L⁵⁹ TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

Background: Hepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. There are no validated biomarkers that can non-invasively monitor excessive production of ECM (i.e., fibrogenesis). Transforming growth factor (TGF)-β, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-β is enveloped by its pro-peptide, the latency-associated protein (LAP). Thus, active TGF-β must be released from the complex for binding to its receptor and inducing ECM synthesis. We recently reported that during the pathogenesis of liver fibrosis, plasma kallikrein (PLK) activates TGF-β by cleavage between R⁵⁸ and L⁵⁹ residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R⁵⁸ (R⁵⁸ LAP-DPs), can be detected mainly around activated HSCs by specific antibodies against R⁵⁸ cleavage edges and functions as a footprint of PLK-dependent TGF-β activation. Here, we describe a sandwich enzyme-linked immunosorbent assay (ELISA) that detects the other by-products, the C-terminal side LAP-DPs starting from residue L⁵⁹ (L⁵⁹ LAP-DPs). We demonstrated that the L⁵⁹ LAP-DPs are a potentially novel blood biomarker reflecting hepatic fibrogenesis. Results: We established a specific sandwich ELISA to quantify L⁵⁹ LAP-DPs as low as 2 pM and measured L⁵⁹ LAP-DP levels in the culture media of a human activated HSC line, TWNT-4 cells. L⁵⁹ LAP-DPs could be detected in their media, and after treatment of TWNT-4 cells with a TGF-β receptor kinase inhibitor, SB431542, a simultaneous reduction was observed in both L⁵⁹ LAP-DP levels in the culture media and the mRNA expression levels of collagen type (I) a1. In carbon tetrachloride- and bile duct ligation-induced liver fibrosis models in mice, plasma L⁵⁹ LAP-DP levels increased prior to increase of hepatic hydroxyproline (HDP) contents and well correlated with a-smooth muscle actin (aSMA) expression in liver tissues. At this time, aSMA-positive cells as well as R⁵⁸ LAP-DPs were seen in their liver tissues. Conclusions: L⁵⁹ LAP-DPs reflect PLK-dependent TGF-β activation and the increase in aSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.