higher density gels (0.73 vs 1.88 ␮g; p ⬍ 0.01). This was
accompanied by an increased percentage of apoptotic cells
in the low-density collagen gels at day 14 (43.3 vs 34.1;
p ⬍ 0.05). If the gels were maintained in the attached
state, which largely prevents contraction, apoptosis was
significantly reduced, suggesting that contraction rather
than matrix composition was a requirement for the increased apoptosis. Taken together, these findings indicate
that the initial matrix composition can lead to differing
outcomes during fibroblast mediated contraction of collagen gels. Such effects may contribute to the altered tissue
structures that characterize the lung in COPD.
of parenchymal lesions associated with emphysema.4 This
study has prompted a surge of new preclinical and clinical
research exploring the molecular basis for the function of
vitamin A within the lung. In this review article, historical
data supporting the role of vitamin A in the differentiation
of lung structure and the maintenance of normal function
will be reviewed. Experimental evidence elucidating the
molecular basis of function via selective gene expression
will be discussed. Data supporting the effects of ATRA on
the repair of experimental models of emphysema will be
presented in the context of their potential therapeutic use
in the treatment of COPD.
Molecular Basis for Action of Retinoic
Acid
Effects of All-Trans-Retinoic
Acid in Promoting Alveolar
Repair*
Paula N. Belloni, PhD; Laura Garvin, BS;
Cheng-Ping Mao, MS; Irene Bailey-Healy, BA; and
David Leaffer, BA
(CHEST 2000; 117:235S–241S)
Abbreviations: ATRA ⫽ all-trans-retinoic acid; PCNA ⫽ proliferating cell nuclear antigen; PDGF ⫽ platelet-derived growth
factor; RA ⫽ retinoic acid; RAR ⫽ retinoic acid receptor; RXR ⫽
retinoid X receptor; SP ⫽ surfactant protein; TGF ⫽ transforming growth factor
is characterized by airway destruction disE talmphysema
to the terminal bronchioles, gradual loss of lung
recoil, decreased alveolar surface area, and impaired gas
exchange, leading to a reduced FEV1.1 These last two
features, impaired gas exchange and reduction in expiratory flow, are characteristic physiologic abnormalities in
patients with emphysema. The most common cause of
emphysema is cigarette smoking, although other potential
environmental toxins also may contribute. These various
insulting agents activate destructive processes in the lung,
including the release of active proteases and free radical
oxidants in excess of protective mechanisms. The imbalance in protease/antiprotease levels leads to the destruction of the elastin matrix and alveolar structure with
progressive loss of lung recoil. Removing the injurious
agents (ie, quitting smoking) slows the rate of damage;
however, unlike the response after acute lung injury, the
damaged alveolar structures do not repair and lung function is not regained.
The relationship between vitamin A status and airway
obstruction has been examined in cross-sectional studies.2,3 These studies established an inverse relationship
between plasma retinol status and the degree of airway
obstruction (assessed by FEV1). Recent preclinical studies
suggest that an analog of vitamin A, all-trans-retinoic acid
(ATRA), may promote the repair and/or realveolarization
*From Roche Bioscience, Department of Respiratory Diseases,
Palo Alto, CA.
Correspondence to: Paula Belloni, PhD, Roche Bioscience, Respiratory Diseases, 3401 Hillview Ave, Palo Alto, CA 94308
Retinoids are a class of compounds structurally related
to vitamin A that comprise natural and synthetic compounds. Retinoic acid (RA) and its other naturally occurring retinoid analogs (9-Ci-RA, all-trans-3– 4 didehydroRA, 4-oxo-RA, and retinol) are pleiotropic regulatory
compounds that modulate the structure and function of a
wide variety of inflammatory, immune, and structural
cells. These compounds function like hormones to regulate epithelial cell proliferation, pattern formation in developing tissues, morphogenesis in the lung, and cellular
differentiation. The current proven clinical uses of selected retinoids are for the treatment of dermatologic
diseases (acne, psoriasis, eczema, and photo-damaged
skin) and specific forms of cancer. Retinoids exert their
biological effects through a series of nuclear receptors that
are ligand-inducible transcription factors belonging to the
steroid/thyroid receptor superfamily.5 The ligand-bound
heterodimer binds to RA response elements in the noncoding region of the target gene to repress or enhance
expression (Fig 1). Retinoids also can modulate gene
expression by binding directly to specific transcription
factors such as AP-1 that interfere with the protein-protein
interactions, similar to the effects of glucocorticoids.6
The retinoid receptors are classified into two families,
the RA receptors (RARs) and the retinoid X receptors
(RXRs), each consisting of three distinct subtypes (␣, ␤,
and ␥). Each subtype of the RAR gene family encodes a
variable number of isoforms arising from differential splicing
of the two primary RNA transcripts. ATRA is the physiologic
hormone for the RARs. It binds with approximately equal
affinity to all three RAR subtypes. The RXRs do not bind
ATRA, but bind instead to the 9-Ci isomer of RA.
Vitamin A Metabolism
Vitamin A is acquired from the diet in the form of
retinyl-esters and ␤-carotene, converted to retinol in the
intestine and stored in the liver after reconversion to
retinyl esters. Retinol released from the liver is transported to target tissues complexed to retinyl binding
proteins, where it can be stored in the form of esters or
converted to the active hormone ATRA. Retinol is converted to RA at the cellular site of action in a highly
controlled metabolic pathway.7 Retinol is first oxidized
to an inactive intermediate, retinal, by members of the
alcohol dehydrogenase family (ie, retinol dehydrogenase),
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Figure 1. Retinoid nuclear hormone receptors.
which is followed by oxidation of retinol to the active
ligand RA by members of the aldehyde dehydrogenases.8
Throughout the metabolic processes, retinoid metabolites
and ATRA remain complexed to retinoid-binding proteins
(retinol binding protein, cellular retinol binding protein,
and cellular retinoic acid binding protein) to protect the
cells from hormonal action. Much of this highly controlled
pathway is autoregulated by local concentrations of
ATRA.9 Either an excess of ATRA or inadequate maintenance of ATRA can have significant pathologic consequences throughout life.
Vitamin A in Lung Development and
Function
Lung development involves the formation of the primordial lung from the foregut and sequential branching
morphogenesis into small airways, which are followed by
three maturation phases: phase 1 is pseudoglandular, with
continued airway branching; phase 2 is canalicular, with
thinning of the epithelium and cell differentiation; and
phase 3 is the terminal saccular stage, with rapid proliferation of interstitial fibroblasts, alveolar budding, septation,
and differentiation of type II and type I epithelia.10,11 On
completion of septation, the alveolar walls become thinner
and apoptotic processes reduce the number of interstitial
fibroblasts.12,13 Throughout this development process, the
lung is composed of the following two primary tissue
layers: the epithelium and the mesenchyme. The mesenchyme produces growth factors (epidermal growth factor,
transforming growth factor [TGF]-␣, human growth factor, fibroblast growth factor-7, and TGF-␤) and matrix
molecules (collagen, elastin, and proteoglycans) that stimulate epithelial cell proliferation and differentiation, promoting branching. Similarly, the epithelium produces
growth factors (platelet-derived growth factor [PDGF],
insulin-like growth factor, TGF-␤2, and proteases such as
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matrix metalloproteinases) as well as cell-cell contacts,
direct fibroblast proliferation, and matrix deposition. RA is
known to be one of the primary morphogens that regulates
the temporal and spatial expression of many of these
factors in both tissue layers.14 –18
Relationships Among RARs, RA, and
Alveolar Septation
ATRA has been shown to modulate various aspects of
cellular differentiation and matrix metabolism by interacting with specific RARs. Expression of the RARs is highly
regulated both temporally and spatially at various times
during lung development. RAR-␣ is associated with instructing epithelial cell differentiation and driving structural changes during the transition from the glandular to
the canalicular stage of development. In contrast, RAR-␤
increases significantly in the terminal saccular stage, with
the induction of both type II and type I epithelial cells.
RAR-␥ tends to be restricted to cells of the mesenchyme
throughout this process.19,20 RA storage granules are most
abundant in the fibroblastic mesenchyme surrounding
alveolar walls, where levels peak prior to alveolar septation.21–24 Depletion of these retinyl-ester stores parallels
the deposition of a new elastin matrix and septation. In
neonatal rats fed a vitamin A-deficient diet or treated with
dexamethasone, alveolar septation is significantly reduced.
At the molecular level, the expression of cellular retinol
binding protein and RAR-␤ messenger RNA is diminished
in the lungs of vitamin A-deficient rat pups.25,26 In contrast, the treatment of neonatal rat pups with ATRA
increases lung alveolarization and can reverse the effects
of dexamethasone.27
The effects of dexamethasone and ATRA on the late
stages of branching morphogenesis have also been demonstrated ex vivo.18,28 In these studies, terminal branching
and type II epithelial cell proliferation were inhibited in
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gestational day 14/15 lungs when cultured in the presence
of dexamethasone and were normalized by costimulation
with ATRA. The authors related the changes in transcription of fibroblast growth factor-7 and human growth factor
to the structural observations induced by dexamethasone
with or without ATRA.
In adult animals deficient in retinol, the conducting
airways undergo squamous metaplasia, the transformation
of the mucociliary epithelium into squamous cells.29 –31
Similar changes are observed in bronchopulmonary dysplasia, a chronic lung disease encountered by infants after
ventilation therapy for respiratory distress. In addition to
delayed septation, lung function is impaired in these
infants by inadequate levels of surfactant phospholipids,
which normally line alveoli. Vitamin A deficiency is
thought to mediate some of the lung pathology associated
with these neonates.
Preclinical studies indicated that the supplementation
of vitamin A-deficient rat pups with physiologic levels of
ATRA not only promotes septation, but also promotes the
expression of surfactant protein (SP) genes.32,33 In this
study, levels of SP-␣ and SP-␤ were correlated directly
with plasma retinol concentrations. Although the molecular basis for action has not been investigated clinically,
results from recent clinical studies suggest that supplementation with retinol enhances the survival rate of these
rat pups.34
Elastin deposition in the saccule wall is instrumental to
alveolar septal formation. Elastin is the primary structural
protein in the alveolar wall that is the basis for its recoil
properties. Tropoelastin is the soluble elastin gene product
that becomes insoluble on polymerization. ATRA has been
shown to effect the transcription of elastin in fetal lung
fibroblasts directly.21–24,35 The critical need for elastin
deposition during septation is borne out in genetic knockout studies. PDGF-␣ null mice are homozygous lethal,
with restriction points before E-10 and one postnatally. In
PDGF-␣-deficient mice that survive, emphysema develops secondary to the failure to septate. The failure to
septate was due to a loss of alveolar myofibroblasts and the
associated elastin fibers.15 The deficiency in myofibroblasts and elastin was restricted to the lung parenchyma,
which appear healthy in the bronchi and blood vessels.
Preclinical investigations of repair mechanisms after acute
lung injury suggest that similar profiles of growth factors
and receptors promote the structural repair of damaged
alveoli.
Tissue Repair and Matrix Deposition in the
Adult Lung
Wound healing occurs in the following three phases:
inflammatory, proliferative, and remodeling.36 The first
phase of inflammation is characterized by an infiltration of
polymorphonuclear neutrophils and macrophages. The
second phase requires fibroblast proliferation, angiogenesis, and the production of a provisional matrix of collagen/
elastin. Wound contraction and reepithelialization constitute the final phase of repair. Evidence supporting the
capacity for self-renewal or repair in adult tissue stems
from studies examining the alveolar microenvironment of
patients or animals after acute lung injury. In acute lung
injury, the process begins with massive inflammatory
infiltration in the alveolar wall after exposure of a noxious
environmental or endogenous biological agent, followed
by significant tissue destruction. Repair is initiated by an
extensive fibroproliferative response, leading to granulation of the alveolar airspaces, which is a classic woundhealing response. The granulation tissue is composed of
fibroblasts, endothelial cells, residual macrophages, and a
provisional collagen matrix.37,38 PDGF (␣- and ␤-chains),
TGF-␤1, and TGF-␤2 are rapidly induced into alveolar
epithelial cells in response to an injury.39 PDGF is a potent
mitogen for mesenchymal cells, whereas TGF-␤ retards
fibroblast growth but promotes matrix deposition. PDGF
receptor-␣ expression is markedly enhanced in lung myofibroblasts within 24 h of injury and subsides prior to the
deposition of fibrotic matrix proteins.40,41 In patients or
animals that survive, there is resolution of the granulation
tissue with subsequent restoration of the gas-exchange
apparatus. The reduction in cell mass occurs via apoptosis,42 which is similar to the final stages of septation in
development,13 as well as in normal wound healing.43 The
effects of RA and PDGF on dermal wound repair are well
documented.36,44 Additional studies are required to determine whether ATRA may activate a similar gene expression cascade in the repair of emphysema.
Retinoid Agonists in the Treatment of
Experimental Emphysema
Numerous studies have demonstrated that the instillation of elastolytic enzymes into the lung can induce
experimental emphysema. Elastase treatment leads to
rapid destruction of the elastin content and to permanent
disruption of the elastin fiber architecture within the
alveolus.45 Airspace enlargement and partial loss of lung
capacity have been measured in the rat. The loss of lung
structure and function in elastase-induced emphysema are
thought to be representative of the changes that occur in
mild-to-moderate human emphysema.
The studies reported by Massaro and Massaro4 suggest
that ATRA can reverse the effects of elastase-induced
damage in the rat. In the reported study, lungs were
damaged by a single instillation of pancreatic elastase.
Three weeks after injury, the rats were treated with ATRA
(0.5 mg/kg) or a vehicle for an additional 14 days. Lung
volumes were determined by volume displacement.
Changes in alveolar structure were determined by the
selector method using serial sections and classic methods
of morphometry. In these studies, the treatment of rats
with elastase plus vehicle resulted in an 18% increase in
alveolar volume and 45% fewer alveoli, relative to healthy
rats or those treated with elastase and ATRA.
We have repeated these studies and analyzed changes
in alveolar area and density using computer-assisted image
analysis. Lung tissue is nearly ideal for computer-assisted
morphometry. Alveoli are represented histologically as
empty spaces surrounded by tissue. The alveolar lining
cells can be stained using standard histochemical dies
(hematoxylin-eosin), so therefore, alveoli can be differentiated clearly from surrounding tissue when the image is
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converted to a gray scale. The image threshold can be set
within a narrow range of pixel intensities, and the areas of
interest are defined by contiguous pixels. As such, areas of
alveolar wall destruction result in larger alveoli and in
fewer alveoli per field.
Using these methods, the average alveolar area in rats
treated with elastase and the vehicle was threefold larger
than that in untreated rats (pixel density, 5,200 vs 1,800,
respectively). The average alveolar area of rats treated
with elastase and ATRA was 3,200 pixels, which represents
an approximately 50% reversal of damage (Fig 2). The
treatment of elastase-injured rats with another nonselective RAR agonist, 9-cis RA, had a similar effect; a 70%
improvement in alveolar area. Lung volumes were deter-
mined in experimental emphysema by volume displacement
and were found to be increased by 15% in elastase-treated
rats, as reported by Massaro and Massaro4; however, in these
studies, lung volumes were not corrected by treatment with
ATRA. The results from these studies suggest that the repair
of the alveolar structure does not necessarily confer an
improvement in elastic recoil.
Cellular Changes in Alveoli Indicative of
Wound-Healing Response
We have performed immunohistochemistry (proliferating cell nuclear antigen [PCNA]) to identify proliferating
cells and in situ hybridization (TUNEL) to mark apoptotic
Figure 2. Experimental emphysema with and without ATRA treatment. IT ⫽ intratracheal;
Veh ⫽ vehicle.
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cells, to further characterize changes in cell turnover in
the peripheral lung tissue in response to ATRA. Areas of
cell proliferation were identified in lung tissues by immunolocalization of PCNA (Fig 3). A low level of staining was
observed in conducting airways of all animals regardless of
treatment, reflecting the normal turnover of the airway
epithelium. However, staining within the peripheral lung
was restricted to the alveoli of rats treated with elastase
followed by ATRA. The proliferation of the alveolar
epithelium was not observed in unchallenged rats with or
without ATRA treatment, suggesting that ATRA promotes
the proliferation of these cells only in response to injury.
Apoptosis occurs during normal wound-healing processes to allow for the elimination of specific cell populations without generating an inflammatory response. Numerous studies in vitro and in vivo suggest that ATRA may
help drive apoptosis. Apoptosis was assessed in experimental emphysema over the 2-week time course of ATRA
treatment using the TUNEL assay. A threefold increase in
the number of apoptotic cells was induced within 24 h of
treatment with ATRA and quickly subsided. Staining was
restricted to cells lining the alveoli and to mononuclear
cells within the alveoli. No significant differences were
observed between naive and treated rats at later times.
Relationship Between Lung Structure and
Function
A primary issue raised in response to the initial publications by Massaro and Massaro4,27 is whether improvements in lung structure would translate to changes in lung
function. We have used both invasive and nonrestrained
plethysmography to assess lung capacity and compliance.
No differences have been detected in compliance with or
without ATRA treatment relative to naive animals. In
contrast, there was a measurable change in the ratio of the
alveolar-arterial oxygen pressure difference to Po2, suggesting there is improved diffusion capacity in ATRAtreated rats. The effects of elastase treatment, with or
without ATRA, on lung function in the rat also have been
reported by Tepper et al.46 In these early studies, lung
volumes (total lung capacity, residual volume, vital capacity, and functional residual capacity) were increased by
elastase treatment, while FEV1 and the diffusing capacity
of the lung for carbon monoxide were decreased. Treatment with ATRA for 2 weeks partially reversed these
changes. Taken together, the results of current studies
suggest that ATRA treatment may promote repair, regenerate, or both damaged alveoli, resulting in improvement
of selected functional parameters.
Summary
An appreciation of the central role of RAs in embryogenesis, tissue homeostasis, and aging was greatly expanded during the last decade by the discovery that its
actions are mediated by a subgroup of nuclear hormone
receptors, the RARs. It is now recognized that ATRA is a
potent embryonic morphogen that has defined roles in the
development and postnatal maintenance of many tissues,
including the lung. While many of the responses to ATRA
both in vitro and in vivo appear to be contradictory, the
Figure 3. Immunolocalization of cell proliferation/PCNA.
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Figure 4. Role of ATRA in alveolar repair. See Figure 2 for abbreviation. EGFr ⫽ epidermal growth
factor receptor; PDGFr ⫽ PDGF receptor; TIMP ⫽ tissue inhibitor matrix metalloproteinase;
CRABP ⫽ cellular retinoic acid binding protein; ROH ⫽ retinol; RoDH ⫽ retinol dehydrogenase;
RAL ⫽ retinal; RalDH ⫽ retinal dehydrogenase; LRAT ⫽ lecithin retinol acyl transferase;
RE ⫽ retinyl ester; REH ⫽ retinyl ester hydrolase.
effects reflect the capacity of this molecule to “normalize”
cellular behavior rather than to stimulate or inhibit them
specifically.
ATRA currently is used clinically to treat promyelocytic
leukemia and is used cosmetically in the treatment and
prevention of photo-aging and epidermal atrophy. ATRA
is thought to reverse epidermal atrophy in photo-aging by
inducing gene expression profiles that are similar to those
observed earlier in development. The initial report by
Massaro and Massaro4 showing that ATRA can reverse
experimental emphysema by inducing new alveoli suggests
that ATRA may have similar activity in the adult lung. The
remarkable effects of ATRA in experimental models of
COPD have stimulated significant hope that ATRA or
selective chemical analogs will bring some benefit to those
with emphysema. If one considers the limited epidemiologic data indication and the inverse relationship between
plasma retinol in smokers and the degree of airway
obstruction, then it may be reasonable to assume that
inadequate levels of RA may contribute to the chronic
injury observed in COPD (Fig 4). The National Institutes
of Health have sponsored clinical proof-of-concept studies
that will be initiated in the year 2000. Results from these
studies, as well as from preclinical projects addressing
more fundamental mechanism driving lung restructuring,
will likely stimulate additional therapeutic approaches to
improve the health of COPD patients.
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Decreases the Expression of
Vascular Endothelial Growth
Factor by Cultured Cells and
Triggers Apoptosis of
Pulmonary Endothelial Cells*
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(CHEST 2000; 117:241S–242S)
Abbreviations: CSE ⫽ cigarette smoke extract; VEGF ⫽ vascular endothelial cell growth factor.
he mechanisms behind the disappearance of lung
T tissue
(“vanishing lung”) in COPD remain enigmatic
in spite of the commonly discussed hypotheses of lung
inflammation and a protease-antiprotease imbalance. We
postulate that there are cellular and molecular programs
that maintain the structure of the adult lung. One or
*From the Departments of Pathology and Medicine, Division of
Respiratory Sciences and Critical Care Medicine, University of
Colorado Health Sciences Center, Denver, CO.
Correspondence to: Rubin M. Tuder, MD, Division of Pulmonary
Science and Critical Care Medicine, University of Colorado
Health Sciences Center, 4200 East 9th Ave, Denver, CO 80262
CHEST / 117 / 5 / MAY, 2000 SUPPLEMENT
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241S