Targeting of macrophages and hepatic stellate cells for the treatment of liver diseases

Dhadhang Wahyu Kurniawan

Research output: ThesisPhD Thesis - Research UT, graduation UT

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Liver fibrosis and its progression to liver cirrhosis and hepatocellular carcinoma (HCC) is a growing health problem affecting millions of people worldwide. This growing global health burden is attributed to hepatitis viral infections (viral hepatitis), metabolic disorders such as obesity and diabetes (non-alcoholic fatty liver disease, NAFLD) and alcohol abuse (alcohol-associated liver disease, ALD), among others. Unfortunately, no clinically approved drugs are available for the treatment of liver diseases. Liver transplantation is the only option available in case of end-stage liver failure (liver cirrhosis).
Macrophages and hepatic stellate cells (HSCs) are known to play a crucial role in the development of liver diseases, both during acute and chronic liver injury. Therefore, in this thesis, we developed and investigated novel therapeutic approaches targeting macrophages and HSCs.
We explored the effects of a small molecule SYK inhibitor, R406 (without and with nanoparticles) in vitro and in vivo. We assessed the implication of the SYK pathway in macrophages and found that inhibition of the SYK pathway using R406 resulted in the inhibition of inflammation markers i.e., nitric oxide (NO) release, and enhanced gene expression of IL-1β, FcγR1, iNOS, CCL2, IL-6, and CCR2, in a dose-dependent manner. We found that both R406-PLGA nanoparticles as well as R406 itself could significantly inhibit the gene expression of several inflammatory markers in vitro. In a subsequent in vivo study, using the MCD-diet NASH mouse model, we found that R406-PLGA nanoparticles significantly ameliorated liver inflammation, fibrosis, and steatosis as compared to free R406.
The selective small molecule SRC kinase inhibitor, KX2-391 inhibited the phosphorylation of SRC and significantly reduced the NO release in RAW macrophages. Gene expression analysis in RAW macrophages and BMDMs evidenced that SRC pathway inhibition mediated by KX2-391 attenuated the expression of different inflammatory markers including iNOS, CCL2, and FcγR1. Additionally, in a precision-cut liver slices (PCLS) model, KX2-391 decreased the gene expression of SRC, iNOS and CCL2 without affecting the cell/tissue viability. Moreover, KX2-391 attenuated hepatocytic lipid accumulation; TGFβ-induced HSCs activation, contractility and collagen expression. KX2-391 attenuated inflammation, fibrosis, and steatosis in both NASH and ASH mouse models. Mechanistic studies further revealed that SRC mediated the effects through the FAK/PI3K/AKT pathway.
We observed that FGF2 was able to inhibit TGFβ-induced HSCs activation (confirmed by reduced collagen-I and α-SMA expression), migration of HSCs (confirmed by wound healing assays) and contraction of HSCs (confirmed by 3D-gel contraction assays). These results laid the foundation for our further studies in which we conjugated FGF2 to superparamagnetic iron oxide nanoparticles (FGF2-SPIONs) to improve the stability and half-life of FGF2 and thereby to improve the therapeutic efficacy of FGF2 for the treatment of liver fibrosis. After performing several in vitro and in vivo experiments, the results revealed that the potency of FGF2-SPIONs conjugate is significantly improved as compared to free FGF2.
In conclusion, the approaches described in this thesis are promising to be explored further for the treatment of liver diseases like NASH or ALD-based liver fibrosis.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • University of Twente
  • Prakash, Jai, Supervisor
  • Storm, Gert, Supervisor
  • Bansal, R. , Co-Supervisor
Award date11 May 2022
Place of PublicationEnschede
Print ISBNs978-90-365-5344-5
Publication statusPublished - 11 May 2022


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