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Published ahead of print on April 19, 2007, doi:10.1165/rcmb.2006-0352OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 193-201, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0352OC

Methionine Aminopeptidase-2 as a Selective Target of Myofibroblasts in Pulmonary Fibrosis

Daniel Kass, Robert Stone Bridges, Alain Borczuk and Steven Greenberg

Division of Pulmonary, Allergy, and Critical Care, Department of Medicine; Department of Pharmacology; and Department of Cell Biology and Pathology, Columbia University College of Physicians and Surgeons, New York, New York

Correspondence and requests for reprints should be addressed to Daniel Kass, M.D., Columbia University, Department of Medicine, 630 West 168th Street, New York, NY 10032. E-mail: dk371{at}columbia.edu


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Idiopathic pulmonary fibrosis (IPF) is a progressive, scarring lung disease characterized by fibroblast accumulation and deposition of collagen. Factors that promote growth and/or survival of fibroblasts are potential therapeutic targets. Methionine aminopeptidase 2 (MetAP2), a member of the aminopeptidase family of proteases, has been implicated in cell proliferation in a variety of cell types, but its expression and function in the lung is not known. By immunohistochemistry, MetAP2 was expressed in many cell types, including fibroblasts, in IPF lungs. Fumagillin, an irreversible inhibitor of the enzymatic activity of MetAP2, attenuated collagen deposition in the bleomycin model of acute lung injury in mice. Treatment with fumagillin caused a selective reduction in the numbers of bromodeoxyuridine (BrdU)-positive myofibroblasts, but not type II alveolar epithelial cells, macrophages, or B- and T-lymphocytes in the lungs of bleomycin-treated mice. Incubation of primary rat lung fibroblasts with either fumagillin or with short interfering RNA that targeted MetAP2 led to reduced proliferation, as assessed by incorporation of BrdU. The profibrotic growth factor, platelet-derived growth factor, increased expression of MetAP2 in rat lung fibroblasts. We propose that MetAP2 plays a role in the proliferation of fibroblasts and myofibroblasts in fibrotic lung diseases and may serve as a novel pharmacologic target in IPF.

Key Words: methionine aminopeptidase 2 • pulmonary fibrosis • bleomycin • fumagillin • myofibroblasts



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Methionine aminopeptidase 2 (MetAP2) may regulate the proliferation of fibroblasts in experimental and human pulmonary fibrosis. An existing agent, fumagillin, targets MetAP2 and may be a potential treatment for idiopathic pulmonary fibrosis.

 
Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by scarring of the lung parenchyma leading to severely compromised gas exchange (1). Microscopically, the lung contains "fibroblastic foci," small aggregates of actively proliferating myofibroblasts and fibroblasts that constitute many microscopic sites of ongoing alveolar epithelial injury and activation (2). Despite a modestly enhanced understanding of the molecular and cellular mechanisms of this disease, the prognosis is poor, with a median survival of 35 mo from the initial patient visit (3). There is no known effective treatment, except possibly lung transplantation (4).

Although the pathogenesis of this disease remains poorly understood, evidence suggests that several classes of proteases play key roles in disease pathogenesis. For example, expression of proteases, such as matrix metalloproteinase (MMP)-7 (5) and MMP-9 (6, 7), as well as cathepsins (8), are increased in lung samples from patients with IPF, and promote development of pulmonary fibrosis and/or extracellular matrix remodeling in experimental models (5, 9, 10). Another class of proteases, comprising the aminopeptidase family, has recently been implicated in several disease models of fibrosis (11–14), and increased aminopeptidase activity was found in bronchoalveolar lavage fluid from patients with IPF (15) and from patients with interstitial lung involvement from collagen vascular disease (16) compared with normal volunteers. One member of this peptidase family, methionine aminopeptidase 2 (MetAP2), which catalyzes the removal of the N-terminal methionine from nascent polypeptides (17), may also function as a regulator of cell proliferation (18, 19), We hypothesized that cell proliferation may contribute to the expansion of fibroblasts and myofibroblasts, the major collagen-producing cells in pulmonary fibrosis. In this study, we demonstrate that MetAP2 is expressed in lungs from normal individuals and from individuals with IPF. In a model of acute lung injury in mice, we also show that pharmacologic inhibition of MetAP2 resulted in decreased pulmonary fibrosis while sparing lung inflammation.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
This protocol was approved by the Institutional Animal Care and Use Committee and the Institutional Review Board of Columbia University.

Reagents
Bleomycin was obtained from Bedford Labs (Bedford, OH). Fumagillin was from EMD Biosciences (San Diego, CA). The Sircol collagen assay was from Biocolor Ltd. (Belfast, UK). Platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, and epidermal growth factor (EGF) were from R&D Systems (Minneapolis, MN). Insulin-like growth factor (IGF)-I, fibroblast growth factor (FGF)-2, and connective tissue growth factor (CTGF) were obtained from Peprotech (Rocky Hill, NJ). Euthanasia 5 solution was from Henry Schein (Melville, NY). DNase and insulin-transferrin-sodium selenite liquid supplement were from Sigma-Aldrich (St. Louis, MO). Rabbit IgG against MetAP2 for immunoblotting and immunofluorescence and rabbit IgG against vimentin were purchased from Abcam (Cambridge, UK). Rabbit IgG against MetAP2 for immunohistochemistry and Rabbit IgG against total ERK were from Invitrogen (Carlsbad, CA). Biotin-conjugated anti-B220 was from Becton-Dickinson (Franklin Lakes, NJ). Rabbit anti-CD3 was from Dako Cytomation (Carpinteria, CA). Rabbit IgG against phospho-ERK was from Cell Signal Technologies (Beverley, MA). Goat IgG against CD68, pro-collagen I, and beta-actin, were from Santa Cruz Biotechnology (Santa Cruz, CA) and a monoclonal antibody (mAb) against bromodeoxyuridine (BrdU) (clone BMC 9318) was from Roche (Indianapolis, IN). Alexa Fluor 488–conjugated goat anti-mouse IgG and Alexa Fluor 488–conjugated streptavidin were from Molecular Probes (Carlsbad, CA). An mAb against {alpha}-SMA (clone 1A4) was from EMD Biosciences (San Diego, CA). Rabbit IgG against pro–surfactant protein-C was from RDI (Concord, MA). Peroxidase-conjugated secondary antibodies and Rhodamine-conjugated streptavidin were from Jackson Immunoresearch (West Grove, PA) and Supersignal West Pico Chemiluminescent system was from Pierce (Rockford, IL). RNeasy Mini Kit, Quantitect SYBR Green PCR Kit, and HiPerfect were purchased from Qiagen (Valencia, CA). Superscript III First-Strand Synthesis System for RT-PCR was from Invitrogen (Carlsbad, CA). Quantitative RT-PCR was performed using the LightCycler 3 (Roche). A SMARTpool reagent targeting rat MetAP2 and nontargeting control RNA oligonucleotides (siCONTROL) were from Dharmacon (Lafayette, CO). Vectastain ABC Universal kit was from Vector Labs (Burlingame, CA).

Mouse Model of Bleomycin-Induced Lung Injury and Histopathology
Female C57Bl/6 mice (6–8 wk old) were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). The mice were anesthetized with isoflurane in an anesthesia chamber. Bleomycin (3 mg/kg) or saline control was administered intranasally. Four days later, fumagillin 0.5 mg/kg (10 µg dissolved in 10 µl of 10% DMSO in RPMI 1640), or vehicle control, was administered intranasally and repeated daily for 10 d. This dosage was chosen, rather than the more commonly used higher doses in rodents (20–100 mg/kg [20–23]), because intranasal delivery afforded localized lung, rather than systemic delivery. Mice were killed on Day 14 with Euthanasia 5 solution, and the lungs were excised for histopathology, immunostaining, and determination of collagen content. Lung tissue was also embedded in paraffin. Routine staining of tissue sections for hematoxylin and eosin and trichrome were performed by the Columbia University histology service.

Sircol Assay for Acid-Soluble Collagen
Lungs excised from mice were snap-frozen in liquid nitrogen followed by lyophilization. Lungs were minced and placed in 0.5 M acetic acid at 4°C overnight. The Sircol assay was performed following the manufacturer's instructions. Briefly, 100 µl of the lung-acetic acid mixture was added to 1 ml of the Sirius Red reagent and incubated for 60 min. The collagen–dye complex was pelleted by centrifugation at 10,000 x g for 10 min, and the precipitated material was dissolved in 0.5 M NaOH. OD540 was recorded using a microplate reader, and results were normalized to dry lung mass.

Isolation of Rat Lung Fibroblasts and Cell Culture
Eight-week-old female Sprague-Dawley or Lewis rats were purchased from Harlan (Indianapolis, IN). Rats were killed with Euthanasia 5 solution. The lungs were perfused with cold PBS, and the parenchyma were excised. Large airways were carefully removed, and lungs were minced and suspended in a 12.5% trypsin solution containing 2.5 mM EDTA for 90 min. After passage through a cell strainer, the suspension was pelleted and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5 ng/ml PDGF, and 10 ng/ml EGF and an insulin-transferrin-selenite (ITS) liquid supplement (Sigma), modified from Ref. 24. The suspension was plated and incubated in 5% CO2 at 37°C and used for experiments after the second passage. Experiments were run no later than the eighth passage.

Induction of MetAP2: Immunoblotting and Quantitative RT-PCR
A quantity of 7 x 104 rat lung fibroblasts (RLFs) was plated in DMEM supplemented with 10% FBS, 5 ng/ml PDGF, and 10 ng/ml EGF and maintained at 37°C in a CO2 incubator. After 16 h, the cells were washed and incubated in DMEM supplemented with 1% FBS for an additional 48 h. The indicated growth factors were then added for 24 h, followed by detergent lysis for protein recovery. In parallel, total RNA was harvested from the cells 8 and 16 h after addition of the above growth factors using the RNeasy Mini Kit according to the manufacturer's protocol. RNA was reverse transcribed into cDNA primed by random hexamers by the Superscript III First-Strand Synthesis System. We quantified MetAP2 and control mRNA using the LightCycler 3. For MetAP2, the upstream primer was 5'-GTGCCAATAAGGCTTCCAAGAAC-3'. The downstream primer was 5'-GGATCTACAATGCCCAAGTCA-3'. Control mRNA differed depending on the stimulus. For example, PDGF induced an increase in GAPDH mRNA (25 and data not shown). For samples incubated with PDGF, we used 18 s ribosomal RNA: the upstream primer was 5'-AAACGGCTACCACATCCAAG-3' and the downstream primer was 5'-CCTCCAATGGATCCTCGTTA-3'. For samples incubated with TGF-beta we used GAPDH; the upstream primer was 5'-GACCCCTTCATTGACCTCAAC-3', and the downstream primer was 5'-CTTCTCCATGGTGGTGAAGA-3'.

Data were quantified with the second derivative maximum method by the LightCycler 3 software. Data were normalized between samples by total copies of control mRNA present. For immunoblotting, 24 h after the addition of growth factors, cells were lysed in 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 5 mM PMSF, 50 µg/ml leupeptin, 50 µg/ml aprotnin, 50 mM Tris-HCl, pH 7.4, and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting as previously described (26). Quantification of band intensity was performed with ImageJ software (http://rsb.info.nih.gov/ij/).

Quantification of BrdU in Rat Fibroblasts and Fluorescence Microscopy
RLFs were plated in DMEM supplemented with 10% FBS on glass coverslips and incubated overnight in a 5% CO2 incubator at 37°C. Cells were washed and incubated in DMEM without FBS for 48 h to synchronize the cells in G0 (27). The media were replaced with DMEM + 10% FBS supplemented with fumagillin dissolved in DMSO or vehicle control and incubated for a further 24 h. Cells were pulsed with BrdU for the final 4 h. In some experiments, cells were transfected with RNA oligonucleotides targeting rat MetAP2 or nontargeting control RNA oligonucleotides (Dharmacon) using the HiPerfect system according to the manufacturer's recommendations. For siRNA experiments, cells were incubated for 72 h and pulsed with 10 µM BrdU for the final 2 h. Cells were fixed in 3.7% formaldehyde, permeabilized in 0.2% Triton X-100, and incubated with 100 U/ml DNase for 60 min at 37°C. Cells were processed for immunofluorescence using an mAb against BrdU or {alpha}-SMA followed by Alexa Fluor 488–conjugated goat anti-mouse IgG and DAPI. Cells were visualized with a Nikon Eclipse TE200 microscope (Nikon, Tokyo, Japan) equipped with epifluorescence. Fields were selected at random and BrdU+ cells were enumerated and scored as a percentage of all cells present/high-power field (hpf).

Immunohistochemistry and Immunofluorescence Staining of Tissue Sections
For immunohistochemistry, lungs were fixed in 10% formaldehyde and embedded in paraffin. Five-micrometer sections were cut and mounted on positively charged glass slides. Sections were deparaffinized with xylene followed by rehydration through ethanol. After antigen retrieval, samples were stained with the indicated antibodies. All antigens were developed with the Vectastain ABC Universal kit and diaminobenzidine. Sections were counterstained with Harris' hematoxylin, dehydrated through graded alcohol series, and mounted with Permount (Fisher, Fairlawn, NJ). Sections were visualized using a x40 objective and an Olympus CHS microscope (Olympus, Tokyo, Japan). Fields were selected at random and scored for number of positively staining cells per field. A minimum of 15 fields were counted per lung (pilot experiments showed that the cumulative percentage of positive cells plateaued at 12–14 fields).

Immunofluorescence was performed on murine lung cryosections, unless otherwise stated. Mice were exposed to bleomycin followed by fumagillin or vehicle control as described above. BrdU, 50 mg/kg, was administered by intraperitoneal injection to mice 24 h before killing. Tissue was embedded in Tissue-Tek OCT compound (Sakura-Finetek, Torrance, CA) and frozen in an isopentane–dry ice slurry. Five-millimeter sections were cut and mounted on positively-charged glass slides. Slides were air-dried and fixed in ice-cold 1:1 methanol:acetone, followed by rehydration in PBS. Tissue sections were incubated in 2 N HCl at 37°C for 1 h to expose the BrdU for immunostaining. An mAb directed against {alpha}-SMA and BrdU was used with the M.O.M. kit and Avidin-Biotin blocking kit (Vector) as directed by the manufacturer. {alpha}-SMA staining was visualized with rhodamine-conjugated streptavidin (Jackson), and BrdU was visualized with Alexa Fluor 488–conjugated streptavidin (Molecular Probes). Lung sections were examined using a Nikon TE200 inverted microscope equipped with epi-fluorescence or a Leica DMIRBE microscope (Leica, Wetzlar, Germany) equipped with DIC optics and epifluorescence. Cells were imaged using Metamorph software. All the cells in 50 hpf per animal were scored for presence or absence of staining for BrdU and {alpha}-SMA, and sections were scored in a blinded fashion. Counterstaining was performed with DAPI. MetAP2 was stained with a rabbit polyclonal antibody (Abcam) overnight at 4°C followed by biotinylated anti-Rabbit IgG and Alexa Fluor 488–conjugated streptavidin.

Statistics
Data were analyzed by ANOVA followed by Fisher's LSD post hoc test using Kaleidagraph 4 (Synergy Software, Reading, PA) unless otherwise noted.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
MetAP2 Expression in Human Lung
Recent data suggest that aminopeptidases may play a role in the pathogenesis of human fibrotic diseases (15, 16). We sought to determine if one member of this class of enzymes, MetAP2, previously implicated in proliferation of endothelial cells (19) and certain tumor cell lines (28) and in angiogenesis (19), was expressed in human lung. Immunohistochemistry for MetAP2 was performed on control lung samples (i.e., histologically normal appearing lung removed from patients undergoing pneumonectomy for lung cancer) and samples from IPF lung. Staining was present in many cell types, particularly Type II epithelial cells, alveolar macrophages, and fibroblasts/myofibroblasts (Figures 1A and 1B). Nonimmune rabbit IgG control showed no detectable staining. To confirm expression of MetAP2 in myofibroblasts in IPF, immunofluorescence was performed for {alpha}-SMA and MetAP2. This revealed co-localization of MetAP2 and {alpha}-SMA in all fields examined (Figures 1C–1F).


Figure 1
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  		Figure 1. Detection of MetAP2 in control and IPF lungs. Immunohistochemistry for MetAP2 was performed on formalin-fixed paraffin-embedded samples. Staining was performed using diaminobenzidine and sections were counterstained with hematoxylin. (A) Control lung; (B) IPF lung. Inset panels show staining using isotype-matched controls. Note staining for MetAP2 in macrophages (arrowhead in A) Type II epithelial cells (short arrow in B), and fibroblasts/myofibroblasts (long arrow in B). IPF lung sections were stained with DAPI to visualize nuclei (C) and antibodies to detect {alpha}-SMA (D) and MetAP2 (E). Arrowheads in E point to cells that stain positively for {alpha}-SMA in D. (F) Overlay of images demonstrates cytoplasmic co-localization of {alpha}-SMA and MetAP2. Original magnifications: x400.

 
Fumagillin Reduces Collagen Content in the Lungs of Bleomycin-Treated Mice
As MetAP2 has been implicated in the regulation of cell proliferation, a process that may contribute to pulmonary fibrosis, we determined whether inhibition of the enzymatic function of MetAP2 attenuates collagen deposition in the bleomycin model of pulmonary fibrosis. Fumagillin is a fungal metabolite (29) that irreversibly inactivates the enzymatic activity of MetAP2 (30, 31). A high-resolution crystal structure of human MetAP2 complexed with fumagillin shows a covalent bond formed between a reactive epoxide of fumagillin and histidine-231 in the active site of MetAP2 (30, 31). Eight-week-old C57BL/6 mice were exposed to bleomycin intranasally on Day 0, followed by either intranasal delivery of 0.5 mg/kg fumagillin or vehicle control starting on Day 4 and repeated daily for 10 d. We verified expression of MetAP2 in normal and bleomycin-injured murine lung by immunohistochemistry (Figure 2A). At Day 14, the acid-soluble collagen content of bleomycin-treated lungs was increased 2.2-fold compared with controls. Fumagillin reduced collagen content by 18.4% of the total, which represented ~ 34% of the bleomycin-induced collagen (Figure 2B). The reduction in collagen content in fumagillin-treated mice was observed without an apparent change in overall inflammation (Figure 2C). We repeated these studies using a 10-fold higher dose of fumagillin as well as a separate experiment extending the period of fumagillin treatment to 21 d after bleomycin administration, and found no additional protection against bleomycin-induced lung fibrosis by fumagillin under these conditions (data not shown).


Figure 2
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  		Figure 2. Fumagillin reduces lung collagen content in bleomycin-exposed mouse lungs. Mice were exposed to bleomycin 3 mg/kg intranasally on Day 0 followed by either intranasal delivery of fumagillin 10 µg in 10% DMSO in RPMI 1640 or vehicle control starting on Day 4 and repeated daily for 10 d. Mice were killed on Day 14. (A) IH for MetAP2 was performed on formalin-fixed paraffin-embedded samples. Staining was performed using diaminobenzidine and sections were counterstained with hematoxylin. Staining for MetAP2 was evident in multiple cell types in the control and bleomycin-exposed lung. Inset shows staining of bleomycin- exposed lung with nonimmune IgG control (magnification: x400). (B) Lungs were dissected, snap-frozen, lyophilized, and acid-soluble collagen content was determined by the Sircol assay. Data represent mean ± SEM, n = 9. Bleomycin induced a 2.2-fold increase in acid-soluble collagen content versus control (*P < 0.001). Fumagillin reduced bleomycin-induced acid-soluble collagen content by 18.4% (**P = 0.03). (C) Hematoxylin and eosin and trichrome staining were performed. Note that fumagillin reduced collagen deposition as seen in the trichrome staining without a significant decrease in the inflammatory infiltrate.

 
Fumagillin Causes a Reduction in Mesenchymal Cells in the Lungs of Bleomycin-Treated Mice
Fumagillin and its analogs have been shown to inhibit cell cycle progression through G1 (32) or to induce apoptosis (33). We reasoned that the mechanism of decreased fibrosis observed with fumagillin might be due, in part, to decreased numbers of cells that were either directly or indirectly responsible for promoting fibrosis. As myofibroblasts are enriched in fibroblastic foci, where the bulk of collagen is deposited in IPF lungs, we identified these cells by using an mAb against {alpha}-SMA, a prototypical marker of myofibroblasts. In control lungs, {alpha}-SMA was abundantly detectable in cells lining large airways and blood vessels, but was otherwise barely detectable in lung interstitium (not shown). Bleomycin treatment alone resulted in a nearly 40-fold increase in the number of {alpha}-SMA staining cells and fumagillin reduced this increase by 44% (Figures 3A and 3B). Lungs from fumagillin-treated mice also exhibited fewer cells expressing vimentin, which is also abundant in mesenchymal cells (Figure 3B). However, fumagillin-exposed lungs did not exhibit reductions in Type II epithelial cells, macrophages, T-cells, or B-cells (Figure 3B), indicating that the effect of fumagillin on cell number was relatively specific to the mesenchymal compartment and appeared to be independent of inflammation.


Figure 3
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  		Figure 3. Fumagillin causes a reduction in {alpha}-SMA– and vimentin-positive, but not pro–SP-C-, CD68-, CD3-, or B220-positive cells in the lungs of bleomycin-treated mice. Fourteen days after administration of bleomycin, lungs of mice treated with fumagillin or vehicle control were dissected and processed for immunohistochemistry using an mAb against {alpha}-SMA. Sections were counterstained with hematoxylin (A). Original magnification: x400. Quantitation of cells stained with antibodies against the indicated markers was performed as described in MATERIALS AND METHODS (B). Data represent mean ± SEM, n = 3.

 
We verified that myofibroblasts express MetAP2 in vivo, since double-label immunofluorescence of lung sections derived from bleomycin-exposed mice showed co-localization of MetAP2 and {alpha}-SMA (Figure 4).


Figure 4
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  		Figure 4. MetAP2 co-localizes with {alpha}–SMA in bleomycin-injured mouse lung. Cryosections of mouse lungs exposed to bleomycin were fixed in acetone and methanol and prepared for immunofluorescence using antibodies against {alpha}-SMA and MetAP2 as described in MATERIALS AND METHODS. Nuclei were visualized with DAPI. Note group of {alpha}-SMA+ cells co-staining for MetAP2. Inset shows staining with nonimmune rabbit IgG. Original magnification: x1,000.

 
Requirement for MetAP2 in Proliferation of Primary Rat Lung Fibroblasts In Vitro
We undertook in vitro experiments to determine a possible mechanism for the reduction in the number of fibroblasts/myofibroblasts caused by fumagillin. We incubated primary rat lung fibroblasts in serum-free medium as described in MATERIALS AND METHODS to synchronize cells in G0. The cells were then released from G0 by addition of 10% FBS with and without fumagillin for an additional 24 h. The cells were pulsed with BrdU, and the percentage of BrdU-positive cells was determined by immunofluorescence. Increasing concentrations of fumagillin led to decreased incorporation of BrdU (Figure 5A). The IC50 observed was 3 nM, which is similar to the IC50 for the enzymatic activity of recombinant MetAP2 (19, 34). To confirm that fumagillin targeted MetAP2 in these cells, we transfected rat lung fibroblasts with MetAP2-targeting or nontargeting siRNA oligonucleotides and measured expression of MetAP2 and BrdU. MetAP2 siRNA resulted in a profound reduction in MetAP2 expression (Figure 5B). Transfection with MetAP2-targeting oligonucleotides led to reduced BrdU incorporation by 63% (Figure 5C). This extent of inhibition was similar to that obtained using RLFs incubated with fumagillin in the absence of MetAP2-targeting oligonucleotides. Addition of fumagillin to cells transfected with MetAP2-targeting oligonucleotides did not lead to further decreases in BrdU incorporation. This suggests that the effect of fumagillin on cell proliferation is due to MetAP2 inhibition.


Figure 5
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  		Figure 5. Fumagillin and MetAP2-targeting siRNA inhibit incorporation of BrdU into RLFs in vitro. (A) Primary RLFs were serum-starved in DMEM for 48 h and incubated with 10% FBS and the indicated concentrations of fumagillin or vehicle control at 37°C for a further 24 h Cells were pulsed with 10 µM BrdU for the final 4 h. BrdU-positive cells were quantified as described in MATERIALS AND METHODS. Data represent mean ± SEM, n = 4. (B, C) Primary rat lung fibroblasts were plated in DMEM + 10% FBS and incubated in the presence (2, 4, 6) or absence (1, 3, 5) of 10 nM fumagillin and either no added oligonucleotides (1 and 2), nontargeting RNA oligonucleotides (3 and 4), or MetAP2 siRNA oligonucleotides (5 and 6) at 37°C as described in MATERIALS AND METHODS. Immunoblotting (B) and BrdU incorporation (C) were determined as described in MATERIALS AND METHODS. (B), Immunoblot reveals undetectable expression of MetAP2 using siRNA oligonucleotides targeting MetAP2, although a prolonged exposure revealed some residual MetAP2 expression (not shown). (C). BrdU+ cells were quantitated per hpf using fluorescence microscopy. Solid bars, vehicle; shaded bars, 10 nM fumagillin. Data represent mean ± SEM, n = 4.

 
To determine whether fumagillin had additional effects on lung fibroblasts other than proliferation, we examined the effect of fumagillin on several parameters of fibroblast function. Fumagillin did not block TGF-beta–induced synthesis of pro-collagen I (see Figure E1 in the online supplement) or expression of {alpha}-SMA (Figure E2). Addition of fumagillin also did not change the morphology of RLFs or affect the known ability of EGF (35) to downregulate {alpha}-SMA expression in these cells (Figure E3).

Induction of MetAP2 by Growth Factors Implicated in Pulmonary Fibrosis
Several growth factors trophic for fibroblasts have been implicated in pulmonary fibrosis, including TGF-beta, PDGF, IGF-I, FGF-2 (reviewed in Refs. 36, 37), and CTGF (38). It is not known whether any of these influence the expression of MetAP2. We incubated rat lung fibroblasts in the presence or absence of growth factors and determined the expression of MetAP2. PDGF led to a 2.1-fold increase in expression of MetAP2 protein (Figures 6A and 6B), which correlated with a 1.6- and a 1.8-fold increase in mRNA for MetAP2 at 8 and 16 h, respectively (P < 0.02, not shown). Addition of TGF-beta led to a 1.3- and 1.5-fold increase in MetAP2 mRNA at 8 and 16 h, respectively (P < 0.02); although there was a trend for an increase in MetAP2 protein by TGF-beta, this was not statistically significant. Thus PDGF, a profibrotic growth factor that is present in IPF lungs (39), induced the expression of MetAP2 in primary rat lung fibroblasts in vitro.


Figure 6
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  		Figure 6. Induction of MetAP2 by growth factors implicated in pulmonary fibrosis. Primary rat lung fibroblasts were grown in DMEM + 1% FBS at 37°C for 48 h followed by addition of TGF-beta (10 ng/ml), PDGF (10 ng/ml), IGF-I (40 ng/ml), FGF-2 (10 ng/ml), or CTGF (50 ng/ml) for an additional 24 h. Cells were lysed in detergent and subjected to SDS-PAGE and immunoblotting for MetAP2 and actin. (A) Immunoblot showing an increase in MetAP2 expression by TGF-beta, PDGF, and IGF-I. (B) Densitometry of immunoblots was performed as described in MATERIALS AND METHODS and expressed as fold-induction compared with cells incubated in the absence of added growth factors (n = 4). Only PDGF increased MetAP2 protein expression to a significant extent (*P = 0.04). Data were analyzed by the Wilcoxon Rank Sign Test.

 
Fumagillin Decreases BrdU Incorporation into Myofibroblasts in Bleomycin-Injured Mouse Lungs
Because we observed that both fumagillin and MetAP2-targeting siRNA decreased incorporation of BrdU in RLFs in vitro, we hypothesized that fumagillin specifically decreases proliferation of myofibroblasts in bleomycin-injured mice in vivo. We labeled bleomycin-injured mice treated with fumagillin or vehicle control with BrdU 24 h before killing and determined the expression patterns of {alpha}-SMA and BrdU (Figures 7A–7D). BrdU+ cells were visualized in all lungs, and the number of these cells increased to the same extent in lungs derived from mice exposed to bleomycin and either vehicle control or fumagillin (Figure 7E, left panel). However, treatment with fumagillin was associated with 60% fewer cells that stained positive for both {alpha}-SMA and BrdU (Figure 7E, middle and right panels), indicating that the effect of fumagillin on cell proliferation was not a global one, but relatively specific for those cells that expressed {alpha}-SMA (i.e., myofibroblasts).


Figure 7
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  		Figure 7. Double-label immunofluorescence for BrdU and {alpha}-SMA in bleomycin-injured mouse lungs. Cryosections of lungs from mice exposed to bleomycin followed by fumagillin or vehicle control and pulsed with BrdU were fixed in acetone and methanol and processed for immunofluorescence using antibodies against BrdU (A) and {alpha}-SMA (B) as described in MATERIALS AND METHODS. (C) Overlay of images A and B. Representative examples of cells that stained positive for both BrdU and {alpha}-SMA (arrowheads in C) or positive for BrdU only (arrows in C); magnification: x400. (D) Overlay of C and a DIC image of the same field. The total number of BrdU+ cells and BrdU+ {alpha}-SMA+ cells were quantified as described in MATERIALS AND METHODS (E). Data are expressed as mean ± SEM, n = 4–7. Data were analyzed by ANOVA followed by Fisher's LSD post hoc test.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In this study, we demonstrated that MetAP2 is expressed in normal lung and in lungs of patients with IPF, and that pharmacologic inhibition of MetAP2 led to reduced collagen deposition and a selective reduction in fibroblasts/myofibroblasts in an experimental model of lung injury. We have shown for the first time that fumagillin was efficacious in this mouse model of lung injury 4 d after the onset of lung injury by bleomycin, a time when acute neutrophilic inflammation has begun to subside (40). We observed that the anti-fibrotic effect of fumagillin in this model is independent of inflammation in the bleomycin model of acute lung injury. This suggests that MetAP2 may be an attractive drug target for IPF in humans, as the diagnosis of IPF is typically made in advanced disease, when fibrosis, but not inflammation, is apparent.

Like MetAP1, the recognized enzymatic function of MetAP2 is to cleave the N-terminal methionine from nascent polypeptides during the early stages of protein synthesis (17). The removal of methionine is essential for further amino terminal modifications (e.g., acetylation by N-{alpha}-acetyltransferase and myristoylation of glycine by N-myristoyltransferase) (41). It is not known how inhibition of this function leads to decreased cell proliferation. Cellular targets of fumagillin and its analogs include myofibroblasts (this study), endothelial cells (19, 42), and various tumor cell lines (28). What is the relevant cellular target of fumagillin in pulmonary fibrosis? Our data point to fibroblasts/myofibroblasts as the relevant target. Other studies have shown that inhibition of MetAP2 by fumagillin and its analogs leads to decreased hepatic (13) and peritoneal fibrosis (14) in animal models as well as decreased proliferation of hepatic stellate cells (13), which are thought to represent the hepatic equivalent of myofibroblasts.

The reduction in collagen deposition that we observed with fumagillin treatment was relatively modest. Although fumagillin appears to target a replication-competent population of collagen-producing cells in bleomycin-induced lung injury, other nonreplicating collagen-producing cells might be present in these lungs and therefore not be sensitive to drugs that affect cell proliferation. Collagen-producing cells in pulmonary fibrosis may accumulate from multiple sources, such as the influx of bloodborne fibrocytes (43, 44) or from epithelial–mesenchymal transition (45). It is possible that these or other collagen-producing cells are either postmitotic or are otherwise insensitive to fumagillin. However, fibrocytes appear to have the capacity to replicate in vitro (46) and, possibly, in vivo (47). Further studies will be needed to elucidate the relative contributions of these cells to pulmonary fibrosis. We have also have not excluded the possibility that fumagillin targets additional cell types present in fibrotic lesions. For example, fumagillin has been shown to inhibit angiogenesis, and angiogenesis may play a role in the development and/or progression of pulmonary fibrosis in humans (48) and animals (49, 50). Areas of neovascularization may be the source of profibrotic growth factors and chemokines (reviewed in Ref. 51). Further studies are needed to determine whether blood vessels in the injured lung are also targeted by fumagillin.

Although the current study has implicated the enzymatic activity of MetAP2 in promoting pulmonary fibrosis, MetAP2 has other recognized nonenzymatic functions. It was first characterized in 1988 as a 67-kD protein that binds to eukaryotic initiation factor-2{alpha} (eIF2-{alpha}), protecting it from phosphorylation and thus maintaining active protein synthesis (17). Pharmacologic inhibition of MetAP2 by fumagillin-type compounds does not affect its ability to protect eIF2-{alpha} from phosphorylation (52). MetAP2 has also been demonstrated to bind and inhibit ERK in myoblasts in vitro (53), and fumagillin itself leads to increased MetAP2 expression (53). However, we found no evidence that ERK was the relevant target for fumagillin in rat lung fibroblasts (Figure E4).

TNP-470, a synthetic fumagillin analog, has advanced into clinical trials in different malignancies (54–57), but dose-limiting neurotoxicity during systemic administration has limited its wide application (58). We chose intranasal delivery as a potential means to limit neurotoxicity in this mouse model of lung injury. Although use of the intranasal route of delivery of fumagillin and/or its analogs in humans has not been reported in clinical trials, the potential for delivery of high, local concentrations of MetAP2 inhibitors might be expected to minimize central nervous system toxicity. Further study will be required to determine the safety and effectiveness of inhaled fumagillin in human lung diseases.


   Acknowledgments
 
The authors thank Dr. Richard Vallee for use of the Leica DMIRBE microscope and Daniel Traum for technical assistance in the preparation of this manuscript. The authors also thank Dr. Byron Thomashow for his generosity and support of their work.


   Footnotes
 
This work was supported by the Stony Wold-Herbert Fund of New York and the Janet and Tony Goldman Fund for Innovative Lung Research.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0352OC on April 19, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 19, 2006

Accepted in final form February 27, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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