Targeted Gene Transfer of Hepatocyte Growth Factor to Alveolar Type II Epithelial Cells Reduces Lung Fibrosis in Rats

Introduction

I diopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with a 50% survival of 2–4 years (Gharaee-Kermani and Phan, 2005). Effective treatment stabilizing or even improving the disease is still missing. Novel therapeutic strategies are therefore urgently needed. It was previously believed that chronic inflammation plays a major role in the development of IPF (Piguet et al., 1990; Gross and Hunninghake, 2001); it is now accepted that rather inefficient and abnormal alveolar wound repair after repeated epithelial injuries may dominate the development of pulmonary fibrosis (Selman et al., 2001; Geiser, 2003). Evidence supports this hypothesis, showing abnormal alveolar epithelial repair in patients with IPF (Selman et al., 2001; Thannickal et al., 2004), particularly in areas of fibroblastic foci, a histological hallmark in IPF. Novel therapeutic strategies therefore aim to support the alveolar repair process in the injured lung. It was previously shown that efficient alveolar epithelial repair may reduce the development of pulmonary fibrosis in the bleomycin-induced lung injury model (Adamson and Bakowska, 1999). Therefore supporting alveolar epithelial repair may represent a promising new strategy in patients with pulmonary fibrosis.

Various cellular and molecular mechanisms have been suggested to be critical in the pathogenesis of pulmonary fibrosis. Some evidence suggests the epithelial–mesenchymal transition (EMT) as an important mechanism that may contribute to the accumulation of fibroblasts and differentiation to myofibroblasts in pulmonary fibrosis (Willis et al., 2005). More recently, persistent activation of intraalveolar procoagulant activity and increased deposition of extravascular fibrin have been shown to increase the fibrotic response in the IPF lung (Antoniou et al., 2007).

Repeated injuries to alveolar type II epithelial cells (AECII) (Sisson et al., 2010) and their failure to reepithelialize is thought to be one of most important factors triggering the development of IPF. In the alveolar epithelial repair process many different cytokines and growth factors are involved. Hepatocyte growth factor (HGF) is a potent mitogenic factor in various organs including the lung (Tsao et al., 1993; Singh-Kaw et al., 1995). In the lung HGF plays an important role both in the developmental stage and in the adult lung; during fetal lung development HGF is implicated in the regulation of mesenchyme–epithelium interactions (Itakura et al., 1997). HGF is a pulmotrophic factor, as it stimulates proliferation of respiratory epithelium during postpneumonectomy compensatory lung growth (Sakamaki et al., 2002). Furthermore, HGF has antiapoptotic effects on epithelial cells (Bardelli et al., 1996) and induces proliferation of AECII. It is therefore a promising candidate to support the repairing capacities of AECII and to improve alveolar epithelial repair in the injured lung. We showed that electroporation-mediated gene transfer of HGF directly to the bleomycin-injured lung reduces pulmonary fibrosis (Gazdhar et al., 2007).

However, to increase efficiency of electroporation-mediated gene transfer and to minimize possible side effects of this treatment a more targeted technique of gene transfer is needed. We therefore developed a noninvasive gene transfer technique that allows us to specifically target AECII by using a surfactant protein C promoter system, and present data showing a marked reduction in pulmonary fibrosis in the bleomycin-induced lung fibrosis model after cell-specific hHGF gene transfer. Moreover, we present possible mechanisms that may contribute to the beneficial effect induced by cell-specific HGF gene transfer to AECII in this model.

Materials and Methods

Results

hHGF is specifically expressed in alveolar type II epithelial cells after electroporation-mediated hHGF gene transfer in vivo

Extracorporeal electroporation-mediated hHGF gene transfer was performed 7 days after intratracheal bleomycin instillation, using pSpC-hHGF. In situ hybridization and immunohistochemistry performed 7 days after electroporation-mediated hHGF gene transfer showed expression of hHGF at the RNA and protein levels. Both mRNA (Fig. 1a, inset: sense probe) and hHGF protein expression (Fig. 1b) were detected and specifically expressed in AECII as shown by costaining experiments performing surfactant C staining (Fig. 1c–e). In accordance with the stereological data (see below) and as shown in Fig. 1e, not all AECII were transfected with hHGF.

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FIG. 1.

hHGF mRNA and protein expression in bleomycin-injured rat lung after electroporation-mediated cell-specific gene transfer of hHGF (pSpC-hHGF). (a) In situ hybridization. Inset: Negative control. (b) Immunohistochemistry, showing positivity specifically for alveolar epithelial type II cells. To confirm alveolar epithelial cell-specific targeting, double immunofluorescence was performed. Distinct cells coexpressing hHGF and SpC were detected: the green channel (c) shows SpC-positive cells, and the red channel (d) shows hHGF-positive cells. There were SpC- and HGF-positive cells (yellow arrows) and other Sp-C positive cells (green arrow). There were other SpC-positive cells (green) that did not express hHGF (merged image) (e). Arrows indicate positive cells. Objective magnification, ×40.

hHGF levels in lung homogenates from pSpC-hHGF- and pSpC-treated rats were measured to quantify hHGF expression in bleomycin-injured lung. In pSpC-hHGF-treated rat lungs, the mean hHGF level was 0.33±0.24 pg/mg of total lung protein, whereas no hHGF could be detected in rats treated with the empty vector (pSpC) 7 days after electroporation-mediated hHGF gene transfer (p<0.01). Rat HGF levels were also increased in the treatment group (26.43±24.50 vs. 9.90±3.23 pg/mg of total lung protein in the control group) as measured by ELISA (mean±SD).

Cell-specific hHGF gene expression in alveolar type II epithelial cells reduces lung fibrosis

Targeted cell-specific hHGF gene transfer markedly reduced lung fibrosis compared with the control group as assessed 14 days after bleomycin instillation and 7 days after hHGF gene transfer. Whereas severe inflammation and fibrotic changes were detected in the lungs of control animals (Fig. 2a), considerable improvement could be observed in hHGF-treated animals (Fig. 2b). The fibrotic changes in the lung were further quantified using the Ashcroft score. Cell-specific gene transfer of hHGF reduced the score from 3.8±0.7 in the control group to 2.2±1.1 in the hHGF-treated group (p=0.02) (mean±SD) (Fig. 2c). In accordance, the collagen content, as assessed by hydroxyproline assay, was significantly reduced in the animals treated by cell-specific hHGF gene transfer compared with the control group (1663±1746 vs. 3829±1694 μg/gm of wet lung tissue; p=0.035) (mean±SD) (Fig. 2d).

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FIG. 2.

Electroporation-mediated cell-specific gene transfer of hHGF reduces bleomycin-induced pulmonary fibrosis. (a) Bleomycin-induced lung injury followed by electroporation-mediated cell-specific gene transfer of pSpC (empty vector, not containing the HGF sequence) 7 days after bleomycin (control). (b) Bleomycin-induced lung injury followed by electroporation-mediated cell-specific hHGF gene transfer (pSpC-hHGF) 7 days after bleomycin [(a) and (b) hematoxylin–eosin (H&E) staining; objective magnification, ×20]. (c) Assessment of lung fibrosis, using the Ashcroft score for grading pulmonary fibrosis after electroporation-mediated cell-specific hHGF gene transfer. n=6 for each group, *p<0.05. (d) Reduction of hydroxyproline content after electroporation-mediated gene transfer of hHGF (pSpC-hHGF) in bleomycin-injured lungs. Values represent means±SEM, n=6 for each group, *p<0.05. Color images available online at www.liebertpub.com/hum

Stereological analysis

General lung architecture

Stereological data are given in Supplementary Table S1. The volume fraction of septal wall tissue [V _V(sep/par)] as well as the mean thickness of alveolar septal walls [ \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\bar {\tau}$$\end{document} (sep)] demonstrated a significant reduction after hHGF gene transfer (Figs. 3A–D and 4A). The total volume of septal tissue per lung [V(sep,lung)] showed a tendency to decrease, whereas the total volumes of the alveoli [V(alv,lung)] and alveolar ducts [V(duct,lung)] were slightly increased after hHGF gene transfer. In addition, the total volume of destructed lung parenchyma [V(des,lung)] showed a trend toward reduction.

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FIG. 3.

Light (A and B) and electron micrographs (C and D) of bleomycin-injured lungs after HGF gene transfer (pSpC-hHGF) (B and D) and controls (pSpC) (A and C). Toluidine blue staining was performed for semithin sections (A and B). The alveolar septal walls were thinner in pSpC-hHGF-treated animals (B and D) than in control animals (pSpC) (A and C). Higher light microscopy magnification revealed in the control group the existence of fibroblastic foci-like structures (fibrobl) covered with cuboidal epithelium (arrowheads). These structures could not be observed after HGF gene transfer (pSpC-hHGF). At the electron microscopy level, the components of the alveolar septal walls can be recognized: the amount of interstitial tissue (ECM, extracellular matrix; IC, interstitial cells) was higher in controls (pSpC) (C) with abundant collagen fibrils (*). The blood–gas barrier between the alveolar space (alv) and capillary lumen (cap) includes the basal lamina (BL) underneath the alveolar epithelium (alvepi) and appeared to be thinner in animals after cell-specific HGF gene transfer (pSpC-hHGF) (D). In both electron micrographs the thin alveolar epithelium consists entirely of alveolar epithelial type I cells; alveolar epithelial type II cells were not visible.

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FIG. 4.

Detailed stereological data: Treatment by targeted cell-specific hHGF gene transfer led to a significant reduction in alveolar septal wall (A) as well as blood–gas barrier thickness (B). The number of alveolar epithelial type II cells (AECII) did not differ between these two groups 2 weeks after bleomycin administration (C), but hHGF gene transfer was associated with a reduced mean volume of AECII (D). Analysis of the composition of alveolar septa revealed a significant reduction of the amount of collagen fibrils within alveolar septal walls (E), and a trend toward lower amounts of total extracellular matrix (ECM) (F) and interstitial cells (G).

Parameters related to alveolar type II epithelial cells

Stereological data are summarized in Supplementary Table S2. Neither the total number of AECII per lung [N(typeII,lung)] (Fig. 4C) nor the total surface area of alveolar epithelium covered with AECII [S(typeII,lung)] differed between the two groups. However, the number-weighted mean volume of AECII [ \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\bar {\nu}$$\end{document} ν_N(typeII)] was significantly smaller in the lungs expressing hHGF (Fig. 4D). Analysis of randomly sampled sections immunohistochemically stained for hHGF demonstrated that 2.22% of the alveolar epithelium was covered with hHGF-positive AECII, corresponding to an average absolute surface area per lung of 64.38 cm². As all AECII together contributed 207.9 cm² to the total alveolar surface area an average of 30.5% of the surface area covered with AECII was covered with hHGF-positive AECII. Thus, cell-specific electroporation-mediated gene transfer results in relevant gene delivery targeted to AECII.

Parameters related to alveolar septal walls

Stereological data are summarized in Supplementary Table S3. The composition of the alveolar septa was analyzed to identify the septal wall components that are responsible for the reduction in thickness of the alveolar septa after cell-specific hHGF gene transfer (Fig. 4E–G). The total amount of collagen fibrils [V(coll,sep)] was significantly reduced within the alveolar septal walls after hHGF gene transfer (Fig. 4E). Regarding the amount of ECM [V(ECM,sep)] and interstitial cells [V(intcell,sep)], there was a trend toward reduction (Fig. 4F and G). Neither the total volume of the alveolar epithelium [V(alvepi,sep] nor the total volume of capillaries [V(cap,sep)] differed between the two groups. Taken together, the reduction in the thickness of the alveolar septal walls in the hHGF-expressing group was due to a reduction in the interstitial components, in particular the ECM and collagen fibrils. Furthermore, a statistically significant decrease in the blood–gas barrier thickness [ \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\bar {\tau}$$\end{document} (bg)] (p=0.028) was associated, showing an improvement in structural features important for efficient gas exchange after hHGF gene transfer (Fig. 4B).

hHGF reduces TGF-β₁ levels in the lungs

Because TGF-β₁ is a major profibrotic factor, we determined TGF-β₁ levels in the lung tissue homogenate after HGF gene transfer. TGF-β₁ levels were reduced in animals treated with pSpC-hHGF compared with the control animals (10.7±6.52 vs. 27.3±12.80 pg/mg of total lung protein; p<0.05) (mean±SD). Moreover, we found a clear correlation between hHGF and TGF-β₁ levels after hHGF gene transfer (p<0.01, correlation coefficient r=–0.98; linear regression R ²=0.81) (Fig. 5), indicating that reduced TGF-β₁ levels may contribute to the antifibrotic effect of HGF after cell-specific electroporation-mediated gene transfer.

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FIG. 5.

TGF-β₁ levels were elevated in the group treated with empty vector; however, in the hHGF-treated group the TGF-β₁ levels were reduced. A correlation analysis of TGF-β₁ and hHGF levels in the treated group clearly indicate that the level of TGF-β₁ was reduced when the HGF level was higher, showing a significant negative correlation (p<0.01, correlation coefficient r=–0.98; linear regression: R ²=0.81).

hHGF regulates EMT and increases alveolar epithelial cell proliferation

To study the effect of hHGF on the epithelial–mesenchymal transition in the bleomycin-injured lung, we stained for two typical markers indicating modulation of the EMT: α-smooth muscle actin (α-SMA) for (myo)fibroblasts and the adhesion protein E-cadherin for epithelial cells.

α-SMA was markedly decreased in pSpC-hHGF-treated animals (Fig. 6h), whereas highly positive signals were observed in the control group (Fig. 6g). E-cadherin immunoreactivity was localized mainly at the cell membrane in the control lungs (pSpC) (Fig. 6a; and Fig. 6c at higher magnification), whereas in the pSpC-hHGF-treated lungs E-cadherin-positive signals shifted from the cell membrane to the cytoplasm (Fig. 6b; and Fig. 6d at higher magnification). These results indicate that cell-specific hHGF gene transfer may modulate the EMT, resulting in a decrease in (myo)fibroblasts in the fibrotic lung tissue.

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FIG. 6.

E-cadherin was observed by immunostaining on the cell surface in control animals (pSpC) (a and c), whereas E-cadherin shifted to the cytoplasmic compartment after cell-specific hHGF gene transfer (pSpC-hHGF) (b and d). (c) and (d) present higher magnifications of (a) and (b), respectively, after digital zoom for better visualization. Cell proliferation was enhanced after hHGF gene transfer as shown by Ki67 staining (arrows) (f); few Ki67-positive cells were seen in the control animals (e). α-Smooth muscle actin (α-SMA; myofibroblast marker) immunoreactivity was markedly reduced after cell-specific HGF gene transfer (h), compared with controls (g). Objective magnification: (a and b) ×100; (c–f) ×40; (c and d), ×2 digital zoom for a clear view. Color images available online at www.liebertpub.com/hum

Furthermore, hHGF-treated lungs showed increased Ki67 positivity in AECII (Fig. 6f) compared with the control group (Fig. 6e), indicating that HGF induces alveolar epithelial cell proliferation in the bleomycin-injured lung.

hHGF induces fibrinolytic activity and reduces alveolar fibrin deposition

Because dysbalance in alveolar coagulation and fibrinolysis was shown to contribute to the development of pulmonary fibrosis, we studied the presence of fibrin in bleomycin-injured lungs after HGF gene transfer. Alveolar fibrin depositions were markedly reduced after hHGF gene transfer (Fig. 7b) compared with controls (Fig. 7a), based on MSB staining (arrows indicate extravascular fibrin). We further studied the expression of uPA and PAI-1 after HGF gene transfer as selected markers of alveolar coagulation and fibrinolysis. Analysis of mRNA expression of uPA by real-time RT-PCR revealed that uPA was increased after cell-specific hHGF gene transfer (p=0.02) (Fig. 8a). Moreover, the protein level of PAI-1 was markedly reduced after hHGF gene transfer compared with control (681.2±392.3 vs. 11,608±7502 pg/ml; p=0.002) (mean±SD) (Fig. 8b). Upregulation of uPA and downregulation of PAI-1 indicate a profibrinolytic environment in the injured lung after hHGF gene transfer that may contribute to the reduced accumulation of fibrin that was observed after HGF gene transfer.

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FIG. 7.

Extravascular and intercellular fibrin deposition was observed in the control group (a); fibrin deposition was seen in the interstitial space, in the thickened alveolar septa, and was found in the intercellular spaces. In contrast, the hHGF-treated group showed marked reduction in fibrin deposition in the same areas; furthermore, methyl blue staining of the collagen in the hHGF-treated group was also markedly reduced (b). Objective magnification, ×40. Color images available online at www.liebertpub.com/hum

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FIG. 8.

Urokinase plasminogen activator (uPA) mRNA levels were significantly increased after hHGF gene transfer as compared with the control group (pSpC) (*p=0.02) (a), whereas plasminogen activator inhibitor-1 (PAI-1) levels were markedly reduced after hHGF gene transfer compared with controls (*p=0.002, n=6) (b).

Discussion

We demonstrate that targeted cell-specific, electroporation-mediated gene transfer of hHGF specifically to AECII is an efficient nonviral gene transfer method in the bleomycin-induced lung injury and fibrosis model in the rat. Cell-specific hHGF gene transfer performed 7 days after bleomycin-induced lung injury resulted in considerable reduction of lung fibrosis. Stereological assessment showed a marked reduction in the thickness of the alveolar septal walls, mainly by reduction of collagen fibrils and ECM in the interstitial space. Cell-specific hHGF gene transfer induces downregulation of profibrotic TGF-β₁ modulation of the EMT and an increase in profibrinolytic activity by upregulation of uPA and marked downregulation of PAI-1 in the bleomycin-injured lung. These effects, among others, may significantly contribute to the antifibrotic effect observed after cell-specific hHGF gene transfer in the bleomycin-injured rat lung.

IPF is a fatal lung disease characterized by accumulation of excessive collagen, (myo)fibroblasts, and remodeling of extracellular matrix, leading to destruction of lung architecture and a decline in lung function (du Bois and Wells, 2001; Gross and Hunninghake, 2001). AECII are considered to be precursor cells of the alveolar epithelium and are characterized by their ability to rapidly proliferate and differentiate into AECI. In the IPF lung, however, AECII become hyperplastic and show evidence of abnormal epithelial repair, in particular in areas of fibroblast foci (Adamson et al., 1988, 1990). One report suggests that targeted injury of AECII induces fibrosis in the mouse model (Sisson et al., 2010). Hence inefficient restoration of the alveolar epithelium is crucial for the development of pulmonary fibrosis, and thus enhancement of alveolar epithelial repair after injury is a promising novel strategy to treat pulmonary fibrosis. Here, we aimed at overexpression of an epithelial growth factor, hHGF, targeting specifically the AECII. hHGF was expressed in AECII by means of a surfactant protein C promoter, in order to support alveolar epithelial repair after bleomycin-induced lung injury.

HGF is a potent mitogen and induces epithelial cell proliferation, motility, and morphogenesis and stimulates DNA synthesis in AECII (Shiratori et al., 1995; Day et al., 2002). Its therapeutic potential in the bleomycin lung injury model has also been reported (Mason et al., 1994; Watanabe et al., 2005; Gazdhar et al., 2007). However, the short half-life of the HGF protein diminishes its potential as a future treatment. Gene therapy is a promising novel approach for lung disease, but the lack of safe and nontoxic, reproducible techniques has been a major problem. We therefore developed a novel, nonviral gene transfer technique using electroporation-mediated gene transfer in vivo.

Reports have demonstrated that the physical method of in vivo electroporation-mediated gene transfer to normal and injured lung is a promising and safe technique (Gazdhar et al., 2006, 2007). However, generalized expression of hHGF in several lung cell types (including, e.g., bronchial epithelial cells) is a matter of concern because malignant transformation may occur after long-term expression. We therefore developed a cell-specific gene transfer technique specifically targeted to AECII by putting hHGF under the control of the SpC promoter in order to locally support alveolar epithelial repair induced by AECII and to avoid unwanted transgene expression in other cell types in the lung. In our previous study we have shown that electroporation-mediated gene transfer of hHGF attenuated bleomycin-induced lung fibrosis (Gazdhar et al., 2007). The procedure performed in that study, however, had some technical drawbacks due to the invasive surgical procedures involved (thoracotomy), which would make a future clinical translation difficult. Hence in this present study a modified noninvasive protocol involving placement of electrodes over the chest wall with increased number of pulses was applied, as reported (Machado-Aranda et al., 2005). By using this modified protocol we were able to achieve relevant hHGF transgene expression specifically in AECII. With this approach we were able to achieve the desired cell-specific gene transfer because HGF was detected only in the SpC-positive AECII and not in any other cell types in the lung. Moreover, it has been shown that targeted cell-specific gene transfer of hHGF in the healthy lungs of rats was not associated with any side effects, although alteration of the lung structure at the ultrastructural level were found; in particular, neither pulmonary emphysema nor signs of malignant transformation were found (Leuenberger et al., 2012).

Stereological analysis revealed that 30% of the alveolar surface area covered with AECII stained positive for HGF, asserting the efficiency of extracorporeal electroporation-mediated cell-specific gene transfer. After HGF gene transfer to AECII we observed significant and biologically relevant levels of hHGF in the rat lung tissue homogenate. Although the levels of hHGF were low, we observed reduced collagen content improving histological and stereological parameters of the bleomycin-injured lung, assuming that relatively low levels of hHGF expressed in the local microenvironement of an injured alveolus may be sufficient to induce repair processes resulting in the antifibrotic effects observed.

Under the influence of HGF the volume of destructed lung parenchyma and septal wall tissue as well as its mean thickness were reduced, demonstrating that HGF leads to reduction of fibrosis. Furthermore, TGF-β₁ levels were markedly reduced after HGF treatment, with a significant negative correlation between hHGF and TGF-β₁ levels. TGF-β₁ is a multipotent profibrotic cytokine, regulating tissue morphogenesis and extracellular matrix production. It is involved in induction of fibrosis in various organs including the lung (Sime and O'Reilly, 2001). Elevated levels of TGF-β₁ have been reported in the lungs of patients with IPF (Bergeron et al., 2003). TGF-β₁ acts as a major switch in tissue repair and remodeling and also has been implicated to play an important role in the epithelial–mesenchymal transition (EMT) (Zavadil and Bottinger, 2005). Our data indicate that the antifibrotic effect of electroporation-mediated HGF gene transfer may be at least in part related to a TGF-β₁-dependent mechanism.

However, the beneficial effect of cell-specific HGF gene transfer on pulmonary fibrosis may be the result of several cellular and molecular mechanisms. The reduction of profibrotic TGF-β₁ after HGF gene transfer may result in the modification of antifibrotic processes such as EMT or upregulation of fibrinolytic activities as shown in our model. EMT is a complex process in which fully differentiated and functional epithelial cells transform into a mesenchymal phenotype (Kasai et al., 2005). A new emphasis has been put on the alveolar epithelium as a major site of EMT in the progress of pulmonary fibrosis; also, there is evidence indicating the epithelial origin of fibroblasts in lung fibrosis models (Adamson et al., 1988; Willis et al., 2006). TGF-β₁ induces EMT by modulating E-cadherin expression leading to destabilization of structural integrity of the pulmonary epithelium. E-cadherin is an epithelial marker that plays an important role in maintaining cell–cell integrity of pulmonary epithelium and its polarization (Takeichi, 1991); loss of or downregulation of E-cadherin leads to destabilization of structural integrity of the pulmonary epithelium. TGF-β₁ decreases E-cadherin expression, hence inducing EMT.

In this study we demonstrate that E-cadherin expression was observed on the epithelial membrane of AECII in untreated lungs. However, after HGF gene transfer we observed a cytosolic shift of E-cadherin from the cell membrane to the cytoplasm, as reported previously (Kim et al., 2007), resulting in reduced cell–cell adhesion and increased cell proliferation as evident by increased Ki67-positive AECII. Similar internalization of E-cadherin in response to HGF has also been demonstrated (Howard et al., 2011). Furthermore, α-SMA expression was markedly reduced after HGF gene transfer. We therefore assume that HGF is inducing epithelial cell proliferation, but preventing EMT as evidenced by reduced α-SMA-positive cells in the interstitial space (Shibamoto et al., 1994; Grotegut et al., 2006). These in vivo data are in accordance with an in vitro study using primary murine AECII (Shukla et al., 2009), in which HGF was shown to inhibit TGF-β₁-induced generation of the (myo)fibroblast phenotype, indicating that HGF modulates TGF-β₁-induced EMT in the lung.

HGF also contributes to angiogenesis (Hoot et al., 2010) and is known to induce VEGF expression in some cell types (Gille et al., 1998). VEGF is a potent angiogenic factor and is involved in lung development and physiology (Voelkel et al., 2006). The role of VEGF in pulmonary fibrosis, however, is controversial (Medford, 2005); studies have revealed that inhibition of VEGF or VEGF receptors attenuates bleomycin-induced lung fibrosis (Hamada et al., 2005; Chaudhary et al., 2007). In our study we did not see any significant increase in VEGF levels as measured in total lung homogenate. These data are in accordance with a clinical study in which VEGF levels were measured in the bronchoalveolar lavage of patients with IPF and did not differ between patients with IPF and control subjects (Simler et al., 2004). In addition, stereological analysis did not reveal a relevant increase in the total surface area of capillary endothelium within septal wall tissue after electroporation-mediated HGF gene transfer, indicating that relevant angiogenesis did not occur after HGF overexpression in our model.

In the course of fibrotic lung disease a dysbalance of coagulation and fibrinolysis was observed (Chambers, 2003, 2008). Upregulation of procoagulatory mediators and simultaneous downregulation of fibrinolytic factors result in a procoagulatory state leading to accumulation of fibrinous matrix in the injured alveoli. PAI-1, for instance, has been identified as a key regulator of pulmonary fibrosis as overexpression of PAI-1 leads to augmentation of fibrosis in the bleomycin model, whereas absence of PAI-1 was protective in the same model (Eitzman et al., 1996). We therefore studied the expression of fibrinolytic mediator uPA and its inhibitor, PAI-1. We show that cell-specific HGF gene transfer resulted in upregulation of uPA and downregulation of PAI-1, supporting a fibrinolytic microenvironement in the alveolar space after HGF gene transfer (Gong et al., 2003). Therefore, our data suggest that the antifibrotic effect of HGF in the bleomycin-induced lung injury model relates to modification of the balance between coagulation and fibrinolysis in the injured lung.

In conclusion, we provide a promising new cell-targeted gene transfer technique resulting in efficient hHGF gene transfer specifically to AECII, resulting in a reduction of bleomycin-induced lung injury and fibrosis in the rat model. Although gene therapy to the human lung is still far from clinical application, electroporation-mediated, targeted gene transfer to the lung or other organs may represent an efficient and safe, nonviral gene transfer technique that needs to be further developed by technical modifications and improvements for future clinical application.