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a Lung and Airway Laboratory, Federal University of Rio Grande do Sul School of Medicine, Hospital de Clínicas de Porto Alegre, Brazil
b Department of Anesthesiology, Federal University of Rio Grande do Sul School of Medicine, Hospital de Clínicas de Porto Alegre, Brazil
c Department of Pathology, Federal University of Rio Grande do Sul School of Medicine, Hospital de Clínicas de Porto Alegre, Brazil
d Gene Therapy Center, Federal University of Rio Grande do Sul School of Medicine, Hospital de Clínicas de Porto Alegre, Brazil
Received 19 August 2007; received in revised form 18 December 2007; accepted 19 December 2007.
* Corresponding author. Address: Hospital de Clínicas de Porto Alegre, Serviço de Cirurgia Torácica, Rua Ramiro Barcelos 2350, Sala 2050-M1, 90035-003 Porto Alegre, RS, Brazil. Tel.: +55 51 2101 8684; fax: +55 51 2101 8684. (Email: msaueressig{at}hcpa.ufrgs.br).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Abbreviations: VEGF = vascular endothelial growth factor • DNA = deoxyribonucleic acid • phVEGF165 = naked plasmid enclosed human VEGF165 • cDNA = complementary DNA • bp = base pairs • mRNA = messenger ribonucleic acid • RT-PCR = reverse transcription-polymerase chain reaction • GAPDH = glyceraldehydes-3-phosphate dehydrogenase • RU = relative optical density unit • PBS = phosphate-buffered saline
Key Words: Gene therapy • Angiogenesis-inducing agents • Pulmonary surgical procedures • Models • Animal • Vascular endothelial growth factor
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Procedures such as lung transplantation, tracheobronchoplasty and pneumonectomy interrupt bronchial circulation for about 2 weeks. As a consequence, airway viability during this period would depend solely on mixed-venous blood provided by the collateral pulmonary circulation [4]. In lung transplantation, the importance of the bronchial circulation has been recognized not only due to the persistence of airway complications in up to 12% of allografts [5,6], but also to the fact that a correlation has been reported between obliterative bronchiolitis and small airway ischemia [7,8].
Nevertheless, surgical attempts to restore the bronchial circulation have been abandoned because no evidence of clinical benefit was obtained [9]. Currently, therapeutic angiogenesis using gene therapy to promote protracted expression of specific angiogenic proteins brings new hope as an alternative to surgery.
Vascular endothelial growth factors (VEGF) are the main angiogenic cytokines studied in the last 10 years [10]. VEGF165, the most abundant isoform of VEGF, is a secreted endothelial cell specific mitogen. In fact, experimental studies [11,12] and randomized controlled trials of angiogenic therapy with naked plasmid DNA encoding human VEGF165 (phVEGF165) [13,14] have already achieved promising results.
Accordingly, the present study was undertaken to investigate the efficacy of an angiogenesis gene therapy approach to promote the early re-establishment of systemic arterial perfusion in canine bronchi.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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General anesthesia was induced with a combination of atropine sulfate (0.25 mg/kg) s.c. and thiopental sodium (15 mg/kg) and atracurium (0.4 mg/kg) intravenously. The dogs were then intubated with a 7 mm orotracheal tube and placed under mechanical ventilation with tidal volume of 10 ml/kg and a respiratory rate of 16 breaths/min. Anesthesia was maintained with isoflurane (0.5–1.3%) i.n. and an inspiratory oxygen tension (FiO2) of 0.5.
Interruption of circulation in the bronchus was achieved following the experimental model of complete transversal bronchotomy described by Baile et al. [15]. Briefly, the chest was opened by a left thoracotomy through the fifth intercostal space, and the pericardial sac around the left hilum was completely incised. Then, a bronchotomy was performed at the left mainstem bronchus by completely cutting the airway transversally. The section was performed two cartilaginous rings proximal to the upper lobe orifice, thus dividing the airway into proximal and distal bronchus (deprived of systemic arterial circulation). Immediately after airway transection, either the gene therapy or saline solution was delivered to the distal bronchus. The bisecting left mainstem bronchus was then reanastomosed using interrupted 4.0 propylene sutures (Johnson & Johnson, São José dos Campos, Brazil). The skin was closed with interrupted 3.0 nylon sutures, and a 14 Fr chest tube was installed. This chest tube was removed after the thoracotomy wound was closed.
On the third postoperative day, the animals were euthanized using tetracainchloride (T-61) (0.3 ml/kg) intravenously. Nine animals (four VEGF and five controls) were euthanized for investigation of VEGF165 gene expression in the distal bronchus. The other seven dogs (four VEGF and three controls) were sacrificed for histological and immunohistochemical analysis (Table 1
). All analyses were performed blinded.
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Transfection of the left mainstem bronchus was carried out using 50 µg of naked plasmid phVEGF165 dissolved in sterile saline (final volume of 200 µl) and slowly injected into the submucosa of the membranous portion of the distal bronchus, using a syringe with 27 G needle. In the control group, only saline (200 µl) was administered.
2.3 RNA isolation and reverse transcription-polymerase chain reaction
The efficacy of gene transfer was determined by estimating the levels of VEGF165 transgene mRNA. For that, human VEGF165 gene expression in the distal bronchus was evaluated at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR).
Samples retrieved from the membranous portion of the distal bronchus, near the site of gene injection, were immediately frozen in liquid nitrogen for extraction of total RNA using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The concentration and purity of total RNA from the samples were determined by spectrophotometry at 260 nm. After that, the samples were stored at –80 °C.
The VEGF165 mRNA in these samples was reverse-transcribed to single strand cDNA. Additionally, synthesis of the canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was carried out as an internal control. Two pairs of primers were designed based on human VEGF165 mRNA (GenBank accession # AF486837) and canine GAPDH mRNA (GenBank accession # AB038240) sequences (VEGF165 sense 5'-TTCATGGATGTCTATCAGCG-3', antisense 5'-GCTCATCTCTCCTATGTGCT-3'; GAPDH sense 5'-GCCAACATCAAATGGGGTGA-3', antisense 5'-CATATTTGGCAGCTTTCTCC-3'). The size of the RT-PCR product for VEGF165 and GAPDH was 234 bp and 470 bp, respectively.
Synthesis and amplification reactions of the cDNA from each sample were accomplished using 250 ng of total mRNA, 12.5 µl of 2x Reaction Mix (0.4 mM of each dNTP and 2.4 mM MgSO4) (Invitrogen Corp, Carlsbad, CA), 1 µl of each primer (10 µM), 0.5 µl of Superscrip II RT/Platinum Taq Mix (Invitrogen Corp, Carlsbad, CA) and distilled water to complete the final volume of 25 µl per tube for reaction. In a thermocycler, the reaction tube underwent initial denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min and final extension at 72 °C for 10 min. The RT-PCR products were analyzed with 1.5% agarose gel electrophoresis, and the results were quantified by gray-scale optical density with the UVIDocMw digital imaging system 10.01 software (Uvtec, Cambridge, England). The results of gene expression were represented as the VEGF/GAPDH optical density ratio, for each animal, and expressed as relative optical density units (RU).
2.4 Injection of microvascular dye
Microvascular latex dye (Labsynth, Diadema, Brazil) was injected in seven dogs (four VEGF and three controls) after euthanasia to evaluate the presence of systemic arterial perfusion in the distal bronchus. Briefly, the 50% green-colored dye was injected under 150 mmHg controlled pressure into the thoracic aorta using a 14 G intravenous catheter.
2.5 Histological analysis
The left mainstem bronchus was harvested immediately after injection of microvascular dye, and the specimen was fixed in formalin. After fixation, the airway was divided in the anastomosed region into a proximal and a distal portion. An additional transversal section was performed at the distal bronchus 6 mm from the anastomosis to minimize the effects of surgical trauma on the analysis. These paraffin-embedded specimens were cut into 5 µm thick sections, stained with hematoxylin–eosin for histological examination, and processed for immunohistochemistry.
Thereafter, the identification of capillary vessels filled with microvascular dye in high-power fields (400x magnification) was accomplished by one trained observer blinded for experimental groups. The number of submucosal dye-filled vessels per section was counted manually.
2.6 Immunohistochemical assay
Expression of VEGF protein after gene transfer was confirmed by immunostaining. Briefly, paraffin-embedded specimens of the distal bronchus were cut into 5 µm thick sections and submitted to deparaffinization. After rinsing with phosphate-buffered saline to block endogenous peroxidase activity and nonspecific binding, the sections were incubated in 5% hydrogen peroxide for 20 min and then pretreated with 10% normal horse serum. Specimens were incubated with polyclonal anti-human VEGF antibody 1:400 (Santa Cruz Biotechnology, Santa Cruz, CA), during 1 h at room temperature. Bound primary antibody was detected with the biotin–streptavidin–peroxidase method (Dako, Glostrup, Denmark).
Digital photomicrographs of the VEGF-positive airway epithelium were taken from four randomly chosen fields (400x magnification) per section and transformed into 256 grayscale to estimate the intensity of VEGF protein expression with optical densitometry using the UVIDocMw® digital imaging system version 10.01. The results were described as optical density units (DU).
2.7 Statistical analysis
Results were expressed as mean ± standard deviation. Statistical significance was evaluated using Wilcoxon Mann–Whitney test for comparison between two means. The relationship between the number of submucosal dye-filled vessels and the VEGF protein expression in the distal bronchus was also analyzed through calculation of Spearman's linear correlation coefficient (r
s). Statistical significance was established at p
< 0.05. All statistical analyses were performed with PEPI 3.0 (J.H. Abramson & Paul M., Gahlinger, Salt Lake City, USA).
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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3.1 Reverse transcription-polymerase chain reaction
The VEGF/GAPDH optical density ratio in the VEGF group was significantly higher than in the control group (p
< 0.01) (Fig. 1
). As a result of the greater VEGF gene expression in VEGF-treated distal bronchi, the mean VEGF/GAPDH optical density ratio was 17.2 times greater than in the control group. In other words, the intensity of VEGF expression indirectly estimated by optical density reached a mean of 86% of GAPDH expression in VEGF-treated bronchi, compared to only 5% calculated for the control group (Table 1, Fig. 2
).
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In all specimens of the VEGF-treated distal bronchus, submucosal dye-filled vessels were observed. In these sections, 56.25 ± 83.07 submucosal dye-filled microvessels were identified, with diameter ranging from 10 µm (capillaries) to 50 µm (arterioles) (Fig. 3 ). On the other hand, in the control group, microvascular dye was not observed in any of the specimens examined.
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Although local injection of recombinant angiogenic proteins [17–19] has been previously tested to induce tracheal revascularization, and despite the existing evidence showing the ability of VEGF165 to stimulate endothelial cell proliferation within 48 h [16], there have been no studies so far describing the use of gene therapy with phVEGF165 to induce early bronchial revascularization.
In this sense, the present study was the first to document the early re-establishment of systemic arterial perfusion, that is, bronchial circulation, in an airway segment using gene therapy with phVEGF165. The observation of dye-filled vessels in an airway deprived of bronchial circulation led us to conclude that VEGF165-induced angiogenesis was capable of restoring transanastomotic vascular communication, allowing the microvascular dye injected into the thoracic aorta and, consequently, through bronchial arteries, to reach bronchial capillaries not less than 6 mm away from the anastomosis.
Our analysis of gene expression reveals an increase in VEFG165 RNAm synthesis, indicating that transfection was successful. In this study, the effectiveness of therapeutic angiogenesis resulted from the correct choice of target cells for gene transfer and from the method of phVEGF165 delivery. Despite the high cellular differentiation rate and low proliferation rate, the airway epithelium is more susceptible to gene transfer than other types of cells present in lungs [20], in addition to being the main endogenous source of lung VEGF [21]. The effectiveness of our gene transfer strategy to this epithelium is confirmed by the 17.2-fold increase in VEFG165 gene expression in the distal bronchial specimens transfected with phVEGF165.
The present immunohistochemical findings suggest that even a modest increase in VEGF expression after transfection with a naked plasmid would be sufficient to locally re-establish the bronchial circulation after a few days. At the same time, our study shows a positive and significant correlation between the intensity of VEGF protein expression and the number of dye-filled vessels reconnected to the systemic arterial circulation. Thus, it is possible that an additional elevation in gene expression levels could promote a more consistent, and perhaps more precocious, angiogenesis. In future works, the quantitative analysis of protein expression with enzyme-linked immunoassay (ELISA) and the use of viral vectors that enable more intense transfection may provide an answer to this question.
Once again, the intrinsic characteristics of the target tissue of gene therapy in this study may have facilitated VEGF protein synthesis. It is known that the respiratory epithelium suffers no damage even in the absence of arterial perfusion because the oxygen supply to this epithelium predominantly originates from the airway lumen [20]. As non-ischemic cells have a higher VEGF synthesis capacity [21], the guarantee of sufficient oxygen supply to the bronchial epithelium has probably contributed to the success of therapeutic angiogenesis.
Also, in our study, gene transfer specifically to the respiratory epithelium was made possible by the use of a novel method of phVEGF165 administration to the bronchial submucosa, that is, close to the target cells. In contrast, techniques of plasmid instillation in the airway lumen, employed in most studies [22–24], do not ensure gene transfer to the airway and may disrupt the cDNA. These studies also show limitations of the viral vector for efficient transfection of epithelial cells through the apical membrane, since junctional complexes establish a polarization of respiratory epithelial cells. However, the administration of our naked plasmid directly to the bronchial submucosa served to avoid potential physical barriers in the airway lumen, such as mucus. We believe that gene transfer was probably achieved through a different pathway than that used by viral vectors, that is, the basolateral membrane of the bronchial epithelium. In this sense, our method of gene therapy delivery may have yielded a higher number of plasmids per target cell, which would be extremely advantageous for protocols based on naked plasmids, which usually present a low transfection rate.
The main limitation of our study was the large standard deviations. This makes the evaluation of clinical significance more difficult, even in the presence of statistical significance. Another deficiency may be the experimental model employed to interrupt bronchial circulation, which did not consider other factors affecting surgical procedures such as lung transplant.
In the meantime, the present study is a first step towards the development of a simple and effective technique to promote airway angiogenesis, and as such it clarified some issues concerning the potential for transfection and protein expression of the bronchial epithelium as well as the angiogenic capacity of airway tissue treated with VEGF165 . Future studies with lung transplant models are required to confirm if the early restoration of bronchial circulation can minimize bronchial microcirculation disturbances associated with acute rejection and reperfusion lesion, perhaps impacting the incidence of obliterative bronchiolitis [6,7].
In summary, the successful expression of phVEGF165 and the early restoration of systemic arterial circulation in a short segment of distal bronchus suggest the usefulness of phVEGF165 as a simple method to stimulate angiogenesis. We thus believe that gene therapy with phVEGF165 is a promising strategy to induce angiogenesis and re-establish bronchial circulation in airways.
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Acknowledgments |
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Footnotes |
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A poster was presented at the 15th Brazilian Congress of Thoracic Surgery, Porto de Galinhas, Brazil, March 22–24, 2007.
This work was supported by grants from the Research Incentive Fund at Hospital de Clínicas de Porto Alegre (FIPE-HCPA), Graduate Program in Surgery at the Federal University of Rio Grande do Sul, Rio Grande do Sul State Department of Science and Technology and the Millennium Institute – Gene Therapy Network (MCT-National Research Council CNPq). phVEGF165 was kindly provided by Genentech Inc.
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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