Background/Purpose: Fibroproliferative disorders such as systemic sclerosis (SSc) have no effective therapies and result in significant morbidity and mortality. We recently demonstrated that the C-terminal domain of endostatin known as E4 prevented and reversed both dermal and pulmonary fibrosis. Our goal was to identify the E4 receptor and the mechanism by which E4 abrogates fibrosis.

Methods: To identify E4 binding, we conducted a pull down assay using biotinylated-E4 and neutravidin-beads. Proteins that bound E4 in fibroblasts were identified by mass spectrometry. Binding was confirmed using immunoblotting. To assess the mechanism by which E4 exerts anti-fibrotic effects in vitro, lung fibroblasts were treated with TGF-b with vehicle or E4. The expression levels and activity of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), an inhibitor of uPA, were evaluated by immunoblotting and activity assays, respectively. Since MMP-1 and MMP-3 are downstream effectors of plasminogen activation, we assessed MMP-1 and -3 activity using collagen and casein zymography, respectively. In vivo, bleomycin with vehicle or E4 was administered intratracheally to C57BL/6J male mice to induce lung fibrosis. Bronchoalveolar lavage fluid (BALF) was collected on days 3, 5, 7, and 14 post treatment, and the levels and activity of uPA and PAI-1 were measured. Furthermore, the mRNA levels of uPA and PAI-1 in human whole lung tissue and primary lung fibroblasts from 9 healthy controls (HC) and 32 SSc patients were measured using real-time PCR.

Results: Proteins bound to biotinylated-E4 were identified as enolase-1, known as a plasminogen receptor, and urokinase plasminogen activator receptor (uPAR).  TGF-b reduced uPA levels and increased PAI-1 levels in primary fibroblasts. E4 abrogated these effects and increased the uPA/PAI-1 ratio. Moreover, E4 increased MMP-1 and MMP-3 expression and activity. Knockdown of uPAR abolished the anti-fibrotic activity of E4. Plasminogen exerted anti-fibrotic activity similarly to E4 treatment. In vivo, bleomycin reduced uPA levels and activity, and increased PAI-1 levels and activity in BALF. E4 reduction of PAI-1 preceded the increase in uPA activity, suggesting that a release from inhibition may explain in part the increase in uPA activity. Finally, the uPA:PAI-1 ratio was decreased in both SSc patient lung tissues and fibroblasts, compared to HC. The lowest uPA:PAI-1 ratio was detected in SSc patients with pulmonary fibrosis.

Conclusion: Our findings show that E4 binds enolase-1 and uPAR, suggesting that E4 bridges enolase-1 and uPAR, likely to facilitate activation of enolase-bound plasminogen by uPAR-bound uPA. The anti-fibrotic effect of E4 is mediated, in part, by its increase uPA and activation of plasminogen. Further, E4 induces 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 E4 exerts its anti-fibrotic effects via regulation of the urokinase pathway. Thus, E4 is a promising therapeutic agent for SSc.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-anti-fibrotic-effect-of-endostatin-derived-peptide-is-mediated-by-the-urokinase-pathway-via-binding-to-enolase-1-and-urokinase-plasminogen-activator-receptor/