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HGF reduces advancing lung fibrosis in mice: a potential role for MMP-dependent myofibroblast apoptosis

Division of Molecular Regenerative Medicine, Department of Molecular Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Japan

²Correspondence: Division of Molecular Regenerative Medicine, Dept. of Molecular Regenerative Medicine, Osaka University Graduate School of Medicine, Yamadaoka 2-2-B7, Suita 565-0871, Japan. E-mail: nakamura{at}onbich.med.osaka-u.ac.jp

SPECIFIC AIM

Previous studies have demonstrated that hepatocyte growth factor (HGF) inhibits pulmonary fibrosis in murine models, but molecular mechanisms by which HGF improves lung fibrosis have yet to be fully understood. Because myofibroblasts play a central role in over-deposition of extracellular matrix (ECM) in fibrotic lungs, this study was designed to address c-Met/HGF receptor expression in lung myofibroblasts, mechanisms of apoptosis in myofibroblasts promoted by HGF, and their involvement in a process of reducing lung fibrosis.

PRINCIPAL FINDINGS

1. Delayed treatment with HGF leads to reduced lung fibrosis in mice
Pulmonary fibrosis is a common outcome of chronic respiratory failures and is histologically characterized by hyperplasia of interstitial myofibroblasts to deposit ECM. It is still unclear whether HGF reduces connective tissue areas once lung fibrosis is advancing. Therefore, we gave recombinant human HGF (rh-HGF: 500 µg/kg/12 h, s.c.) to bleomycin (BLM)-treated mice for 2 wk after the onset of lung fibrosis. As a result, there was an apparent decrease in collagen-stained interstitial areas of the HGF group (4W) compared with those in the pretreatment (2W) or in saline groups (4W) (see online data), indicating that progression of lung fibrosis is in part reversed by HGF. To determine potential target cells of HGF in fibrotic murine lungs, we analyzed distribution of the c-Met. In BLM-treated lungs, c-Met expression was extensive, whereas -smooth muscle actin (-SMA)-positive signals were noted mainly on interstitial areas. Most of the -SMA-stained interstitial cells were also immunopositive for c-Met (see online data), demonstrating that myofibroblasts can be a direct target for HGF. Thus, our attention was directed to determine whether HGF affects interstitial myofibroblasts.

2. MMP-related mechanisms in HGF-induced myofibroblast apoptosis
We next examined the effects of HGF on myofibroblast survival, focusing on fibronectin and MMP-2/-9 (enzymes for degradation of fibronectin), because: 1) myofibroblast apoptosis is needed for regression of tissue fibrosis; and 2) fibronectin is important for cell survival that depends on anchorage to ECM. In a culture model using myofibroblast-like MRC-5cells, HGF increased active MMP-2 and MMP-9 levels in the supernatants of myofibroblasts (Fig. 1 A). We asked whether HGF-induced MMPs affect myofibroblast survival. When recombinant human MMP-2 and/or MMP-9 were added to myofibroblast-like MRC-5 cells, significant increases in TUNEL-positive cells were seen, and MMP-9 was more potent than MMP-2 in enhancing apoptosis (Fig. 1B ). HGF increased the number of apoptotic cells, whereas addition of MMI270, a broad-spectrum inhibitor for MMPs, diminished HGF-mediated apoptosis by 75% (Fig. 1B ).

View larger version (35K): [in this window] [in a new window]   Figure 1. HGF-induced MMPs participate in apoptosis, associated with decreased FN-CCB domain and repressed FAK activation. A) Changes in MMP-2/-9 levels in culture medium of myofibroblast-like MRC5 cells, incubated for 96 h with rh-HGF (10, 30, and 100 ng/mL). Culture supernatants were subjected to ELISA systems to measure active MMP-2/-9 levels. All data are shown as mean ± SD (n=6). *P <0.05 and **P <0.01 vs. vehicle control. B) MMP-dependent apoptosis in the myofibroblast-like MRC5 cells. Left: enhancing effects of recombinant MMP-2/-9 on apoptosis, noted in myofibroblast-like MRC5 cells at 96 h after addition of MMPs (50 µM). Right: effect of MMI270 (a MMP-inhibitor) on the HGF-induced apoptosis. Apoptotic changes were enhanced in the myofibroblast-like MRC5 cells at 96 h after addition of HGF under serum-starved conditions, whereas MMI270 suppressed such apoptotic responses by 75%, as evidenced by TUNEL staining. C) Reducing effect of HGF on FN-CCB accumulation involved in cell survival. Left: effect of anti-FN-CCB IgG (E2E) on apoptosis of myofibroblast-like MRC5 cells. The cells were incubated under serum-free conditions for 96 h with anti-FN-CCB IgG or normal IgG. Apoptotic changes were evaluated using TUNEL staining. Right: effects of HGF or HGF plus an MMP-inhibitor (MMI270) on fibronectin (FN) and FN-CCB expressions around the myofibroblast-like MRC5 cells, incubated for 96 h under serum-free conditions. The bottom number means a relative value of the band density when basal levels are calculated as 1. D) Changes in FAK and phosphorylated FAK levels in myofibroblast-like MRC5 cells at 96 h after addition of MMI270, HGF, or HGF plus MMI270.

The CCB domain in fibronectin (i.e., FN-CCB) is a key bridge between cells and fibronectin. In our MRC5 model, anti-FN-CCB IgG increased the number of apoptotic cells (Fig. 1C ), showing that the cell-substratum interaction mediated by FN-CCB is important for myofibroblast survival. Immunoblot analysis revealed that FN-CCB accumulation in cultures of myofibroblast-like MRC5 cells was reduced by HGF, whereas MMP inhibition with MMI270 restored such a suppressive effect by HGF (Fig. 1C ), suggesting that HGF-induced MMPs are critical for breakdown of FN-CCB domain involved in cell survival. We then focused on intracellular FAK, since FAK activation via fibronectin-mediated signals is critical for cell survival. During a culture period of 96 h, HGF inhibited FAK expression and phosphorylation, whereas these effects were released when MMI270 was added together with HGF. Thus, our data indicate that the decrease in cell-substratum interaction mediated by FN-CCB/FAK signals is involved in HGF-mediated apoptosis, and is associated with increased MMP activities.

3. Induction of MMP-dependent myofibroblast apoptosis by HGF involved in regression of lung fibrosis in mice
We returned to the animal model to determine whether: 1) HGF has apoptotic effects on myofibroblastosis in vivo; and 2) MMPs play a critical role during the apoptotic process, using the MMP-inhibitor, MMI270 (Fig. 2 A). HGF administrations increased active MMP-9 and MMP-1 (proteases for degradation of interstitial fibronectin and collagen, respectively) levels in the BLM-treated lungs to more than a 2-fold level of the saline control, whereas MMI270 repressed the HGF-mediated increases in active MMP-1/-9 levels (Fig. 2B ). Together with the increase in MMP-9 levels, HGF increased the number of cells double-positive for TUNEL and -SMA in the lung compared with findings in saline-injected controls (Fig. 2C ), suggesting that HGF stimulated apoptosis in myofibroblasts of fibrotic lungs. A combination of HGF and MMI270 significantly suppressed HGF-mediated apoptotic effects on myofibroblasts (Fig. 2C, D ). Likewise, HGF reduced interstitial areas (occupied by FN-CCB, myofibroblasts and collagens), but these antifibrotic effects of HGF were suppressed by the concomitant MMI270 administrations (Fig. 2D ). Consistent with immunohistological evidence, the lung hydroxyproline level was reduced by HGF, whereas it increased in mice treated with HGF plus MMI270 over levels in mice treated with HGF alone (Fig. 2E ). In general, myofibroblast behavior is regulated by interstitial ECMs, whereas epithelial cell survival depends on basement membrane ECMs. In our study, HGF decreased interstitial ECMs, whereas basement membrane ECMs remained intact in HGF-treated lungs (not shown). This may be one of the reasons why HGF selectively induced anoikis-like cell death (apoptosis induced by a loss of anchorage to extracellular substrates) -like apoptosis in interstitial myofibroblasts, but not in epithelial cells. Together with antifibrotic findings, HGF accelerated alveolar and endothelial repairs and all these events were associated with improved exercise activity (not shown).

View larger version (53K): [in this window] [in a new window]   Figure 2. Effect of HGF and MMP inhibition on myofibroblast apoptosis in BLM-treated mouse lungs. A) Protocol for MMP inhibition (with MMI270) in the HGF supplement toward BLM-treated mice. B) In vivo effect of MMI270 on active MMP-9 and MMP-1 levels in mouse lungs treated with BLM. Data are shown as a mean value ± SD (n=6). *P <0.05, **P <0.01 vs. saline group, and #P <0.05 vs. HGF group. C) Changes of the myofibroblast apoptosis in the BLM-treated lungs by HGF or HGF plus MMI270. The incidence of myofibroblast apoptosis was expressed as a ratio of double-positive cells in more than 1000 -SMA-positive interstitial cells. D) Histochemical findings of myofibroblast apoptosis and interstitial fibrosis in the lungs. Upper: changes of interstitial FN-CCB deposition by HGF alone or HGF plus MMI270 (x260). Middle: detection of apoptosis in interstitial myofibroblasts. The myofibroblasts were visualized as cytoplasmic red (i.e., -SMA-positive) signals, whereas apoptosis was detected as nuclear brown signals (in an expanded frame) (x360). Bottom: changes in lung fibrosis by HGF or combined MMP inhibition (type I collagen staining; x200). E) Comparison of lung hydroxyproline levels among saline-, HGF-, and HGF plus MMI270-injected groups.

CONCLUSIONS AND SIGNIFICANCE

Over-accumulation of myofibroblasts is an unavoidable step during development of chronic organ disorders (including lung fibrosis), especially during the expansion of ECM-deposited areas. Under such fibrotic situations, HGF sequentially regulates molecular and cellular events to lessen fibrosis: 1) HGF initially targets myofibroblasts and enhances production of MMP enzymes (such as MMP-9/-1); 2) these MMPs reduce accumulation levels of myofibroblast-surrounding ECM proteins (such as CCB-containing fibronectin or collagens); and 3) loss in ECMs leads to apoptotic cell death in lung myofibroblasts, as noted in anoikis. Together, deletions of both myofibroblasts and ECMs via HGF/c-Met activations in the lung interstitium would provide a space for remaining parenchymal epitheli and vessels to regenerate, even if parenchymal spaces had been replaced by myofibroblasts (Fig. 3 ). However, we cannot exclude the possibility that MMP-independent pathways may be involved in HGF-mediated apoptotic mechanisms of lung myofibroblasts, because the present MMP-inhibitor has a broad spectrum with undetermined specificity and its effect on HGF-induced antifibrosis was significant but not complete.

View larger version (29K): [in this window] [in a new window]   Figure 3. Hypothetical model for HGF-mediated resolution of lung fibrosis. Under persistent injuries, infiltrated macrophages (M) produce and release TGF-ß1, then resident fibroblasts are activated and transformed into myofibroblasts (MyoFB). In this process, myofibroblasts newly acquire the c-Met/HGF receptor, whereas HGF production is suppressed, allowing for progression of pulmonary fibrosis. Therefore, supplement of exogenous HGF is helpful to halt or reverse progression of lung fibrosis: 1) HGF initially targets interstitial myofibroblasts; 2) MMP production is enhanced in the cells, resulting in degradation of ECMs (including fibronectin CCB domain); and 3) the myofibroblasts became apoptotic, along with a loss of surrounding ECMs. To cover the loss in myofibroblast-accumulated areas, remaining epithelial and endothelial cells can proliferate under HGF/c-Met-activated conditions. Eventually, HGF supplement leads to recovery from respiratory dysfunctions in chronic lung diseases via a shift from a fibrosis-dominant to a regeneration-dominant response.

We recently obtained similar evidence that HGF is apoptotic toward hepatic and renal myofibroblasts (unpublished data), whereas delayed treatment with HGF reduces experimental liver cirrhosis and renal sclerosis, concomitantly with a remarked decrease in myofibroblast-deposited areas. Our study provides a clue as to how regression vs. progression of tissue fibrosis is molecularly regulated and how to deal with chronic organ failures.

FOOTNOTES

¹ Present address: Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Dongdan Santiao 5, Beijing 100005, China.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1535fje;

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This Article Full Text (PDF) All Versions of this Article: 19/6/580 04-1535fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mizuno, S. Articles by Nakamura, T. Search for Related Content PubMed PubMed Citation Articles by Mizuno, S. Articles by Nakamura, T.