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Ann Thorac Surg 2004;77:458-463
© 2004 The Society of Thoracic Surgeons

Original article: general thoracic

Induction of pulmonary angiogenesis by adenoviral-mediated gene transfer of vascular endothelial growth factor

Virginie Lambert, MDa*, René Michel, MD, CMb, Guy-Michel Mazmanian, MDc, Elisabeth M. Dulmet, MDa, André Capderou, MD, PhDa, Philippe Hervé, MDa, Claude Planché, MDa, Alain Serraf, MD, PhDa

a Department of Pediatric Cardiac Surgery, Marie-Lannelongue Hospital, Le-Plessis-Robinson, France
c Laboratory of Experimental Surgery, Marie-Lannelongue Hospital, Le-Plessis-Robinson, France
b Department of Pathology, McGill University, Montreal, Quebec, Canada

Accepted for publication August 6, 2003.

* Address reprint requests to Dr Lambert, Department of Pediatric Cardiac Surgery, Marie-Lannelongue Hospital, 133 Avenue de la Résistance, 92350 Le-Plessis-Robinson, France
e-mail: vlambert{at}ccml.com


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: We hypothesized that gene transfer of vascular endothelial growth factor (VEGF) mediated by an adenovirus vector might induce pulmonary artery angiogenesis in a lamb model of pulmonary artery hypoplasia.

METHODS: Thirteen fetal lambs had left pulmonary artery banding at 106 days of gestation. Following birth, 3 groups were divided: VEGF group (n = 5) and ß-GAL group (n = 4) received an adenoviral vector encoding respectively for human VEGF165 and for galactosidase A. A control group (n = 4) had neither gene nor virus. Viral suspensions were selectively instilled in the left bronchus 6.5 days after birth. Five nonoperated lambs constituted the normal group. Euthanasia was performed at 30 days of age. Gene transfer was confirmed by blue coloration of left lung obtained with Xgal solution in an additional experiment. Histomorphometric evaluation was performed. All groups were compared with ANOVA test and paired test was used to compare right and left lung in each animal.

RESULTS: Left lung was similarly hypoplastic in all operated lambs. Left pulmonary artery hypoplasia present in all operated groups was significantly less pronounced in VEGF group. The number of pleural arteries was similarly increased in left lung of all operated lambs. Left lung arterial density was higher in VEGF group than in all other groups. The percentage of parenchyma of left lung was lower in ß-GAL group than in all others, partially returned to normal in VEGF group.

CONCLUSIONS: In this model, transbronchial VEGF gene transfer induces pulmonary angiogenesis, proximal pulmonary artery growth and contributes to lung parenchyma recovery.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Lung and pulmonary artery (PA) hypoplasia is observed in various congenital diseases such as tetralogy of Fallot with or without pulmonary atresia. The feasibility of a complete surgical repair depends on the development of pulmonary vessels. Therapeutic angiogenesis describes the induction or stimulation of neovascularization for the treatment or prevention of pathologic clinical situation characterized by local hypovascularity [1]. Vascular endothelial growth factor (VEGF) was shown to play an important role in angiogenesis and to improve blood flow and collateral development in ischemic limb and myocardium using gene therapy [2]. VEGF protein and messenger RNA have been detected in distal airway epithelial cells in the developing lung, suggesting that VEGF plays a role in the development of the pulmonary capillary bed [3]. We hypothesized that gene transfer of VEGF mediated by an adenovirus (Ad) vector might induce a pulmonary artery angiogenesis in an experimental lamb model of pulmonary artery hypoplasia.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1985).

Surgical procedure
The study was carried out with 13 survival lambs (pré-Alpes) operated on in utero between 103 and 108 days of gestation (median 106 days, normal term 145 days). Pregnant ewes were sedated with intravenous pentobarbital sodium (15 mg/kg) and anesthetized by halothane. Under sterile conditions, hysterostomy was performed through an anterior midline incision, and the left forelimb of the fetus was delivered through the uterine incision. The fetal skin was incised after spinal anesthesia with 1% xylocaine. A thoracotomy was performed in the fourth left intercostal space and the left PA was isolated. A tie 5-mm wide was placed around the left PA post ductus arteriosus and was tightened carefully on a dilator to obtain a PA stenosis with a lumen size of 2 mm. After thoracotomy closure, the fetus was replaced in the amniotic fluid. After recovery from anesthesia, ewes were placed in a warm pen with food and water. Natural delivery occurred.

Gene therapy
Adenovirus vector
The replication-deficient vector Ad.VEGF is an E1a-, partial E1b- partial E3-Ad vector with an expression cassette in the E1 position containing the CMV immediate early promotor/enhancer driving the cDNA for the 165-residue form of human VEGF [4, 5]. Ad.RSVß-gal (similar to Ad.VEGF but with the E. coli lacZ gene encoding for ß-gal in the expression cassette) was used both as a control vector and to assess gene transfer (cells expressing ß-gal can be detected by a nuclear-dominant blue color). The administration of Ad vectors was performed in lambs 3 to 7 days after delivery (median 6.5 days) under a brief general anesthesia: the viral solution was instilled through a tube placed selectively into the left bronchus; the correct placement of the tube was controlled by auscultation. The lung was ventilated for 3 minutes to allow distribution of the virus. After recovery from anesthesia, lambs were placed with their mother until euthanasia.

Experimental groups
The operated lambs were divided into three groups: the VEGF group (n = 5) and the ß-GAL group (n = 4) consisted of lambs that received respectively 1 x 1010 plaque-forming units (pfu) of Ad.VEGF and 1 x 1010 pfu of AdRSV.ß-gal, diluted in 300 µL of 0.9% NaCl solution; the control group (n = 4) was not exposed to the Ad vector. A normal group consisted of 5 non operated lambs which underwent no gene transfer and were paired for age (median 29 days, range 23 to 45 days) and weight (median 10 kg, range 8 to 15.5 kg) at the time of euthanasia; one of these lambs was excluded because the results were not in accordance with others: although this lamb was not operated on and was not in contact with the virus of experimentation, there was a loss of parenchyma in the left lung and a high number of pleural arteries in the right lung; we suspected that it had a extraneous viral infection before euthanasia and we could not include it in the normal group.

Evaluation of transbronchial gene transfer
We evaluated in vivo gene transfer and expression of lacZ gene in an additional lamb, which had received AdRSVß-gal and was killed 3 days following infection. The lungs were fixed (5 minutes, 21°C) in 1% formaldehyde and 0.2% glutaraldehyde. The presence of the lacZ gene product, ß-gal, was determined by staining the lungs with the Xgal reagent for 6 hours [6]. A blue coloration of the left lung was observed (Fig 1), the right one was used as control.



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  		Fig 1. Both images show a dorsal view of fixed lungs after Xgal staining: a blue coloration of the left lung is observed.

 
Morphologic analysis
Preparation of the lungs
The lambs were killed at a median age of 30 days (range 23 to 45 days) by intravenous injection of pentothal (1 g) and KCl (2 g) after administration of heparin (300 U/kg). The heart and lungs were removed from the thorax through a median sternotomy and were prepared for histology. A saline solution was infused into PA until the left atrial effluent was grossly clear of blood. Pulmonary arteries diameter was measured with a calibrator introduced up to lobar branching, downstream of the banding in the left PA. The diameter was expressed in mm/kg1/3. The pulmonary veins were ligated and the lung was fixed with 10% buffered Formalin instilled at 20 mm Hg of pressure through the main stem bronchus and PA, and kept for 24 hours on a constant pressure system [7]. The lung volume was obtained using fluid displacement (Archimedes principle) for each fixed lung and expressed in mL/kg. The lung was cut into 1-cm slices, 10 random samples per lung were cut from the slices and embedded in paraffin by standard histologic techniques. Five-micrometer–thick sections were cut from the blocks and stained with elastic Van Gieson stain.

Morphometric measurements
The morphometric measurements were performed on five random slides selected on each lung of each animal; the pathologist reviewing the sections was blinded to the experimental group until the end of the analysis.

Percentage of parenchyma
Morphometry was performed using standard point-counting technique [8]. The lung was evaluated as parenchyma and nonparenchyma. Parenchyma included alveoli, alveolar ducts, respiratory bronchioles, and their adjacent vessels. Nonparenchyma included all other structures: bronchioles, bronchi and their adjacent vessels, connective tissue and veins. The slides were examined using Olympus BX50 optical microscope equipped with a grid of 100 points. At a magnification of x2, we systematically analyzed four fields (400 points) per slide. For each compartment, the points were summed. A total of 14,400; 13,600; 10,800; and 9,600 points were respectively counted in normal, control, ß-GAL, and VEGF groups. The results for each lamb were averaged and expressed as a percentage of parenchyma in the lung.

Arterial density in parenchyma
The PA more than 15 µm diameter were counted in the parenchyma. The different color of the dyes distinguished arteries with external and internal elastic lamina from the veins with only external one. If there was any doubt as to whether a vessel was an artery or a vein, it was excluded. At a magnification of x20, 20 fields per slide corresponding to an area of 1 cm2 were analyzed. The arterial density corresponded to the number of PA per cm2 of parenchyma area analyzed. We examined a total of 36, 34, 27, and 24 cm2 respectively for normal, control, ß-GAL, and VEGF groups.

Number of pleural arteries
The pleural arteries more than 15 µm diameter were counted in the visceral pleura at a magnification of x20. Lymphatic ducts and veins were excluded. A total of 315, 440, 320, and 240 mm of pleura were examined respectively in normal, control, ß-GAL, and VEGF groups. The results were expressed by the number of arteries per millimeter of pleura (n/mm).

Statistical analysis
Data are expressed as mean ± SD. Analysis of variance for paired data (left versus right lung) was performed. ANOVA with PLSD Fisher test was used to test for differences among the 4 groups in each lung. Differences between the right and left lung for each group were compared by paired Student's t tests. A p value of less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Gene transfer was assessed by blue staining of left lung obtained with Xgal reagent in the additional experiment. No lambs died after inclusion in the study. The morphologic and morphometric results are summarized in Table 1. The left lung volume was significantly lower in all operated lambs than in normal group with no difference in any of the banded lambs. After dissection of the main pulmonary artery, the banding was well placed at the origin of left PA in all operated lambs. Downstream of the banding, the left PA was hypoplastic: the left PA size was significantly lower than the right PA in each operated group and than the left PA of normal group. The left PA hypoplasia was significantly less pronounced in VEGF group than in control and ß-GAL groups. The number of pleural arteries was significantly higher in the left lung of operated groups with no difference in any of them. The arterial density in parenchyma for the left lung was significantly higher in VEGF group than all other groups and the difference was close to the significance when compared with the contralateral right lung (p = 0.06). The left lung showed fibrosis in lambs of ß-GAL group and the percentage of parenchyma in this lung was significantly lower than in the right lung and than in the left lung of normal and control groups. The percentage of parenchyma in the left lung of VEGF group was significantly lower than in the left lung of normal and control groups but higher than in the left lung of ß-GAL group.


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  		Table 1. Morphological and Morphometric Results

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
This preliminary study suggests that transbronchial adenomediated-virus VEGF gene transfer induces PA angiogenesis in this experimental model of PA and lung hypoplasia. VEGF is an important factor for endothelial cell proliferation and a key regulator of blood vessel development in embryos; its effects on collateral vessel formation after gene transfer are shown in experimental model of ischemic limb or myocardium [9, 10] and are investigated in human as therapeutic angiogenesis in peripheral arterial diseases [11]. To the best of our knowledge, the effects of VEGF gene transfer on pulmonary vascularization was not explored.

The experimental model used in this study was designed to simulate PA hypoplasia observed in patients with congenital disease such as tetralogy of Fallot. Left PA and lung hypoplasia were present in all operated lambs. The fetal operation was performed at the canalicular period of gestation (80 to 120 days) corresponding to a rapid expansion of peripheral airways units and growth of the pulmonary vasculature [12]. In autopsy studies, a small lung volume was noted in patients with tetralogy of Fallot [13] and with unilateral congenital dysplasia of lung [14] and was associated with low pulmonary blood flow. Lung hypoplasia was observed after a complete interruption of blood flow by ligation of left PA in lamb fetus performed at the same stage of gestation [15, 16], due to a decrease in the volume of the future airspaces and parenchymal tissue, without any change in non parenchymal tissue [15, 16]. We have not observed a decrease in percentage of parenchyma nor in arterial density in parenchyma in the left lungs of control group versus normal group. It suggests that a reduced but persisting blood flow is sufficient to maintain parenchyma development and that the lung hypoplasia, observed in our study, is related to the decrease in the same proportion in airways and vessels. These findings were observed in lamb models after banding of main PA [17] and in patients with tetralogy of Fallot [13, 18].

Recent studies have confirmed that the vascular component of the lung develops in tight coordination with the potential air spaces [19]. Gebb and associates have demonstrated that the lung epithelium is required to maintain proliferation and differentiation of the developing vasculature within the mesenchyme, suggesting that tissue interactions are critical to normal development of the pulmonary circulation [20]. Molecular genetic studies suggest a mutual regulation of these processes mediated by VEGF [21]. The temporal and spatial expression of flk-1 mRNA which encodes an endothelial cell specific VEGF receptor in fetal and neonatal rat lung was defined: flk-1 positive cells were observed in the lung at every prenatal stage through birth and clusters of flk-1 positive cells were localized in the mesenchyme closely to the developing epithelium [20]. These findings raise the possibility that up regulating transcription of VEGF mRNA may result in up regulation of endothelial cell receptors for VEGF. Moreover, high levels of VEGF protein was found in the developing human lungs: Shifren and associates [22] demonstrated that VEGF is widely expressed in the midgestation human fetus and is localized primarily to epithelial cells and myocytes and not endothelial cells, suggesting that VEGF is a paracrine factor secreted by nonendothelial cells, which modulates activities in adjacent vascular endothelium. Acarregui and associates [3] showed localization of VEGF protein to the basement membrane region of distal airway epithelial cells. These findings, if applicable to our animal model, support our hypothesis that administration of VEGF gene in lungs by gene transfer stimulates endothelial receptors for VEGF and leads to the formation of new pulmonary vessels as observed in systemic circulation [23]. The two processes implicated in the development of new vessels of systemic circulation, vasculogenesis and angiogenesis, were shown to be similar to the processes observed in the development of lung vasculature [24]. In our lambs, we have observed a significant increase in arterial density in the parenchyma of lungs in contact to the VEGF gene, versus all other groups, suggesting an angiogenic effect of VEGF gene transfer. Recently, VEGF was one implicated in arterial venous malformation [25]: we have noted no morphologic anomalies of PA, suggesting that the blood vessels counted were not shunt vessels between PA and pulmonary veins but might improve flow to alveolar capillaries. Furthermore, we observed a significant growth of proximal left PA in VEGF groups versus other operated groups: we speculate that the development of new distal PA led to a decrease in vascular resistances and an increase in blood flow; the PA growth may be a consequence of an increase in shear stress secondary to an increase in trans-stenotic gradient through the banding.

Replication-deficient recombinant adenovirus are used as vectors for transferring exogenous genes [4] and are the most advanced method for gene delivery to airway epithelium [26]. Aerosol delivery of Ad-vectors is of particular interest in regard to gene therapy with potential advantages of more uniform respiratory delivery, less invasive approach and ease of repetition. Other techniques have been used to transfer genes to the lungs in vivo such as the Ad-vectors administration in the pulmonary circulation: the exogenous gene transferred through PA is expressed in all categories of endothelium and epithelium of the lung including the endothelium of pulmonary and bronchial circulation [6, 27]. In our study, administration of Ad-vectors through airways was interesting for two reasons: first VEGF gene was directly transferred to the target cells as VEGF protein was shown in previous study to be produced and elaborated by distal airway epithelial cell and localized at the basement membrane of type II and type II precursors in the developing human lung [3]; second, airway administration of Ad-vectors seemed to be more selective than by pulmonary circulation avoiding that the Ad-vectors reached the bronchial circulation: the increase in bronchial circulation assessed by an increasing number of pleural arteries was observed in the left lung of all operated lambs, similar to the VEGF group as in the others. Angiogenesis in the bronchial circulatory system has been described in patients with congenital absence of unilateral PA [14] and in a lamb model after unilateral PA ligation [28]. In our study, we hypothesized that the development of bronchial arteries is due to the PA banding and not to the VEGF action.

Several drawbacks of Ad-vectors have been identified [29]: current Ad-vectors are associated with only transient, 2 to 4 weeks transfer expression; it has been demonstrated that first generation Ad-vectors evoke a strong cellular immune response, resulting in destruction of target cells by cytotoxic T lymphocytes. This immune reaction may explain the significant loss of parenchyma in the left lung of the ß-GAL lambs. A partial recovery of parenchyma was observed in the VEGF group supporting the potential role of VEGF in lung protection or recovery from acute lung injury after adenoviral infection. This is in accordance with some clinical studies: in preterm infants with lung injury, toward the end of the post natal week, and during recovery from acute respiratory distress, Lassus and colleagues [30] found that pulmonary concentrations of VEGF were at the same magnitude as those that induce proliferation and differentiation of human fetal airway epithelial cells in vitro [3]. In adult patients with acute respiratory distress syndrome, epithelial lining fluid VEGF levels have shown inversely correlated with lung injury [31].

The most important limitation of this study is that the effective transfection of the VEGF gene was not demonstrated. We didn't look for VEGF protein, mRNA or DNA because, as shown previous studies performed in dog pericardium [32] and rat retroperitoneal adipose tissue [33], VEGF cDNA expression decreased after 5 days following administration of adenovector, and VEGF protein was not detected after 10 days; capillary proliferation persisted at 28 to 30 days. Thus, it is possible that, when we performed euthanasia 3 to 4 weeks after viral infection, no transfected VEGF-gene was expressed in the lung. Although transfection of VEGF was not demonstrated at a molecular level in this study, we found an angiogenic effect assessed by an increased arterial density in VEGF group, statistically significant when compared with non-VEGF groups. Further investigation is required to assess VEGF expression at earlier points following transfection. A second limitation is that the morphology of PA was not analyzed. A decreased medial muscular layer was observed in fetal experimental model of pulmonary stenosis and in patients with tetralogy of Fallot [13, 17, 18]; an altered perfusion pressure of the pulmonary circulation or a small change in oxygen tension in the blood flow were suggested to explain a decreased stimulus to development of the pulmonary vascular bed [17, 18]. Further investigation could be interesting to determine whether PA wall may be altered by the unilateral banding and restored by the transfected VEGF gene.

In summary, in this model of PA and lung hypoplasia, VEGF gene transfer mediated by Ad vectors and administrated by airways induced PA angiogenesis and proximal PA growth. Also it contributes to parenchyma recovery after adenoviral infection. Our findings favor the notion that angiogenic activity of VEGF applied to lung is sufficiently potent to constitute a therapeutic effect and future studies are planned to prove that VEGF transfection was effective in this model. Further investigation may clarify the extent to which use of VEGF may be appropriate for additional treatment of selected patients with pulmonary hypoplasia associated with congenital disease.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
We are grateful to Dr Patricia Lemarchand (Necker-Enfants-Malades Hospital) for providing VEGF165. This work was supported by "la Fondation de l'Avenir" and the Marie-Lannelongue Hospital. We thank Chantal Verriest, Frédéric Seccatore, Sylvie Plante, Annette Popineau, and Annick Denoncourt for their technical assistance.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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