Am J Physiol Lung Cell Mol Physiol 292: L778–L787, 2007.
First published December 1, 2006; doi:10.1152/ajplung.00352.2006.
Elastin protein levels are a vital modifier affecting normal lung development
and susceptibility to emphysema
Adrian Shifren,1 Anthony G. Durmowicz,2 Russell H. Knutsen,3 Eiichi Hirano,3 and Robert P. Mecham3
Departments of 1Internal Medicine, 2Pediatrics, and 3Cell Biology and Physiology,
Washington University School of Medicine, Saint Louis, Missouri
Submitted 9 September 2006; accepted in final form 26 November 2006
by destruction of
alveolar septae resulting in pathological alveolar air space
enlargement (50). The etiology of emphysema is most
frequently related to cigarette smoking, although not all
smokers develop clinical disease (28). Current hypotheses
of the pathogenesis of emphysema center on imbalances in
protease/antiprotease or oxidant/antioxidant levels, among
others. In most cases, pathways of tissue damage in cigarette
smoke-induced injury involve the destruction of elastin-rich
structures in the lung.
The mechanical properties of the lung are determined principally by the connective tissue networks laid down during
development. These extracellular matrices are composed
chiefly of elastin, collagen, and proteoglycans, and it is elastin
that is primarily responsible for providing reversible distensibility during respiratory cycling. Within the parenchyma, elastin is distributed extensively within the alveoli, including the
alveolar septae, septal junctions, and along the septal-free
edges. Organization of this elastin architecture begins during
the pseudoglandular stage of lung development, increases
throughout the canalicular and saccular stages, and peaks
during alveolation, an event that in mice begins a few days
after birth. Alveogenesis requires the participation of alveolar
myofibroblasts at the tips of the developing alveolar septae,
and active elastin expression by these myofibroblasts suggests
that elastic fiber deposition and proper elastic fiber formation
are critical for alveolar septation and normal alveolar development (5, 11, 31, 38). Consistent with this hypothesis is the
observation that mice deficient for elastin (Eln⫺/⫺) demonstrate arrested perinatal development of terminal airway
branches, resulting in dilated distal air sacs with attenuated
tissue septae, a condition characteristic of emphysema (55).
Elastic fibers are composite structures containing at least two
morphologically distinguishable components: amorphous elastin and microfibrils. To build a functional fiber, the production
of these components must occur in a well-defined temporal
sequence during tissue development. Many proteins have been
localized to microfibrils or to the elastic fiber-microfibril interface and are thought to influence elastic fiber assembly.
These proteins include the fibrillins, microfibril-associated glycoproteins, latent transforming growth factor (TGF)-␤-binding
proteins (LTBPs), and fibulins, among others (reviewed in Ref.
26). This compositional complexity also accounts for why
elastic fibers are difficult to repair when damaged. Fiber
reconstruction requires the coordinated reexpression of all of
the molecules that constitute the elastic fiber as well as the
cross-linking enzymes. This is an inefficient process in adult
tissue, most often resulting in production of elastin that either
does not polymerize or does not organize into a functional
three-dimensional fiber.
The central role of elastin in the pathology of emphysema is
apparent from the numerous disease models that have been
studied over the years. Intrapulmonary instillation of elastolytic enzymes, for example, confirmed that destruction of
existing elastic fibers leads to emphysema-like lesions (24, 27,
46). There is also evidence that functional variants of elastin
may increase lung susceptibility to cigarette smoke-induced
damage by altering elastic fiber assembly and integrity (25).
More recently, experiments in mice have shown that deficiency
in elastin or in the elastic fiber proteins fibrillin-1,
LTBP-3 and -4, fibulin-4 and -5, and EMILIN (elastin microfibril interface located protein) all result in emphysema-like
lung morphology at birth (6, 35, 36, 49, 51, 56, 58). This
congenital or developmentally acquired emphysema results
largely from abnormal elastin deposition or altered elastic
fiber-mediated growth factor signaling during development
(36, 58) and is fundamentally different from the destruction of
Address for reprint requests and other correspondence: R. P. Mecham, Dept.
of Cell Biology and Physiology, CB 8228, Washington Univ. School of
Medicine, 660 S. Euclid, St. Louis, MO 63110 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
smoking
PULMONARY EMPHYSEMA IS CHARACTERIZED
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Shifren A, Durmowicz AG, Knutsen RH, Hirano E, Mecham
RP. Elastin protein levels are a vital modifier affecting normal lung
development and susceptibility to emphysema. Am J Physiol Lung
Cell Mol Physiol 292: L778–L787, 2007. First published December 1,
2006; doi:10.1152/ajplung.00352.2006.—Cigarette smoking is the
strongest risk factor for emphysema. However, sensitivity to cigarette
smoke-induced emphysema is highly variable, and numerous genetic
and environmental factors are thought to mitigate lung response to
injury. We report that the quantity of functional elastin in the lung is
an important modifier of both lung development and response to
injury. In mice with low levels of elastin, lung development is
adversely affected, and mice manifest with congenital emphysema.
Animals with intermediate elastin levels exhibit normal alveolar
structure but develop worse emphysema than normal mice following
cigarette smoke exposure. Mechanical testing demonstrates that lungs
with low levels of elastin experience greater tissue strains for any
given tissue stress compared with wild-type lungs, implying that
force-mediated propagation of lung injury through alveolar wall
failure may worsen the emphysema after an initial enzymatic insult.
Our findings suggest that quantitative deficiencies in elastin predispose to smoke-induce emphysema in animal models and suggest that
humans with altered levels of functional elastin could have relatively
normal lung function while being more susceptible to smoke-induced
lung injury.
ELASTIN AND EMPHYSEMA SUSCEPTIBILITY
MATERIALS AND METHODS
Animals. C57BL/6J mice bearing a heterozygous deletion of exon
1 in the elastin gene (Eln⫹/⫺) (30) backcrossed for more than five
generations in the C57BL/6J background were used for all studies.
Eln⫹/⫹ littermates from Eln⫹/⫺ crosses were used as wild-type (WT)
controls. hBAC⫺/⫺ mice (human Eln transgene positive, mouse Eln
gene null) were generated using a human bacterial artificial chromosome (BAC) clone (CTB-51J22) containing the complete human
elastin gene. BAC circular DNA was injected into fertilized mouse
oocytes from C57B1/6xCBA mice, which were implanted into the
uterus of pseudopregnant foster mothers. After birth, founders expressing the transgene were identified using RT-PCR. Of the six
founder lines, line 3 was found to have the highest transgene expression level and was used throughout this study. Eln gene expression
levels, based on RT-PCR, were approximately one-third of those
found in WT mice (manuscript in preparation). Transgene-positive
animals were mated to WT (C57BL/6) mice for more than five
generations to stabilize the line. All housing and surgical procedures
were performed in accordance with institutional guidelines. All experimental procedures were approved by the Washington University
institutional Animal Studies Committee.
Lung volume estimation. Three-month-old WT, Eln⫹/⫺, and
hBAC⫺/⫺ mice were euthanized with a lethal dose of intraperitoneal
pentobarbital. Lungs were dissected from the thoracic cavity after
ligating the trachea prior to transection to prevent fluid entering the
lungs. Lungs were rinsed with saline to remove any blood, and excess
fluid was removed by blotting. Lungs were then weighed. The trachea
was cannulated, and, without deaeration, the lungs were inflated with
formalin at 25 cm of water pressure. Lungs were then reweighed.
Calculated differences in weight are directly proportional to the
volume of infused formalin. Lung volume was calculated by multiplying the difference in lung weight by the specific gravity of
formalin.
AJP-Lung Cell Mol Physiol • VOL
Lung elastin, hydroxyproline, and total protein content measurements. Three-month-old mice from all three genotypes were euthanized with pentobarbital. Lungs were removed, dissected free of the
extra-pulmonary airways and connective tissue, washed, weighed
(wet weight), and snap-frozen on dry ice. Frozen lungs were lyophilized and reweighed (dry weight). Ratios of dry lung weight-to-body
weight were then analyzed. Dried left lungs were hydrolyzed with 6
N HCl at 110°C for 72 h. Total protein, hydroxyproline, and desmosine and isodesmosine levels in protein hydrolysates were determined
using a Beckman 6300 amino acid analyzer as previously described
(4). Each group analyzed contained a minimum of three animals.
Static lung compliance assessment. Three-month-old mice from
all three genotypes were euthanized with pentobarbital, and the
tracheas were cannulated through a neck incision. The lungs were
not flushed with solution to avoid altering surfactant and thereby
the alveolar air-liquid interface. Lungs were removed en bloc with
the cannula in situ and placed in a humidity chamber to avoid
desiccation. The cannula was connected via a three-way stopcock
system to a graduated syringe and a modified pressure arteriograph
(Pressure Myograph System P-100; Danish Myotechnology,
Copenhagen, Denmark) used previously in our laboratory (12, 54).
The equal volume method of determining lung history was used.
Briefly, all lungs were preconditioned with 3 inflation-deflation
cycles using 0.5 ml of air for each cycle. Starting at the residual
volume (no inflation, trachea open to atmospheric pressure) the
lungs were inflated in 0.1-ml increments (range: 0.1–1.2 ml) every
30 s to a maximum inflation volume of 1.2 ml [total lung capacity
(TLC) in C57Bl/6J mice: ⬃1.0 ml; Ref. 23]. Thereafter, lungs were
allowed to passively deflate in 0.1-ml decrements every 30 s.
Pressure and volume on inflation and deflation were recorded using
Myoview Acquisition software (Danish Myotechnology) and used
to produce a static pressure-volume curve. Expiratory curves are
represented. Specific compliance was determined by calculating
the slope (⌬V/⌬P) of the expiratory curve at functional residual
capacity (FRC)(⬃0.25 ml) (29, 41), whereas chord compliance
was calculated over 100-␮l increments at inflation volumes ranging from 200 ␮l to 1,100 ␮l. Each group analyzed contained a
minimum of five animals.
Respiratory system compliance assessment. Mice from all three
genotypes were anesthetized using sodium pentobarbital, and the
tracheas were cannulated through a neck incision. Regular quasisinusoidal mechanical ventilation was achieved using a volumecontrolled model 687 ventilator (Harvard Apparatus, Holliston,
MA) delivering a respiratory rate of 160 breaths/min and a tidal
volume of 5 ml/kg. PEEP was kept at 0 throughout. Briefly, the
whole-body pressure plethysmography chamber (PLY3111 for
mice; Buxco Electronics, Sharon, CT), was calibrated for both
pressure and flow. Pressure was calibrated with the use of a manual
manometer after excluding leaks. The preamplifier signal was
adjusted to 0 V and 20 cmH2O pressure was applied to the system.
Peak steady voltage was defined as 20 cmH2O. Flow was calibrated
by excluding leaks, zeroing the preamplifier voltage, and steadily
injecting 1 ml of air into the sealed chamber. Peak flow was
determined by the software and accepted if the flow met the
effective range for the chamber (10 –20 ml/s). Each ventilated
mouse was placed in the plethysmography chamber, and an esophageal catheter was placed for monitoring changes in pleural pressure. The chamber-pressure-time wave was continuously measured
via a transducer connected to a computer data acquisition system
running Biosystem XA software (Buxco Electronics) (15, 29).
Real-time readouts of all measured variables (respiratory system
compliance, airway resistance, peak airway pressures, and peak
expiratory flows) were provided by the data acquisition system,
and all animals were carefully monitored over a period of 3 min
during which all readings were recorded. The pressure equation for
the lung states that total measured pulmonary pressure (Ptot) is the
sum of the elastic (Pel) and resistive (Pres) pressures of the
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normal, mature fibers seen in acquired emphysema. Genetic
disorders affecting many of these same elastic fiber proteins
have also been linked to emphysema-like disease in humans.
Marfan syndrome, for example, caused by dominant mutations
in the fibrillin-1 gene, results in emphysema in a subset of adult
Marfan syndrome patients. Similarly, mutations in fibulin-4
and -5 have recently been linked to severe emphysema-like
lung disease in some individuals (17, 22, 53). Mutations in
elastin that result in the synthesis and incorporation of mutant
tropoelastin into the extracellular matrix have been linked to
emphysema in individuals with cutis laxa (39, 53).
Whereas the reports cited above describe emphysematous
changes in the lung as a result of alterations in elastin quality,
in this report we show that elastin quantity is a more important
modifier of susceptibility to disease than has been heretofore
appreciated. Lower than normal levels of elastin resulting from
genetic or environmental abnormalities, or elastic fibers that
are functionally compromised because of mutations within
elastin or elastic fiber genes, may convey a predisposition to
emphysema when the lung is challenged by insults such as
cigarette smoke or oxidant injury. Furthermore, genetic disorders that compromise elastic fiber deposition and function
during lung maturation may contribute to neonatal alveolar
septation defects that manifest as developmental emphysema.
The identification of elastin and elastic fiber proteins as modifiers of predisposition to emphysema is important in identifying underlying mechanisms leading to human disease and in
the discovery of markers that can be used as predictors of
disease susceptibility.
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segments subtending alveolar walls at both ends (9, 16, 37). Chord
length measurement was chosen over mean linear intercept as it
has the advantage of being independent of alveolar septal thickness. A minimum of 10 fields per slide, from each of 4 slides per
animal (total ⫽ 40 fields per animal) were measured. Each group
analyzed contained a minimum of six animals.
Macrophage immunohistochemistry. Macrophage staining was performed with rat anti-mouse Mac-3 antibody (BD Pharmingen, San
Diego, CA). Primary antibody (1 ␮g/ml) or diluting buffer (negative
control) was applied to the sections and incubated overnight in a
humidity chamber at 4°C. The sections were developed with the
Vector ABC Kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine applied for 30 – 60 s (16, 47). The slides were counterstained with 2% Gill’s hematoxylin (Fisher Scientific). Lung fields for
analysis were selected using a random number generator and random
starting points. The number of Mac-3-positive cells in each field was
counted manually in a blinded fashion. Counts included both tissue
and alveolar macrophages. A minimum of 6 fields per slide, and 4
slides per animal (total ⫽ 24 fields per animal) were counted. Each
group analyzed contained a minimum of six animals. All counts were
normalized to alveolar wall length to account for tissue loss in
emphysematous lungs.
Statistical analyses. Data analysis was performed using one-way
analysis of variance (ANOVA), to determine the differences between
⬎2 groups of data, and Student’s t-tests to determine differences
between pairs of data. All results are presented as mean values ⫾ SE.
P values ⬍0.05 were chosen as the threshold for statistically significant differences.
RESULTS
Elastin content in Eln⫹/⫺ and hBAC⫺/⫺ lungs is reduced.
Three groups of mice with differing levels of lung elastin
(Table 1) were used in this study. All animals were on the
C57BL/6 background and were maintained under identical
conditions. Mice bearing a heterozygous deletion of exon 1 of
the elastin gene (Eln⫹/⫺) (30) have lung elastin levels that are
45% lower than WT mice (P ⫽ 0.01). The third group of mice
was generated using a BAC containing the complete human
elastin gene. The BAC transgene was expressed in founder
animals, which were then bred with Eln⫹/⫺ mice to produce
mouse elastin null/human elastin transgene-positive animals
(hBAC⫺/⫺). These mice are unique in that the human elastin
transgene rescues the perinatal lethal elastin null (Eln⫺/⫺)
Table 1. Pulmonary elastin content is reduced in Eln⫹/⫺
and hBAC⫺/⫺ mice
Elastin content, pmol
(desmosine ⫹ isodesmosine)/g
lung weight
% Elastin
Collagen content, nmol
hydroxyproline/g lung weight
% Collagen
WT
Eln⫹/⫺
hBAC⫺/⫺
118⫾5
100
65⫾2*
55
40⫾3**
37
65⫾2
100
65⫾3
100
58⫾2***
90
Values are means ⫾ SE. Left lungs from all 3 genotypes were weighed,
hydrolyzed, and analyzed for total elastin (desmosine and isodesmosine) and
collagen (hydroxyproline) content. Elastin content is expressed as picomole of
desmosine and isodesmosine per gram of wet lung weight, and collagen
content as nanomole of hydroxyproline per gram of wet lung weight; n ⫽ 3
mice per group. *P ⫽ 0.01 vs. wild-type (WT) controls; **P ⫽ 0.006 vs. WT
controls; ***P ⫽ 0.03 vs. WT controls. Eln⫹/⫺, mice bearing a heterozygous
deletion of exon 1 in the elastin gene; hBAC⫺/⫺, mice that are human Eln
transgene-positive, mouse Eln gene null.
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respiratory system (Ptot ⫽ Pel ⫹ Pres). This equation can be
expanded to: C ⫽ V/(P ⫺ F ⫻ R), where C ⫽ compliance, V ⫽
volume, P ⫽ pressure, F ⫽ flow, and R ⫽ resistance. From this
formula, we understand that compliance is directly related to
volume and resistance but inversely related to pressure. Each group
analyzed contained a minimum of four animals.
Stress-strain measurements. Three-month-old mice from all three
genotypes were euthanized with sodium pentobarbital. The lungs were
removed from the thoracic cavity, placed into balanced physiological
saline (130 mM NaCl, 15 mM NaHCO3, 5.5 mM dextrose, 4.7 mM
KCl, 1.2 mM MgSO4 䡠 7H2O, 1.2 mM KH2PO4, 0.026 mM EDTA,
and 1.6 mM CaCl2, pH 7.2) on ice, and separated into individual
lobes. Lung parenchymal strips were obtained by cutting tissue strips
from the long axis of each lobe. Tissue strips were fixed to adjustable
metal posts mounted on a force transducer (Danish Myotechnology).
A central region along the attached tissue strip length was chosen for
diameter and length measurements. Tissue strip length was adjusted
manually using a micrometer attached to one of the metal posts. The
starting length (inter-post distance; l0) and cross-sectional area (A0)
for each strip were determined after 3 preconditioning cycles from 0
to 0.1 mm. At the starting length, the force was set to 0 and the strips
were stretched from 0 to 0.4 mm (or until yield strength was exceeded) in steps of 0.02 mm for 45 s/step to allow for force equilibration at each new length. At 45 s the force was recorded. All
measurements were performed in a heated tissue bath containing
balanced physiological saline at 37°C within 1 h of death. The length
and force measurements were normalized to obtain stress (␴) and
stretch ratio (␭) as ␴ ⫽ F/A0; ␭ ⫽ (l ⫹ l0)/l0, where l is stretched
length, l0 is starting length of the strip, F is measured force, and A0 is
cross-sectional area of the strip corresponding to l0. The elastic
modulus (Y) of each tissue was calculated by determining the slope of
the stress-strain curves (⌬␴/⌬␭) over known changes in measured
length (l). Each group analyzed contained a minimum of four animals
and utilized at least one strip per lobe from each animal.
Smoke exposure. Three-month-old WT and Eln⫹/⫺ mice were
exposed to cigarette smoke from two filtered 2R4F University of
Kentucky research cigarettes (cigarette weight 1,060 mg, filter ventilation 28%, total particulate matter per cigarette ⫽ 13.6 mg) per day,
6 days per week, for a total of 6 mo with the use of a nose-only
smoking apparatus adapted for mice (16, 48). Cigarettes were presented sequentially, and mice remained in the smoking chambers until
all smoke flow had ceased following the burning of both cigarettes.
Each group contained a minimum of six animals.
Morphometric analysis of alveoli. Mice from all three genotypes
were euthanized, and the lungs were inflated (without deaeration)
through the trachea to 25 cm water pressure with 10% neutral
buffered formalin (Fisher Scientific, Pittsburgh, PA). Lungs were
removed en bloc, formalin-fixed for 24 h, and embedded in paraffin
(59). Serial 5-␮m-thick parasagittal sections were stained with
hematoxylin and eosin (H&E), Hart stain, and periodic acid-Schiff
stain. For chord length measurements, H&E sections were utilized
exclusively. Lung images were sampled in a blinded fashion from
all lobes at ⫻200 magnification using a random number generator
and random starting points. Only sections showing obviously gross
preparation artifact or distortion were excluded from analysis.
Images were digitally captured in eight-bit grayscale using a Zeiss
Axioskop microscope with an inline Zeiss Axiocam camera and
Axiovision software (all Carl Zeiss, Thornwood, NY). Chord
length analysis was performed using NIH Image software (version
1.63; National Institutes of Health, Bethesda, MD) with a chord
length macro (available at http://rsb.info.nih.gov/nih-image). Binarized, inverted lung micrographs were manually thresholded and
subjected to a logical “AND” operation with horizontal and vertical grids of parallel lines. The line lengths overlying alveolar
spaces were measured and averaged to yield mean chord length.
Chord lines touching the edges of the image fields were excluded
from analysis to ensure that mean chord length included only line
ELASTIN AND EMPHYSEMA SUSCEPTIBILITY
animals (manuscript in preparation). hBAC⫺/⫺ animals have
lung elastin levels that are ⬃65% lower than those found in
WT animals (P ⫽ 0.006). Lung collagen levels (Table 1)
expressed as a measure of wet lung weight demonstrated no
differences between WT and Eln⫹/⫺ animals, but hBAC⫺/⫺
lungs showed significantly decreased collagen levels (P ⫽
0.03).
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Pulmonary structure is similar in WT and Eln⫹/⫺ mice.
Lungs from WT and Eln⫹/⫺ mice were inspected macroscopically for evidence of gross anomalies. Ex vivo appearance of
WT and Eln⫹/⫺ lungs was identical (Fig. 1A), and no differences in lobe number or major airway branching pattern were
noted between the genotypes (data not shown). Lung weights
normalized to body weights showed a trend towards decreased
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Fig. 1. Lung development is dependent on pulmonary elastin content. Lungs from 3-mo-old wild-type (WT; labeled as Eln⫹/⫹), Eln⫹/⫺, and hBAC⫺/⫺ mice were
inflation-fixed with 10% neutral buffered formalin at 25 cmH2O and removed en bloc (A). Ruler scale ⫽ centimeters. Representative hematoxylin and eosin
(H&E)-stained sections (B) are shown for all 3 groups of animals. Scale bar ⫽ 200 ␮m. Chord length measurements (C) from WT (white bar), Eln⫹/⫺ (black
bar), and hBAC⫺/⫺ (gray bar) mice were used to quantify alveolar size. N ⫽ 6 mice per group. Error bars represent SE. *P ⬍ 0.0001 vs. WT controls. Eln⫹/⫺,
mice bearing a heterozygous deletion of exon 1 in the elastin gene; hBAC⫺/⫺, mice that are human Eln transgene positive, mouse Eln gene null, generated using
a human bacterial artificial chromosome (BAC) clone (CTB-51J22) containing the complete human elastin gene and the human elastin gene promoter.
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ELASTIN AND EMPHYSEMA SUSCEPTIBILITY
lung weight in Eln⫹/⫺ mice that did not reach statistical
significance (P ⫽ 0.17). Analysis of lung volume data showed
evidence of increased lung volume in Eln⫹/⫺ animals (0.94 ⫾
0.02 ml) compared with WT littermate controls (0.90 ⫾ 0.02
ml), but the difference was not statistically significant (P ⫽
0.29). There was no obvious difference in large airway morphology between Eln⫹/⫺ and WT controls as assessed by
histochemical staining. Alveolar architecture (Fig. 1B) appeared identical in WT and Eln⫹/⫺ mice, and there was no
difference in chord length between 3-mo-old WT and Eln⫹/⫺
animals (31.9 ⫾ 1.0 vs. 31.9 ⫾ 1.4 ␮m; Fig. 1C).
Eln⫹/⫺ mouse lungs are more susceptible to cigarette
smoke-induced lung injury. To determine how elastin insufficiency impacts the ability of the lung to tolerate injury, WT
and Eln⫹/⫺ animals were exposed to cigarette smoke twice
per day for 6 mo. Cigarette smoke exposure was well
tolerated by both genotypes over the entire 6-mo exposure.
One WT animal developed a pulmonary carcinoma and was
excluded from all analyses. Figure 2 shows representative
lung fields from smoked and nonsmoked WT and Eln⫹/⫺
mice with corresponding chord length measurements. Agematched nonsmoking controls from both strains showed no
significant difference in their chord lengths (WT ⫽ 31.4 ⫾
0.7 ␮m, and Eln⫹/⫺ ⫽ 31.5 ⫾ 0.9 ␮m; P ⫽ 0.37). These
values were designated as baseline chord length. The lack of
change in chord length between 3 and 9 mo in either group
of nonsmoking animals indicates that degenerative change is
not a factor in this model over the time course studied.
Emphysema was readily apparent in both strains of smoked
mice as evidenced by destruction of both alveolar sac and
alveolar ductal walls (Fig. 2A). However, air space enlargement secondary to alveolar septal destruction in Eln⫹/⫺
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Fig. 2. Eln⫹/⫺ mice develop greater air space
enlargement following cigarette smoke exposure than do WT mice. After exposure to 6
mo of cigarette smoke, mouse lungs were
inflation-fixed with 10% neutral buffered formalin at 25 cmH2O and evaluated for alveolar
enlargement. Representative H&E-stained
sections (A) are shown for all 4 groups of
animals. Scale bar ⫽ 200 ␮m. Chord length
measurements (B) were used to quantify alveolar space enlargement and are reported for
smoking and nonsmoking WT (white bars)
and Eln⫹/⫺ (black bars) mice. N ⫽ 6 mice per
group. Error bars represent SE. *P ⬍ 0.0001
vs. respective nonsmoking genotype controls;
**P ⬍ 0.001 vs. WT smokers.
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ELASTIN AND EMPHYSEMA SUSCEPTIBILITY
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Fig. 4. Pulmonary compliance is inversely related to lung elastin content.
Static pressure-volume curves (A) were constructed by inflating ex vivo lungs
from WT (solid line), Eln⫹/⫺ (dashed line), and hBAC⫺/⫺ (dotted line) mice in
0.1-ml increments and measuring the resulting pressures. N ⫽ 5 mice per
group. Error bars represent SD. Chord compliance (B) was calculated at 0.1-ml
intervals for WT (white bars), Eln⫹/⫺ (black bars), and hBAC⫺/⫺ (gray bars)
animals, allowing for comparison at low volumes, mid-volumes, and high
volumes. N ⫽ 5 mice per group. *P ⬍ 0.01 vs. WT controls. **P ⬍ 0.001 vs.
WT controls and ⬍0.003 vs. Eln⫹/⫺ mice. Respiratory system compliance (C)
was evaluated in ventilated WT (white bar), Eln⫹/⫺ (black bar), and hBAC⫺/⫺
(gray bar) mice using whole body plethysmography. N ⫽ 4 mice per group.
Error bars represent SE. *P ⬍ 0.0001 vs. WT controls; **P ⬍ 0.0001 vs.
Eln⫹/⫺ animals. Cresp, respiratory system compliance; Cstat, chord compliance.
Fig. 3. Smoke-induced recruitment of pulmonary macrophages is augmented
in Eln⫹/⫺ mice compared with WT controls. Lung sections from smoked and
nonsmoked WT (white bars) and Eln⫹/⫺ (black bars) mice were immunostained for macrophages using Mac-3 antibody. Lung macrophage counts are
expressed as macrophages per high-powered field adjusted for alveolar wall
length. N ⫽ 6 mice per group. Error bars represent SE. *P ⬍ 0.01 vs.
respective nonsmoking genotype controls; **P ⫽ 0.02 vs. WT smokers.
AJP-Lung Cell Mol Physiol • VOL
staining techniques, were identical to those in both Eln⫹/⫺and
WT animals.
Lung compliance is increased in mice deficient in elastin.
Figure 4A shows the expiratory static pressure-volume curves
of isolated WT, Eln⫹/⫺, and hBAC⫺/⫺ mouse lungs. At all
given volumes, the pressure-volume curve in Eln⫹/⫺ lungs is
shifted to the left compared with WT controls, indicating that
the lung elastic recoil pressure in Eln⫹/⫺ lungs is reduced. The
hBAC⫺/⫺ mouse shows the lowest elastic recoil pressures of all
three genotypes and similarly has the greatest lung compliance,
irrespective of lung volume. Calculation of specific lung com292 • MARCH 2007 •
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mice (43.3 ⫾ 1.2 ␮m) was more extensive than that in WT
controls (38.1 ⫾ 0.9 ␮m) (P ⬍ 0.0001; Fig. 2B).
Eln⫹/⫺ mouse lungs demonstrate an augmented inflammatory response to cigarette smoke. Macrophages play an important role in the pathogenesis of emphysema and account for
many known features of the disease (13, 14, 16, 44). Macrophages are known to localize to the sites of alveolar wall
destruction, and correlation between macrophage numbers and
disease severity is well documented (10). The pulmonary
macrophage response to 6 mo of cigarette smoke exposure in
both genotypes of mice is shown in Fig. 3. Comparison of
macrophage number normalized to alveolar surface length
revealed no significant difference between Eln⫹/⫺ and WT
nonsmoking controls (3.2 ⫾ 0.31 vs. 3.2 ⫾ 0.34 cells/mm
alveolar wall). However, in smokers, normalized macrophage
number was significantly increased in both Eln⫹/⫺ (12.4 ⫾
0.44 cells/mm alveolar wall) and WT lungs (10.5 ⫾ 0.48
cells/mm alveolar wall) (P ⬍ 0.01). Whereas both genotypes
showed a marked influx of macrophages into the lung, the
response in Eln⫹/⫺ mice was 1.25⫻ greater than smoked WT
controls (P ⫽ 0.02).
Low levels of lung elastin result in congenital emphysema.
In contrast to the normal size and lung morphology of WT and
Eln⫹/⫺ mice, 3-mo-old hBAC⫺/⫺ mice demonstrated grossly
enlarged thoraces and lungs with evidence of marked volume
increase (Fig. 1A). Analysis of lung volume data showed that
lung volume in the hBAC⫺/⫺ mice (1.41 ⫾ 0.01 ml) was
significantly greater than both the WT and Eln⫹/⫺ animals
(P ⬍ 0.0001). Microscopic examination revealed obvious air
space enlargement (Fig. 1B), and chord length analysis yielded
a mean chord length of 57.4 ⫾ 1.9 ␮m, an increase of 165%
compared with WT control mice (P ⬍ 0.0001) (Fig. 1C).
Closer inspection showed that enlargement of alveoli subtending alveolar ducts occurred in addition to that occurring in the
alveolar sacs. Larger airways, as evaluated by histochemical
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Fig. 5. Lung tissue stiffness increases with increasing pulmonary elastin
content. Stress-strain curves were obtained by stretching isolated lung tissue
strips from WT (solid line), Eln⫹/⫺ (dashed line), and hBAC⫺/⫺ (dotted line)
mice in 20-␮m increments and measuring the resulting forces. N ⫽ 4 mice per
group. Error bars represent SE.
tionship between elastin dosage and mechanical stiffness with
WT tissue stiffness (100% elastin dosage; Y ⫽ 9.25 N/cm2)
being ⬃1.7⫻ greater than Eln⫹/⫺ tissue stiffness (⬃55%
elastin dosage; Y ⫽ 5.16 N/cm2), which is ⬃1.4⫻ greater than
hBAC⫺/⫺ tissue stiffness (⬃35% elastin dosage; Y ⫽ 3.35
N/cm2) (P ⫽ 0.004). Thus the incremental elastic modulus (Y)
of lung tissue is decreased as elastin content is lowered,
indicating that the lung tissue stiffness is reduced when lung
elastin is lacking.
DISCUSSION
A critical elastin protein level is required for normal lung
development. Elastin is known to have a pivotal role in lung
development. In mice deficient for elastin (Eln⫺/⫺), or in mice
deficient in key growth factors such as platelet-derived growth
factor that have an effect on the onset of elastin expression,
perinatal development of terminal airway branches is arrested,
resulting in distal air sacs that are dilated with attenuated tissue
septae (3, 55). Our data show that mice with elastin levels
about half those found in WT animals (Eln⫹/⫺) have lungs that
are macroscopically normal, with normal lobe numbers and
major airway branching patterns (see also Ref. 55). On microscopic examination, neither the pulmonary parenchyma nor the
resident inflammatory cells offer an obvious difference from
the WT controls, and chord length measurements show no
difference between the two genotypes. This is in contrast to
what was found for the hBAC⫺/⫺ animals where lung morphology was grossly abnormal. These animals, with approximately one-third normal elastin expression levels, manifest
with severe congenital emphysema that is present from birth
and is not progressive. In many respects, the lungs in these
animals resemble the dilated and thinner-walled lungs found in
elastin null (Eln⫺/⫺) animals (55) even though the hBAC⫺/⫺
animals are rescued from the perinatal lethality observed with
the Eln⫺/⫺ phenotype. The finding that development up to the
terminal bronchiole level is microscopically normal and that
alveoli at both the alveolar duct and alveolar sac levels are
perturbed suggests a defect in alveolar septal development and
alveolar growth, processes that are known to be driven by Eln
expression (31). Based on this data, we conclude that low
levels of elastin become a limiting factor in normal lung
development. It appears that a minimum amount of cross292 • MARCH 2007 •
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pliance at FRC showed that lung compliance for WT ⫽ 0.03
ml/cmH2O, Eln⫹/⫺ ⫽ 0.05 ml/cmH2O, and hBAC⫺/⫺ ⫽ 0.09
ml/cmH2O (P ⬍ 0.01).
Chord compliance (Fig. 4B), calculated from the slopes of
the curves (⌬V/⌬P) over 100-␮l increments, indicate that at
low lung volumes, Eln⫹/⫺ lungs have much greater compliance
than WT lungs. However, at high volumes, this difference is no
longer evident. Similarly, at both low and high lung volumes
the compliance of Eln⫹/⫺ lungs tends to remain constant,
whereas compliance of the WT lungs increases at higher
volumes. hBAC⫺/⫺ lungs demonstrate the greatest chord compliance at any given lung volume. In addition, chord compliance in these mice tends to remain constant at all measured
lung volumes.
Whole body plethysmography was used to measure respiratory system compliance (Cresp) in mechanically ventilated
animals (Fig. 4C). The chest wall and thoracic structures in
mice are extremely compliant, with almost all of the elastic
recoil of the respiratory system attributable specifically to the
lung (23, 29). Since chest wall compliance is not a significant
factor in our model, respiratory system compliance measurements in WT and Eln⫹/⫺ animals (0.01 ⫾ 0.008 and 0.02 ⫾
0.001 ml/cmH2O; P ⬍ 0.001) support our static compliance
findings that lung compliance at FRC in Eln⫹/⫺ animals is
approximately double that found in WT controls. Similarly,
hBAC⫺/⫺ animals had the greatest Cresp (0.03 ⫾ 0.001 ml/
cmH2O; P ⬍ 0.0001) as would be expected in animals with
grossly emphysematous lungs.
Tidal volumes in WT (0.15 ⫾ 0.006 ml) and Eln⫹/⫺ (0.15 ⫾
0.006 ml) mice were identical (P ⫽ 0.45) as would be expected
with volume-controlled ventilation. The pressure equation predicts the maximum pressure change during tidal volume ventilation in WT lungs (1.59 ⫾ 0.010 cmH2O) to be greater than
that in Eln⫹/⫺ lungs (1.35 ⫾ 0.007 cmH2O), as was indeed the
case (P ⬍ 0.0001). Pulmonary resistance contributes significantly to airway pressure and may account for observed pressure differences during ventilation. Measurement of pulmonary
resistance demonstrated no difference between WT (0.95 ⫾
0.012 cmH2O䡠s⫺1 䡠ml⫺1) and Eln⫹/⫺ (0.95 ⫾ 0.002
cmH2O䡠s⫺1 䡠ml⫺1) lungs (P ⫽ 0.48), eliminating pulmonary
resistance as a contributing factor to the different lung pressures. These dynamic measurements suggest, therefore, that
measured pressure differences are a function of different lung
compliances. WT mice (0.08 ⫾ 0.0008 cmH2O 䡠ml⫺1 䡠s⫺1)
perform less work of breathing during tidal volume ventilation
than Eln⫹/⫺ mice (0.07 ⫾ 0.0004 cmH2O䡠ml⫺1 䡠 s⫺1) (P ⬍
0.001). Since it requires less energy to ventilate more compliant lungs at similar volumes, the data suggest that WT and
Eln⫹/⫺ lungs operate at similar lung volumes, with the higher
compliance of Eln⫹/⫺ lungs leading to lower physiological
ventilation pressures (as opposed to similar pressures at different lung volumes).
Eln deficiency markedly alters the tissue properties of isolated lung strips. Figure 5 shows the stress-strain curves of
isolated lung tissue strips from WT, Eln⫹/⫺, and hBAC⫺/⫺
lungs at stress and strain amplitudes approximating the range
experienced during tidal volume breathing at FRC. The net
effect of decreasing total lung elastin content is a shifting of the
stress-strain curve to the right.
Calculation of incremental elastic modulus (Y) at intermediate strains indicated that around FRC there is a direct rela-
ELASTIN AND EMPHYSEMA SUSCEPTIBILITY
AJP-Lung Cell Mol Physiol • VOL
volumes (different slopes) with little or no contribution occurring at higher lung volumes since the curves are in fact parallel
(equal slopes). Furthermore, the data in the Eln⫹/⫺ animals
(Fig. 4B) supports the finding that collagen is an important
determinant of lung compliance even at low lung volumes (45,
57), especially when elastin is deficient. hBAC⫺/⫺ lungs show
the greatest chord compliance at all lung volumes (Fig. 4B),
with chord compliance tending to remain constant irrespective
of inflation volume. This suggests that the low levels of elastin
in these animals may have little effect on the mechanical
properties of the lung at any given volume and that collagen
may be the primary matrix determinant of compliance in these
animals.
Studies of lung deflation from TLC have shown that
elastic recoil pressure decreases notably in fluid-filled lungs
compared with air-filled controls, implying that the surface
film contributes significantly to lung elasticity (1, 2). More
recent work, however, has shown that for small pressurevolume changes like those occurring with tidal breathing,
the hysteresis of the surface film is small and that surface
active forces are less important determinants of mechanics
than lung tissue viscoelasticity (43). Similar findings were
observed in experiments comparing isolated lungs with lung
tissue strips lacking an air-liquid interface (40). By measuring dynamic respiratory system compliance at tidal-breathing volumes, the effect of surfactant is effectively negated,
suggesting that the observed differences in our animal
model result almost exclusively from the viscoelastic properties of the lung and are therefore due to the decreased
elastin content of the Eln⫹/⫺ mouse lung tissue. The absolute values for dynamic compliance differ from the static
measurements due to differences in experimental technique
(dynamic measurement within an intact chest static vs. static
measurements on isolated lung). However, the data demonstrate that the relationship between decreased elastin content
and increased lung compliance persist despite differences in
measurement technique.
Eliminating the effects of the air-liquid interface critical for
surfactant function using isolated lung tissue strips in a tissue
bath allowed us to focus primarily on the role of the extracellular matrix in lung mechanics. Previous studies have confirmed that the elastin-collagen fiber network dominates the
macroscopic elastic properties of lung tissue strips (57), making this model relevant to investigating the role of elastin
content in lung mechanics. To ensure the physiological relevance of our results, we utilized stress and strain amplitudes
approximating the range experienced during tidal volume
breathing around FRC. Our data strongly suggest that pulmonary elastin content contributes to lung tissue elasticity across
the range of applied strains corresponding to FRC. Since
incremental loss of lung tissue elastin leads to progressive
decreases in tissue stiffness, one would hypothesize that lungs
with decreased elastin content would be progressively more
compliant if tissue properties are an important determinant of
lung mechanical behavior. This hypothesis is confirmed by
both the static and respiratory system compliance measurements.
Altered elastin gene dosage in human disease. Our studies in
mice relate directly to humans where elastin insufficiency can
result directly from genetic factors such as loss of function
mutations in the elastin gene and indirectly through elastin insuf292 • MARCH 2007 •
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linked elastin is required to sustain the scaffolding needed for
functional alveolar formation and growth. Abnormalities in
lung signaling related to elastin and extracellular matrix composition may also play a role.
Altered Eln gene dosage affects pulmonary responses to
injury. Mice heterozygous for the elastin gene develop worse
emphysema when exposed to cigarette smoke than do WT
mice (Fig. 2), suggesting that levels of elastin are an important
determinant of susceptibility to lung injury. By histological
analysis, Eln⫹/⫺ animals showed an almost twofold increase in
air space enlargement after smoking compared with WT controls. Our data also indicate that Eln⫹/⫺ mice have half the
amount of cross-linked elastin as WT mice, suggesting that
there is less enzyme substrate present for smoke-induced degradative proteases, resulting in more significant fiber destruction given the same degree of injury. Lower than normal
amounts of elastin may also make the lung more susceptible to
injury by mechanisms other than proteolysis, such as oxidant/
antioxidant imbalance (8, 18, 19, 42). It is interesting that
Eln⫹/⫺ mice develop more severe macrophage inflammation
during smoking than do WT mice. Monocyte influx into the
lung is mediated to a large extent by soluble elastin peptides
generated through degradation of insoluble elastin fibers by
macrophage-derived proteases (20). Thus the higher number of
macrophages and the more severe emphysema observed in the
smoking Eln⫹/⫺ animals is consistent with enhanced monocyte
recruitment by elevated levels of elastin peptides.
Another likely factor in the propagation of lung damage in
Eln⫹/⫺ mice is force-induced alveolar destruction resulting
from the altered mechanical properties of the elastin-deficient
lung. This is consistent with the model by Suki et al. (52)
wherein protease-induced degradation of elastic fibers leads to
their mechanical failure and, ultimately, to collapse of the
alveolar wall. Alveolar wall collapse, in turn, results in the
progression of emphysema. It seems likely, given the material
properties of the Eln⫹/⫺ mouse lung (that experiences greater
tissue strain for any given degree of tissue stress) that the
alveolar elastic fibers in Eln⫹/⫺ mice experience more stress
under progressive loading than WT animals, predisposing them
to greater damage following proteolysis as a consequence of
mechanical forces.
Altered elastin gene dosage affects pulmonary mechanical
function. Static pressure-volume analysis showed important
mechanical differences when lungs from Eln⫹/⫺ animals were
compared with WT lungs. The preserved collagen content and
reduced elastin content in the Eln⫹/⫺ lungs results in an altered
collagen-elastin ratio of the lung parenchyma, an alteration that
would be expected to lead to a change in mechanical properties
of the lung. Because elastic fibers account for lung compliance
changes occurring around FRC, with collagen acting to limit
TLC, an Eln⫹/⫺ mouse lung would be expected to show a
decrease in elasticity and therefore an increase in lung compliance, predominantly at lower lung volumes. This is indeed
the case in our animals (Fig. 4B), where inflation of Eln⫹/⫺
mouse lungs generates lower pressures around FRC than the
WT controls. Conversely, around TLC the compliance is similar to that in WT mice, suggesting that the preserved collagen
content of the Eln⫹/⫺ phenotype has the predicted effect on
limiting lung expansion. The data also suggest that the leftward
shift of the entire pressure volume curve in the Eln⫹/⫺ lungs
(Fig. 4A) is primarily due to changes in the curve at low lung
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that a second higher threshold is, in turn, necessary for timely
and efficient response to lung injury. When elastin levels are
below this second critical threshold, the response is not productive and the lungs are more prone to undergo accelerated
alveolar destruction leading to emphysema.
ACKNOWLEDGMENTS
We thank Terese Hall for administrative assistance and Chris Ciliberto
for expertise with animal care and technical support. We also gratefully
acknowledge helpful discussion from Jack Pierce, Robert Senior, and
Jessica Wagenseil. Current address: Anthony G. Durmowicz, Federal Drug
Administration, Division of Pulmonary and Allergy Products, 1098 New
Hampshire Ave., Building 22, Room 3375, Silver Spring, MD 20993-0002.
GRANTS
The research reported in this manuscript was supported by National Heart,
Lung, and Blood Institute Grants HL-53325, HL-71960, and HL-74138 to
R. P. Mecham, and American Lung Association Grant RT-9702-N to A.
Shifren. We also thank the Mouse Core supported by PPG-HL29594 for
assistance with the smoking protocols.
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