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HIGHLIGHTED TOPICS
Lung Growth and Repair
Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623
Submitted 27 February 2004 ; accepted in final form 9 June 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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vascular endothelial growth factor; caspase; cre recombinase; apoptosis
knockout, increased surfactant biosynthesis and improved survival rates (6). Likewise, overexpression of pulmonary IL-13 protected mice from hyperoxic lung injury through a VEGF-dependent pathway, and adenoviral-delivered VEGF has been reported to increase survival from hypoxic pulmonary hypertension (7, 27). Thus VEGF appears to be an important factor in lung injury, repair, and survival. VEGF protects endothelial cells from apoptotic cell death through the coordinate signaling of phosphatidyl 3-kinase/Akt and inhibition of p38 MAPK (12, 13). Furthermore, insufficient maintenance of VEGF levels has been reported to inhibit capillary formation in neonatal mice and to lead to regression of newly formed vessels in the retina, heart, and liver (3, 8, 11). A limited number of studies have examined the consequences of VEGF inactivation in adult organs (30). In patients with severe emphysema, VEGF levels are reduced in the bronchoalveolar lavage, sputum, and lung tissue (18, 21). These findings would suggest that insufficient pulmonary VEGF levels may impede the ability of pulmonary endothelial or epithelial cells to repair from injurious aldehydes and oxidants found in cigarette smoke. Recently, Kasahara and colleagues (19) blocked the action of a VEGF receptor VEGFR-2 (KDR/Flk-1) in rats through chronic administration of the chemical inhibitor SU5416. Loss of VEGFR-2 function resulted in increased alveolar size and septal cell apoptosis without a change in the inflammatory cell profile (5, 19). Furthermore, biopsies of human emphysema tissue revealed apoptosis of both alveolar epithelial and endothelial cells (18). Apoptotic cells have also been detected in mice with emphysema due to elastase instillation (24), and recently the direct administration of the apoptotic enzyme caspase-3 or nodularin, a proapoptotic serine/threonine kinase inhibitor, was found to rapidly and transiently (within hours) induce lung apoptosis that led to changes in alveolar structure and lung mechanics that persisted for 15 days (1).
Through the use of a Cre-LoxP strategy to site-specifically ablate the pulmonary VEGF gene, the importance of VEGF for the maintenance of alveolar structure and lung mechanics may be determined. We were, therefore, interested in the effects of long-term downregulation of pulmonary VEGF on the lung pressure-volume relationship, alveolar size, and apoptosis of alveolar septal cells. Permanent destruction of alveolar septal wall structures and decreased elastic recoil would be consistent with emphysema. As such, structural and mechanical measurements were made at 5 and 8 wk after-infection of VEGFloxP transgenic mouse lungs with an adeno-associated cre recombinase virus (AAV/Cre).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Recombinant AAV/Cre construction and intratracheal delivery.
An AAV helper-free system (Stratagene, La Jolla, CA; Ref. 37) was used for the production of AAV, which expresses cre recombinase (AAV/Cre) or control viruses AAV/EGFP and AAV/LacZ. These recombinant viruses were generated by using the complete reading frame of the cre recombinase gene (14) or control genes in addition to the strong cytomegalovirus promoter and SV40 poly A sequence to create a pAAV-Cre plasmid that carries the gene cassette between two AAV2 long terminal repeats and allows recombinant AAV/Cre to be assembled in 293 packaging cells by a standard calcium phosphate transfection method (CalPhos mammalian transfection kit, BD Sciences Clontech, Palo Alto, CA). Two hundred ninety-three packaging cells were collected 4 days after plasmid transfection, and recombinant AAVs were released by use of a freeze-thaw cell lysis method. Viral titer was estimated by comparison of known amounts of pAAV-Cre plasmid and serial dilutions of slot-blotted viral supernatant hybridized to a 32P dCTP oligo-labeled pAAV-Cre plasmid probe (Prime-It II random primer labeling kit, Stratagene, La Jolla, CA).
VEGFloxP and C57BL/6J mice were anesthetized with halothane and tracheally instilled with 100 µl of AAV/Cre, AAV/EGFP, or AAV/LacZ supernatant (2 x 1010 total viral particles) suspended in MEM (Invitrogen Life Technologies, Carlsbad, CA) immediately followed with a 200-µl bolus of perflubron (Rimar 101, Miteni, Tissino, Italy) to aid in viral delivery and distribution throughout distal lung regions. The mice were then kept at a 45° angle for 
1 min, during which time supplemental 100% oxygen was administered. Mice generally awoke from anesthesia within 1–2 min of halothane being discontinued and were kept in an oxygenated (10 l/min) plastic chamber warmed to 37°C for at least 15 min to aid in recovery.
Lung VEGF, flk-1, PCNA, and caspase-3 levels.
Frozen lung samples were homogenized in 50 mM Tris·HCl, 150 mM NaCl, 1% Triton X-100, and protease inhibitor complete tablet with EDTA (Roche Diagnostics, Indianapolis, IN). Protein concentrations were measured using the Bio-Rad Dc protein assay (Bio-Rad, Hercules, CA). Fifty micrograms of lung protein were denatured in loading buffer (0.25 M Tris pH 6.8, 20% glycerol, 4% SDS, 0.2 M DTT, 0.1% 
-mercaptoethanol, and 0.05% bromophenol blue) by boiling for 5 min and were electrophoresed on a 12% polyacrylamide gel. After electrophoretic transfer to Immobilon-P membrane (Millipore, Bedford, MA), blots were probed with the following primary antibodies: rabbit polyclonal anti-VEGF (147) (sc-507, 1:500 dilution), rabbit polyclonal anti-Flk-1 (C-20) (sc-315, 1:200), goat polyclonal anti-PCNA (C-20, 1:500), or goat polyclonal anti-caspase-3 p20 (L-18) (sc-1225, 1:500), all purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) and detected with either mouse anti-goat IgG-HRP (sc-2354, Santa Cruz Biotechnologies) or anti-rabbit IgG-HRP (NA934, Amersham Life Science, Arlington Heights, IL) and enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech UK Limited, Little Chalfont, UK).
Immunohistochemical localization of VEGF, cre recombinase, and PCNA. The same primary antibodies described above for use in Western analysis were used to detect localized expression by immunohistochemical assay. To prepare paraffin-embedded lung sections, mice were anesthetized with pentobarbital sodium (40 mg/ml ip) and ventilated with 100% oxygen for 10 min. Lungs were then carefully removed and fixed with 4% paraformaldehyde by tracheal instillation at a pressure of 20 cmH2O while simultaneously immersed in paraformaldehyde solution overnight. Fixed lungs were then paraffin embedded and 7-µm were sections prepared on Vectabond (Vector Laboratories, Burlingame, CA)-treated glass slides (Fisherbrand Superfrost/Plus microscope slides). For VEGF detection, sections were deparaffinized, rehydrated, and sequentially blocked with avidin and biotin solutions (Avidin-Biotin Blocking Kit, Vector Laboratories) and 2% normal goat serum (30 min) and incubated with mouse-specific VEGF antibody (1:50 dilution) for 2 h followed by biotinylated anti-rabbit IgG (heavy and light) (1:1,000 dilution, Vector Laboratories) for 30 min. Signal was detected with Vectastain Universal ABC-AP reagent (30 min) and Vector Red substrate (30 min). Sections were counterstained with aqueous hematoxylin (Biomedia, Foster City, CA), dehydrated, cleared, and mounted with VectaMount mounting medium.
For cre recombinase detection in paraffin-embedded sections, an additional antigen retrieval step with 2.5 mg/ml pepsin-Tris solution, pH 2.0 (BioGenex, San Ramon, CA) for 5 min at 37°C was performed. Endogenous peroxides were quenched with 3% H2O2 in methanol (10 min); slides were sequentially blocked with avidin and biotin solutions and 5% normal goat serum and incubated for 1 h with rabbit polyclonal anti-cre antibody at 1:1,000 dilution (Novagen, Madison, WI), followed by anti-rabbit biotinylated secondary antibody. Signal was detected using Vectastain ABC reagent (30 min) and DAB (brown) substrate (6 min). PCNA detection was accomplished with a high-temperature (95°C) antigen-retrieval step with DAKO Target Retrieval Solution (DAKO, Carpinteria, CA) for 40 min. This step was again followed by avidin-biotin blocking steps and an additional block in 20% bovine serum-3% BSA-0.1 M Tris for 30 min. PCNA primary antibody was applied at a 1:25 dilution in 2% serum-1% BSA-PBS for 1.5 h. Specific antibody binding was detected with biotinylated anti-goat IgG (1:1,000), ABC-AP-0.1% Tween 20 reagent, and Vector Red substrate (30 min). Immunohistochemical analysis for each antibody was repeated with sections from three separate WT or VEGFloxP mice.
Estimation of air space enlargement by mean linear intercept. A point-count morphometric technique was used to assess air space enlargement according to a modification of the method of Thurlbeck (32). Lungs from all mice were fixed at 20 cmH2O as described for immunohistochemical studies. Multiple digital images (at least 20 images per lung) were systematically taken at a x10 magnification of the entire cross section of paraformaldehyde-paraffin-embedded lungs. Images were overlaid with a 10 x 10 grid (1 mm2), and the mean linear intercept (MLI) was established from every second image (i.e., in a checkerboard fashion) such that 50% of the entire cross-sectional area was counted. Distribution of MLI for each mouse was assessed by frequency distribution analysis and characterized by use of a Gaussian model.
Evaluation of static lung mechanics. The relationship between airway pressure and lung volume was measured in lungs isolated from WT and VEGFloxP mice at 5 and 8 wk postinfection. Mice were anesthetized with pentobarbital (40–60 mg/kg ip), the trachea was cannulated, the chest was opened, and heparin (100 units) was injected directly into the right ventricle. The heart and lungs were then carefully removed and suspended. Airway pressure was continuously monitored with a force-displacement transducer (Grass model F10E) and chart recorder. A 1-ml tuberculin syringe, graduated every 0.01 ml, was used to incrementally (0.05-ml increments) inflate and deflate the lungs. Airway pressure of 30 cmH2O was used as a target pressure for inflating control lungs, but VEGF-inactivated lungs could not withstand an airway pressure >20 cmH2O.
Caspase-3 enzyme activity.
Caspase-3 activity in lung homogenates was measured by using a caspase-specific peptide conjugated to the chromophore, 
-nitoanaline, and detected by spectrophotometer at 405 nm (Caspase-3 Colorimetric Kit, R & D Systems).
Apoptosis assay. Lung paraffin sections were first labeled with the endothelial cell marker factor VIII-related antigen (Von Willebrand factor), before assay for apoptotic, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. To allow antibody access, the slides were immersed for 5 min in DAKO target retrieval solution prewarmed to 95°C. As described above, sections were avidin-biotin blocked, PBS washed, blocked with 10% normal goat serum (10 min), and incubated with a rabbit polyclonal anti-factor VIII-related antigen (BioGenex, San Ramon, CA) diluted 1:10 in 5% goat serum (30 min). After primary antibody incubation, slides were PBS washed and incubated with biotinylated anti-rabbit antibody (1:500) for 10 min. Each section was again PBS rinsed and incubated with ABC-AP (twice the recommended concentration) for 10 min and detected with Vector Red substrate (30 min). Once a clear endothelial cell signal was confirmed by light microscopy, the slides were PBS rinsed and assayed for the presence of nuclear DNA fragmentation in situ. Each section was incubated with 50 µl of a TUNEL reaction mixture (2.5 µl of TUNEL enzyme, 2.5 µl of TUNEL dilution buffer and 45 µl of TUNEL label; Roche Diagnostics) for 30 min in a 37°C humid chamber. At the end of the labeling period, sections went through PBS rinse steps, were quenched with 3% H2O2, and were blocked in 20% serum-3% BSA-0.1 M Tris (20 min). TUNEL-positive signal was converted by use of a 1:2 dilution of TUNEL POD (Roche Diagnostics) in block solution and detected with a DAB (gray/black) substrate containing nickel (Vector Laboratories).
Statistical analysis. A Student's t-test was used to compare control and experimental group densitometry readings from samples analyzed by Western blot. Control and experimental samples at each time point were electrophoresed on the same gel and transferred for antibody detection. Analysis of variance was used to determine MLI statistical differences between control and VEGF-inactivated lungs, and a Fishers post hoc test was used to distinguish statistical differences at each time point. P < 0.05 was considered significant for all analyses.
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RESULTS |
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-actin levels was observed at either time point (Fig. 1C). In WT mice (Fig. 1Da), VEGF could be detected by immunohistochemistry in large airway epithelial cells and pulmonary artery smooth muscle cells. In the parenchymal region it was seen mostly in the alveolar epithelial cells with less intense expression in fibroblasts. In VEGFloxP mice (Fig. 1Db), diffuse regions of the parenchyma displayed an absence or decreased expression of VEGF compared with WT. By using an anti-cre antibody to detect the distribution of virus-infected cells (Fig. 1E), cre expression was found to be similar in AAV/Cre-infected WT and VEGFloxP mouse lungs with a uniform signal detected throughout the parenchyma and high levels of cre recombinase detected in the large airways (Fig. 1E, a and b). No cre expression was observed in the lungs of mice infected with an adeno-associated virus expressing the enhanced green fluorescent protein (AAV/EGFP, Fig. 1Ec).
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20 cmH2O. At 5 wk, a lung volume of 0.5 ml corresponded to an airway pressure of 21.3 ± 4 cmH2O in WT mice but to only 11.8 ± 3.3. cmH2O in VEGFloxP mouse lungs. At 8 wk postinfection, despite a return in the level of VEGF comparable to WT, lung compliance remained raised, consistent with the increased MLI still observed at this time point. In addition, the variability between animals was reduced compared with that at 5 wk. At 8 wk, inflation to a lung volume of 0.5 ml produced airway pressures of 25.7 ± 2.9 cmH2O in AAV/Cre-infected WT mice, 22.0 ± 0.6 cmH2O in VEGFloxP mouse lungs infected with the reporter virus (AAV/EGFP) and significantly lower values of 9.2 ± 0.3 cmH2O in AAV/Cre-infected VEGFloxP mouse lungs. Thus the AAV/Cre-infected VEGFloxP mouse lungs were inflated to a larger lung volume than control groups at a given pressure at both 5 and 8 wk.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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In the present experiment, the VEGF gene itself [and not the receptor, as in Kasahara et al. (19)] was inactivated through delivery of cre recombinase to the pulmonary airways. VEGF is a well-known and potent mitogen for endothelial cells and has additionally been reported to regulate epithelial cell proliferation (4, 23). Thus both VEGF-producing cells and VEGF-receptor-presenting target cells have the potential of being affected by VEGF-dependent signaling. In the present study, apoptosis was observed in both bronchial epithelial cells and alveolar septal wall cells. This occurred without an accompanying change in cell proliferation. Thus an increase in the rate of cell death without a change in proliferative response led to a net loss of pulmonary cells and an emphysema-like phenotype.
VEGF withdrawal and capillary regression in the lung. Withdrawal of VEGF from newly formed vessels in developing and postnatal organs including the retina, heart, and liver leads to endothelial cell apoptosis and regression of capillary structures (3, 8, 11). The formation of mature vessels (refractory to the withdrawal of VEGF) in the retina is thought to occur through incorporation of pericytes or smooth muscle cells around nascent vessels (3). In addition, we have recently demonstrated in adult mouse skeletal muscle that VEGF is necessary to maintain capillary number under normal activity conditions (30). The present study demonstrates that the lung, an organ containing one of the most abundant amounts of VEGF (26), also requires a sufficient amount of VEGF to continuously maintain the vast network of small blood vessels and capillaries throughout the lung parenchyma. Furthermore, it is the alveolar structures that appear to be most susceptible to the loss of VEGF in this lung-targeted model.
Changes in alveolar structure. Emphysema is characterized by the permanent change in alveolar structure distal to the terminal bronchioles. The transient decrease in VEGF and its receptor VEGFR-2 (KDR) also leads to a concomitant increase in caspase-3 activity and presence of TUNEL-positive cells. Although apoptosis did not persist up to 8 wk after AAV/Cre delivery, changes in alveolar structure (reflected by increased MLI, MLI distribution, and lung compliance) remained. This observation raises the possibility that apoptosis may be a critical step in the development of emphysema. More direct evidence for apoptosis to lead to permanent changes in lung structure was recently reported by Aoshiba et al. (1). Direct delivery of caspase-3 or nodularin (a proapoptotic serine/threonine kinase inhibitor) to the airways led to the rapid initiation of alveolar septal cell apoptosis that was detectable by 2 h and rapidly cleared by 6 h (1). This very transient apoptotic episode resulted in enlargement of the air spaces that lasted for 15 days with only a very modest 14% recovery in mean chord length. Thus lung alveolar septal cell apoptosis possibly due to deficient VEGF levels in emphysema patients may be an early event in the development of air space enlargement and decrease in lung elastic recoil (18, 21, 28).
Absence of pulmonary inflammation.
Cigarette smoke-induced emphysema is characterized by a robust inflammatory response. Current hypotheses implicate matrix metalloproteinase-12 and/or additional matrix metalloproteinases released from macrophages, which initiate release of TNF-, expression of vascular adhesion molecules (e-selectin and VCAM-1), and subsequent neutrophil influx (5, 15). Activation and release of neutrophil elastase then contributes to the breakdown of the extracellular matrix (5). However, similar to experimental VEGFR-2 blockade with the tyrosine kinase inhibitor SU5416 (19) or caspase-3 administration (1), we did not observe an inflammatory response after gene inactivation of pulmonary VEGF. Apoptosis itself would not be expected to generate an immune response. Programmed cell death, or apoptosis, targets cells for phagocytosis as a normal part of development or tissue remodeling in adult organisms. Critical steps in the apoptotic process include DNA fragmentation, cell condensation, and presentation of phosphatidylserine on the membrane surface (9, 16). Recognition of phosphatidylserine by bridging molecules such as SP-A, SP-D, and C1q, along with cells that express phosphatidylserine receptor, allows efficient clearance of apoptotic cells before complete lysis and release of proinflammatory mediators (35). Moreover, in addition to bypassing the innate immune response, apoptotic signals upregulate anti-inflammatory mediators such as transforming growth factor-1 and prostaglandin E2 to suppress inflammation. Our findings suggest that apoptosis without necrosis-triggered inflammation (2) is sufficient to initiate lung remodeling and alter pulmonary mechanics.
It is unclear at this time whether the VEGF deficiency observed in the lungs of cigarette smoke-induced emphysema patients is the cause of apoptosis that leads to alveolar septal wall destruction or is a consequence of cigarette smoke-induced transcriptional inhibition, possibly through reactive oxygen species or toxic aldehyde signaling (22, 29). Toxic agents in cigarette smoke could lead to necrosis and release of proinflammatory agents (36). However, persistent apoptosis could also progress into postapoptotic necrosis, possibly through protease cleavage or oxidative modification of the phosphatidylserine receptor resulting in inefficient cell clearance (16, 34, 35). Furthermore, it is unknown how VEGF inactivation may limit vascular permeability, usually associated with inflammation, and potentially prevent the access of antiproteases (i.e., 1-anti-trypsin, secretory leukocyte proteinase inhibitor, or elafin) that could moderate extracellular matrix breakdown.
An imbalance of proteases and antiproteases in this model of pulmonary VEGF inactivation has yet to be established. However, the role of oxidants has been addressed in the SU5416 VEGFR-2 blockade model. Experiments presented by Tuder et al. (33) suggest that alveolar wall destruction resulting from VEGFR-2 inhibition can be prevented by reducing oxidative stress with the superoxide dismutase mimetic M40419. Antioxidant-dependent preservation of lung structure in SU5416-treated mice was accompanied by increased septal cell proliferation and enhanced phosphorylation of the anti-apoptotic kinase Akt (33). Thus oxidants could influence several cellular pathways with low levels of reactive oxygen species signaling VEGF transcription and cell proliferation and excessive oxidant levels promoting the destructive path to emphysema in the absence of VEGF protection (22, 29, 33).
Overall, this study suggests that VEGF is an important growth factor for the maintenance and protection of normal adult mouse lung. Substantial VEGF gene reduction in adult mice using a Cre-LoxP strategy leads to an emphysema-like phenotype that persisted for at least 8 wk postdeletion. The mechanism by which pulmonary VEGF inactivation leads to permanent changes in lung structure and the contribution of VEGF to cigarette smoke-induced emphysema remains to be fully elucidated.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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B pathway. Int Immunol 12: 1539–1546, 2000. release. Am J Respir Crit Care Med 167: 1083–1089, 2003. and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat Med 8: 702–710, 2002.[ISI][Medline]
and interleukin-1 receptor-deficient mice. Lab Invest 82: 79–85, 2002.[ISI]
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), AAV/Cre-infected VEGFloxP transgenic mice (
), AAV/enhanced green fluorescent protein-infected WT mice (
), and noninfected WT mice (
) 5 (A) and 8 (B) wk after viral delivery. Data represent means ± SD of the pressure-volume value measured during lung deflation; n = 3–4 separate mouse lungs.
