Lung alveoli: endogenous programmed destruction and regeneration

Gloria De Carlo Massaro1, Svetlana Radaeva2, Linda Biadasz Clerch1, and Donald Massaro2

Lung Biology Laboratory, Departments of 1 Pediatrics and 2 Medicine, Georgetown University School of Medicine, Washington, DC 20007-2197


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mammalian alveoli, complex architectural and cellular units with dimensions that are linked to the organism's O2 consumption (VO2), are thought to be destroyed only by disease and not to spontaneously regenerate. Calorie restriction of adult mammals lowers VO2, and ad libitum refeeding returns VO2 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE LUNG'S ONLY KNOWN ESSENTIAL function is to provide sufficient gas-exchange (alveolar) surface to meet the organism's need for O2 uptake (VO2) 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 VO2 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 VO2 (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 VO2 (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 VO2 (21) and quantitatively examined their effect on lung gas-exchange units.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Body mass (A) and lung volume (B). Values are means ± of 3 animals in each group. F1, fed ad libitum for 15 days; CR, fed one-third of the average daily chow consumed during the immediately preceding 4-5 days for 15 days; F2, fed ad libitum for 36 days; CR-RF, calorie restricted for 15 days and then allowed chow ad libitum for 21 days. P < 0.05 vs. each other group.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Alveolar number (Na), average volume of individual alveoli (<A><AC>V</AC><AC>&cjs1171;</AC></A>a), and alveolar surface area (Sa). Values are means ± SE of 3 animals in each group. ○P < 0.05 vs. each other group.



View larger version (120K):
[in this window]
[in a new window]
 
Fig. 3.   Morphology of gas-exchange region in lungs fixed at a transpulmonary pressure of 20 cmH2O. A: animals fed ad libitum for 15 days. B: animals fed one-third of the daily chow consumed during the immediately preceding 4-5 days. C: animals fed ad libitum for 36 days. D: animals fed one-third of the daily chow consumed during the immediately preceding 4-5 days for 15 days and then refed for 21 days. Morphological difference between B and A, C, and D is clear and in accord with quantitative data in Fig. 2B. Scale bars, 50 µm.

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).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Lung DNA amount (A) and fragmentation (B), apoptosis (C), and cell division (D). A: mice were killed at time 0 (n = 18), after 24 h of calorie restriction (CR 24 h, n = 4), after 72 h of calorie restriction (CR 72 h, n = 13 mice), after 72 h of calorie restriction followed by 72 h of ad libitum refeeding (CR 72 h-RF 72 h, n = 10 mice), or after 144 h of ad libitum feeding (Fed 144 h, n = 9 mice). P < 0.008 vs. 0 h; ○P < 0.02 vs. CR 72 h and P > 0.05 vs. Fed 144 h. B: percentage of fragmented DNA was higher in CR 72 h mice than in lungs from mice that were allowed food ad libitum for 72 h (Fed 72 h). P < 0.05 between groups. C: fed and CR as in B (n = 4 mice/group). Apoptosis of alveolar wall cells was estimated using TdT-mediated dUTP nick end labeling (TUNEL) transfer assay in Fed 72 h and CR 72 h mice. P < 0.05 between groups. D: proliferating cell nuclear antigen (PCNA), a marker of dividing cells, was assessed in methanol-fixed cryosections of frozen lungs from Fed 144 h and CR 72 h-RF 72 h mice (n = 4/group). black-triangle P < 0.02 between groups.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 VO2 (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.


    ACKNOWLEDGEMENTS

We thank Z. Opalka for technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barazzone, C, Horowitz S, Donati YR, Rodrigues I, and Piguet P. Oxygen toxicity in mouse lung: pathways to cell death. Am J Respir Cell Mol Biol 19: 573-581, 1998[Abstract/Free Full Text].

2.   Blackburn, GL. Pasteur's quadrant and malnutrition. Nature 409 Suppl: 397-401, 2001[ISI][Medline].

3.   Bravo, R, and Macdonald-Bravo H. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol 105: 1549-1554, 1987[Abstract].

4.   Clerch, LB, Neithardt G, Spencer U, Melendez JA, Massaro GD, and Massaro D. Pertussis toxin treatment alters manganese superoxide dismutase activity in lung. Evidence for lung oxygen toxicity in air-breathing rats. J Clin Invest 93: 2482-2489, 1994[ISI][Medline].

5.   Cruz-Orive, LM. Particle number can be estimated using a disector of unknown thickness: the selector. J Microsc 145: 121-142, 1987[ISI][Medline].

7.   Francavilla, A, Ove P, Polimeno L, Coetzee M, Makowka L, Barone M, Van Thiel DH, and Starzl TE. Regulation of liver size and regeneration: importance in liver transplantation. Transplant Proc 20 Suppl1: 494-497, 1988.

8.   Gadek, JE, and Pacht ER. The protease-antiprotease balance within the human lung: implications for the pathogenesis of emphysema. Lung 168 Suppl: 552-564, 1990[ISI][Medline].

9.   Harkema, JR, Mauderly JL, Gregory RE, and Pickerell JA. A comparison of starvation and elastase models of emphysema in the rat. Am Rev Respir Dis 129: 584-591, 1984[ISI][Medline].

10.   Kam, I, Lynch S, Svanas G, Todo S, Polimeno L, Francavilla A, Penkrot RJ, Takaya S, Ericzon BG, Starzl TE, Evidence that host size determines liver size: studies in dogs receiving orthotopic liver transplants. Hepatology 7: 362-366, 1987[ISI][Medline].

11.   Kaplan, PD, Kuhn C, and Pierce JA. The induction of emphysema with elastase. I. The evolution of the lesion and the influence of serum. J Lab Clin Med 82: 349-356, 1973[ISI][Medline].

12.   Karlinsky, JB, Goldstein RH, Ojserkis B, and Snider GL. Lung mechanics and connective tissue levels in starvation-induced emphysema in hamsters. Am J Physiol Regul Integr Comp Physiol 251: R282-R288, 1986[Abstract/Free Full Text].

13.   Kawasaki, S, Makuuchi M, Ishizone S, Matsunami H, Terada M, and Kawarazaki H. Liver regeneration in recipients and donors after transplantation. Lancet 339: 580-581, 1992[ISI][Medline].

14.   Kerr, JS, Riley DJ, Lanza-Jacoby S, Berg RA, Spilker WC, Yu SY, and Edelman NH. Nutritional emphysema in the rat. Influence of protein depletion and impaired lung growth. Am Rev Respir Dis 131: 644-650, 1985[ISI][Medline].

15.   Kitamura, Y, Hashimoto S, Mizuta N, Kobayashi A, Kooguchi K, Fujiwara I, and Nakajima H. Fas/Fas L-dependent apoptosis of alveolar cells after lipopolysaccharide-induced lung injury in mice. Am J Respir Crit Care Med 163: 762-769, 2001[Abstract/Free Full Text].

16.   Kitchen, I. Letter. Trends Pharmacol Sci 8: 252-253, 1987[ISI].

17.   Liestol, K. "Robust" statistical methods. Scand J Clin Lab Invest 44: 177-181, 1984[ISI][Medline].

18.   Massaro, GD, and Massaro D. Formation of alveoli in rats: postnatal effect of prenatal dexamethasone. Am J Physiol Lung Cell Mol Physiol 263: L37-L41, 1992[Abstract/Free Full Text].

19.   Massaro, GD, and Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema. Nat Med 3: 675-677, 1997[ISI][Medline].

20.   McCance, I. The number of animals. News Physiol Sci 4: 172-176, 1989[Abstract/Free Full Text].

21.   Munch, IC, Markussen NH, and Oritsland NA. Resting oxygen consumption in rats during food restriction, starvation and refeeding. Acta Physiol Scand 148: 335-340, 1993[ISI][Medline].

22.   Murray, CJL, and Lopez AD. Evidence-based health policy---lessons from the Global Burden of Disease Study. Science 274: 740-743, 1996[Free Full Text].

23.   Pauwels, RA, Buist AS, Calverley PM, Jenkins CR, and Hurd SS. Global strategy for the diagnosis, management, and prevention of chronic obstructive lung disease (COLD) workshop summary. Am J Respir Crit Care Med 163: 1256-1276, 2001[Free Full Text].

24.   Sahebjami, H, and Wirman JA. Emphysema-like changes in lungs of starved rats. Am Rev Respir Dis 124: 619-624, 1981[ISI][Medline].

25.   Secor, SM, and Diamond J. A vertebrate model of extreme physiological regulation. Nature 395: 659-662, 1998[ISI][Medline].

26.   Scherle, WA. A simple method for volumetry of organs in quantitative stereology. Mikroscopie 26: 57-60, 1970.

27.   Sherrill, DL, Enright PL, Kaltenborn WT, and Lebowitz MD. Predictors of longitudinal change in diffusion capacity over 8 years. Am J Respir Crit Care Med 160: 1883-1887, 1999[Abstract/Free Full Text].

28.   Shimabukaro, M, Wang MY, Zhou YT, Newgard CB, and Unger RH. Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl-2 expression. Proc Natl Acad Sci USA 95: 2498-2502, 1998[Abstract/Free Full Text].

29.   Shott, S. Statistics for Health Care Professionals. Philadelphia, PA: Saunders, 1990, p. 229-268.

30.   Sinclair, JD. Multiple t-tests are appropriate in science. Trends Pharmacol Sci 9: 12-13, 1988[ISI][Medline].

31.   Subcommittee on Laboratory Animal Nutrition. Nutrient Requirements of Laboratory Animals (3rd ed.). Washington, DC: National Research Council, National Academy of Sciences, 1978, p. 47. (Publ. 10)

32.   Tenney, SM, and Remmers JE. Comparative quantitative morphology of the mammalian lung diffusing area. Nature 197: 54-56, 1963[ISI].

33.   Thet, LA, Delaney MD, Gregorio CA, and Massaro D. Protein metabolism by rat lung: influence of fasting, glucose, and insulin. J Appl Physiol 43: 463-467, 1977[Abstract/Free Full Text].

34.   Thurlbeck, WH. Internal surface area and other measurements in emphysema. Thorax 22: 483-496, 1967[ISI][Medline].

35.   Vlahovic, G, Russell ML, Mercer RR, and Crapo JD. Cellular and connective tissue changes in alveolar septal walls in emphysema. Am J Respir Crit Care Med 160: 2086-2092, 1999[Abstract/Free Full Text].

36.   Wiebe, BM, and Laursen H. Lung morphometry by unbiased methods in emphysema: bronchial and blood vessel volume, alveolar surface area and capillary length. APMIS 106: 651-656, 1998[ISI][Medline].

37.   Wikelski, M, and Thom C. Marine iguanas shrink to survive El Nino. Nature 403: 37-38, 2000[ISI][Medline].

38.   Winer, BJ. Statistical Principles in Experimental Design. New York: McGraw-Hill Kogakusha, 1971, p. 196-201.

39.   Winick, M. Hunger disease. In: Studies by the Jewish Physicians in the Warsaw Ghetto. New York: Wiley, 1979.


Am J Physiol Lung Cell Mol Physiol 283(2):L305-L309
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society