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Published ahead of print on December 30, 2004, doi:10.1165/rcmb.2004-0325OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 185-191, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0325OC

Upregulation of Hypoxia-Induced Mitogenic Factor in Compensatory Lung Growth after Pneumonectomy

Dechun Li, Lucas G. Fernandez, Jeffrey Dodd-o, John Langer, Danming Wang and Victor E. Laubach

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; and Department of Surgery, University of Virginia Health System, Charlottesville, Virginia

Correspondence and requests for reprints should be addressed to Dechun Li, M.D., Ph.D., Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, 600 N. Wolfe St., Blalock 1404A, Baltimore, MD 21205. E-mail: dechunli{at}jhmi.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
After pneumonectomy, the remaining lung increases in size. This process is referred to as compensatory lung growth. Various pathways likely play important roles in this growth response. The molecular mechanisms involved in compensatory lung growth, however, remain poorly understood. Hypoxia-induced mitogenic factor (HIMF), also called FIZZ1 or RELM-{alpha}, possesses mitogenic, vasoconstrictive, angiogenic, and antiapoptotic effects. In this study, we examined the expression of HIMF in mouse lung after pneumonectomy to test the hypothesis that HIMF expression is upregulated during compensatory lung growth. Results showed that HIMF is upregulated from Day 1 after pneumonectomy and peaking at Day 7 in the lung. HIMF upregulation is temporally and spatially related to lung cell proliferation, as demonstrated by expression of proliferating cell nuclear antigen. Immunohistochemical staining and in situ hybridization showed that upregulated HIMF protein and mRNA are mainly distributed in airway epithelium, alveolar type II cells, and endothelial cells of the pulmonary vessels. Intratracheal instillation of recombinant HIMF resulted in widespread cell proliferation, including airway epithelium, alveolar type II cells, and cells in the alveolar septa. These results indicate a new role for HIMF in compensatory lung growth, which is that HIMF may act as a lung-specific growth factor and participate in lung regeneration after pneumonectomy.

Key Words: compensatory lung growth • hypoxia-induced mitogenic factor • mouse • pneumonectomy


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
After pneumonectomy in experimental animals, the remaining lung rapidly increases in size (1–4). This adaptive growth, referred to as compensatory lung growth, results in restoration of essentially normal lung cell populations, alveolar number, and total lung volume, compliance, mass, DNA, protein, alveolar and capillary surface areas, and lung function (5–9). Children and adolescents subjected to unilateral pneumonectomy also develop a varying degree of compensatory growth in the contralateral lung (10). The extent of compensatory growth achieved is reduced with increased age, but if lung tissue is removed sufficiently early in life, there is nearly complete restitution of the lung. Various pathways likely play important roles in this growth response, including changes in lung inflation, blood flow, hypoxia, and release of growth-promoting substances, such as growth factors, nitric oxide, retinoic acid, and adrenal hormones (11–15). The molecular mechanisms involved in compensatory lung growth, however, are not understood. Successful compensatory lung growth is likely to require the coordinated expression of various growth factors and their receptors, directed at the different cell types within the lung, acting through autocrine, endocrine, juxtacrine, or paracrine pathways (15–18). Despite extensive literature relating to experimental pneumonectomy, the mediators of compensatory lung growth in the remaining lung tissue have not been elucidated.

This study focused on the role of hypoxia-induced mitogenic factor (HIMF) in compensatory lung growth after pneumonectomy. From a mouse model of hypoxia-induced pulmonary hypertension, we previously found a highly upregulated gene that we named HIMF (19). HIMF has an identical amino acid sequence to a protein called FIZZ1, identified in a mouse lung inflammation model (20), and RELM-{alpha} in adipose tissue (21). However, the precise functions of HIMF are still largely unknown, especially in the lung. Studies from Stepann and coworkers (20) demonstrated that HIMF (FIZZ1) mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells, in nonneuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the walls of the large and small intestine. During allergic pulmonary inflammation induced by ovalbumin challenge, HIMF (FIZZ1) expression markedly increases in hypertrophic and hyperplastic bronchial epithelium and also appears in alveolar type II cells. Holcomb and colleagues (21) showed that HIMF (RELM-{alpha}) is expressed in adipose tissue, and may play an important role in metabolism and diabetes.

To date, there have been no studies related to the function of HIMF in compensatory lung growth, and its biological function and roles in the pulmonary system are still largely unknown. Previous studies from our laboratory demonstrated that recombinant HIMF has mitogenic, angiogenic, and vasoconstrictive effects that can: (1) induce pulmonary microvascular smooth muscle cell proliferation, (2) induce pulmonary vessel constriction (which is more potent than endothelin-1 and serotonin), (3) induce endothelial cell migration, and (4) promote endothelial cell tubule formation (19). In the developing lung, HIMF has been shown to be highly expressed in the perinatal period and also possesses an antiapoptotic function in cultured embryonic lungs (22). Because HIMF is a secreted and inducible protein in the lung, it is possible to speculate that HIMF may act in an autocrine or paracrine fashion in the lung. However, because the putative receptor for HIMF is still elusive, the exact mechanism and signaling pathways in HIMF-induced biological effects are still unidentified.

In the present study, we tested the hypothesis that HIMF expression is upregulated during the compensatory growth process of the lung. We found HIMF to be highly upregulated in compensatory lung growth, especially during the early hyperplastic period (1–7 d) after pneumonectomy. During that period, the remaining lung cells proliferate vigorously and restore the lost lung parenchyma. Moreover, intratracheal instillation of recombinant HIMF protein induced widespread cell proliferation in the lung, including airway epithelial cells, alveolar type II cells, and vascular endothelial and smooth muscle cells. These findings indicate that HIMF may be a lung-specific growth factor participating in lung cell proliferation and modulation of compensatory lung growth after pneumonectomy.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animal Model of Pneumonectomy
All animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally along with an injection of 0.5 ml saline to alleviate any blood volume loss. Left pneumonectomy and sham surgeries were performed as previously described (11). The sham surgery was essentially a left thoracotomy without removal of the lung to serve as a surgical control group. Each mouse was shaved along the left lateral chest followed by endotracheal intubation with a 24-gauge catheter. All animals were mature, age-matched (14–16 wk) male C57BL6 mice weighing ~ 25 g each (11). Mice were divided into two groups: sham controls (7 d after surgery, S7d, n = 6); and pneumonectomy. The pneumonectomy group was then subdivided into five subgroups on the basis of the postoperative survival times: immediate (Time 0), 1 d, 3 d, 5 d, 7 d, and 14 d (n = 8/group). Preliminary experiments have shown that there was no significant increase of HIMF protein in the sham-operated animal lungs compared with normal mouse lungs at all the designated time points. Thus we used only the Day 7 group of sham animal lungs in the Western blot as an example. All the experimental procedures were approved by the Animal Care and Use Committees of the Johns Hopkins University and the University of Virginia Health System.

Measurement of Total Lung Weight and Lung Weight Index
Mice were subjected to sham operation or left pneumonectomy and the animals were killed at the designated time points. Their lung/body weights were measured as total lung weight (left + right lung weight in sham control animals and right lung weight in the pneumonectomy animals) and lung weight index (LWI) as previously described (23, 24). After measuring body weight and a bilateral anterior sternothoractomy, the animals were rapidly exsanguinated by vena cava division. The total or right lungs were then blotted dry and measured for wet weight (25).

Immunohistochemical Staining for HIMF and Proliferating Cell Nuclear Antigen
Mouse lungs from different time points (Days 0, 1, 3, 7, and 14) after pneumonectomy and sham surgery were collected and fixed by intratracheal instillation under constant pressure (20 cm H2O) in 4% paraformaldehyde for 4 h and processed as paraffin sections. Following deparaffinization, incubation in 3% H2O2 to block endogenous peroxidase activity and three washes with phosphate-buffered saline, the sections were incubated for 1 h with anti-HIMF antiserum (19) or anti–proliferating cell nuclear antigen (PCNA, 1:1,000 dilution; Dako, Carpinteria, CA) followed by a 2-h incubation with goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP; Bio-Rad, Hercules, CA). DAB substrate kit (Dako) was used as the substrate to generate dark brown precipitate in the cells of the tissues (22). For the identification and colocalization of HIMF- and PCNA-positive cells, consecutive sections were stained separately with either anti-HIMF or anti-PCNA antibody. The sections were examined and images taken with a Sony color digital DXC-S500 camera (Sony Electronics, Oradell, NJ), and images were acquired using Image Pro-Express software (Media Cybernetics, Silver Spring, MD).

Western Blot for HIMF, PCNA, and ß-Actin in Lung Homogenates
Tissue collection, homogenization, and protein electrophoresis were performed as described previously (22). Protein (50 µg) from each sample was subjected to 4–20% precast polyacrylamide gel (Bio-Rad) electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). HIMF was detected with 1:1,000 dilution of the anti-HIMF antibody followed by 1:3,000 dilution of goat anti-rabbit HRP-labeled antibody (Bio-Rad). For PCNA (Dako) and ß-actin (Sigma, St. Louis, MO) detections, the primary antibody dilutions were 1:500 and 1:2,000, respectively, and were followed by a 1:3,000 dilution of either goat anti-rabbit (for PCNA) or goat anti-mouse (for ß-actin detection) HRP-labeled antibodies (Bio-Rad). Enhanced chemiluminescence substrate kit (Amersham Health, Arlington Heights, IL) was used for the chemiluminscent detection of the signals with autoradiography film (Amersham).

In Situ Hybridization for Localization of HIMF mRNA in the Postpneumonectomy Mouse Lung
For the detection and localization of HIMF mRNA in the lung, synthetic sense (5'-gactctctcttgcactagtgtca-3') and antisense (5'-ttgacactagtgcaagagagagtc-3') HIMF oligoprobes labeled with 6-carboxyfluorescein were used, respectively, in paraffin sections as reported previously (22). Briefly, following deparaffinization, the slides were treated with 1 µg/ml proteinase K (Roche, Indianapolis, IN) in 100 mM Tris-HCl, pH 8.0, and 50 mM EDTA for 10 min and fixed in 2% paraformaldehyde for 5 min. Sense and antisense probes (500 ng/ml) were added separately in the hybridization buffer (50% formamide, 4x SSC, 100 µg/ml salmon sperm DNA, and 1x Denhardt's solution). After hybridization at 50°C and washing in 0.2x SSC/0.1% SDS, the slides were examined under a Leitz Fluovert fluorescent microscope (Wild Leitz, Wetzlar, Germany) connected to a Sony color digital DXC-S500 camera (Sony Electronics, Oradell, NJ), and images were acquired using Image Pro-Express software (Media Cybernetics, Silver Spring, MD).

Northern Blot Analysis for HIMF mRNA
For the quantitation of HIMF gene transcripts in the lung, total RNA was isolated from the lungs at different time points (Day 1, 3, 7, 14, and from Day 3 sham control animals; n = 3 for each time point). Total RNA (20 µg) from each animal was denatured in loading buffer containing 3.2 M formaldehyde and then subjected to 1% agarose gel electrophoresis. The RNA was transferred to Hybound-N+ nylon membrane (Amersham) and hybridized with 32P-labeled HIMF cDNA probe. The ratio of 18 s and 28 s RNA from each animal lung was used as loading control.

Intratracheal Instillation of HIMF
Recombinant HIMF protein was produced and isolated as described previously (19). To examine potential proliferative effects of HIMF, recombinant HIMF protein was intratracheally instilled into unoperated mouse lungs (200 ng/animal in 40 µl saline) via a 21-gauge catheter. The catheter was positioned in the main trachea (2.5 cm from the front teeth). The vehicle controls were instilled with saline diluted FLAG peptide (200 ng/animal; Sigma) because the recombinant HIMF was FLAG-tagged at its N-terminus. Three and five days later, the mouse lungs were collected, fixed, and processed as paraffin sections for PCNA immunostaining as described above.

Statistical Analysis
Unless otherwise stated, all data are shown as mean ± SEM for each time point. Statistical significance (P < 0.05) was determined by an analysis of variance followed by assessment of differences using Duncan's multiple range test through SigmaStat 2.03 software (Jandel Scientific, Erkrath, Germany).


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Pneumonectomy
As expected, there was significant reduction of the lung tissues in the pneumonectomy animals compared with the sham control animals. Figure 1A shows that there was a significant amount of lung tissue removed by pneumonectomy (P < 0.001, sham 7 d total lung weight compared with Day 0 and Day 1 right lung after pneumonectomy). However, left pneumonectomy resulted in a significant increase in the right lung weight beginning at Day 3 after pneumonectomy. By Day 14, the right lung weight had reached the same level as that in sham control animals. Figure 1B shows that after pneumonectomy, LWI started to significantly increase at Day 3, and reached high levels after 5–14 d (a 33.4% increase of LWI at Day 14 after surgery).



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  		Figure 1. (A) Total lung weight (right + left lungs) in sham control animals 7 d after surgery (S7 d, n = 6) and right lung weight at different days after pneumonectomy. There is significant loss of lung tissue in pneumonectomy animals (P < 0.001, compared with sham 7 d, Day 0, and Day 1 after pneumonectomy). However, at Day 14 after pneumonectomy, the right lung weight has reached the same levels as in the sham controls. (B) Total (right + left) lung (sham 7 d) and right lung weight index (LWI) after left pneumonectomy. The LWI was increased at Day 3 (*P < 0.05) after surgery and reached high levels at Days 5–14 (**P < 0.001, compared with Day 0; one-way analysis of variance, Student-Newman-Keuls method). By Day 14 after pneumonectomy, the LWI does not differ from the sham animals (n = 8 for each time point for the pneumonectomy groups).

 
Western Blot Analysis of HIMF, PCNA, and ß-Actin
Although we previously reported a prominent role of HIMF in adult and developing mouse lung (19, 22), it was not known if HIMF is expressed in the regenerating lung after pneumonectomy. To examine HIMF expression after pneumonectomy, lung protein homogenates were subjected to polyacrylamide gel electrophoresis and subsequently detected by Western analysis using the respective antibodies to HIMF, PCNA, and ß-actin for loading control (Figure 2). As predicted, HIMF protein expression was very low in Day 0 lungs, and sham surgery did not alter this expression. However, pneumonectomy induced significant upregulation of HIMF protein expression. HIMF first emerged at Day 1 with a slight but significant increase (P < 0.05 compared with Day 0 and sham), and gradually peaked to the highest levels at Day 7 (Figures 2A and 2B). At 14 d after pneumonectomy, HIMF level was reduced but was still higher than Day 0 and sham groups. Most interestingly, HIMF expression paralleled with upregulation of PCNA expression in the regenerating mouse lung, which also peaked at Day 7 after pneumonectomy (Figures 2A and 2C).



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  		Figure 2. (A) Representative Western blot analyses of hypoxia-induced mitogenic factor (HIMF), proliferating cell nuclear antigen (PCNA), and ß-actin protein expression in the right lung at different times after sham and left pneumonectomy. There were significant increases of HIMF and PCNA protein expression starting at Day 1 after the surgery and peaking at Day 7. Inclusion of the sham group at Day 7 documents that surgery alone does not induce these proteins. (B and C) Quantitation of HIMF and PCNA protein expression, respectively, by Western analysis (n = 4 for each time point). HIMF expression increased at Day (D) 1 and peaked at D7 (*P < 0.05) compared with Day 0 and sham controls. PCNA increased from D3 and peaked at D7 (*P < 0.05 compared with D0, D1, and sham control groups).

 
HIMF and PCNA Immunohistochemistry
To elucidate the localization of cells expressing HIMF within the lung during compensatory lung growth, immunohistochemical staining of HIMF and PCNA was performed. Immunohistochemical staining of HIMF confirmed the induction of HIMF in the lung after pneumonectomy (Figure 3). Consistent with previous reports of HIMF protein localization in the lung (19, 21), the results showed that HIMF protein was restricted to the airway epithelial cells, alveolar type II cells, and endothelial cells of the pulmonary vessels (Figure 3). Most interestingly, the number of PCNA-positive cells was also enhanced after pneumonectomy, and these cells were also largely airway epithelial cells, alveolar type II cells, and endothelial cells of the pulmonary vessels (Figure 4), indicating the overlapping of HIMF and PCNA temporal and spatial expression. Furthermore, using consecutive sections stained with HIMF and PCNA separately, we demonstrated that HIMF and PCNA are expressed in the same cells (airway epithelial cells and alveolar type II cells) to further support the idea that HIMF may stimulate cell proliferation in an autocrine and paracrine fashion (Figure 5).



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  		Figure 3. Immunohistochemical staining of HIMF in lung sections of sham control (A) and pneumonectomy (B, C, and D) mice 7 d after surgery. HIMF protein expression is induced by pneumonectomy and is mainly localized in airway epithelial cells (arrows), endothelial cells of the vessels (arrowheads in B and C), and alveolar type II cells (arrowheads in D). Scale bars: 60 µm.

 


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  		Figure 4. Immunohistochemical staining of PCNA in lungs from sham (A and C) and pneumonectomy (B and D) mice 7 d after surgery. Increased numbers of PCNA-positive cells (nuclear staining) are observed in airway epithelium (arrows in B), endothelial cells of the pulmonary artery (arrowheads in B), and alveolar type II cells (arrowheads in D), but not in the sham control (A and C). Scale bars: 100 µm.

 


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  		Figure 5. Immunohistochemical staining for HIMF and PCNA in consecutive sections from Day 3 (A and B) and Day 7 (C and D) pneumonectomy lungs. The HIMF- and PCNA-positive cells are colocalized in airway epithelial cells (arrows) and alveolar type II cells (arrowheads). Scale bars: 100 µm.

 
Fluorescent In Situ Hybridization for HIMF
Fluorescent in situ hybridization (FISH), using 6-carboxyfluorescein–labeled sense and antisense HIMF oligoprobes, demonstrated HIMF mRNA upregulation after pneumonectomy (Figure 6). The results showed that, in the sham lung (at Day 7 after surgery), the weakly positive HIMF mRNA signal only appeared in airway epithelial cells and a few alveolar type II cells (Figure 6A). In the lungs from Days 3, 7, and 14 after pneumonectomy, a strong positive HIMF signal was localized in airway epithelia and alveolar type II cells (Figures 6B–6D depict Day 7 lungs). These results agree with the immunohistochemical staining and Western blot results, and indicate that HIMF expression is largely controlled at the transcriptional level. Furthermore, because HIMF was highly upregulated during the proliferating period of lung growth, it may play an important role in the control of hyperplastic lung regeneration.



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  		Figure 6. Fluorescent in situ hybridization (FISH) of HIMF antisense oligoprobe labeled with 6-carboxyfluorescein in mouse lung of 7 d sham control (A) and 7 d after pneumonectomy (B, C, and D). The sham control only has very weak signals, as expected (A). Strong HIMF mRNA–positive signals are present in airway epithelium (arrows in B, C, and D), endothelial cells of the vessels (arrowheads in C), alveolar type II cells (arrowheads in D). Scale bars: 100 µm.

 
Northern Blot for HIMF
Consistent with the FISH results in the regenerating lung, Northern blot analysis showed that the sham control had very low levels of HIMF expression (Figure 7). Pneumonectomy dramatically increased HIMF mRNA at Day 1 and peaked at Day 7 after surgery, and then was reduced to a low level at Day 14 (Figure 7). These changes paralleled the HIMF protein expression (see Figures 2 and 3) and indicate that HIMF gene expression is controlled at the transcriptional level.



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  		Figure 7. Northern blot analysis of HIMF mRNA in lungs of Day 3 sham (S) and pneumonectomy animals at different time points (Days 1, 3, 7, and 14) after surgery. There was a slight increase in HIMF mRNA at Day 1 and a marked increase at Days 3 and 7 after pneumonectomy. 18 s and 28 s RNA were used as loading control.

 
Intratracheal Instillation of HIMF
Lung tissues were collected 3 and 5 d after intratracheal instillation of HIMF into unoperated mice, and the slides were examined for PCNA-positive cells. Immunohistochemical staining of PCNA showed that in the FLAG controls, the PCNA-positive cells were rarely seen in the airway epithelium and in the lung parenchyma (Figures 8A and 8B). However, HIMF instillation induced widespread cell proliferation in the lung, including airway epithelial cells, alveolar type II cells, endothelial cells of the pulmonary vasculature, and the cells of the alveolar septa, both in the Day 3 (Figures 8C and 8D) and Day 5 groups (Figures 8E and 8F). The PCNA-positive cells were mostly seen in the proximal airways and the lung tissues surrounding them. These data support the hypothesis that HIMF stimulates lung hyperplasia after pneumonectomy, possibly by promoting epithelial and parenchymal cell proliferation. In addition, there was no difference detected between the intensity of PCNA staining in the cells of pneumonectomy and intratracheal HIMF-treated lung sections.



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  		Figure 8. Immunohistochemical staining of PCNA in lungs after intratracheal instillation of saline control with FLAG peptide (A and B, after 3 d), or HIMF 3 d (C and D) and 5 d (E and F) after instillation. PCNA-positive nuclear staining was found in airway epithelial cells (arrows in C and E) and in the lung parenchyma (arrows in D and F) of the HIMF-instilled lungs. Vascular endothelial cells also showed PCNA-positive staining (arrowheads in C and E). Scale bars: 100 µm.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The treatment of certain lung diseases or conditions, such as cancer, emphysema, or severe trauma, often requires pneumonectomy or lobectomy. It has been shown, not only in experimental animals but also in humans, particularly in infants or young children, that removal of lung tissue often results in compensatory growth of the remaining lung (1, 2, 18, 26, 27). In addition, various scenarios, such as reduced-size lung transplantation and pulmonary insufficiency, can benefit from the modulation and induction of lung growth (4, 15). Extensive animal studies have enhanced our knowledge of this process, and a better understanding of the mechanisms of lung growth will undoubtedly facilitate lung regeneration to accelerate restoration of lung function. The molecular mechanisms of compensatory lung growth, however, are still largely unknown. Many reports have illustrated the importance of various factors involved in regulation of compensatory lung growth, including mechanical strain (28), hypoxia (29), growth factors (16, 24), retinoic acid (23), and nitric oxide (11). A previous study in our laboratory showed that keratinocyte growth factor (KGF) enhances postpneumonectomy lung growth by alveolar proliferation (24). In that study, it was found that administration of KGF to adult rats resulted in significantly enhanced LWI, lung volume index, cell proliferation index, and total volume of respiratory region after pneumonectomy but not after sham surgery, suggesting that KGF can induce new alveolar formation in mature lung.

In the current study, we examined the expression of HIMF in compensatory lung growth. We found that HIMF protein and mRNA are highly upregulated in the regenerating lung after pneumonectomy, especially in the first 7 d. HIMF protein was localized to airway epithelial cells, alveolar type II cells, and, occasionally, to vascular endothelial cells. Most importantly, HIMF expression paralleled with PCNA expression, and both HIMF and PCNA immunostaining were localized in the same populations of cells. Using consecutive-section staining with HIMF and PCNA, we have identified the cells expressing both proteins simultaneously, including airway epithelial cells and alveolar type II cells. These results strongly support the idea that HIMF, as a lung-specific, inducible growth factor, may stimulate lung cell proliferation in an autocrine or paracrine fashion in the compensatory growing lung. In addition, intratracheal instillation of recombinant HIMF protein induced wide-spread proliferation of airway epithelial cells, alveolar type II cells, and cells in the lung parenchyma. These results support the idea that HIMF is not only a mitogenic factor to pulmonary microvascular smooth muscle cells (19), but is also a lung-specific growth factor that participates in compensatory lung growth after pneumonectomy.

Previous studies have demonstrated that HIMF is also a potent vasoconstrictor that can significantly raise pulmonary arterial pressure (19). HIMF-induced pulmonary arterial pressure increase is more potent than the common vasoactivators, such as endothelin-1, angiotensin II, and serotonin, but less potent than the thromboxane mimetic U-46619. However, we cannot exclude the possibility of a vasoconstrictive effect during upregulation of HIMF in compensatory lung growth. Moreover, HIMF also possesses an angiogenic function that promotes vascular tube formation in a matrigel plug model (19). More recently, we reported that HIMF is developmentally regulated in mouse lung, particularly during the perinatal period, and that HIMF also has an antiapoptotic effect in cultured embryonic lung (22). As we have shown here, intratracheally instilled HIMF stimulated a variety of cell proliferation in the airways and in the parenchyma of the lung (Figure 8). It is conceivable to postulate that HIMF, as a secreted and inducible lung-specific growth factor mainly from airway epithelial cells and alveolar type II cells, may act through its receptor. HIMF upregulation in the postpneumonectomy lung facilitates growth by stimulating proliferation in a variety of cell types via its receptor in an autocrine or paracrine fashion. However, the identification of the HIMF receptor is still elusive, and the detailed mechanism of HIMF upregulation and HIMF-induced cell proliferation remains unclear. As we have shown, HIMF-induced pulmonary microvascular smooth muscle cell proliferation is partially mediated by PI3K/Akt signal transduction pathway (19), but whether Akt activation participates in compensatory lung growth through HIMF receptors is currently unknown.

It has been speculated that the process of compensatory lung growth potentially involves the same signaling mechanisms that participate in normal postnatal alveolarization in the lung (15, 17, 24). HIMF might be one such signaling factor, as we have described the upregulation of HIMF in both compensatory lung growth (the present study) and the developing lung (22). We speculate that HIMF upregulation is a partially reserved process to recapitulate normal postnatal lung maturation, and upregulated HIMF may be a specific mediator of compensatory lung growth by contributing to alveolar formation and lung maturation. Consistent with this speculation, intratracheal instillation of recombinant HIMF stimulated lung cell proliferation, as ascertained by PCNA immunohistochemistry both in the airway epithelial cells and in the lung parenchyma.

In summary, we have shown that HIMF expression is induced in compensatory lung growth and that intratracheal instillation of HIMF protein induced wide-spread cell proliferation in the mouse lung. In addition, HIMF and PCNA expression localize spatially and temporally in airway epithelial cells, alveolar type II cells, and parenchymal cells of the alveolar septa after pneumonectomy. These results suggest that upregulation of HIMF may play an important role in compensatory lung growth after pneumonectomy. The determination of the functional role of HIMF in regenerating lung warrants further investigation that will ultimately contribute to a better understanding of postnatal lung growth.


   Footnotes
 
This work was supported by National Institutes of Health RO1 grants HL75755 (D.L.) and HL67780 (V.E.L.).

Conflict of Interest Statement: D.L. has no declared conflicts of interest; L.G.F. has no declared conflicts of interest; J.D. has no declared conflicts of interest; J.L. has no declared conflicts of interest; D.W. has no declared conflicts of interest; and V.E.L. has no declared conflicts of interest.

Received in original form October 13, 2004

Received in final form December 2, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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