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Respiratory distress and bronchopulmonary dysplasia (BPD) are major problems in preterm infants that are often addressed by glucocorticoid treatment and increased oxygen supply, causing catabolic and injurious side effects. Recombinant human keratinocyte growth factor (rhKGF) is noncatabolic and antiapoptotic and increases surfactant pools in immature lungs. Despite its usefulness in injured neonatal lungs, the mechanisms of improved surfactant homeostasis in vivo and systemic effects on lipid homeostasis are unknown. We therefore exposed newborn rats to 85% vs. 21% oxygen and treated them systemically with rhKGF for 48 h before death at 7 days. We determined type II pneumocyte (PN-II) proliferation, surfactant protein (SP) mRNA expression, and the pulmonary metabolism of individual phosphatidylcholine (PC) species using [D(9)-methyl]choline and tandem mass spectrometry. In addition, we assessed liver and plasma lipid metabolism, addressing PC synthesis de novo, the liver-specific phosphatidylethanolamine methyl transferase (PEMT) pathway, and triglyceride concentrations. rhKGF was found to maintain PN-II proliferation and increased SP-B/C expression and surfactant PC in both normoxic and hyperoxic lungs. We found increased total PC together with decreased [D(9)-methyl]choline enrichment, suggesting decreased turnover rather than increased secretion and synthesis as the underlying mechanism. In the liver, rhKGF increased PC synthesis, both de novo and via PEMT, underlining the organotypic differences of rhKGF actions on lipid metabolism. rhKGF increased the hepatic secretion of newly synthesized polyunsaturated PC, indicating improved systemic supply with choline and essential fatty acids. We suggest that rhKGF has potential as a therapeutic agent in neonates by improving pulmonary and systemic PC homeostasis.

Surfactant deficiency and bronchopulmonary dysplasia (BPD), major obstacles in preterm infants, are addressed with pre- and postnatal glucocorticoids which also evoke harmful catabolic side-effects. Keratinocyte growth factor (KGF) accelerates surfactant production in fetal type II pneumocytes (PN-II), protects epithelia from injury and is deficient in lungs developing BPD, highlighting its potential efficacy in neonates. Neonatal rats were treated with recombinant human (rh)KGF, betamethasone, or their combination for 48 hr prior to sacrifice after which body weight, surfactant, and tissue phosphatidylcholines (PC) were investigated at postnatal d3, d7, d15, and d21. Pneumocyte proliferation, surfactant protein (SP) expression and SP-B/C in lung lavage fluid (LLF) were also determined at d7 and d21 to identify broader surfactant changes occurring at the beginning and end of the initial alveolarization phase. While all treatments increased secreted surfactant PC, BM compromised animal growth whereas rhKGF did not. At d3 rhKGF was more effective in male compared to female rats. Single treatments became less effective towards d21. Neither treatment altered PC composition in LLF. BM inhibited PN-II proliferation and increased surfactant PCs at the expense of tissue PCs. rhKGF however increased surfactant PCs without decreasing other PC species. Whereas SP-B/C gene expression was induced by all treatments, the changes in secreted SP-B/C mirrored those observed for surfactant PC. Our results encourage investigation of the mechanisms by which rhKGF improves surfactant homoeostasis, and detailed examination of its efficacy in neonatal lung injury models with a view to implementing it as a non-catabolic surfactant-increasing therapeutic in neonatal intensive care., (Copyright © 2011 Wiley-Liss, Inc.)

Rationale: Emphysema is characterized by destruction of alveoli with ensuing airspace enlargement and loss of alveoli. Induction of alveolar regeneration is still a major challenge in emphysema therapy., Objectives: To investigate whether therapeutic application of palifermin (DeltaN23-KGF) is able to induce a regenerative response in distal lung parenchyma after induction of pulmonary emphysema., Methods: Mice were therapeutically treated at three occasions by oropharyngeal aspiration of 10 mg DeltaN23-KGF per kg body weight after induction of emphysema by porcine pancreatic elastase., Measurements and Main Results: Airflow limitation associated with emphysema was largely reversed as assessed by noninvasive head-out body plethysmography. Porcine pancreatic elastase-induced airspace enlargement and loss of alveoli were partially reversed as assessed by design-based stereology. DeltaN23-KGF induced proliferation of epithelium, endothelium, and fibroblasts being associated with enhanced differentiation as well as increased expression of vascular endothelial growth factor, vascular endothelial growth factor receptors, transforming growth factor (TGF)-beta1, TGF-beta2, (phospho-) Smad2, plasminogen activator inhibitor-1, and elastin as assessed by quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. DeltaN23-KGF induced the expression of TGF-beta1 in and release of active TGF-beta1 from primary mouse alveolar epithelial type 2 (AE2) cells, murine AE2-like cells LA-4, and cocultures of LA-4 and murine lung fibroblasts (MLF), but not in MLF cultured alone. Recombinant TGF-beta1 but not DeltaN23-KGF induced elastin gene expression in MLF. Blockade of TGF-signaling by neutralizing antibody abolished these effects of DeltaN23-KGF in LA-4/MLF cocultures., Conclusions: Our data demonstrate that therapeutic application of DeltaN23-KGF has the potential to induce alveolar maintenance programs in emphysematous lungs and suggest that the regenerative effect on interstitial tissue is linked to AE2 cell-derived TGF-beta1.

Purpose: Lung allografts are threatened by primary graft dysfunction, infections, and rejection. Novel therapies protecting pulmonary allografts are badly needed. Keratinocyte growth factor (KGF) protects the lung against a variety of injurious stimuli and exerts anti-inflammatory effects. The aim of the study was to test the potential of recombinant truncated KGF (DeltaN23-KGF, palifermin) to attenuate pulmonary allograft rejection., Materials and Methods: Intratracheal instillation of 5 mg/kg DeltaN23-KGF was performed twice in donor rats on days 3 and 2 before explantation of the lung. In control animals, an equivalent volume of vehicle was instilled. Left lungs were transplanted in the fully allogeneic Dark Agouti to Lewis rat strain combination and in the less stringent Fischer 344 to Wistar Kyoto combination. Allograft recipients were additionally treated with DeltaN23-KGF post-transplantation. Graft outcome, leukocytic infiltration, and major histocompatibility complex (MHC) class II antigen expression was analyzed., Results: In both rat strain combinations, DeltaN23-KGF treatment did not improve pulmonary allograft outcome. Graft infiltration by macrophages and T lymphocytes remained unchanged. In addition, we demonstrated that MHC class II antigens were more abundant in KGF-treated allografts compared to control-treated grafts, which probably results in an increased alloreactivity., Conclusion: In conclusion, intratracheal DeltaN23-KGF treatment is not effective to prevent acute pulmonary allograft rejection.

Airway epithelial cells are exposed to environmental toxicants that result in airway injury. Naphthalene (NA) causes site-selective damage to Clara cells in mouse distal airways. N-terminally truncated recombinant human keratinocyte growth factor (DeltaN23-KGF) protects against acute lung injury. The present study investigated whether or not DeltaN23-KGF protects against NA-induced acute Clara cell damage by measuring airway responses specifically and in order to identify underlying molecular mechanisms. Mice were treated with DeltaN23-KGF or PBS 33 h prior to injection of 200 mg.kg body weight(-1) NA. Lung function was analysed by head-out body plethysmography. Distal airways isolated by microdissection were assessed for cell permeability using ethidium homodimer-1. Immunohistochemistry of Clara cell-specific protein in conjunction with a physical dissector was used to quantify Clara cell numbers. RNA was isolated from frozen airways in order to analyse gene expression using quantitative RT-PCR. DeltaN23-KGF prevented NA-induced airflow limitation and Clara cell permeability, and resulted in twice as many Clara cells compared with PBS pre-treatment. DeltaN23-KGF-pre-treated mice exhibited increased expression of proliferating cell nuclear antigen mRNA. Cytochrome P(450) isoform 2F2, which converts NA into its toxic metabolite, was reduced by approximately 50%. The present results demonstrate that pre-treatment with N-terminally truncated recombinant human keratinocyte growth factor protects against naphthalene-induced injury. This suggests that N-terminally truncated recombinant human keratinocyte growth factor exerts its beneficial effect through a decrease in the expression of cytochrome P(450) isoform 2F2.

Primary graft dysfunction, characterised by intra-alveolar oedema, is a major obstacle in pulmonary transplantation. The present study evaluates the potential of keratinocyte growth factor (palmiferin; DeltaN23-KGF) for the prevention of oedema in lung transplants. Intratracheal instillation of 5 mg x kg(-1) DeltaN23-KGF was performed in Lewis rats on days 3 and 2 before explantation. Control animals obtained an equivalent volume of vehicle. Left lungs were isogeneically transplanted and the graft recipients were sacrificed 1 day later for stereological analysis of intra-alveolar oedema and bronchoalveolar lavage. The total protein and phospholipid content, as well as surfactant proteins, were measured. Surfactant activity was analysed with a pulsating bubble surfactometer. In grafts from control treated donors, the fraction of intra-alveolar oedema amounted to 3.4+/-1.1% of the total parenchymal volume. Treatment of donor lungs with DeltaN23-KGF reduced oedema to a fraction of 1.6+/-0.8%. In the lavage fluid of pulmonary grafts from DeltaN23-KGF-treated donors, the total protein content was decreased compared with vehicle-treated lung transplants, whereas phospholipids did not differ. The protein fraction contained increased amounts of surfactant protein-C after DeltaN23-KGF treatment and surfactant function was improved. Treatment of donor lungs with palifermin protects against intra-alveolar oedema formation upon transplantation. This effect appears to be mediated by an improved surfactant homeostasis.

Quantitative (immuno) transmission electron microscopy using design-based stereology was performed on specimens collected by means of systematic uniform random sampling of rat lungs, which were fixed by vascular perfusion to stabilize intra-alveolar surfactant in situ. This procedure ensures that the data recorded are representative of the whole organ. Ultrathin sections of specimens embedded at low temperature in Lowicryl HM20 were labeled by indirect immuno-gold staining for surfactant protein A. We observed that, 3 days after treatment of lungs in vivo with truncated keratinocyte growth factor (DeltaN23-KGF), a potent mitogen of alveolar epithelial type II cells, surfactant protein A associated with the tubular myelin fraction of intra-alveolar surfactant was increased by 47% in comparison with buffer-treated control lungs. Despite the marked type II cell hyperplasia, the relative amount of ultrastructural surfactant subtypes was not significantly affected. Because surfactant protein A reduces the sensitivity to inhibition of the biophysical activity of surfactant by exudating plasma proteins, we propose that pretreatment of lungs with DeltaN23-KGF ameliorates adverse effects observed in acute lung injury following, for example, ischemia and reperfusion., ((c) 2007 Wiley-Liss, Inc)

Administration of recombinant human keratinocyte growth factor (rHuKGF, Delta23N-KGF, palifermin) protects the lung against a variety of injurious stimuli. The exact mechanisms leading to lung protection are unknown. Alterations in the non-neuronal cholinergic system of the lung might be involved, as vital pulmonary functions are regulated by acetylcholine. Here, we investigated the effect of KGF on the expression of nicotinic acetylcholine receptor subunits alpha7, alpha9 and alpha10 in rat lungs. Adult rats were treated via intratracheal instillation with rHuKGF or with an equivalent volume of PBS. The expression of nicotinic acetylcholine receptor subunits was analyzed by real-time RT-PCR, immunoblotting and immunohistochemistry. Treatment with rHuKGF led to a decreased expression of nicotinic receptor subunit alpha7 in the total lung. In contrast, the expression of the receptor subunits alpha9 and alpha10 was up-regulated. In conclusion, nicotinic acetylcholine receptors are differentially regulated by KGF treatment in vivo, which might result in changes in the biological effects of acetylcholine.

Keratinocyte growth factor protects the lung against various injurious stimuli. The protective mechanisms, however, are not yet fully understood. The aim of this study is to determine the influence of keratinocyte growth factor on the pulmonary capacity to synthesize acetylcholine, a potent regulator of pulmonary functions which is potentially involved in lung damage. Rats were treated twice (days 1 and 2) intratracheally with keratinocyte growth factor and analyzed at day 4. The mRNA expression of choline acetyltransferase - the acetylcholine synthesizing enzyme - was analyzed by real-time RT-PCR in the lung and in isolated alveolar epithelial type II cells. Choline acetyltransferase protein was assessed by immunoblotting and immunohistochemistry. Finally, pulmonary acetylcholine content was assessed biochemically. Keratinocyte growth factor-treatment led to decreased levels of choline acetyltransferase mRNA in the lung and in isolated alveolar epithelial type II cells. Accordingly, pulmonary choline acetyltransferase protein levels were reduced and pulmonary acetylcholine content declined from 2.8 nmol (control) to 0.4 nmol acetylcholine per gram of wet weight. In conclusion, the present data show that the potent regulator of pulmonary functions, acetylcholine, is produced by the major pulmonary target cell of keratinocyte growth factor, that is alveolar epithelial type II cells. Acetylcholine synthesis is down-regulated by keratinocyte growth factor administration which might contribute to lung protection and to harmonize surfactant homeostasis under conditions of keratinocyte growth factor-induced alveolar epithelial type II cell hyperplasia.

Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor.