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 R(58) and L(59) residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R(58) (R(58) LAP-DPs), can be detected mainly around activated HSCs by specific
antibodies against R(58) 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 L(59) (L(59) LAP-DPs). We demonstrated that the L(59) LAP-DPs are a potentially novel blood
biomarker reflecting hepatic fibrogenesis.
RESULTS: We established a specific sandwich ELISA to quantify L(59) LAP-DPs as low as 2 pM and measured L(59) LAP-DP levels in the
culture media of a human activated HSC line, TWNT-4 cells. L(59) 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 L(59) LAP-DP levels in the
culture media and the
mRNA expression levels of
collagen type (I) α1. In
carbon tetrachloride- and bile duct
ligation-induced
liver fibrosis models in mice, plasma L(59) LAP-DP levels increased prior to increase of hepatic
hydroxyproline (HDP) contents and well correlated with α-smooth muscle actin (αSMA) expression in liver tissues. At this time, αSMA-positive cells as well as R(58) LAP-DPs were seen in their liver tissues.
CONCLUSIONS: