To the Editors:
Protease-activated receptor (PAR)-4 is a thrombin receptor expressed in lung tissue. Previous in vitro data showed that PAR-4 triggers epithelial-to-mesenchymal transition, which is crucial in pulmonary fibrosis. These observations prompted us to investigate the role of PAR-4 in a murine model of bleomycin induced pulmonary fibrosis. We observed that wildtype and PAR-4 deficient mice developed fibrosis, but there were no differences in the severity of fibrosis or in the expression of fibrotic markers like collagen and fibronectin in PAR-4 deficient mice compared with wildtype controls. Overall, our results demonstrate that PAR-4 does not play a significant role during pulmonary fibrosis and question the importance of PAR-4 in fibrotic disease.
PAR-4 is a G-coupled receptor of the PAR family that is generally recognised as a thrombin receptor [1] although it can also be activated by trypsin, tissue kallikrein and cathepsin G. PAR-4 activation is best known to induce platelet aggregation, but more recent studies suggest that PAR-4 may also target other cell types thereby contributing to (patho)physiology. Indeed, thrombin stimulation of epithelial cells induces epithelial–mesenchymal transition (EMT), as evident from changes in cell morphology and expression levels of epithelial (E-cadherin) and myofibroblast (α-smooth muscle actin) markers [2]. Moreover, PAR-4 activation stimulates interleukin (IL)-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. Considering the importance of both EMT and inflammation for fibrosis, it is thus tempting to speculate that PAR-4 would play a pro-fibrotic role and indeed several recent reviews [3, 4] specifically state that some of the pro-fibrotic effects of thrombin may be mediated through PAR-4. To prove or refute the importance of PAR-4 in fibrotic disease, PAR-4 deficient mice and wildtype controls were subjected to the well-established experimental model of pulmonary fibrosis [5]. To this end, mice were intratracheally inoculated with bleomycin sulfate (1 mg·kg−1 body weight in 45 μL of saline). 14 days later, mice were sacrificed and lungs were removed to analyse fibrotic changes. As shown in figure 1a, the severity of fibrotic changes as indicated by the Ashcroft score was similar in wildtype and PAR-4 deficient animals (score of mean±sem 3.46±0.25 and 2.48±0.50, respectively, p=0.13). In line, collagen deposition as assessed using Masson's trichrome stainings was also similar in wildtype and PAR-4 deficient mice (mean±sem arbitrary units of 2.2±0.9 and 2.0±0.8, respectively, p=0.88; fig. 1b). Finally, we determined pro-fibrotic gene expression in the lungs of bleomycin treated mice and, as shown in figure 1c, no differences in fibronectin, collagen 1 and collagen 3 levels between wildtype and PAR-4 deficient mice were observed. Overall these data thus indicate that PAR-4 does not play a significant role in the regulation of pulmonary fibrosis. Although extrapolation of our data to other fibrotic disorders should be performed with great care, our data argue against an important role of PAR-4 in fibrotic disease and once more stress that in vitro data should not be over interpreted by claiming potential roles in vivo.
Footnotes
Statement of Interest
None declared.
- ©ERS 2012