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Ann Thorac Surg 1997;63:1562-1567
© 1997 The Society of Thoracic Surgeons

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Original Article: General Thoracic

Experimental Bronchiolitis Obliterans Induced by In Vivo HVJ-Liposome–Mediated Endothelin-1 Gene Transfer

First Department of Surgery, Institute for Cellular and Molecular Biology, and Division of Organ Transplantation, Biomedical Research Center, Osaka University Medical School, Osaka, Japan; and Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Accepted for publication February 11, 1997.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

 
Background. Bronchiolitis obliterans (OB) is a lesion that results when injury to small conducting airways is repaired by a proliferation of fibrous granulation tissue. Bronchiolitis obliterans has emerged as a main cause of morbidity and mortality in the setting of lung and heart-lung transplantation. Endothelin-1 (ET-1), initially discovered as a vasoconstrictive peptide, has a mitogenic activity on vascular smooth cells and airway epithelial cells. Overproduction of endothelin has been reported in patients with OB or chronic rejection after lung transplantation. It is still undetermined whether locally overexpressed ET-1 has a potential impact in the pathogenesis of OB.

Methods. We locally overexpressed ET-1 using ultraviolet irradiation-inactivated hemagglutinating virus of Japan (HVJ)-liposome–mediated in vivo gene transfer. Plasmid DNA of prepro-ET-1 and high mobility group 1 protein were coencapsulated in liposomes, and were introduced into airway epithelial cells by HVJ-mediated membrane fusion. Control animals received instillation of HVJ-liposome with an empty expression cassette. To confirm the efficiency of transfection, HVJ liposome with ß-galactosidase gene was introduced. The expression of ET-1 and ß-galactosidase was assessed by immunohistochemistry.

Results. Bronchial epithelium alveolar cells and alveolar macrophage were stained blue (X-Gal) 1 week after in vivo gene transfer of ß-galactosidase gene, indicating ß-gal activity. In animals 1 to 2 weeks after in vivo transfection of prepro-ET-1 gene, hyperplastic connective tissue plaque was seen in the alveolar duct and small conducting airway, indicating histologically distinctive bronchiolitis obliterans. Strong ET-1–like immunoactivities were seen in the airway epithelial, hyperplastic connective tissue, and alveolar cells. No histopathologic changes were seen in the control animals.

Conclusions. These results suggested that ET-1 may play an important role in the pathogenesis of OB. The effective pharmacologic antagonist or inhibitor may possibly control the progression of disease in patients of OB.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

 
Fibroproliferative diseases of the lung, which include pulmonary fibrosis and bronchiolitis obliterans (OB), are chronic, progressive, and sometimes fatal disorders. These heterogeneous disease entities are characterized by overgrowth of mesenchymal cells, fibroblasts, and connective tissues. Idiopathic pulmonary fibrosis has an aggressive course and is usually fatal despite the conventional medical therapy. It became the first target of lung transplantation reported by Cooper and associates [1]. Ironically, however, posttransplantation OB affects 30% to 50% of recipients and is a frequently fatal complication unless retransplantation is performed [2–4].

See also 1527 and 1556.

It is thus desirable to elucidate the molecular mechanism in the pathogenesis of these fibroproliferative diseases. Overexpression of various cytokines or growth factors, including transforming growth factor ß [5], tumor necrosis factor [6], and platelet-derived growth factor (PDGF) [7, 8], is believed to contribute to the progression of fibroproliferative disease. Recently, Yoshida and associates [9] have successfully made a histologically distinctive interstitial pneumonia by locally overexpressing transforming growth factor ß and PDGF using hemagglutinating virus of Japan (HVJ) liposome-mediated gene transfer.

Endothelin-1 (ET-1), initially discovered as a vasoconstrictive peptide [10], has a mitogenic activity on vascular smooth cells and airway epithelial cells, and its release is regulated by various cytokines [11]. Three known members of the mammalian endothelin family, ET-1, ET-2, and ET-3, are produced in various tissues [12]. They act on two distinct subtypes of G-protein–coupled receptors termed ET_A and ET_B, which are expressed on a wide variety of vascular and nonvascular target cells, eliciting contraction and proliferation of vascular smooth muscle cells, and stimulation of c-fos and c-myc [13]. In the present study we focused our attention on the action of ET-1 that promotes mitogenesis in airway smooth muscle cells [14] and epithelial cells [15]. In clinical settings, overproduction and overexpression of ET-1 has been reported in the lung of patients with pulmonary fibrosis [16].

Systemic overexpression of ET-1 with a transgenic mouse is an alternative method; however, it is difficult to interpret the physiologic role of ET-1 because various compensations may attenuate the role of the molecule during development. Physiologic analysis of the ET-1 gene knockout mice revealed paradoxical hypertension [17]. The HVJ-liposome–mediated in vivo gene transfer is safe and has high efficiency of transfection [9, 18–20]. In this sense, it is a simplified and optimal model to assess the effect of locally overexpressed targeted molecules [9, 21] in adult animals compared with the knockout or transgenic model. To elucidate the potential role of ET-1 in lung fibroproliferative diseases including OB, we have locally introduced the prepro-ET-1 gene into rat lung tissue.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

 
Construction of Plasmid
pME18Sf-prepro-endothelin-1 (a gift from Dr N. Emoto, The Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX) contains human prepro ET-1 complementary DNA (1.2 kb) inserted into pME18Sf vector at BstXI-blant (817, 1175).

As reporter genes, complementary DNA plasmid of ß-galactosidase (a gift from Dr S. Ishii, Institute of Physical and Chenical Research, Tsukuba, Japan) was prepared as described previously [19, 20].

	Preparation of HVJ-Liposome

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

 
The HVJ-liposomes were prepared as described previously [18, 20, 22]. Briefly, phosphatidylserine, phosphatidylcholine, and cholesterol were mixed in a weight ratio of 1:4.8:2. The lipid mixture (10 mg) was deposited on an evaporator. Dried lipid was hydrated in 200 µL of balanced salt solution (balanced salt solution = 137 mmol/L NaCL, 5.4 mmol/L KCL, 10 mmol/L Tris•HCl; pH 7.6) containing ET-1 and ß-galactosidase genes. The control group liposomes did not contain expression genes (balanced salt solution, 200 µL). Liposomes were prepared by shaking and sonication. Purified HVJ (Z strain) was inactivated by ultraviolet irradiation (110 ergs per mm² per second) for 3 minutes just before use. The liposome suspension (0.5 mL, containing 10 mg of lipids) was mixed with HVJ (10,000 hemagglutinating units) in a total volume of 4 mL of balanced salt solution. The mixture was incubated at 4°C for 5 minutes and then for 30 minutes with gentle shaking at 37°C. Free HVJ was removed from the HVJ-liposomes by sucrose density gradient centrifugation. The top layer of the sucrose gradient was collected for use. This preparation method has been optimized to achieve maximal transfection efficiency as reported previously.

	Experimental Protocol

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

 
Twelve-week-old male Wistar rats weighing 250 g (Japan SLC, Hamamatsu, Japan) were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg). After orotracheal intubation, the animals were ventilated with room air at a tidal volume of 2.5 to 3.0 mL and respiratory rate of 90 to 100 breaths/min. After sufficient hyperventilation, HVJ-liposome solution was installed into the lungs of the animals. Mechanical ventilatory support was required 2 hours after instillation. Twelve Wistar rats received an HVJ plasmid DNA containing 20 to 30 µg DNA for prepro-ET-1, total volume of 300 µL intratracheally. Four control animals received instillation of HVJ-liposome with an empty expression cassette. The animals were sacrificed with an overdose of pentobarbital 3, 7, and 14 days after instillation of the ET-1 genes and 7 days after transfection of empty cassettes. All animals received humane care in compliance with "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985). The lungs were expanded and fixed by intratracheal instillation of 4% paraformaldehyde at a constant hydrostatic pressure of 10 cm H_2O. Three-micrometer-thick paraffin-embedded sections cut from each lobe were stained with hematoxylin and eosin. Expression of the exogenous ET-1 gene in the lung was confirmed by immunohistochemical analysis. Polyclonal antiserum against human endothelin-1 (C-terminal of endothelin-1 and big endothelin-1) was used.

To confirm in vivo transfection by HVJ-liposome, the ß-galactosidase gene was introduced into 4 animals, which were sacrificed 7 days after transfection. The lungs were expanded and pressure-fixed with 2% paraformaldehyde and 0.2% glutaraldehyde for 60 minutes and then placed in a bath of 5-bromo-4 chloro-3-indolyl-ß-D-galactoside (X-Gal; Sigma, St. Louis, MO) reagent for 6 hours. Five-micrometer-thick paraffin-embedded sections were counterstained with hematoxylin and eosin or nuclear fast red stain.

	Results

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

 
The HVJ liposome with the ß-galactosidase gene was introduced to confirm the efficiency of transtracheal transfection. Bronchial epithelium (Fig 1A) and alveolar cells and alveolar macrophages (Fig 1B) were stained blue (X-Gal), indicating ß-gal activity.

View larger version (109K): [in this window] [in a new window]   Fig 1. . Rat lung tissues from a Wistar rat 7 days after transtracheal introduction of HVJ liposome with ß-galactosidase gene. Bronchial epithelium (A) and alveolar cells and alveolar macrophages (B) were stained blue (X-Gal), indicating ß-gal activity. (A, x200; B, x400; both before 35% reduction.)

View larger version (109K): [in this window] [in a new window]   Fig 2. . Photomicrographs showing histologic findings of lung tissue from a Wistar rat 7 days after in vivo transfection of HVJ-liposome with an empty expression cassette. No histopathologic changes were seen in the control animals after introduction. (Hematoxylin and eosin; A, x100; B, x200; both before 35% reduction.)

View larger version (121K): [in this window] [in a new window]   Fig 3. . Photomicrographs show histologic changes of lung tissue from Wistar rat on day 3 after in vivo transfection of HVJ-liposome with prepro-ET-1 gene. Histologic section of the lung parenchyma shows alveolar septal thickening due to the interstitial mononuclear cell infiltrate and hyperplasia of the alveolar lining epithelium. No apparent concentric changes were seen in alveolar ducts and small conducting airways. (Hematoxylin and eosin; A, x100; B, x200; both before 35% reduction.)

View larger version (145K): [in this window] [in a new window]   Fig 4. . Photomicrographs showing histologic changes of lung tissue from a Wistar rat on day 7 after in vivo transfection of HVJ-liposome with prepro-ET-1 gene. Hyperplastic connective tissue plaques were seen in the alveolar duct and small conducting airway, identical to human bronchiolitis obliterans (A). Detail of an intrabronchiolar polyp of hyperplastic loose connective tissue (B). (Hematoxylin and eosin; A, x100; B, x200; both before 35% reduction.)

View larger version (128K): [in this window] [in a new window]   Fig 5. . Photomicrographs showing immunohistochemical study of lung tissue from a Wistar rat 7 days after in vivo transfection of HVJ-liposome with prepro-ET-1 gene (A) and HVJ-liposome with an empty expression cassette (B). Strong ET-1–like immunoactivity is seen in airway epithelium or vascular endothelium associated with severe morphologic changes. (Immunohistochemistry; x100 before 35% reduction.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

The distinct histologic findings of OB represent a nonspecific "final common pathway" of tissue reaction at the level of small airway. But the etiology of OB is diverse. As previously reported, OB is idiopathic and occurs late after viral infection, exposure to a toxic substance, or associated with connective tissue disease and after organ transplantation. Posttransplantation OB is a highly specific entity, because OB is believed to result from a prolonged chronic rejection [2–4].

It is generally recognized that primary immunologic stimuli may cause inappropriate production of growth factors or cytokines, which lead to unsuppressed fibroblast proliferation and excessive collagen synthesis. Hertz and associates [8] found an increase in PDGF expression in the lung and bronchoalveolar lavage fluid in patients with OB, and they suggested the role of PDGF in the development of OB. On the other hand, PDGF-deficient mice showed lung emphysema histologically, indicating that PDGF is also essential for normal lung development [23]. Yoshida and colleagues [9] have developed an experimental fibrosing alveolitis, partly akin to those changes in human pulmonary fibrosis, by in vivo transfer of PDGF and transforming growth factor ß genes. But they did not obtain direct evidence that overexpression of PDGF is a primary cause of OB.

Endothelin-1, initially discovered as a vasoconstrictive peptide [10], promotes mitogenesis in airway smooth muscle cells [14] and epithelial cells. In addition, increased ET-1 messenger RNA expression or overproduction was also reported in patients with chronic lung rejection [24]. Schersten and associates demonstrated an increased ET-1 activity in bronchoalveolar lavage fluid from transplanted allografts in humans [25] and experimental animals [26]. By applying the HVJ-liposome–mediated gene transfer, we have obtained direct evidence that histologically distinctive OB can be induced by overexpression of ET-1 genes.

Even though the HVJ-liposome in vivo gene transfer method has transient results, it is a simple and efficient approach to study the unknown biological function of the molecules [21] and to make animal models for human diseases as well as transgenic and knockout animals. Using this technique, foreign genes have been successfully introduced into liver [22], kidney [20], heart [27], and lung [9]. Because HVJ itself is a virus of respiratory system, it has a tropism for the respiratory epithelium like adenovirus. Moreover, the HVJ-liposome method is superior to the adenovirus vector for introduction to the peripheral alveolar cells of the lung (personal communication, Dr M. Yoshida).

This result suggests that ET-1 may have an important role in the pathogenesis of OB, presumably in the "common final pathway." However, the mechanism of mitogenic signaling by ET-1, its regulation of fibroblast proliferation, and its interaction with other cytokines or growth factors remain to be clarified at present. Further studies are needed to ascertain the specific role of ET-1 in the progression of OB.

In summary, we conclude that local overexpression of ET-1 has a key role in the pathogenesis of OB. This evidence suggests the potential clinical implication that a pharmacologic antagonist or inhibitor may be able to control the progression of this disease in patients with OB.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

 
We gratefully acknowledge Dr Satoru Yamamoto, Department of Pathology, Kinki National Central Hospital, for important comments on lung pathology and review of the manuscript.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

 
Address reprint requests to Dr Takeda, First Department of Surgery, Osaka University Medical School, 2-2 Yamadaoka, Suita Osaka 565, Japan (e-mail: stakeda{at}surg1.med.osaka-u.ac.jp).

	References

Top Footnotes Abstract Introduction Material and Methods Preparation of HVJ-Liposome Experimental Protocol Results Comment Acknowledgments References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Shin-ichi Takeda Hikaru Matsuda Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Takeda, S.-i. Articles by Matsuda, H. Search for Related Content PubMed PubMed Citation Articles by Takeda, S.-i. Articles by Matsuda, H. Related Collections Related Articles