Background/Purpose:

Fibrotic disorders such as systemic sclerosis (SSc) result in end-stage organ failure and loss of function, consequently causing high morbidity and mortality. We recently demonstrated that the C-terminal domain of endostatin, known as E4, prevented and reversed both dermal and pulmonary fibrosis. In this study, we investigated the mechanism by which E4 exerts anti-fibrotic effects using pre-clinical models of SSc.

Methods:

To assess the effects of E4, lung fibroblasts were treated with TGF-β with vehicle or E4. The expression levels and activity of matrix metalloproteases (MMP)-1 and MMP-3 were evaluated by using real-time PCR, immunoblotting (IB), and collagen and casein zymography, respectively. Furthermore, the mRNA and protein levels of the pro-fibrotic proteins, connective tissue growth factor (CTGF) and insulin-like growth factor binding protein (IGFBP)-3, were measured using real-time PCR and IB, respectively. In vivo, bleomycin with vehicle or E4 was administered intratracheally to 6 to 8-week-old C57BL/6J male mice to induce lung fibrosis. Lung and bronchoalveolar lavage fluid (BALF) were collected 7 days post-treatment, and the levels and activity of hepatocyte growth factor (HGF) were measured using real-time PCR and immunoblotting.

Results:

The mRNA levels of MMP-1 and MMP-3 were increased by E4. Similarly, secreted MMP-1 and MMP-3 protein levels were upregulated in culture supernatants. Furthermore, MMP-1 and MMP-3 activity was increased as assessed by zymography. TGF-β increased the expression levels of CTGF and IGFBP-3 in primary human fibroblasts, and E4 abrogated these effects. In vivo, bleomycin reduced HGF levels in BALF and lung tissues. E4 peptide partially blocked these effects.

Conclusion:

Our results demonstrate that E4 increased MMP-1 and MMP-3 levels and activity. The ability of E4 to reverse fibrosis can thus be explained, in part, by its increase of MMP, thus promoting extracellular matrix (ECM) degradation. In addition, E4 reduced levels of the pro-fibrotic mediators CTGF and IGFBP-3 while increasing levels of the anti-fibrotic and anti-inflammatory factor HGF. Our findings suggest that the anti-fibrotic effects of E4 peptide are multi-pronged and include decreasing levels of essential pro-fibrotic mediators, increasing levels of an anti-fibrotic mediator, and promoting ECM degradation.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-fibrotic-mechanisms-of-endostatin-derived-peptide-are-the-result-of-reduction-in-pro-fibrotic-mediators-and-promotion-of-extracellular-matrix-degradation/