Induction of Compensatory Lung Growth in Pulmonary Emphysema Improves Surgical Outcomes in Rats Running Head: HGF for Lung Volume Reduction Surgery Norihisa Shigemura, MD; Yoshiki Sawa, MD, PhD; Shinya Mizuno, PhD; Masamichi Ono, MD, PhD; Masato Minami, MD; Meinoshin Okumura, MD, PhD; Toshikazu Nakamura, PhD; Yasufumi Kaneda, MD, PhD; Hikaru Matsuda, MD, PhD From the Department of Surgery (N.S., Y.S., M.O., M.M., M.O., H.M.), Division of Molecular Regenerative Medicine, Course of Advanced Medicine (S.M., T.N.), and Department of Gene Therapy Science (Y.K.), Osaka University Graduate School of Medicine, Osaka, Japan Correspondence to: Hikaru Matsuda, MD, PhD, Department of Surgery, Osaka University Graduate School of Medicine, E1, 2-2 Yamadaoka, Suita, Osaka 565-0871, JAPAN Tel: +81-6-6879-3152 Fax: +81-6-6879-3163 E-mail: n-shige@blue.ocn.ne.jp Subject code: 56 (COPD: surgical management) Word count for the body of manuscript: 4484 AJRCCM Articles in Press. Published on March 11, 2005 as doi:10.1164/rccm.200411-1518OC Copyright (C) 2005 by the American Thoracic Society. Shigemura N 1 Abstract Rationale and Objectives Although lung volume reduction surgery (LVRS) has been widely employed as a therapeutic strategy for pulmonary emphysema, the procedure carries significant disadvantages, including significant operative mortality and a limited duration of effective response. Pulmonary resection is known to elicit compensatory growth in remnant lung tissues, however, it remains unclear whether and how compensatory growth occurs and contributes to clinical outcomes after LVRS. The goal of the present study was to characterize the role of hepatocyte growth factor (HGF) in compensatory lung growth following LVRS in a rat model of elastase-induced emphysema, since HGF is a potent pulmotrophic factor responsible for the regeneration of lung parenchyma in damaged lungs, including following a pulmonary resection. Methods and Main Results Unexpectedly, LVRS did not cause apparent increases in the endogenous HGF profiles of emphysematous lungs. Further, the lowered HGF production reflected a histologically inferior regenerative capacity in remnant lungs and was linked with impaired pulmonary functional recoveries after LVRS. When HGF was exogenously supplemented by gene transfection into emphysematous lungs simultaneously with LVRS, compensatory lung growth (as evidenced by increased lobe weight, and alveolar regeneration and angiogenesis) was significantly enhanced as compared with rats that underwent LVRS alone. Consequently, pulmonary function and gas exchange were also significantly improved. Conclusions We concluded that the induction of compensatory growth by growth factors following LVRS may be a new strategy to further improve clinical outcomes of LVRS in patients with pulmonary emphysema. (Word count: 239) Keywords: emphysema; lung volume reduction surgery; gene therapy; growth factor Shigemura N 2 Introduction Conservative estimates predict that chronic obstructive pulmonary disease (COPD), including emphysema will be the third leading cause of death by the year 2020 (1). Over the past two decades, lung volume reduction surgery (LVRS) has been increasingly used as a palliative treatment for patients with severe emphysema, who may have no other effective treatment options (2). Under adequate patient selection criteria, LVRS can lead to improved pulmonary function and quality of life. Despite the potential benefits, however, LVRS still yields significant operative mortality and results in short-term benefits at best (3). Thus, establishment of new treatment strategies to overcome the current limitations of LVRS is urgently required for the effective treatment of the growing number of patients with emphysema. Recently, we have focused on the fact that pulmonary resection induces compensatory growth in remnant lung tissues, in examinations of small animals and infant patients (4, 5). Following surgical removal of a lung lobe, alveolar epithelial cells initiate proliferation into the remaining lobes, which is associated with an increase in lung organ weight (6). This phenomenon is considered to be a beneficial response to compensate for a loss in functional lobes. During the adaptable events, regenerated epithelial cells reconstitute the alveolar network, which is called alveolar septation (7, 8). Considering that extensive destruction of the alveolar networks is a characteristic of emphysema, induction of alveolar septation by therapeutic challenge may contribute to or improve LVRS-mediated outcomes in emphysema patients. However, it is still unknown whether and how LVRS induces or enhances compensatory growth even in emphysematous lung tissues. LVRS involves resection of only the most severely affected regions of the lung. Since the remnant portions of the lung are still severely diseased, patients who have Shigemura N 3 undergone LVRS are at higher risk of death due to pulmonary dysfunction. Previous studies of patient outcomes after LVRS have focused primarily on the chest wall and diaphragmatic mechanics in respiration (9-11). However, relatively little is known about the function of the remnant diseased lung. Hepatocyte growth factor (HGF) has been identified as a mitogen for mature hepatocytes and cloned (12, 13), and there is ample evidence that it plays an essential part in parenchymal repair in various organs (14, 15). In the lungs, HGF is a potent morphogenetic and mitogenic factor during organogenesis or in cases of acute injuries (16-20). In a murine model of lung reduction, endogenous HGF levels were found to rapidly increase, followed by alveolar cell division, while neutralizing antibody to rodent HGF blocked alveolar regeneration during compensatory growth (21), suggesting a role of HGF as an intrinsic ligand to drive alveolar septation in compensatory lung growth following LVRS (21, 22). These backgrounds led us to hypothesize that HGF may have beneficial effects on pathophysiologic conditions of emphysematous lung tissues that have undergone LVRS. To test our hypothesis, we used rodent models of emphysema and attempted to determine whether LVRS alters endogenous HGF production and alveolar regeneration in emphysematous tissues or inversely, whether supplementation of exogenous HGF accelerates compensatory growth-related phenotypes. Based on our results, we discuss the possibility that a combined treatment of LVRS with HGF supplementation may be a new therapeutic option that surpasses the current limitations of LVRS alone (3, 22). Shigemura N 4 Materials and Methods Emphysema Induction Emphysema was induced in anesthetized 3-month-old Sprague-Dawley rats by means of a single intratracheal instillation of porcine pancreatic elastase (PPE; Roche Diagnostics), 25 U/100g body weight, diluted in 0.8 mL of normal saline solution, or an equal volume of saline alone as control. After the instillation, rats were extubated and returned to the animal care facility and managed routinely until a week after induction. LVRS Technique To perform pulmonary resection, the animals were anesthetized, intubated, and ventilated again. Then, right lower lobectomy (RLL) was performed on Day 7 after elastase induction. RLL has been shown to elicit compensatory growth in remnant lungs (23, 24), and our preliminary data also indicated that RLL induced compensatory growth in mice (21). Thus, considering for the safety and stable survival rates in the context of emphysema during the experiments, we used here the RLL-undergone rats as a conceptual model to mimic LVRS in pulmonary emphysema. Right lateral thoracotomy was performed. The lower lobe on the right was resected after clamping near the hilum, and the stumps were ligated with 1-0 silk and 6-0 polypropylene ties. Ventilation was transiently held while the lobe was resected and the ties were secured. A single 18-gauge 1.16-inch intravenous catheter was placed into the right hemithorax through the separate intercostal stab incisions and placed to - 2cm H 2 O suction. After closing the incision, the animals were awakened and extubated. All animals were without persistent air leak following cessation of Shigemura N 5 positive-pressure ventilation. The chest tubes were removed when the animals showed signs of attempting to ambulate. Assessment of Remnant Lung Growth after RLL Lung Weight Determination. Animals that underwent RLL were sacrificed at 1 and 2 weeks after the surgery to measure the weight of remnant right lung (including upper, middle, and cardiac lobes). After a mid-line thoracotomy, the remaining right lung was excised en-bloc, and its weight was measured. Morphological Changes. In order to identify the morphological changes, immunohistochemical staining was performed with antibodies against proliferating cell nuclear antigen (1:50)(PCNA; Santa Cruz), and factor (1:3)(DAKO). Morphological changes after pulmonary resection were accurately evaluated using the same parameters as previously described in our report on the quantitation of proliferating alveolar cells and factor analysis (25). Further, the radial alveolar count (RAC) index was used as described previously to estimate morphological changes in alveolar epithelial cells (26). Measurement of HGF in Tissue and plasma Measurement of tissue and plasma HGF concentrations was performed using an enzyme-linked immunosorbent assay (ELISA) kit for rodent HGF (Institute of Immunology) from five rats at each time point as described previously (21, 27). Additionally, correlations between the PCNA index as a parameter of alveolar regeneration and tissue HGF levels in the remnant right lungs were assessed. Shigemura N 6 Assessment of Alveolar Gas Exchange and Exercise Tolerance To assess alveolar gas exchange at rest, arterial blood gas analysis was performed with blood samples from ascending aorta using ABL TM 505 system (Radiometer). These data were obtained with a ventilator using oxygen supplementation (FiO2=0.3) at 60 breaths/min. Treadmill testing was also performed to determine the adequacy of cardiopulmonary capacity under exercise stress using a small animal treadmill system consisting of an acrylic plastic chamber with a small rodent animal treadmill (Shizume Medical) as described previously (28). Cardiopulmonary functional capacities were then determined using the values of maximum running speed and O 2 uptake (VO 2max ) during the treadmill test. Plasmid DNA and HVJ Envelope Vector An HGF expression vector was prepared by inserting human HGF cDNA into the Not site of the pUC-SR expression vector plasmid as described previously (29). A control expression vector without the HGF gene was also constructed. Hemagglutinating virus of Japan (HVJ, also known as Sendai virus) was amplified as described previously (30). In Vivo Gene Transfer via the dorsalis penis superficialis vein Rats treated with elastase were divided into three groups: control rats (PPE-control group), rats undergoing RLL alone (Surgery group) and rats undergoing combined treatment of RLL and gene transfection with HGF (HGF group). RLL was performed seven days after induction of elastase. After RLL was complete, gene transfection via the dorsalis penis superficialis vein was performed on rats from the Surgery and HGF groups with the HVJ envelope-plasmid complex (0.5 mL, including 100 g of Shigemura N 7 cDNA). The expression vector with HGF cDNA was transfected into the HGF rats and the empty vector without HGF was transfected into the rats in the Surgery group. Five rats in each group were sacrificed for histopathological and pulmonary blood perfusion analysis at one and two weeks after the treatment. Furthermore, long-term follow-up assessment for pulmonary function was performed at 14 days and at 1, 2, 4, and 6 months after elastase induction. All experimental procedures were carried out with great care, according to guidelines for animal welfare and DNA studies, set up by Osaka University Graduate School of Medicine. Analysis for Expression Levels of HGF after the Transfection After transfection, we used a rabbit polyclonal antibody against human HGF (1:200) (Institute of Immunology) for immunohistochemical analysis as described previously (27). This antibody specifically detects human HGF but not rat HGF (25). In addition, the concentrations of human exogenous HGF and rat endogenous HGF in lung tissue were also measured by ELISA at 1, 2, 3, and 4 weeks after the transfection to verify its expression levels. Laser Doppler Blood Flow Analysis Lung surface blood perfusion was evaluated using a Laser Doppler Image (LDI) analyzer (Moor Instruments) as described previously (25). Statistical Analysis Data are expressed as the mean SEM. The means of the different groups were compared using one-way analysis of variance. Unpaired Students t test was used for Shigemura N 8 statistical analysis, and a P value of < 0.05 was considered to be statistically significant. Results Characterization of Morphological and Physiological Phenotypes in Elastase- induced Rats We initially characterized morphological and physiological phenotypes in our emphysema model during the experimental periods. In the elastase-treated rats, pathological findings, such as airspace enlargement and progressive destruction of alveolar wall structures become evident in a time-dependent manner (Fig 1A-A). To quantify the alveolar injuries, we measured RACs in emphysematous lung tissue at each time point. The RAC values decreased rapidly as early as Day 3, and then gradually decreased thereafter (Fig 1A-B). Further, consistently with the histological changes, the elastase-treated rats manifested a progressive loss in alveolar gas exchange and exercise tolerance: both PaO 2 and VO 2max decreased rapidly right after the elastase induction and thereafter decreased gradually in a time-dependent manner (Fig 1A-C). Changes in the Remaining Lung Weight after RLL To determine whether there is difference in compensatory lung growth after pulmonary resection (RLL) between normal and emphysematous lungs (PPE group), we initially determined lung weight (expressed as the ratio of wet lung weight to body weight). Lung weight after RLL was significantly lower in the PPE group than in the normal rats at one week after surgery (5.20.9 versus 6.70.6, P<0.01) despite Shigemura N 9 equivalent weights of the resected specimens (Table 1). This difference was even more evident at the two-week time point. In addition, there was a marked difference in the weight between the remaining right lung at two weeks after RLL and the whole right lung without RLL in the PPE group, as compared with in the normal rats. Alveolar Regeneration and Pulmonary Angiogenesis after RLL To histologically characterize the differences in compensatory lung growth, we investigated whether there was a difference in the capacity of alveolar regeneration after pulmonary resection. This investigation revealed that the number of PCNA- positive alveolar cells was significantly lower in the PPE group than in normal rats at one week (2.00.3/mm 2 versus 5.51.6/mm 2 , P<0.01) (Fig 1B, Left) and at two weeks after surgery. Further, the number of factor -positive pulmonary capillaries was counted to evaluate angiogenesis in pulmonary vasculature. Likewise, the number of factor -positive pulmonary capillaries was significantly lower in the PPE group than in normal rats at one week after surgery (3.00.5/mm 2 versus 12.0 1.8/mm 2 , P<0.01) (Fig 1B, Right). These data suggest that emphysematous lungs possess a lower capacity of alveolar regeneration and pulmonary angiogenesis, which may be linked with insufficient compensatory lung growth after RLL in emphysema. Restoration of Alveolar Gas Exchange and Exercise Tolerance with Regenerative Effects Along with the above histological changes with regenerative effects, rats in the PPE group consistently manifested a greater loss in the recovery of pulmonary function at one week after surgery: PaO2 and VO 2max in the PPE group were approximately half that of preoperative values and were significantly lower than in normal rats (PaO2: 50 Shigemura N 10 6 % versus 7512 % , P<0.01; VO 2max : 407 % versus 7713 % , P<0.01) (Fig 2). There was no further improvement in PaO2 and VO 2max in the PPE group and they were still significantly lower at two weeks. Endogenous HGF Levels and Alveolar Regeneration after RLL Using the successful emphysema model, we initially verified the HGF expression levels in lung tissue and plasma after the elastase induction. The endogenous HGF levels in lungs of the elastase-induced rats increased as early as on Day 1 and reached a peak on Day 5. Thereafter, they decreased to a level below pretreated values (Fig 3A). Interestingly, the local HGF levels on Day 7 were significantly lower than those of the pretreated (normal) rats (189 ng/g tissue versus 455 ng/g tissue, P<0.01) Likewise, circulating HGF levels in the elastase-induced rats increased to a maximum on Day 3 and then decreased rapidly below the normal levels after Day 7 (data not shown). We next examined whether expressions of endogenous HGF levels may be modulated under the pathophysiological conditions after RLL. Intrinsic tissue HGF levels in normal rats after RLL reached a peak on Day14 (Fig 3B-A) before beginning to decrease. However, HGF levels on Day 28 were still higher than pretreatment levels (654 ng/g tissue versus 435 ng/g tissue , P<0.01). In contrast, HGF levels in the PPE group reached a peak on Day 1, and then rapidly decreased to a level below preoperative values between 7 and 28 days after RLL. These values at each time point were significantly lower than those of normal rats (on Day 7; 248 ng/g tissue versus 13211 ng/g tissue, P<0.01). Similarly, circulating HGF levels in the PPE group peaked on Day 1, and thereafter, decreased rapidly to normal levels between Day 3 Shigemura N 11 and Day 28. Of note, HGF value for plasma and lung was significantly lower in the PPE group than in the normal rats at each time point (P<0.01). We next explored whether these endogenous HGF levels are linked to alveolar regeneration after RLL since HGF is a potent pulmonary regenerative factor. To this end, we counted the number of PCNA-positive alveolar cells in remaining lung tissue at each time point. Tissue HGF levels correlated well with the alveolar regeneration, as evidenced by the PCNA index (Fig 3B-B). Furthermore, considering that the value of the PCNA index in non-treated and non-surgical rats is an indication of the natural alveolar cell turnover (Index = 3.5), the difference in the regenerative capacity between normal and emphysematous lungs was noteworthy. Interestingly, normal rats showed active alveolar regeneration (Index > 3.5), whereas the emphysematous rats experienced regeneration failure (Index < 3.5) after RLL. Successful Expression of HGF Supplementation and Its Regenerative Effects after RLL in Emphysema To determine whether HGF supplementation may compensate for the failure in alveolar regeneration after RLL in emphysematous lungs, we examined the effectiveness of a combined treatment involving RLL and gene transfection with HGF. At 7 days after transfection of the plasmid, the exogenous HGF (that is, human HGF) was clearly detected around the alveolar epithelium and the pulmonary vascular areas, as evidenced by immunohistochemistry using antibody reactive for human (but not rat) HGF (Fig 4-A). By the quantitative analysis using ELISA, human HGF could be detected at as high as 9.30.5 ng/g tissue in lung tissue of rats transfected with human HGF vector at one week whereas human HGF protein could not be detected in control rats. Interestingly, an increase in rat endogenous HGF was also observed in Shigemura N 12 rats transfected with human HGF vector at almost 10-fold higher levels than with control vector (P < 0.01). Further, those rat HGF expression levels remained at considerably high levels up to 3 weeks after transfection (Fig 4-B). Importantly, no HGF gene expression could be detected in other organs, including brain, liver, kidney and spleen (data not shown), thus suggesting that the present method with an HVJ- envelope leads to selective and persistent expression of exogenous HGF at local sites in emphysematous lung tissues. Concomitant with the successful expression of exogenous HGF, histological examination consequently demonstrated that the number of PCNA-positive alveolar cells was significantly higher in the HGF group than in the Surgery or PPE groups as early as a week after treatment (5.81.2 /mm 2 versus 2.30.5 and 1.71.0 /mm 2 , P<0.01) (Fig 5A-A). These differences between the HGF group and the other two groups persisted at the two-week time point. As a consequence of these regenerative effects on alveolar structures, the lungs from rats that underwent combined treatment of RLL and gene transfection with HGF had a decreased area of air space enlargement and alveolar wall destruction as compared with those in the Surgery groups (Fig 5A- B), which was also verified by the quantitative assessment using the RAC index (Fig 5A-C). Enhanced Angiogenesis with Concomitant Improvement of Exercise Tolerance and Gas Exchanges Angiogenesis. A marked increase in the number of factor -positive pulmonary capillaries was observed in the HGF-treated group when compared with the PPE- control and Surgery groups, and the difference was significant as early as a week after treatment (10.21.1 /mm 2 versus 3.00.7 and 1.80.8/mm 2 , P<0.01) (Fig 5B-A). Shigemura N 13 At two weeks after surgery, the number of positive capillaries in the HGF group increased further, however, there was almost no change in the Surgery group. Laser Doppler Analysis for Lung Blood Perfusion. Representative images obtained a week after treatment are shown in Figure 5B-B. As can be seen, the blood perfusion in the remaining right lung showed a greater increase with better expansion in the HGF group than in the Surgery group, while the unoperated left lung showed nearly no change. Long-Term Follow-up for the Effects of Combined Treatment on Pulmonary Function. PaO 2 in the natural course of PPE-control group decreased rapidly on Day 1 and thereafter decreased gradually until 6 month after elastase induction (Fig 6-A). Although the PaO 2 was lower in the Surgery group than in the PPE-control group on Day 14 (7 days after surgery, 608 mmHg versus 8515 mmHg, P<0.01), the PaO 2 value was higher in the Surgery group than in the PPE-control group for the first time at 2 months (718 mmHg versus 605 mmHg, P<0.01). In contrast, PaO 2 in the HGF group was higher as early as Day 14 and continued to increase gradually, with the levels significantly higher than in the Surgery and PPE-control groups at each time point until 6 months (at 6 months: 11110 mmHg versus 895 and 524 mmHg, P<0.01). Similarly, VO 2max was significantly higher immediately after surgery in the HGF group as compared to the Surgery and PPE-control groups and continued to increase over 6 months (at 6 months: 7210 mL/kg/min versus 485 and 317 mL/kg/min, P<0.01) (Fig 6-B). Overall, these data indicate that the combined treatment of LVRS and HGF supplementation led to attenuation of pulmonary function impairment over a period of at least six months, as a result of successful alveolar and vascular regenerations. Shigemura N 14 Discussion Pulmonary emphysema is histologically characterized by the destruction of alveolar network structures associated with a progressive loss of vascular beds, with the results being progressive respiratory dysfunction. Surgical resection of the emphysematous lobules improves the pathological conditions (9, 11), however, the effect is not long- lasting. Using a rat model of pulmonary emphysema, we found that alveolar regeneration was insufficient in the emphysematous tissues, which was associated with a lowered production of HGF even after surgical treatment. However, when exogenous HGF was supplemented in the affected lungs together with surgical reduction, alveolar and vascular repairs were accelerated, leading to the promotion of compensatory lung growth in the remnant lung tissue, followed by improvements in clinical findings. Indeed, induction of alveolar septation in adults remains a major therapeutic challenge (22). Further, we cannot exclude the possibility that HGF regulates VQ mismatch through amelioration of blood supply by the effects of the strong angiogenesis, which might result in improvement of pulmonary function. However, some physiologic studies indicated that such growth does occur to some extent in humans, primarily in infants and young children (5, 31), suggesting the possibility that compensatory lung growth occurs in human adult lung after the injury. Therefore, the present results led us to consider the importance of an HGF supplementation strategy for improving the outcomes of LVRS, via enhancement of alveolar regeneration and angiogenesis simultaneously in emphysematous lung tissues following surgery. Our previous report demonstrated that lung compensatory growth occurs under an endogenous HGF-mediated system (21). Based on those findings, we focused on the alveolar regenerative capacity in emphysematous lungs after surgical resection and Shigemura N 15 attempted to determine whether endogenous HGF production is associated with regenerative events following LVRS. One of the possible mechanisms responsible for increased HGF production is that inflammatory mediators following lung injury, such as tumor necrosis factor-alpha and interleukin-1 enhanced HGF gene expression (32, 33). Taken together, these findings seem to show that up-regulation of HGF would have been strongly induced in elastase-induced emphysematous lungs after LVRS. However, unexpectedly, surgical resection did not cause apparent increases in endogenous HGF, which was associated with a poor level of regeneration of alveolar architectures in the present elastase-induced rat model. Pulmonary emphysema is clinically characterized by local and systemic hypoxia (1), while several in vitro studies have demonstrated that hypoxia down-regulates HGF gene expression in several types of cells (34, 35). Further, TGF-beta system has been shown to be up- regulated under hypoxic stress (36, 37), whereas TGF-beta inhibits HGF production (34). Thus, we speculated that surgical challenge in emphysema fails to stimulate HGF production to a sufficient level, resulting in compensatory growth failure. Our results were similar to those of a previous report, which found that HGF production after a hepatectomy was limited in cirrhotic livers, whereas hypoxia was predominant (38, 39). To overcome this dilemma, we attempted local administration of exogenous HGF (i.e., human HGF) during the surgical treatment, using an HVJ-envelope method. In our model, the exogenously administrated HGF gene was found to be expressed, especially around the interstitial vessels. It is noteworthy that in addition to the increase in human HGF, HGF concentrations in rat lung tissue were also significantly increased to approximately 10-fold as compared with the control group, and they remained at considerably high levels for as long as 3 weeks. We speculated that the Shigemura N 16 exogenous HGF augmented the secretion and/or production of endogenous HGF by an auto-induction of HGF by HGF itself, as the same effects were previously observed in cultured fibroblast-like and endothelial cells (private communications by S.M. and T.N.) and in vivo (40, 41). Under these HGF-supplemented conditions, compensatory growth was accelerated to a level sufficient to improve clinical findings and offset surgical damage. Furthermore, we recently found that HGF supplementation per se accelerated tissue repair of the injured site in the emphysematous tissues, under LVRS-free conditions in elastase-treated rats (unpublished data). Thus, such a direct repair system by HGF may be involved in the improved clinical outcomes even after the LVRS treatment. Considering that endogenous HGF production can be suppressed under hypoxia, supplementation with exogenous HGF or its gene seems to be a reasonable strategy for overcoming the physiological dilemma in treatment of pulmonary emphysema. Gene transfection with an HVJ envelope vector has been used for HGF supplementation (30). This method allows for local expression of the HGF gene without increases in plasma HGF, thereby providing more long-lasting effects than recombinant treatment and avoiding unwanted systematic influences (data not shown). In addition, this technique is much more useful than the administration of recombinant HGF, which requires multiple daily injections, because of the relatively short half life (< 10 minutes) of recombinant HGF. Indeed, the effects provided by one injection using our method persisted for several months. A longer duration of pulmonary functional response may be possibly obtained by additional administration of an HVJ envelope vector containing the HGF gene and could be easily performed using a peripheral intravenous injection method, with long-term expression regulated by expression together with a suicide gene (42). Although additional detailed Shigemura N 17 investigations including analysis of the safety issues and the effects of mutation in the fusion glycoproteins of HVJ are required, the ease and effective duration of this technique makes it potentially ideal for clinical treatment of chronic diseases like COPD. The national emphysema treatment trial (NETT) study estimated that the duration of pulmonary response to LVRS was approximately two years in humans, and indicated that extension of the period of therapeutic response beyond that time and decreasing surgical mortality were critical factors for improved patient outcomes (3). As reported previously, at least several months is required for LVRS to produce a therapeutic effect toward respiratory function in rodent models, because of the delayed initiation of diaphragm-related mechanical mechanisms (43), suggesting a time lag between the surgery and onset of clinical improvements. Our finding that HGF supplementation rapidly improved respiratory function in the emphysematous rats within a few weeks after LVRS was in contrast to such a time lag, as alveolar and vascular regeneration by HGF may have provided a de novo microenvironment to enhance gas exchange via reconstruction of alveolar networks. These results suggest the potential role of HGF supplementation as a bridge therapy until a diaphragm- related mechanical mechanism can be developed that works well on the chest wall and diaphragmatic mechanics during respiration. Consequently, in addition to the near- immediate advantages described above, the outcomes of a long-term follow-up study of pulmonary physiology also demonstrated a superior effect of HGF supplementation with LVRS over LVRS alone that persisted for as long as six months. Based on the previous and present findings, we suppose that the application of HGF supplementation with LVRS, via sequential two steps using regenerative and mechanical mechanisms, may be helpful to overcome the current LVRS-related Shigemura N 18 limitations. A tentative hypothesis regarding a combined treatment of the surgery with growth factors has been proposed for the treatment of other pulmonary diseases (21, 44). In the current study, we found for the first time that compensatory growth is suppressed in emphysematous lungs following surgical resection, which was associated with a decrease in HGF production, whereas HGF gene supplementation enhanced compensatory growth and improved pathophysiologic conditions in rats following LVRS for pulmonary emphysema. Although our technique of LVRS in this study may not conform wholly to the currently performed LVRS in human subjects, the changes identified after RLL can be induced as compensatory growth in remnant lungs. Thus, we consider that the RLL-undergone rats could mimic LVRS as a conceptual model in pulmonary emphysema and answer the goal of this study to determine whether the induction of compensatory lung growth in emphysema may ameliorate the morbidities or not. 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Circulation 2002; 106: 120-124. Shigemura N 25 Figure Legends Figure 1A. Rats with elastase-induced emphysema exhibit marked emphysematous changes with focal airspace enlargement and progressive destruction of alveolar wall structures in a time-dependent manner. (A) Hematoxylin and eosin-stained lung tissue sections obtained from non-treated (Control) and elastase-induced rats on Days 7, 14. (B) Radial alveolar count (RAC) was used to quantify the apparent decrease in alveolar number in emphysematous tissue at each time point. Each value represents the meanSEM of values obtained using five rats at each time point. *P<0.01 versus Day 0 (Control). (C) Changes in pulmonary function tests at rest (arterial blood gas: PaO2) and under exercise stress (treadmill test: maximal oxygen uptake (VO 2max )). Each value represents the meanSEM of values obtained using five rats at each time point. *P<0.01 versus Day 0 (control) Figure 1B. Rats with elastase-induced emphysema (PPE) possess a lower capacity of alveolar regeneration and pulmonary angiogenesis than those with normal lungs. (A) Changes in the number of PCNA-positive alveolar cells in the remaining lung at one and two weeks after RLL. Top: Representative photomicrographs subjected to immunohistochemical staining using an anti-PCNA antibody. Bottom: Semiquantification of these histological findings (mean SEM, n=5). *P<0.01 versus normal group. (B) Changes in the number of vascular density. Vascular density was determined as the number of factor -positive capillaries less than 100m in diameter per square millimeter. Top: Distribution of capillary vessels in the lung following RLL. Bottom: Semiquantification of these histological findings (mean SEM, n=5). *P<0.01 versus normal group. Figure 2. Pulmonary functional recoveries following RLL as compared with normal Shigemura N 26 lungs: (A) Changes in alveolar gas exchange at rest (arterial blood gas: PaO2). (B) under exercise stress (treadmill test: maximal oxygen uptake (VO 2max )). Each value represents the mean SEM of values obtained using 5 rats at each time point respectively. Figure 3A. Time course of changes in endogenous HGF expression levels in lung tissue following emphysema induction with elastase. Data are mean SEM using 5 rats at each time point. Figure 3B. Time course of changes in rat endogenous HGF levels following RLL. (A) Alterations of HGF levels in lung tissue and plasma, as measured by ELISA. Data are mean SEM using 5 rats at each time point in each group. (B) Link of PCNA index used as an indicator of alveolar DNA synthesis in relation to HGF levels in lung tissue. Multiple linear regression analyses were undertaken. The natural alveolar cell turnover in non-treated rats was equivalent to 3.5 in PCNA index. Figure 4. Expression of HGF levels after the transfection in lung tissue following RLL. (A) Immunohistochemical staining for human HGF in the lung 7 days after gene transfection. Human HGF was detected around the alveolar epithelium and the pulmonary vascular areas in HGF-treated group. (B) Concentrations of human (a) and rat endogenous HGF (b) in lung tissue at 1, 2, 3, and 4 weeks after gene transfer. Control indicates rats transfected with empty vector (Con); HGF, rats transfected with human HGF vector (HGF); N.D., not detected. Each value represents the mean SEM of values obtained using five rats. *P<0.01. Figure 5A. Beneficial effects of HGF gene supplementation on the regeneration of alveolar structures following RLL. (A) Changes in the number of PCNA-positive alveolar cells in the lung at one and two weeks after the transfection. Three groups were designed. Two of them were the controls: rats treated with elastase (PPE-control Shigemura N 27 group) and rats undergoing RLL alone after elastase-induction (Surgery group), and the other group was rats undergoing combined treatment of LVRS and HGF supplementation (HGF group: RLL+HGF-HVJ). Semiquantification of the histological examinations (mean SEM, n=5). *P<0.01 versus Surgery group (RLL). (B) Hematoxylin and eosin-stained lung tissue at two weeks after the surgery. (C) Changes in the number of RAC. Each value represents the mean SEM of values obtained using five rats. *P<0.01 versus Surgery group. Figure 5B. Therapeutic effects of the transfection on lung blood perfusion. (A) Changes in the number of vascular density. Vascular density was determined as the number of factor -positive capillaries less than 100m in diameter per square millimeter. Semiquantification of the histological findings (mean SEM, n=5). *P<0.01 versus Surgery group (RLL). (B) Representative Laser Doppler Image analysis of lung blood perfusion at 7days after the transfection. Figure 6. Long-term follow-up for the effects of the treatments on pulmonary function. Amelioration of PaO2 and cardiopulmonary capacity under exercise stress by HGF supplementation compared with the control groups (PPE-control and Surgery groups), as evaluated by PaO2 (A) and VO 2max in treadmill test (B). Each value represents the mean SEM of values obtained using five rats. Shigemura N 28 Shigemura N 29 Shigemura N 30 Shigemura N 31 Shigemura N 32 Shigemura N 33 Shigemura N 34 Shigemura N 35 Shigemura N 36 Shigemura N 37