Abstract

Background: ECM1 depletion results in excessive latent TGF-β1 activation and lethal liver fibrosis in mice. In patients suffering from chronic liver diseases, ECM1 expression gradually decreases with increasing severity of fibrosis. We investigated the underlying mechanisms of how ECM1 contributes to tissue homeostasis in healthy livers and what changes occur during CLD progression.

Methods: RNAseq of WT and ECM1-KO murine livers was performed to detect ECM1-KO-related changes in gene expression. Functional assays were performed using immortalised and primary hepatic stellate cells, WT and ECM1-KO mice, and liver tissue from patients suffering from CLD.

Results: DESeq shows that expression of thrombospondins (TSPs), including TSP-1, matrix metalloproteinases, ADAMTS proteases, increased concomitantly with LTGF-β1 activation and fibrosis following ECM1-KO. In the LX-2 and phHSCs, ECM1 prevented TSP-1-, ADAMTS1-, MMP-2- and -9-mediated LTGF-β1 activation, as assessed by the concentration of active TGF-β in conditioned media through using the MFB-F11 TGF-β reporter cell line and the expression changes of hepatic fibrosis markers. IF staining for TGF-β1 LAP-D (R58) further demonstrated that protease-mediated LTGF-β1 activation is inhibited by the ECM1 in LX-2 HSCs. Co-IP analyses revealed that ECM1 interacts with all four of the tested LTGF-β1-activating proteases and, for MMP-2 and -9, binding occurred preferentially to their respective activated state. Next, in vitro interaction assays showed that ECM1 abrogates TSP-1- and ADAMTS1-mediated LTGF-β1 activation through an interaction with their respective KRFK or KTFR sequence. Further, ECM1 abolishes MMP-2/9-mediated LTGF-β1 activation through inhibiting their proteolytic activity. In mice, AAV-mediated overexpression of ECM1 in hepatocytes protected from KRFK-mediated LTGF-β1 activation and subsequent hepatic fibrosis. Importantly, KTFR injection was able to rescue the ECM1-KO phenotype and thus revert liver injury in mice affected by ECM1 depletion. We also found that in patients suffering from CLD, a loss ECM1 expression was accompanied by induction of TSP-1, ADAMTS1, MMP-2, and MMP-9, all of which mediate LTGF-β1 activation.

Conclusion: ECM1 exerts its hepatoprotective effect through an inhibition of protease-mediated excessive LTGF-β1 activation by interacting with activating motifs and directly reducing proteolytic activity. During progression of CLD, ECM1 expression decreases and therewith liver tissue homeostasis is disturbed as protease-mediated LTGF-β1 activation becomes unhinged, resulting in increased active TGF-β1 bioavailability, elevated fibrogenic signaling, and ultimately the worsening of hepatic fibrosis. Our findings indicate that delivering ECM1 (or respective phenocopying peptides) to the liver when it is lost can serve as a novel yet safer TGF-β1 directed therapy.