- Email: [email protected]
Retinoic acid enhances lung growth after pneumonectomy
Retinoic Acid Enhances Lung Growth After Pneumonectomy Aditya K. Kaza, MD, Irving L. Kron, MD, John A. Kern, MD, Stewart M. Long, MD, Steven M. Fiser, MD, Richard P. Nguyen, BS, Curtis G. Tribble, MD, and Victor E. Laubach, PhD Department of Surgery, Division of Thoracic and Cardiovascular Surgery, University of Virginia Health System, Charlottesville, Virginia
Background. We sought to identify the role of retinoic acid (RA) upon lung growth. RA has a role in perinatal lung development, and we hypothesized that exogenous RA would enhance postpneumonectomy compensatory lung growth. Methods. Utilizing the postpneumonectomy rat model, we studied the impact of RA upon contralateral lung growth. Adult Sprague-Dawley rats were divided into three groups. Group S underwent a sham left thoracotomy, group P underwent left pneumonectomy, and group R underwent left pneumonectomy with administration of exogenous RA (0.5 g/g/day intraperitoneally). We then quantitated right lung growth after 10 and 21 days. Lung weight and volume were expressed as a ratio to the final body weight (lung weight and volume indi-
ces, LWI and LVI). Epidermal growth factor receptor (EGFR) expression was quantitated using Western blot analysis. Cellular proliferation index (CPI) was determined using BrdU immunostaining. Results. LWI, LVI, CPI, and EGFR expression at 21 days were significantly higher in group R versus S and P. At the 10-day interval, both LWI and LVI were significantly higher in group R versus S and P. Conclusions. RA administration markedly enhances lung growth after pneumonectomy, as evidenced by increases in LWI, LVI, and CPI. Upregulation of EGFR expression was associated with these effects.
T
growth after pneumonectomy and that this correlated with an upregulation of the epidermal growth factor receptor [6]. With this background, we sought to understand the effects of RA in the process of lung growth. The rat postpneumonectomy lung growth model was used because of its reliability, reproducibility, and its establishment as a lung growth model. Various classic experiments have shown the rapid and restorative nature of lung growth after pneumonectomy [7–10]. Our laboratory has shown that adult lungs, when transplanted into immature recipients, exhibit hyperplastic growth [11]. This growth was believed to be due to the various humoral factors in the immature recipient. The present study, through the use of RA, seeks to test our overall hypothesis that adult regenerative lung growth can be augmented beyond that which occurs in response to pneumonectomy.
he role of retinoic acid (RA) in lung growth has been elucidated in various classic experiments. RA is a product of Vitamin A metabolism and has been shown to be essential for the normal development of lung. RA has been shown to be one of the important factors controlling fetal lung development [1]. Maternal Vitamin A stores have been shown to be essential for fetal pulmonary organogensis. Rats fed a diet deficient in Vitamin A had abnormal tracheal morphology [2]. This phenomenon was noted to be a reversible one; rats initially fed a Vitamin A-deficient diet had a reversal of the abnormal pulmonary morphology once Vitamin A was returned to their diet [3]. Massaro and Massaro [4] recently showed that treatment of newborn rats with RA influenced the postnatal formation of alveoli, and this is believed to be the first evidence of alveolar replication in the so-called terminally differentiated lungs. They also demonstrated that administration of RA caused a reversal in the pulmonary pathology of emphysematic rats [5]. Although the phenomenon of compensatory lung growth has been well described, little is known about the modulation of such growth. We have recently reported that the administration of epidermal growth factor enhances lung Presented at the Forty-seventh Annual Meeting of the Southern Thoracic Surgical Association, Marco Island, FL, Nov 9 –11, 2000. Address reprint requests to Dr Laubach, Department of Surgery, University of Virginia Health System, Lane Rd, MR4, Room 3111, Charlottesville, VA 22908-1359; e-mail: [email protected].
© 2001 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 2001;71:1645–50) © 2001 by The Society of Thoracic Surgeons
Material and Methods Animals Adult male Sprague-Dawley rats weighing approximately 400 grams each were utilized for all experiments. Animal acquisition was under the supervision of the Department of Comparative Medicine and a licensed veterinarian. Facilities for animal care are accredited by the American Association for Accreditation of Laboratory Animal Care. The facility is approved by the Institutional 0003-4975/01/$20.00 PII S0003-4975(01)02478-X
1646
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
Animal Care and Use Committee. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and “The Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Science and published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Operative Model Rats were divided into 3 groups (S, P, and R), and each group was subdivided into 2 groups based on postoperative study time (10 or 21 days). There were 8 animals in each subgroup to allow for adequate tissue samples for molecular and morphometric analysis. All animals were anesthetized with a combination of ketamine and xylazine injected intraperitoneally followed by endotracheal intubation with a 16F gauge catheter. They were then ventilated with room air using a pressure-regulated rodent ventilator (Kent Scientific, Litchfield, CT), placed in the right lateral decubitus position, and shaved and prepped in a sterile fashion. Animals in group S underwent a sham thoracotomy on the left side. Animals in group P underwent a left pneumonectomy. Animals in group R underwent a left pneumonectomy with the administration of exogenous retinoic acid (0.5 g/g/day intraperitoneally, a generous gift from Hoffman-La Roche, Nutley, NJ). After the sham left thoracotomy (group S), the chest was closed after an expiratory sigh using 3-0 silk suture and skin closed using surgical staples. Animals in groups P and R underwent a posterolateral thoracotomy; the left lung was freed from the inferior pulmonary ligament. The lung was then delivered into the surgical wound, the hilum tied with a 4-0 silk ligature, and the lung excised. The chest was closed as described above. Animals were allowed to recover from surgery and extubated after initiation of spontaneous respirations. The animals then received postoperative analgesia in the form of buprenorphine injected intramuscularly every 12 hours for the first 24 hours. The animals were allowed to feed ad libitum and maintained in a controlled environment. After the designated time interval, the animals were anesthetized, weighed, intubated via a tracheostomy, and exposure of the thoracic organs was obtained by a bilateral anterior sternothoracotomy. The animals were rapidly exsanguinated by vena caval division. The right lung in half the animals was removed, patted dry, weighed, snap frozen in liquid nitrogen, and stored at ⫺80°C for molecular analyses. The remaining animals received intratracheal instillation of 70% ethanol to a pressure of 20 cm H2O. The right lung was then removed with the fixative in place, ligated at the hilum and stored in 70% ethanol for 24 hours. Lung volume was obtained by volume displacement technique as described by Scherle [12]. The lung volumes (mL) and lung weights (g) were expressed as a ratio to the final body weights of the animals (g) to correct for the variability in animal sizes. The lung was then paraffin embedded and random sections obtained for morphometric analysis.
Ann Thorac Surg 2001;71:1645–50
Morphometry Lung sections were H & E stained and used for morphometric analysis. Lung morphometry was carried out using the point counting technique described by Gil [13] and the three-level sampling technique described by Davies [14] and Wandel and colleagues [15]. The volumes of the various respiratory regions were determined using a 42-point test reticule (lattice with 85 m grid lines) attached to a Nikon Eclipse E400 microscope. This technique is briefly described below. The first level of analysis, which is usually performed under gross inspection, was not performed due to the small size of the rat lung and the lack of accurate differentiation at the gross level. The second level of analysis was performed at 50⫻ magnification. The number of lattice points that fell on intra-acinar air space and their intervening tissue was designated as Pr. Points that corresponded with extraacinar airways and vessels less than 0.5 mm in diameter were ignored. Intra- and extra-acinar airspace refers to the areas in the lung that correspond to the space inside and outside of an alveolar unit, respectively. The volume of the respiratory region (Vvr) was calculated using the following equation:
Vvr ⫽ 共Pr/42兲ⴱ100. The third level of analysis was performed at 200⫻ magnification. The number of lattice points that overlap the respiratory airspaces was designated as Pra. The volume of the respiratory airspace (Vra) was calculated using the following equation:
Vra ⫽ 共pra/42兲ⴱ100. The next level of analysis was also performed at 200⫻ magnification. The number of test lines intercepting the airspace-epithelial interface was designated as Is. The alveolar surface density (Sv) was then determined using the following equation:
Sv ⫽ 2/dⴱ共Is/Pp兲 where d ⫽ length of the test grid line (85 m) and Pp ⫽ total number of test points on the lung parenchyma ⫽ 42. The relative values of the various respiratory compartments in the lung were then calculated. The total volume of respiratory region, a measure of the volume of the alveoli and the intervening tissue, was calculated as follows:
共TVvr兲 ⫽ VvrⴱVL. The total volume of the respiratory airspace, a measure of alveolar volume in the lung without the intervening tissue was calculated as follows:
共TVvra兲 ⫽ VvraⴱVvrⴱVL. (VL ⫽ total volume of the lung.)
Western Analysis Total lung protein (120 g), quantitated using the Bradford method [16], was fractionated on a 7.5% (w/v) sodium dodecyl sulfate polyacrylamide gel, and trans-
Ann Thorac Surg 2001;71:1645–50
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
1647
Fig 1. Initial and final total body weight (iTBW and fTBW) at 10- and 21-day intervals. *p ⫽ 0.007 for iTBW versus fTBW. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
ferred to nitrocellulose using an electrophoretic transfer cell (Bio-Rad, Hercules, CA). The blot was blocked and incubated with primary epidermal growth factor receptor (EGFR) antibody (1:300, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at room temperature, followed by washing with 50 mM Tris HCl pH 7.4, 150 mM NaCl, 0.1% Tween. The blot was then incubated for 1 hour with secondary antibody coupled to horseradish peroxidase and washed as before. Protein bands were visualized by chemiluminescence (ECL, Amersham, Arlington Heights, IL) and quantitated by computerized densitometry. Preliminary studies using A431 cell lysate, not shown, indicate that the bands on the Western blot co-migrate with the 170kDa EGFR in A431 cell lysate.
5-Bromo-2⬘-deoxyuridine (BrdU) Labeling and Detection BrdU, a thymidine analogue, is incorporated into DNA during the S phase (DNA synthesis) of the cell cycle. Cells that have incorporated BrdU can then be detected by immunohistochemistry. The percentage of stained cells can then be counted to yield the proliferation index. BrdU (50 mg/kg) was injected intraperitoneally 2 hours prior to lung harvest. For immunohistochemistry, the VectaStain ABC-AP kit (Vector Laboratories, Burlingame, CA) was utilized using anti-BrdU monoclonal antibody (1:100, Dako Corp, Carpinteria, CA). Slides were processed as instructed, counterstained with nuclear fast red, and evaluated using light microscopy. Proliferation indices were determined in peripheral lung tissue using the ratio of the number of labeled nuclei among 1,000 total counted nuclei. Endothelial cells and cells from large airways were excluded from this process. This technique allows us to calculate the percentage of alveolar cells (mostly type II pneumocytes) that exhibit cell division, thus helping determine if RA has a mitogenic effect on the lung tissue.
Statistical Methodology Measurements are reported as the mean ⫾ standard error of the mean (SEM). Two-way analysis of variance (ANOVA) and contrast analysis were used to determine if a difference exists between study groups. A p value of 0.05 or less is used to indicate significant differences. Bonferroni multiple comparison test was used when appropriate.
Results Changes in Total Body Weight The initial and final total body weight of the animals in the 3 groups at 10 and 21 days are illustrated in Figure 1. We did not notice significant difference between the initial body weight and the final body weight in any of the groups except the sham thoracotomy animals at 21 days (S21, p ⫽ 0.007).
Lung Volume Index (LVI) The results of the LVI are illustrated in Figure 2. Pneumonectomy alone significantly increased LVI at both 10 and 21 days ( p ⬍ 0.001) when compared with the sham group. At 10 days, the LVI was significantly higher in the RA-treated group when compared with the sham thoracotomy and untreated pneumonectomy groups (17.68 ⫾ 0.82 versus 10.93 ⫾ 0.46 and 14.82 ⫾ 0.68, p ⬍ 0.001). Similarly, at 21 days, the RA-treated group had a significantly higher LVI when compared with the sham thoracotomy and untreated pneumonectomy groups (25.27 ⫾ 0.66 versus 10.16 ⫾ 0.17 and 14.03 ⫾ 0.12, p ⬍ 0.001).
Lung Weight Index (LWI) The results of the LWI are illustrated in Figure 3. LWI was significantly increased in the pneumonectomy group at 21 days when compared with the sham group ( p ⬍ 0.001). At 10 days, the RA-treated group had a signifi-
1648
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
Fig 2. Lung volume index at 10- and 21-day intervals. @p ⬍ 0.001 versus S10; *p ⬍ 0.001 versus S10 and P10; &p ⬍ 0.001 versus S21; # p ⬍ 0.001 versus S21 and P21. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
cantly higher LWI when compared with the sham thoracotomy and untreated pneumonectomy groups (3.98 ⫾ 0.13 versus 2.81 ⫾ 0.07 and 3.09 ⫾ 0.11, p ⬍ 0.001). Similarly, at 21 days, the RA-treated group had a significantly higher LWI when compared with the sham thoracotomy and untreated pneumonectomy groups (4.44 ⫾ 0.03 versus 2.37 ⫾ 0.05 and 3.86 ⫾ 0.03, p ⬍ 0.001).
Cellular Proliferation Index (CPI) The lungs from animals at the 21-day interval were analyzed to determine the CPI, and the results are illustrated in Figure 4. Pneumonectomy caused a significant rise in CPI when compared with the sham group ( p ⬍ 0.001). Animals that received RA had a significantly higher CPI when compared with the sham thoracotomy and untreated pneumonectomy groups (5.33 ⫾ 0.57 versus 0.95 ⫾ 0.06 and 3.69 ⫾ 0.36, p ⬍ 0.001).
Ann Thorac Surg 2001;71:1645–50
Fig 4. Cellular proliferation index (% of dividing cells) at 21-day interval. #p ⬍ 0.001 versus S21; *p ⬍ 0.001 versus S21 and P21. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
when compared with the sham thoracotomy and untreated pneumonectomy groups (5.62 ⫾ 0.73 versus 2.79 ⫾ 0.62 and 3.36 ⫾ 0.22, p ⫽ 0.017).
Morphometric Analysis Morphometric analysis was used to determine the alveolar surface density (Sv), volume of respiratory regions (TVvr), and volume of respiratory airspaces (TVvra) in the lung. These results are shown in Table 1. At 10 days, the RA-treated group had a significantly lower Sv when compared with the sham and pneumonectomy groups. At 10 days, the treatment group had a significantly higher TVvr when compared with the other 2 groups, but TVvra in the treatment group was only higher than the sham thoracotomy group. At 21 days, there was no difference in Sv among the 3 groups, but both TVvr and TVvra were significantly higher in the RA-treated group when compared with the other 2 groups.
Epidermal Growth Factor Receptor (EGFR) Expression
Comment
Western analysis was used to measure the expression of EGFR, and the results are illustrated in Figure 5. At 10 days, the RA-treated group had a trend towards a higher expression of EGFR when compared with the sham thoracotomy and untreated pneumonectomy groups, however, this was not statistically significant (data not shown). At 21 days, the RA-treated group had a significant upregulation in EGFR
RA and its precursor Vitamin A are essential for pulmonary morphogenesis. Maternal Vitamin A deficiency has
Fig 3. Lung weight index at 10- and 21-day intervals. *p ⬍ 0.001 versus S10 and P10; @p ⬍ 0.001 versus S21; #p ⬍ 0.001 versus S21 and P21. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
Fig 5. Western blot analysis for the detection of epidermal growth factor receptor (EGFR) at 21 days. The Western blot is shown at top (EGFR, 170kDa). The computerized densitometry is shown below. *p ⫽ 0.017 versus S21 and P21. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
Ann Thorac Surg 2001;71:1645–50
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
Table 1. Morphometric Analysis for Sham, Pneumonectomy, and Pneumonectomy Group With Exogenous RA at 10- and 21-day Intervals Variable S10 P10 R10 S21 P21 R21
Sv
TVvr
TVvra
220.9 ⫾ 13.3 222.1 ⫾ 9.7 171.2 ⫾ 9.2a 194.8 ⫾ 7.0 200.6 ⫾ 5.5 181.2 ⫾ 7.1
4.28 ⫾ 0.09 5.36 ⫾ 0.22 5.98 ⫾ 0.16b 4.08 ⫾ 0.10 5.74 ⫾ 0.09 9.76 ⫾ 0.26d
2.39 ⫾ 0.15 3.28 ⫾ 0.19 3.01 ⫾ 0.22c 2.26 ⫾ 0.16 3.28 ⫾ 0.13 5.18 ⫾ 0.22e
b c p ⫽ 0.007 vs S10 and P10. p ⬍ 0.001 vs S10 and P10. p ⫽ 0.003 vs d e p ⬍ 0.001 vs S21 and P21. p ⬍ 0.001 vs S21 and P21. S10.
a
P ⫽ pneumonectomy; R ⫽ pneumonectomy group with exogenous RA; RA ⫽ retinoic acid; S ⫽ sham; Sv ⫽ alveolar surface density (alveoli/cm); TVvr ⫽ total volume of respiratory region (mL); TVvra ⫽ total volume of respiratory airspace (mL).
been shown to result in improper fetal lung development and branching [2], and retinoids have been implicated in the development of the tracheal and bronchopulmonary structures in the lung. RA treatment stimulates type II pneumocyte proliferation in cell cultures [17], which is similar to the mitogenic response that we observed in this in vivo model. The role of RA in lung development, however, is not limited to the antenatal period, as it has been shown to induce alveolar development in the postnatal period [4]. In this study, we wanted to determine the role of RA in postpneumonectomy compensatory lung growth. Our results indicate that the administration of exogenous RA enhances contralateral lung growth after pneumonectomy beyond what occurs normally. We studied the growth response at two time points, 10 days and 21 days. The earlier time point was chosen to study molecular mediators that might be upregulated during the growth period. The later time point was chosen to study morphometric and cellular responses at the conclusion of lung growth, since it has been shown that postpneumonectomy lung growth reaches a plateau between 10 and 21 days [6]. At both time points, we noted an increase in the LWI and LVI in the RA-treated group when compared to both the sham thoracotomy group and the untreated pneumonectomy group. CPI was elevated in the RA-treated group when compared with the other 2 groups at 21 days. This growth response was associated with an upregulation of EGFR expression at the 21-day interval in the RA-treated rats. This association between RA and EGFR has been elucidated in various studies. One such study, by Schuger and colleagues [18], showed that an interaction between EGFR and RA was responsible for the effects of RA on lung development. We have recently shown that the administration of epidermal growth factor augments lung growth after pneumonectomy due to upregulation of its receptors by an auto-regulatory process [6]. Similarly, we now show that the effect of RA on postpneumonectomy lung growth is associated with the EGFR signaling pathway. Morphometric analysis revealed that the administration of RA enhances the volume of respiratory region and respiratory airways at both 10 and 21 day intervals. The
1649
alveolar surface density, however, is lower in the RAtreated group at 10 days, and returns to normal levels at 21 days. We can postulate that RA enhances lung volume in the early time point by alveolar hypertrophy, and that at the later time point the hypertrophic response is replaced by alveolar hyperplasia, which in turn restores the alveolar surface density. Vitamin A pretreatment has been shown to lower the incidence and severity of nitrofen-induced congenital diaphragmatic hernia secondary to an enhancement of lung growth and maturation [19], which shows the efficacy of RA in the treatment of lung injury. Our study provides a novel means of modulating postpneumonectomy compensatory lung growth. A better understanding of the various key modulators of lung growth has enormous clinical application.
The authors express their appreciation to Ms Kimberly Shockey, Mr Anthony Herring, Ms Sheila Hammond, and Dr Paul Davies for their invaluable technical assistance. This research was supported by National Institutes of Health grants RO1 HL48242 and T32 HL07849, and by NICHD/NIH through cooperative agreement U54 HD28934.
References 1. Masuyama H, Hiramatsu Y, Kudo T. Effect of retinoids on fetal lung development in the rat. Biol Neonate 1995;67: 264–73. 2. Wolbach SB, Howe PR. Tissue changes following deprivation of fat-soluble A vitamin. J Exp Med 1925;42:753–77. 3. Wolbach SB, Howe PR. Epithelial repair in recovery from vitamin A deficiency. J Exp Med 1933;57:511–26. 4. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats [see comments]. Am J Physiol 1996;270:L305–10. 5. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats [see comments; published erratum appears in Nat Med 1997;3:805]. Nat Med 1997;3:675–7. 6. Kaza AK, Laubach VE, Kern JA, et al. Epidermal growth factor augments post-pneumonectomy lung growth. J Thorac Cardiovasc Surg 2000;120:916–22. 7. Brody JS. Time course of and stimuli to compensatory growth of the lung after pneumonectomy. J Clin Invest 1975;56:897–904. 8. Buhain WJ, Brody JS. Compensatory growth of the lung following pneumonectomy. J Appl Physiol 1973;35:898 –902. 9. Cagle PT, Thurlbeck WM. Postpneumonectomy compensatory lung growth. Am Rev Respir Dis 1988;138:1314–26. 10. Rannels DE, Rannels SR. Compensatory growth of the lung following partial pneumonectomy. Exp Lung Res 1988;14: 157– 82. 11. Binns OA, DeLima NF, Buchanan SA, et al. Mature pulmonary lobar transplants grow in an immature environment. J Thorac Cardiovasc Surg 1997;114:186–94. 12. Scherle W. A simple method for volumetry of organs in quantitative stereology. Mikroskopie 1970;26:57– 60. 13. Gil J. Models of lung disease. New York: Marcel Dekker, 1990. 14. Davies P. Morphologic and morphometric techniques for the
1650
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
detection of drug- and toxin-induced changes in lung. Pharmacol Ther 1991;50:321–36. 15. Wandel G, Berger LC, Burri PH. Morphometric analysis of adult rat lung after bilobectomy. Am Rev Respir Dis 1983; 128:968–72. 16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248–54. 17. Nabeyrat E, Besnard V, Corroyer S, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithe-
Ann Thorac Surg 2001;71:1645–50
lial cells: relation with the IGF system. Am J Physiol 1998; 275:L71–9. 18. Schuger L, Varani J, Mitra R, Jr, Gilbride K. Retinoic acid stimulates mouse lung development by a mechanism involving epithelial-mesenchymal interaction and regulation of epidermal growth factor receptors. Dev Biol 1993;159: 462–73. 19. Thebaud B, Tibboel D, Rambaud C, et al. Vitamin A decreases the incidence and severity of nitrofen-induced congenital diaphragmatic hernia in rats. Am J Physiol 1999;277: L423–9.
DISCUSSION DR JOHN R. ROBERTS (Nashville, TN): I enjoyed your study very much. I wonder if you had a control study from your pneumonectomy group at a later date? What I am getting at is, are you postulating that the retinoic acid gets you to a baseline improvement in lung growth sooner, or that it gets you lung growth that you would not have otherwise?
DR KAZA: Thank you for the question. It has been shown in traditional studies that postpneumonectomy compensatory lung growth reaches a peak in the 2nd and 3rd week in rats. So that is basically the historical cohort that serves as a control for this experiment. Based on these experimental findings, we believe that retinoic acid enhances postpneumonectomy lung growth beyond that noted in untreated pneumonectomy animals.
INVITED COMMENTARY This study poses an interesting question: Can the mature lung residual after pneumonectomy be induced to “grow” in a way that yields more functional lung parenchyma? The authors assess the efficacy of retinoic acid (RA), a Vitamin A metabolite, to stimulate parenchymal regeneration after pneumonectomy in a rodent model. In this model, the residual lung hypertrophies during the first 3 weeks after left pneumonectomy, yielding a 60% increase in weight of the right lung. Here the authors demonstrate a striking, rapid increase in lung size, weight, and respiratory airspace volume in animals receiving RA compared to sham thoracotomy animals. Pneumonectomized animals exhibit intermediate increases in lung volumes, and in expression of epidermal growth factor receptor protein. The RA effect is similar to what they have seen previously in this model using epidermal growth factor. The result would be much more interesting if accomplished in more mature animals, or more importantly in an animal model where mesenchymal growth does not continue for the lifetime of the animal. Young adult rats are still in a fairly steep growth phase, and lung growth in this system would not be expected to faithfully model any biologically important phenomenon relevant to adult humans. Further, since rats grow continuously throughout their life span, the possible clinical relevance of any
© 2001 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
model using this species (or standard farm pigs, which behave similarly) is questionable. These issues impose severe constraints on the value of this model, and on the potential for clinical application of the authors’ findings. It is also of concern that RA treatment tended to be associated with persistent inhibition of weight gain, raising toxicity issues. Nevertheless, proliferation of type II pneumocytes in the setting of a return of alveolar surface density to normal suggests that RA triggered an impressive acceleration in compensatory lung parenchymal growth. A pharmacologic approach to enhance lung growth after resection is biologically interesting. If this “new” lung represents functional tissue, as perhaps measured by diffusing capacity, and if this phenomenon can be duplicated in mature animals, this observation could become clinically important. Richard N. Pierson III, MD Department of Surgery Nashville VA Medical Center, and Department of Cardiac and Thoracic Surgery Vanderbilt University Medical Center Nashville, TN 37232-5734
0003-4975/01/$20.00 PII S0003-4975(01)02532-2
Background. We sought to identify the role of retinoic acid (RA) upon lung growth. RA has a role in perinatal lung development, and we hypothesized that exogenous RA would enhance postpneumonectomy compensatory lung growth. Methods. Utilizing the postpneumonectomy rat model, we studied the impact of RA upon contralateral lung growth. Adult Sprague-Dawley rats were divided into three groups. Group S underwent a sham left thoracotomy, group P underwent left pneumonectomy, and group R underwent left pneumonectomy with administration of exogenous RA (0.5 g/g/day intraperitoneally). We then quantitated right lung growth after 10 and 21 days. Lung weight and volume were expressed as a ratio to the final body weight (lung weight and volume indi-
ces, LWI and LVI). Epidermal growth factor receptor (EGFR) expression was quantitated using Western blot analysis. Cellular proliferation index (CPI) was determined using BrdU immunostaining. Results. LWI, LVI, CPI, and EGFR expression at 21 days were significantly higher in group R versus S and P. At the 10-day interval, both LWI and LVI were significantly higher in group R versus S and P. Conclusions. RA administration markedly enhances lung growth after pneumonectomy, as evidenced by increases in LWI, LVI, and CPI. Upregulation of EGFR expression was associated with these effects.
T
growth after pneumonectomy and that this correlated with an upregulation of the epidermal growth factor receptor [6]. With this background, we sought to understand the effects of RA in the process of lung growth. The rat postpneumonectomy lung growth model was used because of its reliability, reproducibility, and its establishment as a lung growth model. Various classic experiments have shown the rapid and restorative nature of lung growth after pneumonectomy [7–10]. Our laboratory has shown that adult lungs, when transplanted into immature recipients, exhibit hyperplastic growth [11]. This growth was believed to be due to the various humoral factors in the immature recipient. The present study, through the use of RA, seeks to test our overall hypothesis that adult regenerative lung growth can be augmented beyond that which occurs in response to pneumonectomy.
he role of retinoic acid (RA) in lung growth has been elucidated in various classic experiments. RA is a product of Vitamin A metabolism and has been shown to be essential for the normal development of lung. RA has been shown to be one of the important factors controlling fetal lung development [1]. Maternal Vitamin A stores have been shown to be essential for fetal pulmonary organogensis. Rats fed a diet deficient in Vitamin A had abnormal tracheal morphology [2]. This phenomenon was noted to be a reversible one; rats initially fed a Vitamin A-deficient diet had a reversal of the abnormal pulmonary morphology once Vitamin A was returned to their diet [3]. Massaro and Massaro [4] recently showed that treatment of newborn rats with RA influenced the postnatal formation of alveoli, and this is believed to be the first evidence of alveolar replication in the so-called terminally differentiated lungs. They also demonstrated that administration of RA caused a reversal in the pulmonary pathology of emphysematic rats [5]. Although the phenomenon of compensatory lung growth has been well described, little is known about the modulation of such growth. We have recently reported that the administration of epidermal growth factor enhances lung Presented at the Forty-seventh Annual Meeting of the Southern Thoracic Surgical Association, Marco Island, FL, Nov 9 –11, 2000. Address reprint requests to Dr Laubach, Department of Surgery, University of Virginia Health System, Lane Rd, MR4, Room 3111, Charlottesville, VA 22908-1359; e-mail: [email protected].
© 2001 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 2001;71:1645–50) © 2001 by The Society of Thoracic Surgeons
Material and Methods Animals Adult male Sprague-Dawley rats weighing approximately 400 grams each were utilized for all experiments. Animal acquisition was under the supervision of the Department of Comparative Medicine and a licensed veterinarian. Facilities for animal care are accredited by the American Association for Accreditation of Laboratory Animal Care. The facility is approved by the Institutional 0003-4975/01/$20.00 PII S0003-4975(01)02478-X
1646
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
Animal Care and Use Committee. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and “The Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Science and published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Operative Model Rats were divided into 3 groups (S, P, and R), and each group was subdivided into 2 groups based on postoperative study time (10 or 21 days). There were 8 animals in each subgroup to allow for adequate tissue samples for molecular and morphometric analysis. All animals were anesthetized with a combination of ketamine and xylazine injected intraperitoneally followed by endotracheal intubation with a 16F gauge catheter. They were then ventilated with room air using a pressure-regulated rodent ventilator (Kent Scientific, Litchfield, CT), placed in the right lateral decubitus position, and shaved and prepped in a sterile fashion. Animals in group S underwent a sham thoracotomy on the left side. Animals in group P underwent a left pneumonectomy. Animals in group R underwent a left pneumonectomy with the administration of exogenous retinoic acid (0.5 g/g/day intraperitoneally, a generous gift from Hoffman-La Roche, Nutley, NJ). After the sham left thoracotomy (group S), the chest was closed after an expiratory sigh using 3-0 silk suture and skin closed using surgical staples. Animals in groups P and R underwent a posterolateral thoracotomy; the left lung was freed from the inferior pulmonary ligament. The lung was then delivered into the surgical wound, the hilum tied with a 4-0 silk ligature, and the lung excised. The chest was closed as described above. Animals were allowed to recover from surgery and extubated after initiation of spontaneous respirations. The animals then received postoperative analgesia in the form of buprenorphine injected intramuscularly every 12 hours for the first 24 hours. The animals were allowed to feed ad libitum and maintained in a controlled environment. After the designated time interval, the animals were anesthetized, weighed, intubated via a tracheostomy, and exposure of the thoracic organs was obtained by a bilateral anterior sternothoracotomy. The animals were rapidly exsanguinated by vena caval division. The right lung in half the animals was removed, patted dry, weighed, snap frozen in liquid nitrogen, and stored at ⫺80°C for molecular analyses. The remaining animals received intratracheal instillation of 70% ethanol to a pressure of 20 cm H2O. The right lung was then removed with the fixative in place, ligated at the hilum and stored in 70% ethanol for 24 hours. Lung volume was obtained by volume displacement technique as described by Scherle [12]. The lung volumes (mL) and lung weights (g) were expressed as a ratio to the final body weights of the animals (g) to correct for the variability in animal sizes. The lung was then paraffin embedded and random sections obtained for morphometric analysis.
Ann Thorac Surg 2001;71:1645–50
Morphometry Lung sections were H & E stained and used for morphometric analysis. Lung morphometry was carried out using the point counting technique described by Gil [13] and the three-level sampling technique described by Davies [14] and Wandel and colleagues [15]. The volumes of the various respiratory regions were determined using a 42-point test reticule (lattice with 85 m grid lines) attached to a Nikon Eclipse E400 microscope. This technique is briefly described below. The first level of analysis, which is usually performed under gross inspection, was not performed due to the small size of the rat lung and the lack of accurate differentiation at the gross level. The second level of analysis was performed at 50⫻ magnification. The number of lattice points that fell on intra-acinar air space and their intervening tissue was designated as Pr. Points that corresponded with extraacinar airways and vessels less than 0.5 mm in diameter were ignored. Intra- and extra-acinar airspace refers to the areas in the lung that correspond to the space inside and outside of an alveolar unit, respectively. The volume of the respiratory region (Vvr) was calculated using the following equation:
Vvr ⫽ 共Pr/42兲ⴱ100. The third level of analysis was performed at 200⫻ magnification. The number of lattice points that overlap the respiratory airspaces was designated as Pra. The volume of the respiratory airspace (Vra) was calculated using the following equation:
Vra ⫽ 共pra/42兲ⴱ100. The next level of analysis was also performed at 200⫻ magnification. The number of test lines intercepting the airspace-epithelial interface was designated as Is. The alveolar surface density (Sv) was then determined using the following equation:
Sv ⫽ 2/dⴱ共Is/Pp兲 where d ⫽ length of the test grid line (85 m) and Pp ⫽ total number of test points on the lung parenchyma ⫽ 42. The relative values of the various respiratory compartments in the lung were then calculated. The total volume of respiratory region, a measure of the volume of the alveoli and the intervening tissue, was calculated as follows:
共TVvr兲 ⫽ VvrⴱVL. The total volume of the respiratory airspace, a measure of alveolar volume in the lung without the intervening tissue was calculated as follows:
共TVvra兲 ⫽ VvraⴱVvrⴱVL. (VL ⫽ total volume of the lung.)
Western Analysis Total lung protein (120 g), quantitated using the Bradford method [16], was fractionated on a 7.5% (w/v) sodium dodecyl sulfate polyacrylamide gel, and trans-
Ann Thorac Surg 2001;71:1645–50
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
1647
Fig 1. Initial and final total body weight (iTBW and fTBW) at 10- and 21-day intervals. *p ⫽ 0.007 for iTBW versus fTBW. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
ferred to nitrocellulose using an electrophoretic transfer cell (Bio-Rad, Hercules, CA). The blot was blocked and incubated with primary epidermal growth factor receptor (EGFR) antibody (1:300, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at room temperature, followed by washing with 50 mM Tris HCl pH 7.4, 150 mM NaCl, 0.1% Tween. The blot was then incubated for 1 hour with secondary antibody coupled to horseradish peroxidase and washed as before. Protein bands were visualized by chemiluminescence (ECL, Amersham, Arlington Heights, IL) and quantitated by computerized densitometry. Preliminary studies using A431 cell lysate, not shown, indicate that the bands on the Western blot co-migrate with the 170kDa EGFR in A431 cell lysate.
5-Bromo-2⬘-deoxyuridine (BrdU) Labeling and Detection BrdU, a thymidine analogue, is incorporated into DNA during the S phase (DNA synthesis) of the cell cycle. Cells that have incorporated BrdU can then be detected by immunohistochemistry. The percentage of stained cells can then be counted to yield the proliferation index. BrdU (50 mg/kg) was injected intraperitoneally 2 hours prior to lung harvest. For immunohistochemistry, the VectaStain ABC-AP kit (Vector Laboratories, Burlingame, CA) was utilized using anti-BrdU monoclonal antibody (1:100, Dako Corp, Carpinteria, CA). Slides were processed as instructed, counterstained with nuclear fast red, and evaluated using light microscopy. Proliferation indices were determined in peripheral lung tissue using the ratio of the number of labeled nuclei among 1,000 total counted nuclei. Endothelial cells and cells from large airways were excluded from this process. This technique allows us to calculate the percentage of alveolar cells (mostly type II pneumocytes) that exhibit cell division, thus helping determine if RA has a mitogenic effect on the lung tissue.
Statistical Methodology Measurements are reported as the mean ⫾ standard error of the mean (SEM). Two-way analysis of variance (ANOVA) and contrast analysis were used to determine if a difference exists between study groups. A p value of 0.05 or less is used to indicate significant differences. Bonferroni multiple comparison test was used when appropriate.
Results Changes in Total Body Weight The initial and final total body weight of the animals in the 3 groups at 10 and 21 days are illustrated in Figure 1. We did not notice significant difference between the initial body weight and the final body weight in any of the groups except the sham thoracotomy animals at 21 days (S21, p ⫽ 0.007).
Lung Volume Index (LVI) The results of the LVI are illustrated in Figure 2. Pneumonectomy alone significantly increased LVI at both 10 and 21 days ( p ⬍ 0.001) when compared with the sham group. At 10 days, the LVI was significantly higher in the RA-treated group when compared with the sham thoracotomy and untreated pneumonectomy groups (17.68 ⫾ 0.82 versus 10.93 ⫾ 0.46 and 14.82 ⫾ 0.68, p ⬍ 0.001). Similarly, at 21 days, the RA-treated group had a significantly higher LVI when compared with the sham thoracotomy and untreated pneumonectomy groups (25.27 ⫾ 0.66 versus 10.16 ⫾ 0.17 and 14.03 ⫾ 0.12, p ⬍ 0.001).
Lung Weight Index (LWI) The results of the LWI are illustrated in Figure 3. LWI was significantly increased in the pneumonectomy group at 21 days when compared with the sham group ( p ⬍ 0.001). At 10 days, the RA-treated group had a signifi-
1648
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
Fig 2. Lung volume index at 10- and 21-day intervals. @p ⬍ 0.001 versus S10; *p ⬍ 0.001 versus S10 and P10; &p ⬍ 0.001 versus S21; # p ⬍ 0.001 versus S21 and P21. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
cantly higher LWI when compared with the sham thoracotomy and untreated pneumonectomy groups (3.98 ⫾ 0.13 versus 2.81 ⫾ 0.07 and 3.09 ⫾ 0.11, p ⬍ 0.001). Similarly, at 21 days, the RA-treated group had a significantly higher LWI when compared with the sham thoracotomy and untreated pneumonectomy groups (4.44 ⫾ 0.03 versus 2.37 ⫾ 0.05 and 3.86 ⫾ 0.03, p ⬍ 0.001).
Cellular Proliferation Index (CPI) The lungs from animals at the 21-day interval were analyzed to determine the CPI, and the results are illustrated in Figure 4. Pneumonectomy caused a significant rise in CPI when compared with the sham group ( p ⬍ 0.001). Animals that received RA had a significantly higher CPI when compared with the sham thoracotomy and untreated pneumonectomy groups (5.33 ⫾ 0.57 versus 0.95 ⫾ 0.06 and 3.69 ⫾ 0.36, p ⬍ 0.001).
Ann Thorac Surg 2001;71:1645–50
Fig 4. Cellular proliferation index (% of dividing cells) at 21-day interval. #p ⬍ 0.001 versus S21; *p ⬍ 0.001 versus S21 and P21. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
when compared with the sham thoracotomy and untreated pneumonectomy groups (5.62 ⫾ 0.73 versus 2.79 ⫾ 0.62 and 3.36 ⫾ 0.22, p ⫽ 0.017).
Morphometric Analysis Morphometric analysis was used to determine the alveolar surface density (Sv), volume of respiratory regions (TVvr), and volume of respiratory airspaces (TVvra) in the lung. These results are shown in Table 1. At 10 days, the RA-treated group had a significantly lower Sv when compared with the sham and pneumonectomy groups. At 10 days, the treatment group had a significantly higher TVvr when compared with the other 2 groups, but TVvra in the treatment group was only higher than the sham thoracotomy group. At 21 days, there was no difference in Sv among the 3 groups, but both TVvr and TVvra were significantly higher in the RA-treated group when compared with the other 2 groups.
Epidermal Growth Factor Receptor (EGFR) Expression
Comment
Western analysis was used to measure the expression of EGFR, and the results are illustrated in Figure 5. At 10 days, the RA-treated group had a trend towards a higher expression of EGFR when compared with the sham thoracotomy and untreated pneumonectomy groups, however, this was not statistically significant (data not shown). At 21 days, the RA-treated group had a significant upregulation in EGFR
RA and its precursor Vitamin A are essential for pulmonary morphogenesis. Maternal Vitamin A deficiency has
Fig 3. Lung weight index at 10- and 21-day intervals. *p ⬍ 0.001 versus S10 and P10; @p ⬍ 0.001 versus S21; #p ⬍ 0.001 versus S21 and P21. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
Fig 5. Western blot analysis for the detection of epidermal growth factor receptor (EGFR) at 21 days. The Western blot is shown at top (EGFR, 170kDa). The computerized densitometry is shown below. *p ⫽ 0.017 versus S21 and P21. (P ⫽ pneumonectomy; R ⫽ pneumonectomy with administration of retinoic acid; S ⫽ sham thoracotomy.)
Ann Thorac Surg 2001;71:1645–50
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
Table 1. Morphometric Analysis for Sham, Pneumonectomy, and Pneumonectomy Group With Exogenous RA at 10- and 21-day Intervals Variable S10 P10 R10 S21 P21 R21
Sv
TVvr
TVvra
220.9 ⫾ 13.3 222.1 ⫾ 9.7 171.2 ⫾ 9.2a 194.8 ⫾ 7.0 200.6 ⫾ 5.5 181.2 ⫾ 7.1
4.28 ⫾ 0.09 5.36 ⫾ 0.22 5.98 ⫾ 0.16b 4.08 ⫾ 0.10 5.74 ⫾ 0.09 9.76 ⫾ 0.26d
2.39 ⫾ 0.15 3.28 ⫾ 0.19 3.01 ⫾ 0.22c 2.26 ⫾ 0.16 3.28 ⫾ 0.13 5.18 ⫾ 0.22e
b c p ⫽ 0.007 vs S10 and P10. p ⬍ 0.001 vs S10 and P10. p ⫽ 0.003 vs d e p ⬍ 0.001 vs S21 and P21. p ⬍ 0.001 vs S21 and P21. S10.
a
P ⫽ pneumonectomy; R ⫽ pneumonectomy group with exogenous RA; RA ⫽ retinoic acid; S ⫽ sham; Sv ⫽ alveolar surface density (alveoli/cm); TVvr ⫽ total volume of respiratory region (mL); TVvra ⫽ total volume of respiratory airspace (mL).
been shown to result in improper fetal lung development and branching [2], and retinoids have been implicated in the development of the tracheal and bronchopulmonary structures in the lung. RA treatment stimulates type II pneumocyte proliferation in cell cultures [17], which is similar to the mitogenic response that we observed in this in vivo model. The role of RA in lung development, however, is not limited to the antenatal period, as it has been shown to induce alveolar development in the postnatal period [4]. In this study, we wanted to determine the role of RA in postpneumonectomy compensatory lung growth. Our results indicate that the administration of exogenous RA enhances contralateral lung growth after pneumonectomy beyond what occurs normally. We studied the growth response at two time points, 10 days and 21 days. The earlier time point was chosen to study molecular mediators that might be upregulated during the growth period. The later time point was chosen to study morphometric and cellular responses at the conclusion of lung growth, since it has been shown that postpneumonectomy lung growth reaches a plateau between 10 and 21 days [6]. At both time points, we noted an increase in the LWI and LVI in the RA-treated group when compared to both the sham thoracotomy group and the untreated pneumonectomy group. CPI was elevated in the RA-treated group when compared with the other 2 groups at 21 days. This growth response was associated with an upregulation of EGFR expression at the 21-day interval in the RA-treated rats. This association between RA and EGFR has been elucidated in various studies. One such study, by Schuger and colleagues [18], showed that an interaction between EGFR and RA was responsible for the effects of RA on lung development. We have recently shown that the administration of epidermal growth factor augments lung growth after pneumonectomy due to upregulation of its receptors by an auto-regulatory process [6]. Similarly, we now show that the effect of RA on postpneumonectomy lung growth is associated with the EGFR signaling pathway. Morphometric analysis revealed that the administration of RA enhances the volume of respiratory region and respiratory airways at both 10 and 21 day intervals. The
1649
alveolar surface density, however, is lower in the RAtreated group at 10 days, and returns to normal levels at 21 days. We can postulate that RA enhances lung volume in the early time point by alveolar hypertrophy, and that at the later time point the hypertrophic response is replaced by alveolar hyperplasia, which in turn restores the alveolar surface density. Vitamin A pretreatment has been shown to lower the incidence and severity of nitrofen-induced congenital diaphragmatic hernia secondary to an enhancement of lung growth and maturation [19], which shows the efficacy of RA in the treatment of lung injury. Our study provides a novel means of modulating postpneumonectomy compensatory lung growth. A better understanding of the various key modulators of lung growth has enormous clinical application.
The authors express their appreciation to Ms Kimberly Shockey, Mr Anthony Herring, Ms Sheila Hammond, and Dr Paul Davies for their invaluable technical assistance. This research was supported by National Institutes of Health grants RO1 HL48242 and T32 HL07849, and by NICHD/NIH through cooperative agreement U54 HD28934.
References 1. Masuyama H, Hiramatsu Y, Kudo T. Effect of retinoids on fetal lung development in the rat. Biol Neonate 1995;67: 264–73. 2. Wolbach SB, Howe PR. Tissue changes following deprivation of fat-soluble A vitamin. J Exp Med 1925;42:753–77. 3. Wolbach SB, Howe PR. Epithelial repair in recovery from vitamin A deficiency. J Exp Med 1933;57:511–26. 4. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats [see comments]. Am J Physiol 1996;270:L305–10. 5. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats [see comments; published erratum appears in Nat Med 1997;3:805]. Nat Med 1997;3:675–7. 6. Kaza AK, Laubach VE, Kern JA, et al. Epidermal growth factor augments post-pneumonectomy lung growth. J Thorac Cardiovasc Surg 2000;120:916–22. 7. Brody JS. Time course of and stimuli to compensatory growth of the lung after pneumonectomy. J Clin Invest 1975;56:897–904. 8. Buhain WJ, Brody JS. Compensatory growth of the lung following pneumonectomy. J Appl Physiol 1973;35:898 –902. 9. Cagle PT, Thurlbeck WM. Postpneumonectomy compensatory lung growth. Am Rev Respir Dis 1988;138:1314–26. 10. Rannels DE, Rannels SR. Compensatory growth of the lung following partial pneumonectomy. Exp Lung Res 1988;14: 157– 82. 11. Binns OA, DeLima NF, Buchanan SA, et al. Mature pulmonary lobar transplants grow in an immature environment. J Thorac Cardiovasc Surg 1997;114:186–94. 12. Scherle W. A simple method for volumetry of organs in quantitative stereology. Mikroskopie 1970;26:57– 60. 13. Gil J. Models of lung disease. New York: Marcel Dekker, 1990. 14. Davies P. Morphologic and morphometric techniques for the
1650
KAZA ET AL RETINOIC ACID AND LUNG GROWTH
detection of drug- and toxin-induced changes in lung. Pharmacol Ther 1991;50:321–36. 15. Wandel G, Berger LC, Burri PH. Morphometric analysis of adult rat lung after bilobectomy. Am Rev Respir Dis 1983; 128:968–72. 16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248–54. 17. Nabeyrat E, Besnard V, Corroyer S, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithe-
Ann Thorac Surg 2001;71:1645–50
lial cells: relation with the IGF system. Am J Physiol 1998; 275:L71–9. 18. Schuger L, Varani J, Mitra R, Jr, Gilbride K. Retinoic acid stimulates mouse lung development by a mechanism involving epithelial-mesenchymal interaction and regulation of epidermal growth factor receptors. Dev Biol 1993;159: 462–73. 19. Thebaud B, Tibboel D, Rambaud C, et al. Vitamin A decreases the incidence and severity of nitrofen-induced congenital diaphragmatic hernia in rats. Am J Physiol 1999;277: L423–9.
DISCUSSION DR JOHN R. ROBERTS (Nashville, TN): I enjoyed your study very much. I wonder if you had a control study from your pneumonectomy group at a later date? What I am getting at is, are you postulating that the retinoic acid gets you to a baseline improvement in lung growth sooner, or that it gets you lung growth that you would not have otherwise?
DR KAZA: Thank you for the question. It has been shown in traditional studies that postpneumonectomy compensatory lung growth reaches a peak in the 2nd and 3rd week in rats. So that is basically the historical cohort that serves as a control for this experiment. Based on these experimental findings, we believe that retinoic acid enhances postpneumonectomy lung growth beyond that noted in untreated pneumonectomy animals.
INVITED COMMENTARY This study poses an interesting question: Can the mature lung residual after pneumonectomy be induced to “grow” in a way that yields more functional lung parenchyma? The authors assess the efficacy of retinoic acid (RA), a Vitamin A metabolite, to stimulate parenchymal regeneration after pneumonectomy in a rodent model. In this model, the residual lung hypertrophies during the first 3 weeks after left pneumonectomy, yielding a 60% increase in weight of the right lung. Here the authors demonstrate a striking, rapid increase in lung size, weight, and respiratory airspace volume in animals receiving RA compared to sham thoracotomy animals. Pneumonectomized animals exhibit intermediate increases in lung volumes, and in expression of epidermal growth factor receptor protein. The RA effect is similar to what they have seen previously in this model using epidermal growth factor. The result would be much more interesting if accomplished in more mature animals, or more importantly in an animal model where mesenchymal growth does not continue for the lifetime of the animal. Young adult rats are still in a fairly steep growth phase, and lung growth in this system would not be expected to faithfully model any biologically important phenomenon relevant to adult humans. Further, since rats grow continuously throughout their life span, the possible clinical relevance of any
© 2001 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
model using this species (or standard farm pigs, which behave similarly) is questionable. These issues impose severe constraints on the value of this model, and on the potential for clinical application of the authors’ findings. It is also of concern that RA treatment tended to be associated with persistent inhibition of weight gain, raising toxicity issues. Nevertheless, proliferation of type II pneumocytes in the setting of a return of alveolar surface density to normal suggests that RA triggered an impressive acceleration in compensatory lung parenchymal growth. A pharmacologic approach to enhance lung growth after resection is biologically interesting. If this “new” lung represents functional tissue, as perhaps measured by diffusing capacity, and if this phenomenon can be duplicated in mature animals, this observation could become clinically important. Richard N. Pierson III, MD Department of Surgery Nashville VA Medical Center, and Department of Cardiac and Thoracic Surgery Vanderbilt University Medical Center Nashville, TN 37232-5734
0003-4975/01/$20.00 PII S0003-4975(01)02532-2