Published ahead of print on September 28, 2006, doi:10.1164/rccm.200603-406OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200603-406OC
Lung Tissue Engineering Technique with Adipose Stromal Cells Improves Surgical Outcome for Pulmonary EmphysemaDivision of Cardiothoracic Surgery, Department of Surgery, Division of Molecular Regenerative Medicine, Course of Advanced Medicine, and Medical Center for Translational Research, Osaka University Graduate School of Medicine, Osaka, Japan Correspondence and requests for reprints should be addressed to Yoshiki Sawa, M.D., Department of Surgery, Osaka University Graduate School of Medicine, E1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: sawa{at}surg1.med.osaka-u.ac.jp
Rationale and Objectives: Hepatocyte growth factor (HGF) is a potent regenerative factor generated after a lung injury, and HGF supplementation after surgical reduction has been shown to enhance compensatory growth in remnant lungs and improve pathophysiologic conditions in a rat model of emphysema. Adipose tissue–derived stromal cells (ASCs) produce a large amount of angiogenic factors, including HGF. After lung volume reduction surgery (LVRS), we treated rats by implanting HGF-secreting ASCs with a scaffold onto the remnant lung tissue to determine the usefulness of this technique for treating respiratory dysfunction. Methods and Main Results: Cells were isolated from rat inguinal adipose tissue and characterized by flow cytometry. ASCs were cultured on a polyglycolic acid felt sheet as a sealant material, and were shown to secrete significantly greater amounts of HGF than other angiogenic factors. Next, ASCs on polyglycolic acid felt sheets were used to cover the cut edge of the remaining lungs after LVRS for emphysema in rats. One week after implantation of the ASCs, both alveolar and vascular regeneration were significantly accelerated as compared with the rats that underwent LVRS alone. Consequently, gas exchange and exercise tolerance were also significantly restored, with these good results persisting for more than 1 mo. Conclusions: The present findings demonstrate the therapeutic potential of cell therapy using ASCs with a scaffold for selective delivery of HGF to remnant lungs, which resulted in enhancement of compensatory growth, after surgical resection. This approach may provide a new strategy for lung tissue engineering to improve LVRS outcome.
Key Words: adipose tissue • angiogenesis • hepatocyte growth factor • lung • pulmonary emphysema • tissue engineering
Lung volume reduction surgery (LVRS) has been shown to improve the exercise capacity of patients with severe emphysema (1), although it does not offer survival advantages over medical therapy and only a small portion of patients can enjoy its current benefits (2). A number of improvements in LVRS strategies are needed, and if the procedure can be performed in a more effective manner, its indications may be expanded, with more patients taking advantages of its benefits (3). Hepatocyte growth factor (HGF) has been discovered to be a potent mitogen for mature hepatocytes (4, 5). In lung, HGF is an essential ligand to elicit lung repair and growth in vitro and in vivo (6, 7). We recently obtained findings regarding tissue regeneration biology for emphysema, and successfully demonstrated that compensatory growth is suppressed in emphysematous lungs after surgical resection, which is associated with a failure in endogenous HGF production in remnant lung tissue. In contrast, supplementation of exogenous HGF via a gene transfection leads to enhanced compensatory growth after LVRS for emphysema, leading to clinical improvements (8). However, an effective means to deliver HGF specifically to injured lungs remains to be determined. Adipose tissue has been traditionally used in thoracic surgery settings as a reinforcing material for damaged lungs, such as omentum filling transposition for empyema surgery and pericardial fat pad flap for bronchial stumps (9). Furthermore, adipose tissue was also reported to contain an ample source of pluripotent cells, such as hematopoietic progenitors and spare mesodermal stem cells, which will differentiate into osteogenic, chondrogenic, myogenic, and neurogenic lineages (10–14). Notably, adipose tissue–derived stromal cells (ASCs) were recently suggested to secrete multiple angiogenic growth factors, including HGF (15). In the present study, we developed cultured ASCs taken from rat inguinal subcutaneous fat tissues on a commercially available, bioabsorbable sealant used as a scaffold, and demonstrated that the ASCs attached and expanded on the sealant could secrete ample amounts of HGF. Furthermore, transplantation of the cultured ASCs with a sealant onto the remnant lungs after surgical reduction enhanced tissue repair and regeneration through the induction of HGF expression in a rat model of emphysema, which was followed by the amelioration of pulmonary function. Our results indicate that adipose tissue–derived cells may have therapeutic potential through HGF supplementation to enhance compensatory growth after surgical resection in emphysema.
Cell Culture and Transfection of ASCs with Green Fluorescent Protein ASCs were isolated from Lewis rat (weight, 200 g) inguinal subcutaneous adipose tissue and cultured according to the method of Mizuno and colleagues (11) and Bjorntorp and colleagues (16) with minor modifications. ASCs were labeled for 20 to 30 min on ice with manufacturer-recommended concentrations of fluorescence-labeled antibodies against CD44, CD45, CD90, or the isotype control, which all came from BD Biosciences (Tokyo, Japan). Cells were subsequently washed using routine procedures and analyzed on a fluorescence-activated cell sorter instrument (FACSCalibur; BD Biosciences). Then, ASCs at passage 1 were infected with retrovirus green fluorescent protein (retro-GFP) virus (107 viral particles/ml medium) in Dulbecco's modified Eagle's medium polybrene overnight and cultured for an additional 3 d before implantation. Thereafter, we followed the directions for "Retroviral Gene Transfer and Expression" provided by Clontech (Mountain View, CA). The transfection efficiency of ASCs for retro-GFP was greater than 95%, as assessed by flow cytometry.
ASCs Implanted onto Sealant Material (PGAF) as a Scaffold
Determination of Angiogenic Growth Factors in ASCs with PGAF-conditioned Media The 7-d–conditioned media from ASCs with PGAF (passage 3) were analyzed for angiogenic factors, including vascular endothelial growth factor (VEGF), HGF, and basic fibroblast growth factor (bFGF), using ELISA kits by Pierce Technology (New York, NY) and Institute of Immunology Co. (Tokyo, Japan). Data are expressed as the mean ± SEM in picograms of the secreted factor per 106 cells.
Emphysema Induction and LVRS Technique with PGAF Sealant
Measurement of HGF in Tissues
Immunohistochemical (PCNA, Factor VIII) and Laser Doppler Blood Flow Analysis for Pulmonary Perfusion Lung surface blood perfusion was evaluated using a laser Doppler image (LDI) analyzer (Moor Instruments, Cambridge, UK). Blood flow measured by the LDI analyzer correlated well with capillary density, as we previously demonstrated during assessment of rat lung angiogenesis (17).
Pulmonary Function Test (Arterial Blood Gas, Treadmill Test)
Statistical Analysis
Characterization of ASCs and Attachments of Cells to PGAF Freshly isolated ASCs, obtained from inguinal subcutaneous fat pads of emphysema rat models, exhibited a heterogeneous population of fibroblast-like cells morphologically, were adherent, and showed significant expansion in the media (Figure 2A). Furthermore, the cells expressed similar CD marker profiles, with high expressions of CD44 and CD90, and the absence in CD45 (Figure 2B), as reported previously (11). The ASCs were transduced with a GFP-expressing retrovirus for tracking purposes and all were positive for GFP after transduction (positive rate, 97.6%; Figure 2C). After the third passage of cell expansion, 5 x 107 ASCs were seeded and cultured on PGAF sheets, with medium, for 5 d in noncoated dishes. We found a large number of GFP-positive cells attached firmly along the PGAF fibers, which showed a round shape (Figure 3).
ASCs as a Source of Pulmotrophic Factors After confirming that the GFP-positive ASCs had attached to the PGAF sheets, we found that the cells secreted large amounts of HGF (20,557 ± 628 pg/106 cells), and smaller amounts of VEGF (2,511 ± 1,228 pg/106 cells) and bFGF (430 ± 256 pg/106 cells; Figure 4).
Enhanced Induction of Endogenous HGF Production in Emphysematous Lungs by Exogenous HGF via ASC Implantation after RLL At 7 d after the RLL surgery and implantation of ASCs on a PGAF sheet in the remaining lung, a significant increase in total HGF expression level, including both ASC-derived (exogenous) and resident-produced (endogenous) HGF, was observed in the rats transplanted with ASCs, which was nearly sevenfold greater than in the control rats without ASCs (surgery alone, p < 0.01). Furthermore, HGF expression remained at considerably high levels for up to 3 wk after surgery in the ASC-treated rats (Figure 5). Importantly, no significant increase in HGF expression levels could be detected in other organs, including the brain, liver, kidney, and spleen (data not shown), suggesting that the present method of ASC implantation with a PGAF scaffold leads to selective and persistent HGF expression at higher levels in emphysematous lung tissues.
Alveolar Regenerative and Angiogenic Effects of HGF in Emphysematous Lungs after Surgery and ASC Implantation on PGAF Sheet Next, we examined if implanted ASCs were able to alter alveolar regeneration in vivo by providing HGF supplementation, because HGF has been shown to be a potent pulmotrophic factor. We detected PCNA-positive cells among the intraalveolar, septal, and endothelial cells around the injured alveolar networks more frequently in ASC-treated rat lungs than in the control lungs (surgery-alone group), whereas the number of PCNA-positive alveolar cells in the ASC-treated rats was also greater (5.1 ± 0.9/mm2 vs. 1.9 ± 1.1/mm2; Figure 6A). To evaluate lung angiogenesis in vivo, an immunohistochemical examination of anti–factor VIII was performed. A marked increase in the number of factor VIII–positive pulmonary capillaries was observed in rats implanted with ASCs as compared with the control group (Figure 6B), and the difference was significant (11.6 ± 0.5/mm2 vs. 2.0 ± 1.7/mm2, p < 0.01).
Restoration of Pulmonary Function Concomitant with Enhanced Alveolar and Vascular Regeneration Laser Doppler analysis of lung blood perfusion. To determine if APC implantation altered microcirculation in the lungs, blood perfusion on the lung surface was measured with an LDI analyzer. Representative images, obtained 1 wk after surgery, are shown in Figure 7A (right). Blood perfusion levels in the remaining right lung in the ASC-treated group were remarkably increased.
Effects of ASC implantation after RLL on pulmonary function. The PaO2 value in the ASC-treated group at 1 wk after surgery and implantation of ASCs on PGAF sheets was significantly higher than that in the surgery-alone group (75 ± 7 vs. 54 ± 8 mm Hg, p < 0.05; Figure 7A, left).  O2max was also significantly higher in the ASC-treated rats as early as 1 wk after surgery (57 ± 3.7 vs. 29 ± 5.3 ml/kg/min, p < 0.05; Figure 7B). At 4 wk after surgery, the significantly elevated levels of PaO2 and exercise tolerance parameters remained unchanged. Overall, ASC implantation on PGAF sheets after RLL restored impaired gas exchange and exercise tolerance capacity via alveolar and vascular regeneration.
Current tissue engineering techniques generally use a procedure for cell implantation with growth factors on an extracellular matrix (as a scaffold) for reconstruction of organ or tissue morphology and physiologic functions. However, in the field of thoracic surgery, it remains unclear whether a strategy of tissue engineering combined with surgery provides therapeutic results to attenuate or reverse respiratory dysfunction. Using a rat model of pulmonary emphysema, we found that a combination of implantation of adipose tissue–derived HGF-producing cells (ASCs), with a sealant material and LVRS, produced beneficial effects toward progressive alveolar and vascular destruction as well as respiratory failure. The novel finding of the present study is the successful development of a new delivery system of multipotent regenerative factors specifically to injured lungs via cell transplantation for treatment of chronic pulmonary disease. Because both the adipose tissue and sealant used are currently used in clinical thoracic surgery settings (9, 18), the present combination seems to be a practical therapy that shows promise for therapy. Previous studies have shown that lung compensatory growth occurs under an endogenous HGF-mediated system, which is also closely related to the regenerating conditions seen in remnant lungs after LVRS (8). However, emphysema-induced hypoxia may down-regulate HGF gene expression (19–21), leading to the current limitations in outcomes after LVRS. Thus, supplementation with HGF is considered to elicit a successful outcome after LVRS in emphysematous lungs. An efficient method to deliver HGF locally to lung tissue remains to be developed. Although several delivery systems, such as via the airway (22), pulmonary artery (23), and a transgene pathway (8), have been reported in animal experiments, which are not easily applied in clinical situations. In a previous study, we used a liposome-mediated cDNA delivery system for HGF supplementation; however, that method may restrict long-term gene expression, because the exogenous gene is not incorporated into genomic DNA (8), indicating the possibility of transient expression with a possible degradation of the gene. To overcome that problem, we used ASCs in the present technique to provide long-term maintenance of HGF levels, because a large number of ASCs are easily obtainable from subcutaneous adipose tissues, and the transplantation method is safe and not antigenic. Furthermore, surgical techniques using fat pads from around organs such as the omentum transposition or pericardial fat tissue are reported to be effective for reinforcement of damaged lung tissues (24, 25). As expected, local HGF production was successfully sustained at a sufficient level with the combination of LVRS and ASC implantation. We considered that those good results may have been due to supplementation with ASC-derived (exogenous) HGF and the enhancement of resident (endogenous) HGF production by exogenous HGF, because supplementation with HGF is able to stimulate intrinsic HGF production in an autoinducible way, as reported in our previous study (8) and other in vivo experiments (26, 27). Such direct and indirect effects may contribute to an increase in total HGF levels in emphysematous lungs. Recently, adipose tissue has been shown to contain stem cell–like or progenitor cell–like cell populations, suggesting that it is a novel source of cells used in cardiovascular cell therapy (13–15). Indeed, we found that cultured ASCs after some passages retained their mesenchymal pluripotency, together with their expression of CD44 and CD90, as described previously (12). Furthermore, several studies have revealed that HGF promotes transdifferentiation and migration of stem cell–like cells in vitro (28, 29) and in vivo (30, 31). Interestingly, HGF promotes alveolar and endothelial repairs by recruiting bone marrow–derived stem cells, even under emphysematous conditions (31). Thus, we cannot exclude the possibility that ASCs may in part contribute to tissue regeneration via recruitment of stem cell populations, although further studies are needed to clarify this notion (32). In addition, ASCs were reported to produce and secrete bFGF and VEGF, both of which may play an important role in pulmonary alveologenesis (33–36), suggesting that such pulmotrophic ligands participate in lung tissue repair in cooperation with HGF. It is important to discuss the merits of using a sealant for cell anchorage. In the present study, we used PGAF sheets, which are used widely in other thoracic surgery procedures. PGAF is a suture reinforcement material made of bioabsorbable components, which is completely absorbed by the surrounding tissues within a certain period of time and which has become the standard reinforcement tool for thoracic surgery (18). Because tissue regeneration in the lungs is achieved via a paracrine mode of HGF-mediated mechanism (37, 38), our method, which uses this HGF-producing sealant material, may mimic the paracrine mode. Remarkably, angiogenic effects were observed directly under the PGAF sheets attached to the surface of the remaining lung tissues. Furthermore, the good results persisted for more than 1 mo with consistently high levels of HGF; this finding is consistent with a previous proposal that an ideal future therapy for chronic pulmonary diseases like COPD will maintain a stable and long-lasting expression of HGF, in addition to prolonging the effects of HGF (37). Another merit of PGAF is prevention of air leakage from the cut edge of the remnant lung tissue after a pulmonary resection, which is a complication predicted to become a common issue in future combination strategies used for thoracic surgery and tissue engineering technologies. Overall, the concept of using a combination of ASCs and PGAF as a new method for releasing regenerative factors may be applicable to tissue engineering techniques for other damaged organs. To the best of our knowledge, this is the first report demonstrating successful use of a therapeutic strategy for emphysema with ASCs and sealant together as a tissue engineering technique, and the findings may provide important information for improving LVRS outcomes. Our data also suggest that elucidation of stem cell biology and application of bioengineering technologies will lead to other therapeutic advantages for currently nontreatable pulmonary diseases.
The authors thank Masako Yokoyama for her excellent techniques and contributions in viral transfection experiments and Dr. Toshiya Komatsu (Department of Pathology, Takarazuka Municipal Hospital) for his invaluable pathologic assistance.
Originally Published in Press as DOI: 10.1164/rccm.200603-406OC on September 28, 2006 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 22, 2006; accepted in final form September 5, 2006
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