INTRODUCTION

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Hepatocyte growth factor (HGF) has been purified from the serum of partially hepatectomized rats (1), rat platelets (2), and human plasma (3). Notwithstanding the original concept of HGF as a potent mitogen for hepatocytes (1), it has been found to be a multifunctional growth factor, produced by mesenchymal cells such as endothelial cells (5, 6), fibroblasts (6, 7), and macrophages (6), and acts on a variety of epithelial cells and organs (8) as a mitogen (1, 4), motogen (9), and morphogen (10, 11). Thus, HGF is now recognized as a humoral mediator of epithelial-mesenchymal interactions (12). Recent findings indicate that HGF prevents apoptosis during embryogenesis and organogenesis in mice targeted with the HGF gene (13, 14). There is no species specificity among human, rat, and mouse with regard to biologic activities (15).

Administration of recombinant HGF following liver and kidney insults with hepatotoxins or renotoxins in vivo resulted in markedly elevated DNA synthesis in hepatocytes or renal tubular cells, suppression of hepatitis or renal damage, and stimulation of liver regeneration or reconstruction of the normal renal tissue structure (15). These findings suggest that HGF acts as a pleiotropic factor for various organs.

We reported that higher HGF levels were detected in the bronchoalveolar lavage fluid (BALF) of patients with idiopathic pulmonary fibrosis (19). Yanagita and coworkers reported that the HGF concentration in the sera of patients with lung diseases were higher than in those of normal controls (20, 21).

In vitro, HGF stimulates DNA synthesis in alveolar type II cells (22, 23). The receptor for HGF (c-met protooncogene product [24]) was expressed in alveolar type II cells but not in macrophages (22, 23), and the HGF-mRNA was detected in macrophages but not in type II cells (19, 22). In acute lung injury caused by hydrochloric acid (HCl), DNA synthesis in alveolar type II cells and increased HGF activity in the lung were observed in vivo (20).

The information noted above suggests that HGF may act as a pulmotrophic factor. To determine whether exogenously administered HGF can protect against lung injury, human recombinant HGF was given to mice with severe lung injury induced by bleomycin (BLM), and the protective effect of HGF, administered simultaneously or after BLM, on this injury was analyzed.

	METHODS

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Animals and Materials

Female C57BL/6 mice (specific pathogen free), 8 to 10 wk of age, were purchased from Charles River Japan, Inc. (Yokohama, Japan), and were maintained at constant temperature (22° C), humidity (40%), and light cycle (8:00 A.M. to 8:00 P.M.) with food and water ad libitum. Human recombinant HGF (hrHGF) was provided by Dr. Nakamura (Osaka University School of Medicine, Osaka, Japan). BLM was supplied by Nihonkayaku Co. (Tokyo, Japan). Other reagents are of analytic grade.

Administration of hrHGF and BLM

Administration of HGF and BLM were performed by constant intraperitoneal (intraperitoneally [i.p.]) and subcutaneous (subcutaneously [s.c.]) infusion, respectively, through osmotic minipumps (Model 1007D and 2001, respectively; Alza Co., Palo Alto, CA) as specified below. In mice anesthetized with thiopental sodium (150 mg/kg i.p.), the pump with BLM was implanted subcutaneously on the back, slightly posterior to the scapulae, and the pump with HGF or vehicle was implanted intraperitoneally through a midline skin incision, about 0.5 cm long, in the lower abdomen posterior to the rib cage. After the mice were killed, the pumps were examined to determine if they had delivered the entire dosage of their contents in each mouse.

Determination of Tissue HGF Concentration

Three groups of three mice were used. Group A was treated with HGF (50 µg/mouse dissolved in 100 µl of saline) by constant i.p. infusion through a minipump (Model 1007D), and BLM (100 mg/kg dissolved in 200 µl of saline) by constant s.c. infusion from an osmotic minipump (Model 2001). Group B was treated using the same protocol as group A except that the amount of HGF per mouse was 333 µg instead of 50 µg. Group C was treated with BLM alone. Mice were killed 3 d after initial treatment. Tissue of lung, liver, and kidney were homogenized on ice in four volumes of buffer composed of 10 mM Tris-HCl (pH 7.5), 2 M NaCl, 0.01% Tween-80, 1 mM phenylmethyl-sulfonyl fluoride, and 1 mM ethylenediaminetetraacetic acid. After centrifugation at 105,000 × g at 4° C for 1 h, the supernatant was used for HGF quantification by an enzyme-linked immunosorbent assay (ELISA) system (Institute of Immunology, Tokyo, Japan) (18). A mouse monoclonal antibody against hrHGF was used according to the manufacturer's protocol. This mouse monoclonal antibody has little cross- reactivity against mouse HGF; hence, mouse HGF was not detected in this ELISA system.

Animal Treatment

Simultaneous administration of low dose HGF with BLM. To induce fibrotic changes in the lungs of mice, C57BL/6 mice received BLM by a continuous s.c. infusion from an osmotic minipump (Model 2001), from Day 0 to Day 7, as originally described by Harrison and coworkers (25). The minipump was loaded with BLM (100 mg/kg, dissolved in 200 µl of saline). Mice received HGF (50 µg/mouse, dissolved in 100 µl of saline) by a continuous i.p. infusion through a minipump (Model 1007D), from Day 0 to Day 7, simultaneously with BLM. Two and 4 wk after initial treatment, the mice were killed with thiopental sodium injection (450 mg/kg i.p.), thoraces were opened, lungs were removed, and protective effects of HGF for fibrotic changes induced by BLM were analyzed (Figure 1).

View larger version (22K): [in this window] [in a new window]   Figure 1.   Experimental design of three different administration regimens of HGF and BLM. Mice in all groups received BLM by a continuous subcutaneous infusion from an osmotic minipump for 7 d, from Day 0 to 7. HGF was administered in three different ways as follows: (1) simultaneous administration of low dose HGF (50 µg/mouse, continuous i.p. administration for 7 d, from Day 0 to Day 7). Mice were killed 2 and 4 wk after initial treatment of BLM. (2) Simultaneous administration of high dose HGF (total dosage of 280 µg/mouse of HGF over 14 d; 140 µg by continuous i.p. infusion for 7 d, from Day 0 to Day 7, and followed by bolus i.p. injections of 20 µg daily for 7 successive days, from Day 8 to Day 14). Mice were killed 4 wk after initial treatment of BLM. (3) Delayed administration of high dose HGF (total dosage of 280 µg/ mouse of HGF over 14 d; 140 µg by continuous i.p. infusion for 7 d, from Day 14 to Day 21, and followed by bolus i.p. injections 20 µg daily for 7 successive days, from Day 22 to Day 28). Mice were killed 4 wk after initial treatment of BLM.

Simultaneous administration of high dose HGF with BLM. The mice were injured by BLM from Day 0 to Day 7 as described above. At the same time, the mice received a total dosage of 280 µg/mouse of HGF over 14 d; 140 µg by continuous i.p. infusion through a minipump for 7 d followed by bolus injections of 20 µg/mouse/d for 7 successive days. Mice were killed at 4 wk after initial treatment of BLM. The lungs were removed and analyzed.

Delayed administration of high dose HGF after BLM insults. The mice in the delayed HGF administration group first received BLM (100 mg/kg, from Day 0 to Day 7) as described above. Then on Day 14, the mice were treated with a total dosage of 280 µg/mouse of HGF over 14 days; 140 µg by continuous i.p. infusion through a minipump from Day 14 to Day 21 followed by bolus injections of 20 µg/mouse/d from Day 22 to 28. Mice were killed 4 wk after initial treatment of BLM. The lungs were removed and analyzed.

Assessment of the Protective Effect of HGF on Lung Injury Induced by BLM

Histologic examination. The left lung was fixed by simple immersion in 10% buffered formaldehyde under constant negative pressure (10 cm water pressure) for 48 h in preparation for histologic examination. Inflation-fixation method was avoided because of technical problems to apply only to the left mouse lung. The fixed lung was sectioned sagittally, embedded in paraffin, and stained with elastica-Masson.

Quantitative evaluation of histologic findings. For the quantitative histologic analysis of fibrotic changes induced by BLM, a numerical fibrotic scale (Ashcroft scale [26]) was used. A numerical fibrotic score (Ashcroft score) was obtained as follows. The severity of the fibrotic changes in each lung section was assessed as a mean score of severity from observed microscopic fields. More than 25 fields within each lung section were observed at a magnification of ×100 in each successive field, and each field was assessed individually for the severity of fibrotic changes and allotted a score from 0 (normal) to 8 (total fibrosis) using a predetermined scale of severity (numerical fibrotic scale). After examination of the whole section, the mean of the scores from all fields was taken as the fibrotic score. In order to prevent observer's bias, all histologic specimens were randomly numbered and interpreted in a blinded fashion. Each specimen was scored independently by two or three observers, including a histopathologist; finally, the mean of their individual scores was considered as the fibrotic score.

Hydroxyproline analysis. Whole collagen content of the right lung was estimated by an assay of hydroxyproline (27). Briefly, after acid hydrolysis of the lung with 6 N HCl at 110° C for 16 h in a sealed glass tube (Iwaki, Tokyo, Japan), hydroxyproline content was determined by high performance liquid chromatography (28), and normalized by saline-treated control mice.

Statistical analysis. All values are shown as mean ± SEM. Statistical analysis of the results was performed with ANOVA and post hoc analysis with Newman-Kuels procedure (29). p Values lower than 0.05 were considered significant.

	RESULTS

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Tissue HGF Concentration

To assess the distribution of intraperitoneally administered exogenous hrHGF, tissue hrHGF concentrations in mice were determined by ELISA (Table 1). The tissue HGF concentration after 3 d of continuous i.p. administration showed that only a small fraction was distributed. The lung contained less hrHGF per gram of tissue than liver and kidney. In addition, less proportional increase of hrHGF in the lung between group A (hrHGF 50 µg/mouse/7 d) and group B (hrHGF 333 µg/mouse/7 d) was observed in contrast to those in the liver or kidney.

Protective Effect of hrHGF Administered Simultaneously with BLM

Morphologic findings. Murine lung injury was induced by BLM released from an osmotic minipump subcutaneously implanted in the mouse. The dose of hrHGF used in this study (50 µg/mouse/7 d i.p.) was comparable to those in the experiments in which the hepato- or renotrophic effects of HGF was evaluated (5 to 10 µg/mouse/d i.v.) (15).

Histologic findings in control mice treated with BLM alone showed that fibrotic changes were progressive at 2 and 4 wk after treatment. Examination 2 wk after initial treatment revealed focal fibrotic lesions primarily in subpleural and occasionally in perivascular areas with consolidation of lung parenchyma, loss of alveolar architecture, and increased cellularity with alveolar macrophages (Figure 2A). At 4 wk, fibrotic changes became more severe, expanding to the central areas of the lobes, involving the perivascular and peribronchiolar regions, and showing much more confluence and uniformity of appearance in subpleural areas (Figure 2C).

View larger version (123K): [in this window] [in a new window]   Figure 2.   Microscopic findings of the murine lungs 2 and 4 wk after initial treatment of BLM and low dose HGF. (A) BLM (100 mg/kg, continuous s.c. administration from Days 0 to 7) alone at 2 wk; (B) BLM and low dose HGF (50 µg/mouse, continuous i.p. administration from Days 0 to 7) at 2 wk; (C ) BLM alone at 4 wk; (D) BLM and low dose HGF at 4 wk. Lungs were stained with elastica-Masson. Original magnification: ×10.

In contrast to the findings in mice treated with BLM alone, histologic findings at 2 wk with simultaneous administration of HGF (50 µg/mouse/7 d) revealed that fibrotic lesions were less focal in the subpleural areas, and fibrotic changes were milder in degree than in mice treated with BLM alone (Figure 2B). After 4 wk, focal fibrotic changes, which did not expand to the central areas of the lobe, were observed in subpleural areas and lung tissue outside of the subpleural areas appeared normal except for increased cellularity with alveolar macrophages (Figure 2D).

Quantitative evaluation of histologic change. The overall grades of the fibrotic changes of the lungs were obtained by the numerical score (Ashcroft score) 2 and 4 wk after treatment. The scores in the mice treated with BLM with simultaneous HGF and with BLM alone were 2.0 ± 0.2 and 2.6 ± 0.3 at 2 wk, and 3.7 ± 0.4 and 4.9 ± 0.3 at 4 wk, respectively (Figure 3A). All mice survived the experiment (n = 6 in each group). However, one mouse, treated with HGF and killed on day 28, received an insufficient dosage of HGF because of technical problems with the intraperitoneal minipump, and was excluded from this study. A significant difference in the scores between two groups was demonstrated at 4 wk by ANOVA and post hoc analysis with Newman-Kuels procedure (p < 0.05).

View larger version (26K): [in this window] [in a new window]   Figure 3.   Evaluation of fibrotic changes in the lung by numerical fibrotic score and measurement of hydroxyproline content in the lung treated with BLM and low dose HGF. (A) Evaluation of fibrotic changes by a numerical fibrotic scale (Ashcroft scale) in the murine lung treated with BLM and low dose HGF. Fibrotic score (Ashcroft score [26]) was obtained with a continuous numerical scale for determining the degree of fibrotic changes in the lung. Grade was scored on a scale from 0 to 8, with use of the average of microscopic field scores. The average score of the group treated with BLM and low dose HGF (hatched bars) was lower than that with BLM alone (closed bars) both at 2 and 4 wk after initial treatment of BLM. There was a significant difference between these two groups at 4 wk when examined by ANOVA and post hoc analysis with Newman-Kuels procedure (p < 0.05). (B) Measurement of hydroxyproline content in the lung treated with BLM and low dose HGF. BLM (100 mg/kg, continuous s.c. administration) and vehicle (saline) (closed bars) or BLM and low dose HGF (50 µg/ mouse, continuous i.p. administration) (hatched bars) were administered for 7 d. Lungs were removed 2 and 4 wk after initial treatment of BLM. Hydroxyproline content in the right lung was measured and normalized to those obtained from mice treated neither by BLM nor HGF (saline treated control mice).

Assessment of fibrosis by hydroxyproline content. Collagen content was assessed by measuring hydroxyproline in the right lung 2 and 4 wk after initial treatment. The hydroxyproline content was lower in lungs treated with BLM and simultaneous HGF (Figure 3B). Control C57BL/6 mice which were treated with saline showed minor increases in pulmonary hydroxyproline content (data not shown). In this context, hydroxyproline content in the right lungs, normalized by saline-treated control mice, 2 and 4 wk after treatment was 127.5 ± 4.9% and 182.8 ± 8.3% in mice treated with BLM and HGF, and 140.9 ± 9.9% and 205.1 ± 28.0% in mice with BLM alone, respectively. This reduction of the hydroxyproline content was not statistically significant.

Comparison of Simultaneous and Delayed Administration with High Dose HGF on Lung Injury Induced by BLM

Morphologic changes. Simultaneous administration of BLM and HGF (50 µg/mouse/7 d) partly suppressed the fibrotic change. A higher dose of HGF (total, 280 µg/mouse/14 d) given either simultaneously with BLM or 2 wk later, showed at 4 wk that the suppressive effect was obvious irrespective of simultaneous or delayed administration of HGF (Figure 4). Fibrotic changes in the lungs in both groups treated with higher dose HGF were limited to the subpleural areas and some parts of the perivascular or peribronchiolar areas. Other areas showed almost normal alveolar architecture. There were no specific histologic differences noted between these two groups.

View larger version (91K): [in this window] [in a new window]   Figure 4.   Microscopic findings of the murine lung injured by BLM and treated with simultaneous or delayed administration of high dose HGF. (A) With BLM alone; (B) with BLM (100 mg/kg, continuous s.c. administration from Days 0 to 7) and simultaneous high dose HGF (total dosage of 280 µg/mouse of HGF over 14 d; 140 µg by continuous i.p. infusion for 7 d, from Day 0 to Day 7, and followed by bolus i.p. injections of 20 µg daily for 7 successive days, from Day 8 to Day 14); (C ) with BLM and delayed high dose HGF (total dosage of 280 µg/ mouse of HGF over 14 d; 140 µg by continuous i.p. infusion for 7 d, from Day 14 to Day 21, and followed by bolus i.p. injections 20 µg daily for 7 successive days, from Day 22 to Day 28). The lungs were removed at 4 wk after initial treatment of BLM, and stained with elastica-Masson. Original magnification: ×40.

Quantitative evaluation of histologic changes. Quantitative evaluation by a numerical fibrotic score (Ashcroft score) confirmed the suppressive effect of HGF on fibrotic changes when administered in either a simultaneous or delayed fashion. No mice died during the experiment. In this context, significant differences in the scores were observed when comparisons were made (1) between the group treated with BLM alone (n = 5) and that treated with BLM and simultaneous HGF (n = 5) (4.1 ± 0.2 versus 2.6 ± 0.4, p < 0.01), and (2) between the group treated with BLM alone and that treated with BLM and delayed HGF (n = 5) (4.1 ± 0.2 versus 2.4 ± 0.2, p < 0.01) (Figure 5A). The fibrotic scores of mice with higher dose HGF was significantly smaller than that of mice with low dose HGF (p < 0.05).

View larger version (26K): [in this window] [in a new window]   Figure 5.   Evaluation of fibrotic changes in the lung by numerical fibrotic score and measurement of hydroxyproline content in the lung treated with BLM and simultaneous or delayed administration of high dose HGF. (A) Evaluation of fibrotic changes by numerical fibrotic scale (Ashcroft scale). Both of averages of numerical fibrotic scores of the group treated with BLM and simultaneous high dose HGF (hatched bars), and those treated with BLM and delayed high dose HGF (hatched bars) were significantly lower than those with BLM alone (closed bars) 4 wk after initial treatment of BLM. (B) Measurement of hydroxyproline content in the lung treated with BLM and high dose HGF. Hydroxyproline content in right lung of both groups treated with BLM and simultaneous (hatched bars) or delayed (hatched bars) administration of high dose HGF was significantly lower than those with BLM alone (closed bars) 4 wk after initial treatment of BLM. Hydroxyproline content in the right lung of mice was normalized to those in saline-treated controls. Statistical analysis of the results was performed with ANOVA and post hoc analysis with Newman-Kuels procedure.

Assessment of fibrosis by hydroxyproline content. Mice treated with high dose HGF and BLM were used for assessment of collagen content by measuring hydroxyproline in the right lung 4 wk after initial treatment. The hydroxyproline content in the lungs was significantly lower in groups treated with BLM and simultaneous or delayed administration of high dose HGF in comparison to the group treated with BLM alone (p < 0.001, p < 0.001) (Figure 5B). When normalized with saline-treated control mice, hydroxyproline content in the lungs were 121.8 ± 8.1%, and 113.2 ± 6.2%, and 162.7 ± 4.6% in mice with simultaneous and delayed administration of high dose HGF, and BLM alone, respectively.

	DISCUSSION

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Mode of HGF Action in Repair Process of Damaged Respiratory Epithelial Structures

Organotoxins induce parenchymal cell death, and cause subsequent pathologic remodeling of normal structure, resulting in functional insufficiency of organs. HGF, as a multifunctional and essential cytokine transducing a superb signal for normal morphogenesis and regeneration, functions as an antiorganotoxic agent in several organs (12, 15, 30, 31). In the present study, we hypothesized that exogenous HGF may act as a pulmotrophic factor that prevents respiratory epithelial cell death as well as promoting ordered regeneration of the peripheral re-spiratory tract. In mice with simultaneous low dose HGF during BLM administration, there was a significant difference in suppressed fibrosis by morphology at 4 wk in comparison with BLM-treated mice, although collagen deposition assayed by hydroxyproline in mouse lung treated with HGF and BLM contained a reduced amount, but not significant. With high dose HGF, both simultaneous and delayed, HGF not only effectively suppressed fibrotic changes, but actually ameliorated them, even when administered 2 wk after the start of BLM treatment. Moreover, with high HGF dose, significant repression of fibrotic changes was confirmed by assessment of hydroxyproline. To date, very few reports have described the effective suppression of the fibrotic process when agents are administered after the onset of damage. Only prior and/or simultaneous administration of steroid hormones (32) or antibodies to cytokines such as anti-TNF- antibody are capable of preventing damage (33, 34). Significance of this consequence in the clinical setting is obvious, because the treatment should be effective despite the fact that the clinical manifestation of lung injury or chronic inflammation usually becomes apparent after the latent period following insults.

Route of HGF Administration and the Concentration of HGF in the Tissue

In our experimental design, both BLM and HGF were administered by continuous infusion through osmotic minipumps. Despite the fact that excessive amounts of HGF (50 µg/mouse/7 d for low dose, and 280 µg/mouse/14 d for high dose) were used, only a small fraction of HGF may have reached the injured organs, because HGF binds to heparin in the peritoneum as well as to connective tissues along the route of diffusion. In vitro, HGF stimulates DNA synthesis in alveolar epithelial cells in a concentration-dependent manner, reaching a maximum at an HGF concentration of 5 to 10 ng/ml (35). In our study, the tissue concentration of HGF in the lung at Day 3 was 0.63 ± 0.27 ng/g tissue when low dose HGF (50 µg/7 d) was used, and 2.0 ± 0.04 ng/g tissue when higher dose HGF (333 µg/7 d) was used. Thus, we assumed that these HGF concentrations were capable of stimulating alveolar epithelial DNA synthesis.

A continuous osmotic infusion pump was chosen because of the difficulty with repeated intravenous administrations of HGF. Regarding the drug delivery system, however, the continuous infusion as well as the strong binding of HGF to heparin (1, 2) in the connective tissue allows for constant chronic dosing of HGF in mice. Because high local concentrations of HGF have not caused any serious adverse effect to date, these results provide a potential option for future treatment, perhaps even direct expression of HGF cDNA through gene therapy.

Lung Injury, Fibrosis, and Therapeutic Concepts for Repair

With regard to the mechanisms of lung damage and disordered remodeling (36, 37), therapeutic strategies can be conceptualized into three categories: (1) to counteract active inflammatory reactions caused by pro-inflammatory agents, (2) to avoid apoptotic loss of epithelial cells and stimulate ordered tissue repair, and (3) to eliminate chronic stimuli for fibrosis.

Current therapeutic approaches are mostly confined to the early inflammatory phase. Steroid hormones are effective in suppressing the activity of immune effector cells (38), anti-oxidant agents such as glutathione (GSH) are effective against oxidative stimuli (39), and antiproteases are active against proteolytic damage (40, 41). However, in addition to suppression of the pro-inflammatory and pro-injurious agents, strong therapeutic support for the repair of epithelial cells is essential. Potent growth factors for alveolar type II epithelial cells have been characterized, such as epidermal growth factor (EGF), transforming growth factor-, (TGF-), keratinocyte growth factor (KGF), acidic and basic fibroblast growth factor (aFGF, bFGF), and HGF (22, 23, 35, 42). Panos and colleagues have demonstrated that HGF and KGF induce DNA synthesis in alveolar type II cells, and HGF stimulates DNA synthesis more strongly than KGF (35). Similar results were reported by Shiratori and coworkers, i.e., HGF is more potent in stimulating DNA synthesis than aFGF, TGF-, or EGF (23). In our study, simultaneous administration of HGF protected against lung injury induced by BLM, suggesting that HGF stimulated DNA synthesis in type II epithelial cells and promoted the turnover of damaged epithelial cells. This in turn would diminish inflammatory changes and suppress fibrotic changes in the lung. Recently, the possibility that HGF may be involved in the prevention of apoptosis was reported (13, 14). Matsumoto and colleagues have described HGF as a potent survival factor for pheochromocytoma PC12 cells in culture (45). Thus, HGF may have protective activity in damaged respiratory epithelial cells through an anti-apoptotic mechanism.

TGF-1 is a very potent inducer of tissue fibrosis due to its chemotactic and stimulatory activity for matrix synthesizing cells (46). After administration of BLM, TGF-1 mRNA and protein in lung tissue were elevated and peaked at 7 to 10 d (47). Subsequently, collagen and other extracellular matrix proteins peaked at 10 to 14 d (47, 49, 50). The timing of delayed administration of HGF in this study coincided with the time course of TGF-1 involvement. HGF suppressed the fibrotic changes in lung even at this stage. Although HGF expression is dramatically inhibited by TGF-1 (51, 52), exogenous HGF may affect the function of TGF-1 directly or indirectly and may mitigate the tendency toward lung fibrosis (53). In this context, as a therapeutic approach, it is of interest to examine the synergistic effect of HGF and anti-TGF-1 agents (48, 54).

In summary, our results suggest a substantial therapeutic benefit of HGF in both early and delayed BLM lung injury. Further long-term studies will be necessary to confirm the clinical efficacy of this therapy.