Lung Biology Laboratory, Departments of 1 Pediatrics and 2 Medicine, Georgetown University School of Medicine, Washington, DC 20007-2197
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ABSTRACT |
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Mammalian alveoli, complex architectural
and cellular units with dimensions that are linked to the organism's
O2 consumption (O2), are
thought to be destroyed only by disease and not to spontaneously
regenerate. Calorie restriction of adult mammals lowers
O2, and ad libitum refeeding returns
O2 to pre-calorie-restriction values. We
took advantage of these relationships and tested the hypothesis in
adult mice that calorie restriction (two-thirds reduction for 2 wk)
followed by ad libitum refeeding (3 wk) would cause alveolar
destruction and regeneration, respectively. Calorie restriction
diminished alveolar number 55% and alveolar surface area 25%.
Refeeding fully reversed these changes. Neither manipulation altered
lung volume. Within 72 h, calorie restriction increased alveolar
wall cell apoptosis and diminished lung DNA (~20%). By 72 h of refeeding, alveolar wall cell replication increased and lung DNA rose to amounts in mice that were never calorie restricted. We
conclude that adult mice have endogenous programs to destroy and
regenerate alveoli, thereby raising the danger of inappropriate activation but the possibility of therapeutic induction, if similar programs exist in humans.
oxygen consumption; calorie restriction; apoptosis; cell replication
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INTRODUCTION |
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THE LUNG'S ONLY KNOWN
ESSENTIAL function is to provide sufficient gas-exchange
(alveolar) surface to meet the organism's need for O2
uptake (O2) and CO2 release.
Gas-exchange units (alveoli) are destroyed in common lung diseases,
including chronic obstructive pulmonary disease (COPD), the sixth
leading cause of death worldwide, which is expected to rise to the
third leading cause of death by 2020 (22). Alveolar
destruction in COPD is thought to be produced by proteases released
from inflammatory cells in the alveolus (8). However,
therapy aimed at lessening protease activity by smoking cessation or by
the use of anti-inflammatory drugs and protease inhibitors
(23) fails to slow the loss of gas-exchange function
(27). This suggests that present concepts of the mechanism
of destruction of alveolar septa are incomplete. Furthermore, until
recently (19), remediation of emphysema by the therapeutic
induction of alveolar regeneration had not been pursued; even now, the
available potential therapies for remediation are limited (FORTE:
NHLBI-NIH Clinical Trials Database).
In mammals, the liver is the only organ found to regenerate complex
architectural units matching structure to systemic functional need
(10); however, the evidence that liver size diminishes to
match functional demand is not convincing (7, 13). By contrast, the following factors, considered together, indicate that
adult mammals might have endogenous programs of destruction and
regeneration of gas-exchange units that link complex structure to
systemic function: 1) Gas-exchange surface area is directly proportional to the organism's O2
across the entire range of mammalian body mass (32).
2) Calorie restriction, common in nature (25)
and a past and present problem among humans (1), diminishes
O2 (21),
lessening the need for gas-exchange surface area. 3) Calorie
restriction doubles the rate of lung proteolysis (33),
suggesting that lung tissue is destroyed, thereby avoiding the energy
burden of maintaining unneeded tissue and simultaneously providing
substrate to maintain muscle and for gluconeogenesis, which is needed
to provide glucose for brain metabolism. 4) Refeeding after
calorie restriction elevates
O2
(21), increasing the need for gas-exchange surface. To
determine whether the lung has endogenous programs to destroy and
regenerate alveoli, we took advantage of calorie-related changes in
O2 (21) and quantitatively examined their effect on lung gas-exchange units.
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METHODS |
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Animals and experimental manipulations. We housed adult male C57BL/6 mice (Jackson Laboratory) in individual cages and, in initial studies, divided them into four groups: One group (F1) was allowed food (Ralston Purina Laboratory Chow 5001) ad libitum for 15 days, and a second group (F2) was allowed food for 36 days; a third group (calorie restricted) was provided one-third of their individual daily food consumption measured daily over 4-5 days immediately before the onset of calorie restriction for 15 days; and the fourth group was subjected to calorie restriction for 15 days and then refed ad libitum for 21 days. The duration of calorie restriction and refeeding was arbitrary, except calorie restriction was influenced by preliminary observations that the rate of loss of body mass had become asymptotic by 15 days; for refeeding, we knew that all-trans retinoic acid induced alveolus formation within 12 days (19) and simply added several more days to that time. In other studies, we subjected mice to calorie restriction for 72 h and killed some; others were refed ad libitum for 72 h. The retinol content of Rodent Laboratory Chow 5001 is 22 IU/g. The measured ad libitum chow intake per mouse was ~4.2 g/day. Decreasing this amount by two-thirds provides a daily retinol intake of 30 IU. The minimal daily requirement of retinol for mice is 1-2 IU (31). Therefore, retinol deficiency, which requires many months on a retinol-free diet to establish (31), should not have been a factor in the present studies. All mice were continuously allowed tap water ad libitum and were housed in the animal care facilities of Georgetown University on a 12:12-h light-dark cycle at an ambient temperature of 21-22°C. Animals were killed by cutting the great vessels of the abdomen after the induction of a surgical level of anesthesia (no response to toe pinch) with xylazine plus ketamine. All procedures were approved by the Georgetown University Animal Care and Use Committee and comply with US Department of Agriculture and National Institutes of Health guidelines.
Lung fixation and morphometric procedures. Lungs were fixed in cold 2.5% cacodylate-buffered glutaraldehyde at a transpulmonary pressure of 20 cmH2O (18, 19), and their volume was measured by volume displacement (26).
We examined serial sections of lung to distinguish alveoli from alveolar ducts (18, 19). The selector method (5) was used to choose alveoli for analysis, the volume of individual alveoli was determined, and the number of alveoli was calculated as previously described in detail (18). These determinations were made without knowledge of the experimental manipulation to which the mice were subjected. Our selection of three animals per group was determined by a power analysis based on the variance of prior estimates of these parameters and was done in the spirit of using just sufficient animals to answer a question (20).Other measurements. DNA was measured fluorometrically using calf thymus DNA as standard (4). Two assays were used to detect cleavage of DNA. A kit, In Situ Death Detection POD, was used according to the manufacturer's (Roche) directions to detect DNA fragmentation in alveolar wall cells by the TdT-mediated dUTP nick end labeling (TUNEL) transfer assay. Two individuals each counted 500 cells/slide without knowledge of the experimental group from which the slides were made. Their values were combined, and the means ± SE of each experimental group were calculated. DNA fragmentation was estimated by a second method; we used a Polytron to disrupt lung tissue and separated the homogenate by centrifugation (14,000 g for 20 min at 4°C) into a pellet (unfragmented DNA) and a supernatant fraction (fragmented DNA) (28). We assayed each fraction for total DNA.
We used a rabbit polyclonal antibody against a recombinant protein of human origin representing full-length proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology) to identify PCNA-positive cells (3). In methanol-fixed cryosections of frozen lungs, the percentage of PCNA-positive alveolar wall cells on slides from two tissue blocks per mouse was determined. Two individuals each counted 500 cells/slide without knowledge of the experimental group from which the slides were made. The data were combined and expressed as means ± SE.Statistical analysis. Means ± SE were calculated for each group, and a Mann-Whitney nonparametric analysis was used to assess the statistical significance of values between groups (29). Scientists commonly disagree about the most appropriate test to determine statistical significance (16, 30, 38). We chose a nonparametric robust method (17), because we did not assume a normal distribution. We elected to use multiple t-tests, because the P value is a descriptive statistic (30, 38).
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RESULTS |
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Body mass did not differ among groups at the onset of the study
(not shown), but calorie-restricted mice weighed considerably less,
when killed, than mice in all other groups (Fig.
1A). Although lung volume
measured at a transpulmonary pressure of 20 cmH2O was the
same in all groups (Fig. 1B), calorie-restricted mice had
55% fewer alveoli than F1 mice (Fig.
2A). This difference was due
to destruction of alveolar walls as indicated by the 1.9-fold greater
volume of an average alveolus (Figs. 2B and
3) and the 25% lower alveolar surface
area (Fig. 2C) without a difference in lung volume between
calorie-restricted and other mice (Fig. 1B). The volume of
alveolar wall tissue was 0.12 ± 0.01 (SE) cm3
(n = 3) in F1 mice and 0.08 ± 0.00 (n = 3) in calorie-restricted mice (P < 0.05). Mean alveolar volume, number, and surface area were the same
in F1 and F2 mice (Fig. 2), even though
F1 and F2 mice were killed 3 wk apart.
Refeeding calorie-restricted mice resulted in alveolar regeneration,
shown by the return of alveolar volume, number, and surface area to
values in F1 and F2 mice (Figs. 2 and 3)
without an increase of lung volume (Fig. 1).
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To test whether lung cell loss and replication accompanied
calorie-related alveolar turnover, we measured the effect of calorie restriction and calorie restriction followed by ad libitum refeeding on
the quantity of lung DNA. Within 24 h of onset of calorie
restriction, DNA amount in the lung began to fall and by 72 h of
calorie restriction had declined ~20% (Fig.
4A). At 72 h after
reinstitution of ad libitum feeding of calorie-restricted mice,
the amount of DNA in the lungs was significantly greater than in
calorie-restricted mice but not different from lungs of mice that had
never been calorie restricted (Fig. 4A). The fall in DNA was
at least partly due to apoptosis, as evidenced by the higher
percentage of fragmented DNA in homogenates of lungs of
calorie-restricted mice than in homogenates of lungs of fed mice (Fig.
4B) and by the 2.5-times-greater percentage of
TUNEL-positive (3) alveolar wall cells in
calorie-restricted than in fed mice (Fig. 4C). The
percentage of TUNEL-positive alveolar wall cells in fed mice is higher
than reported values (1, 15), and we do not have an
explanation for the difference. The higher percentage of cells
immunoreactive to PCNA, an index of cell replication (3),
in alveoli of calorie-restricted 72-h-refed mice compared with fed
144-h mice (Fig. 4D) shows that refeeding increased replication of
alveolar wall cells, consistent with the return of lung DNA to the
amount in mice that were never calorie restricted (Fig. 4A).
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DISCUSSION |
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Calorie restriction in rodents increases the distance between alveolar walls (Lm) and diminishes alveolar surface area (9, 12, 14, 24), features of human (34) and experimental (11) pulmonary emphysema that have been believed to represent a lung abnormality, eg., nutritional emphysema (9, 12, 14, 24); these authors did not suggest that the decrease of Lm during refeeding might reflect alveolar regeneration (14, 24). Furthermore, because Lm includes air space of alveoli and alveolar ducts, differences in Lm do not identify the site of the change, i.e., alveolus and/or alveolar duct, which is needed to determine whether alveolar regeneration occurred. To avoid the ambiguity of the Lm measurement, we distinguished alveoli from alveolar ducts by analysis of serial lung sections (18, 19). Therefore, because calorie restriction and calorie restriction followed by ad libitum refeeding did not alter lung volume (Fig. 1B) and because our estimates of the volume of individual alveoli are unbiased (5, 18, 19) and do not include the volume of alveolar ducts, our results unambiguously show that calorie restriction causes a loss of alveoli. In our experiments, the same considerations indicate that refeeding causes alveolar regeneration.
During food deprivation, which diminishes
O2 (21), the endogenous
program of alveolar destruction would eliminate the cost of maintaining
unneeded tissue, simultaneously producing substrate (33)
to maintain brain and muscle, thereby offering a survival advantage.
This, we believe, reflects the same principle as the shrinkage of long
bones of iguana during periods of food scarcity (37).
Furthermore, the evidence from the tragedy of the Warsaw Ghetto
suggests that starvation of humans causes loss of alveoli
(39), indicating that the calorie restriction-induced program of alveolar destruction is conserved in humans. If this is so,
it raises the possibility that, unrelated to calorie intake, it is
inappropriately triggered in COPD by signals not responsive to current
therapy, accounting for the inexorable loss of gas-exchange function in
this disease (27). Supporting this notion is the recent
evidence that, in human pulmonary emphysema, alveolar septal walls are
destroyed in an all-or-nothing manner (35, 36). This
finding is counter to the present paradigm of focal destruction in
septa by the very local release of proteases by neutrophils and
mononuclear cells but does not exclude the possibility that local
damage triggers a programmed all-or-nothing destruction of an entire
alveolar septum.
Understanding the molecular basis for the calorie-related programs of alveolar destruction and regeneration may result in much needed therapy to slow the inexorable loss of alveoli and induce their regeneration in COPD and other diseases associated with alveolar destruction.
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ACKNOWLEDGEMENTS |
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We thank Z. Opalka for technical assistance.
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FOOTNOTES |
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This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-37666, HL-20366, HL-59432, and HL-47413. G. D. Massaro and D. Massaro were Senior Fellows of the Lovelace Respiratory Research Institute (Albuquerque, NM) when this work was performed. D. Massaro is Cohen Professor, Georgetown University School of Medicine.
Present address of S. Radaeva: NIAA-Flow Lab, Rm. 19, National Institutes of Health, 12501 Washington Ave., Rockville, MD 20852.
Address for reprint requests and other correspondence: G. D. Massaro, Lung Biology Laboratory, Georgetown University School of Medicine, 3900 Reservoir Rd., NW, Washington, DC 20007-2197 (E-mail: massarog{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.
March 15, 2002;10.1152/ajplung.00035.2002
Received 23 January 2002; accepted in final form 7 March 2002.
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