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Published ahead of print on January 31, 2003, doi:10.1165/rcmb.2002-0228OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 88-97, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0228OC

Oxidative Stress and Apoptosis Interact and Cause Emphysema Due to Vascular Endothelial Growth Factor Receptor Blockade

Rubin M. Tuder, Lijie Zhen, Chung Y. Cho, Laima Taraseviciene-Stewart, Yasunori Kasahara, Daniela Salvemini, Norbert F. Voelkel and Sonia C. Flores

Department of Pathology, Division of Cardiopulmonary Pathology, and Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; Division of Pulmonary and Critical Care Medicine, and Department of Medicine, COPD Center, University of Colorado School of Medicine, Denver, Colorado; Department of Respirology (B2), Chiba Medical School, Chiba, Japan; Metaphore Corporation, St. Louis, Missouri; and Webb-Waring Institute, Denver, Colorado

Address correspondence to: Rubin M. Tuder, M.D., Division of Cardiopulmonary Pathology, Department of Pathology, Johns Hopkins University School of Medicine, 720 Rutland Ave., Ross 519, Baltimore, MD 21205. Email: Rtuder{at}JHMI.EDU


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously demonstrated that a failure of pulmonary endothelial cell survival induced by vascular endothelial growth factor (VEGF) receptor blockade results in lung alveolar septal cell apoptosis and emphysema. Because apoptosis and oxidative stress may be pathobiologically linked, we hypothesized that oxidative stress has a central role in alveolar septal cell apoptosis and emphysema induced by VEGF receptor blockade. When compared with control animals, rats treated with the VEGF receptor blocker SU5416 showed increased alveolar enlargement, alveolar septal cell apoptosis, and expression of markers of oxidative stress, all of which were prevented by the superoxide dismutase mimetic M40419. The preservation of lung structure in SU5416+M40419-treated lungs was associated with increased septal cell proliferation, and enhanced phosphorylation of the prosurvival and antiapoptotic Akt, when compared with SU5416-treated lungs. Consistent with a positive feedback interaction between oxidative stress and apoptosis, we found that apoptosis predominated in areas of oxidative stress, and that apoptosis blockade by a broad spectrum caspase inhibitor markedly reduced the expression of markers of oxidative stress induced by SU5416 treatment. Oxidative stress and apoptosis, which cause lung cellular destruction in emphysema induced by VEGF receptor blockade, may be important mediators common to human and experimental emphysema.

Abbreviations: 4 hydroxy-2-nonenal, 4-HNE • 8 hydroxy-2' deoxyguanosine, 8-HG • centrilobular alveoli, CL • control, CTL • dinitrophenyl hydrazone, DNPH • proliferating cell nuclear antigen, PCNA • peripheral alveoli, PL • terminal deoxynucleotidyl transferase-mediated dUTP end-labeling, TUNEL • vascular endothelial growth factor, VEGF • VEGF receptor 2, VEGFR2


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Emphysema due to chronic cigarette smoking is a significant cause of mortality and morbidity worldwide. The presence of increased lung inflammation in the lungs of smokers, and the evidence that patients with {alpha}1-antitrypsin develop emphysema, led to the hypotheses of inflammation and protease/antiprotease imbalance to explain emphysema (1). We have developed a rodent model of inflammation-independent emphysema based on the blockade of vascular endothelial growth factor (VEGF) receptor signaling. This model provides an alternative explanation for emphysema based on the hypothesis that a failure of the lung cellular and molecular maintenance program may significantly contribute to the unique characteristics of pulmonary emphysema (2).

Several lines of evidence indicate that VEGF, which is abundantly expressed in the normal lung (3), has a prosurvival and antiapoptotic role in endothelial cells. VEGF withdrawal causes apoptosis of cultured endothelial cells (4) and results in vessel regression and apoptotic endothelial cell death in vivo (5). The evidence of lung airspace enlargement in newborn mice treated with a soluble VEGF receptor (6), and the reports of emphysema induced by endothelial cell immunization (7) or lung-specific deletion of VEGF using a Cre-Lox approach (8), all support the data obtained with our model (2). Disruption of survival signals of pulmonary capillary endothelial cells leads to a failure of normal alveolar septal structure and the data with the model are in accordance with the prior observations of almost avascular alveolar septa in emphysematous lungs and decreased expression of VEGF and VEGF receptor 2 (VEGFR 2) in emphysematous lungs (9).

The role of apoptosis in the lung destruction in emphysema is increasingly being recognized. Emphysema triggered by the VEGF receptor blocker SU5416, which is characterized by the presence of apoptotic alveolar septal cells, is prevented by a broad spectrum caspase inhibitor (2). Furthermore, apoptosis is involved in the pancreatic elastase instillation model (10). Finally, human emphysematous lungs have increased numbers of apoptotic septal cells when compared with normal lungs (9, 11).

Oxidative stress has a central role in the regulation of cell function and maintenance of homeostasis. Low levels of oxidative stress mediate cell growth and cellular signaling, such as seen with VEGF stimulation of growth of cultured endothelial cells (12). On the other hand, excessive levels of oxidants cause injury and disease, and have been implicated in the pathogenesis of emphysema (13). When high levels of pro-oxidants result in altered cell signaling due to post-translational modification of tyrosine-containing receptors and enzymes, and damage structural proteins, lipids, and DNA, they trigger apoptosis (14). A vicious circle might be established, because cells undergoing apoptosis display increased oxidative stress, which further contributes to the apoptosis (15).

We propose that oxidative stress and apoptosis are both necessary and integral players in the pathobiology of the development of emphysema. Specifically, we hypothesize that VEGF receptor blockade triggers lung oxidative stress, which is causally involved in alveolar septal cell death in this emphysema model. The present studies demonstrate that a superoxide dismutase mimetic protects against the development of apoptosis and emphysema induced by VEGF receptor blockade. In addition, our finding that caspase inhibition reduces lung expression of markers of oxidative stress emphasize the importance of a positive feedback loop between apoptosis and oxidative stress, which, when triggered by interruption of alveolar capillary endothelium survival signals, results in alveolar destruction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The animal protocol has been approved by the Johns Hopkins University Animal Care and Use Committee. Male Sprague-Dawley rats (3 mo old, 300 g) were purchased from a commercial vendor and experiments were performed in the same shipment lot of rats. SU5416 (provided by SUGEN, Inc., South San Francisco, CA) was administered once, at 2 mg/kg, subcutaneously, at the beginning of the experiment. VEGF receptor blockade by SU5416 causes airspace enlargement in rats after 3 wk of a single injection (2). M40419 (Metaphore Pharmaceuticals, Inc., St. Louis, MO) is a macrocyclin ligand, 1,4,7,10,13-pentaazacyclopentadecane, which catalyzes the dismutation of O2- near the catalytic rate of manganese superoxide dismutase, i.e., > 2 x 107 M-1 s-1. A related compound has been used successfully to inhibit inflammatory injury (16). M40419 was dissolved in phosphate-buffered saline (PBS) and administered intraperitoneally 3 times a week, at 2 mg/kg, the upper therapeutic dosage based on prior rodent experimentation (16). Experiments were performed with six animals in each of the following groups: (i) SU5416 {3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin 2-one}+PBS group; (ii) SU5416+ superoxide dismutase mimetic M40419; (iii) M40419+CMC (0.5% carboxymethylcellulose sodium, 0.9% sodium chloride, 0.4% polysorbate 80, 0.9% benzyl alcohol in deionized water); and (iv) control, vehicle-treated (CMC+PBS) group. The experimental details of studies involving the broad spectrum caspase inhibitor zAsp-CH2-DCB (Z-Asp-2, 6-dichlorobenzoyloxymethylketone, an IL-1ß converting enzyme inhibitor III) have been reported in detail elsewhere (2). In these studies, the animal protocol with respect to SU5416 administration and animals was identical to the one used in the present study.

Tissue Processing
After completion of the treatment period, the lungs were expanded with low melting point agarose at 25 cm of water pressure as previously described (2), subsequently fixed in buffered 10% formaldehyde, and embedded in paraffin.

Morphologic and Morphometric Analyses
Sections (5 µm) were stained with hematoxylin and eosin. Alveolar diameter and alveolar septal perimeter were determined by computer-assisted morphometry with the Image Pro Plus software (Media Cybernetics, Silver Spring, MD). Images (five per lung section, three sections per rat) from the upper left lung lobe were acquired with a x10 lens.

Apoptosis Assays
Terminal deoxynucleotidyl transferase-mediated dUTP end-labeling (TUNEL) was performed as reported before (2). TUNEL-positive cells were determined using a macro, written by one of the authors (C.Y.C.), using Image Pro Plus. Images were captured with Nikon Eclipse E800 microscope with a x20 lens (Nikon, Melville, NY) for quantification. Then the counts were normalized by alveolar perimeter using Image Pro Plus' perimeter macro. All of the counts were rechecked by manual counts.

Active Caspase-3 assay was performed with anti–Active Caspase-3 (BD PharMingen, San Diego, CA) as described before (2). Primary antibody or isotype-matched negative control was applied over the sections and incubated for 16 h at 1:4,500 dilutions in 4°C humidity chamber. The reaction was developed with the Vector ABC kit with diaminobenzidine applied for 5 min. The slides were counterstained with methyl green. The number of active caspase-3–positive cells was counted with a macro, using Image Pro Plus, which was validated by manual counts. The counts were normalized by alveolar perimeter.

Single-stranded DNA immunohistochemical assay was performed with the mouse anti-ssDNA (mono, F7–26; Chemicon, Temecula, CA). Sections of paraffin-embedded lungs were incubated in saponin (0.1 mg/ml in PBS), and proteinase K (20 µg/ml in PBS), followed by incubation with pronase E (20 µg/ml in Tris buffer) for 20 min each, at room temperature. Sections were then processed in 50% formamide preheated in water bath to 60°C for 20 min, followed by washing with ice-cold PBS for 5 min. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 5 min, and nonspecific staining was blocked with 3% nonfat dry milk in PBS. Two hundred microliters of monoclonal anti–single-stranded DNA antibody at 1:10 dilution in 1% nonfat dry milk in PBS was applied for 15 min at room temperature. Antibody binding was developed with a peroxidase-conjugated anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA), used at 1:20 dilution in antibody diluent (DAKO, Carpinteria, CA) for 15 min, and development with diaminobenzidine as chromogen substrate with nickel added (Vector laboratory), for 5 min.

DNA laddering was determined by a PCR-based technique as described before (9), using the ApoAlert LM-PCR Kit (CLONTECH, Palo Alto, CA). The bands obtained by ethidium bromide staining of agarose gel were scanned and the second lane from the bottom of the gel was arbitrarily chosen for quantification by densitometry. Samples from all groups were processed, amplified and separated by gel electrophoresis simultaneously.

Quantification of Proliferating Alveolar Septal Cells
Immunostaining for proliferating cell nuclear antigen (PCNA) was performed with DAKO Envision plus monoclonal kit (DAKO), following the manufacturer's instructions. Quantification of alveolar septal cells immunoreactive for the PCNA was determined by computer-assisted morphometry as outlined for the apoptosis assays.

Western Blot and Immunoprecipitation for Akt and VEGFR 2
The following antibodies were used: Akt-1 (Upstate Biochem, Lake Placid, NY), Phospho-Akt (Upstate Biochem), Phosphotyrosine (Upstate Biochem), FLK-1 (A32) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse horseradish peroxidase (Amersham, Piscataway, NJ), anti-rabbit horseradish peroxidase (Amersham). The procedures have been described elsewhere (2).

Markers of Oxidative Stress
A TISSUE-TEAROR homogenizer (BioSpec Products, Bartlesville, OK) set at 50% of maximal speed was used to homogenize 100 mg of tissue in 500 µl of a buffer containing 10 mM HEPES, 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.6 mM MgSO4, 1.1 mM ethylenediamine tetraacetic acid pH 7.4 supplemented with a protease inhibitor cocktail, 0.2 mM phenylmethyl sulfonylfluoride and 50 mM DTT for 10–20 s on ice. Triton X-100 was added to 0.1% lysate incubated on ice for 5 min and clarified by centrifugation for 10 min at 12,000 x g at 4°C. Supernatant was collected, and 50-µl aliquots were stored at -80°C. Protein concentration was determined by the Bradford assay method (BioRad, Hercules, CA).

The slot-blot method was based on the one described by Robinson and coworkers (17). Total lung protein samples (1.5 µg) were diluted in a buffer containing 85.7 mM Tris, 0.857 mM ethylenediamine tetraacetic acid, 20 mM NaOH, pH 8.5 and applied to a polyvinyl difluoride membrane (PVDF; BioRad) membrane cut to fit a plexiglass slot blot apparatus (GIBCO-BRL; Carlsbad, CA). The membrane and two sheets of thick blotting paper were equilibrated in 100% MeOH (methanol) for 5 min, followed by incubation in TBS-MeOH (20 mM Tris, 500 mM NaCl, pH 7.4, 20% methanol) for 5 min and placed on the apparatus, with the membrane facing upward. The sample was applied and allowed to bind for 20 min with no vacuum. The liquid was then gently aspirated by application of a low vacuum to avoid drying the membrane. The membrane was then incubated sequentially in 100% MeOH for 1 min, TBS-MeOH for 5 min, and 2N HCl for 5 min and immersed in dinitrophenyl hydrazone (DNPH) solution (100 µg/ml DNPH in 2N HCl) for exactly 5 min. The membrane was rinsed in 2 N HCl, and then washed three times (5 min each) in 2 N HCl, followed by seven washes (5 min each) in 100% MeOH. The dry membranes were rehydrated in TBS for 5 min, and nonspecific sites blocked by incubation in TBS-5% nonfat dry milk overnight at 4°C. Membranes were washed three times in TBS-0.05% Tween for 5 min each, and incubated with a rabbit polyclonal anti-DNPH antibody (Molecular Probes, Eugene, OR) diluted 1:25,000 in TBS-5% milk, 1% Tween for 1–3 h at room temperature. The membrane was incubated with 1:5,000 dilution of a goat anti rabbit horseradish peroxidase-coupled secondary antibody (enhanced chemiluminescence; Amersham, Piscataway, NJ) in TBS-5% milk-1% Tween for 1 h at room temperature, then washed six times for 5 min each with TBS-5% milk-1% Tween and once in TBS-0.05% Tween. Signal detection was accomplished using enhanced chemiluminescence followed by exposure to X-ray film (Fuji, Stamford, CT). Autoradiographic images were acquired with a Kodak DC 290 Digital Camera (Kodak, Rochester, NY) and quantified by densitometry with a Kodak 1D Image Analysis software. Membranes were stripped and equal loading of proteins assessed by colloidal gold staining (BioRad). Multiple exposures ensured that the detection was within the linear range.

Oxyblot
Lung protein lysate was resolved by SDS polyacrylamide gel electrophoresis in 5–15% gradient pre-cast gels (BioRad), electroblotted in a buffer containing 25 mM Tris, 192 mM glycine, and 10% methanol onto a PVDF membrane. The membranes were successively washed in methanol and 2N HCl, and the bound proteins derivatized in situ with DNPH as described above. Washes, incubation, and detection of the DNPH-protein adducts were performed as described for the slot-blot method.

Total Lung Glutathione
Total lung glutathione (reduced glutathione and glutathione disulfide) quantification was performed with the Glutathione Quantification Kit (Dojindo Mol. Tech, Inc., Kunamoto, Japan). In brief, lung tissue (50 mg/lung) was homogenized in 5-sulfosalicylic acid, and the particulate cellular debris was removed by centrifugation (8,000 x g) for 10 min. The internal standards consist of varying serial dilutions of glutathione (100, 50, 25, 12.5, 3.13, 1.56, and 0 µM). After 20–30 min reaction, total glutathione was calculated by regression analysis using the GSH calibration curve using glutathione standards (slope = 45.3, correlation coefficient = 0.98).

Immunohistochemical detection of 4 hydroxy-2-nonenal modified proteins (4-HNE) and 8-hydroxy-2'deoxyguanosine (8-HG): Paraffin-embedded sections were processed as described previously. The primary antibody, mouse anti–8-HG (QED Bioscience, San Diego, CA) or rabbit anti–4-HNE (Alpha Diagnostic, San Antonio, TX) antibody was applied at 1:250 or 1:1,000 dilution, and negative controls consisted of isotype-matched control or rabbit serum, respectively. Biotinylated universal secondary antibody and Elite ABC reagent were applied at room temperature for 30 min each. After washing with Tris-buffered saline with 0.05% Tween 20 (Sigma, St. Louis, MO), Vector RED substrate was used as chromogen. Co-localization studies between active caspase 3 and 8-HG were performed in three lungs/each group. First, the reaction for active caspase-3 was performed as described previously, followed by the procedure for 8-HG localization. To address the role of apoptosis blockade by a broad spectrum caspase blocker on oxidative stress caused by SU5416, paraffin blocks of lungs (n = 3/group) treated with SU5416, SU5416-zAsp-CH2-DCB, or vehicle from experiments reported in Ref. 2 were retrieved, and subjected to 8-HG and 4-HNE immunohistochemistry as described above.

Quantification of 8-HG or 4-HNE staining in images captured in a "blinded fashion" was performed with Image Pro. Control-treated lungs were used to set up the lower threshold of detection, followed by masking of background staining, and measurement of the overall area occupied by cells expressing 8-HG or 4-HNE. The area of positive staining was normalized by alveolar septal perimeter. In the colocalization studies, quantification of active caspase 3 and 8-HG was performed in centrilobular (CL), i.e., alveolar structures present along alveolar ducts immediately downstream of a terminal bronchiole, and in peripheral lung tissue (PL), i.e., alveolar structures located away from airways. Three lungs each in the SU5416- and in the SU5416+M40419 groups were used for quantification. The operations were performed by a macro function.

Statistical Analyses
Statistical analysis was performed by analysis of variance (ANOVA), with the selection of the most conservative pairwise multiple comparison method, using the program SigmaStat for the PC platform. Statistical significance of pairwise comparisons was set at P < 0.05 level. Bars in the graphs represent 1 standard deviation of the mean.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of the MnSOD Mimetic M40419 on Emphysema Caused by VEGF Receptor Blockade
We have previously reported that VEGF receptor blockade with SU5416 resulted in a progressive increase of overall protein nitration in lungs, nitration of MnSOD (a key mitochondrial antioxidant enzyme whose activity can be downregulated by nitration [18]), nitrotyrosine immunoreactivity in lung sections, DNPH-modified carbonyl proteins, and isoprostanes (19). To address whether oxidative stress plays a role in the emphysema caused by SU5416 treatment (2), M40419, a MnSOD mimetic, was given to SU5416-treated rats for 3 wk, starting on the day of SU5416 administration. Emphysema induced by VEGF receptor blockade was inhibited by M40419 (Figures 1A–1E). SU5416-treated lungs showed an increased alveolar diameter over that of the other groups (Figure 1E), with ~ 20% more alveoli with an alveolar diameter within the 90–120 µm range than that of SU5416+M40419-, M40419-only, or vehicle-treated lungs. This airspace enlargement in the SU5416-treated lungs occurred at the expense of alveoli within normal diameter range (70–90 µm). Approximately 4% of alveoli in this group measured more than 120 µm in diameter, whereas no alveolar measurement in the SU5416+M40419 or control groups fell in this extreme tertile (Figure 1F). The tertile range distribution of alveolar measurements in the SU5416+M40419 lungs was identical to that of the control group (Figure 1F).



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  		Figure 1. Lung histomorphometry. SU5416-treated (a) lungs show increased alveoli in comparison with SU5416+M40419-treated (b), M40419-alone (c), or vehicle-treated (control) lungs (d) (bar = 100 µm). (e) Alveolar diameters of SU5416 (SU), SU5416+M40419 (SU+M), M40419 (M), and control (CTL) lungs. Fifteen fields we measured/lung (total = 90 measurements/group). Shown are medium, 25th–75th (bar ends) and 5th–95th (dots) range of individual measurements. (SU versus SU+M, SU versus M, SU versus CTL, P < 0.05, by ANOVA, with all pairwise multiple comparison procedures by the Dunn's Method). (f) Frequency of alveolar diameter measurements. Results are displayed as the fraction of total alveolar measurements which fell in the normal tertile (70–90 µm in diameter), or moderately enlarged tertile (90–120 µm), or markedly enlarged tertile (> 120 µm in diameter). The vast majority (90%) of normal alveoli fall in the lower tertile, whereas SU5416 treatment results in reduction of normal size alveoli, and a pronounced increase in alveoli falling in the mid and higher tertile ranges. M40419 treatment, alone or in combination with SU5416, results in alveolar sizes similar to control lungs. Black bars, SU; hatched bars, SU+M; striped bars, M; open bars, CTL.

 
Effect of MnSOD Mimetic M40419 on Indices of Lung Apoptosis and Cell Proliferation
Because SU5416-induced emphysema involves increased alveolar cell apoptosis (2), we assessed whether the mimetic might prevent SU5416-mediated apoptosis. Lungs treated with the combination M40419 and SU5416 had less active caspase 3– (Figures 2A–2E) and TUNEL-positive cells (results not shown) than SU5416 alone. Active caspase 3–positive cells preferentially localized to the centrilobular region, which was the site of airspace enlargement in SU5416-treated lungs (Figure 2A). Oligonucleosomal DNA fragmentation (Figure 2F) and detection of single-stranded DNA, which specifically highlights apoptotic cells with single-stranded DNA breaks (see ONLINE SUPPLEMENT), confirmed the protective effects of M40419 on SU5416-induced alveolar septal cell apoptosis.



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  		Figure 2. Expression of markers of apoptosis. Active caspase 3 expression (arrows) in SU5416- (a, b), and SU5416+M40419-treated lungs (c, d). Note the preferential centrilobular (CL), localization of active caspase 3–positive cells (arrows), which becomes more infrequent and scattered in peripheral (PL) lung tissue in SU5416-treated lungs. The centrilobular alveolar ducts and alveoli can be traced to a proximal terminal bronchiole (b). The cells expressing active caspase 3 are distributed along alveolar septa and in alveolar corners at the confluence of adjacent alveolar septa. SU5416-M40419–treated lungs show significantly less active caspase 3–positive cells (c and d). (a and c: bar = 50 µm; b and d: bar = 10 µm). (e) Quantification of active caspase 3 immunostaining, with results corresponding to analysis of 15 microscopic fields each (x20 magnification) of two lungs in the SU5416 (SU)- and SU5416+M40419 (SU+M)-treated groups and three lungs each in the M40419 (M)-treated and control (CTL) groups. Positive cells are expressed as per alveolar perimeter (expressed in µm) (SU versus SU+M, SU versus CTL, SU versus M, SU versus CTL, P < 0.05, by ANOVA, with all pairwise multiple comparison procedures by the Dunn's Method). (f) DNA oligonucleosomal laddering, showing increase in DNA fragments in SU5416-treated lungs (SU) as compared with SU5416+M40419 (SU+M), M40419 (M), and control (CTL) lungs. Shown are representative two lung samples of six lungs per group.

 
M40419 significantly increased alveolar septal cell expression of the proliferation marker PCNA in rats that received SU5416 as compared with SU5416-only treated lungs (Figure 3).



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  		Figure 3. Lung alveolar cell proliferation. M40419 leads to increased cell proliferation in lungs treated with SU5416 (b), as compared with SU5416-alone–treated lungs (a) as assessed by immunohistochemical detection of proliferating cellular nuclear antigen (PCNA). (c) M40419-only–treated lung. (d) Control lung. (e) Quantification of PCNA-positive cells in SU5416-treated (SU), SU5416+M40419 (SU+M), M40419 (M), and control (CTL) lungs. Data are based on analyses of 20 microscopic fields (x20 magnification per lung, two lungs per group). Positive cells are expressed as per alveolar perimeter (expressed in µm) (SU versus SU+M, SU versus CTL, M versus CTL, P < 0.05, by ANOVA, with all pairwise multiple comparison procedures by the Dunn's Method).

 
VEGF signaling protects against endothelial cell apoptosis through recruitment of PI3 kinase to a multimeric complex with VEGFR 2, followed by activation of Akt (20). We next asked whether oxidants are involved in SU5416-mediated disruption of survival signals. SU5416-treated lungs showed reduced levels of phosphorylated Akt, as previously shown by us (2). The use of the MnSOD mimetic resulted in significantly higher levels of phosphorylated Akt in SU5416-treated lungs when compared with SU5416 alone (Figure 4). The levels of phosphorylated Akt were similar in lungs treated with the combination of M40419 and SU5416 and those in the control or M40419-only treated groups. M40419 treatment led to increased levels of phosphorylated VEGFR 2 in SU5416-treated lungs, approaching statistical significance with a P = 0.07 by ANOVA (results not shown).



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  		Figure 4. Phosphorylated Akt (phosphoAkt) expression obtained by immunoprecipitation (Ippt) with anti-Akt antibody and Western blot (WB) with phosphoAkt antibody. Upper panel shows representative images of two lungs per group (SU5416+M40419 [SU+M], SU5416 [SU], M40419 [M], and control [CTL] lungs. Quantification by scanning densitometry was obtained from data of six lungs per group and shown in lower panel (SU versus SU+M, P < 0.5, ANOVA, with pairwise multiple comparison procedures by the Tukey Test).

 
Effect of the MnSOD Mimetic M40419 on Lung Markers of Oxidative Stress
Figure 5A shows that SU5416 treatment for 3 wk increased the levels of total carbonyl proteins (63,390 ± 4,372 SD) scanning densitometric units as evidenced by increased DNPH-immunoreactivity by slot blot analysis. When the MnSOD mimetic M40419 was given with the VEGF receptor blocker, levels of carbonyl proteins (48,315 ± 5,330 SD) were lower than even in the control vehicle– (55,662 ± 1,793 SD) or mimetic alone–treated lungs (60,378 ± 6,624 SD). To determine whether some particular proteins are more susceptible to oxidation, immunoblot analyses with an anti-DNPH antibody were performed. An overall increase in reactivity of multiple carbonyl proteins was observed (data not shown) and a protein of ~ 200 kD was found to have higher carbonyl content in the SU5416-treated lungs when compared with control (Figures 5B). Interestingly, M40419 inhibited the increase in carbonyl content of this protein in SU5416-treated lungs.



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  		Figure 5. M40419 decreases expression of markers of oxidative stress in SU5416-treated lungs. (a) Upper panel: slot-blot analysis of carbonylated proteins after derivatization by DNPH and reaction with anti-DNPH modified proteins. Shown are four lungs in SU5416+M40419- and SU5416-treated groups. Lower panel: oxyblot analysis showing increased carbonylation of proteins of 200 and 28 kD in SU5416-treated lungs (SU, n = 4 lungs) when compared with SU5416+M40419 (SU+M, n = 5), M40419 alone (M, n = 5) and control (n = 4) lungs. (b) Quantification of bands by scanning densitometry is shown in bottom graph (SU versus SU+M, SU versus M, and SU versus CTL, P < 0.05, ANOVA, with pairwise multiple comparison performed with the Tukey test).

 
M40419 administration to SU5416-treated rats resulted in a trend toward increased lung levels of total glutathione, whereas M40419 alone or SU5416 alone expressed similar glutathione levels as in control lungs (Table 1). The comparison among groups was however not statistically significant by one factor ANOVA with analyses on ranks.


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  		TABLE 1 Glutathione lung levels and quantification of 8-HG and 4-HNE immunohistochemical expression in SU5416-treated (SU), SU5416+M40419 (SU+M), M40419 (M) and vehicle control (CTL) lungs

 
Immunohistochemistry reliably detects adducts formed by oxidant attack on macromolecules and allows for precise localization of lung cellular sites of oxidative stress (21). In agreement with the markers of oxidative stress based on whole lung lysates (Figure 5), SU5416-treated lungs had the highest level of localization of 4-HNE (also a marker of protein modification by lipid peroxides) and 8-HG, a marker of DNA damage by oxidative stress (Figure 6). The immunohistochemical signal was localized along alveolar septa (Figure 6C, insert), with a clear predominance of expression along centrilobular alveolar ducts when compared with that in the periphery of the alveolar lobule. In SU5416+M40419-treated and control lungs, 8-HG and 4-HNE were predominantly localized in macrophages and type II cells, and infrequently along alveolar septa (Figures 6E–6H). Quantification of 8-HG and 4-HNE expression confirmed the significantly higher level of oxidative stress in SU5416-treated lungs when compared with all other groups (Table 1).



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  		Figure 6. Immunohistochemical detection of 8-hydroxy-2-guanosine modified proteins (a, c, e, g) and 4-hydroxy-2-nonenal (b, d, f, h) in neuronal cells and glial plaques of brain sections with Alzheimer's disease (positive control, a and b), SU5416-treated lungs (c and d), SU5416+M40419-treated lungs (e and f), and control lungs (g and h). Bar = 10 µm. Note the increased expression of both markers of oxidative stress (red reaction product) in SU5416-treated lungs, in particular, along alveolar ducts (arrows in all panels). In inset in c, a type II pneumocyte is shown with a strong reactivity for 8-hydroxy-2'-guanosine. Lungs treated with SU5416+M40419 expressed markers of oxidative stress similar to that in control lungs, predominantly in alveolar macrophages (arrowheads) and focally along alveolar septa (arrows). Type II cells (dashed arrows), located at the corners of converging alveolar septa, expressed the oxidative stress markers albeit less abundantly than in SU5416-treated lungs. Figures are representative of one lung of three stained lungs (bar = 10 µm).

 
In immunohistochemical co-localization studies of SU5416-treated lungs revealed that active caspase 3 and 8-HG were preferentially co-distributed along centrilobular lung tissue when compared with the periphery of lung lobule (Figures 7A–7D). There was a significant correlation between intensity of 8-HG expression and numbers of active caspase 3 positive alveolar cells in SU5416-treated lungs (Figure 7E). When compared with the latter lungs, co-localization studies confirmed the lower levels of apoptosis and 8-HG in SU5416+M40419 lungs, and a uniform coexpression of both markers in different regions of the pulmonary acinus. These findings were confirmed morphometrically (results not shown).



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  		Figure 7. (a and b) Co-localization of active caspase 3–positive cells and 8-HG in the centrilobular lung (a) or periphery of lung lobule (b) of a SU5416-treated lung. The peripheral alveolar lung parenchyma (PL) shows less intense 8-HG expression (red reaction product) and fewer active caspase 3–positive cells (arrows, black reaction product) than that seen in centrilobular lung tissue (CL). Note that in (b), focal, more intense, 8-HG staining predominated in lung fields closer to centrilobular areas (CL, arrowheads) and in pulmonary artery walls (V). Figures are representative of three SU5416-treated lungs (bar = 50 µm). (c and d) Quantification of active caspase 3–positive cells (c) and 8-HG expression (d) in centrilobular (CL) or perilobular (PL) regions of SU5416-treated lungs. Active caspase 3–positive cells (c) and 8-HG expression (d) are significantly higher in CL alveolar structures when compared with PL regions (P < 0.01, Mann-Whitney Rank Sum Test). Data are based on analyses of a total of 36 microscopic fields (x40 magnification, n = 3 lungs). Positive cells are normalized by alveolar perimeter (expressed in µm). Regression analysis demonstrates that there is a significant positive correlation between number of active caspase 3–positive cells and 8-HG expression in SU5416-treated lungs. Each circle represents a pair measurement obtained in a lung field.

 
Apoptosis Blockade with the Broad Spectrum Caspase Inhibitor zAsp-CH2-DCB Reduces Oxidative Stress Induced by VEGF Receptor Blockade
The results described thus far demonstrate that, in the VEGF receptor blockade–induced emphysema, SU5416 administration causes lung oxidative stress, and that a superoxide dismutase mimetic prevents alveolar cell apoptosis and emphysema. To address whether lung cell apoptosis and oxidative stress are part of a mutually positive feedback loop due to SU5416 receptor blockade, i.e., whether apoptosis is causally associated with oxidative stress, we assessed lung oxidative stress by immunohistochemical localization of 8-HG and 4-HNE in sections of lungs (n = 3 per group) treated with SU5416, SU5416+zAsp-CH2-DCB, or vehicle (2). Control lungs had only focal alveolar cell and macrophage reactivity for the markers of oxidative stress (Figure 8). Alveolar septal cell localization of 8-HG and 4-HNE in lungs treated with SU5416 and zAsp-CH2-DCB was significantly reduced when compared with SU5416-treated lungs, yet increased when compared with control lungs (Figures 8 and 9).



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  		Figure 8. 8-HG immunostaining of SU5416-treated (a), or SU5416+zAsp-CH2 (b), and control lungs (c). Apoptosis blockade with zAsp-CH2 resulted in marked reduction of septal 8-HG immunoreactivity along alveolar septa (arrows) in SU5416-treated lungs (b) when compared with SU5416-alone (a) (arrows). Control lungs had patchy 8-HG expression (c). Representative images of one of three lungs per group.

 


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  		Figure 9. Quantification of 8-HG (left) and 4-HNE (right) immunostaining of control lungs, SU5416-treated, or SU5416+zAsp-CH2, with three lungs per group, 12 fields per lung (CTL versus SU, CTL versus SU+Zasp-CH2, and SU versus SU+Zasp-CH2, P < 0.001, ANOVA, with pairwise multiple comparison performed with the Dunn's test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings underscore the critical role of oxidative stress and apoptosis in alveolar destructive enlargement caused by VEGF receptor blockade. The requirement of the quiescent pulmonary microcirculation on VEGF survival signals and its sensitivity to oxidative stress may account for the lung's unique enhanced susceptibility to VEGF receptor blockade. VEGF is required for endothelial cell protection against apoptosis in models of rapidly growing vessels during fetal development or tumor angiogenesis (5). Furthermore, it is well recognized that apoptotic death due to growth factor withdrawal is mediated by oxidative stress (4, 6, 22).

Oxidative stress leads to free radical attack on DNA, lipids, and proteins. When oxidants react with membrane phospholipids, lipid peroxides are produced. Their decomposition products react with proteins leading to "carbonylated" proteins, which, when detected by their reaction with DNPH or by means of detection of 4-HNE–modified proteins, are a measure of lipid peroxidation. We have previously observed that the pattern of elevation of indices of oxidative stress coincides temporally with the peak of DNA oligonucleosomal fragmentation (Day 3–7 [2]), and, in the 2 wk thereafter, the persisting oxidative stress followed the pattern of expression of active caspase 3 in SU5416-treated lungs (2). These observations suggest to us a mechanistic link and, perhaps, a positive feedback loop between lung alveolar cell apoptosis due to VEGF blockade and oxidative stress. This link was further corroborated by the finding of centrilobular co-localization of cells expressing active caspase 3 and undergoing oxidative stress assessed by 8-HG immunostaining. Furthermore, SU5416 lungs had a significant correlation between number of apoptotic cells and localization of 8-HG. In contrast, a marked reduction of markers of oxidative stress was found in lungs treated with the VEGF receptor blocker and the apoptotic inhibitor zAsp-CH2-DCB. This bidirectional relationship between apoptosis and oxidative stress adds a novel perspective on the role of apoptosis in disease because most studies have emphasized the angle of oxidative stress causing apoptosis, with far less data on the effect of apoptosis leading to oxidative stress (13, 23).

The initiating events early in the course of VEGF receptor blockade in the lung, which trigger an amplification feedback loop of oxidative stress and apoptosis, may be causally related to incipient endothelial cell apoptosis or to endothelial cell dysfunction due abrogation of VEGF signaling. Indeed, recent evidence indicates that, in vitro, VEGF upregulates MnSOD (24) and BCL-2 expression (20), which have been shown to increase cell survival and reduce oxidative stress (15). In addition, VEGF signaling inhibition may lead to oxidant/antioxidant imbalance by means of decreased activities of endothelial cell nitric oxide synthase and prostacyclin synthase (25, 26). Nitric oxide can scavenge O2- and blocks caspase activation (27), and prostacyclin induces glutathione synthesis (28), an important intracellular and extracellular antioxidant.

The interruption of VEGF survival signals may be interpreted by lung cells as "danger signal," resulting in oxidative stress as a downstream response (14). In the process of apoptosis, as effector caspases are activated, free radicals may be produced by nonmitochondrial sources (for example, xanthine oxidoreductase and aldehyde oxidase; NADPH or NADH oxidoreductases; cytochrome P-450, nitric oxide synthase, and arachidonic acid metabolizing enzymes [29]) or due to progressive mitochondrial dysfunction with dissipation of the mitochondrial membrane potential and participation of the permeability transition pore signals. Imbalance between oxidants and antioxidants, i.e., oxidative stress, may reset the threshold of lung injury and amplify apoptotic processes in emphysema.

In the present study, there was a significant co-localization of oxidative stress and apoptosis to centrilobular regions vis-à-vis the periphery of the alveolar lobule in SU5416-treated lungs, whereas most animal models, including the intrabronchial elastase instillation and cigarette smoke inhalation models, are characterized by panlobular involvement (1, 30). The mechanisms involved in the centrilobular destruction in cigarette smoke–induced human emphysema remain unknown. Our data suggest that the alveolar capillary endothelial cells may be heterogeneous with respect to their requirement of VEGF as a survival factor, and the centrilobular vascular bed may ultimately drive the unique susceptibility of this region's alveolar septa to emphysematous destruction.

Preventing oxidative stress with M40419, a MnSOD mimetic, had a significant protective effect on lung structure despite ongoing VEGF receptor blockade. Furthermore, we observed identical protection with the superoxide mimetic MnTE-2-PyP5+ pentachloride (19). The fact that two chemically distinct free radical scavengers have similar protective effects against SU5416-induced emphysema argues against the possibility of direct binding and inactivation of SU5416 by M40419. Oxidative stress abrogation with M40419 treatment led to a shift of the net balance of apoptosis and cell proliferation when compared with VEGF receptor blockade alone, thus preventing the development of emphysema. It is conceivable that apoptosis of alveolar endothelial cells destabilizes the tight cellular organization and interdependence of the alveolar septum and may thus increase the susceptibility to further cellular injury, with damage to alveolar epithelial or septal myofibroblast cells (14). Apoptosis-triggered oxidative stress may itself increase expression and activate proteases as demonstrated in epithelial detachment assays with human amniotic membranes and in cultured endothelial cells (31, 32). Proteases would then act downstream of oxidative stress and apoptosis in alveolar septal destruction.

Our data indicate that inhibition of oxidative stress has a broader biological impact than singly decreasing apoptosis in that it also increased alveolar cell proliferation after VEGF receptor blockade. Because we did not find an excess of alveolar septal cells or a polyalveolated pattern in the MnSOD mimetic+SU5416-treated lungs, the observed cellular proliferation may have compensated for some degree of early apoptosis (2). The resetting of oxidative stress to physiologic levels by the SOD mimetic may have relayed growth and repair signals to lung cells (14), as highlighted by our finding of increased expression of active lung Akt in the animals treated with M40419. By blocking oxidative stress in the setting of VEGF receptor blockade, we might have increased the antioxidant defenses in the lung and interfered with a cell injury cascade triggered by dysfunction of lung capillary endothelial cells. Preliminary evidence supports the idea that the MnSOD mimetic can be used to partly reverse established emphysema due to VEGF receptor blockade, as shown with all-trans retinoic acid treatment in emphysema induced by pancreatic elastase instillation (33).

Consistent with the requirement of tightly regulated levels of oxidative stress for proper lung cell signaling, administration of M40419 alone led to decreased cell proliferation and reduction of phosphorylated Akt levels when compared with control rats. However, these alterations were not pathophysiologically relevant. Based on our data, it is necessary to have a heightened oxidative stress to achieve protection by MnSOD mimetic supplementation.

Several of the findings of the present study may have relevance to cigarette smoking–induced human emphysema. Chronic cigarette smoking leads to increased levels of markers of oxidative stress because every cigarette smoke puff contains more than 1015 oxidant molecules (34). Many of the oxidative markers in our model of emphysema caused by VEGF receptor blockade, such as increased detection of nitrotyrosine, 8-HG, 4-HNE, MnSOD nitration, isoprostanes, and carbonyl proteins are also present in human emphysematous lung (R. M. Tuder, unpublished observations, and Ref. 35). In addition, cigarette smoke extract downregulates VEGF expression by epithelial cells, causes apoptosis of endothelial cells, and decreases VEGF-dependent survival of cultured endothelial cells (R. M. Tuder, unpublished observations). Most importantly, emphysematous lungs have increased numbers of apoptotic alveolar septal cells (9, 11) and decreased expression of VEGF and its receptor 2 (9). We conjecture that chronic cigarette smoke may act as source of oxidative stress and may interrupt VEGF-dependent maintenance signals in the lung.

Our concept of a failure of the lung cellular and molecular maintenance program by VEGF receptor blockade, which triggers lung oxidative stress, apoptosis, and emphysema, provides a framework to test the mechanisms of lung damage in other emphysema models and after chronic cigarette smoke inhalation. Oxidative stress, as an amplifier and modifier of the lung cell response to interruption of cell maintenance signals, together with alveolar septal cell apoptosis, may constitute a positive feedback loop common to human and experimental emphysema. Interruption of this feedback loop with caspase inhibitors or free radical scavengers may arrest lung destruction and may lead to improved lung repair in this disease.


   Acknowledgments
 
This work was supported by NIH grants to R.M.T. (1RO1 HL60195) and S.C.F. (2RO1 HL59785). The authors thank SUGEN, Inc. for providing SU5416, Drs. Gregg Semenza and Peter Henson for reviewing the manuscript, and Amy Allred for the expert secretarial help.


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

Received in original form October 4, 2002

Received in final form December 25, 2002


   References
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 Introduction
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 Results
 Discussion
 References
 

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