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Ann Thorac Surg 2001;72:380-385
© 2001 The Society of Thoracic Surgeons

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Original article: general thoracic

Epidermal growth factor receptor up-regulation is associated with lung growth after lobectomy

^a Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health System, Charlottesville, Virginia, USA

Accepted for publication April 25, 2001.

Address reprint requests to Dr Laubach, Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health System, Charlottesville, VA 22908
e-mail: vel8n{at}virginia.edu

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. We hypothesized that compensatory lung growth after lobectomy is characterized by a combination of cellular hyperplasia and hypertrophy and that up-regulation of epidermal growth factor receptor (EGFR) is involved in these processes.

Methods. Age-matched mature pigs were divided into four groups. The control group (group C) did not have operation. Two groups underwent left upper lobectomy and were studied 2 weeks (group L2) or 3 months (group L3) later. The last group underwent a sham left thoracotomy, and the left lower lobe was harvested 2 weeks later for EGFR analysis. Left lower lobes were studied using wet weight, cell proliferation index through immunostaining for 5-bromo-2'-deoxyuridine, morphometry, and Western blot analysis for EGFR. Content of protein and DNA (deoxyribonucleic acid) in the lung tissue was also determined.

Results. Left lower lobe weights were elevated in both groups L2 and L3 compared with group C. We noted a significant rise in the proliferation index, with a concomitant increase in EGFR expression, in group L2 compared with group C. In group L3, there was an increase in the protein to DNA ratio compared with group C.

Conclusions. We conclude that compensatory lung growth after lobectomy comprises an early increase in the cell proliferation index (ie, cellular hyperplasia) and a late increase in the protein to DNA ratio (ie, cellular hypertrophy). The early proliferative phase is associated with EGFR up-regulation.

	Introduction

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Lung tissue has been shown to exhibit rapid and restorative growth after pneumonectomy, a finding that has been well demonstrated in experimental models of postpneumonectomy lung growth [1–3]. This phenomenon also occurs in humans, especially in children who have undergone either pneumonectomy or lobectomy [4, 5]. In many mammalian species, pneumonectomy or lobectomy results in rapid growth of the remaining lung. This growth, referred to as compensatory lung growth, leads to restoration of total lung volume, compliance, mass, DNA (deoxyribonucleic acid), protein, alveolar number, and normal lung cell populations [6].

The mechanisms that signal and regulate the process of compensatory lung growth are not completely understood. The process of compensatory lung growth was thought to occur by hyperinflation of alveoli. To gain a better understanding of the sequence of cellular events that occurs during this process, we investigated the cellular changes that prompt this growth. Various questions remain unanswered about the process of compensatory lung growth. At what time do cells proliferate? When do the cellular elements exhibit hypertrophy? Is there any specific growth factor receptor expression associated with this process? Morphometric analysis of this process would help researchers understand the status of the respiratory compartment in the lung as well as the alveolar surface density, and this, in turn, could help them draw conclusions about the status of alveoli during the compensatory growth process.

Using the porcine model, we compared left lower lobe (LLL) growth in control animals with growth in animals that underwent left upper lobectomy. We studied the compensatory lung growth response after left upper lobectomy, and using various molecular methods, we sought to explain the specific nature of this growth response.

	Material and methods

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Adult Hanford miniature pigs were used for all experiments. Animal acquisition was under the supervision of the Department of Comparative Medicine and a licensed veterinarian. 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" published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Operative model
The animals were divided into four groups. All animals used in the experiments were adult (age > 5 months) male swine. Those in the first group served as controls (group C) (n = 5; body weight, 38.2 ± 4.2 kg) and did not have operation. Animals in two groups underwent a left upper lobectomy and were allowed to survive for 2 weeks (group L2) (n = 5; initial body weight, 40.4 ± 3.5 kg, and final body weight, 41 ± 2.5 kg) and 3 months (group L3) (n = 5; initial body weight, 34 ± 5.8 kg, and final body weight, 54.4 ± 5.9 kg). Animals in the final group underwent a sham left thoracotomy, which included the division of the inferior pulmonary ligament, and were allowed to survive for 2 weeks (group S) (n = 3; initial body weight, 36.7 ± 3.1 kg, and final body weight, 37.7 ± 2.1 kg). The growth of the LLL was analyzed in groups C, L2, and L3. Growth factor receptor expression was studied in all four groups using Western blot analysis.

Animals in groups L2 and L3 underwent left upper lobectomy. A chest tube was placed in the thoracic cavity and was removed on postoperative day 1. Animals received buprenorphine hydrochloride postoperatively for analgesia. They were then allowed to feed ad libitum and were maintained in a controlled environment.

At the designated time, animals in groups C, L2, and L3 underwent LLL harvest. The lobes were patted dry, and lobar weights were obtained. A section of the LLL was removed, snap frozen in liquid nitrogen, and stored at -80°C for molecular analysis. Another piece of lung tissue was taken, weighed and subsequently dried in a warm incubator to obtain dry weight. The wet weight was then expressed as a ratio to the dry weight to obtain the wet to dry weight ratio. From a column height of 25 cm H₂O, 70% ethanol was instilled intrabronchially into the lungs. Samples of peripheral lung tissue were obtained for morphometric analysis and for determining the cell proliferation index.

In addition, the LLL was harvested from animals in group S 2 weeks after the sham thoracotomy. Molecular analysis of the tissue was performed to investigate the role of surgical stress and postoperative inflammation on growth factor receptor expression, and the results were compared with findings in groups C, L2, and L3.

Morphometric analysis
Approximately nine 1-cm³ tissue blocks were taken by a random sampling technique from peripheral lung and processed for analysis. Sections were stained with hematoxylin and eosin for morphometric analysis, which was performed in a blinded fashion in our laboratory. Lung morphometry was carried out using the point counting technique described by Gil [7] and the three-level sampling technique described by Davies [8] and Wandel and colleagues [9]. The volumes of the various respiratory regions were determined using a 42-point test reticule (lattice with grid lines 85 µm long) attached to a Nikon Eclipse E400 microscope. The technique is briefly described here. The first level of analysis was performed at 50x magnification. The number of lattice points that fell on intraacinar air spaces and the intervening tissue was designated as Pr. Points corresponding to extraacinar airways and vessels less than 0.5 mm in diameter were ignored. The volume of the respiratory region (Vvr), which represents the percent of unit lung tissue occupied by alveoli and interalveolar tissue, was calculated using the following equation:

The second level of analysis was performed at 200x magnification. The number of lattice points that overlapped the respiratory air spaces was designated as Pra. The volume of the respiratory air space (Vra), which represents the percent of unit lung tissue occupied by alveoli, was calculated using the following equation:

The next level of analysis was also performed at 200x magnification. The number of test lines intercepting the air space–epithelial interface was designated as Is. The alveolar surface density (Sv), which represents the number of alveoli per centimeter was determined using the following equation: where d = length of the test grid (85 µm) and Pp = the total number of test points on the lung parenchyma, or 42).

DNA and protein determinations
Isolation of protein and DNA from lung tissue was performed using TRIzol reagent (Life Technologies, Gaithersburg, MD). Briefly, lung tissue was homogenized in TRIzol reagent and centrifuged at 15,000 rpm. Total RNA (ribonucleic acid) was precipitated from the aqueous phase with isopropyl alcohol, washed with 70% ethanol, and dissolved in RNAse-free water. Total DNA was precipitated from the interphase and organic phase with ethanol. It was washed first in a solution containing 0.1 mol/L sodium citrate in 10% ethanol, then washed in 70% ethanol, and finally dissolved in 8 mmol/L NaOH. Protein was purified from the pellet. The DNA samples were assayed for quality and concentration by a 260:280 ratio in a spectrophotometer. Protein concentrations were measured using the Bradford method [10] with bovine serum albumin as the standard. The protein and DNA quantities per unit lung tissue were then expressed as a ratio.

5-Bromo-2'-deoxyuridine labeling and detection
5-Bromo-2'-deoxyuridine (BrdU), a thymidine analogue, is incorporated into DNA during the S phase (DNA synthesis) of the cell cycle, and cells that have incorporated BrdU can then be detected by immunohistochemistry. The percentage of stained cells can be calculated to yield the proliferation index. The Brdu (50 mg/kg) was injected intravenously 2 hours prior to lung harvest. For immunohistochemistry, the VECTASTAIN ABC-AP kit (Vector Laboratories, Burlingame, CA) was used with anti-BrdU monoclonal antibody (1:100 ratio) (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 per 1,000 total counted nuclei. Nuclei in the immediate vicinity of conducting airways and vasculature were excluded from the analysis. Results were expressed as the cell proliferation index, defined as the percentage of nuclei labeled with BrdU (ie, percentage of cells dividing).

Western blot analysis
Total lung protein (120 µg) was fractionated on a 7.5% (wt/vol) sodium dodecyl sulfate polyacrylamide gel and transferred to nitrocellulose using an electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA). The blot was blocked and incubated with primary epidermal growth factor receptor (EGFR) antibody (1:300 ratio) (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour to 3 hours at room temperature, followed by washing with 50 mmol/L Tris HCl, pH 7.4, 150 mmol/L NaCl, and 0.1% Tween. The blot was 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. The EGFR protein was confirmed by its known molecular weight of 170 kDa [11] and also by Western blot analysis of EGFR expression in A431 human carcinoma cells (data not shown).

Statistical methodology
Measurements are reported as the mean ± the standard deviation. The error bars in the figures represent the standard error of the mean. Analysis of variance was used to determine whether a difference existed between study groups. A p value of 0.05 or less indicated significant differences. Bonferroni’s multiple comparison test was used when appropriate.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
LLL weight
The LLL weights in the lobectomy groups at 2 weeks (196.9 ± 23.5 g) and 3 months (181.3 ± 44.5 g) were significantly increased compared with group C (121.6 ± 19.5 g) (p = 0.013) (Fig 1). To determine if this gain in lobar weight was due to edema, we calculated wet to dry weight ratios on the lobar tissue from the three groups and failed to see a difference (5.6 ± 0.3, 5.8 ± 0.6, and 5.9 ± 0.7 for groups C, L2, and L3, respectively). Thus, we concluded that the gain in lobar weight is indeed true growth.

View larger version (27K): [in this window] [in a new window]   Fig 1. Left lower lobe weight in grams. (C = control group; L2 = left upper lobectomy group studied at 2 weeks; L3 = left upper lobectomy group studied at 3 months; *p = 0.013 versus groups L2 and L3.)

View larger version (29K): [in this window] [in a new window]   Fig 2. Cell proliferation index (% of cells that are dividing). (C = control group; L2 = left upper lobectomy group studied at 2 weeks; L3 = left upper lobectomy group studied at 3 months; *p = 0.001 versus groups C and L3.)

View larger version (31K): [in this window] [in a new window]   Fig 3. Epidermal growth factor receptor (EGFR) expression in left lower lobe. (A) Representative Western blot and (B) densitometry findings. (C = control group; L2 = left upper lobectomy group studied at 2 weeks; L3 = left upper lobectomy group studied at 3 months; S = sham thoracotomy group studied at 2 weeks; *p < 0.001 versus groups C, S, and L3.)

View larger version (30K): [in this window] [in a new window]   Fig 4. Protein to DNA (deoxyribonucleic acid) ratio. (C = control group; L2 = left upper lobectomy group studied at 2 weeks; L3 = left upper lobectomy group studied at 3 months; *p = 0.038 versus group C.)

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

In our experiment, the compensatory process reached significance starting 2 weeks after lobectomy. This is illustrated by the significant increase in the LLL weight in group L2 compared with the weight in group C; this increase in lobar weight is sustained at the 3-month interval. The growth peak at 2 weeks coincides with a peak in the cell proliferation index. However, the increase in the proliferation index noted in the 2-week group returns to control levels at the 3-month interval; this could represent a return to baseline mitogenic activity in the lung tissue. The increase in the cell proliferation index represents a hyperplastic (pneumonocyte proliferation) response in the lung during the early phase of compensatory lung growth. The protein to DNA ratio, which is indicative of cell size, peaks at 3 months. This indicates that there is cellular hypertrophy, which restores cell size after the early proliferative stage. However, other researchers [12] failed to notice this increase in the protein to DNA ratio because their study of compensatory growth was limited to the early postoperative time. On the basis of these experimental data, we conclude the following: there is an initial phase of cell division and proliferation, followed by a period of cellular hypertrophy, which restores the newly divided cells in the lung to their normal size.

The rapid proliferative response noted in our experiment is similar to one reported by Brody [2] in a study of lung growth after pneumonectomy. The classic studies in compensatory lung growth identified the existence of a hyperplastic response. However, we believe that the finding of cellular hypertrophy in this process is unique. Morphometric analysis revealed that the relative size of the various respiratory compartments in the lung as well as the alveolar surface density remains constant. Alveolar surface density represents the number of alveoli per unit lung tissue. This information can be combined with the increase in lung tissue noted in the compensatory growth groups, and we can conclude that the total number of alveoli increases during the compensatory growth process starting at 2 weeks. The process of compensatory lung growth does not involve just alveolar distention as alluded to in classic studies but also alveolar proliferation and subsequent restoration of cellular size.

Epidermal growth factor (EGF) has been shown to play an important role in prenatal and postnatal lung development. Along with EGFR, EGF modulates epithelial maturation and regeneration, and the lung has been shown to harbor EGFRs on various cell types such as ciliated cells and alveolar cells. Raaberg and co-workers [13] demonstrated EGF expression in type II pneumonocytes from a few of days prior to birth and throughout life. Ruocco and associates [14] postulated that in addition to being mitogenic, EGF also functions in a paracrine fashion to help in epithelial maturation of the lung tissue.

We also examined the effect of surgical stress and postoperative inflammatory response on EGFR expression. We used group S for this analysis and failed to see any up-regulation of EGFR in the group compared with group C. Up-regulation of EGFR was noted during the process of compensatory lung growth in our experiment. This up-regulation was limited to the early interval; thus, it could potentially represent a rapid humoral response to lobectomy. In this study, there was a temporal correlation between the increase in the cell proliferation index and the EGFR up-regulation. However, additional studies need to be done to establish a causal relationship.

The up-regulation of EGFR could represent a novel avenue for the modulation of adult lung growth in various settings. We [15] have shown recently that the administration of exogenous EGF has a significant impact on lung growth after pneumonectomy. Identification of such molecular mediators in compensatory lung growth will aid in the development of potential therapies for lung injury and end-stage lung disease through the stimulation and control of healthy lung growth.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
This research is supported by National Institutes of Health grants RO1 HL48242 and T32 HL07849 and by the National Institute of Child Health and Human Development, National Institutes of Health, through cooperative agreement U54 HD28934. We thank Mr Anthony Herring, Ms Sheila Hammond, and Paul Davies, PhD, for their invaluable technical assistance.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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