Idiopathic pulmonary fibrosis: immunohistochemical analysis provides fresh insights into lung tissue remodelling with implications for novel prognostic markers.

AIM: This study explored the cellular and biological interrelationships involved in Idiopathic Pulmonary Fibrosis (IPF) lung tissue remodelling using immunohistochemical analysis. METHODS AND RESULTS: IPF and control lung tissues were examined for localisation of Epithelial Mesenchymal Transition (EMT), proliferation and growth factor markers assessing their relationship to key histological aberrations. E-cadherin was expressed in IPF and control (Alveolar type II) ATII cells (>75%). In IPF, mean expression of N-cadherin was scanty (<10%): however 4 cases demonstrated augmented expression in ATII cells correlating to histological disease status (Pearson correlation score 0.557). Twist was expressed within fibroblastic foci but not in ATII cells. Transforming Growth Factor- ß (TGF-ß) protein expression was significantly increased in IPF ATII cells with variable expression within fibroblastic foci. Antigen Ki-67 was observed within hyperplastic ATII cells but not in cells overlying foci. Collagen I and α-smooth muscle actin (α-SMA) were strongly expressed within fibroblastic foci (>75%); cytoplasmic collagen I in ATII cells was present in 3 IPF cases. IPF ATII cells demonstrated variable Surfactant Protein-C (SP-C). CONCLUSIONS: The pathogenesis of IPF is complex and involves multiple factors, possibly including EMT. Histological analysis suggests TGF-ß-stimulated myofib rob lasts initiate a contractile response within established fibroblastic foci while proliferating ATII cells attempt to instigate alveolar epithelium repair. Marker expression (N-cadherin and Ki-67) correlation with histological disease activity (as reflected by fibroblastic foci extent) may emerge as future prognostic indicators for IPF.

Simvastatin inhibits growth factor expression and modulates profibrogenic markers in lung fibroblasts.

Simvastatin is best known for its antilipidemic action and use in cardiovascular disease due to its inhibition of 3-hydroxy-3-methylglutaryl CoenzymeA (HMG CoA) reductase, a key enzyme in the cholesterol synthesis pathway. Inhibition of biological precursors in this pathway also enables pleiotrophic immunomodulatory and anti-inflammatory capabilities, including modulation of growth factor expression. Connective tissue growth factor (CTGF) and persistent myofibroblast formation are major determinants of the aggressive fibrotic disease, idiopathic pulmonary fibrosis (IPF). In this study we used human lung fibroblasts derived from healthy and IPF lungs to examine Simvastatin effects on CTGF gene and protein expression, analyzed by RT-PCR and ELISA, respectively. Simvastatin significantly inhibited (P < 0.05) CTGF gene and protein expression, overriding the induction by transforming growth factor-beta1, a known potent inducer of CTGF. Such Simvastatin suppressor action on growth factor interaction was reflected functionally on recognized phenotypes of fibrosis. alpha-smooth muscle actin expression was downregulated and collagen gel contraction reduced by 4.94- and 7.58-fold in IMR90 and HIPF lung fibroblasts, respectively, when preconditioned with 10 microM Simvastatin compared with transforming growth factor-beta1 treatment alone after 24 h. Our data suggest that Simvastatin can modify critical determinants of the profibrogenic machinery responsible for the aggressive clinical profile of IPF, and potentially prevents adverse lung parenchymal remodeling associated with persistent myofibroblast formation.

Connective tissue growth factor expression and induction by transforming growth factor-beta is abrogated by simvastatin via a Rho signaling mechanism.

Connective tissue growth factor (CTGF), a potent profibrotic mediator, acts downstream and in concert with transforming growth factor (TGF)-beta to drive fibrogenesis. Significant upregulation of CTGF has been reported in fibrogenic diseases, including idiopathic pulmonary fibrosis (IPF), and is partly responsible for associated excessive fibroblast proliferation and extracellular matrix deposition, but no effective therapy exists for averting such fibrogeneic events. Simvastatin has reported putative antifibrotic actions in renal fibroblasts; this study explores such actions on human IPF-derived and normal lung fibroblasts and examines associated driving mechanisms. Simvastatin reduces basal CTGF gene and protein expression in all fibroblast lines, overriding TGF-beta induction through inhibition of the cholesterol synthesis pathway. Signaling pathways driving simvastatin's effects on CTGF/TGF-beta interaction were evaluated using transient reporter transfection of a CTGF promoter construct. Inhibition of CTGF promoter activity by simvastatin was most marked at 10 muM concentration, reducing activity by 76.2 and 51.8% over TGF-beta-stimulated cultures in IPF and normal fibroblasts, respectively. We also show that geranylgeranylpyrophosphate (GGPP), but not farnesylpyrophosphate, induces CTGF promoter activity following simvastatin inhibition by 55.3 and 31.1% over GGPP-negative cultures in IMR90 and IPF-derived fibroblasts, respectively, implicating small GTPase Rho involvement rather than Ras in these effects. Indeed, the specific Rho inhibitor C3 exotoxin significantly (P < 0.05) suppressed TGF-beta-induced CTGF promoter activity in transfected lung fibroblasts, a finding further supported by transfection of dominant-negative and constitutively active RhoA constructs, thus demonstrating that simvastatin through a Rho signaling mechanism in lung fibroblasts can modulate CTGF expression and interaction with TGF-beta.