD. Nevertheless, these are interesting
an altered turnover of SP-A in type II pneumocytes hypotheses that need further exploration.
in patients with COPD, or both. A number of stud-
ies have shown that SP-A has the potential to stimu-
late macrophages and to increase phagocytosis of Acknowledgments
various bacteria, viruses, allergens, and apoptotic Author contributions: Dr Vlachaki: contributed to the acquisition,
cells. Thereby, functioning as opsonin could also analysis, and interpretation of data.
enhance the uptake of SP-A fractions by alveolar Dr Koutsopoulos: contributed to the acquisition, analysis, and
interpretation of data.
macrophages.2,20 Dr Neofytou: contributed to the acquisition, analysis, and
Our study has some limitations. First, although the interpretation of data.
study subjects were well characterized, for feasibility Ms Siganaki: contributed to the acquisition, analysis, and
interpretation of data.
reasons the lung tissue specimens were obtained only Dr Drositis: contributed to the acquisition of data.
from subjects undergoing resection for lung cancer. Dr Moniakis: contributed to the acquisition of data.
Although it is known that pulmonary malignancy Dr Schiza: contributed to the acquisition of data.
Dr Siafakas: contributed to interpretation of data and critically
could affect surfactant expression,33,34 all subjects revised the article.
included in this study had the same comorbidity (ie, Dr Tzanakis: contributed to interpretation of data and critically
lung cancer). Second, surgical lung biopsies were not revised the article.
Dr Tzortzaki: contributed to conception and design of the study,
obtained from patients with more advanced COPD. the analysis and interpretation of data, and drafted the submitted
This could have led to underestimation of our results article.
because our data suggested that these patients prob- Financial/nonfinancial disclosures: The authors have reported
to CHEST that no potential conflicts of interest exist with any
ably could express even lower SP-A levels. Third, the companies/organizations whose products or services may be
median age of the patients with COPD was higher discussed in this article.
than that of the smokers without COPD (P 5 .03),
although no significant correlation between total SP-A
expression and age was found. Interestingly, a previ- References
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