Cyclosporin A has anti‐fibrotic effects in human lung fibroblasts and in bleomycin‐induced lung fibrosis model mice

Abstract

In pulmonary fibrosis, normal tissues have been replaced by scar tissues, which results in tissue dysfunction. The scar tissues are mainly composed of myofibroblasts differentiated from fibroblasts. It is known that transforming growth factor‐β1 (TGFβ1) plays a central role in this mechanism. Myofibroblasts express α‐smooth muscle actin (αSMA) and extracellular matrix proteins such as collagens and fibronectin. Since the expressions of these proteins are up‐regulated in the lungs of pulmonary fibrosis patients, the proteins are widely used as markers of fibrosis.Pirfenidon and anti‐inflammatory drugs such as hydrocortisone and immunosuppressants (cyclosporin A (CsA), FK506, rapamycin, cyclophosphamide and azathioprine) are now used clinically for treatment of pulmonary fibrosis. However, the detailed effects of these drugs are still unclear because many types of cells are involve in this disease. Therefore we first investigated the effects of these drugs on αSMA expression in WI‐38 cells, a human lung fibroblasts. Interestingly, among the drugs noted above, only CsA (3–10 μM) significantly suppressed the expression of αSMA induced by TGFβ1. Like CsS, treatment with FK506 completely inhibited calcineurin activity in the lysates of WI‐38 cells, but FK506 had no effect on the αSMA expression. TGFβ1 activates at least 4 transcriptional factors; Smad, activating protein‐1, hypoxia inducible factor 1α (HIF1α) and nuclear factor of activated T‐cells to induce αSMA expression. CsA did not change phosphorylation state of Smad3, a main signaling molecule of TGFβ receptors, ERKs nor JNK when cells were treated with TGFβ1. Then, we next examined the effect of CsA on HIF1α pathway.HIF1α is usually degraded soon after the production in normoxia while it is stable in hypoxia. It is reported that TGFβ1 stabilizes HIF1α expression in normoxia in renal cells. In WI‐38 cells, TGFβ1 also stabilized HIF1α expression from 3 h. We also examined half‐life of HIF1α with or without 10 μM CsA.According to our results in vitro, it was expected that CsA would have anti‐fibrotic effect when it is delivered directly to lungs. To confirm this, 3 mg/kg bleomycin induced lung fibrosis model mice (C57BL/6) were administered 0.5 mg/kg CsA intratracheally on 7, 8, 9 days after bleomycin administration. The left lungs of each mice were obtained 14 days after bleomycin administration and examined the numbers of cells in bronco alveolar lavage fruid, αSMA and HIF1α expressions, TGFβ1 mRNA expressions, hematoxin‐eosin staining, masson tricrome staining and immunohistochemistry.Our results suggest that CsA has antifibrotic effects both in vitro and vivo when it is delivered directly to the lung cells especially lung fibroblasts. One of the novel mechanism of which is inhibiting HIF1α pathway activated by TGFβ1 in fibroblasts.Support or Funding InformationThis work is partly funded by Sasakawa Scientific Research Grant from The Japan Science Society.