Am J Physiol Lung Cell Mol Physiol 309: L1239–L1272, 2015.
First published September 11, 2015; doi:10.1152/ajplung.00268.2015.
Recent advances in the mechanisms of lung alveolarization and the
pathogenesis of bronchopulmonary dysplasia
Diogo M. G. Silva,1,2 Claudio Nardiello,1,2 Agnieszka Pozarska,1,2 and Rory E. Morty1,2
1
Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center (UGMLC), Member of the
German Center for Lung Research (DZL), Giessen, Germany; 2Department of Lung Development and Remodelling, Max
Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
Submitted 5 August 2015; accepted in final form 9 September 2015
Silva DM, Nardiello C, Pozarska A, Morty RE. Recent advances
in the mechanisms of lung alveolarization and the pathogenesis of
bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol
309: L1239 –L1272, 2015. First published September 11, 2015;
doi:10.1152/ajplung.00268.2015.—Alveolarization is the process by
which the alveoli, the principal gas exchange units of the lung, are
formed. Along with the maturation of the pulmonary vasculature,
alveolarization is the objective of late lung development. The terminal
airspaces that were formed during early lung development are divided
by the process of secondary septation, progressively generating an
increasing number of alveoli that are of smaller size, which substan-
tially increases the surface area over which gas exchange can take
place. Disturbances to alveolarization occur in bronchopulmonary
dysplasia (BPD), which can be complicated by perturbations to the
pulmonary vasculature that are associated with the development of
pulmonary hypertension. Disturbances to lung development may also
occur in persistent pulmonary hypertension of the newborn in term
newborn infants, as well as in patients with congenital diaphragmatic
hernia. These disturbances can lead to the formation of lungs with
fewer and larger alveoli and a dysmorphic pulmonary vasculature.
Consequently, affected lungs exhibit a reduced capacity for gas
exchange, with important implications for morbidity and mortality in
the immediate postnatal period and respiratory health consequences
that may persist into adulthood. It is the objective of this Perspectives
article to update the reader about recent developments in our under-
standing of the molecular mechanisms of alveolarization and the
pathogenesis of BPD.
bronchopulmonary dysplasia; persistent pulmonary hypertension of
the newborn; pulmonary hypertension; lung development; alveolar-
ization; hyperoxia
THE TRANSPORT OF OXYGEN from the atmosphere into the blood-
stream, and conversely carbon dioxide from the bloodstream
into the atmosphere, is the primary function of the lung. To
optimize gas exchange, the barrier across which gas exchange
takes place, the alveolo-capillary barrier, must have a surface
area as large as possible and must be as thin as possible. Thus
the objective of lung development is to generate this optimized
gas-exchange structure (131, 358). The development of the
mammalian lung is broadly divided into two phases, namely,
early lung development (357), when the core lung structure
containing the branching conducting airways with their atten-
dant vasculature develops. This is followed by the structural
refinement of the gas-exchange surface, through the generation
of alveoli, the principal gas-exchange units of the lung. The
Address for reprint requests and other correspondence: R. Morty, Dept. of
Lung Development and Remodelling, Max Planck Institute for Heart and Lung
Research, Parkstrasse 1, D-61231 Bad Nauheim, Germany (e-mail:
rory.morty@mpi-bn.mpg.de).
http://www.ajplung.org 1040-0605/15 Copyright © 2015 the American Physiological Society
Perspectives
alveoli, which were first described by Marcello Malpighi in
1661 (210, 211), are generated by a process of secondary
septation that occurs during late lung development and are
uniquely designed to facilitate gas exchange (364). A recent
report has indicated that these two phases of lung development
are intimately coordinated (61).
Perturbations to lung development result in lungs that are
defective for gas exchange. This is exemplified in the clinical
setting by bronchopulmonary dysplasia (BPD) (169, 218), a
common complication of preterm birth originally described by
Northway and coworkers in 1967 (258). Preterm infants with
compromised respiratory function attributable to respiratory
distress syndrome (RDS) require oxygen supplementation, of-
ten by mechanical ventilation. In these infants, infection,
inflammation, oxygen toxicity, and volu- and baro-trauma
from ventilation, together with other factors, stunt the post-
natal maturation of the lung (154). This maturational stunt-
ing includes blunted alveolarization and the generation of a
dysmorphic pulmonary vasculature, accompanied by aber-
rant pulmonary vascular wall remodeling, causing pulmo-
nary hypertension (125). BPD is associated with significant
morbidity and mortality in the neonatal intensive care unit,
and survivors exhibit long-term consequences that persist
into adulthood. Similar disturbances to alveolar structure
and the pulmonary vasculature are noted in infants with
congenital diaphragmatic hernia (CDH) (317, 344). The
improved medical management of preterm birth has inter-
estingly led to an increase in the incidence of BPD (158,
159). This highlights the need to better understand normal
and aberrant late lung development. In particular, the mo-
lecular mechanisms underlying the associated blunted al-
veolarization and disturbed vascular development and ho-
meostasis associated with BPD remain largely unclear.
To this end, there is much activity within the lung develop-
ment and pediatric communities to better understand the mo-
lecular mechanisms of normal lung development (242) and the
factors that influence the postnatal maturation of the mamma-
lian lung (366). It is the objective of this Perspectives article to
update the reader about advances in our understanding of lung
alveolarization reported in the literature since the beginning of
2013. It is intended that this update will pick up where the last
update, published in 2013 (206), ended. This Perspectives
article will first address recent developments in methodologies,
including new state-of-the-art approaches to quantify the lung
architecture, as well as an overview and critique of the current
animal models employed to study alveolarization. These ele-
ments of this Perspectives article will be followed by a report
on novel players in normal and aberrant late lung development,
either identified by transgenic mouse approaches or pharma-
L1239
Perspectives
L1240 ADVANCES IN LUNG ALVEOLARIZATION AND BPD
cological interventions in animal models of arrested lung
development. Recent advances in our understanding of the
long-term sequelae of arrested alveolarization and the process
of neoalveolarization in developed lungs, as well as recent
advances in cell therapy for arrested alveolarization, will also
be addressed.
Quantitative Assessment of Lung Structure
Being able to robustly quantify structural changes to the lung
architecture is central to the study of normal and pathological
lung development (361). Historically, this has been performed
by many investigators using paraffin-embedded lung tissue,
which is then assessed by light microscopy, usually with the
determination of the mean linear intercept (MLI, or the related
mean chord length, Lm) or radial alveolar count (RAC) as a
surrogate for alveolar size and, hence, alveolar number. Addi-
tionally, the septal thickness has been directly determined,
usually at the “thinnest” point of the septal wall. While pro-
viding a wealth of meaningful and valuable data, this approach
is facing increasing valid criticism. Among the critiques are the
lack of randomization in the sampling, the introduction of
artifacts associated with nonuniform tissue dehydration and
rehydration (“shrinkage”) during classical fixations protocols,
and subsequent paraffin embedding. Another concern is the
range in hydrostatic pressure used for pressure fixation via the
trachea, with some investigators preferring 20 cmH2O,
whereas others employ 25 cmH2O. Presently, there is much
enthusiasm in the field for the implementation of improved
fixation and embedding techniques and stereological ap-
proaches to address these problems and to provide robust
quantitative data.
A very important study comparing three methods of lung
tissue preparation was published by Schneider and Ochs (309),
in which lung tissue was embedded in paraffin, in glycol
methacrylate alone (“simple”), or in glycol methacrylate with
osmium tetroxide and uranyl acetate. The authors documented
a 40% shrinkage of cut face areas in the paraffin-embedded
tissue, a 30% shrinkage with the simple glycol methacrylate,
and ⬍3% shrinkage with glycol methacrylate with osmium
tetroxide and uranyl acetate. The authors also documented the
superiority of glutaraldehyde-based fixative protocols. This
study clearly demonstrated the need to move from a paraffin-
based to a glycol methacrylate with osmium tetroxide and
uranyl acetate-based tissue-embedding system, with glutaral-
dehyde-based fixation. With this in mind, two review articles
have been published in the pages of the American Journal of
Physiology Lung Cellular and Molecular Physiology recently,
highlighting the challenges presently faced by investigators
interested in the quantitative determination of lung structure
and solutions to these challenges. These articles take a prob-
lem-based approach to illustrate basic principles of stereology
(267) and to highlight a stereological approach to the study of
lung structure (248). The stereological and morphological
analyses of lung tissue, including recent developments, have
also recently been reviewed (246, 247, 361).
Using a stereological approach, Herring and coworkers
(134) studied postnatal alveolar growth in humans aged 2 mo
to 15 yr and demonstrated that alveolar growth continues well
into adolescence. Furthermore, by comparing these data with
historical data derived from rats and Rhesus macaques, the
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00268.2015 • www.ajplung.org
investigators confirmed that Rhesus macaques represent a bet-
ter model for postnatal lung growth in humans than do rats.
Furthermore, the studies revealed that the kinetics of postnatal
lung growth and alveolarization in rats were more comparable
to prenatal lung growth and alveolarization in humans. This
idea that alveolarization persists into adolescence has been
supported by limited radiological evidence that alveolariza-
tion in school-age survivors of extreme prematurity and
term-born children is similar, suggesting that “catch-up”
alveolarization took place in the preterm group (252), al-
though the suitability of 3He diffusion MRI to reach this
conclusion has been questioned (271). As such, the idea of
catch-up alveolarization remains controversial. Together,
these observations reinforce the utility of combining state-
of-the-art lung structural analyses with alternative (radio-
logical or physiological) supportive approaches.
Presently, the use of stereological approaches to quantify
structures in the developing lung is used by relatively few
groups. This methodology has been pioneered by Tschanz and
coworkers (341) to identify a biphasic formation of new alveoli
during postnatal rat lung development, in which a bulk forma-
tion of new alveoli was observed between postnatal day 4 (P4)
and P21. This was followed by a second phase of continued
alveolarization between P21 and P60. The stereology approach
has also been applied to assess total alveoli number and mean
septal wall thickness after pharmacological interventions in
developing mouse pup lungs in a BPD model that relies on
exposure to hyperoxia (described in detail in the next section)
(207, 238) and to assess lung vessel wall thickness during
normal lung development in transgenic mice (205). Presently,
no stereological approach is available for the quantification of
capillaries in the lung. The development of such a methodol-
ogy is highly desirable, given the drawbacks of the currently
used surrogates. These surrogates include number of platelet/
endothelial cell adhesion molecule (PECAM), or von Wille-
brand factor (vWF), stained vessels per microscopic high-
power field. In those approaches, differences in lung inflation,
artifacts of fixation and embedding, and biased selection of
fields may impact the robustness of the data. The stereological
analysis of lung structure is both technically complicated (in
terms of lung sample preparation) and time consuming, which
has limited the enthusiasm of the lung community to embrace
the methodology despite the clear advantages to acquiring
robust, unbiased data. Looking to the future, the time con-
straints may be circumvented by automation of the tissue
section analysis. Efforts in this direction are already underway
by the Tremblay group (307); however, there is most likely a
long and difficult road ahead before this goal is attained to the
satisfaction of the stereology community.
Animal Models of Arrested Alveolarization
The study of lung development is entirely reliant on the use
of animal models, and a broad range of animal models has been
employed in the past (139). All the presently available BPD
animal models have been recently reviewed in the American
Journal of Physiology Lung Cellular and Molecular Physiol-
ogy, including the use of mice (33), rats (264), rabbits (80),
lambs (15), and baboons (377). Although it is generally ac-
knowledged that the large animal models, notably preterm
lambs and preterm baboons, probably represent the best mod-

ADVANCES IN LUNG ALVEOLARIZATION AND BPD
els of human disease, there is considerable cost and ethical
debate associated with the use of these models. For this reason,
the mouse and rat models have found the most widespread use.
The key advantages of the mouse and rat models are the
relatively low costs, as well as access to transgenic strains,
particularly for mice. Additionally, the small size of newborn
rodent pups translates to lower costs (because of the lower
quantity required) of pharmacological agents delivered. How-
ever, critical evaluation of the rodent models also reveals key
concerns that are becoming more apparent. The mouse and rat
BPD models are often promoted because mouse and rat pups
are born in a stage of lung development comparable to the
stages in which premature humans are born that go on to
develop BPD. As such, these are seen as “relevant” models.
However, it is also true that these term mouse and rat pups are
born with lungs that are already fully competent for gas
exchange, which is not the case for premature human infants.
Furthermore, another significant drawback of the term mouse
and rat models is the difficulty with prolonged mechanical
ventilation, which can be undertaken for short periods (up to
about 24 h) (33, 38) but not beyond. In this sense, the lamb and
baboon models are far superior although these models are also
complicated by high costs and ethical considerations. The
recent demonstration that infant baboons infected with respi-
ratory syncytial virus (RSV) develop clinical and pathological
changes that parallel those of human infants (269) highlights
the potential utility of the baboon model, given the relationship
between RSV and BPD in a clinical setting, in which infants
with BPD are particularly susceptible to RSV infection (57).
Another key concern, largely with the rodent models, is a
lack of standardization. This is particularly evident with the
hyperoxia models. In the many studies reported in this Per-
spectives article, 1) investigators employed a wide variety of
mouse strains, including BALB/c, C57BL/6, FVB/N, C3H/
HeN, as well as mixed backgrounds. However, there is noto-
rious variability in the susceptibility of mouse strains to hy-
peroxia-induced lung injury (147, 365), making comparisons
of studies performed with different strains very difficult. 2)
This is exacerbated by the use of a broad range of oxygen
concentrations in the hyperoxia-based models, in which term
mouse pups are exposed to a range of hyperoxia levels,
between 40% O2 and 100% O2 in the inspired air. 3) A further
confounding factor impacting standardization is the duration
and window of hyperoxia exposure, in which term mouse pups
may be exposed to hyperoxia from the day of birth for up to 28
days. Alternatively, term mouse pups are exposed for a specific
window (for example, P4 to P8) of hyperoxia, followed by
variable durations of normoxia. This variability in the hyper-
oxia models extracted from the studies reported in this Per-
spectives article, published between 2013 and 2015, is made
clear in Fig. 1. There is a pressing need for a systematic
comparison of the effects of different oxygen levels (assessed
over the same time range) on lung development. Additionally,
it remains important to assess the critical time window in
which lung development is exquisitely susceptible to oxygen
injury, should such a window exist. It may well be that oxygen
exposure over the first few hours of postnatal life (in mice) is
sufficient to irreversibly perturb lung development, but this
remains to be experimentally demonstrated. This idea is sup-
ported by the observations of Firsova and coworkers (102) that
exposure of newborn mouse pups to 90% O2 over the first 4
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L1241
days of life led to a persistent disturbance to alveolarization
(assessed by MLI) that was evident 56 days after birth. It is
clearly important to better understand how much oxygen is
required, and when to provoke a particular structural abnor-
mality in the lung and what impact this has on the magnitude
of that disturbance (346). To this end, Wang and coworkers
(355) have examined the impact of exposure to 40% O2 and
70% O2 over the first 7 days of life, followed by recovery in
room air for 14 days, in which no differences were observed in
alveolar structure or number (assessed by RAC) comparing the
40% O2 and 70% O2 groups. However, the alveolar structure
was not assessed at the conclusion of hyperoxia exposure (P7).
Another issue of standardization relates to nomenclature, in
which some investigators consider P1 as the day of birth,
whereas others report P1 as the day after birth. Clearly, there is
much room for improvement of the standardization and report-
ing of experimental protocols and nomenclature in rodent
models of BPD.
Efforts are underway to further characterize and optimize the
hyperoxia-exposure protocols in animal models of BPD.
Rieger-Fackeldy and coworkers (300) demonstrated the impact
of abrupt vs. gradual weaning of FVB/N mouse pups from
hyperoxia (85% O2) to normoxia (21% O2). Interestingly, a
gradual weaning (10% reduction in O2 levels per day, over 6
days) resulted in double the number of alveoli compared with
age-matched controls subjected to abrupt return to room air.
These data indicate that, not only the oxygen levels and the
duration of exposure, but also the steepness of the oxygen
gradient posthyperoxia exposure can influence alveolarization.
These ideas have also received attention in the pioneering work
of Michael O=Reilly’s group (52), which continues to explore
the role of different oxygen levels on lung structural develop-
ment, particularly in relation to influenza A virus infection.
There is also a need to further develop alternative animal
models of BPD, not based (exclusively) on hyperoxia. The
impact of cell stretch and lung distention is likely to be a key
cause of damage to the developing lung (151), highlighting the
need for increased use of ventilation-based models to study the
impact of mechanical ventilation on the developing lung.
Along these lines, Ratner and coworkers (294) demonstrated
that an 8-h period of aggressive mechanical ventilation (tidal
volume 15 ␮l/g) of 5-day-old mouse pups with room air caused
pronounced mitochondrial dysfunction accompanied by
blunted alveolarization (measured by RAC) that was evident
10 days later, which are important findings given the increasing
appreciation for mitochondria being more than just a “power-
house” (311). However, using the same protocol employing
gentle mechanical ventilation (tidal volume 8 ␮l/g), no effect
on alveolarization or mitochondrial dysfunction was noted.
Similarly, Young and coworkers (379) demonstrated that lung
elastase activity can be locally regulated by stretch during
alveolar septation, which, together with the other studies re-
ported here, highlights the usefulness of mechanical ventilation
models of BPD.
The rabbit is often seen as a compromise between the
advantages of small rodents (rats and mice) and larger verte-
brates (sheep and nonhuman primates). The rabbit affords the
possibility of preterm delivery of rabbit kittens, followed by
hyperoxia exposure or other interventions. Additionally, the
rabbit lung is anatomically closer to human lungs than are rat
or mouse lungs. The development of a preterm [at embryonic
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L1242 ADVANCES IN LUNG ALVEOLARIZATION AND BPD

Prenatal Postnatal (days)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 28 56

[163]
[130]
[156,173, 369]
[299]
[323]
[326] 10%
[362]
21%
[270]
[207] 40%
[37, 217, 351, 352] 50%
[27] 60%
[292]
[9, 32, 220]
70%
[239] 75%
[120] 80%
[187]
85%
[9, 50, 156, 288]
[19]
90%
[11] 95%
[19] 100%
[19, 231, 342]
[83]
LPS Injection
[149]
[13]
[324]
[270]
[284]
[355]
[62]
[276]
[319]
[64]
[305]
[23]
[288, 300]
[300]
[236]
[324]
[335]
[8]
[51]

Fig. 1. Spectrum of oxygen levels and oxygen-exposure windows that are employed in the hyperoxia models of bronchopulmonary dysplasia in rodents. Only
hyperoxia-exposure protocols published between 1 January 2013 and 30 June 2015 are illustrated. The legends at right indicate the color scheme used to describe
the different oxygen concentrations applied, as a percentage (vol/vol) of the inspired air (for example, 21% indicates an FIO2 of 0.21).  indicate prenatal or
postnatal bacterial LPS administration. The numbers in parentheses after each bar indicated the corresponding citation in the references from which the
oxygen-exposure protocol was extracted.

day (E)28 vs. the E31 term date] rabbit model, followed by
hyperoxia exposure (95% O2 for 7 days), has been carefully
described by Richter and coworkers (299), highlighting the
potential utility of this model for studies on arrested alveolar-
ization. Further refinement of this model has also been under-
taken (215), demonstrating that caesarean delivery of preterm
rabbit kittens at E29 vs. E28 was accompanied by reduced
mortality although the E29 group maintained a reduction in
alveoli number and thus remained suitable for modelling struc-
tural changes to the lung associated with preterm birth. These
efforts to further develop the rabbit model are also being
facilitated by transcriptional studies in rabbit lungs from pre-
term rabbit kittens exposed to hyperoxia (306).
So long as rodents cannot be ventilated for prolonged peri-
ods, the preterm lamb model remains the method of choice.
This model has recently been used to demonstrate the useful-
ness of high-frequency nasal ventilation to maintain adequate
gas exchange and to promote alveolarization in preterm lambs
(260), which would not have been possible with the rodent
ventilation approach. To this end, the ventilated preterm lamb
model continues to be developed (49). Collins and coworkers
(73) have clarified the need for the optimization of this model,
given the different transforming growth factor (TGF)-␤ re-
sponses of Merino ewes to intrauterine LPS vs. Ureaplasma
parvum exposure. Ureaplasma is commonly isolated from
pregnant mothers with chorioamnionitis and thus may pose an
increased risk for preterm birth and the development of BPD in
the neonate (114, 161). The preterm lamb model has also been
used to study ventilation mechanics and most recently the
utility of recruitment maneuvers before mechanical ventilation
of preterm lambs to limit ventilator-induced lung injury (140).
In that study, Hillman and coworkers (140) found that, irrespec-
tive of the functional residual capacity recruited, the recruitment
maneuvers did not alter the acute phase and proinflammatory
responses to mechanical ventilation at birth; however, alveolar
development was not quantified in that study.
Antenatal inflammation can lead to pulmonary inflamma-
tion, with important consequences for pre- and postnatal lung

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ADVANCES IN LUNG ALVEOLARIZATION AND BPD
development (179), although chorioamnionitis as a risk factor
for BPD remains controversial (180, 333). With antenatal
inflammation in mind, several animal models of BPD have
been developed to include an intrauterine inflammation com-
ponent, which is translatable to the clinic, as described above
for intrauterine Ureoplasma infection in sheep (73). Related
intrauterine inflammation models have been reported in rats to
study the utility of antenatal glucocorticoids to influence lung
development in the background of antenatal inflammation
(378). Studies have also examined NF-␬B function in the
endothelium (329). Further work in preterm lambs examined
inflammatory signaling and glucocorticoid-growth factor sig-
naling in fetal sheep lungs (73, 74). These are important and
clinically relevant models that demand more widespread use.
To date, BPD has not been modeled in other large verte-
brates apart from nonhuman primates and lambs; however,
work has been initiated in piglets, with a recent report by
Bartlett and coworkers (31) on the proteomic analysis of
bronchoalveolar lavage fluid and epithelial lining fluid in
newborn piglets. These authors suggest that the relative simi-
larity of human and porcine lungs supports the further devel-
opment of animal models in the porcine system. This idea has
been reinforced by Caminita and coworkers (55), who have
elegantly demonstrated that, between gestational days E98 and
E102, preterm piglets exhibited ventilation inadequacies as
well as risks for the development of RDS that mimic those of
preterm infants born during the saccular phase of lung devel-
opment. The investigators concluded that these piglets were
compatible with modern neonatal intensive care management
approaches and thus represented an alternative to nonhuman
primates and lambs.
Although use of the primate models is steadily declining,
largely attributable to cost and ethical issues, macaques have
recently been used to evaluate the impact of ozone on postnatal
lung maturation in nonhuman primates by Murphy and co-
workers (250), who noted that susceptibility to ozone damage
is age dependent and that prior ozone exposure modulates the
response to subsequent ozone exposure. These data have
clearly translatable information to how children living in pol-
luted metropolitan areas during key stages of lung development
may respond to ozone exposure in later life.
Returning to rodents, intrauterine growth restriction (IUGR)
also impacts alveolarization, and thus IUGR is also occasion-
ally employed as a BPD model. Zana-Taieb and coworkers
(383) compared two IUGR models, namely protein deprivation
and nitric oxide synthase (NOS) inhibition. The investigators
concluded that only the protein-deprivation approach yielded a
sustained impairment of alveolarization (assessed by MLI and
RAC) in rat pups, whereas NOS inhibition blunted early lung
development before alveolarization. The same investigators
followed up with a transcriptional profiling study in the pro-
tein-deprivation IUGR model (384) and identified the peroxi-
some proliferator-activated receptor (PPAR) pathway as a
likely key mediator of impaired alveolarization. Prenatal
growth restriction also forms the basis of a new double-hit
mouse model of BPD, where pregnant mothers are exposed
between E14 and E18 to hypoxia (10% O2), after which
offspring were exposed to 75% O2 for the first 14 days of life.
Although not yet in widespread use, this model was used to
demonstrate the therapeutic potential of umbilical cord mono-
nuclear cells to promote proper lung alveolarization (assessed
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L1243
by stereology, using airspace volume density and surface
density of septa as surrogates for alveolarization) (239), and,
on the basis the expression of surfactant protein and other
genes, this model is considered to closely mimic the clinical
situation (120). This concept of fetal priming has also been
observed in the rodent hyperoxia model using maternal diabe-
tes as a first hit, in which streptozotocin-induced diabetes in
rats before pregnancy appeared to modulate the effects of
hyperoxia on septal wall thickness in pup lungs, although the
precise implications of this are not clear (173). In terms of
other new models, the observation that maternal deprivation
(that is, separation of mother and pups) in rats also led to a
pronounced blunting of lung development (assessed by MLI),
ostensibly mediated by dipeptidyl peptidase IV, suggests that
maternal deprivation may represent a new (stress-induced) rat
model of BPD (150). This idea possibly intersects with the
recent observation that maternal stress during pregnancy alters
the predisposition of pups to allergy susceptibility, in which a
role for glucocorticoids was implicated (193). This idea has yet
to be tested with regard to lung parenchymal and vascular
development. Additionally, Ayala de la Peña and coworkers
(26) have suggested that the expression of a KrasG12D variant
in the urothelium may represent a new BPD model, an idea
based on lung histopathology assessed at P1. The utility of this
transgenic mouse strain as a BPD model awaits further evalu-
ation, and further genetic models will be discussed later in this
Perspectives article.
As an alternative to animal models, several recent reports
have detailed the used of ex vivo lung culture (277), epithelial-
mesenchymal cocultures (122), in vitro-generated human plu-
ripotent stem cell-derived lung organoids (90), 3D multicell
microtissue culture models of airway smooth muscle (363),
and a biomaterial-templated alveoli culture system (189) for
the study of alveolarization and alveolar biology. Although not
yet in widespread use, these systems potentially provide useful
alternatives to in vivo studies, in which high-throughput
screening of pharmacological or genetic interventions could be
conducted to identify pathways relevant to alveolarization and
to limit the use of experimental animals. Among these ap-
proaches is the coculture of fetal (but not adult) mesenchymal
cells with A549 cells (a human lung epithelial cell line), in
which three-dimensional peaks formed that had a similar cel-
lular orientation to alveolar structures in vivo (122). Addition-
ally, there is evidence that secondary septation continues in ex
vivo lung slices maintained in culture (277). This provides an
alternative model for the in vitro analysis of signaling path-
ways and pharmacological or genetic interventions to study
secondary septation. Clearly, this model lacks input from
physiological blood flow and cyclic stretch from breathing
motions, suggesting areas where this potentially interesting in
vitro approach could be further refined. Along these lines, the
reproducible uniform equibiaxial stretch of precision-cut lung
slices has recently been demonstrated (82).
Last, concerning the analysis of gene expression in tissues
harvested from animal models, several investigators have sys-
tematically addressed the selection of reference genes in real-
time PCR analysis. Mehta and coworkers (234) assessed the
utility of Tuba1a, Actb, Gapdh, Rn18S, and Hist4h4 and
identified Tuba1a as the least variable in terms of expression
levels over the course of mouse lung development (E11.5 to
P28). Unfortunately, other popularly used references genes,
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L1244 ADVANCES IN LUNG ALVEOLARIZATION AND BPD
including Hmbs and Polr2a, were not evaluated. In a related
study, Bouhaddioui and coworkers (43) screened reference
genes for the normalization of microRNA gene expression and
concluded from the panel of Sno135, Sno142, Sno202, Sno234,
and Sno251 that the expression of Sno234 was the most stable
over the course of mouse lung development studied (E15.5 to
P30) although other popularly used reference genes, such as
the U6 small nuclear 1 RNA (Rnu6-1), were not assessed.
Imaging Lung Structure and Function
The imaging of the lung in real time is an emerging area of
interest and technological development, leading to a special
call by the American Journal of Physiology Lung Cellular and
Molecular Physiology for papers, “Real-time visualization of
lung function: from micro to macro” (244). In response to this
call, a review article that broadly described the imaging of the
lung using a spectrum of tomographic and X-ray approaches
was recently published (116). Recent advances in lung imaging
include the development of noninvasive MRI approaches to
quantify lung volume changes in mice and the application of
this approach to monitor interventional studies, in this case, the
impact of the somatostatin analog pasireotide (SOM230) on the
progress of bleomycin-induced lung injury (91). This method-
ological approach has not yet been extended to the study of
postnatal lung development in mice. Very recently, a study has
emerged (354) that demonstrated that pulmonary MRI can
reveal quantifiable and significant differences between patients
with BPD, premature patients without BPD, and full-term
controls. These new data suggest that pulmonary MRI might be
implemented to individually phenotype disease, which would
be most useful for clinical care and to predict outcome.
Other types of imaging are also emerging for the study of
lung development and BPD. For example, optical cryoimaging
has been employed to address the mitochondrial redox state of
mouse lungs during lung development in Bcl-2-deficient mice,
in which the authors determined that Bcl-2 deficiency was
associated with oxidative stress that correlated with perturbed
alveolarization (221). Thus these authors have added fluores-
cent imaging to our lung development methodology toolbox.
Additional recent reports reveal the exciting potential of con-
trast-enhanced micro-CT in rodents (77) and automated intra-
vital microscopy to study vascular function (129); however,
these approaches have not yet been applied to developing
lungs. Other tried and tested imaging approaches continue to
support studies on lung vascular development, for example,
barium angiography, which allows the extraordinary visualiza-
tion of vascular pruning in the rat hyperoxia model of BPD
(342).
Although not imaging per se, the three-dimensional recon-
struction of serial sections of lungs from patients with BPD has
been reported by Galambos and coworkers (111) and facili-
tated the identification of intrapulmonary anastomoses in af-
fected lungs. These investigators employed the same method-
ology to describe curious pathological perturbations to lung
vascular structure in patients with alveolar capillary dysplasia
with misalignment of pulmonary veins (ACDMPV) (112, 113)
and CDH (2). This approach has not yet been employed in
animal models of arrested lung development.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00268.2015 • www.ajplung.org
New Mediators and Modulators of Impaired Alveolarization
The past 2.5 yr have seen the addition of several new
mediators of alveolarization that have been identified in animal
models of BPD. Among these is soluble guanylate cyclase
(sGC), in which lung development was particularly impaired
after hyperoxic injury in mouse pups carrying a targeted
deletion of the gene encoding the ␣1 subunit of sGC (27).
These data support the idea of activating sGC as a management
strategy in hyperoxia-induced lung injury. Along similar lines,
McKenna and coworkers (231) demonstrated that sustained
NF-␬B activation, through overexpression of the NF-␬B inhib-
itor-␤ (I␬-B␤), protected mice against the deleterious effects of
hyperoxia (95% O2) on alveolarization (assessed by MLI and
RAC), suggesting that the potentiation of NF-␬B activity may
also represent a management strategy in hyperoxia-induced
lung injury. Although not yet studied in a BPD model, over-
expression of the receptor for glycation end products (RAGE)
blunted alveolarization (assessed by MLI) in mouse pups,
adding RAGE to the catalog of negative regulators of alveo-
larization (101). Although the receptor for RAGE is expressed
in alveolar type I cells (304), other studies have indicated that
RAGE expression in the lung is suppressed by exposure of rat
pups to hyperoxia (198); as such, it remains unclear whether
RAGE plays a functional role in limiting alveolarization in
experimental BPD. Further studies on RAGE in lung develop-
ment are clearly indicated, particularly in light of the recent
report that RAGE also acts as a mediator of endothelial
activation (214).
Isoforms of hypoxia-inducible factor (HIF) continue to re-
ceive attention as mediators of lung development, including
HIF-1␣ (335), HIF-2␣ (270), and HIF-3␣ (146). The expres-
sion of HIF-1 is known to be downregulated in patients with
BPD, leading Tibboel and coworkers (335) to overexpress a
constitutively active HIF-1␣ subunit (HIF-1␣⌬ODD) in the
distal lung epithelium. This increased lung vessel density
upregulated the expression of proangiogenic factors and drove
increased alveolarization in mice maintained in room air.
However, contrary to expectations, when these HIF-1␣⌬ODD-
expressing C57BL/6 mice were exposed to hyperoxia (80% O2
for 28 days), lung function worsened, and the surfactant and
lung architecture abnormalities (assessed by MLI and RAC)
seen in hyperoxia-exposed wild-type mice persisted. Opposite
results were obtained by Vadivel and coworkers (342), who
delivered wild-type HIF-1␣ via adenovirus in the rat hyperoxia
model (95% O2 for 14 days) and, in doing so, conferred
striking protection against the deleterious impact of hyperoxia
on alveolarization (assessed by MLI), which was comparable
to that of room air-exposed rat pups. The reasons for the
discordant observations reported by Tibboel and coworkers
(335) vs. Vadivel and coworkers (342) might be due to the
epithelial-restricted overexpression of constitutively active
HIF-1␣ in the Tibboel study. This idea is supported by the
dramatically improved vascularization seen in the Vadivel
study, which might suggest that the HIF-1␣ must be delivered
to the endothelium to confer protection and promote lung
maturation in the background of hyperoxia.
MicroRNA are also emerging as candidate mediators of
fibroblast (41, 148) and pulmonary artery smooth muscle cell
behavior (157), alveolarization, and BPD (165); however, to
date, this idea is supported only by the observed changes in

ADVANCES IN LUNG ALVEOLARIZATION AND BPD
expression of some microRNA species that correlate with
arrested alveolarization. These candidate microRNA species
have recently been reviewed (165, 249, 375), and integration of
microRNA pathways into disease processes by modeling (310)
and systems biology approaches (36, 127, 235) continues to be
developed; however, the field awaits the first demonstration of
a causal role for any microRNA species in arrested late lung
development. Narasaraju and coworkers (251) have suggested
a role for miR-150 in blunted alveolarization associated with
hyperoxia, using a miR-150⫺/⫺ mouse strain, which, after
hyperoxia exposure (95% O2 from P1 to P10), exhibited
transient rescue of alveolarization (assessed by MLI) seen at
P6, but this rescue did not persist to P10. Capillary density was
apparently improved in this model; however, the strain back-
ground of the miR-150⫺/⫺ mouse strain was not declared, and,
as such, the appropriateness of the C57BL/6J background as a
control in these studies cannot be assessed. Some studies in
clinical BPD material have indicated that the expression of
miR-206 is downregulated in the lungs of patients with BPD
(388); however, miR-206 has yet to be causally implicated in
arrested alveolarization. A role in alveolarization for long
noncoding RNA, which are involved in early lung develop-
ment (132), is yet to be addressed.
The mechanisms leading to cell death in the lungs in patients
with BPD and experimental (hyperoxia-based) animal models
of BPD have also been addressed. One notable recent finding
is that exposure to hyperoxia leads to elevated interferon-␥
levels, which in turn drive endoplasmic reticulum stress-de-
pendent cell death in a manner that relies on cyclooxygenase-2
(COX-2) (72). This represents a novel cell-death pathway that
is relevant to clinical and experimental BPD. These findings
support the exploration of COX-2- and DNA-damage-induc-
ible transcript 3 (Ddit3; also called Chop)-based approaches in
the management of BPD, and, indeed, efforts along these lines
are already underway, with the report of Masood and cowork-
ers (220), who employed a highly selective COX-2 inhibitor
5,5-dimethyl-3-(3-fluorophenyl)4-(4-methylsulfonyl)phenyl-
2(5H)-furanone in the rat hyperoxia model (60% O2 from P1 to
P14). In that study, COX-2 inhibition blunted inflammatory
infiltration provoked by hyperoxia and improved lung vascu-
larization and alveolarization (assessed by MLI). Conflicting
data have been obtained in an alternative study by Britt and
coworkers (50), who could also demonstrate the ability of both
aspirin and the COX-2-specific inhibitor celecoxib to limit
pulmonary inflammation provoked by hyperoxia (85% O2 from
P1 to P14) in C3H/HeN mice; however, pharmacological
inhibition of COX-2 did not improve lung structure (assessed
by number of alveoli per high-power field) in the background
of hyperoxia. These data were supported using Cox2⫹/⫺ and
Cox2⫺/⫺ C57BL/6 mice, which were also protected against
pulmonary inflammation provoked by hyperoxia; however,
genetic ablation of COX-2 expression did not improve lung
structure in the background of hyperoxia. The reasons for these
divergent observations are not clear; however, it is apparent
that no impact of COX-2 inhibition (or gene ablation) was seen
at the higher oxygen level, highlighting a possible influence of
the model (different O2 levels) on the outcome. Additionally,
one study was performed in rats, whereas the other was
performed in mice. As such, although the data clearly implicate
COX-2 as a candidate druggable target to limit lung inflam-
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00268.2015 • www.ajplung.org
Perspectives
L1245
mation in the background of hyperoxia, it remains unclear
whether an impact on alveolarization accompanies this effect.
Sex differences are emerging as possible confounders in
studies on clinical and experimental BPD (121), as recently
discussed in the context of pulmonary hypertension (182), and
it is important that the sex of experimental animals is consid-
ered in the design of experimental studies and interpretation of
data. There is a growing body of evidence that both androgens
and steroidogenic enzymes may influence postnatal lung de-
velopment (339). The importance of considering sex bias in
studies in animal models of BPD is underscored by the obser-
vations that adult (C57BL/6J) mice exhibit sex-specific dimor-
phic effects on inflammation and antioxidant enzyme expres-
sion in response to hyperoxia (196), with male animals exhib-
iting greater lung injury, neutrophilic inflammation, and
apoptosis, all of which are also characteristic of clinical BPD.
In support of this idea, Martin and coworkers (219) reported a
pronounced impact of hyperoxia on estradiol metabolism in
postnatal airway smooth muscle cells. Additionally, the effects
of glucocorticoids on alveolar type II cell function in vitro were
sex dependent (167). To date, no study has addressed sex
differences in animal models of BPD, and this represents a
priority area for research for future work in the field, particu-
larly given the differences in the kinetics of structural changes
in the ageing lung for male vs. female Rhesus macaques (133).
Gasotransmitters including NO, carbon monoxide (CO), and
hydrogen sulfide (H2S) are also emerging as mediators of late
lung development and, possibly, arrested lung development
associated with BPD. NO has a long history of use in prema-
ture infants with or at risk for BPD (168, 340). One recent
report has indicated that treatment of newborn rats with high-
dose (20 ppm) inhaled NO improved lung growth, most likely
through increased expression of hemeoxygenase-1 and VEGF/
VEGF receptor 2 (VEGFR2), as well as matrix metalloprotei-
nases (MMPs). Interestingly, increased gene expression was
seen over the course of NO administration but was lost after
discontinuation of NO (89). The immediate implication of this
study is not clear because NO administration was made in
room air to healthy, properly developing rat lungs. Interest-
ingly, 20 ppm NO inhalation yielded an ⬃20% increase in
RAC, suggesting that healthy lungs are indeed capable of
hyperalveolarization. In a different line, continued work on the
inhibition of phosphodiesterase (PDE)5 by Park and coworkers
(270) demonstrated that sildenafil modulation of the NO/cGMP
pathway promoted alveolarization (assessed by Lm) in the
background of hyperoxia. These data highlight PDE5 as a
candidate druggable target to drive alveolarization, and this has
stimulated additional studies on the interaction between differ-
ent cGMP- or cAMP-dependent agonists in the pulmonary
vasculature in newborn rats (96). Interestingly, working in
adult mice, Czövek and coworkers (79) have demonstrated the
utility of VIP to stimulate the NO pathway and preserve lung
function and structure in the background of hyperoxia in adult
rats. The modulation of NO signaling with VIP in neonates
awaits investigation. Future work on inhaled NO approaches in
hyperoxia-based animal models will have to take into account
the impact of hyperoxia on S-nitrosothiol accumulation and
L-type amino acid transporter 1 expression and function, given
the recent important report from Richard Auten’s group that
the oxidative stress generated by hyperoxia blocks a major
route of inhaled NO action (45, 46).
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L1246 ADVANCES IN LUNG ALVEOLARIZATION AND BPD
The alternative gasotransmitter CO has also received atten-
tion by Anyanwu and coworkers (23), who reported the pro-
tective effects of CO administration concomitant with hyper-
oxia (75% O2, from P3, for up to 21 days) exposure in
C57BL/6J mice on alveolar development. These studies were
supported with hemeoxygenase-1 knockout mice, which were
demonstrably more susceptible to the injurious effects of hy-
peroxia. This phenomenon has also been reported by Yang and
coworkers (374), who suggested that the ␤-catenin/heteroge-
neous nuclear ribonucleoprotein K axis mediated the protective
effects of hemeoxygenase-1 during hyperoxia exposure. The
protective effects of CO were attributed to the modulation of a
broad spectrum of inflammatory cytokines, in which the ex-
pression of proinflammatory cytokines was suppressed, and the
expression of anti-inflammatory cytokines (IL-10) was in-
creased by CO treatment. These studies highlight the CO/
hemeoxygenase-1 axis as a potentially exciting lung-protective
axis and also, together with work by Luo and coworkers (201),
emphasize a role for regulators of the Wnt/␤-catenin system in
regulating lung development. Indeed, given the recent reports
of the ␤-catenin system regulating epithelial repair (185, 385)
and smooth muscle responses to hypoxia (381), ␤-catenin is
emerging as an exciting mediator of the postnatal lung re-
sponse to injury, which might impact lung maturation.
The third gasotransmitter H2S has also received attention. In
pioneering work by Vadivel and coworkers (343), exogenous
administration of H2S to rats in the hyperoxia model (95% O2
from P1 to P14) improved lung alveolarization (assessed by
MLI), limited hyperoxia-provoked increases in pulmonary ar-
teriole medial wall thickness, restored the pulmonary arterial
acceleration time/right ventricular ejection time ratio, and par-
tially restored the Fulton index. These data support a protective
role for H2S administration, primarily related to restoration of
vascular remodeling and pulmonary hemodynamics in the
hyperoxia model. In a subsequent study by Madurga and
coworkers (207), daily administration of the H2S donor
GYY4137 in the mouse hyperoxia model (85% O2 from P1 to
P14) protected C57BL/6 mice against the damaging impact of
hyperoxia (85% O2 from P1 to P10) on alveolarization (as-
sessed by stereology). This appeared to be correlated with
blunted inflammatory responses provoked by hyperoxia, per-
haps mediated by increased IL-10 levels. Taken together, these
reports underscore H2S as a mediator of lung development that
warrants further study. This idea was recently reinforced by
another report by Madurga and coworkers (205), who dem-
onstrated that two of the three endogenous H2S-generating
enzyme systems, cystathionine ␤-synthase (Cbs) and cysta-
thionine ␥-lyase (Cth), are mediators of normal lung alveo-
larization and vascular development. These studies are not
conclusive, however, because, although the necessity of Cbs
and Cth for alveolarization was documented using knockout
mice, it remains unclear whether the defective alveolariza-
tion and vascular development seen in knockout mouse pups
was due to a lack of H2S production or a toxic buildup of
enzyme substrates. Furthermore, the contribution of the
third H2S-generating enzyme, mercaptopyruvate sul-
furtransferase, awaits study.
Singh and coworkers (316) have recently added cigarette
smoke to the list of potential inducers of arrested alveolariza-
tion. Interestingly, gestational (maternal) but not postnatal
exposure to cigarette smoke blunted alveolar development in
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00268.2015 • www.ajplung.org
both BALB/c and C57BL/6 mice. This could be attenuated by
administration of mecamylamine, a nicotinic acetylcholine
receptor antagonist, to pregnant mothers during gestation,
which concomitantly with cigarette smoke exposure attenuated
the impact of cigarette smoke on alveolarization (assessed by
MLI) in mouse pups. Some components of cigarette smoke
have also been studied in isolation; Thankur and coworkers
(331) administered benzo[a]pyrene to pregnant rats at E17,
E18, and E19. After delivery, newborn pups were exposed to
hyperoxia (85% O2 from P1 to P14). Maternal benzo[a]pyrene
exposure appeared to impact alveolarization; however, lung
structure was not quantified in that study. Additionally, the
translatability of the very high benzo[a]pyrene dose (25 mg/kg
per day, for 3 days) is unclear, as the comparability of this dose
to benzo[a]pyrene levels in smoking mothers is not reported.
Still much remains to be investigated about how exactly
cigarette smoke impacts lung development; however, some
lead studies, such as the observation that cigarette smoke
drives hyperplasia and extracellular matrix (ECM) production
by fetal airway smooth muscle cells (347), are laying important
groundwork in this area. Studies have not been confined to
tobacco-containing cigarettes, with a recent report (229) that
exposure of newborn C57BL/6J mouse pups to E-cigarette
vapor (containing nicotine in propylene glycol vehicle) once
per day on days 2 and 3 of life, followed by twice daily on days
4-10 of life, led to an attenuation of alveolarization (assessed
by MLI), compared with mice exposed to propylene glycol
vehicle alone. The alveolarization defect was accompanied by
detectable systemic levels of cotinine. These data add to the
argument that exposure to both tobacco and E-cigarettes is an
important consideration in postnatal lung maturation and as-
sume further importance in light of recent reports that perinatal
nicotine can induce transgenerational asthma (297) and that
nicotine drives epithelial-mesenchymal transition through
Wnt/␤-catenin signaling in human airway epithelial cells
(390). Apart from cigarette smoke, nanoparticles are another
environmental factor that can impact lung development. Nano-
particles are present in cosmetics, paint, sunscreens, and food
(287). Using titanium oxide nanoparticles (average diameter
8 –10 nm), Ambalavanan and coworkers (22) reported that
exposure of mouse pups at P4 to single or multiple doses of
nanoparticles provoked an increase in the expression of proin-
flammatory mediators and blunted lung development (assessed
at P14; by MLI and RAC). In a related study, Chan and
coworkers (60) described the muted expression of Cyp1A1, a
cytochrome P-450 superfamily member, in neonatal, but not
adult rats, after exposure to ultrafine particular matter, which is
a key component of vehicle exhaust. These data underscore the
need for further investigation into the public health impact of
exposure of children to nanoparticles and particulate matter
during periods of postnatal lung growth.
A genome-wide transcriptional analysis approach has been
used by Bhattacharya and coworkers (37) in the mouse hyper-
oxia model to identify the aryl hydrocarbon receptor (Ahr) as
a candidate key regulator of responses to hyperoxia in
C57BL/6 mouse pups. This observation is interesting because
Shang and coworkers (387) have reported that Ahr is necessary
to protect fetal human pulmonary microvascular endothelial
cells against hyperoxic injury. Genome-wide association stud-
ies (GWAS) have been used by Nichols and coworkers (254)
to evaluate the role played by genetic background in the

ADVANCES IN LUNG ALVEOLARIZATION AND BPD
response of mouse pups from 36 inbred mouse strains to
hyperoxia (95% O2 from P1 to P3). These investigators iden-
tified the muscarinic acetylcholine receptor M2 gene (Chrm2)
as a susceptibility gene, and mouse strains carrying a Chrm2
single-nucleotide polymorphism (SNP; causing a P265L sub-
stitution in Chrm2) exhibited reduced hyperoxia-induced in-
flammation. Importantly, that study employed hyperoxia-in-
duced lung inflammation as an endpoint readout, and similar
GWAS studies using perturbations to lung architecture have
not yet been performed. GWAS have been less successful in
patients with BPD, in whom, in one recent report, no new
SNPs were identified that could be associated with BPD;
furthermore, this study could not validate any previously iden-
tified SNPs (356). In another GWAS involving material from
patients with BPD, no new SNPs were identified, but Amba-
lavanan and coworkers (20) highlighted the miR-219 pathway
[which includes platelet-derived growth factor (PDGF)-␣] and
the IGF family pathway as areas that warrant further study in
BPD. Studies on DNA methylation patterns are also emerging
(78), which have implicated epigenetic regulation of growth
factor, ECM, and antioxidant enzyme expression during nor-
mal lung development and epigenetic (dys)regulation of de-
toxifying enzymes and growth factors in clinical BPD (126).
Interestingly, whole-genome expression studies in plasma sam-
ples in patients with BPD revealed alteration of the expression
of nearly 10% of the genome in affected patients (279).
Recent studies have also shed new light on classical medi-
ators and pathways that remain controversial players in lung
development and candidate druggable targets and pathways in
BPD. Lykkedegn and coworkers (203) thoughtfully appraised
the impact of vitamin D on lung maturation in a systematic
review and highlighted the discord between observations made
in animal models, in which vitamin D dramatically stimulated
lung maturation, whereas clinical data currently do not support
a role for vitamin D in the prevention or treatment of infant
RDS or BPD. This report highlights a need for further studies
on vitamin D deficiency or insufficiency as a risk factor for
RDS and BPD and supported further investigations into the
optimal dosing (in terms of dose and timeframe) of vitamin D
and its analogs for the managements of infant RDS and BPD.
To this end, Mandell and coworkers (213) reported that ante-
natal vitamin D administration improved survival and lung
structure (assessed by RAC) after intra-amniotic endotoxin
exposure in rats [which is also known to modulate airway
reactivity in mice following hyperoxia exposure (71)], suggest-
ing a potential role for vitamin D in the prevention of arrested
alveolarization in this rat model. Along these lines, Yurt and
coworkers (382) demonstrated a biphasic response to cholecal-
ciferol nutritional supplementation, in which both alveolar
development (assessed by RAC and MLI) as well as PPAR-␥
and CCAAT/enhancer binding protein-␣ expression in the lung
peaked at a supplementation of 500 U/kg cholecalciferol,
which were all reduced at both 250 U/kg and 1,000 U/kg.
These data highlight the concerns about dose in vitamin D
supplementation studies. In the context of the impact of nutrition
on lung development, Mayor and coworkers (222) identified
maternal high-fat diet as a risk factor for alveolar simplification
(assessed by MLI), possibly related to placental inflammation.
These studies provide a firm basis for further work on the role of
maternal nutrition on postnatal lung maturation.
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Perspectives
L1247
In addition to these newly identified mediators of alveolar-
ization, several recent studies have identified changes in the
expression of further candidate mediators that accompany
disturbances to lung alveolarization, which have yet to be
validated in interventional or transgenic mouse studies. These
candidate mediators include neuronal Per Arnt Sim domain
protein 3 (274) and SOD-3 (283). SOD-3 is an antioxidant that
is curiously targeted to the ECM and epithelial lining fluid of
the lung and is not regulated by nuclear factor, erythroid-
derived 2-like 2 (Nfe2l2, also called Nrf2) (283) and adds
SOD-3 and thioredoxin-1 (66) to the list of antioxidants that
might play a role in perturbed lung alveolarization in response
to hyperoxia (103, 337). This idea is all the more interesting
given the recent report by Gupta and coworkers (124) that loss
of SOD-2, studied in Sod2⫹/⫺ C57BL/6 mice, did not impact
arrested alveolarization (assessed by changes in alveolar area),
changes in Fulton index, or pulmonary arteriole medial wall
thickness that were caused by hyperoxia (75% O2, from P1 to
P14) exposure, leading these authors to conclude that other
antioxidant systems may compensate the loss of SOD-2 or that
a single sod allele generated sufficient SOD to elicit a wild-
type response.
Epithelial Cells and Epithelial Cell Transdifferentiation
Epithelial growth and differentiation and epithelial interac-
tions with the ECM are key events in alveolarization. Using a
lung epithelium-specific [surfactant-associated protein C
(Sftpc)-driven] deletion of integrin-␤1, Plosa and coworkers
(281) demonstrated a pivotal role for integrin-␤1 in branching
morphogenesis and alveolarization. The alveolarization defect
was accompanied by abnormal epithelial differentiation, as
well as blunted secondary septation (assessed by MLI), exces-
sive ECM production, hypercellularity of the mesenchyme, and
persistent alveolar inflammation. Using a clodronate approach,
the investigators further demonstrated that disturbed alveolar-
ization after loss of integrin-␤1 in the epithelium was second-
ary to persistent inflammation. These investigators proposed
that loss of integrin-␤1 in the epithelium increased epithelial
production of chemokine (C-C motif) ligand 2 (Ccl2), chemo-
kine (C-X-C motif) ligand 1 (Cxcl1), and reactive oxygen
species (ROS), which drove increased recruitment of macro-
phages, blunting alveolar development. These observations add
to a growing body of data, discussed in detail below, implicat-
ing inflammation as a key mediator of both normal and aber-
rant lung alveolarization. Lafemina and coworkers (181) ad-
dressed a role for a related cell-communication and adhesion
molecule, claudin 18, in postnatal lung growth. Claudin 18 is
reported to be a lung-specific tight junction protein that is
abundant on alveolar type I cells. Genetic ablation of claudin
18 expression had no impact on newborn mouse lung structure
but did stunt postnatal lung maturation (assessed by MLI). The
precise role played by claudin 18 in postnatal lung growth is
not yet clear; however, loss of Cldn18 did upregulate the
expression of amphiregulin, sonic hedgehog (Shh), and elastin
and downregulated the expression of fibroblast growth factor
(FGF) receptor (FGFR)4, adrenomedullin, and VEGF-A, hint-
ing at a possible mechanism. Along these lines, p66Shc (188),
IL-1␤ (142), and homeobox proteins Hox3a and Hox5a (42)
have also recently been added to the list of new mediators of
lung epithelial cell differentiation that impact the structural
Perspectives
L1248 ADVANCES IN LUNG ALVEOLARIZATION AND BPD
development of the lung although the underlying mechanisms
remain to be fully elucidated. Additionally, lung epithelial
TNF-␣-converting enzyme (TACE) was implicated by Xu and
coworkers (373) as a regulator of lung saccular formation, in
which genetic ablation of epithelial TACE retarded fetal lung
development but not postnatal alveolarization. As yet, the
molecular mechanisms at play in this process have not been
defined.
The dynamics of type II to type I cell differentiation are
currently a hot topic in lung developmental biology, and Hou
and coworkers (143) have provided data to suggest that hyper-
oxia drives type II cell transdifferentiation in neonatal rat pups
although the relevance to disease and the molecular mecha-
nisms at play have not been clarified. Zhao and coworkers
(389) made the striking observation that the balance between
TGF-␤ and bone morphogenetic protein signaling directs the
transdifferentiation of type II cells into type I cells, perhaps
hinting at a mechanism to explain the observations of Hou and
coworkers (143). Further to this idea, Ghosh and coworkers
(117) identified antagonism between Wnt3a and Wnt5a as a
mediator of alveolar epithelial cell phenotype and proposed
that IGF-1 stimulated type II to type I cell transdifferentiation
through activation of Wnt5a. This process also drove cell
wound repair in vitro. These ideas are integrated into a scheme
in Fig. 3, are emphasized by the demonstration by Keith
Tanswell’s group that the IGF/IGF receptor (IGFR)1 system is
a key regulator of alveolarization (192), and tie into earlier
work by Tom Mariani’s group (318), in which epithelial/
mesenchymal cross talk and IGF-1 contributed to FGFR-
dependent alveolar elastogenesis and proper airspace forma-
tion. The ability to drive repair is likely to be relevant to the
lung response to ventilation because ventilation and cyclic
stretch have recently been described by Kroon and coworkers
(176) to drive alveolar type II cell (but not fibroblast) death by
the extrinsic apoptotic pathway. These and other data reinforce
the idea that promoting epithelial repair is a candidate strategy
to manage aberrant alveolarization following hyperoxia and
ventilator injury to the lung. Consistent with this idea, Kim and
coworkers (166) identified tripartite motif-containing 72
(Trim72) as a mediator of alveolar repair in ventilated adult
mouse lungs, in which overexpression of Trim72 protected
against, and genetic ablation of Trim72 increased, susceptibil-
ity to ventilator-induced lung injury. The molecular mecha-
nisms of the protection conferred by Trim72 are not known;
however, these observations made in adult mice highlight
Trim72 as a molecule that warrants investigation in neonatal
models of hyperoxic and ventilator-induced lung injury.
Several investigators have recently built on the key finding
of Barkauskas and coworkers (30), who demonstrated that type
II cells are stem cells in the adult lung. Among key develop-
ments in this fast-moving field are observations recently made
by Ochieng and coworkers (162), who documented that induc-
tion of sex determining region Y-box 2 (Sox2) in type II cells
in vivo could “deprogram” type II cells, which reverted to a
less differentiated cell type that expressed the stem cell mark-
ers lymphocyte antigen 6 complex, locus A (also called Sca1),
and fucosyltransferase 4 (also called Ssea1), and coexpressed
Sftpc and secretoglobin, family 1A, member 1 [Scgb1a1; also
called club (formerly Clara) cell protein 10], which are char-
acteristics of bronchoalveolar stem cells. The ability of Sox2 to
regulate transformation-related protein 63 and GATA-binding
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protein 6 accounted, at least in part, for the effects of Sox2 on
stem-cell phenotype (266). This finding most likely has rele-
vance for the regeneration lung tissue, perhaps by modulating
Sox2 levels. Along the lines of this thinking, Jain and cowork-
ers (155) demonstrated a surprising plasticity of type I cells,
which up until now have been considered terminally differen-
tiated, as well as a bidirectional relationship between differen-
tiated distal alveolar epithelial cells in a process regulated by
TGF-␤. With these ideas in mind, Desai and colleagues (86)
have challenged the view that type I cells arise from type II
cells, providing data to suggest that type I and type II cells arise
directly from a bipotent progenitor in the prenatal lung but can
arise from type II cells in the postnatal lung, a stem cell
function that is activated by type I cell injury. The next few
years will no doubt see much progress in our understanding of
the interrelated plasticity of type I and type II cells, and this
work has received further clinical impetus by the recent report
that alveolar stem cell failure may be related to telomere
dysfunction (17).
Work in primary type I and type II cells continues to be
hampered by the lack of 1) availability of type I cells for in
vitro studies and 2) efficient delivery of genes to alveolar type
II cells, other than by viral-mediated transduction. Some prog-
ress has been made on both fronts over the past two years. Kim
and coworkers (166) have reported the enrichment of alveolar
type I cell preparations for in vitro studies by panning with
commercially available anti-rat podoplanin (T1␣) IgG. Grzesik
and coworkers (123) have also recently reported the ability to
deliver genes on plasmids to primary alveolar epithelial cells
by nucleofection technology, and Ramachandran and cowork-
ers (291) have described the efficient delivery of RNA inter-
ference oligonucleotides to polarized airway epithelia in vitro.
Although none of these methodologies has yet found wide-
spread use, these important advances will no doubt facilitate
further studies with alveolar type I and alveolar type II cells in
the future. Work is also currently ongoing to develop unbiased
protein screening approaches to detect quantitative changes in
protein expression in type II cells in response to hyperoxia
using isobaric labeling (isobaric tags for relative and absolute
quantitation) with tandem mass spectrometry (35). To date, this
approach remains at the proof-of-principle stage.
Fibroblasts and Fibroblast Differentiation
McGowan and McCoy (227) have examined the molecular
pathways that direct fibroblast migration during alveologen-
esis. These investigators reported data that suggest that Shh,
which is also known to play a role in lung fibrosis (209), directs
the uniform orientation of lung fibroblasts and organizes the
migration of lung fibroblasts toward the distal secondary sep-
tum. These investigators also made the observation that a
larger proportion of PDGF receptor (PDGFR)␣-expressing
lung fibroblasts bore more primary cilia than other cells present
in the alveolus did, and this may impart increased migratory
capacity to these cells and all these cells to respond to PDGF-A
chemotactic gradients. Lu and coworkers (200) further ex-
plored upstream mediators of Shh signaling and, using
Eya1⫺/⫺ and Six1⫺/⫺ knockout mice and Eya1⫺/⫺/Six1⫺/⫺
double-knockout mice, identified that the transcription factors
Eya1 and Six1 act together to regulate saccular development by
modulating Shh levels.

ADVANCES IN LUNG ALVEOLARIZATION AND BPD
The characterization of mesenchymal progenitors in the
embryonic lung as well as the postnatal lung continues to be an
area of intensive efforts. Recent advances include the demon-
stration by El Agha and coworkers (92, 93) that Fgf10-express-
ing cells represent a pool of mesenchymal progenitors in both
the embryonic and the postnatal lung, possibly suggesting that
Fgf10-positive cells might be useful for the development of
stem cell-based therapies for parenchymal lung diseases (65).
Furthermore, using tracing based on the expression of the
Wilm’s tumor suppressor gene, a reliable marker for cells
derived from the mesothelium, Cano and coworkers (56) de-
scribed the extraordinary heterogeneity of mesenchymal-de-
rived cells during lung morphogenesis. These studies continue
to set the stage for our understanding of the original of “the
cellular players” in late lung development.
Fibroblast differentiation and the existence and function of
fibroblast subsets are an exciting area of intensive work in the
lung development field. Lipofibroblasts in particular have re-
ceived much attention. This fibroblast subset is characterized
by the presence of lipid bodies although the presence of
lipofibroblasts in the human lung remains controversial. Using
light and electron microscopy, Tahedl and colleagues (327)
reported that, although lipofibroblasts could be detected in
adult rodent lungs, lipofibroblasts were not noted in any other
mammalian lungs examined, including human, dog, goat, and
horse lungs, among others. Furthermore, lipofibroblasts were
not seen in postnatal human lungs although this cell type was
abundant during postnatal mouse lung development. Ahlbrecht
and McGowan (7) subsequently pointed out that these data
conflicted with reports of lipofibroblasts in human lungs (298)
and suggested that alternative methodologies to identify lipo-
fibroblasts in autopsy tissue may play a role in whether lipo-
fibroblasts are detected or not. Clearly, additional work re-
mains to be done to clarify the existence of lipofibroblasts at
particular time points in the development of the lung across
several species and also to clarify a functional role for lipofi-
broblasts in alveolarization.
The presence or role of the lipofibroblast and a role for FGF
and PDGF signaling in arrested lung development associated
with BPD (228) and CDH (104 –108) have also received recent
attention, with one recent report highlighting the temporal
commitment of the PDGFR-␣⫹ cell lineage to lipofibroblasts
and myofibroblasts during late lung development (259). Con-
tinuing in this line, McGowan and McCoy (228) also suggested
that a transition from lipofibroblasts to myofibroblasts in BPD
drives thickened alveolar septa attributable to collagen depo-
sition. These investigators further suggested that PDGFR-␣-
driven Sox9 expression maintained the lipofibroblast state, and
that Sox9 expression was lost with the acquisition of the
myofibroblast state. As such, targeting PDGFR-␣ downstream
signaling pathways (involving Sox9) may represent a strategy
to drive fibroblasts from the myofibroblast back to the fibro-
blast phenotype, which would limit profibrotic activity and
possibly drive lung (re)generation. These ideas are presented
schematically in Fig. 2. These data have a clinical implication
as well, given the report of Popova and colleagues (284), who
documented that mesenchymal stem cells from patients with
BPD hold stable alterations to PDGFR-␣ gene expression that
drive hypoalveolarization.
Several new mediators of fibroblast motility, proliferation,
and ECM production have recently been identified, including
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Perspectives
L1249
the tumor-associated calcium signal transducer 2 (224, 225)
and cysteine-rich secretory protein LCCL domain containing 2
(also Lgl1) (386). How these mediators function to impact lung
development awaits clarification. Other elements of fibroblast
behavior have also received attention; for example, MMP-9
was identified as a mediator of fibroblast contractility in a
TGF-␤-dependent mechanism (171). The lung mesenchyme is
regarded as a regulator of lung development (223), and coming
work in this area will no doubt address roles for fibroblast
subsets in directing alveolarization, not only through the pro-
duction of the lung ECM “skeleton”, but also through cross
talk between the mesenchyme, in particular, as a regulator of
epithelial cell behavior (152, 348). Looking ahead, Li and
coworkers (190) have recently reported that progenitors of
secondary crest myofibroblasts are developmentally committed
in the early lung mesoderm and highlighted the utility of the
Gli1-CreERT2 line for the study of formation of the alveoli.
ECM Production and Remodeling
Alveolarization occurs through a complex process of cellular
and ECM interactions (226) assisted by mesenchymal and
other progenitors and physical forces such as breathing mo-
tions. Recent work addressing roles for the ECM in lung
development has examined both the production of ECM com-
ponents (Fig. 3), as well as subsequent assembly and remod-
eling of the ECM, building on the pioneering work of Richard
Bland and Kurt Albertine and colleagues (16, 38 – 40). One
recent study by Hilgendorff and coworkers (138) reported that
elastin haploinsufficient C57BL/6 mice exhibited perturbed
lung capillary growth and attenuated elastin deposition in the
developing parenchyma although no alveolarization defect was
noted (assessed by Lm). This study provides further impetus for
the study of collagen dynamics as well as elastin and collagen
cross linking in alveolarization.
In this line, there is currently much interest in the balance
between increased ECM production and impaired ECM deg-
radation, in the context of fibrotic lung disease (97). This topic
has been reviewed in detail recently, while asking whether
idiopathic pulmonary fibrosis is a disease of impaired ECM
degradation, rather than the classical view of increased ECM
production (232). This idea may be extended to lung develop-
ment. Several recent studies have addressed the posttransla-
tional processing of collagen and elastin fibers in experimental
and clinical BPD. Studies have included the examination of
enzyme systems that direct the cross linking of collagen and
elastin fibers, which include the lysyl oxidase, lysyl hydroxy-
lase, and transglutaminase enzyme families. The expression of
lysyl oxidases (namely Lox and LoxL1) (178), lysyl hydrox-
lyases (namely lysyl hydroxylase 2, also called Plod2) (369),
and transglutaminase 2 (368) has been associated with BPD in
clinical subjects and with arrested alveolarization in the hyper-
oxia-based (85% O2 for 14 days) C57BL/6 mouse model of
BPD. Studies employing pharmacological interventions or
knockout mouse strains are required to causally implicate these
enzyme families in aberrant lung development. To this end,
Mižíková and coworkers (238) set out to test a pathogenic role
for lysyl oxidases in arrested alveolarization associated with
hyperoxia exposure. Inhibition of lysyl oxidases with ␤-amino-
propionitrile (BAPN) in developing C57BL/6 mouse pups in
the hyperoxia BPD model was without effect on alveolariza-
Perspectives
L1250 ADVANCES IN LUNG ALVEOLARIZATION AND BPD

Mechanical
ventilation

O2

Activation of
MMP-2/9

Increase of
Periostin Glucorticoids
TGF-β signaling

FIBROBLAST

Loss of Sox9
Hif-2α Decreased sCG activity
activation Gain of Sox9 PDGF-A

(MYO)FIBROBLAST
Increased
miR-210 Trop2
expression Crispld2

Increased expression Increased

Blocked Mnt Increased Increased
of ECM remodeling expression of
expression proliferation migration
enzymes ECM components

Blocked
miR-206
expression
Fibronectin Collagen Elastin

BAPN
Lysyl oxidase
Transglutaminases
Lysyl hydroxylase

Arrested Increased ECM

alveolarization stability

Fig. 2. Integration of selected, recently identified signaling pathways that can be induced by either hyperoxia or mechanical ventilation, which might impact
fibroblast function during alveolarization. The scheme only details new developments that were published between 1 January 2013 and 30 June 2015. Crispld2,
cysteine-rich secretory protein LCCL domain containing 2; MMP-2/9, matrix metalloproteinase 2 and 9; TGF-␤, transforming growth factor-␤; Sox9, sex
determining region Y-box 9; sCG, soluble guanylate cyclase; Trop2, trophoblast antigen 2; HIF-2␣, hypoxia-inducible factor 2␣; ECM, extracellular matrix; miR,
microRNA; BAPN, ␤-aminopropionitrile; MNT, MAX network transcriptional repressor.

tion (assessed by stereology) at P10 and worsened lung devel-
opment at P20, perhaps indicating a need for a careful balance
of lysyl oxidase activity during organogenesis (238). Indeed,
BAPN proved to be very toxic in mouse pups, limiting the
suitability of BAPN as an interventional tool in developing
animals (238), although BAPN is well tolerated in adult mice
when organogenesis is complete (253). Contrasting data were
obtained by Mammoto and coworkers (212), who reported that
lysyl oxidase inhibition with BAPN improved alveolarization
(assessed by MLI) in the mouse hyperoxia model. Both groups

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ADVANCES IN LUNG ALVEOLARIZATION AND BPD
BMP signaling
O2
IGF-I IL-1β
Wnt5a
Wnt3a
Alveolar epithelial
Transdifferentiation
type II cell
TGF-β signaling
employed C57BL/6 mice and a 10-day hyperoxia-exposure
period; however, Mižíková (238) employed 85% O2 and a
stereological analysis, whereas Mammoto (212) employed
75% O2 and a morphometric (MLI) analysis. Whether these
differences explain the discordant observations is unclear. A
weakness of the Mammoto study (212) is that the investigators
supplemented maternal nutrition with BAPN and assumed
maternal transmission to pups through the breastmilk; how-
ever, no data on BAPN levels or lysyl oxidase activity levels in
the lungs of pups were provided. As such, it is not known
whether lysyl oxidase activity levels were impacted at all in
hyperoxia-exposed neonates. The investigators claimed to have
repeated the intervention with intraperitoneal application of
BAPN every other day to mouse pups and apparently obtained
comparable effects on protecting the developing lung from
hyperoxia-induced arrest of alveolarization. However, these
data are neither provided in the manuscript proper nor in the
online supplement. These studies highlight the need 1) to
demonstrate the delivery of pharmacological agents to the
target organs or at least to demonstrate that pharmacological
agents that were applied did have the desired impact in the
target organs; and 2) to provide supporting data, in this case,
the intraperitoneal application of BAPN in the Mammoto study
(212), which could have been compared with the analogous
study by Mižíková (238).
Other ECM components such as periostin, a ligand for ␣v␤3
and ␣v␤5 integrins to support the adhesion and migration of
epithelial cells, have also received attention (9, 44). Both
studies localized periostin to myofibroblasts and clearly indi-
cated that periostin (Postn) expression is driven by hyperoxia.
Ahlfield and coworkers (9) demonstrated that hyperoxia-in-
duced arrest of alveolarization has not been impacted (assessed
by MLI) by global loss of periostin in C57BL/6J Postn⫺/⫺
mice, when mice were exposed to 60% O2 or 85% O2 between
P1 and P7 and assessed at P7. Opposing data were obtained by
Boyzk and coworkers (44), who reported that exposure of
Postn⫺/⫺ mice to 75% O2 from P2 or P3 for 14 days fully
protected against hyperoxia-induced arrest of alveolarization
(assessed by Lm). These investigators also described a circle of
periostin driving TGF-␤ and TGF-␤ driving periostin expres-
sion (highlighted in Fig. 2). Given the opposing trends noted in
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Perspectives
L1251
Trim72
Fig. 3. Some recently described pathways by
which hyperoxia might modulate type II cell-
type I cell transdifferentiation. The proposed
roles for bone morphogenetic protein (BMP)
Wound healing signaling, IGF, IL-1␤, TGF-␤ signaling, tripar-
tite motif-containing 72 (Trim72), Wnt3a and
Wnt5a, are indicated. The scheme only details
new developments that were published between
Alveolar epithelial 1 January 2013 and 30 June 2015.
type I cell
the two studies, it remains unclear whether periostin partici-
pates in aberrant alveolarization. There are several possible
explanations for the opposing trends observed by the two
groups of investigators. It is clear that different oxygen levels
and different exposure protocols were employed. Additionally,
two different mouse strains and backgrounds have been em-
ployed, in which Ahlfield and coworkers (9) employed a
mouse strain (301) in which exon 1 of the periostin gene was
insertionally inactivated with a LacZ cassette. Additionally,
this strain was backcrossed over eight generations onto a
C57BL/6J background. In contrast, Boyzk and coworkers (44)
employed the B6;129-Postntm1Jmol/J strain, in which the exons
4 –10 of the periostin gene were replaced with a neomycin
resistance cassette (268), and the strain was maintained on a
mixed C57BL/6 and 129 background. As a further potential
confounding variable, periodontal disease was present in
Postn⫺/⫺ mice, wild-type mice, and knockout animals fed with
powdered chow by Boyzk and coworkers (44). These studies
exemplify intermodel comparisons that arrive at opposite con-
clusions, possibly attributable to the use of different oxygen
injury protocols or different background strains, as highlighted
in Animal Models of Arrested Alveolarization, or attributable to
different approaches to targeting genes in model animals.
Changes in ECM abundance in the airways have recently
been correlated with lung function in equine heaves (314), and
the influence of mechanical ventilation on ECM production in
neonatal lungs has also recently been addressed. Kroon and
coworkers (177) demonstrated that mechanical ventilation of
8-day-old mouse pups with low (3.5 ml/kg), moderate (8.5
ml/kg), or high (25 ml/kg) tidal volumes impacted ECM gene
expression. Increased expression of the tropoelastin, fibulin 5,
lysyl oxidase-like 1, and tenascin C genes was noted after an
8-h high tidal volume ventilation with room air. This was
correlated with an altered appearance of parenchymal elastin
fibers and reduced vessel density; however, lung structure was
not quantified. These data demonstrate the power of mechan-
ical ventilation to drive ECM gene expression, but the impact
of altered gene expression reported in this study on alveolar-
ization remains unclear.
The ability to therapeutically modulate ECM production by
fibroblasts continues to be an area of interest in terms of
Perspectives
L1252 ADVANCES IN LUNG ALVEOLARIZATION AND BPD
influencing aberrant late lung development in premature neo-
nates. Among the key reports that have emerged in the past 2.5
yr are the exciting finding that glucocorticoids such as flutica-
sone can have divergent effects on ECM production by fibro-
blasts, in which fluticasone drove decorin production in airway
fibroblasts but inhibited biglycan and procollagen production
by parenchymal fibroblasts (47). These data also highlight the
idea of compartmentalized responses to therapy in different
cell types. More generally, it has also recently emerged that
administration of dexamethasone to newborn mice over the
first 4 days of postnatal life impaired alveolarization, ostensi-
bly by the suppression of tenascin C and elastin expression
(303). Glucocorticoids have also recently been demonstrated to
modulate TGF-␤ signaling by redirecting TGF-␤ activity from
the TGF-␤ receptor 1 (Tgfbr1)/smooth muscle actin/mothers
against decapentaplegic (Smad)2/3 axis to the activin A recep-
tor type II-like 1 (Acvrl1)/Smad1 axis in lung fibroblasts. This
may represent a relevant drug-pathway interaction in lung
disease and development (312); however, ECM component
expression was not addressed in that study. Preliminary evi-
dence has also been provided for some new players in the
regulation of ECM production and reorganization during
lung development, including the serine peptidase chymot-
rypsin-like elastase 1 (197) and cathepsin K (170) and the
possible interaction between integrins and the ECM (128);
however, the relevance of these candidates in alveolariza-
tion remains to be demonstrated.
Growth Factors
Along with physical forces (stretch and breathing motions),
the ECM, and coordinated transcription factor expression,
growth factors are credited with regulatory roles in alveolar-
ization (243). Belcastro and coworkers (32) have added to a
growing wealth of evidence implicating disturbances to growth
factor dynamics in arrested alveolarization. Using a hyperoxia-
based mouse model of BPD (60% O2 for 14 days), the
investigators confirmed the increased parenchymal expression
of TGF-␤ in hyperoxia-exposed mouse lungs and further dem-
onstrated that in vivo inhibition of TGF-␤ signaling with
SB431542 concomitant with hyperoxia exposure limited prox-
imal TGF-␤ signaling (as assessed by Smad3 phosphoryla-
tion), and this was accompanied by an increase in secondary
crest formation. These investigators further documented that
increased peroxynitrite, thought to be generated by macro-
phages, drove increased TGF-␤ production. Furthermore, the
peroxynitrite caused nitration of IGFR1, thereby preventing
binding of IGF-1 to its cognate receptor and hence limiting
downstream IGF-1 signaling, with deleterious consequences
for alveolarization. In support of a role for IGF-1 in alveolar-
ization, Galvis and coworkers (115) described that the repres-
sion of IGF-1 expression by the histone methyl transferase
Ezh2 prevented basal cell differentiation in the developing
lung. However, this idea has not yet been assessed in postnatal
lung maturation. This study, along with the work of Brechbuhl
and coworkers (48), adds the IGF system and the EGF system
to the cocktail of modulators of basal cell differentiation and
proliferation.
The TGF-␤ system, which is activated by exposure to a
hyperoxic environment (18), continues to receive attention as a
key mediator of alveolarization (359) and has recently been
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identified as the mediator of epithelial-to-mesenchymal transi-
tion in the lungs of rat pups exposed to hyperoxia (85% O2
from P1 to P7) (350). Using transgenic mice that overexpress
TGF-␤ or mice with genetic ablation of the type II TGF-␤
receptor (Tgfbr2) in the lung epithelium, Sureshbabu and
coworkers (323) described a pivotal role for the TGF-␤/Tgfbr2
axis in mediating arrested alveolarization in the background of
hyperoxia. To this end, the ability of several pharmacological
agents to limit hyperoxia-induced arrest of alveolarization has
been attributed to the ability of these agents to blunt hyperoxia-
induced TGF-␤ signaling, as was the case with curcumin (305)
and nutritional supplementation with docosahexaenoic acid
(345) and an activin A receptor type IIB-Fc antagonist (194).
Other studies have addressed drug-cell interactions in the
context of growth factors; for example, Schwartze and cowork-
ers (312) demonstrated that glucocorticoids modulated TGF-␤
signaling in lung fibroblasts and could shift TGF-␤ signaling
from the Tgfbr1/Smad2/Smad3 axis to the Acvrl1/Smad1 axis
and, in doing so, promote lung myofibroblast differentiation.
Given the widespread use of steroids in the management of
patients with BPD, these studies reveal a drug-cell interaction
of potential clinical significance. The interaction between
TGF-␤ signaling and glucocorticoids has also been studied by
Collins and coworkers (74) in the preterm lamb model in the
context of intrauterine inflammation, in which betamethasone
was documented to blunt TGF-␤ signaling in fetal lungs. The
potential importance of these observations is underscored by
the report of Alanis and coworkers (12), who described that the
boundary separating the two distinct lung compartments, the
proximal conducting airways and the peripheral gas exchange
regions, the bronchoalveolar duct junction, may undergo a
proximal or distal positional shift after premature activation or
loss of glucocorticoid signaling. Other modulators of TGF-␤
signaling in developing lungs have been identified, including
all-trans-retinoic acid, which dampened aberrantly high
TGF-␤ signaling in a caloric restriction model of BPD in rats
(1, 199). Some recent observations made in early lung devel-
opment, such as the control of epithelial differentiation by the
hippo pathway, in which yes-associated protein 1 regulates the
ability of epithelial progenitor cells to respond to TGF-␤-
induced cues (208), await exploration in a postnatal context.
Inflammation
Inflammation is a common complication of BPD, both in a
clinical setting and in experimental animal models that use
inflammation as an injurious stimulus to mimic chorioamnio-
nitis (34). The role played by inflammation in limiting lung
development in other BPD models, notably those based on
hyperoxia and mechanical ventilation, remains relatively un-
explored. The direct (parenteral) attenuation of inflammation
has been undertaken with daily, subcutaneous administration
of an IL-1 receptor antagonist (IL-1Ra) in two slightly different
BPD models (257). Interestingly, in a perinatal inflammation
plus hyperoxia model, in which pregnant dams were first
treated with LPS and then newborn pups were exposed to 85%
O2 for 28 days, no impact of IL-1Ra administration on lung
development (assessed by calculation of the surface area-to-
volume ratio using ImageJ software) was noted. However, in a
“gentler” alternative model, in which, instead of 85% O2,
newborn mouse pups were exposed to 65% O2, a dramatic

ADVANCES IN LUNG ALVEOLARIZATION AND BPD
improvement in lung structure was noted. Similarly, adminis-
tration of resolvin D1 and lipoxin A4 every third day to
C57BL/6 pups exposed to 95% O2 from P1 to P10 improved
alveolarization (assessed by MLI and RAC), which was asso-
ciated with reduced proinflammatory cytokine gene expression
(217). These data indicate 1) the potential benefit of attenuating
inflammation on alveolarization in BPD models and 2) that the
selection of oxygen levels may dramatically influence the
outcome of interventional studies. It is clear that broadly
blunting inflammation in BPD models improves alveolariza-
tion. Future work must address how inflammation modulates
alveolarization.
Jagarapu and coworkers (153) evaluated leukadherin-1
(LA1; applied by daily intraperitoneal injection), a novel ago-
nist of leucocyte surface integrins, and CD11b/CD18, which
blunts transendothelial leukocyte migration to sites of injury in
the hyperoxia-based (85% O2 from P1 to P14) rat model of
BPD. LA1 administration decreased neutrophil and macro-
phage infiltration into the lungs, provoked hyperoxia, improved
alveolarization (assessed by MLI and RAC) and angiogenesis,
and limited aberrant pulmonary vascular remodeling. These
data clearly demonstrate that inflammation plays a key role in
aberrant alveolarization in response to hyperoxia. Identical
observations were made by Drummond and coworkers (88),
who applied AMD3100, an antagonist of Cxcr4 in a similar rat
hyperoxia model (90% O2 from P1 to P16, AMD3100 applied
between P5 to P15, in a therapeutic regimen). Cxcr4 antago-
nism also decreased neutrophil and macrophage infiltration
into the lungs, provoked hyperoxia, and improved alveolariza-
tion (assessed by MLI) and angiogenesis, and limited aberrant
pulmonary vascular remodeling. A role for epithelial integrins
in macrophage recruitment has already been described earlier
in this Perspectives article, relating the work of Plosa and
coworkers (281) using an epithelial-specific (Sftpc-driven)
deletion of integrin-␤1, which promoted macrophage recruit-
ment and blunted alveolarization (assessed by MLI) in mice,
ostensibly through increased proinflammatory cytokine (Ccl2
and Cxcl1) and ROS production by lung epithelial cells. These
data highlight the regulatory cross talk between the epithelium
and inflammatory cells during alveolarization and add to a
growing body of data highlighting the connection between
inflammatory signaling and ROS production (130). Further
studies by Carver and coworkers (58) have linked innate
immune signaling and regulatory developmental programs in
the lung, in which a link between NF-␬B signaling and the
expression of FGF-10, a regulator of lung development, was
demonstrated. The authors suggested that NF-␬B-driven in-
flammatory pathways may suppress the expression of growth
factors and other mediators of alveolarization, which would
negatively impact lung structure. These data take on increased
importance in light of the documented regulation of lung
epithelial stem cell growth by TGF-␤ and FGF-10 (93, 233).
Interestingly, in contrast to the ideas of Carver and coworkers
(58), Hou and coworkers (144), using BAY 11–7082, which is
an inhibitor of NF-␬B signaling, have indicated that it may be
desirable to promote some elements of NF-␬B inflammatory
signaling to suppress damaging chemokine (C-X-C motif)
ligand 2 (Cxcl2) [also called macrophage inflammatory protein
(MIP)-2] production. Clearly much remains to be learned about
the contribution of NF-␬B signaling to postnatal lung matura-
tion.
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Perspectives
L1253
Efforts have been made to further delineate the specific
inflammatory players that impact lung maturation. Among
recent studies, Stouch and coworkers (320) identified I␬B as a
mediator of fetal macrophage maturation and suggested that
the maturation of fetal lung macrophages into mature alveolar
macrophages might include proinflammatory features, hitherto
unrecognized aspects of macrophage activation. Syed and
Bhandari (326) have revealed that macrophage polarization
was skewed in favor of an M1 phenotype after hyperoxia
exposure (100% O2 for 7 days). These studies support a role for
macrophage polarization in promoting aberrant alveolarization
(assessed by Lm) in response to hyperoxia and add to a growing
body of data defining very diverse roles for macrophages in
lung disease (6). This idea was recently elaborated upon by
Eldredge and coworkers (94), who report existence of an
anti-inflammatory population of CD11b⫹ mononuclear cells
that are protective against hyperoxia-induced injury to neonatal
lungs; however, only survival and inflammation were assessed
in that study, without consideration of changes to the lung
architecture. A follow-up study is eagerly awaited, particularly
considering the emerging roles of inflammatory cell types and
subpopulations in lung development and disease (6, 367), to
identify causal roles for specific inflammatory cell subsets in
lung development. By way of examples, Podolin and cowork-
ers (282) recently demonstrated that T-cell depletion protects
against alveolar destruction attributable to chronic cigarette
smoke exposure in mice, but the depletion of T cells has yet to
be evaluated in the context of lung development.
Efforts to modulate regulators of inflammation in experi-
mental BPD have also received attention, with McGrath-
Morrow and coworkers (230) focusing on Nfe2l2 (also called
Nrf2), which was formerly demonstrated to be protective in
hyperoxia-induced arrest of alveolarization (assessed by Lm).
Interestingly, using sulforaphane, an inducer of Nfe2l2, the
investigators demonstrated that, although inflammation could
be attenuated by Nfe2l2 induction, alveolar growth was not
improved, possibly attributable to proliferation-suppressive ef-
fects of Nfe2l2. This study makes the important point that
caution should be exercised when anti-inflammatory strategies
may have the side effect of growth inhibition during a critical
phase of rapid postnatal growth and highlights the need for
additional studies both on Nfe2l2 and other molecules that
protect against oxidative stress, such as Sirt1 (376). An inter-
esting angle on the Nfe2l2 line has been developed in adult
mice by Kawamura and coworkers (163), who reported that the
anti-inflammatory effects of hydrogen gas [2% (vol/vol)] in the
background of hyperoxia (98% for 60 h) were Nfe2l2 depen-
dent. The use of hydrogen gas to limit hyperoxia-induced
damage in neonates has not yet been reported. Another regu-
lator of inflammation that has received recent attention is
macrophage migration inhibitory factor (MIF) (321, 322), in
which it was demonstrated that both too much (overexpression
in transgenic mice) and too little (MIF knockout mice) MIF
arrested alveolarization (assessed by Lm) in both room air-
exposed and hyperoxia-exposed mice. Thus the correct balance
of MIF levels in the developing lung was essential for proper
lung development. Administration of a small molecular MIF
agonist to wild-type mice in the hyperoxia BPD model partially
restored normal lung alveolarization, further supporting the
idea that modulating inflammation represents a viable approach
for the restoration of lung development in BPD.
Perspectives
L1254 ADVANCES IN LUNG ALVEOLARIZATION AND BPD
In addition to a role for inflammation in modulating alveo-
larization, innate immunity has also been addressed in the
context of BPD, in which where Raffay and coworkers (288)
demonstrated that hyperoxia exposure (85% O2 for 14 days,
with a posthyperoxia recovery phase) influenced club (for-
merly Clara) cell function, evident by decreased secretoglobin,
family 3A, member 1 (Scgb3a1) and Scgb1a1 protein expres-
sion, which would have implications for the innate immune
response. These studies may indicate a mechanism by which
infants with BPD are more susceptible to lung infections.
Along the lines of this study, Cai and coworkers (54) demon-
strated that administration of a recombinant-related secretoglo-
bin, secretoglobin, family 3A, member 2 (Scgb3a1), to preg-
nant mice promoted the development of terminal air sacs in
preterm (E17.5) pups (as assessed by RAC). Together, these
data might suggest that the decreased expression of secreto-
globin in the hyperoxia model observed by Raffay and col-
leagues may have also directly negatively impacted terminal
air-sac development or alveolarization, in addition to limiting
innate immune responses.
Pulmonary Vascular Development and PPHN
Pulmonary vascular development and vascular endothelial
and smooth muscle cell function are perturbed in a spectrum of
lung developmental abnormalities, including BPD (21, 245,
319), CDH (3), ACD (172, 302), and persistent pulmonary
hypertension of the newborn (PPHN) (84, 183, 237). Under-
standing the molecular regulation of pulmonary vascular de-
velopment remains a major challenge (273). The past 2 yr have
seen the identification of several new mediators of pulmonary
vascular development, including Wntless (75) and Sox17
(184), which play a role in embryonic vessel development, but
a function for either mediator in normal or aberrant late lung
development is not yet clear. In contrast, the semaphorin/
neuropilin 1 axis was demonstrated to play a role in fetal, but
not postnatal, pulmonary vessel development (160), and pig-
ment epithelium-derived factor appeared to be involved in
perturbed vascular development associated with hyperoxia
(69). Additionally, antagonism of PDE5 with sildenafil pro-
moted pulmonary vascular development and alveolarization,
ostensibly via activation of the phosphatidylinositol-4,5-bis-
phosphate 3-kinase/Akt/mammalian target of rapamycin path-
way and enhanced HIF-1␣ and HIF-2␣ activity (270). Other
candidate regulators of lung vessel cell behavior have also been
identified in vitro, including heat-shock protein 70 (5), the
oxygen-sensitive voltage-gated potassium channel Kv1.5
(202), and AMPK (330); however, a role for these molecules in
pulmonary vascular development awaits clarification.
Along with reports that the transcription factor forkhead box
(Fox)M1 regulates pulmonary inflammatory responses to hy-
peroxia (371), FoxF1 has also been recently implicated as a
candidate regulator of aberrant pulmonary vascular development
in a spectrum of congenital diseases. FoxF1 haploinsufficiency is
characteristic of ACDMPV. Therefore, a transcriptional profiling
study of lung from Foxf1⫹/⫺ mice was undertaken by Sen and
coworkers (313), who reported that FoxF1 regulated the expres-
sion of a spectrum of genes, including multiple collagens and
members of the IGF pathway. These data contribute to our
further understanding of the mechanisms underlying disturbed
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00268.2015 • www.ajplung.org
pulmonary vascular development in BPD, CDH, and ACD,
including ACDMPV.
Further advances have also been made in our understanding
of the molecular mechanisms at play that drive PPHN. Among
these, Delaney and coworkers (85) reported that serotonin
(5-hydroxytryptamine) contributed to elevated pulmonary vas-
cular resistance in an experimental sheep model of PPHN via
the 5-HT2A receptor. The angiotensin system has also received
attention in the context of postnatal lung development, in
which Castro and coworkers (59) describe the downregulation
of angiotensin-converting enzyme (ACE) expression in the
lungs of patients with BPD, suggesting a pathophysiological
contribution of diminished ACE levels to the development of
clinical BPD. This idea is supported by Wagenaar and cowork-
ers (352), who reported that stimulation of the MAS oncogene
with cyclic (Ang)1–7 or stimulation of the type II angiotensin
receptor with [D-lysine]cyclic(Ang)1–7 in the rat hyperoxia
model (100% O2 for 10 days, from P1) partially protected
against hyperoxia-induced damage to alveolarization, with an
increased number of alveolar crests and decreased septal
wall thickness observed. Additionally, the arteriolar medial
wall thickness and Fulton index were partially normalized.
Collectively, these observations support a role for dysregu-
lation of the angiotensin system in clinical and experimental
BPD. In another study, Nijmeh and coworkers (256) re-
vealed the existence of high proliferative potential colony-
forming cells (HPP-CFC) in the adventitial vaso vasorum of
the pulmonary arteries in a hypoxia-induced PPHN model in
calves. These authors proposed that, given the high prolif-
erative potential and vasculogenic capacity of HPP-CFC,
these cells may be pathogenic mediators and perhaps targets
for the management of pulmonary vascular remodeling
associated with hypoxia exposure in neonates.
Several lines of evidence point to PPAR-␥ as a key contrib-
utor to perturbed vascular development and homeostasis in
PPHN. Wolf and colleagues (370) demonstrated that increased
levels of endothelin (ET)-1, which are indeed observed in
clinical PPHN, decreased PPAR-␥ signaling and blocked pul-
monary artery endothelial cell tube formation. PPAR-␥ ago-
nists promoted endothelial tube formation in pulmonary artery
endothelial cells in an NO-dependent manner. These observa-
tions suggest that targeting ET-1 production may restore an-
giogenesis in PPHN. In another study, Wagenaar and cowork-
ers (351) also addressed the ET-1 system using ambrisentan
(an endothelin receptor type A antagonist) in the rat hyperoxia
model (100% O2 from P1 to P10), which improved survival,
promoted alveolar development by normalizing septal wall
thickness (without impacting the number of alveolar crests per
field), limited excessive ECM deposition, and dramatically
improved aberrant remodeling of vessel walls and the heart that
were provoked by hyperoxia exposure. Interestingly, these
positive effects were not seen in a late treatment protocol (pups
were exposed to 100% O2 for 9 days, followed by 9 days of
recovery in room air, with daily ambrisentan application initi-
ated on day 6). A potential connection between the HIF-1␣ and
ET-1, as demonstrated by Larissa Shimoda’s group in smooth
muscle cells in adult rats (280), has not yet been explored in the
context of PPHN.
Returning to PPAR-␥, Gien and coworkers (118) revealed
an interaction between PPAR-␥ and Rho kinase (ROCK) that
increased pulmonary artery smooth muscle cell proliferation

ADVANCES IN LUNG ALVEOLARIZATION AND BPD
and proposed that this interaction was relevant to the aberrant
vascular remodeling seen in PPHN. These data are very inter-
esting considering the observations of Lee and coworkers
(186), who reported that inhibition of ROCK with Y-27632,
not only attenuated the deleterious impact of bleomycin in
alveolarization in rat pups, but also normalized pulmonary
vascular resistance, decreased right-ventricular hypertrophy,
and attenuated vascular remodeling. Thus PPAR-␥ and ROCK
are exciting candidate targets in the management of pulmonary
hypertension associated with BPD, as well as PPHN; however,
the demonstration that some elements of pulmonary artery
contraction are likely Rho independent (98) suggests that other
mediators are also involved. It is worth noting at this junction
that stimulation of PPAR-␥ signaling can rescue alveolariza-
tion and vessel development in the hyperoxia model, in which
Lee and coworkers (187) applied Escherichia coli LPS to the
amniotic sac of pregnant mice. After spontaneous delivery,
pups were exposed to hyperoxia (80% O2 from P1 to P7)
followed by recovery in room air (P8 to P14), which blunted
both alveolarization (assessed by Lm) and vessel density (as-
sessed by visual inspection and PECAM immunohistochemis-
try). Administration of the PPAR-␥ agonist rosiglitazone res-
Perspectives
L1255
cued both vascularization and vascular growth. Taking this
idea further, Morales and coworkers (240) nebulized the
PPAR-␥ agonists rosiglitazone and pioglitazone into the lungs
of 1-day-old rat pups, which were subsequently exposed to
hyperoxia (95% O2 for 3 days, intervention once daily). Neb-
ulized PPAR-␥ agonists protected against hyperoxia-induced
retardation of alveolarization (assessed by MLI and RAC) and
blunted hyperoxia-provoked lung inflammation. Although not
assessed in pulmonary vascular development, in the context of
sickle cell disease-associated pulmonary hypertension, Li and
coworkers (191) described the downregulation of ET-1 by
fenofibrate-stimulated PPAR-␥, which upregulated miR-
199a2, which in turn targeted ET-1 expression. This pathway
may also be relevant to the ET-1/PPAR-␥ axis in the context of
vascular development as well. Thus the PPAR-␥ system rep-
resents an exciting druggable target to drive lung alveolar and
vascular development. These ideas are integrated into a scheme
in Fig. 4. Apart from PPAR-␥/ET-1, AMPK has also recently
been introduced as a candidate target to drive angiogenesis in
utero (330).
In an effort to delineate the impact of congenital heart
defects on lung development, Razavi and coworkers (296)

Normalization of lung development

Proper cascular development and alveolarization

Normalized proliferation Enhanced tube formation

Decreased Increased Fig. 4. Recently identified roles for peroxi-

some proliferator-activated receptor-␥
ROCK eNOS
(PPAR-␥) in pulmonary vascular develop-
activity activity
ment and homeostasis associated with persis-
Rosiglitazone tent pulmonary hypertension of the newborn
Increased
Pioglitazone ACTIVATION (PPHN). Modulation of PPAR-␥ is poten-
Fenofibrate miR-199a2 tially relevant to pulmonary vascular devel-
expression opment and homeostasis in bronchopulmo-
SMC EC
nary dysplasia (BPD) and PPHN. PPAR-␥
can be modulated by activators (green) or by
PPARγγ the suppression of inhibitory pathways (red)
by attenuating the effects of endothelin
SMC EC (ET)-1 mediated by the endothelin 1A
(AT1A) and 1B (ET1B) receptors. The effects
ET1A of PPAR-␥ agonism may impact smooth
INHIBITION ET1B
ET-1 muscle cells (SMC) and endothelial cells
(EC) by modulating endothelial nitric oxide
Increased Decreased synthase (eNOS) or Rho kinase (ROCK) ac-
ROCK eNOS tivity. The scheme only details new develop-
ments that were published between 1 January
activity activity
Ambrisentan 2013 and 30 June 2015.
Bosentan

Hyperproliferation Impaired tube formation

Increased vascular remodeling and decreased angiogenesis

PPHN/BPD

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00268.2015 • www.ajplung.org

Perspectives
L1256 ADVANCES IN LUNG ALVEOLARIZATION AND BPD
employed a model of pulmonary artery stenosis in rats. After
the left pulmonary artery was banded at 21 days of life, lung
structure was assessed at 160 days. Left pulmonary artery
banding resulted in an apparent decrease in alveolar size
compared with sham-operated animals, suggesting that dis-
turbed blood flow to the lung can limit alveolarization. It must
be kept in mind that arterial banding study was performed after
the phase of bulk alveolarization but before the continued
alveolarization described by Tschanz and coworkers (341) that
occurs between P21 and P60. As such, it remains unclear
whether the apparently reduced alveolar size results from a
failure of late alveolar development or from alveolar destruc-
tion in response to blood flow limitation.
The role of perturbed ECM deposition and remodeling,
which result in vessel stiffness, is an exciting and new area of
research in the pulmonary vasculature. Recent work by Nave
and coworkers (253) described aberrant collagen and elastin
cross linking in pulmonary vessels in pulmonary arterial hy-
pertension in adult mice. Additionally, vascular stiffening is
presently being debated as a cause or effect of pulmonary
hypertension in human adults (328). Along these lines, Dodson
and coworkers (87) reported that the main pulmonary arteries
in lambs with PPHN exhibited increased stiffness and exces-
sive ECM remodeling. These exciting data point to hitherto
unrecognized roles for perturbations to vessel stiffness in
proximal (as opposed to distal) pulmonary vessels as a patho-
genic factor in PPHN and highlight the examination of ECM
deposition and the activity of ECM cross-linking enzymes as a
worthwhile avenue to pursue in understanding the mechanistic
basis of PPHN.
A Role for the Small Airways?
The development of the alveoli and the pulmonary vascular
tree has received much attention in the context of late lung
development. However, despite the pioneering reports in the
early 1990s by Marc Hershenson and Julian Solway demon-
strating that exposure to hyperoxia in the immediate postnatal
period causes airway hyperresponsiveness and aberrant airway
remodeling (135, 136) and studies by Shenberger and col-
leagues (315) that demonstrated pulmonary neuroendocrine
cell hyperplasia in developing rats in response to hyperoxia,
the small airway compartment has been very neglected. Given
the intimate relationship that must exist between the develop-
ing airways and the associated parenchyma and vessels (53),
together with many recent advances in airway smooth muscle
cell biology (286), further investigations into airway biology
during normal and aberrant late lung development are war-
ranted. This idea is supported by the predisposition of preterm
neonates to clinical problems related to the airways, such as
wheeze and asthma (50), and the recent exciting observation
from Wolfgang Kübler’s group that upper airway distension
triggers proinflammatory responses in mechanically ventilated
adult C57BL/6 mice (255).
Some recent reports have addressed the role of postnatal
hyperoxia exposure on bronchiolar remodeling that is evident
in adult experimental animals (261–263, 293); however, a
comprehensive assessment of small airway architecture in
neonates has not been undertaken. This appears all the more
important given the recent summary by Hye-Youn Cho and
Steven Kleeberger highlighting the perspective of Nfe2l2 (also
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00268.2015 • www.ajplung.org
called Nrf2) in the airways as a mediator of oxidant damage
responses in the airways (70). The recently described role of
the transcription factors Spdef and FoxA3 in goblet cell meta-
plasia and Th2-mediated inflammatory responses in the post-
natal lung (290) provides impetus for such investigations. A
role for the pulmonary neuroendocrine cells in aberrant lung
alveolarization also awaits attention.
Some studies have started to address the impact of hyperoxia
on small airway structure. Wang and coworkers (355) de-
scribed the differential impact of elevated oxygen levels on
smooth muscle mass and collagen deposition in the developing
airways in mice exposed to 40% O2 or 70% O2 from P1 to P7,
followed by recovery in room air between P8 and P21. Other
reports, for example those documenting the impact of cigarette
smoke on mitochondrial function (24, 25) and the proliferation
and ECM production (347) in airway smooth muscle cells, as
well as the impact of hyperoxia on estradiol metabolism in
postnatal airway smooth muscle (219), highlight the potential
relevance of perturbations to airway development to lung
maturation. Furthermore, Chen and colleagues (67) provide
evidence that endogenous retinoic acid, a key player in lung
development, restricts airway smooth muscle cell differentia-
tion during formation of the airways. Similarly, Xie and co-
workers (372) connected Wnt/␤-catenin and Sox2, two other
key players in lung development already discussed in this
Perspectives article, in airway submucosal gland morphogen-
esis. Clearly, much interesting work in this direction lies ahead.
Long-Term Sequelae of Hyperoxia Exposure
There is currently much interest in the long-term follow-up
of patients with BPD to assess the implications of having had
BPD on morbidity in later life (137, 263). One recent report
suggested that early classification of BPD severity in preterm
infants did not provide useful information about future lung
function, when patient lung function was assessed at 6 and 18
mo after preterm birth (334); however, additional studies are
clearly indicated. Similar studies are also now being conducted
in animal models of BPD, in which the time course to adult-
hood is relatively shorter. Some notable studies have revealed
that perturbed late lung development, for example after hyper-
oxia exposure in the postnatal period, does indeed have con-
sequences for the lung architecture in adult animals. O’Reilly
and coworkers (262) demonstrated that exposure of mouse
pups to 65% O2 over the first 7 days of life resulted in
remodeling of the bronchiolar walls and loss of bronchiolar-
alveolar attachments that was evident at 10 mo of age, leading
the investigators to propose that immediate postnatal hyperoxia
exposure could lead to impaired lung function and airway
hyperreactivity in adulthood. In a related study, these investi-
gators demonstrated that the exposure to hyperoxia caused
persistent alteration to the bronchiolar wall that contributed to
perturbed lung function that had a stronger impact on the male
sex (261). These data thus also contribute to current thinking
that sex plays a key role in lung responses to hyperoxic injury.
These observations are supported by a report from Ramani and
coworkers (293), in which exposure of C57BL/6 mouse pups
to either hyperoxia (85% O2) or normoxia (12% O2) over the
period P2-P14, followed by a 10-wk recovery period under
normoxic (21% O2) conditions, caused stunted alveolar devel-
opment (assessed by MLI and RAC), loss of bronchiolar-

ADVANCES IN LUNG ALVEOLARIZATION AND BPD
alveolar attachments, increased lung compliance, and, in the
case of the hyperoxia group, increased arteriole medial wall
thickening that persisted into adulthood. An important struc-
ture-function correlate has also been reported by Ahlfeld and
coworkers (8), in which mouse pups were exposed to hyper-
oxia (90% O2 from P1 to P7) followed by recovery in room air
from P8 to P56. At P56, an alveolarization (assessed by MLI)
and vessel density defect persisted. Furthermore, the alveolar-
ization defect corresponded with a reduced functional gas
exchange (measured by the diffusing factor for carbon mon-
oxide, DFCO). This report also highlights the usefulness of
combining structural analyses with physiological measures of
lung function, which together provide complementary views
on the same idea, contributing to the robustness of the argu-
ment. To this end, the measurement of pressure-volume curves
in mouse lungs has recently been considered in detail (195).
Experimental studies on susceptibility to viral infection in
later life, after exposure to hyperoxia in the neonatal period
have also been undertaken by Maduekwe and coworkers (204),
who demonstrated that exposure to a particular threshold of
hyperoxia in the neonatal period altered host susceptibility to
influenza virus infection in the long term. Taking this idea
further, Buczynski and coworkers (51) explored a protective
role for SOD during hyperoxia exposure of mouse pups in the
immediate postnatal period (100% O2 between P1 and P4) on
alveolar development quantified in late life (8 –10 wk). Over-
expression of SOD in alveolar type II cells during hyperoxia
exposure conferred protection against hyperoxia-induced dam-
age to lung structure, with a partial normalization of alveolar
development reported, although this was assessed by visual
inspection of lung sections, in which lung structure was not
quantified. Exposure of these mice, as adults, to influenza A
virus indicated that wild-type mice with postnatal hyperoxia
exposure were more sensitive to influenza A virus infection,
exhibiting increased mortality and increased lung collagen
production, which was prevented in transgenic mice overex-
pressing SOD in alveolar type II cells. In sum, these observa-
tions support the idea that immediate postnatal exposure pre-
disposes adults to respiratory viral infection.
Neoalveolarization
Postpneumonectomy lung growth is an increasingly used
model to study lung development in which mice regenerate
lung tissue (neoalveolarization) after surgical resection (pneu-
monectomy) (332). There is currently controversy about the
molecular pathways that drive neoalveolarization. To date,
lung regeneration after pneumonectomy has been attributed to
reinitiation or acceleration of cellular growth, with the proposal
that, analogous to limb regeneration, neoalveolarization would
result from reactivation of lung organogenesis genes (145,
360). In a transcriptional profiling study, Kho and colleagues
(164) make the observation that, rather than the engagement of
early lung organogenesis pathways, postpneumonectomy lung
growth was characterized by the engagement of pathways
driving later stages of lung development (alveolarization)
rather than pathways that direct branching and primary airway
enlargement. In addition to transcriptional control of postpneu-
monectomy lung growth, Ysasi and coworkers (380), using a
unilateral diaphragmatic paralysis approach, have also demon-
strated the critical importance of cyclic stretch associated with
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00268.2015 • www.ajplung.org
Perspectives
L1257
diaphragmatic contraction as a controlling factor in compen-
satory lung growth. On the vascular side, Ackermann and
coworkers (4) have used remarkable imaging approaches to
demonstrate sprouting and intussusceptive angiogenesis as
important mechanisms of lung alveolarization during post-
pneumonectomy lung growth. A key mechanistic finding
was provided by Rafii and coworkers (289), who docu-
mented that platelets drive postpneumonectomy lung regen-
eration by supplying stromal cell-derived factor (SDF)-1,
which activates the SDF-1 receptors Cxcr4 and Cxcr7 on
capillary endothelial cells, which in turn stimulate alveolar
epithelial cell expansion, via the angiocrine membrane-type
metalloproteinase. This idea also hints at worthwhile ave-
nues to pursue for the stimulation of lung maturation in
neonates.
Pharmacological Interventions to Identify New Pathogenic
Pathways
Several interventional pharmacological studies have identi-
fied candidate pathways or molecules that might be targeted in
a translational sense to promote better lung growth or to
identify new pathways relevant to normal or aberrant late lung
development. These targets are summarized in Table 1 and
have identified possible lead compounds for further develop-
ment, with the aim to drive lung maturation by pharmacolog-
ical interventions. Additionally, these studies have highlighted
new pathways relevant to normal and aberrant alveolarization,
which warrant further study.
One area of current intensive investigation is the impact of
caffeine and other xanthine derivatives on lung maturation
(218), highlighted in Fig. 5. Weichelt and colleagues (362)
administered caffeine [10 mg/kg per day, ip; from P6, concom-
itant with the onset of hyperoxia (80% O2 for up to 48 h)
exposure] to rat pups and observed an apparent improvement in
alveolarization by visual inspection of sections (lung structure
was not quantified). Additionally, blunted neutrophil and mac-
rophage infiltration and a normalization of Cxcl1, Cxcl2 (also
called MIP-2), Ccl2, TNF-␣, and IL-6 levels were observed in
the caffeine-treated group in the background of hyperoxia. In
another study, Dayanim and colleagues (83) administered caf-
feine (20 mg/kg on P1, followed by 10 mg/kg per day there-
after) to FVB/N mouse pups in a dosing regimen directly
translated from clinical studies (272, 308). In the latter study,
caffeine administration has only deleterious effects, with in-
creased pulmonary inflammation and increased epithelial apo-
ptosis, as well as worsening of the lung architecture (as
assessed by RAC) in the background of hyperoxia. Clearly,
these two studies yield conflicting results, which may be
attributable to a lack of dose range finding, as acknowledged
by Dayanim and colleagues in their report (83). Interestingly,
both studies reported that caffeine applied under room-air
conditions dysregulated Cxcl1 expression, perhaps suggesting
a proinflammatory activity of caffeine in the lungs under room
air conditions. Comparing these studies is difficult because of
the different animal models and oxygen exposure protocols,
and the impact of caffeine on alveolar architecture in the
Weichelt study (362) remains to be validated because these
investigators did not quantify changes in lung structure, which
were observed by visual inspection only. Clearly, the potential
utility of caffeine to manage lung inflammation and promote
Perspectives
L1258 ADVANCES IN LUNG ALVEOLARIZATION AND BPD

Table 1. Pharmacological interventions in animal models of bronchopulmonary dysplasia and associated persistent
pulmonary hypertension of the newborn undertaken since 1 January 2013
Animal Model

Drug (dose and frequency) Species and Strain Injury Stimulus Molecular/Pathway Targets Lung Pathological Findings Reference
ActRIIB-Fc (5 mg/kg tiw, ip) Mouse C57BL/6 85% O2 P1-P14 Activin A receptor antagonist Improved alveolarization (194)
Blunted PASMC proliferation
Blunted TGF-␤ signaling
Ambrisentan (1-20 mg/kg daily, sc) Rat S/ND 100% O2 P1-P10 ET receptor type A antagonist Improved septal thickness (351)
Suppressed inflammation Improved vascular
structure
AMD3100 (240 ␮g/kg daily, sc) Rat S-D 90% O2 P2-P16 Cxcr4 antagonist Suppressed Improved alveolarization (88)
inflammation Improved vascular
structure
Ascorbylperoxide (infused) Guinea pig Drug application Increased oxidative stress Destruction of alveoli (95)
Hartley
Aspirin (40 mg/kg daily, R/ND) Mouse C3H/HeN 85% O2 P1-P14 Cyclooxygenase-2 inhibitor No improvement in (50)
Suppressed inflammation alveolarization
ATRA (5 mg/kg daily, ip) Rat S-D Nitrofen E9.5 Retinoic acid (primary) Increased Improved alveolarization (109)
leptin signaling
BAPN (15 mg/kg daily, ip) Mouse C57BL/6 85% O2 P1-P10/P20 Lysyl oxidase inhibitor Altered Worsened alveolarization (238)
ECM morphology
BAPN (dose indeterminate) Mouse C57BL/6 85% O2 P1-P10 Lysyl oxidase inhibitor Altered Improved alveolarization (212)
ECM morphology
BAY 11-7082 (10 mg/kg once, ip) Mouse C57BL/6 LPS NF-␬B pathway inhibitor Worsened alveolarization (144)
Increased MIP-2 levels Worsened vascular
structure
Benzo[a]pyrene (25 mg/kg, ip, Rat Fisher 344 90% O2 P1-P14 Increased oxidative stress Worsened alveolarization (331)
E18/19/20) Increased levels of isoprostanes Increased inflammation
Caffeine (10 mg/kg daily, ip) Mouse FVB/N 80% O2 P1-P8 Increased inflammation No improvement in (83)
alveolarization
Caffeine (10 mg/kg, once at P6) Rat Wistar 80% O2 P6-P8 Suppressed inflammation Improved alveolarization (362)
(apparent, not
quantified)
Carbon monoxide (250 ppm, Mouse C57BL/6 75% O2 P3-P21 Suppressed inflammation Improved alveolarization (23)
continuous)
Celecoxib (5 mg/kg daily, R/ND) Mouse C3H/HeN 85% O2 P1-P14 Cyclooxygenase-2 inhibitor No improvement in (50)
Suppressed inflammation alveolarization
Curcumin (5 mg/kg daily, ip) Rat S-D 95% O2 P1-P5 Attenuated TGF-␤ signaling Improved alveolarization (305)
Blunted ECM deposition Improved septal
thickness
cyclic (Ang)1–7 (10–50 ␮g/kg daily, sc) Rat Wistar 100% O2 P1-P10 MAS oncogene receptor agonist Improved septal thickness (352)
Suppressed inflammation Improved vascular
structure
Cyclosporine (15 mg/kg daily, sc) Rat S-D 60% O2 P1-P14 Not reported No improvement in (285)
alveolarization
DFU (10 ␮g/g daily, ip) Rat S-D 60% O2 P1-P14 Cox2 inhibitor Suppressed Improved alveolarization (220)
inflammation
DHA (diet supplement) Mouse C3H/HeN LPS ⫹ 85% O2 P1-P14 Suppressed inflammation Improved alveolarization (345)
GYY4137 (50 mg/kg daily, ip) Mouse C57Bl/6 85% O2 P1-P10 H2S donor Suppressed Improved alveolarization (207)
inflammation Improved septal
thickness
GYY4137 (37 mg/kg daily, ip) Rat S-D 90% O2 P1-P14 H2S donor Blunted PASMC Improved alveolarization (343)
proliferation Improved vascular
structure
5-HT (12–20 ␮g infused) Sheep C-R Fetal d.a.constriction 5-HT2A receptor agonist Increased PVR (85)
ICG001 (10 mg/kg daily, ip) Rat S-D 90% O2 P2-P14 ␤-catenin inhibitor Blunted Improved alveolarization (13)
PASMC proliferation Blunted Improved vascular
ECM production structure
IL-1Ra (10 mg/kg daily, sc) Mouse C57BL/6 LPS ⫹ 65% or 85% O2 (to P28) IL-1 receptor antagonist Improved alveolarization (257)
Suppressed inflammation
iNOS (5 ppm E21-P7) Rat S-D 80% O2 P1-P7 Increased neurotrophin expression Improved alveolarization (276)
Improved vascular
structure
Ketanserin (20 mg infused) Sheep C-R Fetal d.a.constriction 5-HT2A receptor antagonist Decreased PVR (85)
␣-Klotho (60 pmol daily, ip) Rat S-D 90% O2 ⱕ3 days Not reported Reduced edema (295)
LA-1 (1 mg/kg b.i.d., ip) Rat S-D 85% O2 P1-P14 CD11b/CD18 agonist Suppressed Improved alveolarization (153)
inflammation Improved vascular
structure
Lipoxin A4 (2 ng/g, ip, on P1/3/6/9) Mouse C57BL/6 100% O2 P1-P10 Suppressed inflammation Improved alveolarization (217)
Normalized septal
thickness
Liver growth factor (1.7 ␮g biw, ip) Mouse AKR/J Chronic smoke exposure Suppressed inflammation Blunted No improvement in (275)
MMP activity alveolarization
[D-lysine]cyclic(Ang)1–7 (5–20 ␮g/kg Rat Wistar 100% O2 P1-P10 ATII type 2 receptor agonist Improved septal thickness (352)
daily, sc) Suppressed inflammation Improved vascular
structure
Mesd (10 mg/kg, ip, q.a.d.) Rat S-D 90% O2 P1-P14 Lrp5/6 inhibitor Attenuated Wnt/ Improved vascular structure (14)
␤-catenin signaling
Metformin (25-100 mg/kg daily, sc) Rat Wistar 100% O2 P1-P10 Suppressed inflammation Improved septal thickness (68)
Suppressed fibrin deposition Improved vascular
structure
MIF020 (0.4 mg/g daily, P1-P4, id, Mouse C57BL/6 100% O2 P1-P4 MIF agonist Inflammation Improved alveolarization (321)
P5-P14, ip) unaffected
MIF098 (0.4 mg/g daily, P1-P4, id, Mouse C57BL/6 100% O2 P1-P4 MIF antagonist Suppressed No improvement in (321)
P5-P14, ip) inflammation alveolarization
Continued

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 Perspectives
ADVANCES IN LUNG ALVEOLARIZATION AND BPD L1259
Table 1.—Continued
Animal Model

Drug (dose and frequency) Species and Strain Injury Stimulus Molecular/Pathway Targets Lung Pathological Findings Reference
mitoTEMPO (0.7 ␮g/g daily, sc) Mouse C57BL/6 75% O2 72 h at P0 or P4 Not reported Improved alveolarization (81)
Improved vascular
structure
MnTBAP (1-10 mg/kg, ip, P1/2/3) Rat S-D 50% O2 Inter. 12% O2 P1-P14 Superoxide dismutase mimetic Not assessed (62)
Modulated VEGF levels
Blunted MMP-9 levels
PD123319 (0.5-2.0 mg/kg daily, ip) Rat Wistar 100% O2 P1-P10 ATII type 2 receptor antagonist Improved septal thickness (353)
Suppressed inflammation Improved vascular
Blunted ECM deposition structure
Pioglitazone (3 mg/kg, nebulized) Rat S-D 95% O2 P1-P3 PPAR-␥ agonist Improved alveolarization (240)
Resolvin D1 (2 ng/g, ip, P1/3/6/9) Mouse C57BL/6 100% O2 P1-P10 Suppressed inflammation Normalized septal (217)
thickness
Rosiglitazone (3 mg/kg daily, ip) Rat S-D LPS ⫹ 80% O2 P1-P7 PPAR-␥ agonist Improved alveolarization (187)
Improved vascular
structure
Rosiglitazone (3 mg/kg, nebulized) Rat S-D 95% O2 P1-P3 PPAR-␥ agonist Improved alveolarization (240)
SB216763 (10 mg/kg daily, ip) Rat S-D 90% O2 P2-P15 Inhibited GSK3␤ signaling Improved alveolarization (149)
Suppressed inflammation Blunted PH
SB265610 (4 mg/kg daily, ip) Rat S-D Bleomycin Cxcr2 antagonist Suppressed No improvement in (186)
inflammation alveolarization
Sertraline (10 mg infused) Sheep C-R Fetal d.a. constriction Serotonin reuptake inhibitor Increased PVR (85)
Sildenafil (100 ␮g/g daily, ip) Ra S/ND LPS ⫾ 85% O2 P2-P14 Phosphodiesterase 5 (primary) Improved alveolarization (270)
Increased PI3K/Akt/mTOR
axis
D-Sphingosine (1.25 ␮g/g daily, ip) Mouse C57BL/6 80% O2 P1-P28 Inhibited ceramide production Improved alveolarization (336)
Stem cell factor (100 ␮g/kg daily, sc) Rat S-D 90% O2 P2-P15 c-kit agonist Improved alveolarization (236)
Improved vascular
structure
VARA (oral on alternate days) Mouse C57BL/6 85% O2 P1-P14 Retinoic acid pathway (primary) Improved alveolarization (156)
Attenuated oxidative stress
Vitamin D Rat S-D Endotoxin at E20 (intra amniotic) Vitamin D pathway (primary) Improved alveolarization (213)
Increased VEGF/VEGFR axis Improved vascular
structure
Y-27632 (10 mg/kg daily, ip) Rat S-D Bleomycin ROCK inhibitor Suppressed Improved alveolarization (186)
inflammation

ActRIIB-Fc, activin A receptor type IIB-Fc antagonist; Akt, thymoma viral proto-oncogene 1; ATII, angiotensin II; BAPN, ␤-aminopropionitrile; b.i.d., bis
in die (twice daily); biw. biweekly (twice weekly); C-R, Colombia-Rambouillet; DHA, docosahexaenoic acid; Cox2, cycloxygenase-2; d.a., ductus arteriosis;
DFU, 5,5-dimethyl-3-(3-fluorophenyl)4-(4-methylsulfonyl)phenyl-2(5H)-furanone; E, embryonic day; ECM, extracellular matrix; ET, endothelin; GYY4137,
[morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate]; IL-Ra, interleukin-1 receptor antagonist; iNOS, inducible nitric oxide synthase; Inter.,
intermittent; LA-1, leukadherin-1; LRP5/6, low-density lipoprotein receptor–related proteins 5 and 6; MIF, macrophage migration inhibitory factor; MIF020,
4-[4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl]phenol; MIF098, 3-(3-hydroxybenzyl)-5-methylbenzooxazol-2-one; MIP-2, macrophage inflammatory protein 2; MMP,
matrix metalloproteinase; mTOR, mammalian target of rapamycin; P, postnatal day; PASMC, pulmonary artery smooth muscle cell; PI3K, phosphatidylinositol-
4,5-bisphosphate 3-kinase; PPAR, peroxisome proliferator-activated receptor; ppm, parts per million; PVR, pulmonary vascular resistance; q.a.d., quaque alternis
die (every other day); R/ND, route not declared; ROCK, Rho kinase; S-D, Sprague-Dawley; S/ND, strain not declared; TGF, transforming growth factor; tiw.
thrice weekly; VARA, vitamin A and retinoic acid.

lung maturation is exciting, but reports on caffeine toxicity
raised concerns about dose (338), and the observed impact of
early vs. late administration of caffeine to infants (272) high-
lights the need for optimization of treatment protocols. Some
recent evidence suggests that caffeine may also exert an effect
on TGF-␤-driven epithelial-to-mesenchymal transition of lung
epithelial cells (100) and may influence steroid-mediated sur-
factant protein B expression (99); however, the relevance of
this observation to lung alveolarization has not yet been estab-
lished.
The potential application of vitamin A and vitamin A ana-
logs to promote lung maturation continues to receive attention
because retinoid signaling is essential for proper diaphragm
and lung development. Costa and coworkers (76) have added
several retinoid-metabolizing genes to the list of retinoid path-
way genes that are deregulated in human CDH, providing
further impetus for animal model studies. Along these lines,
James and coworkers (156) reported that a combination of
vitamin A and all-trans retinoic acid (in a 10:1 ratio) admin-
istered every second day to newborn mice in the hyperoxia
BPD model (85% O2 for 14 days) improved lung architecture
(assessed by MLI and RAC) and partially normalized lung
compliance and airway resistance. These studies continue to
add ideas to the ongoing debate about the use of and role of
retinoids in lung development and lung structural homeostasis
that started in 1927 with Harry Goldblatt and Maria Benis-
chek’s original observations that vitamin A deficiency causes
lung metaplasia (119). It appears that we have some way to go
yet.
Cell Therapy to Promote Alveolarization
The applicability of stem cells to drive the repair of damaged
lungs and the regeneration of lung tissue to promote that repair
are currently a hot topic in pulmonary research (131, 141, 174,
241), including BPD (265). Some strides forward have been
made in the validation of cell therapy as a viable route to
restore and promote lung maturation in experimental BPD.
Key recent observations include the report of Alphonse and
coworkers (19), who demonstrated that, after exposure to
hyperoxia in vitro, endothelial colony-forming cells (ECFCs)
from human fetal lungs exhibited blunted proliferative and
vessel-formation activity, as did ECFCs from hyperoxia-
treated rat lungs (95% O2, from P1 to P14). Intrajugular

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Perspectives
L1260 ADVANCES IN LUNG ALVEOLARIZATION AND BPD

O2

Modulation of
reactive oxygen
species (ROS) Lung
generation
Adenosine receptor
Acute
Bronchodilatation activation/inhibition

Respiratory cAMP
phosphodiesterase Chronic
drive
modulation
Modulation Inflammation
cell death

Modulation of CAFFEINE
inflammatory
response
Cxcl1 Ccl2

Cxcl2 TNF-α IL-6

Late Early
treatment treatment
Low dose
?
AEC Macrophage Neutrophil
High dose
apoptosis influx influx

Reduced Increased
inflammation inflammation
Reduced Increased
apoptosis apoptosis

Ameliorated
Impaired
histological
alveolarization
damage

Improved Worsened
BPD status BPD status

Fig. 5. Possible mechanisms by which caffeine might attenuate BPD and arrested alveolarization in the developing lung. Caffeine administration has been
examined in early and late applications in clinical settings and experimentally in an acute and chronic administration protocol. Upward arrow indicates an
increased effect. The scheme only details new developments that were published between 1 January 2013 and 30 June 2015. AEC, alveolar epithelial cell; Ccl2,
chemokine (C-C motif) ligand 2; Cxcl1, chemokine (C-X-C motif) ligand 1; MIP-2, macrophage inflammatory protein 2; ROS, reactive oxygen species.

administration of human umbilical cord blood-derived ECFCs
after hyperoxic lung injury in Rag1⫺/⫺ mouse pups (85% O2,
from P4 to P14) restored lung function and also restored
alveolarization (assessed by MLI) and lung vascular growth
and blunted pulmonary hypertension. The low level of engraft-
ment of ECFCs, together with protective effects of ECFC-
conditioned medium, prompted the investigators to suggest
that a paracrine effect conferred the restorative effects of
ECFCs (110). Notably, 10 mo after ECFC administration, the
improvement in lung structure persisted. The same group of
investigators reported the beneficial impact of human umbilical
cord-derived perivascular cells and mesenchymal stem cells
(MSCs) delivered by the intratracheal route in the rat hyperoxia
model, in which alveolarization (assessed by MLI) was rescued
both in the short and long term (6 mo) (278). In a similar study,
Sutsko and coworkers (324) compared whether intratracheal
administration of either MSCs or conditioned medium from
MSCs influenced lung development in the hyperoxia rat model
(90% O2, between P2 and P16). Both MSC and MSC-condi-
tioned medium blunted the impact of hyperoxia on alveolar
development (assessed by MLI) and lung vessel density. Along
these lines, Baker and coworkers (29) applied conditioned
medium from late-outgrowth colony-forming cells, a type of
endothelial progenitor cell, in a bleomycin-based rat model of

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ADVANCES IN LUNG ALVEOLARIZATION AND BPD
BPD. Although application of conditioned medium blunted
heart remodeling (assessed by Fulton index) provoked in the
model, no impact on alveolarization (assessed by RAC) of rat
pup lungs was noted, leading these authors to criticize the
potential benefit of conditioned medium administration in this
model. Why the study of Baker and coworkers (29) could not
demonstrate a beneficial impact of the intervention on alveo-
larization is not clear but may be related to the use of late-
outgrowth colony-forming cells or a different model (bleomy-
cin), compared with the other three studies discussed above.
Along similar lines, Ahn and coworkers (11) evaluated the
relative efficiency of human umbilical cord blood-derived
MSCs, human adipose tissue-derived MSCs, and human um-
bilical cord blood mononuclear cells to protect against blunted
alveolar development induced by hyperoxia (90% O2 for 14
days) in rat pups. The investigators assessed that the human
umbilical cord blood-derived MSCs were most efficient at
promoting improved lung maturation in the background of
hyperoxia, which was correlated with increased VEGF and
hepatocyte growth factor production. The investigators fol-
lowed up with a timing-of-administration study (64) and re-
vealed that the application of umbilical cord blood-derived
MSCs was only beneficial when applied at P3 but not P10
(90% O2, from P1 to P14, followed by 60% O2 from P14 to
P21). In another cord blood study, Mao and coworkers (216)
applied cord blood-derived CD34⫹ cells that had been ex-
panded in vitro, via the intranasal route, to mice in an FVB/N
transgenic mouse model of BPD with club (formerly Clara)
cell-driven expression of Fas ligand. When cord blood-derived
CD34⫹ cells were expanded in vitro in the presence of dexa-
methasone, a moderate improvement in alveolarization (as-
sessed by Lm) was noted. Similarly, the application of human
amnion epithelial cells at P5, P6, and P7 to mouse pups
exposed to 85% O2 from P1 to P14 improved alveolarization
(as assessed by MLI) (349).
Studies have also been undertaken by Ramachandran and
coworkers (292) with single application of bone marrow-
derived c-Kit⫹ cells, which, when applied via the intratracheal
route, led to a significant improvement in both alveolarization
(assessed by MLI) and vessel density in the background of
hyperoxia (90% O2 from P2 to P15, intervention on P8).
Directly in line with this study, Miranda and coworkers (236)
applied stem cell factor (SCF; the ligand of the c-kit receptor)
by daily injection (from P15 to P21) to rat pups exposed to
hyperoxia (90% O2 from P1 to P15, followed by recovery in
room air from P16 to P28). In that study, which represents a
therapeutic (not preventive) protocol, the treatment of oxygen-
injured lungs with SCF increased lung septal density and
alveolarization (assessed by MLI), normalized lung collagen
deposition, and also had a pronounced impact on the vascula-
ture, with partially normalized right ventricular systolic pres-
sure and a fully normalized Fulton index and lung vessel
medial wall thickness. These data identify the c-Kit/SCF axis
as an extremely exciting avenue for the management of stunted
lung maturation and potentially tie in with the idea of c-Kit
delineating a putative endothelial progenitor cell population in
developing human lungs (325).
As highlighted by Stella Kourembanas (175) and Christo-
pher Baker and Steven Abman (28) in two recent editorials,
many questions remain regarding stem cell therapy for the
management of arrested lung development. These include
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00268.2015 • www.ajplung.org
Perspectives
L1261
identifying the correct stem cell type and route of administra-
tion, keeping in mind that a cocktail of multiple stem cell types
may be the best approach, understanding how these cells
promote lung maturation or correct blunted alveolarization,
and clarifying whether stem cell application may have harmful
long-term consequences, such as tumorigenesis. The consider-
able progress made over the past few years in applying stem
cells in animal models that recapitulate the arrested alveolar-
ization associated with clinical BPD has provided impetus for
translation of this idea to the clinical setting. The safety and
feasibility of transplanting allogeneic human umbilical cord
blood-derived MSCs have recently been successfully demon-
strated in preterm infants in a phase I clinical trial
(NCT01297205) (63). The results of a follow-up phase II
clinical trial to assess therapeutic efficacy (NCT01828957), as
well as a long-term follow-up safety assessment study of the
MSC transplant recipients (NCT01897987) (10), are eagerly
awaited.
Synthesis and Concluding Remarks
It is clear that many developments in our understanding of
alveolarization and the pathogenesis of BPD have been made
since the beginning of 2013. It is also clear that many open
questions remain, and new questions have been asked. Notable
advances in methodology used to study lung development have
been made, including the development of new tissue fixation
and embedding protocols and stereology approaches for the
unbiased quantification of lung structure. Further implementa-
tion of these methodologies, moving from RAC or MLI to
stereology measurements, would represent an important ad-
vance in how we study lung structure although concerns about
time and cost are likely to remain barriers to the widespread
implementation of these approaches in the foreseeable future.
This Perspectives article has also highlighted the tremendous
heterogeneity of animal models to study arrested alveolariza-
tion. The hyperoxia-based approaches in particular are prob-
lematic, given the current range of oxygen levels employed,
together with variable periods of oxygen exposure, the combi-
nation of a second hit and a period of recovery under room air
conditions, and posthyperoxia exposure. It appears that there
remains scope for a systematic study, using state-of-the-art
stereological approaches, to clarify whether an optimal injuri-
ous oxygen concentration and window of exposure exist.
Along these lines, it would be important to strike a balance
between the amount of oxygen employed as an injurious
stimulus and the degree of damage to the developing lung that
can be quantified. This is particularly important in pharmaco-
logical or transgenic animal studies in which a potentially
important subtle effect of a pharmacological or genetic inter-
vention on the structural development of an injured lung may
be missed because the injurious stimulus was unnecessarily
severe. In this way, the potential subtle modulation of alveo-
larization by interventions at superoxia levels (ⱖ85% O2) may
not be noted in lung structural analyses. Concerns also persist
about the sex of the experimental animals employed and the
range of strains with variable responses to oxygen injury,
which has resulted in opposite observations being made in
ostensibly the same studies. Also related to mouse strains, there
is increasing concern about the appropriateness of controls
employed in transgenic mouse studies, particularly where
Perspectives
L1262 ADVANCES IN LUNG ALVEOLARIZATION AND BPD
strains are maintained on mixed mouse strain backgrounds. It
is clearly important that, in all experimental studies, the mouse
strain, the backcrossing and background of the transgenic mice,
and the sex of the mice should be clearly reported.
A large number of correlative studies have been reported
since 2013, revealing expression changes in molecules of
interest that correlate with arrested alveolarization. Demonstra-
tion of a causal role for these pathogenic candidates remains to
be reported. Similarly, several transgenic mouse studies have
causally implicated particular proteins or pathways in normal
or arrested alveolarization, and the further delineation of these
pathways to reveal new developmental mechanisms is eagerly
awaited. New technologies (such as the clustered regularly
interspaced short palindromic repeats/Cas9), which will mas-
sively ease genetic manipulation in vitro and in vivo, together
with the continued production of new floxed mouse strains and
driver lines, will open many doors for the further dissection of
molecular pathways that drive normal and aberrant late lung
development. Among these, the role of ECM remodeling,
ECM cell cross talk, and cell-cell cross talk will no doubt
feature in work that will emerge over the coming years, along
with attention to the role of progenitor cells and distinct cell
subpopulations and the regulation of alveolar epithelial cell
plasticity, together with the mechanisms of disturbed vascular
growth and homeostasis. It is clear that much challenging but
exciting work lies ahead.
ACKNOWLEDGMENTS
The authors thank Dr. José Alberto Rodríguez Castillo (Morty Laboratory,
Department of Lung Development and Remodelling, Max Planck Institute for
Heart and Lung Research, Bad Nauheim, Germany) for outstanding assistance
with the preparation of this Perspectives article.
GRANTS
This study was supported by the Max Planck Society (to D. Silva, C.
Nardiello, A. Pozarska, and R. Morty), the German Research Foundation
through grant Mo 1789/1 (to R. Morty), Excellence Cluster 147 “Cardio-
Pulmonary System” (to R. Morty), the Federal Ministry of Higher Education,
Research, and the Arts of the State of Hessen LOEWE Program (to R. Morty),
and the German Center for Lung Research (Deutsches Zentrum für Lungen-
forschung; to D. Silva, C. Nardiello, A. Pozarska, and R. Morty).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
D.M.S., C.N., and A.P. prepared figures; D.M.S., C.N., A.P., and R.E.M.
drafted manuscript; D.M.S., C.N., A.P., and R.E.M. edited and revised man-
uscript; D.M.S., C.N., A.P., and R.E.M. approved final version of manuscript.
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