Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

Emphysema is one of the most important cause of respiratory insufficiency with increasing mortality and morbidity [1]. Lung emphysema is defined pathologically by the destruction of alveolar walls with abnormal permanent enlargement of the air spaces distal to the terminal bronchiole. Precise mechanims contributing to the destruction of alveolar walls remain unclear. Cigarette smoking may induce emphysema by stimulating neutrophils and alveolar macrophages to produce proteases leading to the degradation of the alveolar extracellular matrix and oxidant injury contributing to alveolar destruction [2]. T lymphocytes contribute to the recruitment and activation of these inflammatory cells and may be involved in the apoptosis and destruction of alveolar epithelium [3‐5]. Recent evidence of increased apoptosis of alveolar epithelial and endothelial cells in emphysematous lung suggests that primary alterations of the alveolar epithelium and endothelium might participate in the pathogenesis of the disease [6‐8].

The alveolar epithelium is essential for maintenance of the integrity of the alveolar spaces. Functional restoration of the alveolar epithelium after an injury requires the proliferation and migration of type 2 pneumocytes and their differentiation into type 1 pneumocytes, a tightly regulated phenomenon. Growth factors have been shown to control both phases of the process [9‐12]. Among these factors, the hepatocyte growth factor (HGF), a heterodimeric protein obtained through the cleavage of an inactive precursor, called 90-kD proHGF, and the keratinocyte growth factor (KGF, also named fibroblast growth factor-7, FGF-7) have been identified as key factors in the process of alveolar repair, both in acute or chronic conditions [13‐15]. HGF and KGF respectively act through specific receptors c-met, a membrane bound tyrosine kinase [16] and FGFR2IIIb, also named KGF-R [17]. HGF and KGF productions by lung fibroblasts from emphysema have been shown to be reduced when compared with controls [18]. Recently, Shigemura et al reported that decreased HGF expression due to a failure in sustained endogenous production after injury was associated with emphysema-related histopathologic and physiological changes in a rat model of elastase-induced emphysema [19]. In this animal model, HGF could have a therapeutic effect [20, 19].

We hypothesized that a defective expression of growth factors involved in human alveolar epithelium repair such as HGF and KGF or their specific receptors might participate in the pathophysiology of lung emphysema. We therefore evaluated the expression of HGF, KGF, and their receptors c-met and KGF-R in lung biopsies from patients with emphysema and from non-emphysema patients, according to their smoking status.

This study was approved by the ethics committee of Saint Germain-en-Laye hospital.

The involvement of KGF and HGF in lung repair has been widely documented. Numerous studies in vitro and in vivo have demonstrated that KGF and HGF have protective effects in experimental lung injury [15, 23]. To our knowledge, this is the first study of KGF and HGF lung expression in human emphysema. Proteases, oxidant injury [2], chronic inflammation [3, 5] and apoptosis [6‐8] all contribute to the excessive alveolar wall destruction observed in lung emphysema. We hypothezised that a defect of the lung repair process might be associated with the pathophysiology of lung emphysema. Our results show that lung KGF mRNA and KGF protein are not altered in emphysema. In bleomycin-induced lung injury in rats, KGF and HGF increase in the lung [24]. A few clinical studies have assessed KGF concentrations in acute lung injury. Verghese et al reported that KGF was not increased in lung edema fluid whereas HGF was increased and associated with higher mortality [25]. Stern et al reported that KGF and HGF were increased in bronchoalveolar lavage fluid in acute respiratory distress syndrome and associated with a poor prognosis [13]. Recently, Danan et al observed the highest KGF concentrations in tracheal aspirates from premature infants who survived without bronchopulmonary dysplasia, leading to the conclusion that KGF may prevent injury to lung epithelium and enhanceits repair [26].

This study has some methodological limitations. A limited number of patients was studied in each group, especially in non emphysema groups which were mostly composed of lung biopsies obtained at a site distant from localised carcinoma. Furthermore, the patients could only be evaluated at one time point in the course of their disease. Inclusion of smokers without emphysema allowed the differentiation of emphysema-related and tobacco-related events. Because only one tissue sample from surgically resected material was available for examination, the expression of HGF, KGF and their receptors reflects regional disease activity and may be unrepresentative of the entire lung. Indeed, it is well known that emphysema affects different lung regions to a varying extent. Moreover, we evaluated HGF and KGF in lung homogenates only. Future studies should address the expression of HGF and KGF in a more cell-specific fashion.

In our study, although HGF mRNA lung expression was similar in emphysema and non emphysema patients, a correlation was found between HGF mRNA and the deterioration of pulmonary function tests in emphysema patients. The correlation between airflow obstruction and HGF mRNA level was similarly observed when all smokers with or without emphysema were studied, suggesting that emphysema was not a main determinant of HGF mRNA level in the lung. This strong correlation between airflow obstruction and HGF mRNA in smokers suggests that the increase of HGF mRNA was not related to the presence of emphysema but rather to the degree of airflow obstruction. This observation is supported by the correlation between HGF protein in lung homogenates and the FEV1/FVC ratio in our population. These results are in agreement with the observations of Sauleda et al, who reported that HGF protein concentrations were increased in broncho-alveolar lavage of patients with chronic obstructive pulmonary disease as compared to smokers and non-smoker controls [27]. Interestingly, the increased lung expression of other growth factors (fibroblast growth factors 1 and 2 and their receptors) has already been reported in chronic obstructive pulmonary disease [28].

The mechanisms underlying the correlation between airflow obstruction and HGF mRNA in smokers are unclear. Although speculative, we can propose that the mechanical constraints applied to alveolar tissue secondary to airflow obstruction may stimulate HGF production by alveolar epithelial cells, since Yamamoto et al showed that mechanical stretch induced HGF in alveolar type II cells in vitro [29]. Furthermore, airway inflammation could contribute to increase local HGF expression by neutrophils [22] and macrophages [30]. Interestingly, Aharinejad et al have shown that serum HGF concentrations increased at the time of lung graft rejection, a situation associated with airflow obstruction [31].

As HGF and KGF are key factors in the process of alveolar repair [15], we suggest that their production might be not adapted to the degree of alveolar injury. Indeed, in view of the chronic lung injury observed in emphysema, one could expect an increase of HGF and KGF expression as observed in acute lung injury in rats [24] and in humans [25, 13]. However the direct assessment of HGF content in lung homogenates is technically difficult. Indeed, HGF is a heparin binding growth factor. High concentrations of inactive precursor of HGF (proHGF) are probably bound to proteoglycans of the extracellular matrix and may not be assayed in the lung homogenates by immunoassay. In this study, western blot analysis showed that HGF was only in the mature active form, both in lung biopsies from emphysema and non-emphysema patients.

Recently, HGF has been shown to stimulate pulmonary regeneration and to improve pulmonary function in animal models of elastase-induced lung emphysema [20, 19]. Preserved expression of HGF-R could allow therapeutic use of growth factors in lung emphysema. Further studies are needed to assess the therapeutic potential of HGF and KGF in lung emphysema.

The main results of our study are that: i) KGF and HGF lung expression is preserved in emphysema patients, ii) HGF-R and KGF-R mRNA are consistently expressed in the lung of emphysema patients and are not modified by the smoking status, iii) HGF mRNA correlates with the severity of airflow obstruction in smokers.