Mitsuko Hara

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. 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) α1. 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 α-smooth muscle actin (αSMA) expression in liver tissues. At this time, αSMA-positive cells as well as R58 LAP-DPs were seen in their liver tissues. L59 LAP-DPs reflect PLK-dependent TGF-β activation and the increase in αSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.

While there are many blood and/or tissue biomarkers as well as algorithms clinically used to assess hepatic fibrosis, a good biomarker and therapeutic target of hepatic fibrogenesis, which reflects prefibrotic changes, has not been established. The most fibrogenic cytokine, transforming growth factor (TGF)-β, is produced as a latent complex, in which TGF-β is trapped by its propeptide. On the surface of activated hepatic stellate cells, plasma kallikrein activates TGF-β by cleaving latency-associated protein (LAP) between the R⁵⁸ and L⁵⁹ residues, releasing active TGF-β from the complex. We made specific antibodies that recognize neo-C-terminal (R⁵⁸) and N-terminal (L⁵⁹) ends of LAP degradation products (LAP-DPs) and found that LAP-DPs may serve as a novel surrogate marker of TGF-β activation—namely, generation of active TGF-β—and is thus a therapeutic marker for TGF-β-mediated liver fibrogenesis in patients and can also be used to monitor effects of anti-fibrogenic factors or compounds for discovery of a novel anti-fibrosis drug.

Byproducts of cytokine activation are sometimes useful as surrogate biomarkers for monitoring cytokine generation in patients. Transforming growth factor (TGF)-β plays a pivotal role in pathogenesis of hepatic fibrosis. TGF-β is produced as part of an inactive latent complex, in which the cytokine is trapped by its propeptide, the latency-associated protein (LAP). Therefore, to exert its biological activity, TGF-β must be released from the latent complex. Several proteases activate latent TGF-β by cutting LAP. We previously reported that Camostat Mesilate, a broad spectrum protease inhibitor, which is especially potent at inhibiting plasma kallikrein (PLK), prevented liver fibrosis in the porcine serum-induced liver fibrosis model in rats. We suggested that PLK may work as an activator of latent TGF-β during the pathogenesis of liver diseases in the animal models. However, it remained to be elucidated whether this activation mechanism also functions in fibrotic liver in patients. Here, we report that PLK cleaves LAP between R⁵⁸ and L⁵⁹ residues. We have produced monoclonal antibodies against two degradation products of LAP (LAP-DP) by PLK, and we have used these specific antibodies to immunostain LAP-DP in liver tissues from both fibrotic animals and patients. The N-terminal side LAP-DP ending at R⁵⁸ (R⁵⁸ LAP-DP) was detected in liver tissues, while the C-terminal side LAP-DP beginning at L⁵⁹ (L⁵⁹ LAP-DP) was not detectable. The R⁵⁸ LAP-DP was seen mostly in α-smooth muscle actin-positive activated stellate cells. These data suggest for the first time that the occurrence of a PLK-dependent TGF-β activation reaction in patients and indicates that the LAP-DP may be useful as a surrogate marker reflecting PLK-dependent TGF-β activation in fibrotic liver both in animal models and in patients.