Vol. 282, Issue 3, L345-L358, March 2002
SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and
Plasticity
Invited Review: Pulmonary alveoli:
formation, the "call for oxygen," and other regulators
Donald
Massaro1 and
Gloria D.
Massaro2
Lung Biology Laboratory, Departments of 1 Medicine and
2 Pediatrics, Georgetown University School of Medicine,
Washington, District of Columbia 20007-2197
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ABSTRACT |
The lung's only known essential
function is to provide sufficient alveolar surface to meet the
organism's need for oxygen and elimination of CO2. The
importance of the magnitude of alveolar surface area (Sa) to
O2 uptake (
O2) is supported
by the presence among mammals of a direct linear relationship between
Sa and O2. This match has been achieved,
despite the higher body mass-specific O2
of small organisms compared with large, by a greater subdivision of
alveolar surface, not by a larger relative lung volume in small organisms. This highly conserved relationship between alveolar architecture and O2 suggests the
presence of similarly conserved mechanisms that control the onset,
rate, and cessation of alveolus formation and alveolar size, which are
also influenced by retinoids and thyroid and corticosteroid hormones.
Furthermore, the "call for oxygen" is met at a breathing rate and
tidal volume at which the work of breathing is lowest. Thus there is a
complex, fascinating, but poorly understood, signaling relationship
among O2, the neural regulation of
breathing, and lung architecture, composition, and mechanics.
oxygen consumption; hyperoxia; hypoxia; calorie restriction; corticosteroid hormones; retinoids; thyroid hormone
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INTRODUCTION |
THE LUNG'S ONLY KNOWN
ESSENTIAL function is to provide sufficient gas-exchange surface
to satisfy the organism's need for oxygen and elimination of carbon
dioxide. The central importance of lung architecture to oxygen uptake
or, as elegantly put by Krogh (79), to the "call for
oxygen," is supported by the presence, across the entire
range of mammalian body mass, of a direct linear relationship
between alveolar surface area (Sa) and O2 uptake (O2) (Fig.
1) (143). This match has
been achieved, in spite of the higher body mass-specific
O2 of small organisms compared with
large, by a greater subdivision of the alveolar surface (Fig. 2), not by a larger relative lung volume
in small organisms (143). The highly conserved
relationship among alveolar size, number, Sa, and
O2 suggests the presence of similarly
conserved, but minimally understood, mechanisms that control the onset,
rate, and cessation of alveolus formation as well as the distance
between, and length of, alveolar septa. Furthermore, the organism's
call for oxygen is met at a breathing rate and tidal volume at which the work of breathing, and therefore its energy cost, is at a nadir
(Fig. 3) (2, 40, 105, 109, 122,
128). Thus we propose that there is a complex, fascinating, but
poorly understood, signaling relationship among
O2, the neural regulation of breathing,
and lung architecture, composition, and mechanics.
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Fig. 1.
Interspecies relationship between alveolar surface area (Sa) and
total organismal oxygen uptake (O2).
[From Tenney and Remmers (143).]
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Fig. 2.
The distance between alveolar walls (Alveolar Diameter)
is plotted against the organism's body mass-specific oxygen
consumption (O2). [From Tenney and
Remmers (143).]
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In this paper we review and try to synthesize, interpret, and
understand some recent and old findings about the formation of alveoli,
its regulation, and the plasticity of the architecture of the lung's
gas-exchange region. We do not review development of the fetal lung,
for which there are several recent reviews (61, 107, 150);
we review only selected insights from studies on mutant animals, and we
do not review the lung's architectural response to pneumonectomy.
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FORMATION OF ALVEOLI: TIMING, ARCHITECTURAL METHODS, AND SITES |
To facilitate exposition and to conform to the literature
(5, 25, 26, 95), we call the gas-exchange
structures of the architecturally immature lung alveolar saccules,
their subdivision into smaller units (alveoli) septation, and the
period in which septation occurs the period of septation. The formation
of alveoli other than by septation of alveolar saccules is referred to
as "other," but the architectural mechanism by which the other
occurs is unclear (see below). We discuss evidence that, at
least in part, the regulation of septation and other means of forming
alveoli may differ.
In all mammals of which we are aware, pulmonary alveoli are formed, in
part, by septation of alveolar saccules (3, 5, 17, 25, 26, 28,
33, 41, 43, 44, 46, 83, 154, 156, 157). However, the time in
development during which septation occurs varies considerably among
species in a manner that greatly reflects the newborn's activity
lifestyle. For example, guinea pigs (33) and range mammals
(3, 28, 154), which have great locomotive capacity at
birth, septate in utero; others, e.g., rats (25, 26) and
mice (5), with little locomotive capacity at birth,
septate after birth. Humans septate during the last month of gestation
and during the postnatal period (83, 156, 157). Precisely
when septation ends in humans is uncertain, but it seems to continue
for at least a few months after birth (83, 156, 157).
Burri et al. (25, 26) published some of the first and most
useful modern era studies on septation. Among many important observations, they showed that the distance between alveolar walls (Lm) diminishes during the period of septation
(5, 26). In rats, ~70% of this diminution occurs by
postnatal (PN) day 7 (26), suggesting that
septation is almost complete by then. This interpretation of the 70%
fall in Lm was supported and extended by the use
of procedures that allow estimation of the volume of individual
alveoli. Thus the average volume of individual alveoli in rats falls
sixfold between PN days 1 and 6 (125), but only little more over a period twice as long,
i.e., between PN days 2 and 14 (95)
(Fig. 4). Because the rate at which lung
volume increases does not change during the period of septation
(24), the virtually identical fold fall in volume over 6 days and 12 days indicates septation is complete, or almost complete,
by PN day 7, as was gleaned from the data of Burri et al.
(26).
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Fig. 4.
Age-related changes in the mean volume of individual
alveoli (V) and in the number of alveoli per rat (N). [Values for ages
2 and 14 days from Massaro and Massaro (95); values for
age 44 days from Blanco et al. (13).] Values for age 60 days and age 95 days not previously published.
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Based on the number and volume of individual alveolar saccules present
in 1-day-old rats (before septation) and the number of alveoli in
6-day-old rats (after the onset of septation), Randell et al.
(125) calculated septation of alveolar saccules to account for only about one-third of the alveoli formed between PN days 1 and 6. Calculations from similar measurements
on rats on PN day 2 and PN day 14 indicate that
~25% of alveoli formed during the period of septation result from
septation of the original alveolar saccules (95). These
observations support the notion (93) that during the
period of septation, alveoli are formed by septation of the original
saccules present at birth and by other as yet poorly identified
architectural events, which probably occur at the periphery of the lung.
Beginning in the second PN week and accelerating in the third, alveolar
walls become thinner (25, 26) due, at least in part, to
apoptosis of interstitial cells of the alveolus (7, 22,
136). This timing is stressed to encourage consideration of the
role in these processes, e.g., ending septation, alveolar capillary
remodeling, and apoptosis, of several molecules, already identified, whose expression peaks in rat lung at approximately PN
day 7-9. These molecules include galectin-1 (30,
124), cellular retinoic acid binding protein-I (CRABP-I)
(120), rA5D3, a recently cloned gene (15),
and cGMP phosphodiesterase (57). Bioinformatic and
experimental molecular searches for additional genes whose expression
is briefly elevated or depressed around PN day 7-10 and
that share similar molecular binding sites in their promoter regions
should be fruitful.
In rat (13, 95) and mouse (75), species whose
gas-exchange region has been studied most completely, alveoli continue to form after PN day 14 until about age 40 days (Fig. 4).
However, little about the anatomical process or sites of the
postseptation formation of alveoli has been directly shown. Reports
(18, 46) that the number of generations of conducting
airways diminishes after birth in dogs have suggested these airways may
have been remodeled into gas-exchange airways, thereby increasing the
number of alveoli (so-called retrograde alveolarization). However,
because there are so few terminal airways compared with the number of alveoli, it is unlikely retrograde alveolarization, if in fact it
occurs, would produce many alveoli. More importantly, using rigorous
sampling techniques and morphometric procedures, Randell et al.
(125) did not detect a change in the number of terminal bronchioles in rats during the early PN period. Many additional questions about other means of forming alveoli remain. For example, after the period of septation has ended, are alveoli formed throughout the lung among already formed alveoli, or are they generated, as the
thorax enlarges, in a more peripheral location, i.e., in the subpleural
zone, much as a tree increases in length and crown size by growth from
the peripheral tips of its branches? Reason and indirect evidence
support the periphery as the site of postseptation formation of
alveoli. Thus if after septation of alveolar saccules is complete and
alveoli continue to form throughout the lung by production of septa
among alveoli already formed, alveoli should become smaller, not
larger, after age 14 days (Fig. 4) unless lung volume increases more
rapidly than alveoli are formed, which does not occur
(24). The presence of a uniform turnover
throughout the lung of extracellular matrix after age 14 days would
support the notion of alveolus formation throughout the lung. However, within the limits of the methods used to assess it, matrix turnover is
more rapid among subpleural alveoli than among more central alveoli
(96). This supports the notion the periphery is the site
of the postseptation formation of alveoli. Finally, analysis of changes
in alveolar septal border lengths during PN development of ferrets
suggests septation of already formed alveoli is not a prominent
mechanism for an increase in the number of alveoli and size of alveolar
surface area after the period of septation (155). From
these findings and considerations, we propose that after the period of
septation, alveoli are formed predominantly in the peripheral
subpleural region. We extrapolate from these same considerations that
during the period of septation, the other means of alveolus formation
takes place in the subpleural region.
The cellular and molecular bases for the putative shift in the location
of alveolus formation from throughout the lung, including the
subpleural region, to only the subpleural region, are unclear. The
architectural mechanism(s) for the peripheral formation of alveoli is
also unknown. However, if the lung's gas-exchange region is considered
to be a series of branching tubes, continued branching of the distal
alveoli and septation of the blunt ends of the branches to generate
appropriately sized alveoli as the thorax enlarges are to us, an
attractive possibility as a mechanism of alveolus formation after the
period of septation (93). Supporting this possibility,
cells that store retinol, a precursor of all-trans retinoic
acid (ATRA), which can induce alveolus formation (see below), are
diffusely distributed in the lung during the period of septation
(147) but become concentrated in the subpleural region
after that period has ended (81, 96, 119). It will be of
interest to test whether treatment with ATRA, which induces alveolus
formation in adult rodents (12, 98, 99), causes the in
vivo appearance of lipid interstitial cells throughout the lung.
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THE "CALL FOR OXYGEN" |
Hyperoxia.
The clear relationship among O2 need, alveolar size, and
the magnitude of alveolar Sa from the smallest to the largest mammals (Figs. 1 and 2) (143) suggests that hyperoxia (excess
O2) and hypoxia (or other causes of cellular O2
shortfall) have opposing regulatory effects on the size of the alveolar
Sa. Experimental work supports this notion but must be considered in
light of the fact that most of it has been carried out in rats and with
consideration of the potential harmful effects on cellular function
exerted by hyperoxia and hypoxia through the production of
O2 radicals (50). For example, among several
studies that show hyperoxia diminishes septation (9, 23, 48, 137,
151), those of Randell et al. (125) in which
newborn rats were exposed to 95% O2 from PN day
1 to PN day 6 provide the most quantitative
information, including the demonstration that hyperoxia depresses other
means of forming alveoli as well as septation (125).
However, 95% oxygen severely damages the lung (23, 48, 137,
151) and slows body growth (139); it is, therefore,
uncertain whether the decreased rate of alveolus formation reflects a
need for less gas-exchange surface in an O2-rich
environment, the toxic effect of O2 directly on the lung,
an effect on the lung of systemic O2 toxicity, or a
combination of these possibilities.
The issue of O2 toxicity was somewhat diminished by Burri
and Weibel (27), who began much of the early work on the
relationship between O2 need and lung architecture. They
exposed rats that had already septated but were still growing rapidly
to 40% O2, which is much less damaging than 95%
O2. A key feature of their experiments is that the rats
exposed to 40% O2 increased body mass at the same rate as
air-breathing rats of the same age. In spite of identical rates of body
growth, which suggests the absence of systemic damage due to hyperoxia,
lung growth in O2 rats, as evidenced by total lung volume,
alveolar capillary volume, lung tissue volume, alveolar Sa, and
alveolar capillary Sa, was ~16% less than in air-breathing rats
(27). However, pulmonary oxygen toxicity must still be
considered because at any concentration of inspired O2, the
lung is exposed to a higher PO2 than other tissues. Nevertheless, our operational conclusion regarding the Burri
and Weibel data (27) is that a diminished rate of increase of gas-exchange surface, without a concomitant inhibition of increase of body mass, reflects a need for less alveolar Sa due to a greater delivery of O2 to tissues in an O2-enriched environment.
One of the anonymous reviewers of this manuscript pointed out that the
arterial O2 content at 40% O2 is only about
2% higher than at 21% O2 and asks, "Why should that
tiny increase in O2 content cause the degree of difference
in alveolar architecture reported by Burri and Weibel
(27)?" This is a good, thought-provoking question for
which we lack an equally good answer. Perhaps the most obvious answer
is that our operational conclusion about the Burri and Weibel work is
wrong, and the difference between alveolar and peripheral tissue
O2 tension at these concentrations of O2 is
sufficient to cause alveolar O2 toxicity without
O2 toxicity in the peripheral tissues. Conversely, however,
if it is peripheral tissue(s) that sounds the call for oxygen, the
difference in peripheral tissue PO2 at 20.9%
and 40% inspired O2 may be sufficient to signal the need
for less lung. Such putative tight regulation would be consistent with
the notion of symmorphosis, i.e., sufficient but not excess tissue for
functional need (152). The molecular basis for sensing
O2 need, the location of the sensor(s), and the signaling path(s) constitute a challenging and exciting area of research.
Hypoxia.
The relationship between lung function and a chronically low inspired
PO2 has been intensively studied for many years
(for a range of reviews, see Refs. 8, 34,
51, 67, 76, 82, 110, and 111), mainly because of people native to
high altitude whose forbearers lived for generations at high altitude.
We refer to those individuals as highlanders, and we refer to those of the same race, native to sea level, as lowlanders. It is important to
point out that among highlander populations in different parts of the
world, e.g., South America, the U.S. Rockies, Tibet, and Ethiopia,
those populations that have established high altitude residence
earliest in evolutionary time seem to be most adapted, perhaps
reflecting more time for genetic adaptation (111).
Andean highlanders have a 38% larger residual lung volume
(67), larger, more numerous alveoli (134),
lower maximum expiratory flow rate per lung volume, and lower upstream
airway conductance than lowlanders (20). The last two
characteristics contributed to the notion that gestation and PN
maturation of humans at high altitude results in dysanaptic
lung growth, more specifically, excess growth of the gas-exchange
region compared with the conducting airways (20). Also
contributing to the notion of dysanapsis in this context is the clear
evidence that the adaptive response of healthy pregnant humans and
animals (38, 87, 111), while not complete (54, 64,
111), partially protects the fetus from the low
PO2 of high altitude, which is, therefore,
first fully felt at birth (110, 127). Thus the lung's
conducting zone, which in all mammalian species reported develops
mainly during gestation (21), would be less affected by a
low atmospheric PO2 than the gas-exchange
region of organisms that septate after birth, e.g., rats (25,
26), or mainly after birth, e.g., humans (83, 156,
157). Tenney and Remmers (142) compared alveolar dimensions of guinea pigs bred and raised for many generations at 4,530 m and third-generation sheep resident at 4,390 m with guinea pigs and
sheep native to Hanover, NH (altitude 160 m). Both species septate
in utero (3, 33). They failed to find intraspecific
differences in alveolar dimensions between sea level and
highlander animals (Table 1), supporting
the notion that atmospheric hypoxia does not have a large effect on
alveolar dimensions in organisms that septate in utero.
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Table 1.
The effect of hypoxia on alveolus formation by septation or on
"other" means of forming alveoli depends on when hypoxia is
experienced and whether septation occurs pre- or postnatally
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Dogs brought to high altitude (3,100 m; Leadville, CO) at age 2.5 mo
and maintained at high altitude for 14 mo had greater lung
distensibility, diffusion capacity for carbon monoxide (DLCO), and lung
tissue volume (Vt) 3 mo after return to sea level (Dallas, TX) than did same-aged dogs maintained at sea level
(69). It is of some interest that the higher DLCO
was due to an increased capillary volume (Vc), not higher
membrane diffusion capacity (DMCO). Thus because the elevated
Vt could reflect the higher Vc, there is no
evidence from this study to indicate the young dogs increased Sa.
However, young humans (average age 20 yr) residing at 3,100 m
(42) and native or longtime dwellers at 4,500 m
(126) have a higher DLCO than lowlanders due to a high
DMCO and Vc. The higher DMCO could reflect a larger Sa, but
it could also be due to a thinner alveolar membrane. If the difference
in DMCO between dogs (69) and humans (42) is
due to a higher Sa in human highlanders than in lowlanders, one reason
could be that when the need for an elevated diffusion capacity was
removed in the dogs during 3 mo at sea level, alveoli were destroyed to
match tissue size to functional need, in line with the concept of
symmorphosis (152). This notion is supported by the rapid
calorie-related loss of alveoli when need for Sa is diminished by a
fall of O2 (see below)
(94).
We recognize that gestation, birth, and living under the hypoxia of
altitude differ from the same events in laboratory-generated hypoxia at
sea level. Nevertheless, to examine the effect of hypoxia on the lungs
of organisms that septate after birth, we maintained female rats in
13% O2 for at least 3 wk before they were mated; male rats
were not maintained in 13% O2 because it was very
difficult to breed when both partners, even if acclimated, were kept in 13% O2 (13, 101, 108). Once pregnant, the
rats remained in 13% O2 throughout their pregnancies and
with their pups for varying periods after birth. At age 2 days, the
average volume of alveolar saccules was larger in 13% O2
rats than in air-breathing rats and was, among the measurements made,
the only intergroup difference at that time (Table
2). However, unlike the lack of an effect of hypoxia on septation in guinea pigs and sheep (142),
which septate in utero (3, 33), even though the dams were
acclimatized to it, hypoxia markedly diminished PN septation (Table
3, Fig. 5). And, unlike Andean humans
(134), rat pups exposed to hypoxia had fewer, not more,
alveoli. Thus this issue needs resolution. Removal of rats from 13%
O2 to room air after the period of septation did not result
in spontaneous septation (13, 101). The absence of post
hoc septation indicates, as shown earlier (93), that there
is a "critical" period for septation as there is, for example, in
the development of vision (65). The molecular basis for
the critical period in response to 13% O2 and its
relationship, if any, to the corticosteroid hormone-induced critical
period (93) are unknown. It is also unknown whether
treatment with ATRA prevents hypoxia-induced failed septation or
rescues hypoxia-induced failed septation, as it does with
corticosteroid hormone-produced failed septation (97, 99).
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Fig. 5.
Hypoxia depresses the rate of alveolus formation in rats
during, but not after, the period of septation. Conditions are as
stated in legend to Table 1. N = number of alveoli per
rat;  , air;  , 13% O2.
[From Blanco et al. (13).]
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Diminished septation in 13% O2 rat pups results in fewer
alveolar attachments to small conducting airways (13).
Thus if septation is impaired in highlanders, as suggested by the
abstract that indicates highlanders have large alveoli
(134), it is reasonable to assume that they have fewer
alveolar attachments to conducting airways than lowlanders. The latter,
rather than the presumed hypoxia-stimulated excessive growth of the
gas-exchange region, could be responsible for the premature closure of
conducting airways (20), high residual volume
(67), low maximal expiratory flow rate, and low upstream
conductance (20) present in human highlanders, much as
occurs in chronic obstructive pulmonary disease (36, 63,
131). We make this comparison recognizing that highlanders can
exhibit exceptional exertion at high altitudes and, therefore, have
evolved excellent methods of adaptation (67).
Based on the number and average volume of alveolar saccules present at
age 2 days and the size of alveoli present at age 14 days, it is clear
hypoxia also impairs other means of forming alveoli and to a greater
extent than it impairs septation (Table 3). This other component of
alveolus formation is not trivial (Table 3). After the period of
septation, the rate of alveolus formation in rats remaining in 13%
O2 is the same as in air-breathing rats (Table 2, Fig. 5).
This similarity reflects a decreased rate of alveolus formation in
air-breathing rats after the period of septation, not an increased rate
of alveolus formation in 13% O2 rats (Fig. 5). Thus
alveolus formation during the period of septation seems to have a
hypoxia-depressable component that is not present after the period of
septation, i.e., it is confined to ages ~2-14 days in rats.
We combined three sets of seminal findings by others in an attempt to
explain the age-dependent effect hypoxia has on alveolus formation.
First, because of the direct linear relationship between resting
O2 and Sa across the entire span of
mammalian body mass (Fig. 1) (143), we think the call for
oxygen (79) is the primal, highly conserved regulator of
Sa. This regulation may, at least partly, be mediated by retinoids (see
below). Second, hypoxia decreases O2 in
rats and in many other organisms, including humans, but the depression
diminishes with age (for an excellent review, see Ref.
114). Third, hypoxia-induced increase in minute ventilation (E) is absent in 2-day-old rats but
develops and increases with age (102, 114, 115). We
tentatively propose that the markedly lower
O2 of hypoxic newborn rats decreases the
need for alveolar Sa and, by as yet unidentified signaling pathways,
the formation of alveoli is diminished. Although this occurs without a
lower total lung volume in 13% O2 rats, the
gas-exchange region of early PN hypoxic rats has less tissue than
air-breathing rats of the same age (101). Therefore, in
the face of O2 deprivation, and hence less available energy
(16, 47, 53), the organism's energy needs are low partly
because it makes less lung tissue and partly because there is less to
maintain. This response, as the response to calorie restriction (see
below) (92), may have provided an evolutionary advantage.
With age, the depressive effect of hypoxia on
O2 diminishes (114), and,
therefore, the O2 need increases. The organism compensates,
at least in part, by increasing E. We suggest that
the increasing O2 need, perhaps in concert with the
mechanical events associated with the higher E,
initiates an undefined signaling cascade that prevents the
postseptation slowing of the rate of alveolus formation.
When rats are initially exposed to hypoxia after the period of
septation, they increase Sa by increasing the size of individual alveoli, not by making more alveoli (Table
4) (13). Furthermore, they
increase lung volume, but, unlike early PN 13% O2 rats,
rats exposed to 13% O2 after the period of septation
increase gas-exchange Vt more rapidly than air-breathing
rats. The larger volume of alveolar tissue does not suggest thinner
alveolar walls (13), which would enhance diffusion. This
is different from the lower harmonic mean thickness of the alveolar
wall of guinea pigs native to high altitudes compared with sea level
members of the same species (62) and the high diffusion
capacity in highlander humans (42), which suggest better
adaptation in animal and human highlanders then in lowlander animals.
It is apparent, although functional effects of dwelling at high
altitudes have been studied for many years, that there have been few
studies of lung structure in highlander animals or in humans. However,
as the resolution of noninvasive lung imaging procedures increases,
hopefully supplemented by morphometrically based anatomical studies,
the effect on the lung of being a native highlander, of going to
altitude, and of aging at altitude, may become more clear.
Alveolar plasticity in response to the endogenous call for oxygen.
The use of an altered inspired PO2 to study the
effect the availability of O2 has on alveolar dimensions
has, as discussed above, provided very useful information. However, as
also mentioned, tissue toxicity due to oxygen radicals as a determinant
of the alveolar response to an altered inspired O2 is
difficult to exclude. Treatment with thyroid hormone, or blocking
conversion of thyroxine to triiodothyronine, to alter
O2, is confounded by the action of
thyroid hormone beyond its effect on O2
(6). We believe that a more physiological means of
altering O2, one that occurs in nature
(66) and represents an endogenous alteration in the call
for oxygen, is calorie restriction (CR) and CR followed by refeeding
(CR-RF). CR lowers O2; refeeding after
CR returns O2 to values present before
CR (47, 53).
Sahebjami and Wirman (133) and subsequently others
(58, 74, 77) showed that CR in adult rats (58, 77,
133) and hamsters (74) increases
Lm and diminishes Sa. As may be gleaned from the
titles of their articles (58, 74, 77, 133), they felt
these changes represented starvation or nutritionally induced emphysema. Sahebjami and Wirman (133) and later Kerr et
al. (77) found refeeding reversed these changes but, as
nearly as we can tell from their papers, they did not raise the
possibility that the diminished Lm reflected
alveolar regeneration (77, 133).
The effect of CR and of CR-RF on O2 and
the highly conserved relationship across species between resting
O2 and alveolar dimensions (Figs. 1 and
2) (143) led us to a different interpretation of the early
work on calorie intake and the architecture of gas-exchange structures.
We propose that the changes in alveolar architecture in response to CR
and to CR-RF represent endogenous programs of alveolar destruction and
regeneration, i.e., alveolar turnover induced by calorie-related
changes in O2. Therefore, we extended their work (58, 74, 77,
133) to test the hypothesis that CR activates endogenous
alveolar destruction and that CR-RF induces alveolar regeneration. In
adult mice, CR resulted in ~45% fewer alveoli, less Sa without
diminished lung volume, and a 20% decrease in the amount of
lung DNA due, at least in part, to apoptosis of alveolar wall
cells (94). Refeeding resulted in alveolar wall cell
replication, an increase of lung DNA, and alveolar regeneration (94). The of loss of alveoli with CR is consistent with
the doubled rate of proteolysis in lungs of adult CR rats
(145).
We tentatively suggest the following to explain our findings. CR
diminishes total organismal (47, 53) and lung
(55) O2. The fall in total
O2, perhaps in concert with the lower lung O2, signals a need for less Sa. For
survival during severe CR, the organism requires substrates to maintain
muscle mass and increased gluconeogenesis to provide glucose for brain
metabolism (135). In this setting, we propose that an
endogenous program for increased lung proteolysis (145),
which includes alveolar destruction, is activated and helps provide
needed substrates. This program also diminishes the mass of lung
tissue, thereby decreasing the cost of its maintenance. Refeeding
increases the call for oxygen and, by an as yet unidentified signal(s),
activates a program of alveolar regeneration. The signals, which may
initially be mechanical but must ultimately be molecular, for the
calorie-related endogenous programs of alveolar destruction and
regeneration, are being sought.
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HORMONAL REGULATION OF ALVEOLUS FORMATION DURING THE PERIOD OF
SEPTATION |
Glucocorticosteroid hormones.
In contrast to the detailed information about architectural processes
of alveolus formation during the period of septation, due mainly to
work in Bern (24-27, 156, 157) and which now includes many species (3, 5, 28, 83, 140, 154), little is known about the regulation of septation. Our early thinking was greatly influenced by a consideration of the architectural events needed to
form a septum and by the notion that changes in the concentration of
systemic hormones (59, 60) might regulate the sequence in
which organs, e.g., PN lung and pancreas, attain adult anatomical or
functional characteristics (5, 25, 26, 60). We should bear
in mind that little work on regulation of alveolus formation has been
done other than in rodents.
From the available systemic hormones, we tested the
possibility that glucocorticosteroids might affect (inhibit) the
formation of septa for three main reasons. First, eruption and
elongation of alveolar septa are brought about by forming ridges in
epithelial sheets, which in part requires epithelial cell division. In
addition, new septa must be filled with capillaries and fibroblasts,
which also requires cell replication. Because glucocorticosteroids
inhibit cell division in several tissues (86), including
the lung (93, 112), they might prevent septation. This
idea was strengthened because there is a trough in the serum
concentration of the species' active glucocorticosteroid hormone
during the period of septation, whether septation occurs in utero or
postnatally (59, 70). Furthermore, the serum concentration
of glucorticosteroid hormones begins to increase as septation ends and
as there is acceleration of the thinning of the alveolar wall
(25, 26). This suggests the elevated concentration of the
hormone initiates the end of septation, the onset of accelerated
alveolar wall thinning, and the remodeling of alveolar vessels from a
double capillary to a single capillary system (25, 26).
Treatment of rat pups with dexamethasone, a synthetic
glucocorticosteroid hormone, during the period of septation prevents septation (14, 93) and diminishes the rate of DNA
synthesis and accumulation in the lung (93). The
mechanism(s) by which dexamethasone inhibits septation is unknown, and,
in view of the many genomic (1) and nongenomic
(29) actions of corticosteroid hormones, there is a myriad
of possibilities. Nevertheless, in the spirit of guilt by association
(Ref. 4 and later in this paper), the PN time course of
activity in lung of ornithine decarboxylase (ODC) offers two possible,
not mutually exclusive, mechanisms. The activity of ODC peaks in lungs
of rats during septation (PN days 4-6) but not in
liver, heart, brain, or kidney (146) and is depressed in
lung, but not in liver, by corticosteroid treatment (10)
during the period of septation (26). ODC, whose activity correlates with DNA synthesis (123), catalyzes the
synthesis of polyamines, which are involved in cell replication. Thus
to the extent that proliferation of alveolar wall cells is essential to, but not sufficient for, the formation of a septum, the impairment of septation by dexamethasone could reflect its depression of proliferation of alveolar wall cells by its inhibition of ODC activity.
Because polyamines can regulate communication through gap junctions
(138), the inhibition of septation by dexamethasone could involve more than depression of cell replication. Dexamethasone might
block septation by interfering with intercellular communication through
gap junctions. This notion is supported by studies on other organs
showing that the expression of gap junction protein is developmentally
regulated (19, 71) and that gap junction channels regulate
epithelial-mesenchymal transformation during heart development
(118). To extend this line of thinking, oxidative stress
diminishes the function of gap junctions (78, 90, 116); hypoxia (37), hyperoxia (50), and premature
birth (49) cause oxidative stress and impair septation
(13, 23, 32, 89, 101, 140). Therefore, if corticosteroids,
hyperoxia, hypoxia, and premature birth inhibit septation by
diminishing cell-cell communication through gap junctions, it opens the
possibility of preventing the inhibition of septation during hypoxia,
hyperoxia, or corticosteroid treatment by pharmacologically augmenting
communication through gap junctions.
In addition to blocking septation, dexamethasone treatment
markedly accelerates alveolar wall thinning and changes the cellular composition of the wall (91). Within 2 days of the onset
of treatment of 4-day-old rats with diluent or dexamethasone,
dexamethasone-treated pups have 20% thinner gas-exchange walls, a 32%
lower absolute volume of retinol-storing interstitial fibroblasts, and
a 1.5-fold higher volume of alveolar type II cells than diluent-treated
rats (91). These experiments also provide evidence that
dexamethasone 1) diminishes replication of fibroblasts in
the alveolar wall, thereby diminishing the number of retinol storage
cells (see below for relevance) and 2) impairs conversion of
alveolar type II cells to alveolar type I cells, for which there would
be less need if the gas-exchange surface is increasing at a slower rate
than in diluent-treated pups.
Once septation has been prevented by corticosteroids, discontinuing
them is not followed by spontaneous septation, at least not up to age
60 or 95 days, i.e., 47 (93) and 82 (132)
days after stopping the administration of corticosteroids. Failed
septation is accompanied by the formation of fewer pulmonary arteries
and by pulmonary hypertension (84). Because the
gas-exchange Sa is also diminished, a restricted alveolar capillary bed
could contribute to the pulmonary hypertension. Furthermore, the
magnitude of hypoxia-induced pulmonary hypertension is greater in
dexamethasone-treated rats than in diluent-treated rats
(84). This important paper (84) clearly
demonstrates a long-term functional effect of early events. The effects
of dexamethasone on septation and the pulmonary vessels have important
implications for prematurely born babies treated with corticosteroids
for days or weeks. Such treatment might increase the impairment of
septation that occurs even in the absence of glucocorticosteroid
therapy (68, 89, 140). In addition, because of the anatomy
of the lung in bronchopulmonary dysplasia, these infants have areas of
alveolar hypoxia that could further increase pulmonary vascular resistance.
Thyroid hormones.
Three considerations led us to the hypothesis that thyroid
hormone exerts a regulatory effect on subdivision of the large saccules
that constitute the gas-exchange region of the rat lung at birth.
1) Thyroid hormone concentration in rat serum
(113) and thyroid hormone receptor density in rat lung
(11, 129) begin to increase just before the onset of
septation. 2) Thyroid hormone treatment induces substantial
changes in brain architecture without a detectable effect on
O2 (31, 121). 3)
Thyroid hormone treatment increases DNA synthesis in the lung of
newborn rats (113). Triiodothyronine (T3)
administered to newborn rats at a dose that does alter the
developmental increase in body weight (92) and that at
even higher doses does not increase O2
(141) accelerates the pace of septation, resulting in
smaller alveoli and a greater Sa without affecting lung volume
(92). The combination of a larger Sa without a large lung
volume indicates T3 induces the formation of additional
alveoli. Injection of propylthiouracil, which blocks conversion of
thyroxine to T3 (35), impairs septation without slowing the developmental increase of body mass or lung volume.
Thyroxine treatment, at a lower dose than is required to increase
O2 (141), overcomes the
inhibitory effect of propylthiouracil on septation (92).
These findings indicate thyroid hormone does not accelerate septation
by increasing O2 and raise the
possibility that treatment with thyroid analogs that have little effect
on O2 might induce septation when given
alone, or in combination with, retinoids (see below).
| |
RETINOID REGULATION OF ALVEOLUS FORMATION: MORE GUILT BY
ASSOCIATION |
Several lines of evidence available in the late 1980s and early
1990s led to the notion that retinoids might play a key role in the
formation of pulmonary alveoli during the period of septation. In rats,
this evidence included: 1) the high concentration of cellular retinol binding protein-I (CRBP-I) in lung, but not in liver,
during the period of septation (120); 2)
treatment of adult rats with ATRA upregulates CRBP-I mRNA, whereas
treatment with dexamethasone, which inhibits septation (14, 93,
132), downregulates CRBP-I mRNA (130);
3) the lung's concentration of CRABP-I peaks at
approximately PN day 9-10 (120) (we now
speculate CRABP-I may be an inhibitor of septation or may be involved
in conversion of the double to the single capillary system in the alveolus; see below); and 4) fibroblasts rich in vitamin A
(retinol) storage granules occupy a large fraction of the alveolar wall throughout the lung during the period of septation (147),
a time when alveoli are formed throughout the lung. After the period of
septation, these cells become located mainly in the subpleural region
(81, 96), the site where, we believe, the postseptation formation of alveoli takes place (96). These observations,
as clues that retinoids exert a regulatory effort on septation, were supported by the general knowledge that retinoids play a key role in developmental processes in many tissues (106).
On the supposition of guilt by association, i.e., much lung retinoid
activity during the period of septation, we tested the hypothesis that
treatment of newborn rats with ATRA might prevent the inhibition of
alveolus formation produced by dexamethasone; the hypothesis was not
falsified (97). Furthermore, treatment of rat pups with
ATRA alone causes the formation of more numerous, but smaller, alveoli
without affecting lung volume or alveolar Sa. Briefly (see Ref.
83 for a full discussion of this seeming paradox), the
absence of a higher Sa in rats treated with ATRA alone, compared with
vehicle-treated rats, suggests the presence of a control mechanism that
inhibits the size of alveoli when there is not a
O2-induced need for additional Sa. On
the basis of these findings and the larger alveoli of rats exposed to
hypoxia in the postseptation period (Table 2) (13), we
envision two processes, differently regulated, in the formation of
alveoli: eruption of a septum and subsequent elongation of a septum. We propose that ATRA induces eruption of a septum and determines the
distance between septa; other factors, principally
O2, determine the length of a septum.
Thus with excess eruption of septa in ATRA-treated rats,
without a need for greater Sa, septum length is curtailed. This notion
presupposes a different gradient for the morphogen ATRA among species,
or interspecific differences in cellular sensitivity to ATRA, to
account for the interspecific differences in spacing of septa
(143). Finally, to explain the interspecific differences
in alveolar size, we do not exclude a link between
O2 and alveolar wall ATRA gradients.
Because of the opposing action of ATRA and dexamethasone on CRBP-I in
adult rat lung (130), the effect of treatment with ATRA on
CRBP-I mRNA and CRABP-I mRNA was examined in rats during the period of
septation. ATRA treatment transiently increases the concentration of
CRABP-I mRNA but does not prevent the depression of its mRNA by
dexamethasone (153). ATRA also increases the concentration of CRBP-I mRNA in the lungs of neonatal rats (153) as it
does in lungs of adult rats (130). Of particular interest,
ATRA treatment prevents the depression of CRBP-I mRNA induced by
dexamethasone (153).
The consequences to septation of the timing of the changes in lung
concentration of CRBP-I and CRABP-I and the opposing action of ATRA and
dexamethasone on CRBP-I mRNA concentration in lung are unknown, but it
may be useful to speculate about this in light of some known and
suspected general properties and functions of these proteins. The
cellular concentration of retinoid-binding proteins exceeds those of
the retinoids to which they bind with high affinity, resulting in
exceedingly low intracellular concentrations of free retinoids
(117). Binding of retinoids by CRBP and CRABP helps to
insure specificity of the interaction of retinoids with cellular
dehydrogenases responsible for their metabolism and prevents nonenzymatic isomerization and oxidation of retinoids
(117). Indeed, CRBP-null mice exhibit a sixfold faster
turnover of retinol than wild-type mice, which is consistent with the
notion that retinol is promiscuously metabolized in the absence of
CRBP-I (52). CRBP-I increases cell uptake of retinol,
decreases its esterification, and increases the generation of ATRA and
other biologically active retinoids (117). Therefore, a
high concentration of CRBP-I, as occurs in lungs of untreated rats
during the period of septation (120), should increase the
production of ATRA by lung cells, perhaps in a cell-specific manner.
Because we think septation is mainly over by PN day
7-8, the peak of CRABP-I about then could bind ATRA and end
septation. Furthermore, the high expression of CRABP-I, if it occurred
in only some cell types in the alveolar wall, could determine which
cells respond to ATRA as the lung begins to remodel the alveolar wall,
converting its double capillary system to a single capillary system.
Recent findings expand the potential therapeutic usefulness of ATRA
beyond the prevention of failed septation. ATRA partially rescues
septation previously inhibited by treatment of rat pups with
dexamethasone and in adult mice with a genetic failure of septation
(99). Veness-Meehan et al. (149) found ATRA
does not prevent the hyperoxia-induced inhibition of septation in rat pups but confirmed ATRA prevents the inhibition of septation by dexamethasone. Two subsequent studies showed that rat pups exposed to
hyperoxia and simultaneously treated with ATRA, but not those treated
with vehicle, septate a few weeks after removal from hyperoxia without
post-O2 treatment with ATRA (39, 148). This
suggests that ATRA, perhaps by an antioxidant action, preserves the
lung's ability to septate. Finally, also relevant to potential therapy in humans, ATRA substantially abrogates in adult rats the
elastase-induced loss of elastic recoil, increased lung volume, large
gas-exchange units, diminished number of alveoli, and low alveolar Sa,
i.e., ATRA induces alveolus regeneration (12, 98, 144).
The mechanism(s) and downstream changes in gene expression and
protein-protein interaction by which ATRA affects alveolus formation
are being actively sought in several laboratories.
The induction of alveolus formation by ATRA, of course, led to studies
to identify the retinoid receptors involved. Retinoid receptors are
nuclear receptors of two classes: retinoic acid receptors (RARs) and
retinoid X receptors (RXRs) (88). Three subtypes of RARs
and RXRs have been identified: RAR-
, RAR-
, and RAR-
, and
RXR-, RXR-, and RXR-. At physiological concentrations of
ligand, RARs respond to ATRA and 9-cis retinoic acid and
RXRs respond to 9-cis retinoic acid.
Retinoid agonists, antagonists, and mutant mice are being used to
determine which retinoid receptors are involved in septation. RAR-
/ mutant mice have early onset of septation and, during the period
of septation, form alveoli twice as fast as wild-type mice. As expected
from the results from RAR- mutant mice, a RAR- agonist blocks
septation (100). Thus RAR- is an endogenous inhibitor of septation. However, RAR- / mice generate alveoli after the period of septation at the same rate as wild-type mice. This supports the notion that alveolus formation, during the period of septation and
after, is regulated, at least in part, by different molecular mechanisms. On the basis of the early induction of septation in RAR-
/ mice, it is possible that treatment of very prematurely born
children with a RAR- antagonist (85) would allow the
early onset of septation. Further important information has come from analysis of RAR- mutant mice (104). RAR- gene
deletion diminishes septation, and the additional deletion of one
RXR- allele further impairs septation (104). Because
ATRA induces alveolus formation, RAR- inhibits septation, and
RAR- mutant mice have impaired septation, combined therapy with
appropriate agonists and antagonists might provide the strongest
therapeutic induction of alveolus formation. RAR- was recently
reported (56) to induce "alveolar repair and/or
alveolarization in adult rats." The same report states a RAR-
agonist "has been shown to induce alveolar repair in two rodent
models, pancreatic elastase-induced emphysema in rats, and cigarette
smoke-induced emphysema in mice" (56). Alveolar repair,
i.e., the reestablishment of the integrity of the alveolar air-tissue
barrier after it has been injured (45, 103), occurs spontaneously in many conditions (72, 73), including
elastase-induced emphysema (80). Therefore, the
"repair" ascribed to RAR- must have been induction of the
formation of alveoli.
| |
ACKNOWLEDGEMENTS |
We thank Drs. Linda B. Clerch, Ghenima Dirami, and Le Ann Blomberg
for reviewing the manuscript and for stimulating discussions.
| |
FOOTNOTES |
This work was supported, in part, by National Heart, Lung, and Blood
Institute Grants HL-20366, HL-59432, HL-60115, and HL-37666.
D. Massaro and G. D. Massaro are Senior Fellows of the Lovelace
Respiratory Research Institute, Albuquerque, NM. D. Massaro is
Cohen Professor, Georgetown University.
Address for reprint requests and other correspondence: D. Massaro, Lung Biology Laboratory, Georgetown Univ. School of Medicine, 3900 Reservoir Rd. NW, Washington, DC 20007-2197 (E-mail:
massarod{at}georgetown.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00374.2001
| |
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TOP
ABSTRACT
INTRODUCTION
