Enhanced Effectivity of an ALK5-Inhibitor after Cell-Specific Delivery to Hepatic Stellate Cells in Mice with Liver Injury

Marike Marjolijn Van Beuge,Marie Lacombe,Jai Prakash,Leonie Beljaars,Klaas Poelstra,Eduard Post,Catharina Reker-Smit

doi:10.1371/journal.pone.0056442

Marike Marjolijn Van Beuge, Marie Lacombe + Show 5 more

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https://doi.org/10.1371/journal.pone.0056442

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Abstract

Transforming growth factor-β (TGF-β) is a major pro-fibrotic cytokine, causing the overproduction of extracellular matrix molecules in many fibrotic diseases. Inhibition of its type-I receptor (ALK5) has been shown to effectively inhibit fibrosis in animal models. However, apart from its pro-fibrotic effects, TGF-β also has a regulatory role in the immune system and influences tumorigenesis, which limits the use of inhibitors. We therefore explored the cell-specific delivery of an ALK5-inhibitor to hepatic stellate cells, a key cell in the development of liver fibrosis. We synthesized a conjugate of the ALK5-inhibitor LY-364947 coupled to mannose-6-phosphate human serum albumin (M6PHSA), which binds to the insulin-like growth factor II receptor on activated HSC. The effectivity of the conjugate was evaluated in primary HSC and in an acute liver injury model in mice. In vitro, the free drug and the conjugate significantly inhibited fibrotic markers in HSC. In hepatocytes, TGF-β-dependent signaling was inhibited by free drug, but not by the conjugate, thus showing its cell-specificity. In vivo, the conjugate localized in desmin-positive cells in the liver and not in hepatocytes or immune cells. In the acute liver injury model in mice, the conjugate reduced fibrogenic markers and collagen deposition more effectively than free drug. We conclude that we can specifically deliver an ALK5-inhibitor to HSC using the M6PHSA carrier and that this targeted drug reduces fibrogenic parameters in vivo, without affecting other cell-types.

Highlights

Transforming growth factor b (TGF-b) is a major profibrogenic cytokine during liver fibrosis, playing an important role in various cellular processes such as cell proliferation, apoptosis, differentiation, migration, stimulation of extracellular matrix (ECM) synthesis, and downregulation of ECM degradation [1]
We hypothesize that coupling of an activin-like kinase 5 (ALK5)-inhibitor to mannose-6-phosphate human serum albumin (M6PHSA) will increase its uptake in hepatic stellate cells (HSC) and prevent unwanted effects in hepatocytes and immune cells. We examined this approach in vitro and in vivo to establish whether cell-specific inhibition of ALK5 in HSC can be a potential strategy to treat liver fibrosis
We found that free drug could almost completely inhibit TGF-b-induced Smad phosphorylation (90% reduction) in hepatocytes, whereas the conjugate did not have a significant effect on phosphorylation levels in these cells

Summary

Introduction

Transforming growth factor b (TGF-b) is a major profibrogenic cytokine during liver fibrosis, playing an important role in various cellular processes such as cell proliferation, apoptosis, differentiation, migration, stimulation of extracellular matrix (ECM) synthesis, and downregulation of ECM degradation [1]. The signal via ALK5 is further propagated by phosphorylation of Smad 2/3 transcription factors. The translocation of phosphorylated Smad 2/3 to the nucleus, together with co-transcription factors, leads to transcription of pro-fibrotic genes [1]. TGF-b activates many other pathways which may have pro-fibrotic effects [3]. The inhibition of the TGF-b pathway directly by small molecule inhibitors or via indirect strategies has been investigated as a potential strategy for the treatment of fibrotic diseases. In animal models for liver fibrosis and pulmonary fibrosis, inhibition of the TGF-b pathway has been shown to have anti-fibrotic effects [4,5,6], reducing extracellular matrix deposition and pro-fibrotic cytokines

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