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Circulation Research. 1995;76:701-709

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(Circulation Research. 1995;76:701-709.)
© 1995 American Heart Association, Inc.

Articles

In Vivo Adenovirus-Mediated Gene Transfer Via the Pulmonary Artery of Rats

Susan K. Schachtner, Jonathan J. Rome, Robert F. Hoyt, Jr, Kurt D. Newman, Renu Virmani, David A. Dichek

From the Molecular Hematology Branch (D.A.D.) and Laboratory of Animal Medicine and Surgery (R.F.H.), National Heart, Lung, and Blood Institute, Bethesda, Md; Cardiovascular Section (R.V.), Armed Forces Institute of Pathology, Washington, DC; and the Departments of Cardiology (S.K.S., J.J.R.) and Surgery (K.D.N.), Children's National Medical Center, Washington, DC.

Correspondence to David A. Dichek, MD, Gladstone Institute of Cardiovascular Disease, UCSF, PO Box 419100, San Francisco, CA 94141-9100.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract Gene transfer into the pulmonary vasculature has the potential to be a powerful technique for both investigation of pulmonary pathophysiology and development of genetic therapies for pulmonary vascular disease. To evaluate the potential for in vivo pulmonary arterial gene transfer, we infused adenoviral vectors into the left pulmonary artery of Sprague-Dawley and cotton rats. Access to the left pulmonary artery was obtained by a percutaneous transcatheter approach or through thoracotomy and pulmonary arteriotomy. With the thoracotomy approach, both pulmonary arterial inflow and pulmonary venous outflow were occluded during vector infusion and throughout a subsequent 20-minute dwell period. The success of gene transfer was assessed by staining for evidence of recombinant gene expression in lungs excised at time points ranging from 48 to 72 hours after virus infusion. With the thoracotomy technique, pulmonary gene transfer was successful in 15% of surviving Sprague-Dawley rats and 30% of surviving cotton rats. Percutaneous catheter-based pulmonary gene transfer was not successful. In rats with pulmonary gene transfer, 1% to 8% of total left lung cells expressed the recombinant gene. Recombinant gene expression was found in endothelial cells (0.2% to 18% of total transduced cells), smooth muscle cells (0% to 3%), macrophages (1% to 7%), airway epithelial cells (2% to 50%), and alveolar epithelial cells (38% to 94%). Investigation of the low rate of successful gene transfer in individual animals suggested that insufficient physical contact between the virions and pulmonary cells was the most likely cause. In vivo gene transfer into the rat pulmonary vasculature can be accomplished with adenovirus vectors. The overall efficiency is low, however, and pulmonary arterial infusion of the vectors results in gene transfer primarily into nonvascular cells.


Key Words: pulmonary artery • pulmonary hypertension • gene transfer • gene therapy • adenovirus


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The pulmonary vasculature is a complex vascular bed with biological properties distinct from those of the systemic vasculature. In addition, the pulmonary circulation is the site of well-described but poorly treated pathological processes. Specifically, the responses of the pulmonary vasculature to physiological stimuli (eg, hypoxia, acidemia) and pharmacological manipulation (eg, histamine infusion) differ markedly from those of the systemic vasculature.1 2 Specific pulmonary vascular disease processes include persistent pulmonary hypertension of the newborn, pulmonary vascular obstructive disease in children with congenital cardiac defects, and primary pulmonary hypertension in adults. Each of these disease processes is poorly understood and inadequately treated and remains a significant cause of morbidity and mortality.3 4 5 6

Both the distinct physiological characteristics and the unique pathological processes of the pulmonary vasculature are probably based on local patterns of gene expression. Extensive investigation in animal models of pulmonary hypertension has identified abnormal expression of endothelin-1, elastase, and platelet-derived growth factor in association with the development of pulmonary vascular disease in rats7 8 9 10 11 and alterations in the expression of transforming growth factor–ß and insulin-like growth factor I in sheep with pulmonary vascular disease.12 13 14 In addition, humans with pulmonary vascular disease have increased expression of endothelin-1.15 16 17 18 Although these correlative studies of gene expression and pulmonary pathology are important, in vivo transfer of genetic material into the pulmonary vasculature would offer a more direct approach to elucidating the role of individual genes in the genesis of normal and abnormal pulmonary physiology. In addition, expression of specific genes encoding vasodilators or inhibitors of smooth muscle cell proliferation might provide a powerful local therapeutic approach to pulmonary hypertension.

Adenoviral vectors are unique in that they are capable of mediating high-efficiency in vivo gene transfer into systemic arteries.19 20 21 22 23 24 A recent report25 demonstrated successful adenoviral vector–mediated gene transfer into the pulmonary arteries of sheep; however, no quantification of gene transfer efficiency was given. In the present study, we tested the ability of adenoviral vectors to facilitate the transfer of a recombinant gene to the pulmonary vasculature of the rat, a species that serves as an informative model of human pulmonary vascular disease.26 27 The cellular targets of gene transfer in this model were identified, and the efficiency of gene transfer was quantified.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Adenoviral Vector
We used an adenoviral vector expressing a nuclear-targeted ß-galactosidase (ß-gal) gene (Av1LacZ4) driven by a Rous sarcoma virus promoter. This vector was chosen because it permits easy histological localization of transduced cells and has been used in our laboratory to accomplish high-efficiency in vivo vascular gene transfer in both sheep and rat carotid arteries.20 22 Viral stocks of 1 to 2x1011 plaque-forming units per milliliter (pfu/mL) were prepared as previously described.20 Immediately before use, these stocks were thawed and diluted in normal saline with 2 U/mL porcine sodium heparin (Elkins-Sinn). Heparin was included in all solutions infused through intravascular catheters; this concentration of heparin did not affect the efficiency of transduction by the Av1LacZ4 vector in vitro (data not shown).

Animals
All animal procedures were approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute. Sprague-Dawley rats (250 to 450 g) were obtained from Charles River or Taconic Farms, Inc. Cotton rats (Sigmodon hispidus; 100 to 150 g) were obtained from the National Heart, Lung, and Blood Institute (5 Research Court Section). Sprague-Dawley rats were chosen because they can be used to produce a rat model of pulmonary arterial hypertension.26 Cotton rats were chosen because of their documented susceptibility to human adenoviral infection, which has made them a useful animal model for adenovirus-mediated gene transfer.28 29 30 In all animals, anesthesia was induced with an intramuscular injection of 100 mg/kg ketamine (Fort Dodge Laboratories, Inc) combined with 1 mg/kg acepromazine (Aveco Co, Inc). In procedures confined to percutaneous access, anesthesia was maintained with inhaled isoflurane (A.J. Buck). For procedures that required a thoracotomy, the animals were intubated and ventilated with a rodent ventilator (Kent Scientific Corp) with 1% isoflurane and oxygen at a flow rate of 1 L/min. Breaths were administered at a rate of 70 to 80 per minute with a tidal volume of 6 to 7 mL for Sprague-Dawley rats and 2 mL for cotton rats. As an alternative, a pressure limit of 12 to 14 cm H2O was used for both species.

Gene Transfer Via Percutaneous Pulmonary Arterial Access
Five Sprague-Dawley rats underwent right-side heart catheterization via a cut-down on the right jugular vein. A 3.5F or 5F polyvinylchloride human umbilical venous catheter (Argyle, Sherwood Medical) was modified by bending the distal 1 cm into a 90-degree angle.31 The catheter was advanced through the right side of the heart under fluoroscopic guidance. The animal was then heparinized (100 U/kg) intra-arterially, and the catheter was advanced to a distal pulmonary arterial segment. Wedge position was confirmed by pressure recording and by infusion of nonionic contrast medium (Isovue-200, Squibb Diagnostics) through the catheter. From 1.5 to 3 mL of 2x108 to 2x1010 pfu/mL Av1LacZ4 (total, 6x108 to 3x1010 pfu per animal) was infused through the catheter in 0.1- to 0.2-mL increments over 15 minutes. The catheter was removed, the jugular vein was tied off, the neck wound was closed, and the rat was allowed to recover in a warm, oxygenated environment.

Surgical Approaches
Gene Transfer Into an Isolated Segment of Pulmonary Artery
In one Sprague-Dawley rat, a left thoracotomy was performed by entering the thoracic cavity in the fourth intercostal space. With the aid of a Zeiss operating microscope (x5 to x20, Carl Zeiss, Inc), the single left lung lobe was reflected, and a short segment (approximately 2 mm long) of the left pulmonary artery was isolated between a small vascular clip placed proximally and a 5-0 silk surgical tie placed near the insertion of the left pulmonary artery into the left lung. This technique permitted isolation of the longest possible segment of unbranched pulmonary artery. A small transverse arteriotomy was made in the isolated segment of the left pulmonary artery, through which a silicone elastomer catheter (ID, 0.012 in; OD, 0.025 in; Dow Corning Corporation) attached to a 22-gauge blunt needle was inserted. Then, 60 µL of 1x1011 pfu/mL Av1LacZ4 (total, 6x109 pfu) was infused and allowed to remain in contact with the vessel surface for 20 minutes. The arteriotomy was closed with 10-0 nylon suture, and flow was reestablished. A chest tube (shortened 8F pediatric feeding tube; Baxter Healthcare Corp) attached to a 14-gauge blunt needle was inserted 2 cm caudal to the incision, tunneled through the subcutaneous tissues, and placed between the ribs into the left chest cavity. The thorax was closed, the soft tissues were approximated with 3-0 polyglycolic acid suture, and the skin was closed with staples. The animal was allowed to recover in a warm, oxygenated environment, and the chest tube was removed within 1 hour of the procedure.

Gene Transfer After Surgical Occlusion of Left Pulmonary Artery and Vein
A total of 15 Sprague-Dawley rats and 15 cotton rats underwent occlusion of the left pulmonary artery and vein32 with infusion of virions into the left lung. A left thoracotomy was performed by entering the thoracic cavity in the second to third (cotton rats) or third to fourth (Sprague-Dawley rats) intercostal space. The single left lung lobe was reflected, and the left pulmonary artery and vein were isolated. After arterial inflow was occluded with a small, curved vascular clip, a small transverse arteriotomy was made in the left pulmonary artery distally, and a silicone elastomer catheter was inserted. The catheter was secured in place with a 5-0 silk tie and a straight vascular clip. The left pulmonary vein was occluded with a third vascular clip. One milliliter of 2x1010 pfu/mL (2x1010 pfu per rat) adenoviral vector solution was infused via the catheter over 2 minutes in each Sprague-Dawley rat. In cotton rats, 0.3 mL of 2x1010 pfu/mL Av1LacZ4 (6x109 pfu per rat) was infused. In both species, the volume infused distended the collapsed lung, giving it a translucent appearance. After a 20-minute dwell period, the venous clip was removed, the arteriotomy was repaired with 10-0 suture, and pulmonary arterial flow was reestablished.

Gene Transfer After Surgical Occlusion of Pulmonary Artery, Vein, and Bronchus
In five Sprague-Dawley rats, the procedure was modified to include occlusion of all hilar structures during virus infusion. After insertion of the catheter into the pulmonary artery, a rubber vascular loop was placed tightly around the left hilum, thereby visibly occluding (1) both pulmonary and bronchial arterial flow, (2) pulmonary venous return, and (3) air exchange into the left lung. Occlusion of pulmonary lymphatic drainage could not be confirmed. The viral solution was infused via the catheter and allowed to dwell for 20 minutes; then, the vascular loop was released, the catheter was removed, and the arteriotomy was closed with 10-0 suture.

Skeletal Muscle Transduction
Seven Sprague-Dawley rats from the group that underwent pulmonary artery and vein occlusion were evaluated for susceptibility to transduction with the Av1LacZ4 vector by injection of the viral solution into a peripheral muscle at the time of the pulmonary lung transduction procedure. A 0.5-cm incision was made over the left forelimb triceps muscle, and 0.1 mL of 2x1010 pfu/mL (total of 2x109 pfu) Av1LacZ4 was injected into the muscle with a 25-gauge needle. The muscle injection site was marked with a single 5-0 Prolene stitch, and the wound was closed.

Ex Vivo Transduction of Lung Tissue
In four rats undergoing surgical occlusion of the left pulmonary artery and vein during virus infusion, the right upper lung lobe was separated from the heart-lung block immediately after the animal was killed (as described) and placed into tissue culture medium before fixation of the remainder of the heart-lung block. After placement in culture medium, the lung tissue was cut into 2- to 3-mm-diameter fragments with a scalpel and infected with 0.15 mL of Av1LacZ4 at a concentration of 2x1010 pfu/mL in infection medium (DMEM with 2% fetal bovine serum [FBS]; Biofluids Inc). After 20 (n=2) or 90 (n=2) minutes, the infection medium was replaced with DMEM with 10% FBS, and the lung tissue was maintained at 37°C overnight. Twenty hours after exposure to the virus, the lung tissue was assessed for transgene expression by staining with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal).

Neutralizing Antibody Assay
The protocol of Smith et al33 was used to test rat serum for the presence of neutralizing antibodies to the Av1LacZ4 vector. Briefly, 293 cells (American Type Culture Collection) were plated in 96-well microtiter tissue culture plates at a density of 5x104 cells per well. Serial dilutions, from 1:2 to 1:4096, were made for heat-inactivated test sera obtained from adenovirus-injected cotton and Sprague-Dawley rats. Test sera were obtained from rats in which pulmonary gene transfer was both successful (n=1) and unsuccessful (n=6) using the pulmonary artery and vein occlusion technique. Neutralizing antibody-positive and antibody-negative control mouse sera were provided by Dr Theodore Smith (Genetic Therapy, Inc). Av1LacZ4 (4x105 pfu) was added to each dilution of serum and allowed to incubate at 37°C for 30 minutes. Of these diluted sera, 100 µL was then placed into each well of the tissue culture plate containing 293 cells. Sixty minutes later, the solution was replaced with growth medium (improved minimum essential medium with 10% FBS; Biofluids Inc), and the cells were incubated overnight. Twenty hours later, the cells were fixed and stained with X-gal, and evidence of recombinant gene expression was watched for. Antibodies were considered to be present in dilutions of test sera if the addition of sera to viral stock resulted in loss of gene transfer. The titer was recorded as the highest dilution at which gene transfer was inhibited.

Tissue Fixation and Histochemical Processing
Forty-eight to 72 hours after viral infusion, the rats were killed with an overdose of sodium pentobarbital (300 mg/kg IP). Serum was collected by cardiac puncture. The heart and lungs were resected en bloc, and the lungs were cleared of blood by infusing cold phosphate-buffered saline (PBS) through a catheter positioned in the main pulmonary artery. A portion of the liver also was removed and rinsed in cold PBS. In seven rats, the skeletal muscle previously injected with vector was removed, cut into small pieces, and stored in cold PBS.

All tissue to be stained with X-gal in both in vivo and ex vivo gene transfer experiments was fixed in 2% formaldehyde with 0.2% glutaraldehyde for 30 to 60 minutes, rinsed with PBS, and placed in X-gal reagent34 for 4 hours. The lungs from in vivo experiments were pressure-fixed in a distended state via catheters in the pulmonary artery (100 cm H2O) and trachea (30 cm H2O) and then perfusion-stained in a recirculating X-gal bath. The liver, skeletal muscle, and ex vivo lung tissues were stained by submersion in X-gal reagent.

The virion-exposed left lung and a portion of the unexposed right lung were sectioned transversely into approximately 5-mm-thick segments and examined under the dissecting microscope (x16 to x80; Zeiss) for evidence of ß-gal expression. Lung, liver, and muscle segments were then embedded in paraffin. Sections 5 µm thick were cut from each segment, placed on slides, and counterstained with either hematoxylin and eosin or nuclear fast red stain.

Lung tissue for transmission electron microscopy examination was fixed and stained with X-gal as described and then embedded in epoxy (Ernest F. Fullam Inc). Sections 70 nm thick were cut and viewed unstained with the electron microscope.

Histological and Immunohistochemical Identification of Cell Types
The following morphological criteria were used for identification of cells in histological sections of lungs.35 Cells that were present in the alveolar wall but were not lining the blood vessels were classified as alveolar epithelial cells. These cells were either small, elongated, and thickened in the region of the nucleus or large and cuboidal with cytoplasmic vacuoles (type I or type II pneumocytes). Endothelial cells were identified as thin and elongated and lined a vessel lumen with the nucleus protruding into the lumen. Smooth muscle cells were spindle shaped with eosinophilic cytoplasm and located in the medial layers of blood vessels or airways. Macrophages were large, unattached cells with single, eccentric or oval nuclei, containing many granular inclusions in the cytoplasm. The validity of these morphological criteria was confirmed by immunohistochemical staining of sections of rat lung or by transmission electron microscopy. Antibodies were applied to lung sections at a dilution of 1:3200 for von Willebrand factor (Dako Corp), 1:10 000 for smooth muscle actin (Sigma Chemical Co), and 1:1600 for ED1, an antigen present on rat macrophages, monocytes, and dendritic cells (Harlan). Bound antibody was detected with a secondary antibody conjugated to horseradish peroxidase.

Transmission electron microscopy was performed to both verify the morphological identification of alveolar epithelial cells (pneumocytes) and confirm the presence of characteristic X-gal precipitates in the nuclei of transduced cells.36

Quantitative Analysis of Transgene Expression in the Lung
To quantify the efficiency of pulmonary gene transfer, we cut histological sections from X-gal–stained lungs that had evidence of recombinant gene expression on gross examination. Approximately seven evenly spaced 5-µm-thick sections were cut from each lung. Ten high-power fields ([hpf] x400) were chosen randomly from each histological section. Within each field, both total and X-gal–stained nuclei were counted. Within these approximately 70 hpf (seven sections, 10 fields per section), a total of 10 734±1407 cells (mean±SD) were counted for each animal. Cells containing X-gal–stained nuclei were categorized by cell type according to the criteria described. For each animal, the efficiency of transduction was quantified by three methods. First, the percentage of total pulmonary cells that was transduced was calculated by dividing the total number of transduced cells by the total number of pulmonary cells counted. Second, the percentage of total transduced cells represented by each cell type was determined by dividing the number of positive cells of that type by the total number of transduced cells. Third, to estimate the percentage of each cell type that expressed the recombinant gene, for each animal the total number of transduced cells of each type found in the approximately 70 hpf was divided by the total number of cells of that type in this same number of high-power fields. To perform this third calculation, it was necessary to determine the total numbers of macrophages and endothelial, smooth muscle, airway epithelial, and alveolar epithelial cells per high-power field. This determination was made as follows: histological slides that were stained with cell-type–specific antibodies were used for counting macrophages and endothelial and smooth muscle cells per high-power field. Hematoxylin and eosin–stained slides were used for counting total airway epithelial cells per high-power field. The number of alveolar epithelial cells was estimated by subtracting the calculated number of other cell types from the total number of pulmonary cells. For all calculations of transduction efficiency, mean values were calculated separately for Sprague-Dawley and cotton rats.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Gene Transfer Via Percutaneous Pulmonary Arterial Access
Five Sprague-Dawley rats underwent right-side heart catheterization with infusion of Av1LacZ4 through a catheter wedged in a distal pulmonary arterial segment occluding antegrade flow. No evidence of recombinant ß-gal expression was observed on gross or microscopic examination of the lungs. A portion of the liver from the animal receiving the largest dose of virus (1.5 mL of 2x1010 pfu/mL) was removed and stained with X-gal. Gross examination demonstrated punctate blue staining of the liver surface consistent with ß-gal gene expression. On microscopic examination, dark-blue staining of hepatocyte nuclei was observed. Therefore, it appeared that despite occlusion of the pulmonary arterial inflow, the infused virions were released to the systemic vasculature.

Surgical Approaches
Based on our previous findings, we considered our failure to obtain gene transfer with the percutaneous approach to result from inadequate contact between virions and pulmonary vascular cells. This view was supported by observations made after catheter infusion of radiopaque, nonionic contrast under fluoroscopy. The opacification gradually became less intense over 20 minutes, suggesting that despite occlusion of pulmonary arterial inflow, infused virions could escape via pulmonary veins or lymphatics. We therefore adopted a surgical approach to allow occlusion of pulmonary venous return during virion infusion.

Gene Transfer Into an Isolated Segment of Pulmonary Artery
We first confirmed the susceptibility of rat pulmonary artery endothelium to in vivo adenoviral gene transfer by infusing virions into a small segment of the pulmonary artery isolated between proximal and distal ligatures (see "Materials and Methods"). A high percentage ({approx}30%) of pulmonary artery endothelial cells exposed to the virus under these conditions stained blue with X-gal (Fig 1Down). We therefore concluded that the endothelium of the rat pulmonary artery was susceptible to in vivo adenovirus-mediated gene transfer under the proper conditions.



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  		Figure 1. Photomicrographs of isolated segment of left pulmonary artery exposed to Av1LacZ4 via a surgical technique. Vessel was fixed and stained with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside 3 days after exposure. a, Vessel viewed from the luminal surface demonstrates punctate blue staining of nuclear-targeted ß-galactosidase (ß-gal) expression (original magnification x10). b, Photomicrograph of the same vessel taken after sectioning and counterstaining with nuclear fast red. Dark-blue staining characteristic of nuclear-targeted ß-gal expression is seen in cells lining the pulmonary artery lumen (original magnification x50).

Gene Transfer After Surgical Occlusion of Pulmonary Artery and Pulmonary Vein
Fourteen of 15 Sprague-Dawley rats and 11 of 15 cotton rats survived thoracotomy, occlusion of the left pulmonary artery and pulmonary veins, and infusion of Av1LacZ4 into the left lung. Of note, during infusion the lung became translucent as blood within the lung vasculature was diluted by the viral solution. However, the appearance of the lung changed throughout the 20-minute dwell period, becoming dark red and firm, which is consistent with ongoing inflow of blood. Two of the surviving Sprague-Dawley rats and 3 of the surviving cotton rats had evidence of pulmonary recombinant gene expression as determined by X-gal staining. Examination of light and electron microscopic sections of the X-gal–positive lungs revealed transgene expression in alveolar and airway epithelial cells, macrophages, endothelial cells, and smooth muscle cells (Fig 2Down). In the 2 X-gal–positive Sprague-Dawley lungs, a mean of 2.7% (1.2% and 4.1%) of lung cells expressed the ß-gal gene. A mean of 5.4% (2.3%, 6.2%, and 7.8%) of lung cells stained blue in the 3 cotton rat lungs positive for ß-gal expression. In the Sprague-Dawley rat lungs, a mean of 90% (88% and 92%) of the cells expressing ß-gal activity were alveolar epithelial cells, 2.4% (2.2% and 2.5%) were airway epithelial cells, 2.5% (2.0% and 2.9%) were macrophages, 1.9% (0.2% and 3.6%) were endothelial cells, and 2.9% (2.9% and 2.9%) were smooth muscle cells. The corresponding analysis in the cotton rat lungs revealed that a mean of 68% (38%, 73%, and 94%) of the ß-gal–expressing cells were alveolar epithelial cells, 20% (3.6%, 6.3%, and 50%) were airway epithelial cells, 3.2% (0.8%, 2.1%, and 6.7%) were macrophages, 8.3% (1.9%, 5.1%, and 18%) were endothelial cells, and 0.5% (0.0%, 0.3%, and 1.1%) were smooth muscle cells (Fig 3Down). Nearly all of the transduced smooth muscle cells were found in airway structures, with fewer than 10% found in the media of pulmonary vessels.





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  		Figure 2. Photomicrographs of target cells of in vivo pulmonary artery gene transfer, identified both by staining for recombinant gene expression and by morphological as well as ultrastructural or immunohistochemical criteria. Tissues were removed and stained with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) 48 to 72 hours after gene transfer. Sections in a, c, e, and g were counterstained with nuclear fast red. Sections in d, f, and h were stained with hematoxylin and then immunostained with specific antibodies. a, Photomicrograph of terminal bronchiole and alveoli demonstrating recombinant gene expression in alveolar epithelial cells (pneumocytes). b, Transmission electron micrograph of alveolar epithelial cells. Electron-dense precipitates (small arrows) characteristic of X-gal are visible in the nuclear membrane (arrowhead), providing evidence of recombinant gene expression. Presence of a lamellar body (*) identifies this cell as a type II pneumocyte. c, Photomicrograph of section of bronchus and large vessel demonstrating ß-galactosidase (ß-gal) expression in airway epithelial and arterial endothelial cells. d, Photomicrograph of section from animal in c after immuno- staining to identify endothelial cells. e, Photomicrograph of section of bronchus, illustrating ß-gal expression in airway smooth muscle cells. f, Photomicrograph of section from rat bronchus after immunostaining for smooth muscle actin. g, Photomicrograph of alveoli demonstrating a transduced alveolar macrophage. h, Immunostaining with antibody to ED1 confirms identification of an unattached cell as a monocyte/macrophage. (Original magnification: a, c, d, e, and f, x100; b, x5000; and g and h, x157).



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  		Figure 3. Bar graph of transduced pulmonary cells classified by type of cell, as determined by counting cells in microscopic sections. Heights of bars correspond to the mean percentage of total ß-galactosidase–expressing cells determined for each cell type. Data were obtained from both Sprague-Dawley and cotton rats.

The transduction efficiency per cell type also was estimated, as described (see "Materials and Methods"). In this model, 3 days after gene transfer, the cells in Sprague-Dawley rat lungs were identified as follows (all numbers are approximate and therefore do not total 100%): alveolar epithelial cells, 40%; airway epithelial cells, 20%; macrophages, 10%; endothelial cells, 20%; and smooth muscle cells, 4%. In cotton rat lungs, cell types were identified as follows: alveolar epithelial cells, 40%; airway epithelial cells, 40%; macrophages, 6%; endothelial cells, 20%; and smooth muscle cells, 2%. In Sprague-Dawley rats, a mean of 6% (3% and 8%) of alveolar epithelial cells, 0.4% (0.1% and 0.6%) of airway epithelial cells, 0.6% (0.4% and 0.8%) of macrophages, 0.1% (0.0% and 0.2%) of endothelial cells, and 1% (0.9% and 2%) of smooth muscle cells expressed the recombinant gene. The corresponding estimates in cotton rats revealed that a mean of 10% (7%, 8%, and 14%) of alveolar epithelial cells, 3% (0.3%, 1.0%, and 8%) of airway epithelial cells, 3% (1%, 1%, and 6%) of macrophages, 2% (1%, 2%, and 2%) of endothelial cells, and 1% (0.5%, 0.7%, and 2%) of smooth muscle cells displayed transgene expression.

To evaluate the presence of ectopic gene transfer due to release of virus from the pulmonary circulation, we stained all of the livers from animals in the pulmonary artery and pulmonary vein occlusion experiments with X-gal at the time of pulmonary staining. Three of the 14 surviving Sprague-Dawley rats and 1 of the 11 surviving cotton rats had evidence of recombinant gene expression in liver tissue. The presence of hepatic gene transfer did not correlate with either the presence or the absence of pulmonary gene transfer in individual rats.

Investigation of Variable Animal Susceptibility to Transduction
In both the Sprague-Dawley rat and cotton rat experiments, individual animals in each species had ß-gal expression; however, most animals demonstrated no evidence of recombinant gene expression in either the lung or the liver. Several studies were undertaken on animals undergoing the surgical pulmonary artery and vein occlusion protocol to investigate factors potentially responsible for between-animal variability.

One possible explanation for the variable animal susceptibility to gene transfer was the presence of neutralizing anti-adenovirus antibodies in rat blood from previous virus exposure. To test this hypothesis, we measured the titer of sera from seven rats (three Sprague-Dawley rats and four cotton rats) infused with the Av1LacZ4 vector to determine their ability to inhibit adenovirus-mediated gene transfer; we used an in vitro transduction procedure with Av1LacZ4 placed into cultured 293 cells. Successful in vivo pulmonary gene transfer had been achieved in one of the four cotton rat lungs and none of the three Sprague-Dawley rat lungs. The positive control serum inhibited transduction at all dilutions from 1:2 to 1:4096, but none of the test sera exhibited a neutralizing effect, suggesting that the presence of preformed antibodies could not explain the failure of gene transfer in these rats.

The majority of animals in the present study had no evidence of recombinant gene expression in either the lungs or liver. We considered whether host factors such as variable presence of viral receptors37 could be responsible for failure to obtain gene transfer in individual animals. To investigate the possibility that individual animals were specifically resistant to adenoviral gene transfer, we injected virus into a small area of skeletal muscle in seven of the Sprague-Dawley rats at the end of the pulmonary virion infusion procedure. Animals were killed 2 days later, and sections of the injected skeletal muscle were stained with X-gal. In these seven rats, none of the lungs demonstrated evidence of ß-gal expression; however, all muscle sections stained blue. Although it cannot be excluded that intramuscular injection results in gene transfer by a mechanism different from that occurring after arterial infusion, this finding nevertheless suggests that all rats were susceptible to transduction with Av1LacZ4 and that the negative findings in the lungs were not due to individual animal-specific resistance to adenoviral gene transfer.

Given the uniformly positive results obtained with skeletal muscle gene transfer, we considered whether individual rats could have an organ-specific lack of susceptibility to adenovirus-mediated gene transfer, which, again, could result from the absence of viral receptors. To investigate lung-specific susceptibility to transduction, when four of the Sprague-Dawley rats were killed, the upper right lung lobe of each rat was separated, placed in organ culture, and exposed to virus ex vivo for 20 or 90 minutes. Twenty hours later, the lung lobes were stained with X-gal. No evidence of ß-gal gene expression was present in the lungs that had been exposed for 20 minutes; however, the two lobes exposed to virus for 90 minutes showed several areas of ß-gal expression. All four of the in vivo virus-exposed left lungs of these animals were negative for expression of recombinant ß-gal. These results suggested that the failure to achieve in vivo transduction of many of the rat lungs was not the result of an organ-specific lack of susceptibility to gene transfer but rather of inadequate contact time between the virus and the lung tissue.

Based on these findings, we hypothesized that despite pulmonary venous occlusion, systemic escape of the virions via bronchial vessels or lymphatics might result in only transient virus-cell contact, which would be of inadequate duration to allow gene transfer to pulmonary cells. To test whether this suboptimal contact could be improved by preventing systemic escape of the viral solution, we attempted surgical occlusion of all hilar structures after thoracotomy and cannulation of the pulmonary artery in five Sprague-Dawley rats. Similar to the pulmonary artery and vein occlusion experiments we described, the lung became somewhat translucent during vector infusion; however, in contrast to those experiments, the lung remained translucent throughout the 20-minute dwell period. Three of five animals treated in this manner had significant pulmonary ß-gal expression both grossly and microscopically. However, the lungs appeared grossly damaged and had large areas of hemorrhagic infarct and consolidation. Microscopically, transduced cell types, although not specifically quantified, appeared to be similar to those observed after occlusion of only the pulmonary artery and vein. Unlike the latter method, areas containing transduced cells also contained numerous inflammatory cells, including mononuclear and polymorphonuclear cells (Fig 4Down). Therefore, although this method of occluding the pulmonary artery, vein, and bronchus resulted in more uniformly successful gene transfer and expression, it did not appear to be a useful method of gene transfer due to the concomitant inflammation and tissue damage.



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  		Figure 4. Photomicrograph of lung tissue after gene transfer with hilar occlusion during virion infusion. Tissue was exposed to 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside and then counterstained with hematoxylin and eosin. A severe inflammatory infiltrate is present, consisting of both mononuclear and polymorphonuclear cells. ß-Galactosidase expression is seen in occasional alveolar epithelial cells (original magnification x100).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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We describe both percutaneous and surgical approaches to pulmonary vascular gene transfer in the rat. In contrast to previous pulmonary gene transfer studies in which adenoviral vectors were administered via the airways to achieve expression of marker genes, {alpha}1-antitrypsin, and human CFTR gene in airway epithelial cells,30 38 39 40 41 the ultimate goal of the present study was to achieve reproducible, high-efficiency recombinant gene expression within the cells of the pulmonary vasculature. Our major findings were that (1) percutaneous catheter-based gene delivery is not successful; (2) a surgical approach that includes transient occlusion of both pulmonary artery and vein achieves detectable pulmonary gene transfer in a minority of animals (15% to 30%); (3) nonvascular cells are the primary targets of adenovirus-mediated gene transfer when virions are infused via the pulmonary artery; and (4) the low levels of vascular gene transfer in our system are not due to a lack of susceptibility of pulmonary endothelial cells to transduction, to the presence of preformed neutralizing antibodies, or to animal- or organ-specific resistance to gene transfer.

Previously reported results describing intravascular gene transfer in the carotid arteries of sheep and rats19 20 22 led us to expect significant pulmonary arterial recombinant gene expression. We were therefore initially surprised by the complete lack of pulmonary ß-gal expression after percutaneous pulmonary virion infusion. In retrospect, this lack of gene transfer was likely due to inadequate contact between the virions and the pulmonary endothelium, as the virions were cleared from the lung by ongoing pulmonary blood flow. The success of gene transfer into a surgically isolated pulmonary arterial segment (Fig 1Up) supports the hypothesis that inadequate virion-cell contact is responsible for the failure of percutaneous gene transfer.

It is more difficult to explain the very low rates of pulmonary gene transfer that were found despite surgical occlusion of both the pulmonary artery and vein. The explanation for this low efficiency may reside in the existence of significant anatomic differences between the present pulmonary system and other vascular gene transfer systems. In previously described models of isolated large-vessel gene transfer, virions were incubated in closed vascular compartments, optimizing virion–target cell contact. In the pulmonary delivery system of the present study, virions are introduced into an extensively branched vascular bed with a relatively large surface area (the pulmonary capillary surface area of a rat is approximately 1 m2, as calculated by scaling down the cited42 human surface area of 126 m2). Dilution of the virions in the fluid adherent to this extensive surface area may have detrimental effects on virus–target cell interactions. In addition, as is evident from the observation of gene transfer into nonvascular cells (Figs 2Up and 3Up), the infused virions can escape from the pulmonary vascular space and therefore are further diluted by the pulmonary interstitial fluid. Finally, dilution of virions from an independent bronchial circulation, as well as clearance of virions from the lung via pulmonary lymphatic drainage vessels,43 44 45 would be expected to reduce further the virion concentration within the lungs during the infusion period. Evidence supporting the latter hypothesis is found in the experiments in which the entire left lung hilum was occluded, with probable obstruction of bronchial inflow and bronchial and lymphatic drainage. A relatively high percentage of animals subjected to this procedure (60%) exhibited successful gene transfer and expression. Taken together, it is probable that anatomic and physiological factors peculiar to the pulmonary vasculature combine to minimize opportunities for virion-cell contact and pulmonary vascular gene transfer.

In contrast to the 15% to 30% success rates of the present study, Lemarchand et al25 reported a 75% success rate (13 of 17 surviving sheep) for recombinant gene expression after adenovirus infusion into the pulmonary arteries of sheep with simultaneous occlusion of a lobar artery and vein. The percentage of transduced pulmonary cells overall or of specific cell types was not given. The concentrations of infused virus for rats and sheep were similar, but the anatomic details of the protocols differed significantly. In the sheep model, infusion of virions and occlusion of vessels occurred relatively peripherally. This system of distal virion infusion may be relatively resistant to washout by bronchial arterial flow.43 Species specificity in susceptibility to adenovirus-mediated gene transfer is another factor that might explain the observed differences in rates of successful gene transfer. We have achieved successful adenovirus-mediated gene transfer into sheep arteries22 at concentrations that are not successful at producing significant recombinant gene expression in rat carotid arteries (D.A.D., unpublished data, 1994).

Similar to the findings of Lemarchand et al,25 pulmonary arterial virion infusion in the present study resulted in recombinant gene expression predominantly in nonvascular cells. The results of both studies are consistent with the patterns of extravascular gene transfer that have been described after infusion of adenovirus virions into the cardiac and hepatic capillary beds.24 46 Although not tested directly in any of these studies, it appears that the permeability of capillary endothelial cells to adenovirus virions is different from that of large arterial endothelial cells. Further investigation of the mechanisms underlying this difference in endothelial permeability is warranted as this might eventually permit specific modification of virions to either exploit or avoid egress of virions from the vasculature. These modifications might increase both the versatility and specificity of percutaneous gene transfer. A notable difference between our results and those of Lemarchand et al is the presence of smooth muscle cell gene transfer in the present study. Four of 5 rats with successful gene transfer expressed the recombinant gene in smooth muscle cells (compared with none of 13 sheep in the study of Lemarchand et al). The transduced smooth muscle cells were located primarily in airway structures. Both pulmonary vascular smooth muscle and endothelial cells remain challenging targets for in vivo gene transfer.

Despite the finding of variable and inefficient recombinant gene expression, adenovirus-mediated gene transfer may prove to be a useful technique for both pathophysiological and therapeutic studies of the pulmonary vasculature. Low-level gene transfer models have been used to test the efficacy and safety of potentially therapeutic genes in humans,41 47 and therapeutic effects in the lung have been demonstrated in animal model systems in which gene transfer is not clearly more efficient than that we describe.48 Delivery of genetic material to pulmonary vascular cells provides a unique approach to the study of pulmonary vascular physiology and may eventually serve as a particularly powerful approach for local drug delivery. Given the dismal prognosis of pulmonary vascular disease and the lack of available therapy, continuing efforts to optimize intravascular pulmonary gene transfer are warranted. It is rational to pursue this optimization in a small animal model such as the rat, in which both housing and virion volumes are relatively economical and in which informative models of pulmonary hypertension,9 11 26 adenovirus-mediated gene transfer,30 38 39 and adenovirus-associated lung pathology28 29 49 have been described. The present study provides a basis for further work.


*    Acknowledgments
 
The authors gratefully acknowledge the invaluable assistance of the members of the Laboratory of Animal Medicine and Surgery, NHLBI, with animal protocols and procedures. We also thank Dr Michael E. Burt and members of his research team for instructions on their surgical technique, as well as Sonia Janich and Dr Blake Roessler for the electron microscopic work. In addition, we thank Dr Bruce Trapnell of Genetic Therapy, Inc, for providing the adenoviral vector and Eleonora Dorfman for production and titering of adenoviral stock.

Received October 21, 1994; accepted February 13, 1995.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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