Session Type: Abstract Submissions (ACR)
Background/Purpose: Fibroproliferative disorders such as systemic sclerosis (SSc) have no effective therapies and result in significant morbidity and mortality as a result of organ fibrosis. We recently demonstrated that the C-terminal domain of endostatin known as E4, corresponding to amino acid sequence 133-180, is a promising therapeutic agent for fibrotic disorders. E4 prevented and reversed both dermal and pulmonary fibrosis. Our goal was to delineate the mechanism by which E4 abrogates fibrosis. Preliminary gene expression data from primary human fibroblasts suggested that E4 treatment resulted in increased expression of urokinase (uPA).
Methods: Bleomycin (60 µg/mice) or Bleomycin in combination with E4 (10 ug/ml) was administered intratracheally to 6 to 8-week-old C57BL/6J male mice to induce lung fibrosis. Bronchoalveolar lavage (BAL) fluid was collected on days 3, 5, 7, and 14 post treatment, and the levels and activity of uPA and plasminogen activator inhibitor-1 (PAI-1), an inhibitor of uPA, were measured. Primary human fibroblasts were treated with TGF-b (10 ng/ml) to induce a fibrotic phenotype or TGF-b in combination with E4 (10 ug/ml). The mRNA and protein levels of uPA, PAI-1, matrix metalloproteinase-1 (MMP-1), and MMP-3 were determined using real-time PCR and immunoblotting, respectively. Secreted uPA activity was also measured in fibroblast supernatants. Since MMP-1 and MMP-3 are downstream effectors of uPA, we assessed MMP-1 and -3 activity using collagen and casein zymography, respectively. The mRNA levels of uPA and PAI-1 in human whole lung tissue and lung fibroblasts from 9 healthy control (HC) and 32 SSc patients were examined using real-time PCR.
Results: In vivo, bleomycin reduced uPA levels and activity and increased PAI-1 activity. E4 peptide partially blocked these effects. The reduction of PAI-1 caused by E4 administration preceded the increase in uPA activity, suggesting that a release from inhibition may explain in part the increase in uPA activity. In vitro, TGF-b reduced uPA levels and increased PAI-1 levels in primary fibroblasts. E4 peptide cancelled these effects and increased the uPA/PAI-1 ratio. Moreover, the expression and activity of MMP-1 and MMP-3 were increased by E4 treatment. The mRNA levels of uPA both in whole lung tissue and lung fibroblasts were comparable between HC and SSc patients, however, those of PAI-1 were increased in SSc patients, resulting in a decrease of the uPA/PAI-1 ratio.
Conclusion: Our results demonstrated that E4 increases uPA activity by both increasing uPA levels and activity and reducing PAI-1-mediated inhibition. The ability of E4 to reverse fibrosis can be explained by its ability to induce MMP-1 and MMP-3 levels and activity, thus promoting extracellular matrix degradation. In SSc patients, the uPA/PAI-1 balance shifted toward PAI-1. Taken together, our findings suggest that the anti-fibrotic effects of E4 peptide are mediated, at least in part, by the uPA fibrinolytic system, and that E4 peptide exerts its therapeutic effects in organ fibrosis via regulation of the urokinase pathway.
« Back to 2014 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-anti-fibrotic-effect-of-endostatin-derived-peptide-is-mediated-by-urokinase/