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The transfer of protective genes to the alveolar epithelium can attenuate lung injury if accomplished before its onset. The pathobiology of acute lung injury (ALI) includes formidable hurdles to
gene transfer, including alveoli filled with fluid, inflammatory cells,
and cytokines, all of which may impair gene transfer after the onset of injury. We tested the hypothesis that adenovectors could
efficiently transduce injured alveoli by exposing adult, male
Sprague-Dawley rats to 100% oxygen for 48 or 60 h before endotracheal instillation of either 1 × 109 or 4 × 109 plaque-forming
units of an adenovirus that expresses an Escherichia coli lac Z gene
(ad
-gal) in a surfactant-based vehicle (Survanta). X-gal staining
72 h postinfection revealed transgene expression in all segments
of room air control and hyperoxic lungs infected with either dose
of ad-gal. Net transgene expression in hyperoxic lungs was not
different from room air controls despite the presence of pulmonary edema and severe histologic injury. These findings show that
adenovectors can efficiently transduce the alveoli of acutely injured, edematous lungs. The data indicate that the pathophysiologic processes of ALI do not impair adenoviral-mediated alveolar
gene transfer and provide support for the development of gene
therapies for ALI.
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Keywords: gene therapy; hyperoxia; -galactosidase; pulmonary surfactant; acute lung injury; acute respiratory distress syndrome
A central component of the pathobiology of acute lung injury (ALI) is alveolar epithelial dysfunction (1). This includes increased alveolar permeability, impaired surfactant function, decreased antioxidant function, edema accumulation, and impaired solute transport. Several recent studies have shown that adenoviral-mediated overexpression of protective epithelial proteins such as superoxide dismutase, catalase, interleukin-10 (IL-10) and Na,K-ATPase can improve the lung's ability to withstand injury (2). To affect gene therapy for ALI it will be necessary to transfer genes in the setting of alveolar injury, inflammation, flooding, and collapse. To date, in all prior studies of gene transfer for ALI the lung was transduced before injury; it remains unknown if efficient lung gene transfer can be accomplished subsequent to the onset of acute injury.
It has been previously shown that rats exposed to acute hyperoxia (100% O2 for 60 h) develop a diffuse lung injury that is characterized by increased alveolar permeability, pulmonary edema, and a high death rate due to respiratory failure (6). This model is associated with alveolar flooding (6, 9) and the production of inflammatory mediators that may impair gene transfer (10). In the current study, we tested the hypothesis that adenoviruses could efficiently transduce the alveolar epithelium of rats with a severe lung injury caused by acute hyperoxia.
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INTRODUCTION
METHODS
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Hyperoxic Exposure
The use of animals for these studies was approved by the Northwestern University Institutional Animal Use and Care Committees. Adult, male Sprague-Dawley rats (275 to 285 g) were exposed to > 95% O2 for 48, 60, 64, or 68 h without interruption as previously described (8).
Adenovirus Delivery to Rat Lungs
Adenovirus animals received either 1 × 109 (low-dose) or 4 × 109
plaque-forming units (PFU) (high-dose) of human type 5 (E1a
/E3)
adenoviruses containing either a nuclear localizing Escherichia coli lac
Z gene coupled to a human cytomegalovirus promoter (ad-gal) or no
complementary DNA (cDNA) (adNull) (14). The surfactant-based method of adenovirus delivery to rat lung has been described previously (7, 14) with the following differences: the total volume of surfactant delivery vehicle was reduced to 600 µl to compensate for reduced
lung volumes caused by hyperoxia-induced pleural effusions; intraperitoneal ketamine (12 to 24 mg/kg) and acepromazine (3 to 6 mg/
kg) were used for sedation; and rats were mechanically ventilated
(tidal volume [VT] = 3 ml × 50 breaths/min; fraction of inspired oxygen [FIO2] = 0.21) for approximately 3 to 5 min to attenuate hypoventilation after intubation.
Study Groups
Thirty-four rats were exposed to 60 h of hyperoxia. Four animals were
used for wet/dry weight determinations and confirmation of lung injury after 60 h of hyperoxia (hyperoxic control) and four after 24 h of
recovery in room air. Ten animals receiving ad-gal (six given 4 × 109
and four given 1 × 109 PFU) and three rats given adNull survived intubation, virus instillation, and 72 h of recovery (13 of 26). These animals were compared with room air control rats given ad-gal (four
given 1 × 109 and four given 4 × 109 PFU), adNull (n = 4), or no virus/vehicle (n = 4, room air control). All rats exposed to 64 or 68 h of
hyperoxia died (n = 10/group) during virus delivery and were not included in the results of this study. To control for selection bias based
on survival from hyperoxia and virus delivery, an additional 16 rats
were exposed to hyperoxia for 48 h; four of these rats were used for
wet/dry weight determinations. Of the remaining 12 rats, seven survived infection with ad-gal and 72 h of recovery.
Animal Sacrifice Protocol
Lungs were removed as previously described (3, 14). Blood for hemoglobin determination was collected. In some animals, the right upper
and posterior lobes were removed for the wet/dry weight ratio and tissue hemoglobin measurements. The remaining right lung was used for
-galactosidase activity measurements and the left lung was for X-gal
staining and histologic study.
Wet/Dry Weight Determinations
To provide an estimate of extravascular lung water, wet lung weights were corrected for tissue blood volume and divided by dry lung weights as described elsewhere (8, 15).
Documentation of in Situ -galactosidase Expression
Left lungs were fixed with 0.2% glutaraldehyde/2% formaldehyde in phosphate-buffered saline (PBS) for 24 h at 4° C, then underwent PBS lavage and instillation of X-gal solution for 6 h at 37° C, as described previously (3, 16), before a final wash with PBS, instillation of buffered formalin, paraffin imbedding, and sectioning. Quantitation of transgene expressing cells was accomplished by counting the number of cells with perinuclear blue color in 10 randomly selected high-power fields (hpf) (×200) of longitudinal sections of left lungs.
Quantitative -galactosidase Activity
Right lung segments were homogenized in 3 ml of PBS. Seventy-five
microliters of homogenate was used for measurement of -galactosidase activity using a spectrophotometric assay (Stratagene, La Jolla, CA). Activity was corrected for tissue lysate protein concentration (Bio-Rad, Hercules, CA) and is expressed as units/mg protein.
Statistical Analysis
Differences among groups were assessed using Student's t test. Statistical significance was defined as p < 0.05.
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General Appearance
All of the rats survived hyperoxia but appeared lethargic and tachnypneic. Hyperoxic rats lost an average of 6.81 ± 4.53 g of body weight during the 60 h of hyperoxia. Thirteen hyperoxic rats died during sedation and intubation (13 of 26) after 60 h, and 7 of 12 died after 48 h of hyperoxia. All four animals used for wet/dry weight determinations immediately after 60 h of hyperoxia had bilateral pleural effusions (approximately 4 to 7 ml total volume). Animals studied after 24 h of recovery had small, bilateral effusions; no effusions were noted in rats studied 72 h posthyperoxia. Small pleural effusions (< 0.5 ml) were noted in the four rats used for wet/dry weight measurements after 48 h of hyperoxia.
Wet/Dry Weight Ratios
To obtain a measure of the amount of extravascular lung water, the right upper and posterior lobes were collected before bronchoalveolar lavage for measurement of wet/dry weight ratios. As can be seen in Figure 1, the wet/dry weight ratios, corrected for blood volume, of uninfected hyperoxic lungs (48 h: 5.13 ± 0.84, n = 4; 60 h: 5.16 ± 0.52, n = 4) were significantly greater than room air controls (3.64 ± 0.28, n = 4, p < 0.02 versus uninfected hyperoxic lungs). The lungs of rats allowed to recover for 24 h had corrected wet/dry weight ratios that were not different from room air controls (4.46 ± 0.14, n = 4, p = 0.11 versus room air control, p = 0.03 versus hyperoxic control). Correction for blood volume did not significantly affect wet/dry ratios in any group.
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Distribution of Gene Transfer
X-gal staining of lungs was used to provide a qualitative measure of gene transfer (Figure 2). The X-gal solution (pH = 7.9) used in this study was intended to optimize function of the E. coli lac Z gene and limit endogenous -galactosidase activity (16). As shown in Figure 2, hyperoxic lungs that were infected with 4 × 109 PFU of ad-gal after 60 h of hyperoxia and
stained with X-gal 72 h later have evidence of -galactosidase
activity in all regions of the lung. The pattern of staining was
not visibly different from similarly treated room air lungs infected with ad-gal. Photographs of left lungs imbedded in
paraffin (Figure 3) similarly demonstrate uniform distribution
of -galactosidase activity in both room air and hyperoxic
lungs. As can be seen in the representative photomicrographs
in Figure 4, most alveolar lobules have evidence of -galactosidase activity. The distribution of transgene expression in
lungs from rats exposed to 48 h of hyperoxia was not discernibly different from the 60-h animals. Lungs infected with 1 × 109 PFU of ad-gal had less intense transgene expression than
did lungs infected with 4 × 109 PFU. No -galactosidase activity was noted in any of the adNull infected controls.
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Histologic Lung Injury
To confirm the presence of lung injury, we obtained lung sections from uninfected hyperoxic control rats immediately after 60 h of hyperoxia. As shown in Figure 5 (left photomicrograph), hyperoxia produced marked airspace and interstitial
injury consistent with reports from other investigators regarding the effects of acute hyperoxia on the lung (6). Histologic
review of hyperoxic lungs infected with ad-gal for 72 h (Figure 5, right photomicrograph) showed continued signs of injury and transgene expressing cells within areas of injury.
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Quantitative Assessment of Gene Transfer
Enumeration of transgene expressing cells
The number of cells
with blue color was enumerated in 10 high-power microscope
fields randomly selected from longitudinal sections of left lungs
from 3 to 6 rats/group. The number of transgene expressing
cells in lungs infected with 4 × 109 PFU of ad-gal after 48 or
60 h of hyperoxia was not different from similarly infected
room air controls (Figure 6A). The number of transgene positive cells in lungs infected with 1 × 109 PFU was 70 to 80% of
lungs infected with the higher dose.
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Room air; 
hyperoxia.
-galactosidase activity (Figure 6B).
Gene transfer was further quantified by measurement of -galactosidase activity in
right lung homogenates from 3 to 4 animals/group. Activity
(units/µg protein) in the high-dose, room air ad-gal lungs
(17.7 ± 2.9 units/µg protein, n = 4) was slightly greater than in
high-dose, hyperoxic ad-gal lungs (14.6 ± 3.4 units/µg protein, n = 4, p = 0.042 versus room air ad-gal). Activity in the
low-dose animals was not different between the two groups and was approximately 30 to 35% of that noted in lungs infected with 4 × 109 PFU of ad-gal (room air, 5.23 ± 1.4 and
hyperoxia, 5.12 ± 0.08 units/µg protein, n = 4/group). Quantitative -galactosidase activity in low-dose room air and hyperoxic adNull rats was not different from uninfected room air
and hyperoxic controls.
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Impairment of surfactant function (17), epithelial solute transport (9, 18, 19), alveolar barrier function (20), and antioxidant function (21) are central elements of ALI. Recent data indicate that gene transfer can improve the function of these and other biologic systems in experimental animal models (2, 22). Each of these prior studies suggests a role for gene transfer in ALI; however, in all prior studies of gene transfer for ALI the alveolar epithelium was transduced before initiation of lung injury. No data are available to indicate if gene transfer can be effected in the presence of acute alveolar injury and flooding. Consequently, we undertook the current study to test if an adenovector could efficiently transduce the alveolar epithelium after the development of alveolar injury and pulmonary edema.
Acute hyperoxia is a well-characterized, free radical-mediated lung injury model that produces diffuse endothelial and
epithelial cytotoxicity, increased alveolar permeability, and
pulmonary edema (6, 9). Importantly, hyperoxia has a mean
lethal dose (LD50) of approximately 72 h and as such is highly
lethal for adult rats (6). This model has also been associated
with increased concentrations of inflammatory cytokines such
as tumor necrosis factor-alpha (TNF-
), interleukin-1 (IL-1),
and interferon gamma (IFN-
) in the alveolar airspace. Each
of these cytokines has been shown to diminish the efficiency
of adenoviral-mediated gene transfer (10, 11, 26). Our findings
of increased lung water (wet/dry ratios, Figure 1), large bilateral pleural effusions, histologic injury (Figure 5), weight loss,
and significant mortality during virus delivery reconfirm that
this model causes severe lung injury in rats.
The results of this study demonstrate that a surfactant-based delivery system is capable of widespread vector delivery
to injured lungs. The photographs in Figures 2 and 3 show gene
transfer to all regions of the lung that is not discernibly different between the room air and hyperoxic groups. Quantitative
measurement of -galactosidase activity (o-nitrophenyl--D-galactopyranoside [ONPG] hydrolysis, Figure 6A) was slightly
greater in room air than in hyperoxic ad-gal lungs; however,
the number of transgene positive cells/hpf was not (Figure 6B).
These data suggest that the gene transfer scheme employed in
this study combined with a high dose of virus (4 × 109 PFU)
was capable of transducing injured lungs with near-equal efficiency to that of uninjured lungs. The observation of gene transfer within areas of injury (Figure 5, right photomicrograph) could be the result of gene transfer to areas of lung that
were minimally affected by hyperoxia and could indicate that
the histologic changes surrounding areas of transgene expression are the result of host responses to adenoviral infection.
However, the diffuse nature of lung injury noted in uninfected
hyperoxic controls (Figure 5, left photomicrograph) studied
immediately after hyperoxia makes this hypothesis unlikely.
Thus, we believe that the transgene expression noted within
areas of lung injury supports the hypothesis that adenovectors
can transduce injured alveoli.
Our finding of equal gene transfer efficiency between room
air and hyperoxic lungs suggests that adenoviral gene transfer is not impaired by lung injury. However, it is plausible that 4 × 109 PFU is more than sufficient to transduce all alveolar cells
as well as overcome injury-associated barriers to gene transfer, such as loss of virus resulting from phagocytosis or inactivation. Thus, if this number of live viral particles represents a
"supramaximal" dose, we may not be able to discern if hyperoxia affects transduction efficiency. To address this possibility,
we conducted additional experiments using a lower dose of
adenovirus (1 × 109 PFU). As shown in Figures 2 and 3, this
dose of virus produced gene transfer in all regions of the lung;
however, the intensity of X-gal staining and -galactosidase
activity in the low-dose lungs was less (
30 to 35%) than in
high-dose lungs and was not different between room air and
hyperoxic groups, suggesting that this lower dose is unlikely to
be a maximal dose of virus.
An additional concern in our studies of 60 h of hyperoxia
may be related to selection bias based on the severity of lung
injury. Specifically, it is conceivable that rats that survived
vector delivery were less ill than the animals that did not, and
as such, their lungs may have had fewer impediments to gene
transfer. To control for this concern, we conducted experiments using a shorter duration of hyperoxia to produce a more
modest lung injury. The mortality associated with sedation,
intubation, and vehicle instillation in this group was the same
( 60%) as rats treated with 60 h of hyperoxia; thus we were
unable to control for this concern. However, we believe that
the dose-dependent reductions of -galactosidase activity and
the similarity of activity between room air and control groups
in both the high-dose and low-dose experiments lend support for our hypothesis that adenoviral-mediated gene transfer is
not impaired in injured lungs.
Studies of gene transfer to abnormal airway epithelia have identified numerous barriers to gene transfer, including mucins, bacteria, nonspecific inflammation, proinflammatory cytokines, and abluminal location of adenovirus receptors (11, 27). Impediments to alveolar gene transfer may similarly exist. Bastian and coworkers have shown that bronchoalveolar lavage fluid from normal humans impairs adenoviral infection of HEK293 cells in vitro, independent of anti-adenovirus antibody titers, suggesting that antibody-independent mechanisms in the alveolus could impair gene transfer (32). Other limitations of alveolar gene transfer in lung injury could include mechanical factors due to altered lung compliance and alveolar collapse, which could limit access to the alveolar epithelium or result in nonhomogeneous distribution of vehicle/vector. Interestingly, Weiss and coworkers have reported that adenovectors, delivered using a perfluorocarbon-based vehicle, can transduce the alveolar epithelium of granulocyte macrophage colony-stimulating factor (GM-CSF) knockout mice with chronic filling of alveoli with protein (33). This report suggests that these adenovectors may not be limited by proteinaceous exudates in the injured airspace. Recent studies indicate that basolateral localization of adenovirus fiber receptors limits adenovirus infection of the bronchial epithelium (31) and that methods which increase the permeability of the bronchial epithelium improve the efficiency of adenovirus-mediated gene transfer, presumably by providing adenoviruses access to their receptors (30). The location of these receptors in the alveolar epithelium has not been reported; however, if basolaterally positioned, it may be that the increased alveolar permeability associated with hyperoxic lung injury might similarly improve the "infectability" of the alveolar epithelium and compensate for other processes that would be expected to impede gene transfer.
We have previously shown that the surfactant-based delivery scheme used in this study is capable of highly efficient gene transfer to the alveolar epithelium of normal rats (14). Other groups have similarly shown that surfactant-based vehicles
containing surfactant-associated proteins significantly enhance
peripheral lung gene transfer (34). Surfactants have several
unique biophysical properties that may allow them to overcome barriers to gene transfer, including rapid distal dispersion
(37), improved clearance of mucous, and reopening of collapsed, fluid-filled alveoli (38). Debs and coworkers have shown
highly efficient gene transfer and prolonged transgene expression in cultured alveolar epithelial cells using a liposomal-based method, which raises the interesting hypothesis that the
ability of these cells to produce and recycle surfactant may improve their ability to be transduced by lipid-based vehicles
(39). However, surfactants are not the perfect vehicles for all
types of gene transfer as they do not facilitate naked DNA
gene transfer in mouse lungs, lung epithelial cells, or lung fibroblasts and they impair liposomal-mediated gene expression (probably as a result of disruption of the liposomes) (40, 41). Interestingly, the number of transgene positive cells in the low-dose lungs (Figure 6B) in the current study was only slightly
less than in hyperoxic lungs infected with the high dose of virus
(63 versus 78 blue cells/hpf). We take this to mean that the
greater -galactosidase activity in the high-dose lungs is due to
more viral genomes/cell, not wider distribution of gene transfer. These findings have caused us to speculate that the dispersion characteristics of bovine surfactant may be a rate-limiting
factor in achieving 100% transfection efficiency in rat lung.
We have previously reported that overexpression of Na,K-ATPase subunit genes in the alveolar epithelium accelerates pulmonary edema clearance in normal lungs, protects the lung from injury, and improves survival from hyperoxia (3, 14). Recently, Stern and colleagues showed that plasmid-mediated transfer of a Na,K-ATPase construct rapidly reduces lung water in rats with a mild form of lung injury caused by intraperitoneal administration of thiourea (42). Danel and others have shown that adenoviral-mediated overexpression of antioxidant genes such as catalase and superoxide dismutase protects the lung from injury (2, 4, 23). Other groups have proposed gene transfer to improve alveolar barrier function (20), improve antioxidant function (4), enhance surfactant production (22), or modulate host inflammatory responses to infection (5). Each of these prior studies suggests a role for gene transfer as a modality to improve or restore alveolar epithelial function during ALIs such as the acute respiratory distress syndrome, pneumonia, severe left heart failure, and radiation pneumonitis.
A growing body of data indicates that prophylactic transfer of protective genes to the alveolar epithelium can, in experimental models, ameliorate ALI. Previously, the development of therapeutic gene transfer strategies was limited by concerns that the pathophysiologic processes associated with ALI would preclude efficient transduction of the alveolar epithelium. The results of this study indicate otherwise and show, for the first time, that adenovectors can efficiently transduce severely injured alveoli. We believe that these data lend further support for the development of gene therapies for acute lung injuries.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Phillip Factor, D.O., Pulmonary and Critical Care Medicine, Evanston Northwestern Healthcare, 2650 Ridge Rd., Evanston, IL 60201. E-mail: pfactor{at}northwestern.edu
(Received in original form January 5, 2001 and accepted in revised form November 5, 2001).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.orgAcknowledgments: The authors thank Dr. J. Sznajder for use of the environmental chamber. The authors would also like to acknowledge Linda Devine for histologic aid and Scott Haller for guidance with digital imaging.
Supported by the American Heart Association, the Evanston Northwestern Healthcare Research Institute, HL-48129, and HL-66211.
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